Final Program - Cancer Research Institute

MEETING PROGRAM
INTERNATIONAL immunotherapy SYMPOSIA SERIES
of the Cancer REsearch Institute
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
oCTOBER 3-5, 2011 • MILLENNIUM CONFERENCE CENTER • nEW YORK, NY
Cancer Immunotherapy 2011
1. SPONSORS
Millennium Conference Center
SCHEDULED ACTIVITIES AND LOCATIONS
Registration and Continental Breakfast
Ticket Lobby
• Daily, 8:00 a.m.
Plenary Lectures Hudson Theatre • Daily, Continuous
Refreshment Breaks
Tiffany Foyer
• Monday, October 3,
10:30 a.m. and 3:30 p.m.
• Tuesday, October 4,
10:30 a.m. and 3:30 p.m.
• Wednesday, October 5,
10:30 a.m.
Lunch
Gallery 8
• Monday, October 3, 12:00 p.m.
• Tuesday, October 4, 12:00 p.m.
3. SPEAKERS
4. POSTERS
5. ATTENDEES
Poster Session
Gallery 8
• Tuesday, October 4, 5:00 p.m.*
2. AGENDA
6. ABOUT CRI
7. UPCOMING
MEETINGS
*Immediately following last plenary presentation of the day
8. NOTES
INTERNATIONAL immunotherapy SYMPOSIA SERIES
of the Cancer REsearch Institute
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
sponsored by
Cancer Immunotherapy 2011:
Immune Effector Mechanisms in Tumor Immunity
October 3-5, 2011, New York City
Monday, October 3, 2011
Session 1: The Role of Antibodies
9:00 a.m.
Jeffrey V. Ravetch, The Rockefeller University, New York, NY
Enhancing anti-tumor immunity
9:30 a.m.
Patrick A. Baeuerle, Micromet, Inc., Rockville, MD, and Munich, Germany
Engagement of effector T cells for cancer therapy using bispecific BiTE antibodies
10:00 a.m.
Ana Carrizosa Anderson, Brigham and Women’s Hospital and Harvard Medical School,
Boston, MA
Tim-3 and PD-1 blockade in anti-tumor immunity
10:30 a.m.
Refreshment Break
11:00 a.m.
Mark X. Sliwkowski, Genentech, Inc., South San Francisco, CA
Multiple approaches to targeting ErbB/HER receptors in solid tumors
11:30 a.m.
Ronald Levy, Stanford University School of Medicine, Stanford, CA
Taking antibodies and vaccines to a new level in the therapy of lymphoma
12:00 p.m.
Lunch
Session 2: Myeloid Derived Cells and Their Positive and Negative Effects on the Immune
Response
2:00 p.m.
Irving L. Weissman, Stanford University School of Medicine, Stanford, CA
Cancer stem cells, programmed cell removal, and CD47
2:30 p.m.
Steven M. Albelda, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA
Role of neutrophils in anti-tumor immunity
3:00 p.m.
Alberto Mantovani, Istituto Clinico Humanitas IRCCS, University of Milan, Milan, Italy
Tumor-associated macrophages in tumor progression and as therapeutic targets
3:30 p.m.
Refreshment Break
4:00 p.m.
Vincenzo Bronte, Verona University, Verona, Italy
Post-translational chemokine modification prevents intratumoral infiltration of antigenspecific T cells
4:30 p.m.
Jeffrey W. Pollard, Center for the Study of Reproductive Biology and Women’s Health,
Albert Einstein College of Medicine, New York, NY
Monocyte and macrophage diversity promotes tumor progression and metastasis
Tuesday, October 4, 2011
Session 3: Lymphocytes and Their Positive and Negative Effects on the Immune Response
9:00 a.m.
Tyler Jacks, Massachusetts Institute of Technology, Cambridge, MA
Use of mouse models to study immune responses to cancer
9:30 a.m.
Veronika Groh, Fred Hutchinson Cancer Research Center, Seattle, WA
Roles of NKG2D and its ligands in cancer
10:00 a.m.
Khashayarsha Khazaie, Robert H. Lurie Comprehensive Cancer Center, Northwestern
University, Chicago, IL
T-regulatory cell subsets and etiology of colon cancer
10:30 a.m.
Refreshment Break
11:00 a.m.
Willem W. Overwijk, The University of Texas M.D. Anderson Cancer Center, Houston, TX
Persisting antigen-rich vaccination sites induce CD8+ T cell sequestration, dysfunction
and deletion
11:30 a.m.
Carl H. June, Abramson Cancer Center, University of Pennsylvania School of Medicine,
Philadelphia, PA
Adoptive cell therapies with CARs and TCRs
12:00 p.m.
Lunch
Session 4: Metabolism in Cancer and Immunity
2:00 p.m.
W. Nicholas Haining, Dana-Farber Cancer Institute and Harvard Medical School,
Boston, MA
Molecular mechanisms of T cell exhaustion in humans
2:30 p.m.
Yongwon Choi, University of Pennsylvania School of Medicine, Philadelphia, PA
TRAF6 and the fate of T cells
3:00 p.m.
Tak W. Mak, Campbell Family Institute for Breast Cancer Research, Princess Margaret
Hospital, University of Toronto, Toronto, Canada
Regulation of immune homeostasis in immunotherapy: To kill or be killed
3:30 p.m.
Refreshment Break
4:00 p.m.
George Coukos, Ovarian Cancer Research Center, University of Pennsylvania School of
Medicine, Philadelphia, PA
Traffic lights linking tumor tolerance to hypoxia
4:30 p.m.
Ross L. Levine, Memorial Sloan-Kettering Cancer Center, New York, NY
Role of mutations in epigenetic and metabolic pathways in myeloid malignancies
5:00 p.m.
Poster Session
Wednesday, October 5, 2011
Session 5: Advances in Clinical Immunotherapy
9:00 a.m.
Bruce W.S. Robinson, University of Western Australia, Sir Charles Gairdner Hospital,
Perth, Australia
Effective human chemo-immunotherapy – Roles of cross presentation, CD40, Tregs
and PD1
9:30 a.m.
Paul M. Sondel, Carbone Cancer Center, The University of Wisconsin, Madison, WI
Antibody dependent cell-mediated cytotoxicity as clinical therapy
10:00 a.m.
Sacha Gnjatic, Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer
Center, New York, NY
Immunogenicity of NY-ESO-1 polypeptide vaccine: Role of poly-ICLC adjuvant in rapid
induction of integrated type-1 immune response
10:30 a.m.
Refreshment Break
11:00 a.m.
Charles G. Drake, Johns Hopkins University School of Medicine, Baltimore, MD
PD-1 blockade in cancer immunotherapy
11:30 a.m.
F. Stephen Hodi, Dana-Farber Cancer Institute, Boston, MA
Combinatorial immune therapy approaches to the treatment of metastatic melanoma
12:00 p.m.
Meeting Ends
C A L L F O R PA P E R S
Cancer Immun
de Carvalho F, et al. Evaluation of LAGE-1 and NY-ESO-1 expression
1424-9634Academy of Cancer Immunology
Cancer Immunity (30 June 2011) Vol. 11, p. 2
Submitted: 10 March 2011. Resubmitted: 3 June 2011. Accepted: 9 June 2011.
Copyright © 2011 by Terry H. Wu
110602
Article
homology
for
immunotherapy.
Cancer
Immunity
2011;11:1.
Progression of cancer from indolent to aggressive
despite antigen retention and increased expression of
interferon-gamma inducible genes
Terry H. Wu1*, Karin Schreiber1*, Ainhoa Arina1, Nikolai N. Khodarev2, Elena V. Efimova2, Donald A. Rowley1, Ralph R.
Weichselbaum2 and Hans Schreiber1
Department of Pathology, The University of Chicago, Chicago, IL 60637, USA
Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL 60637, USA
These authors contributed equally to this work
1
2
*
Communicated by: PK Srivastava
Many cancers escape host immunity without losing tumor-specific
rejection antigens or MHC class I expression. This study tracks the
evolution of one such cancer that developed in a mouse following
exposure to ultraviolet light. The primary autochthonous tumor was
not highly malignant and was rejected when transplanted into naïve
immunocompetent mice. Neoplastic cells isolated from the primary
tumor were susceptible to the growth-inhibitory effects of IFNγ in
vitro, but expressed very low levels of MHC I antigen and were
resistant to tumor-specific T cells unless they were first exposed to
IFNγ. Serial passage of the primary tumor cells in vivo led to a highly
aggressive variant that caused fast-growing tumors in normal mice.
In vitro, the variant tumor cells showed increased resistance to the
growth-inhibitory effects of IFNγ but expressed high levels of
immunoproteasomes and MHC I molecules and were susceptible to
tumor-specific T cells even without prior exposure to IFNγ.
approaches for eradicating either earlier or advanced stages of
cancers. Improving this understanding was the goal of the
current study.
Results
Derivation of 4102 primary and variant cell lines
The primary 4102 tumor was induced in a C3H mouse by
chronic UV irradiation (Figure 1). Two fragments of the
primary tumor were cultured briefly (for less than 20 days) to
establish the PRIM1 and PRIM2 cell cultures and aliquots of
these cell populations were stored in liquid nitrogen for
subsequent analyses. A suspension of PRIM1 cells caused a
tumor in a nude C3H mouse. When fragments of this tumor
were subsequently transplanted, nine of ten normal C3H mice
rejected the tumor challenge. The only tumor that developed in
Keywords: mice, cultured tumor cells, melanoma, MHC, IFN- a normal mouse was adapted to culture as EarlyPRO cells and
was serially transplanted to normal mice for three additional
gamma, cytotoxic T lymphocytes, immunoproteasome
passages. In each transplant generation, all of the normal mice
developed progressive lethal tumors, indicating that the
EarlyPRO cells had already acquired a heritably stable
Introduction
Tumor progression is the continuous increase in malignant progressor phenotype. After three successive transplantations,
behavior and is typical for cancers of mice and men (1-3). Clear the growing tumor was adapted to culture and cloned to
and most dramatic examples are the development of progressor generate LatePRO. In a repeat experiment, the same frequency
variants from regressor tumors (4, 5). Regressor tumor cells at of progressor variant developed when normal mice were
any challenge dose are rejected by normal naïve hosts (6), except challenged with tumor fragments generated in nude mice by
for infrequent variants which arise from the original tumor (4, injection of PRIM1 cancer cells: in 5 mice challenged bilaterally
5). These variants have always been found to be due to heritable with tumor fragments from the nude mouse, only 1 of the 10
changes in the cancer cells within the same lineage (7-9). inocula developed a tumor.
Aggressively growing cancers may lack T cell-recognized target
antigens, show altered proteasomal processing and/or have The 4102 cell lines share a unique tumor-specific p53 mutation
reduced expression of MHC I molecules (for review see 10),
Mutations in p53 are one of the earliest genetic changes
suggesting the power of adaptive T cell immunity in cancer associated with human UV-induced skin carcinogenesis and are
surveillance. This notion is reinforced by experiments showing found in the earliest premalignant UV-induced lesions (12). We
that a mixture of anti-CD8, -CD4 and -IFNγ antibodies have shown that unique p53 mutations are convenient and
promoted the outgrowth of tumors in euthymic mice injected reliable markers to track the origin of tumor cell lines (9). To
subcutaneously (s.c.) with methylcholanthrene (11). However, confirm that the 4102 tumor cell lines are derived from a
detailed analyses of progressor variants developing from UV- common precursor, the expressed p53 sequences from PRIM1,
induced regressor tumors cells have shown that the majority of PRIM2, and LatePRO were analyzed by RT-PCR followed by
variants evolved without loss of target antigen or MHC direct sequencing. PCR primers were designed to span exons 5
expression. Understanding principle mechanisms involved in through 8 to include the majority of p53 mutation hot spots. All
the progression of these relatively frequent antigen-retention of the sequences derived from the 4102 tumor cell lines
variants should help develop better, mechanistically sound contained a C to T transition mutation that changed amino acid
www.cancerimmunity.org
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Cancer Immunity is an open access journal committed
to reflecting the broad and growing influence of immunology
on the study of cancer and on the search for ways to control
the disease. Papers, Reviews, Commentaries, and Reflections
on both laboratory and clinical studies are invited.
Publish with us.
www.cancerimmunity.org
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
SESSION ONE
The Role of Antibodies
Enhancing Anti-Tumor Immunity
JEFFREY V. RAVETCH, The Rockefeller University, New York, NY
Recent clinical success in the antigen independent activation of cytotoxic T cells has
highlighted the potential of immune activation as a therapeutic strategy for the treatment of
neoplastic diseases. However, such nonspecific activation can result in considerable toxicity as
a consequence of cytokine release from activated T cells. An additional barrier to effective
immune cell activation in a tumor setting is the observation that the tumor microenvironment is
immunosuppressive, preventing effective activation of cytotoxic cells to effectively target the
tumor cell. Ideally, effective immunotherapy would capture the specificity of antigen-driven T
cell responses through the T cell receptor (TCR), by activating the antigen-presenting cells
(APCs) responsible for restricted T cell responses directed at a tumor target. Antigen delivery
to APCs will result in antigen processing and the presentation of peptides on major
histocompatibility complex (MHC) molecules to T cells expressing the cognate TCR. However,
activation of those T cells requires that an additional signal be delivered concurrently. These
additional signals can result from the enhanced expression of costimulatory molecules,
including CD80 and CD86 on APCs triggered by adjuvants, such as microbial products or
CD40 ligation, among others. The tumor microenvironment likely prevents these co-stimulatory
molecules from effectively activating tumor specific T cells and thus fails to initiate an antitumor response capable of eradicating the tumor cells. Our goal is to develop approaches
whereby tumor-specific T cells can be stimulated in vivo to result in their activation and thereby
in the elimination of carcinoma cells.
To address this goal we have focused on CD40, a member of the TNF receptor (TNFR) super
family, a molecule expressed on antigen presenting cells (APCs), such as dendritic cells and B
cells, and shown to be essential for immune activation. While agonistic CD40 antibodies have
been developed for immunotherapy, their clinical efficacy has been disappointing. We have
found that coengagement of the Fc domain of agonistic CD40 monoclonal antibodies (mAbs)
with the inhibitory Fc receptor FcRIIB is required for immune activation. Direct comparison of
mAbs to CD40 enhanced for activating FcR binding, hence capable of cytotoxicity, or for
inhibitory FcRIIB binding, revealed that enhancing FcRIIB binding conferred
immunostimulatory activity and considerably greater anti-tumor responses. This unexpected
requirement for FcRIIB in enhancing CD40-mediated immune activation has direct
implications for the design of agonistic antibodies to TNFR as therapeutics .
The FcRIIB pathway required for agonistic CD40 antibody activities may be general to other
tumor necrosis factor receptor (TNFR) family members. For example, Fas-mediated toxicity,
triggered by agonistic Fas antibodies, requires FcRIIB. Similarly, DR4, DR5, and CD30
agonistic antibodies show greater anti-tumor activity in vivo when their Fc’s are capable of
FcRIIB engagement. Finally, the recent results showing that an agonistic CD40 antibody
(clone FGK45) has anti-tumor activity in a mouse model of pancreatic ductal adenocarcinoma
and can enhance the characteristic APC indicators such as MHC class II, CD80, and CD86
expression of stromal macrophages support an immune stimulatory component in its antitumor activity. Thus, the results presented here establish a new model for immune activation of
agonistic TNFR antibodies through FcRIIB coengagement that should inform the rational
design of novel therapeutic antibodies.
References:
Li, F. & Ravetch, J.V. (2011) Inhibitory Fc receptor engagement drives adjuvant and anti-tumor
activities of agonistic CD40 antibodies, Science 333, 1030 (2011).
Engagement of Effector T Cells for Cancer Therapy
Using Bispecific BiTE Antibodies
PATRICK A. BAEUERLE, Micromet, Inc., Rockville, MD, and Munich, Germany
Bispecific antibodies can transiently link tumor cells with otherwise inactive cytotoxic T cells in
patients for induction of potent redirected lysis of tumor cells. One example is blinatumomab
(MT103), a CD19/-CD3-bispecific BiTE for the treatment of human B cell-derived
malignancies. Blinatumomab and other BiTE antibodies were shown to activate T cells in a
highly conditional manner that is strictly dependent on the presence of target cells.
Blinatumomab has commenced a pivotal study for the treatment of adult patients with therapyrefractory acute lymphocytic leukemia (ALL). A phase 2 study in ALL patients has shown an
80% complete molecular response rate at a dose level of 15 micrograms/squaremeter per day.
Blinatumomab has also shown high response rates in non-Hodgkin’s lymphoma (NHL) patients
with follicular and mantle cell lymphoma, and first signs of efficacy in patients with diffuse large
B cell lymphoma. Centrally confirmed complete and partial responses according to Cheson
criteria were seen in NHL patients treated at a dose of 60 micrograms/squaremeter per day.
The presentation will update on the clinical development of blinatumomab in leukemia and
lymphoma.
MT110 is a novel BiTE antibody recognizing the pan-carcinoma antigen EpCAM (CD326),
which is expressed on a large variety of human adenocarcinoma, and on cancer-initiating or
stem cells derived thereof. MT110 is in a phase 1 study with gastrointestinal, lung, breast,
prostate, ovarian, and esophageal cancer patients. A murine EpCAM/CD3-specific version of
the BiTE antibody, called muS110, has shown a robust therapeutic window in mice with no
damage to EpCAM-expressing normal epithelia. A series of new BiTE antibodies for solid
tumor treatment are being developed in collaboration with large biopharma partners, including
MedImmune/AstraZeneca, Bayer Schering Pharma, Boehringer Ingelheim and Sanofi-aventis.
References:
Lutterbuese R et al. (2010) Proc Natl Acad Sci USA 107:12605-12610.
Baeuerle PA, Reinhardt C (2009) Cancer Res 69:4941-4944.
Haas C et al. (2009). Immunobiol 214 : 441-453.
Dreier T et al. (2002) Intl J Cancer 100: 690-697.
Hoffmann P et al. (2005) Intl J Cancer 115: 98-104.
Offner S et al. (2005) Mol Immunol 43: 763-771.
Brischwein K et al. (2007) J Immunother 8: 798-807.
Bargou R et al. (2008) Science 321: 974-977.
Schlereth B et al. (2005) Cancer Res 65: 2882-2889.
Brischwein K et al. (2005) Mol Immunol 43: 1129-1143.
Amann M et al. (2008) Cancer Res 68: 143-151.
Amann M et al. (2009) J Immunother 32: 452-467.
For more references, abstracts and poster presentations, please visit www.micromet.com.
Tim-3 and PD-1 Blockade in Anti-Tumor Immunity
ANA CARRIZOSA ANDERSON, Brigham and Women’s Hospital and Harvard Medical School,
Boston, MA
The immune response plays an important role in fighting cancer; however, mechanisms of
immune suppression in tumor-bearing hosts hinder productive anti-tumor immunity. T cell
dysfunction or exhaustion is one such mechanism. PD-1 has been identified as a marker of
dysfunctional or exhausted T cells in chronic disease states and blockade of PD-1/PD-1L
interactions has been shown to partially restore function to exhausted T cells. Recently, Tim-3
has also been identified as a marker of exhausted T cells. We have found that Tim-3 is
expressed on both CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) in multiple different
solid tumors. Interestingly, all CD8+ Tim-3+ TILs co-express PD-1 and Tim-3+PD-1+ TILs
represent a predominant fraction of the CD8+ T cells infiltrating tumors. CD8+Tim-3+PD-1+ TILs
exhibit the most severe exhausted phenotype as defined by failure to proliferate, produce IL-2,
TNF and IFN. We have further found that combined immunotherapeutic targeting of the Tim3 and PD-1 pathways is more effective in controlling tumor growth than targeting either
pathway alone. Indeed, 50% of the mice receiving combination immunotherapy exhibited
complete tumor regression. We have examined whether combined targeting of Tim-3 and PD1 restores effector function to TILs and have found that culture of TILs in vitro in the presence
of anti-Tim-3 plus anti-PD-L1 antibodies augments IFN production. In addition, T cells isolated
from the tumor draining lymph nodes of mice treated in vivo with anti-Tim-3 and anti-PD-L1
antibodies exhibit restored tumor antigen-specific T cell responses compared to those isolated
from mice treated with control immunoglobulins. Collectively, our data support that combined
targeting of the Tim-3 and PD-1 signaling pathways is highly effective in restoring anti-tumor
immunity.
Multiple Approaches to Targeting ErbB/HER Receptors in Solid Tumors
MARK X. SLIWKOWSKI, Genentech, Inc., South San Francisco, CA
Ligand-Dependent HER2. Inappropriate activation of HER2 occurs in human breast cancers
containing HER2 gene amplification. Although not yet clinically validated, ligand-dependent
HER2, which does not require gene amplification, is also thought to be a target in a number of
solid tumors. Pertuzumab may have utility in the treatment of these malignancies since it is
highly effective in blocking HER2’s function as a co-receptor with EGFR and HER3. HER3 is
an important component of the HER signaling machinery and is regarded as a dysfunctional
member of the HER family of receptor tyrosine kinases. Although HER3’s extracellular domain
binds heregulin (HRG), it must heterodimerize with another receptor tyrosine kinase to initiate
signaling. The most common and preferred heterodimerization partner for HER3 is HER2.
Pertuzumab antagonizes this interaction and thus prevents it from associating with HRGbound HER3.
Ligand-Independent HER2. Herceptin® (trastuzumab) is the backbone of HER2-directed
breast cancer therapy and benefits patients in both the adjuvant and metastatic settings.
Clinical observations indicate that not all patients benefit from trastuzumab-based therapies.
Moreover, a subset of patients progress following an initial response. The nature of both innate
and acquired resistance is an area of active research in both the laboratory and the clinic. It is
clear that the oncogenic potential of HER2 in HER2-amplified breast cancer is primarily, if not
solely, mediated by its ability to inappropriately activate HER3 and the PI3K pathway. The antiproliferative activity of trastuzumab is directly linked to its ability to disrupt the constitutive,
ligand-independent HER2-HER3-PI3K signaling complex that forms as a result of HER2
overexpression.
Two-In-One HER3/EGFR Antibody. Extensive crosstalk among ErbB/HER receptors
suggests that blocking signaling from more than one family member may be essential to
effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule
MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used
structural and mutational studies to understand how a single antigen recognition surface binds
two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by
inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage
immune effector functions. Compared to monospecific anti-HER antibodies, MEHD7945A was
more broadly efficacious in multiple tumor models, showing for the first time that combined
inhibition of EGFR and HER3 with a single antibody is beneficial.
Taking Antibodies and Vaccines to a New Level in the Therapy of Lymphoma
RONALD LEVY, Stanford University School of Medicine, Stanford, CA
Originally, the approach to immunotherapy of lymphoma concentrated on the unique idiotype
target present on each patient’s tumor. This customized approach has given way to an attack
on more generic targets, present on both normal and malignant B cells.
Rituximab, a monoclonal antibody against CD20, has been a great success, now being widely
adopted and having changed the practice of oncology clinical care world-wide. However, few
patients with lymphoma are being cured and successor antibodies against the same target
have not improved the efficacy of current therapy. By understanding the major killing
mechanism of rituximab, ADCC, we now have the opportunity to take its efficacy to a new level
by combining it with a second antibody against activation molecules on the killer cells, such as
41BB.
Vaccination with idiotype proteins has been tested in large scale phase III clinical trials and has
failed to show efficacy to prolong remission duration in patients with lymphoma. Subsets of
patients have been identified who may indeed have benefited from such vaccines. Also, novel
forms of idiotype vaccines have been developed that are more potent and more likely to be
efficacious.
In the meantime, a new approach to therapeutic vaccination has been developed, based on
the injection of TLR ligands directly into tumors and the subsequent elicitation of anti-tumor T
cell responses and regression of systemic tumors. This novel in situ vaccination approach can
be enhanced by antibodies against immunologic check points, such as CTLA4, Ox40 and PD-1.
The future of immunotherapy will certainly involve the combination of antibodies, vaccines and
small molecules against signaling pathways unique to malignant cells.
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
SESSION TWO
MYELOID DERIVED CELLS AND
THEIR POSITIVE AND NEGATIVE EFFECTS
ON the IMMUNE RESPONSE
Cancer Stem Cells, Programmed Cell Removal, and CD47
IRVING L. WEISSMAN, Stanford University School of Medicine, Stanford, CA
Self renewal is the principal property that distinguishes stem cells from their daughter cells;
when stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by
differentiation). The balance between self-renewal and differentiation is what generates and
then maintains tissues and enables them to respond to injury or other stressors. Studies
identifying hematopoietic stem cells (HSC) and progenitors have made hematopoiesis one of
the best systems for studying the molecular changes in cell fate decision-making and
oncogenesis. Further, it serves a paradigm for finding preclinical and clinical platforms for
tissue and organ replacement and regeneration, Stem cell isolation and transplantation is the
basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated.
Poorly regulated self-renewal can lead to the genesis of cancer stem cells (CSC), the only
cells within a tumor or leukemia that have the ability to self renew and therefore, the cells that
maintain the cancer. Thus, it is predicted that CSC elimination is required for cure. This
prediction necessitates profoundly different approaches to cancer research, compelling
investigators to prospectively isolate CSCs and to characterize the molecular pathways
regulating their behavior in order to identify targeted and truly effective therapies.
Role of Neutrophils in Anti-Tumor Immunity
STEVEN M. ALBELDA, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, and Zvi Fridlender, Hadassah Medical Center, Jerusalem, Israel
Non-malignant cells are a major part of the tumor microenvironment. These cells include
endothelial cells, pericytes, fibroblasts, and a variety of leukocytes such as lymphocytes,
dendritic cells, and myeloid cells. The most well-characterized of these myeloid cells are tumor
associated macrophages (TAMs). It is now well established that TAMs become polarized
toward an “M2” phenotype in the tumor microenvironment where they can support tumor
growth. However, another less well studied infiltrating cell is the neutrophil, which can also
heavily infiltrate cancers. We define neutrophils as CD11b+ cells that also express high levels
of the marker Ly6G with low expression of the markers F4/80 and Ly6c.
We have postulated that a similar polarization paradigm holds for tumor-associated neutrophils
(TANs). This hypothesis is based on data generated by studying the effect of TGFbeta
blockade on the myeloid populations in murine tumors. Tumor-bearing mice were treated with
an inhibitor of TGFBR1 (SM16) that prevents activation of SMADs. TGF-beta blockade led to
increased tumor necrosis with leukocyte infiltration. The infiltrating cells were CD11b+ and had
increased tumor cytotoxicity due to an augmented ability to make reactive oxygen species
(hydrogen peroxide). However, to our surprise, these infiltrating cells were not macrophages,
but intra-tumoral neutrophils. As we further characterized these neutrophils from anti-TGFbetatreated tumors compared to neutrophils from control tumors, we found that they: (1) had a
more hypersegmented morphology, (2) showed increased mRNA levels of proinflammatory
mediators such as TNFalpha, ICAM1, and CCL3, (3) decreased levels of immunosuppressive
mediators such as arginase, VEGF, and CCL2, and (4) supported, rather than inhibited, antitumor CD8 T cell activity. We thus proposed that these neutrophils had an anti-tumor “N1”
phenotype as opposed to the pro-tumor “N2” phenotype seen in control tumors. This
hypothesis was supported by experiments showing that depletion of N2 neutrophils slowed
tumor growth while depletion of N1 neutrophils (i.e. in the setting of SM16 treatment) increased
tumor growth. Stated another way, tumor-associated neutrophils can be shaped by the tumor
microenvironment to a pro-tumor (N2) or anti-tumor (N1) phenotype.
With this paradigm in mind, it is clear that there are a number of other “non-immune” activities
of N2 neutrophils that can support tumor growth. These include: (1) support of tumor
angiogenesis by release of VEGF and matrix metalloproteases, especially TIMP-free MMP-9
(that can activate growth factors), (2) augmentation of invasion, extravasation, and metastasis,
and (3) direct augmentation of tumor cell growth via uptake of neutrophil elastase.
To further characterize TAN, we performed transcriptomics on isolated Ly6G+ cells. We
compared mRNA from 4 groups of cells (all CD11b+/F480-/Ly6G+) isolated from: bone marrow
(BMN), spleens (granulocytic fraction of myeloid-derived suppressor cells, G-MDSC),
untreated tumors (N2 TAN) and tumors treated with TGFbeta blockade (N1 TAN). By principal
component analysis, the four groups are clearly separated. We first looked at the relationship
between BMN, G-MDSC and N2 TAN. BMN and G-MDSC appear to be much more similar to
each other than to N2 TAN. BMN and G-MDSC have higher levels of structural, respiratory
burst, and granule protein mRNAs than N2 TAN. N2 TAN have major changes in immune
system genes, such as APC activity, cytokine and chemokine levels. For example, mRNA
levels of CCL7, CCL8, CCL12, CCL17, CXCL1, CXCL2, CXCL9, and CXCL16 are markedly
increased. We next looked at N1 vs. N2 TAN. Although N1 and N2 TAN are more like each
other than they are like BMN and G-MDSC, there are still many changes: there were 138
genes with greater than a 10 fold difference. Almost all genes were higher in N1 TAN (except
Gpr114, CCL17) than in N2 TAN. Most inflammatory pathways were increased in N1 TAN
including inflammatory responses, responses to stress, and chemokine activity. A striking
difference was seen in chemokines where 15 of 28 tested chemokines were increased in N1
TAN, including CCL2, CCL4, CCL7, CCL8, CXCL10, and CXCL13. The only two chemokines
upregulated in N2 TAN were CXCL1 and CCL17. CCL17 is interesting, as it functions as
chemokine for CD4 T-regulatory cells. Other mRNA’s upregulated in N1 vs N2 TAN array were
TNFalpha, ICAM-1, and CCL3. VEGF and CCL5 were decreased.
Finally, TAN may play an important role in human tumors. Increased numbers of TAN are
associated with a poor prognosis in renal cell cancer and in head and neck cancer. We have
begun to analyze TAN in human lung cancers. We note a large amount of heterogeneity,
however, on average, about 5-6% of total cells digested from lung cancers stain for the
neutrophil marker CD66b. We are currently isolating CD66b-positive myeloid cells from lung
cancers, adjacent normal lung and blood and plan to conduct a genomic analysis similar to our
work in mice.
In conclusion, our data suggest, that like TAM, there are different functional populations of
TAN: a more pro-tumor (N2) phenotype and a more anti-tumor (N1) phenotype. Tumor
TGFbeta is central in the recruitment and differentiation (“education”) process of TAN. This
new paradigm suggests that TAN are a double edge sword, capable of being pro- or anti-tumor
depending on tumor microenvironment.
Tumor-Associated Macrophages in Tumor Progression
and as Therapeutic Targets
ALBERTO MANTOVANI, Istituto Clinico Humanitas IRCCS, University of Milan, Milan, Italy
Macrophages are key orchestrators of chronic inflammation. They respond to
microenvironmental signals with polarized genetic and functional programs. M1 macrophages,
which are classically activated by microbial products and interferon-, are potent effector cells
that kill microorganisms and tumors. In contrast, M2 cells, tune inflammation and adaptive
immunity; promote cell proliferation by producing growth factors and products of the arginase
pathway (ornithine and polyamines); scavenge debris by expressing scavenger receptors;
promote angiogenesis, tissue remodeling and repair. M1 and M2 cells represent simplified
extremes of a continuum of functional states. Available information suggests that TAM are a
prototypic M2 population. M2 polarization of phagocytes sets these cells in a tissue remodeling
and repair mode and orchestrate the smouldering and polarized chronic inflammation
associated to established neoplasia. Recent studies have begun to address the central issue
of the relationship between genetic events causing cancer and activation of protumor
inflammatory reactions. Rearrangement of the RET oncogene (RET/PTC) is a frequent,
causative and sufficient event in papillary carcinoma of the thyroid. It was recently observed
that RET/PTC activates a proinflammatory genetic programme in primary human thyrocytes,
including in particular chemokines and chemokine receptors. These molecules are also
expressed in vivo and more so in metastatic tumors. These results highlight a direct
connection between an early, causative and sufficient oncogene rearrangement and activation
of a proinflammatory program in a human tumor.
Therapeutic targeting of cancer promoting inflammatory reactions is in its infancy, and its
development is crucially dependent on defining the underlying cellular and molecular
mechanisms in relevant systems. Chemokines are prime targets for interfering with tumor
promotion by inflammatory reactions. Ongoing efforts along this line are encouraging.
References:
Mantovani A., Allavena P., Sica A., Balkwill F. Cancer-Related Inflammation. Nature 454: 436444, 2008.
Mantovani A. Cancer. Inflammation by remote control. Nature 435: 752-753, 2005.
Balkwill F., Charles K.A., Mantovani A. Smouldering and polarized inflammation in the initiation
and promotion of malignant disease. Cancer Cell 7: 211-217, 2005.
Mantovani A., Sozzani S., Locati M., Allavena P., Sica A. Macrophage polarization: tumorassociated macrophages as a paradigm for polarized M2 mononuclear phagocytes. 2002.
Trends Immunol..23: 549-555.
Balkwill F. and Mantovani A., 2001, Inflammation and cancer: back to Virchow? Lancet 357:
539-545.
Post-Translational Chemokine Modification Prevents
Intratumoral Infiltration of Antigen-Specific T Cells
VINCENZO BRONTE, Verona University, Verona, Italy
The goal of all immunotherapeutic approaches against solid tumors is the induction and
expansion of tumor infiltrating T lymphocytes (TILs) capable of invading tumor mass and killing
transformed cells. Unfortunately, in many tumors TILs are unable to reach the core of the
tumor and concentrate at the border of the neoplastic lesion. Several barriers can limit
complete trafficking and migration of lymphocytes within cancerous tissues. We evaluated the
possibility that reactive nitrogen species (RNS) could affect chemokine biology and contribute
to keep TILs distant from the tumor core. Chemokines are small cytokines with selective
chemoattractant properties. Deregulated expression of chemokines and their receptors is a
signature of many diseases, including autoimmunity and chronic inflammation, as well as
immunodeficiency and cancer. We found that the chemoattractants CXCL12, CCL21, and
CCL2 lost their ability to recruit T lymphocytes when exposed to peroxynitrite, a RNS produced
within tumor microenviroment. However, the modified CCL2 chemokine retained its capacity of
recruiting myeloid cells. These data indicate that RNS-altered chemokines modify the tumor
microenvironment and favor immune escape by attracting tumor-promoting myleoid cells while
restraining access to antitumor T lymphocytes. Based on our findings, drugs controlling the in
situ production of RNS might be useful to aid immunotherapeutic approaches for the treatment
of cancer, by creating a favorable tumor environment for lymphocyte recruitment and
activation. Following extensive in vitro and in vivo screenings, we developed novel small
molecules aimed at interfering with multiple, interconnected metabolic pathways leading to
RNS generation in the tumor microenvironment. Pre-conditioning of tumor microenvironment
with novel drugs that inhibited RNS production facilitated CTL invasion of the tumor and
promoted an effective cancer immunotherapy. These results unveil an unexpected mechanism
of tumor evasion and introduce new avenues for immunotherapy of tumors.
Monocyte and Macrophage Diversity Promotes
Tumor Progression and Metastasis
JEFFREY W. POLLARD, Center for the Study of Reproductive Biology and Women’s Health,
Albert Einstein College of Medicine, New York, NY
Clinical and experimental evidence indicate that macrophages promote cancer initiation and
malignant progression. Macrophages enhance malignancy at the primary tumor site by
stimulating angiogenesis, inducing tumor cell migration, invasion and intravasation and by
suppressing anti-tumor immunity. At metastatic sites macrophages promote tumor cell
extravasation, survival and subsequent growth. Each of these activities is stimulated by a
different population of macrophages (Qian and Pollard, 2010).
Lineage tracing studies show that the primary and metastatic tumors recruit different
populations of monocytes partially explaining the macrophage diversity. At the metastatic site
the recruitment of CCR2 expressing monocytes requires tumor synthesized CCL2, the ligand
for CCR2. Inhibition of CCL2 reduces this monocyte recruitment and subsequent differentiation
into macrophages and this in turn reduces tumor cell extravasation and metastatic growth
(Qian and Pollard, 2011).
References:
Qian BZ, Pollard JW (2010) Macrophage diversity enhances tumor progression and
metastasis. Cell 141:39-51.
Qian, B., Li, J., Zhang, H., Kitamura, T., Zhang, J., Snyder, L.A., Campion, L.R., Kaiser, E.A.,
Pollard, J.W. (2011) CCL2 recruited Inflammatory Monocytes Facilitate Breast Tumor
Pulmonary Metastasis. Nature 475: 222-225.
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
SESSION THREE
LYMPHOCYTES AND
THEIR POSITIVE AND NEGATIVE EFFECTS
ON the IMMUNE RESPONSE
Roles of NKG2D and its Ligands in Cancer
VERONIKA GROH, Thomas Spies, Fred Hutchinson Cancer Research Center, Seattle, WA
The stimulatory NKG2D receptor and its stress-induced ligands promote tumor immunity at
early stages of tumor development. In advanced tumors, however, persistent NKG2D ligand
expression negatively imprints on local and systemic T and NK cell responses thereby
facilitating tumor immune evasion and disease progression. Negative immune regulation by
NKG2D ligands involves ligand-induced NKG2D downmodulation and population expansions
of immuno-suppressive NKG2D+CD4 T cells. We will discuss data that define an additional
strategy whereby sustained NKG2D engagement broadly affects T cell and NK cell responses
through simultaneous impairment of multiple CD3-dependent receptor functions. In vitro and
complementing ex vivo results obtained with T and NK cells from cancer patients implicate a
chain of events that includes NKG2D downmodulation, paracrine Fas ligand (FasL) production,
caspase activation, and, finally, caspase-mediated CD3 degradation, thus establishing an
NKG2D-initiated mechanism that may promote far-reaching lymphocyte tolerization. In addition
to promoting immune evasion, however, persistent NKG2D ligand expression might constitute
yet another, perhaps more fundamental, tumor survival asset. Surprisingly, we have found that
cancer cells themselves express NKG2D receptors and may exploit the presence of its ligands
for self-stimulation of tumor growth. Ligand engagement of NKG2D on tumor cells activates
oncogenic signaling pathways, stimulates cell growth and proliferation, and induces epithelial–
mesenchymal transition (EMT), a cellular reprogramming process that promotes tumor cell
dissemination. In a preliminary assessment, the frequency of NKG2D positive cancer cells
significantly correlates with clinical criteria of tumor progression, suggesting that cancers may
co-opt NKG2D receptor expression for their own benefit.
T-Regulatory Cell Subsets and Etiology of Colon Cancer
KHASHAYARSHA KHAZAIE, Robert H. Lurie Comprehensive Cancer Center, Northwestern
University, Chicago, IL
Endowed with T-cell suppressive properties, Treg are considered to be pathogenic in cancer
and a major obstacle to tumor immune surveillance. However, potent anti-inflammatory
properties of Treg may account for recently observed longer survival of cancer patients that
have high densities of tumor infiltrating Treg. We demonstrate differential expansion of distinct
Treg subsets in human colon cancer patients. Expression of intermediate levels of RORt and
high levels of Foxp3 defined Treg that expand in a tumor dependent manner but are unable to
suppress inflammation. Treg of similar characteristics expanded in mouse models of polyposis.
Ablation of RORt produced Treg with stable anti-inflammatory properties. Resulting mice had
attenuated inflammation and almost complete protection against polyposis. Our results show
that expression of RORt distinguishes Treg with inherently pathogenic properties from those
with cancer protective functions. Thus, protective immunity can be achieved by therapeutic
targeting of the RORt expressing pathogenic Treg subset.
Persisting Antigen-Rich Vaccination Sites Induce
CD8+ T Cell Sequestration, Dysfunction and Deletion
Yared Hailemichael, Zhimin Dai, Nina Jaffarzad, Yang Ye, Miguel A. Medina, Xue-Fei Huang,
Stephanie M. Dorta-Estremera, Nathaniel R. Greeley, Giovanni Nitti, Zhi Qiang Wang, Wencai
Ma, Brian Rabinovich, Nahum Puebla-Osorio, Kimberly S. Schluns, Richard E. Davis, Patrick
Hwu, and WILLEM W. OVERWIJK, The University of Texas M.D. Anderson Cancer Center,
Houston, TX
To understand why cancer vaccine-induced T cells often fail to shrink tumors, we studied
immune responses in mice vaccinated with gp100 peptide emulsified in incomplete Freund’s
adjuvant (IFA), commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed
tumor-specific CD8+ T cells, which accumulated not in tumors but at the persisting, antigenrich vaccination site. Once there, primed T cells underwent antigen-driven apoptosis, resulting
in systemic tolerance. Treatment with anti-CD40 antibody, TLR7 agonist and interleukin-2
reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting,
water-based vaccine formulation shifted T cell localization towards tumors, inducing superior
anti-tumor activity. Short-lived vaccine formulation also reduced systemic T cell dysfunction
and promoted memory formation. Persisting peptide/IFA vaccine depots, currently used to
vaccinate cancer patients, can induce specific T cell sequestration at vaccination sites followed
by dysfunction and deletion; short-lived depot formulations may overcome these limitations
and result in greater therapeutic efficacy of peptide-based cancer vaccines.
Adoptive Cell Therapies with CARs and TCRs
CARL H. JUNE, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA
Adoptive T cells engineered to express high affinity TCRs and chimeric antigen receptors
(CARs) has promise for a number of malignancies as an approach to overcome tolerance (1,
2). The majority of non-Hodgkin's lymphomas, acute lymphoblastic leukemias and chronic
lymphocytic leukemias (CLL) express CD19, which is also expressed by normal B cells but not
by hematopoietic stem cells or other tissues. Thus CD19 represents an attractive target for
immunotherapy. Our preclinical studies show that combining robust T cell culture systems with
lentiviral vector modified human T cells expressing CD19-specific Chimeric Antigen Receptor
(CART-19) has potent anti-leukemic efficacy in mice bearing established leukemic xenografts.
4-1BB-containing signaling endodomains enhance this activity. In an ongoing feasibility and
safety clinical trial, three patients with advanced treatment refractory CLL have been treated
(3). CD3+CD45+ cells in leukapheresis products (range 2.3%-4.5%) were positively selected
with anti-CD3/anti-CD28 magnetic beads prior to CART-19 lentiviral vector transduction and
expansion. Patients were infused with a total of 0.3-5 x 109 total T cells, with 5%-27% of cells
expressing CART-19. Two of three patients remain in complete remission beyond 8 months
post infusion. The third patient had a very significant but partial response; he required
corticosteroids 18 days after infusion, during an ongoing response for symptoms presumably
related to cytokine release. We observed significant in vivo expansion in two of three patients
accompanied by long-term persistence in blood and migration to bone marrow, and delayed
onset tumor lysis syndrome accompanied by elevated levels for a broad range of cytokines.
Clinical responses were documented by normalization of blood counts, resolution of
adenopathy, and clearance of MRD when assessed by flow cytometry and deep sequencing
for IgH rearrangements, cytogenetics, and FISH studies in the bone marrow. On-target
toxicities have been observed in all patients, including cytokine release but not cytokine storm,
B cell depletion, plasma cell depletion and hypogammaglobulinemia requiring replacement
serotherapy. Confirmation of these observations that T cells expand and traffic to tumor sites,
stimulate synergistic antitumor immune activity, and persist long term in vivo potentially offers
significant therapeutic and economic advantages over existing therapies and should be
confirmed in larger numbers of patients, however, the therapeutic index with potent cell based
CAR therapies will depend to a large extent on the specificity of the target.
References:
1. Kohn D.B., Dotti G., Brentjens R., Savoldo B., Jensen M.C., Cooper L.J., June C.H.,
Rosenberg S.A., Sadelain M., Heslop H.E. 2011. CARS on Track in the Clinic: Report of a
Meeting Organized by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN)
Sub-Committee on Cell and Gene Therapy. Washington D.C., May 18, 2010. Mol Ther 19:
432-8
2. Johnson L.A., Morgan R.A., Dudley M.E., Cassard L., Yang J.C., Hughes M.S., Kammula
U.S., Royal R.E., Sherry R.M., Wunderlich J.R., Lee C.C., Restifo N.P., Schwarz S.L., Cogdill
A.P., Bishop R.J., Kim H., Brewer C.C., Rudy S.F., Vanwaes C., Davis J.L., Mathur A., Ripley
R.T., Nathan D.A., Laurencot C.M., Rosenberg S.A. 2009. Gene therapy with human and
mouse T cell receptors mediates cancer regression and targets normal tissues expressing
cognate antigen. Blood 114: 535-46
3. Kalos M., Levine B.L., Porter D.L., Katz S., Grupp S.A., Bagg A., June C.H. 2011. T cells
expressing chimeric receptors establish memory and potent antitumor effects in patients with
advanced leukemia. Science Translational Medicine 3: 95ra73
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
SESSION FOUR
METABOLISM IN CANCER
AND IMMUNITY
Molecular Mechanisms of T Cell Exhaustion in Humans
W. NICHOLAS HAINING, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA
CD8+ T cells in tumors and chronic viral infections like HIV develop functional defects such as
loss of cytokine secretion and decreased proliferative potential that are collectively termed
exhaustion. However the molecular basis of T cell exhaustion is not well understood.
Tools to capture global sets of complex biological data (such as measurements of DNA
sequence, or quantification of RNA transcript, protein or metabolite abundance) have become
widely available and commonly used. These tools can reveal how the component processes
within a cell contribute to its biological function in a way that is not evident from examining
each individually. We and others have developed techniques to generate gene expression
profiles of rare populations of antigen-specific T cells using cDNA microarrays to create broad
molecular phenotypes of MHC-peptide-tetramer+ CD8+ T cells responding to a range of acute
and chronic antigenic challenges.
In order to identify mechanisms associated with T cell exhaustion in chronic viral disease, we
studied gene expression profiles of HIV-specific T cells from infected individuals with chronic
elevation of viral load in the absence of anti-retroviral therapy (progressors) and compared
them to individuals with spontaneous control of viral replication (controllers). This allowed us to
study virus-specific T cells at the extremes of functional competence. We found that the global
pattern of gene expression in HIV-specific T cells from progressors compared to controllers
was highly similar to that seen in mouse LCMV-specific T cells responding to chronic versus
acute viral infection. Moreover, analysis of the genes that distinguish between exhausted
CD8+ T cells and their more functional counterparts identified a new role for the transcription
factor BATF in suppressing proliferation and cytokine secretion, possibly by modulating AP-1
target genes.
Our data suggests that genome-wide signatures can provide enriched pools of candidate
genes as a starting point for functionally annotating the immunological genome. However,
connecting the constituent individual parts of a signature and long-term immunity still will
require mechanistic investigation and active experimentation. The development of genomescale shRNA and open reading frame libraries has now provided the tools to scan the genome
for genes associated with aspects of the cell phenotype at high throughput. The ability to
define signatures of immune responses in memory B and T cells using genome-scale
approaches will enable increasingly comprehensive pictures of cell lineage, differentiation state
and activation status to emerge.
TRAF6 and the Fate of T Cells
YONGWON CHOI, University of Pennsylvania School of Medicine, Philadelphia, PA
CD8 T cells are critical constituents of the cell-mediated branch of the adaptive immune
response, whose extensive study has revealed life cycle phases from naïve surveillance to
effector expansion, effector contraction, and finally memory maintenance. The molecular
processes that regulate each of these phases are of great interest and are now beginning to
be understood. One area of specific interest is the regulation of cellular metabolism as T cells
transition from anabolism during expansion to catabolic restrictions during contraction. We
have recently found that CD8 T cells lacking the adapter protein TRAF6 mount a normal
primary response to bacterial infection, but then suffer hyper-contraction, resulting in a
profound T cell memory deficit. Intriguingly, we were able to link this defect to a previously
unrecognized requirement for induction of metabolic fatty acid oxidation (FAO). Here we will
further discuss the link(s) between TRAF6, FAO, and CD8 T cell memory, and whether these
relationships are shaped by external factors encountered by T cells during immune responses.
Tumor Metabolism: Back to the Future
TAK W. MAK, Campbell Family Institute for Breast Cancer Research, Princess Margaret
Hospital, University of Toronto, Toronto, Canada
Tumors arise and eventually metastasize due to the cumulative effects of multiple mutations
on multiple key genes. Oncogenes undergo mutations that cause them to become active when
they shouldn’t, and tumor suppressor genes (TSGs) sustain damaging alterations that
obliterate their protective functions. TSGs include genes that normally control cellular
differentiation, regulate cell growth and the cell cycle, participate in DNA repair, and govern
pathways leading to programmed cell death or survival. Knowledge of the roles of these genes
in preventing or promoting tumor formation has enabled molecular oncologists to seek
mechanistically-based drugs for cancer treatment. Originally, the “Oncogene Revolution”
prompted these investigators to concentrate on the development of agents that block cell
growth and cell cycle progression. Although therapeutics based on this approach have had
some success in the clinic, it has become increasingly clear that to be effective, anti-cancer
agents must also target molecules involved in the metabolism, metastasis and death of tumor
cells as well as proteins crucial for tumor angiogenesis. Our laboratory has spent much of the
last two decades identifying molecular pathways in cancer cells that can potentially be
targeted. Our work has reached a fundamental level in that we are now turning our sights on
molecules that prevent cancer cells from dying. Several major intracellular signaling pathways
involving a plethora of known and unknown genes promote tumor cell survival. One of the
most important of these pathways is driven by PI3’-kinase. In addition to its role in cellular
survival, this lipid kinase activates a diverse array of signaling pathways affecting cell mobility,
protein synthesis, proliferation, metabolism and hypoxia. Our laboratory identified DJ-1
(PARK7) as an important regulator of this pathway. More recently, mutations have also been
found in the isocitrate-dehydrogenase genes in brain cancers and leukemias. In this
presentation, I will discuss recent data from our and other laboratories suggesting that PI3’kinase-mediated signaling in tumor cells is also intimately involved in mediating the Warburg
Effect, the phenomenon whereby a cancer cell produces much of its energy through glycolysis
rather than mitochondrial oxidation of pyruvate. In addition, I will describe our laboratory’s
efforts to identify non-glucose energy sources in tumors.
Traffic Lights Linking Tumor Tolerance to Hypoxia
GEORGE COUKOS, Ovarian Cancer Research Center, University of Pennsylvania School of
Medicine, Philadelphia, PA
Chemokines play a critical role in controlling leukocyte trafficking and polarizing immune
responses. Specific chemokines recruiting CD4+CD25+FoxP3+ T regulatory cells (Treg) have
been previously proposed but the precise tumor microenvironment conditions that lead to their
expression are not well characterized. Tumor hypoxia is recognized as one of the major
contributors to cancer progression and treatment failures. We sought to understand the effect
of tumor cell hypoxia on chemokine regulation and peripheral tolerance mechanisms in the
tumor microenvironment. We identified CCL28 chemokine as one of the chemokine genes
mostly upregulated by hypoxic ovarian cancer cells. Hypoxic tumor cells were found to
upregulate CCL28 via HIF-1alpha and recruited preferentially CD4+CD25+FoxP3+ T
regulatory cells (Treg) from peripheral blood mononuclear cells (PBMC) than non-hypoxic
cells. Treg recruitment was mediated by the cognate receptor CCR10. In human tumor
xenografts, CCL28 was upregulated in areas of hypoxia in vivo, while in human ovarian cancer
we found a significant correlation between CCL28 and HIF-1 alpha proteins. Human ovarian
cancers overexpressing CCL28 were associated with shorter survival. Mouse ovarian tumors
overexpressing CCL28 were found to progress more rapidly, which was due to increased
CCR10+ Treg recruitment and it was specifically abrogated by Treg depletion with CD25
antibody. CCR10 cell depletion was also associated with significant tumor growth suppression.
Treg accumulation was associated with tolerogenic and proangiogenic tumor milieu. Besides
the expected function of suppressing T effector cells, CD4+CD25+ Treg were found to secrete
increased amounts of vascular endothelial growth factor A (VEGF-A) at baseline and under
hypoxia, which specifically promoted endothelial network expansion. Depletion of CCR10+
cells resulted in significant suppression of VEGF expression and tumor angiogenesis. In
conclusion, our results demonstrate that hypoxia promotes Treg recruitment and that Treg
besides mediating immune tolerance are also implicated in tumor angiogenesis through
production of VEGFA. Thus, immune tolerance and angiogenesis are intimately connected at
many levels and cooperate to sustain tumor growth.
Role of Mutations in Epigenetic and Metabolic Pathways in Myeloid Malignancies
ROSS L. LEVINE, Memorial Sloan-Kettering Cancer Center, New York, NY
Although mutational studies have identified somatic alterations in a substantial proportion of
patients with leukemia, the full complement of alterations which contribute to leukemic
transformation have not been delineated. In addition, clinical studies have shown that
hypomethylating agents and histone deacetylase inhibitors have clinical activity in
hematopoietic malignancies. We therefore hypothesized that epigenetic alterations contribute
to the pathogenesis of acute leukemias, either through selection of somatically acquired
epigenetic alterations or through acquisition of somatic mutations in genes which control the
epigenetic state in leukemic cells. Given recent mutational studies have identified somatic
mutations in genes known to regulate chromatin state and promoter methylation in acute
leukemia cells; we therefore have sought to investigate the effects of specific mutations on the
epigenetic state and target gene expression in leukemia cells.
Our initial efforts have focused on the role of TET2 mutations in the pathogenesis of myeloid
malignancies. We and others have found that loss-of-function, somatic mutations in TET2 are
common in patients with myeloproliferative neoplasms (MPN), myelodysplastic syndrome
(MDS), and acute myeloid leukemia (AML) and are associated with adverse outcome in normal
karyotype AML. Recent work by others has shown that TET2 belongs to a family of enzymes
which catalyze the conversion of methylcytosine to hydroxymethylcytosine; accordingly TET2mutant AML is associated with a low of hydroxymethylation and an increase in promoter
cytosine methylation. We have gone on to perform loss-of-function studies using in vitro and in
vivo models. Loss of TET2 in vitro leads to increased self-renewal and impaired hematopoietic
differentiation. Consonant with the in vitro data, loss of TET2 using a hematopoietic specific
conditional knockout mouse leads to increased stem/progenitor cell proliferation, a marked
advantage in competitive long-term transplant assays, and the development of myeloid
malignancies in vivo. Collectively these studies implicate TET2 in regulating stem cell self
renewal and hematopoietic differentiation.
Genetic studies in AML revealed that TET2 mutations were mutually exclusive with
neomorphic mutations in IDH1 and IDH2. This suggested these alleles converge on a shared
pathway in AML pathogenesis. Accordingly, AML with TET2 or IDH1/2 mutations are both
characterized by aberrant hypermethylation, and by perturbed expression of genes important
in normal hematopoietic differentiation. Recent work has shown that mutant IDH alleles are
characterized by neomorphic production of the oncometabolite 2-HG. Functional studies
showed that 2-HG blocks the ability of TET2, an alpha-ketoglutarate dependent enzyme, to
convert methylcytosine to hydroxymethylcytosine, and IDH mutant alleles impair differentiation
and enhance self-renewal in a manner similar to TET2 loss. Taken together, these studies
demonstrate that TET2 and IDH1/2 mutations converge on a metabolic/epigenetic pathway
which enhances self-renewal in stem/progenitor cells and increases susceptibility to myeloid
transformation.
cANCER iMMUNOTHERAPY 2011
IMMUNE EFFECTOR MECHANISMS
IN TUMOR IMMUNITY
SESSION FIVE
ADVANCES IN
CLINICAL IMMUNOTHERAPY
Effective Human Chemo-Immunotherapy –
Roles of Cross Presentation, CD40, Tregs and PD1
BRUCE W.S. ROBINSON, University of Western Australia, Sir Charles Gairdner Hospital,
Perth, Australia
Cross presentation and anti-tumor immunity: CD8+ T cells can exert strong anti-tumor effects but
only if they are presented with tumor antigens in an appropriate context. There are several possible
ways that tumor antigens can be presented to host CD8+ T cells – either cross presentation, direct
presentation (by the tumor cell itself) or by other less well clarified pathways. Cross presentation refers
to a process whereby antigen presenting cells (APC) capture, process and present exogenous antigens
on MHC class I molecules to CD8+ T cells.
Tumor antigen cross presentation by dendritic cells (DCs) to CD8+ T cells in draining lymph nodes
(DLN) is a key component of anti-tumor immunity. It represents the central checkpoint for the
generation of strong anti-tumor immunity. We have shown, using a number of different tumor types,
mouse strains, antigens and tumor sites that the cross presentation of tumor antigens is (i) restricted to
DLN, (ii) efficient compared to non cell-associated antigens, (iii) continuous for dominant epitopes, (iv)
samples cryptic tumor antigens, (v) increases with increased tumor load, including antigen delivered by
apoptosis induction, (vi) sequentially exposes new epitopes, (vii) is not significantly increased by CD4,
IL2, CD40, TLR3 or TLR7 signaling, (viii) can occur via MHC exchange (ix) cross-primes CTL in lymph
node only, (x) is affected by the method of tumor cell death and (xi) requires a CD40 signal to drive CTL
out of the node.
Chemotherapy and CD8 proliferation in patients: It is impossible to study cross presentation directly
in cancer patients. CD8 activation was analyzed as a surrogate. Surprisingly, baseline CD8 T-cell
proliferation (Ki67) before the initiation of chemotherapy in patients with lung malignancies (lung cancer
and mesothelioma) have an inverse correlation with survival (median survival in patients with increased
v decreased Ki67+ CD8 v median value = 6 v 11 months). In contrast, the % of proliferating CD8 cells
following the first cycle, ie. as the CD8 numbers rebounded, correlated with eventual survival following
chemotherapy (median survival in patients with increased v decreased Ki67+ CD8 = 16 v 8 months).
Furthermore, a shift in BCl2 expression suggested a shift in antigen response repertoire rather than just
homeostatic proliferation. Attempts to define antigen specific reactivity using tumor-related peptides
(mesothelin, hTERT and WT1) show limited responses (10% of patients only).
Cross-presentation and cancer chemo-immunotherapy: Many chemotherapies kill cells by
apoptosis and apoptotic cells are a good source of cross-presented antigen. In addition, some
chemotherapies alter host immunity in a way that favors anti-tumor responses eg. preferential depletion
of Tregs. Work from our laboratory revealed that the massive tumor cell apoptosis induced by
chemotherapy leads to increased levels of cross-presentation, unmasking of tumor neo-antigens and
increasing the amount of antigenic material available for cross-priming. Furthermore some
chemotherapies, especially gemcitabine and pemetrexed, require an intact immune system for maximal
effect. As this cooperation is clearly generally insufficient for cancer eradication (otherwise cancers
would melt away after chemotherapy), we evaluated which immune approaches turn this crosspresenting event into a tumor-destructive event. Activating antiCD40 is the most powerful of these.
AnitCD40 plus chemotherapy in patients: Two of the most ‘immunogenic’ chemotherapies are
gemcitabine and pemetrexed. Both synergize with antiCD40 and induced regression of established
tumors. Post gemcitabine immunotherapy using an agonistic anti-CD40 antibody cured 50-80% of
established day 20+ tumors in mice and these studies have informed our clinical trial program. We
have now treated 11 patients who have mesothelioma with a combination of pemetrexed and the
agonistic antiCD40 antibody CP-870,893 iv (0.1 – 0.2 mg/kg for 3-6 cycles). 5 patients have completed
all 6 cycles. Responses have not been excess to that which might be expected with the chemotherapy
alone, though durable stable disease has been observed and latest results will be presented. Common
side effects are a cytokine release syndrome, back pain and vomiting which resolve within 24 hours.
Lymphopenia, thrombocytopenia and thromboses are occasionally seen.
Cross presentation within tumors: When studying how DCs cross present tumor antigens most
attention has focused on ‘priming’ events in the DLN. There is some evidence that cross presentation
within tissues may also be an important component of effector CD8+ T cell responses (effector and/or
memory T cells) in those tissues. This requirement may explain the failure of tumor immunotherapies
that do manage to achieve strong CTL response – when they reach their effector site these CTLs may
not receive necessary signals from APCs within that tumor site to maintain a damaging phenotype.
Injection of CFSE-labeled HA-specific CD8+ T cells directly into the HA-expressing tumors of mice
resulted in no local proliferation of these cells, that is, there was no evidence of TCR engagement in the
tissues. In line with this, TiDCs purified from AB1HA tumors were unable to cross present HA antigen to
CD8+ T cells ex vivo. This data suggests that (i) TiDCs represent a non-cross presenting subtype, or,
(ii) that the tumor itself may be actively suppressing cross presentation by TiDCs. Antigen uptake
(fluorescent beads), processing of soluble antigens (Bodipyl DQ-OVA, recombinant HA protein) and
presentation of soluble peptide are intact, suggesting that TiDCs exhibit most other APC functions
Importantly, we have recently observed that the blockaded cross presentation pathway of DCs within
tumors can be reversed by some apoptosis-inducing chemotherapies, such as gemcitabine (GEM).
Given that GEM has a profound capacity to synergize with DC-activating agents such as CD40 or TLR7
to induce tumor regression, which is associated with intra-tumoral CD8+ T cell infiltration, the possibility
that it might do so by reversing a block in TiDC cross presentation has been evaluated. After GEM
therapy, TiDCs become able to cross present tumor antigen ex vivo. Thus our assumption that the
relevant immune events during chemoimmunotherapy are occurring in the draining lymph nodes is not
correct- chemo-induced cross presentation within tumors plays a role.
Chemotherapy and Tregs in patients: In patients with lung malignancies a higher proportion of Treg
are actively proliferating compared to other CD4+ cells (18+1 v 7+2%). Baseline Treg % correlated with
survival (median survival 6 v 12 months for Treg % < v > 7.3%). Pemetrexed chemotherapy does not
alter Treg number appreciably but cyclophosphamide (CTX) therapy reduced the former to 6% after
each cycle with a rebound to >15% before the next cycle. The CTX-induced Treg depletion is patient
specific. Continuous dosing appears to avoid rebound in Treg numbers.
In conclusion, chemoimmunotherapy is a promising approach to enhancing the effectiveness of
cancer immunotherapy in patients, but as each tumor type, each chemotherapy and each
immunotherapy has different antitumor immune mechanisms, careful analysis of the underlying anticancer immune responsiveness is required.
References:
1. Kurts C., Robinson B.W.S., Knolle P.A. Cross-priming in health and disease. Nature Reviews
Immunology, 2010 10: 403-414.
2. Lake R.A., Robinson B.W.S, Immunotherapy and chemotherapy- a practical partnership. Nature Rev
Cancer, 2005;5:397-405.
3. Steer H.J., Lake R.A., Nowak A.K., Robinson B.W. Harnessing the immune response to treat cancer.
Oncogene. 2010; 29:6301-13.
Antibody Dependent Cell-Mediated Cytotoxicity as Clinical Therapy
PAUL M. SONDEL, Carbone Cancer Center, The University of Wisconsin, Madison, WI
We have pursued immunotherapy approaches designed to eradicate persistent microscopic
cancer for patients in remission; our goal is to prevent relapse. This approach is based on 2
principles related to antibody dependent cell-mediated cytotoxicity (ADCC): (1) Certain tumor
reactive monoclonal antibodies (mAbs) can selectively bind to tumor cells (in vitro and in vivo)
marking them for ADCC by Fc Receptor-bearing cells of the innate immune system; (2)
activation of innate immune cells makes them more potent at mediating ADCC.
We have used the chimeric ch14.18 anti-GD2 mAb in combination with IL2 to activate NK cells
and GM-CSF to activate neutrophils and macrophages. This clinical effort involves a long
collaboration with Dr. Ralph Reisfeld, as well as clinical collaboration with Dr. Alice Yu and the
Children’s Oncology Group (COG). A recent COG Phase III study showed that children with
neuroblastoma receiving the ch14.18 mAb + IL2 + GM-CSF regimen following remission had
an improved event free survival (66% vs 46% p=0.012)(1). These data suggest that other
tumor reactive mAbs might also be considered for testing in the setting of minimal residual
disease, and in combination with cytokines that activate ADCC (such as IL2 and GM-CSF).
We are also investigating a genetically engineered form of this agent, hu14.18-IL2, created by
Drs. Steve Gillies and Ralph Reisfeld, that links IL2 to the humanized form of this same mAb.
This hu14.18-IL2 immunocytokine (IC) works far more effectively to destroy GD2+ tumors in
mice than the naked mAb (even when combined with IL2). Our preclinical work with this
immunocytokine and a separate tumor-reactive mAb linked to IL2, suggest that this
improvement in anti-tumor efficacy may be due to a novel role for IL2 receptors in responding
to ICs (3, 4). Namely, the ICs are far more effective at inducing activated immune synapses
between IL2-receptor bearing NK cells and tumor cells than are IL2, the naked mAb or a
mixture of IL2 and the mAb. These enhanced interactions by ICs are abrogated when the IL2
receptors are blocked (with an anti-IL2R mAb).
Our recent COG Phase II trial of this IC showed antitumor activity in children with relapsed
neuroblastoma (4). As predicted from our preclinical testing in mice, antitumor effects were
more readily apparent in children with non-bulky disease (p = 0.03). As preclinical testing
suggested an important role for NK cells in IC action, we evaluated these children for their
killer inhibitory receptors (KIR) and KIR-ligand repertoires. Genotyping showed an association
between antitumor effects in this trial and “autologous mismatch” for KIR and KIR-ligands (5).
Conclusions:
1. Ch14.18 mAb + IL2 + GM-CSF improves event-free survival for neuroblastoma
2. Activating ADCC effectors (with IL2 + GM-CSF) may augment clinical ADCC
3. ICs are more potent preclinically than the combination of mAb + IL2
4. ICs may induce additional ADCC pathways, including a new, polarizing role, for IL2Rs
5. Clinical benefit (preliminary result) for autologous KIR/KIR-L mismatch in our phase II trial of
IC suggests NK cells are responsible
Future Goals:
1. Combine hu14.18-IL2 with GM-CSF and retinoic acid (as done for ch14.18)
2. Clarify the role of KIR/KIR-L mismatch in NK-mediated clinical immunotherapy
3. Compare the roles of IL2R and FcRs in clinical ADCC using ICs
References:
1. Yu A.L., Gilman A.L., Ozkaynak M.F., London W.B., Kreissman S., Chen H., Smith M., Anderson B.,
Villablanca J., Matthay K.K., Shimada H., Grupp S.A., Seeger R., Reynolds C.P., Buxton A., Reisfeld
R.A., Gillies S.D., Cohn S.L., Maris J.M., Sondel P.M. Chimeric Anti-GD2 Antibody with GM-CSF, IL2
and 13-cis Retinoic Acid for High-risk Neuroblastoma: A Children’s Oncology Group (COG) Phase 3
Study. New England J. Med. 335:1324-1334, 2010.
2. Gubbels J.A.A., Gadbaw B., Buhtoiarov I.N., Horibata S., Kapur A.K., Patel D., Hank J.A., Gillies
S.D., Sondel P.M., Connor J., Patankar M.S.. Ab-IL2 Fusion Proteins Mediate NK Cell Immune
Synapse Formation by Polarizing CD25 to the Target Cell-Effector Cell Interface. Cancer Immunol
Immunother. 2011 Jul 27. [Epub ahead of print] PubMed PMID: 21792658
3. Buhtoiarov I.N., Neal Z.C., Gan J., Buhtoiarova T.N., Patankar M.S., Gubbels J.A.A., Hank J.A.,
Yamane B., Rakhmilevich A.L., Reisfeld R.A., Gillies S.D., Sondel P.M. Differential internalization of
hu14.18-IL2 immunocytokine by NK and tumor cell: impact on conjugation, cytotoxicity and targeting.
Journal of Leukocyte Biology, 89:625-638, 2011. PMID: 21248148
4. Shusterman S., London W.B., Gillies S.D., Hank J.A., Voss S., Seeger R.C., Reynolds C.P., Kimball
J., Albertini M.A., Wagner B., Gan J., Eickhoff J., DeSantes K.D., Cohn S.L., Hecht T., Gadbaw B.,
Reisfeld R.A., Maris J.M., Sondel P.M. Anti-tumor activity of hu14.18-IL2 in relapsed/refractory
neuroblastoma patients: a Children’s Oncology Group (COG) phase II study. J. Clin. Oncology,
28(33):4969-4975; 2010. PMID: 20921469
5. Delgado D.C., Hank J.A., Kolesar J., Lorentzen D., Gan J., Seo S., Kim K.M., Shusterman S., Gillies
S.D., Reisfeld R.A., Yang R., Gadbaw B., DeSantes K.D., London W.B., Seeger R.C., Maris J., and
Sondel P.M. The Role for Genotypes of Killer Ig-Like Receptors (KIRs), Their Ligands, and Fcγ
Receptors on Responses of Neuroblastoma Patients to Hu14.18-IL2: A Children’s Oncology Group
Report. Cancer Research, 70:9554-9661, 2010; PMCID:PMC299644, PMID: 20935224
Immunogenicity of NY-ESO-1 Polypeptide Vaccine:
Role of Poly-ICLC Adjuvant in Rapid Induction of Integrated Type-1 Immune Response
SACHA GNJATIC, Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer
Center, New York, NY
Many vaccine formulations of cancer-testis antigen NY-ESO-1 have demonstrated induction of
measurable specific immunity, but to this day, none has been shown to consistently and
reliably generate integrated high-avidity antibody (Ab), CD4+ and CD8+ T cell responses in
every vaccinated patient. Long peptides are efficiently presented to both CD4+ and CD8+ T
cells after internalization and intracellular processing by antigen presenting cells in vitro. To
investigate their immunogenicity in vivo and the effect of vaccine adjuvants, we conducted a
clinical trial with overlapping long peptides (OLP) from NY-ESO-1 in ovarian cancer patients
who received either NY-ESO-1 OLP alone (n=4), OLP in Montanide (n=13), or OLP in
Montanide plus poly-ICLC (polyinosinic-polycytidylic acid-stabilized by lysine and
carboxymethylcellulose) (n=11). High-avidity NY-ESO-1-specific CD4+ T cells were detected in
patients vaccinated with OLP+Montanide±polyICLC but they were found deleted after
vaccination with OLP alone. NY-ESO-1-specific integrated Ab, CD4+ and CD8+ induction was
undetectable after OLP alone vaccine, but found together in 4/13 patients vaccinated with
OLP+Montanide and 10/11 patients vaccinated with OLP+Montanide+polyICLC. Inclusion of
poly-ICLC not only enhanced and accelerated the induction of immune responses, but also
increased their polyclonality and durability. Poly-ICLC also enhanced NY-ESO-1-specific IFN-producing CD4+ T cell responses while suppressing IL-4- and IL-9-producing CD4+ T cell
responses. These results show that OLP can induce consistent, rapid, high-quality, and
durable integrated antibody, CD8+ and CD4+ T cell responses when given with proper adjuvant
in nearly all vaccinated patients.
PD-1 Blockade in Cancer Immunotherapy
CHARLES G. DRAKE, Johns Hopkins University School of Medicine, Baltimore, MD
Programmed Death-1 (PD-1), and its two B7 family ligands, B7-H1 (PD-L1) and B7-DC (PDL2) play important roles in microbial immunity, tumor immunity and autoimmunity. PD-1
engagement has been shown to induce T cell apoptosis as well as to inhibit proliferative
responses and cytokine release in vitro in response to TCR signaling. Direct evidence that PD1 represents an inhibitory receptor came from analysis of PD-1 knockout mice, which may
develop strain-specific autoimmunity later in life. Most of the inhibitory roles of PD-1 on T cell
responses have been attributed to its interaction with B7-H1. In vivo, B7-H1:PD-1 interactions
have been documented to inhibit T cell effector responses to both tumors as well as to normal
peripheral tissues. It is indeed possible that the up-regulation of B7-H1 by tumors that serves
to protect them from immune attack is a reflection of the normal expression of B7-H1 within
peripheral tissues, where expression during acute injury serves to protect tissues from
bystander immune attack. Clinically, blocking PD-1 with a specific monoclonal antibody has
resulted in objective responses in patients with renal cell carcinoma, melanoma, colorectal
cancer and other tumor types. Phase II studies to optimize dosing are ongoing in renal cell
cancer. Interestingly, PD-1 blockade has relatively subtle effects on the kinetics of a primary
CD8 T cell response to vaccination, whereas combining PD-1 blockade with blockade of the
checkpoint mediated by LAG-3 results in more dramatic effects on CD8 T cell trafficking and
effector function. These results are reflected in several animal models, where dual blockade of
PD-1 and LAG-3 can result in more significant delays in tumor outgrowth than blockade of
either molecule alone. Taken together, these data suggest a more personalized approach to
cancer immunotherapy, in which several patient-specific checkpoints are blocked
simultaneously.
Combinatorial Immune Therapy Approaches
to the Treatment of Metastatic Melanoma
F. STEPHEN HODI, Dana-Farber Cancer Institute, Boston, MA
CTLA-4 blockade with ipilimumab demonstrated a survival advantage in advanced melanoma.
We have demonstrated that ipilimumab induces an immune mediated tumor vasculopathy. In
addition to angiogenic effects, VEGF is a potent immune suppressor of antigen presenting
cells. As a result, we performed the first combination study to investigate potential synergies of
ipilimumab and bevacizumab. In a phase I study evaluating the combination, 22 patients have
been treated. Side effects have included giant cell arteritis(1), hypophysitis(3), thyroiditis(4),
grade 3-4 hepatitis(2), bilateral uveitis(2), and grade 2 colitis(2); 5 pts required systemic
steroids and stopping treatment. All toxicities resolved. Best overall response to date includes
8 partial responses and 6 stable disease. Post-treatment biopsies in 12 pts revealed activated
vessel endothelium with extensive T cell trafficking not seen with ipilimumab alone, as well as
non-productive central angiogenesis. Peripheral blood monitoring revealed marked increase in
CD4/CCR7/CD45RO central memory cells in majority of patients, not seen with ipilimumab
alone. Ipilimumab plus bevacizumab can be safely administered and provides a signal of
clinical activity that provides the basis for further exploration.
POSTER SESSION
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Acosta-Alvear, Diego** .................P1
Amin, Rupesh** ............................P2
Arnold, James**............................P3
Bakowski, Malina** .......................P4
Beemiller, Peter** .........................P5
Behar, Marcelo** ..........................P6
Belousov, Pavel ............................P7
Berezhnoy, Alex ...........................P8
Bergsbaken, Tessa** ....................P9
Blaisdell, Adam...........................P10
Blander, Julie..............................P11
Braga, Walter .............................P12
Brasel, Kenneth ..........................P13
Buckwalter, Matthew ..................P14
Budhu, Sadna.............................P15
Buhrman, Jonathan* ...................P16
Burchill, Matthew** .....................P17
Byrne, Katelyn ............................P18
Cameron, Garth..........................P19
Cappello, Paola ..........................P20
Carroll, Kyla ................................P21
Ceccato, Christina ......................P22
Chapman, Caroline.....................P23
Chaudhry, Ashutosh** ................P24
Chaumeil, Julie** ........................P25
Chen, Emily*...............................P26
Chen, Weisan .............................P27
Chou, Jeffrey** ...........................P28
Chow, Melvyn* ...........................P29
Cooper, Anthony** ......................P30
Crane, Courtney .........................P31
da Silva, Inês ..............................P32
Daugherty, Matthew**.................P33
Deng, Weiwen** .........................P34
Diaz, C. Marcela .........................P35
Dougan, Stephanie .....................P36
Draganov, Dobrin .......................P37
Duan, Mubing*............................P38
Dubey, Purnima..........................P39
Dutoit, Valerie .............................P40
Dzhagalov, Ivan** .......................P41
Eikawa, Shingo...........................P42
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Elinav, Eran** .............................P43
Engel, Alex** ..............................P44
Fan, Xiaozhou ............................P45
Feng, Yongqiang** .....................P46
Fleenor, Courtney* .....................P47
Fox, Jamie* ................................P48
Franz, Bettina .............................P49
Graves, K. Nicole .......................P50
Gross, Gideon ............................P51
Grotzke, Jeffrey** .......................P52
Gudlur, Aparna** ........................P53
Guo, Chunguang** .....................P54
Hadad, Uzi .................................P55
Hailemichael, Yared ...................P56
Hamilton, Melisa .........................P57
Han, Jin-Hwan** .........................P58
Head, Jonathan ..........................P59
Hearth, Chandana ......................P60
Hirschhorn-Cymerman, Daniel ...P61
Huang, Alex ................................P62
Huang, Wendy Jia Men** ...........P63
Ito, Hiroaki** ...............................P64
Jacobsen, Kristen*......................P65
Jensen, Kirk** .............................P66
Junqueira, Caroline ....................P67
Kamphorst, Alice**......................P68
Kodgire, Prashant** ....................P69
Kornbluth, Richard ......................P70
Kovacsovics, Magdalena ............P71
Kreymborg, Katharina** ..............P72
Kueh, Hao Yuan** ......................P73
Kuttruff, Sabrina .........................P74
Lechner, Melissa ........................P75
Lee, Hee Eun .............................P76
Lee, Jung-Hoon** .......................P77
Lee, Sang Yull ............................P78
Leonhardt, Ralf** ........................P79
Lesokhin, Alex ............................P80
Lorenzi, Julio ..............................P81
Loskog, Angelica ........................P82
Lu, Haihui** ................................P83
Maeda, Yuka ..............................P84
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Mandruzzato, Susanna...............P85
Mangsbo, Sara ...........................P86
Manzo, Teresa ...........................P87
Mathew, Rebecca** ....................P88
Matsushita, Hirokazu ..................P89
Matsuzaki, Junko ........................P90
Mayya, Viveka** .........................P91
McKenzie, Brienne .....................P92
Menager, Mickael** ....................P93
Meng, Feilong** ..........................P94
Messina, Nicole* .........................P95
Mim, Carsten** ...........................P96
Moake, Matthew* ........................P97
Monu, Ngozi** ............................P98
Mukherjee, Sulakshana** ...........P99
Muller, Alexander .....................P100
Nagai, Takeshi .........................P101
Naito, Masayasu .......................P102
Napetschnig, Johanna** ...........P103
Ndjamen, Blaise** ....................P104
Ngiow, Shin Foong* ..................P105
Nirschl, Christopher ..................P106
Nistico, Paola ...........................P107
Ogembo, Javier** .....................P108
Ohue, Yoshihiro........................P109
Pearce, Oliver** ........................P110
Pentcheva-Hoang, Tsvetelina ...P111
Perica, Karlo* ...........................P112
Petrácková, Martina..................P113
Pukkila-Worley, Read** ............P114
Rakhmilevich, Alexander ..........P115
Reboldi, Andrea** .....................P116
Sabado, Rachel ........................P117
Saccheri, Fabiana ....................P118
Sag, Duygu ..............................P119
Saint Fleur, Ashley* ..................P120
Santos, Luara ...........................P121
* CRI-Funded Predoctoral Student
** CRI-Funded Postdoctoral Fellow
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Schaer, David ...........................P122
Schietinger, Andrea** ...............P123
Sellars, MacLean** ...................P124
Seth, Rashu** ...........................P125
Simpson, Tyler .........................P126
Singh, Manisha.........................P127
Smigiel, Kate* ...........................P128
Spendlove, Ian .........................P129
Spiegel, David ..........................P130
Srinivasan, Nithya ....................P131
Steinel, Natalie* ........................P132
Stone, Jennifer .........................P133
Stromnes, Ingunn** ..................P134
Sugiyama, Daisuke...................P135
Teng, Grace**...........................P136
Tiyanont, Kittichoat** ................P137
Toettcher, Jared** ....................P138
Trifari, Sara** ............................P139
van den Broek, Maries .......P140-142
Vigdorovich, Vladimir** .............P143
Vincent, Isaah...........................P144
Wang, Xin Xiang** ....................P145
Weir, Genevieve .......................P146
Wen, Haitao** ...........................P147
Wesley, Johnna ........................P148
Wheeler, Lee*...........................P149
Wilson, John** ..........................P150
Wood, Laurence .......................P151
Xiao, Hui** ................................P152
Yan, Qingrong** .......................P153
Yang, Chenghua** ....................P154
Yassin, Mohammed* ................P155
Zanzinger, Kai ..........................P156
Zemp, Franz .............................P157
Zou, Tao* .................................P158
Zurkova, Kamila .......................P159
P1 - Selective Inhibition of IRE1 Triggers Apoptosis in Multiple Myeloma Cells
1
4
4
4
2
**DIEGO ACOSTA-ALVEAR , Luis M. Gomez , Olivia Farias , Sebastian Bernales , Marc A. Shuman , and
1,3
Peter Walter
1
2
3
Department of Biochemistry and Biophysics, Department of Medicine and Howard Hughes Medical Institute, University of
4
California, San Francisco, CA, USA; Fundacion Ciencia Para la Vida, Santiago, Chile
Inositol-requiring enzyme 1 (IRE1) is the most conserved sensor of the protein folding status in the endoplasmic
reticulum (ER). IRE1 activates the transcription factor X-box binding protein 1 (XBP1) via unconventional
splicing, establishing control of a core gene-regulatory program in charge of adjusting the capacity of the
secretory apparatus. In this way, the IRE1-XBP1 pathway modulates the cell physiology to adapt to a
heightened ER function, like that of professional secretory cells. As such, XBP1 is required for plasma cell
differentiation. XBP1 has also been identified as a critical factor driving the progression of multiple myeloma
(MM), a plasma-cell malignancy, in a transgenic mouse model (Carrasco DR, et al. 2007), and its activity has
been reported as a poor prognosis indicator in MM patients (Bargatuni T, et al. 2010). Thus, IRE1-XBP1
signaling constitutes an integral part of the physiology of MM, likely acting as a cytoprotective response, which
may confer a survival advantage to MM cells. We have characterized IRE1 signaling in a panel of eight MM cell
lines and we show that these cells have a remarkably fast and robust IRE1-dependent ER stress response. The
high expression of both IRE1 and active XBP1 allows these cells to withstand perturbations of ER function,
suggesting that MM cells heavily rely on IRE1 for their normal physiology and survival. Indeed, selective
inhibition of the nuclease function of IRE1 with small molecule modulators disrupts XBP1 splicing in MM cells
and triggers apoptosis. Importantly, the cytotoxic effect resulting from IRE1 inhibition is selective toward MM
cells but not other transformed cells of the B-cell lineage. Thus, we posit that the IRE1-XBP1 pathway is
essential for the survival of MM and that inhibition of XBP1 activation presents itself as an attractive target for
developing novel therapies for MM.
P2 - Artificial Tethering of Chromatin Modifying Enzymes and Nuclear Structural Proteins to
Endogenous Cellular Genes
**RUPESH H. AMIN and Mark Groudine
Fred Hutchinson Cancer Research Center, Basic Sciences Division, Seattle, Washington
Insertion of an array of bacterial repressor protein binding sites into a chromosomal site allows for visualization
and tracking of the array in live cells via binding of a fluorescent protein-bacterial repressor fusion. Similarly,
such an array can be tethered to chromatin modifying enzymes or nuclear structural proteins (i.e., nuclear lamin
proteins or nuclear pore components) to alter the transcription or nuclear localization of neighboring genes.
Previously such experiments have been done on artificial gene constructs and in a limited manner. Using a
gene trap approach we have now begun to apply this technology to endogenous mouse genes on a genome
wide scale. Our gene trap consists of a fluorescent protein driven by the trapped cellular gene promoter and an
array of tet operator binding sites. Tethering GFP to the operator array allows visualization and tracking of the
trapped locus in live cells by fluorescence microscopy—thus correlating transcriptional output to nuclear
location. Additionally, tethering various chromatin modifying enzymes or structural proteins to this array can alter
the transcriptional output of the particular gene in either a positive or negative manner. We are thus able to
assess the importance of various chromatin modifications and nuclear localization to transcription and the
maintenance of transcriptional programs. Future work will be directed at testing the effects of these tethering
proteins on cellular genes during differentiation from embryonic stem cells to B lymphocytes.
P3 - Tumor Stromal Cells Expressing Fibroblast Activation Protein-α Play a Pivotal Role in the
Suppression of Anti-Tumor Immunity
**JAMES NOBLE ARNOLD, Matthew Kraman*, Paul J. Bambrough*, and Douglas T. Fearon
The University of Cambridge, The Cancer Research UK Cambridge Research Institute, The Li Ka Shing Centre, Cambridge,
United Kingdom; *These authors contributed equally to this work
Therapeutic vaccination against cancer-associated antigens has largely been ineffective. The observation of
‘concomitant immunity’ suggests that the tumor stroma, which is a mixture of hematopoietic and mesenchymal
cells, is sufficient to suppress an otherwise effective immune reaction within the tumor. A stromal cell type,
expressing the type II membrane protease, fibroblast activation protein- (FAP), was first identified in the stroma
of human cancers 20 years ago by Old and colleagues. However, little was known about the cells that express
FAP or their role in the tumor microenvironment. FAP+ stromal cells in ectopic Lewis lung carcinoma (LL2)
tumors were characterized as a population of stromal cells of mesenchymal (CD45-) and of hematopoietic
(CD45+) origin, and each accounted for 1% of total tumoral cells respectively. The mesenchymal CD45- FAP+
cells had a myofibroblast phenotype, expressing α-smooth muscle actin, as well as type I collagen and CD90.
The hematopoietic CD45+ FAP+ cells were a subpopulation of macrophages, accounting for approximately 10%
of total tumoral macrophages, which expressed, as well as FAP, the markers F4/80, CD11b, CD16/32, and
CCR2, but not Gr-1. They also expressed mannose receptor, as did all tumoral macrophages, consistent with
an M2 macrophage phenotype. However, tumoral mRNA levels for IL-4 and IL-13, which polarize macrophages
to an M2 phenotype, were at least an order of magnitude lower than for IFN-. To assess whether the FAPexpressing populations had a function in suppressing anti-tumor immunity, a bacterial artificial chromosome
transgenic mouse was generated in which FAP+ stromal cells expressed the primate diphtheria toxin receptor to
permit their conditional ablation by the administration of diphtheria toxin. Depletion of FAP+ stromal cells from
immunogenic, ovalbumin-expressing LL2 tumors caused acute hypoxic necrosis of the tumor, which was
suppressed with concomitant administration of neutralizing monoclonal antibodies to IFN- and TNF-.
Therefore, the FAP+ stromal cells have a non-redundant immune suppressive function in the tumor
microenvironment that prevents immune control of tumor growth.
P4 - Microsporidia Pathogenesis in the Nematode C. elegans
**MALINA A. BAKOWSKI and Emily R. Troemel
University of California, San Diego, La Jolla, CA
Microsporidia are highly reduced intracellular parasites closely related to fungi. They are environmentally
ubiquitous and increasingly recognized as medically relevant pathogens with more than 14 different
microsporidia species found to infect humans. Unfortunately, treatment options for these infections are limited.
Moreover, due to difficulties in culturing microsporidia in the laboratory (they must grow inside host cells), the
molecular interactions of microsporidia and host remain largely unexplored.
Nematocida parisii is a species of microsporidia and a natural pathogen of the nematode C. elegans. N. parisii
infects the worm’s intestinal cells and shortens its host’s lifespan. Conveniently, due to the nematode’s genetic
tractability and transparency, the C. elegans/N. parisii infection model is an excellent tool to study
microsporidian pathogenesis. Using this system we plan to discover N. parisii virulence factors as well as
immunity pathways in C. elegans that respond to this intracellular infection.
To this end, in collaboration with the Broad Institute and as part of the Microsporidian Genomes Consortium we
have recently sequenced the genome of N. parisii and performed RNA-seq analysis to determine worm and
microsporidia transcriptomes during distinct stages of infection. Based on these data we have chosen candidate
virulence factors as predicted secreted N. parisii proteins and are currently testing the effects of their
overexpression in C. elegans intestinal cells.
This work will lead to greater understanding of microsporidia pathogenesis by identifying microsporidia virulence
factors and host pathways targeted by microsporidia during infection. It may also yield valuable information
about the C. elegans immune response to intracellular invaders. Furthermore, because of the similarity between
worm and human intestinal epithelial cells, understanding N. parisii infection in worms may shed light on
mechanisms of microsporidia pathogenesis in humans.
P5 - T Cell Receptor Microclusters Centralization in Immune Synapses is Controlled by a Unique
Mechanism Relying on Both Actin Polymerization and Depolymerization
**PETER BEEMILLER and Matthew F. Krummel
University of California San Francisco, San Francisco, CA
T cell receptors (TCRs) are activated when T cells form specialized cell-cell interfaces—immune synapses—
with antigen presenting cells (APCs) bearing agonist peptides that bind the specific TCRs expressed on the T
cell. TCR activation leads to the aggregation of TCRs into micron-sized signaling microclusters that flow
centripetally within the interface. The formation and centralization of TCR microclusters regulate the activation of
T cells, determining their responses to disease-associated peptides, including cancer antigens. TCR signaling
and flows are dependent on the actin cytoskeleton, but the biomechanics are not fully understood, and the
factors that link TCRs to the actin cytoskeleton and stimulate flow remain unknown. Vav1, a known factor in T
cell activation, is capable of binding proteins in TCR signaling complexes and is linked to the actin cytoskeleton
through several mechanisms, including activation of the Rho-family GTPases Cdc42 and Rac. We proposed
that ligated TCRs recruited Vav1 to microclusters to locally activate Cdc42 and Rac, triggering actin
polymerization directly at sites of TCR signaling. We examined the localization of Vav1 in synapses using a
supported lipid bilayer approach that facilitates the analysis of synapses with high spatiotemporal resolution.
Though Vav1 partitioned into centripetally flowing microclusters, Vav1 microclusters were distinct from TCR
microclusters. When we examined the GTPases, we found that neither Cdc42 nor Rac localize to TCR
microclusters, or any particular region of the immune synapse. Furthermore, Cdc42 and Rac activation
appeared dispensable for synapse processes, as expression of dominant negative isoforms did not impair TCR
microcluster formation or centralization. In accord with this, when we analyzed synapse dynamics in T cells
expressing a mutant Vav1 isoform that cannot activate small GTPases, we observed that TCR microcluster and
synapse formation were not impaired. Intriguingly, though, we noticed that TCR microcluster flows in the
synapse were distinct from the flows of other microclusters, undergoing both inward and outward directed flows
depending on their position in the synapse. The generation of inward TCR flows required actin
depolymerization, which established an actin-poor central void that partitioned synapses. This indicates that
TCR-independent actin polymerization controls TCR flows in immune synapses into regions of low actin filament
density.
P6 - Stimulus-Specific Signal Dynamics Provide an Opportunity for Stimulus-Specific Inhibition of
Pleiotropic Signaling Hubs
**MARCELO BEHAR and Alexander Hoffmann
Signaling Systems Laboratory, Dept. Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, and San
Diego Center for Systems Biology, La Jolla, CA
Control of aberrant signals in disease is the aim of many therapeutic approaches. However, many promising
drug targets are highly pleiotropic signaling hubs that transduce signals from multiple pathways. Our previous
work showed that stimulus-specific signaling dynamics could provide sufficient signal insulation to allow for
faithful transduction of distinct signals via a single hub. Here, we address whether perturbations (that may be
induced by therapeutic agents) targeting such a hub could be identified that affect signaling in response to one
stimulus but not another. We first explored this possibility with simple conceptual signaling modules typically
present in signaling networks that are capable—to varying degrees—of faithful stimulus-specific transduction.
Our analysis revealed that perturbations that affect time scales or dose levels relevant for the transduction of
one pattern of signal dynamics but not others allowed for stimulus-specific effects that were robust with respect
to the perturbation strength. We then extended our analysis to the clinically important IB-NF-B signaling
module where we are able to identify perturbations likely to primarily affect the NF-B response to cytokines
(TNF) with minimal effects on the response to pathogen components (LPS), and vice versa. Together, our
results demonstrate that signal dynamics can be treated as a virtual pharmacological target and reveal
principles potentially useful for informing the development of therapeutic agents aimed at pleiotropic signaling
hubs.
P7 - Autoantibody Signature of Differentiated Thyroid Tumors: Immunoproteomic Identification of
Tumor Antigens Expressed in Two Thyroid Cancer Cell Lines
1,2
,2
2
3
3
PAVEL V. BELOUSOV , Yan S. Kim , Andrey A. Tvardovsky , Arthur T. Kopylov , Sergei A. Moshkovskii ,
4
4
1
1,2
Kirill V. Lanshchakov , Vladimir E. Vanushko , Alexey Yu Sazykin , Sergei A. Nedospasov , and Dmitry V.
1,2
Kuprash
1
2
Faculty of Biology, Lomonosov Moscow State University; Engelhardt Institute of Molecular Biology, Russian
3
4
Academy of Sciences; Institute of Biomedical Chemistry, Russian Academy of Medical Sciences; National
Center of Endocrinology; Moscow, Russia.
The repertoire of self-antigens eliciting immune responses in patients with well-differentiated thyroid tumors is
largely unexplored. To begin systematic analysis of this issue, we performed serological proteome analysis
using sera of patients with benign and malignant thyroid neoplasms to identify protein targets of humoral antitumor immune responses expressed in papillary and follicular thyroid cancer cell lines (K1 and FTC133,
respectively). Combined autoantigenic profile of two cell lines comprised several dozens of reproducibly reactive
protein spots in two-dimensional Western blot analysis, 15 of them being recurrently reactive in thyroid
neoplasms vs. healthy donors, malignant vs. benign (or vice versa) fashion, and/or associated with a particular
tumor histotype. NanoLC-ESI-MS/MS analysis of tryptic fragments was performed to identify targets of thyroid
tumor-specific immune responses. Although the molecular chaperones (particularly heat shock proteins) was
the top subgroup enriched in the identified protein set (5 out of 15 antigens), other identified proteins with
miscellaneous functions seem to be more relevant to the biology and diagnostic/therapeutic aspects of these
neoplasms; possible causes of immunogenicity and diagnostic potential of identified antigens will be discussed.
Identified autoantibody signature may provide novel tools for differential diagnostics of benign and malignant
thyroid tumors, as well as give some clues about molecular pathogenesis of differentiated thyroid neoplasms.
P8 - Enhanced Memory Development in Mice by Aptamer Targeted mTOR Inhibition in CD8+ T Cells
ALEXEY BEREZHNOY, Iris Castro, Thomas Malek, and Eli Gilboa
University of Miami, Miller School of Medicine, Miami, FL
Recent studies have highlighted the importance of immunological memory, especially CD8+ T cell memory, in
mediating protective immunity in chronic infectious diseases and in cancer. New insights into the molecular
mechanism underpinning effector/memory differentiation of CD8+ T cells have uncovered several intrinsic
pathways that regulate this process. For example, inhibition of mTOR activity in mice with rapamycin redirected
the activated CD8+ T cells to differentiate into memory cells, which was accompanied by enhanced protective
anti-tumor immunity. Notwithstanding, systemic delivery of pharmacological agents could compromise their
immune promoting properties due to their often pleiotropic effects. Rapamycin, for example, also promotes the
development of immune suppressive CD4+ T cells and tolerogenic dendritic cells.
To this end we have used RNAi technology to inhibit intracellular mediators of T cell differentiation that were
targeted to CD8+ T cells using an oligonucleotide aptamer that binds to 4-1BB expressed on activated CD8+ T
cells. We have shown that a 4-1BB targeted raptor siRNA, a key component of the mTORC1 complex,
downregulated mTOR activity in about 60% of adoptively transferred OVA-primed OT-I cells, which was
accompanied by a significant increase in memory precursor effector cells (MPEC) exhibiting a central memory
phenotype. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory
response as judged by a substantial increase of proliferating OT-I cells following a second antigenic exposure.
Consistent with the hypothesis that targeted mTOR inhibition in CD8+ T cells will obviate undesirable effects
elicited by non-targeted mTOR inhibition with rapamycin, treatment of mice with rapamycin, but not with 4-1BBtargeted siRNA, compromised the cytotoxic effector function of the reactivated OT-1 cells.
Aptamer-targeted mTOR inhibition in CD8+ T cells was also able to enhance the differentiation of adoptively
transferred Pmel-1 cells that was as or more effective than treatment with rapamycin, and 4-1BB-targeted
inhibition of Blimp-1 and T-bet enhanced the formation of memory OT-I cells, though not as effectively as
inhibition of mTOR.
Potentiating vaccine-induced CD8+ T cell memory using cell-targeted inhibition of intracellular mediators of T
cell differentiation could contribute to the development of increasingly effective immune-based methods to
control tumor progression in cancer patients.
P9 - Activation of Caspase-1 in CD8+ T Cells
**TESSA BERGSBAKEN and Michael J. Bevan
University of Washington, Seattle, WA
CD8 T lymphocytes play an important role in controlling a variety of infections and certain malignancies. CD8 T
cells undergo a characteristic phase of rapid expansion after antigen encounter, but only a small number of
these cells survive to form a long-lived memory population, and questions remain about the signals that dictate
memory formation. Previous studies have shown that caspase-1 mRNA is dramatically upregulated in effector
CD8s at the onset of the contraction phase, but the role of this protease in lymphocyte function has not been
addressed. Caspase-1 is initially made as an inactive precursor and can be processed and activated in
response to a variety of stimuli. Caspase-1 cleaves and activates inflammatory cytokines and can mediate cell
death; however, low levels of caspase activation have also been implicated in cell survival. To examine the
activation of caspase-1 in vivo, effector CD8 T cells from infected mice were labeled directly ex vivo using a
fluorescently labeled inhibitor of active caspase-1. A small but significant portion of effector CD8s had activated
caspase-1 when compared to CD8 T cells from naïve mice, and caspase-1 activation peaked at the onset of
contraction. In addition, CD8 T cells that had undergone caspase-1 activation had increased expression of
CD127, indicating these cells were destined to become memory cells. In vitro activation of CD8 T cells was used
to examine how the strength of TCR signaling influenced caspase-1 activation. CD8 T cells were stimulated with
peptides over a range of TCR affinities. Low level TCR stimulation, either using lower affinity peptides or
reduced concentrations of higher affinity peptides, stimulated maximal caspase-1 activation. CD8s that had
undergone caspase-1 activation were phenotypically distinct from the caspase-1 negative population; caspase1+ cells were slightly smaller in size, had downregulated CD62L and CD25 expression, and their mitochondria
produced higher levels of reactive oxygen species (ROS) when compared to those that had not activated
caspase-1. ROS have been implicated in both caspase-1 activation and cell death; however, CD8s with high
ROS levels and active caspase-1 retain viability and the ability to proliferate both in vitro and in vivo. Caspase-1
has been implicated in regulating the metabolic function of cells via autophagy, cholesterol production, and
glycolysis, and we hypothesize capase-1 may be regulating the metabolic function of CD8 T cells.
P10 - Evolving Inflammatory Characteristics of the Tumor Microenvironment in a Mouse Model of
Endometrial Carcinoma
1
3
1
3
ADAM BLAISDELL , Takiko Daikoku , Chin SIean Tay , Sudhansu K Dey , and Adrian Erlebacher
1
1,2
2
Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York,
3
NY; Division of Reproductive Sciences, The Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of
Cincinnati College of Medicine, Cincinnati, OH
One mechanism by which tumors suppress the tumor-specific adaptive immune response involves recruitment
of immunosuppressive leukocyte populations such as myeloid-derived suppressor cells (MDSCs). How these
leukocytes interact in situ with the main perpetrators of adaptive immunity – namely dendritic cells (DCs) and T
cells – remains poorly understood. To address these questions, we took advantage of the recently established
cre
lox/lox
PR Pten
mouse model of autochthonous endometrial cancer, in which conditional deletion of the tumor
suppressor gene Pten within the uterus leads to rapid and synchronous tumorigenesis throughout the entire
organ. Surprisingly, pre-malignant but not malignant lesions contained a greater density of DCs and T cells
compared to non-tumor-bearing controls. Furthermore, although pre-malignant and malignant lesions exhibited
+
+
a significant accumulation of Ly6G CD11b cells with a phenotype similar to granulocytic MDSCs, this
+
+
accumulation was far more pronounced in pre-malignant lesions. The vast majority of Ly6G CD11b cells were
recruited into the neoplastic epithelium itself, while most DCs were detected in close proximity but excluded from
the epithelium, suggesting a possible interaction between these two leukocyte populations. Indeed, antibody+
+
hi
hi
mediated depletion of Ly6G CD11b cells increased the proportion of phenotypically mature (MHCII CD86 )
+
+
DCs, suggesting that these Ly6G CD11b cells may suppress DC maturation and the subsequent priming of
tumor-specific T cells. Furthermore, while the dissipation of inflammation that occurred with tumor progression
correlated with a loss of COX2 expression, it was unexpectedly independent of Treg tissue densities. Together
these results suggest a causative link between COX2 expression, MDSCs, and adaptive immunity that define
the overall inflammatory characteristics of the evolving tumor microenvironment. Future studies will focus on the
+
+
cellular and molecular mechanisms by which Ly6G CD11b cells within the tumor microenvironment manipulate
DC and T cell phenotypes, and how these altered phenotypes might further cross-regulate the inflammatory
characteristics of the tumor.
Supported by a grant from the American Cancer Society (RSG-10-158-01-LIB to A.E.).
P11 - Superior Tumor Immunotherapy by Simultaneous Targeting of Toll-Like and Nod-Like Receptors
1
1
2
Johan Garaude , Andrew Kent , Nico Van Rooijen , and J. MAGARIAN BLANDER
1
1
Immunology Institute, Department of Medicine, and The Tisch Cancer Institute, Mount Sinai School of Medicine, New York,
2
NY; Vrije Universiteit, VUMC, Department of Molecular Cell Biology, Faculty of Medicine, Van der Boechorststraat,
Amsterdam, The Netherlands
Toll-like receptor (TLR) ligands are increasingly being used as adjuvants in cancer vaccine trials with the goal of
mobilizing effective anti-tumor immune responses. Despite some success, considerable challenges remain that
necessitate further improvements in vaccine design. Here we show that expression of the TLR ligand flagellin
within tumor cells constitutes an effective anti-tumor vaccination strategy that relies on simultaneous
engagement of TLR5 and Nod-like receptors (NLRs) NLRC4/Naip5 along with co-delivery of tumor antigen and
flagellin into antigen processing compartments ideally suited for antigen presentation. Although TLR5 signaling
was critical for mediating rapid macrophage-dependent clearance of flagellin-expressing tumor cells in vivo,
+
+
surprisingly both TLR5 and NLRC4/Naip5 were equally important for priming anti-tumor CD4 and CD8 T cells
and suppressing tumor growth. Vaccination with irradiated flagellin-expressing tumor cells prevented tumor
development, and disrupting flagellin recognition by TLR5 or NLRC4/Naip5 impaired protective immunization
against an existing or subsequent tumor. Our findings delineate a new strategy for anti-cancer immunotherapy
consisting of introducing into tumor cells microbial structures with dual TLR and NLR stimulatory activity. This
ensures recognition of tumor-derived antigen within the inflammatory context of microbial recognition, and
additionally mobilizes both the phagocytic and cytosolic pathways of innate immune defense against the tumor.
P12 - Overexpression of CTLA-4 in the Bone Marrow of Patients with Multiple Myeloma as a Sign of
Local Accumulation of Immunosuppressive Tregs – Perspectives for Novel Treatment Strategies
1
1
1
2
WALTER M.T. BRAGA , Antonio C. Carvalho , Andre L. Vettore , Djorge Atanackovic , and Gisele W. B.
1
Colleoni
1
Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil;
Hamburg, Germany
2
University Medical Center Hamburg-Eppendorf,
Introduction: Multiple myeloma (MM) development involves a series of genetic alterations and changes in the
bone marrow (BM) microenvironment, favoring the growth of the tumor and failure of local immune control. T
regulatory (Treg) cells play an important role in the maintenance of self-tolerance and modulation of overall
immune responses against infections and tumor cells. T helper 17 (Th17) cells, on the other hand, have a
critical function in eradicating extracellular pathogens and tumor cells. The balance between Treg and Th17
cells may be essential for maintaining homeostasis of anti-tumor immunity. The aim of our study was to
characterize the expression of Treg- (FOXP3 and CTLA-4) and Th17-related (RORt) genes in total MM bone
marrow samples to assess the local immune milieu as a potential therapeutic target in this still incurable
disease. Material and Methods: Expression of FOXP3, CTLA-4, and RORt was determined by quantitative
real-time PCR (qPCR) in BM aspirates of 55 MM patients, 4 patients with monoclonal gammopathy of
undetermined significance (MGUS), 5 patients with solitary plasmacytoma (SP), and 5 healthy BM donors.
Genes were considered overexpressed when their expression was at least two times higher in myeloma BM
than in normal samples. Results: RORt showed a non-significant overexpression in MM patients compared to
controls. FOXP3, on the other hand, showed overexpression in 63% of MM cases and a 3.6-fold higher
expression in MM patients compared to controls, but the difference was not significant. CTLA-4 expression was
14.6-fold higher in MM patients compared to controls (p=0.03, Mann-Whitney test) and was overexpressed in
70% of MM cases. Importantly the CTLA-4/RORt ratio was significantly higher in MM samples compared to
controls (p = 0.009), while this difference was not significant when controls were compared to MGUS and SP
patients, suggesting that the immunological imbalance worsens with the progression of disease. Conclusions:
CTLA-4 (cytotoxic T lymphocyte antigen-4), an intracellular and membrane inhibitory protein expressed on Treg
cells, modulates T cell activation and is critical in maintaining immune tolerance to self-antigens. Monoclonal
antibodies targeting CTLA-4 have improved the survival of patients with metastatic melanoma in clinical trials.
The myeloma-related overexperssion of CTLA-4 and the increased CTLA-4/RORt ratio suggest an
accumulation of immunosuppressive Tregs in the tumor microenvironment and/or an immediate involvement of
this gene in the development and progression of myeloma. If protein expression confirms gene expression
imbalance between Treg and Th17 subpopulations, therapeutic approaches that specifically target CTLA-4expressing Treg myeloma cases may provide a new treatment strategy for this disease. (Supported by CNPq,
Brazil.)
P13 - A Preclinical Model for Autologous Cellular Immunotherapy Targeting CA9-Expressing Tumors
KENNETH BRASEL, Craig Meagher, Marykay Ligocki, Lauren Cerretti, Sam Li, and James Trager
Dendreon Corp, Seattle, WA
Sipuleucel-T is an FDA approved autologous cellular immunotherapy for asymptomatic or minimally
symptomatic, metastatic castrate-resistant prostate cancer; in controlled clinical studies sipuleucel-T
demonstrated a prolongation in overall survival. Sipuleucel-T consists of patients’ own peripheral blood
mononuclear cells (PBMC), including antigen presenting cells (APC), which have been cultured and activated in
the presence of a recombinant antigen composed of prostatic acid phosphatase (PAP), a prostate-specific
antigen, linked to granulocyte-macrophage colony stimulating factor (GM-CSF). This approach is being applied
to other cancer-associated antigens, but to our knowledge no group has yet reported the preparation of a
cellular immunotherapeutic composed of PBMC in a preclinical setting. Here we describe a method for use of
mouse PBMC to elicit a T cell response to human Carbonic Anhydrase IX (hCA9) and to protect mice from
tumors expressing hCA9. Mouse PBMC were cultured with recombinant hCA9 fused to murine GM-CSF (hCA9GM) or with GM-CSF alone; the cultured cells were injected intraperitoneally into congenic mice. Alternately, the
mice were immunized by injection of hCA9 in complete Freund’s adjuvant (CFA). Under both protocols, mice
were immunized biweekly. Two weeks after the third immunization, serum and spleens were collected from
euthanized cohorts and used to assess humoral and cellular responses to hCA9. Remaining mice were
inoculated with hCA9-expressing tumor cells, and tumor growth followed over time. Mice immunized with hCA9GM-cultured PBMC, or with hCA9 in CFA mounted robust T cell responses to CA9, though only the latter group
developed humoral responses. Mice immunized with PBMC cultured in hCA9-GM (but not GM alone) were
protected against hCA9-expressing renal carcinoma (RENCA) tumor cells in a dose-dependent manner.
Protection against RENCA tumor cell challenge was similar whether mice were immunized with cultured PBMC
or with hCA9 in CFA. The degree of protection provided by this cellular immunotherapy was markedly greater in
the murine bladder tumor model MBT-2 engineered to express CA9. Many complete tumor rejections were
observed in this setting. In conclusion, we show potent immune and anti-tumor responses to an autologous
cellular immunotherapy prepared directly from mouse PBMC.
P14 - Migration of Donor Lymphocytes is Required for Efficient Cross-Priming of Cell-Associated
Antigen
MATTHEW R. BUCKWALTER, Lucie Colineau, Hélène Jusforgues-Saklani, and Matthew L. Albert
Immunobiologie des Cellules Dendritiques, Immunology Department, Institut Pasteur, Paris, France
Uptake and processing of cell-associated antigen by host antigen presenting cells (APCs) results in cross+
presentation of peptide epitopes to antigen-specific CD8 T cells. Previous experimental models and current
vaccination protocols in humans have utilized the transfer of donor leukocytes as a source of antigen. Using two
models of antigen cross-presentation, we performed a component analysis in order to define those cells capable
of efficient antigen transfer. To our surprise, migratory cells were more efficient at delivering antigen to host
+
APCs and eliciting CD8 T cell cross-priming. Interestingly, live donor T and B cells could be recovered from the
draining lymph node, although each population demonstrated distinct kinetics of migration and capacities to
persist following inoculation. Selectively inhibiting donor cell migration from the inoculation site blocked antigen
cross-priming without, however, interfering with host APC movement. Furthermore, we establish that
+
persistence of living donor cells at the site of immunization was required for achieving robust CD8 T cell crosspriming. Histological analysis revealed a localization of donor cells within the periphery of the lymph node,
potentially determining the host APC subset available to engulf and cross-present antigen. These results may
help advance the development and optimization of cell-associated vaccine strategies.
P15 - An In Vitro System for Quantitatively Assessing Suppressive Effects of the Tumor
Microenvironment on Antigen-specific CD8+ T Cell Killing of Cognate Antigen-Expressing Tumor Cells
1
1
1
2
1
1
SADNA BUDHU , David Schaer , Taha Merghoub , John Loike , Alan Houghton , Jedd Wolchok , and Samuel
2
C. Silverstein
1
2
Sloan-Kettering Institute for Cancer Research, New York, NY; Columbia University College of Physicians and Surgeons,
New York, NY
In a previous study we demonstrated that the efficiency with which in vitro-activated ovalbumin (ova) peptidespecific mouse CD8 OT-1 T cells kill cognate peptide-expressing mouse B16 melanoma cells in collagen fibrin
gels in vitro and in melanomas in vivo is dependent on the CD8 T cell concentration and is described by an
equation (Eq. 1, bt/b0 = e-kpt + gt) originally derived to describe neutrophil bactericidal activity in vitro and in
vivo. Using this equation, we determined the Critical antigen-specific CD8+ T cell Concentration (CTC) (the
concentration of antigen-specific CD8 T cells required to hold constant the concentration of a growing population
of tumor cells). The CTCs were ~3.5x105 in vitro activated OT-1 T cells/ml collagen-fibrin gel for OT-1 T cell
killing B16 cells in vitro and ~3x106 in vitro activated OT-1 T cells/ml or gm tumor cells for OT-1 T cell killing B16
cells in tumors in vivo. This 10-fold difference in CTC stimulated us to use our collagen-fibrin gel system to
investigate the factors that hinder CD8+ T cell killing of tumor cells in vivo. We compared the efficiency with
which in vitro-activated OT-1 cells kill ova-expressing B16 melanoma cells alone vs. ova-expressing cognate
antigen-expressing B16 melanoma cells in dissociated cell suspensions of B16 melanomas formed by
inoculating C57Bl/6 mice with the same ova-expressing B16 melanoma cells 8-10 days previously. We found
that OT-1 cells killed tumor-derived ova-B16 cells about half as efficiently as they killed the cell culture-derived
ova-B16 cells used to produce these tumors. Our findings suggest that suppressor cells and substances
produced by these cells reduced the OT-1 cells’ cytolytic activity, that this assay can be used to determine the
magnitude of suppression produced by leukocytes, stromal cells, and the substances they secrete within the
tumor parenchyma, and identify these cells and secretory substances.
P16 - Enhanced T Cell Function by Focusing Mimotope Activated T Cells Against a Self-Tumor Antigen
*JONATHAN BUHRMAN and Jill Slansky
Integrated Department of Immunology, University of Colorado Denver at National Jewish Health, Denver, CO
One approach that enhances the T cell response to tumors is vaccination with peptide mimics of tumor epitopes,
or mimotopes. Mimotopes function by activating T cells that cross-react with the native tumor antigen. We have
been targeting the AH1 epitope of the mouse colon carcinoma CT26 to develop such mimotopes. Using a
tumor-specific CD8+ T cell clone expanded in culture, we identified mimotopes that protect mice from tumor
growth and other mimotopes that do not afford protection. Protective mimotopes elicit more AH1 cross-reactive
cells than non-protective mimotopes, and the responding T cells from protective vaccines more closely resemble
the naturally responding T cell repertoire against AH1. We hypothesized that we can use naturally responding T
cells in the tumors to identify multiple mimotopes optimized to activate those T cells and that vaccination with a
mixture of these mimotopes will facilitate enhanced tumor protection. We first sought to determine if a mixture of
previously identified mimotopes would facilitate tumor protection in our model. Surprisingly, we discovered that
despite eliciting large numbers of AH1-specific cells, mice were not protected from tumor challenge when
vaccinated simultaneously with five previously identified mimotopes. Very few of the mimotope-specific cells, as
detected by tetramer staining, cross-reacted with the native AH1. We hypothesized that we may refocus the
immune response against AH1, and away from the mimotopes, by boosting our mix peptide vaccine with AH1.
Importantly, prime-boost vaccination with AH1 alone does not prevent tumor growth in our system. Indeed,
priming the immune response with mimotopes, followed by a native antigen boost, resulted in an increase in
tumor protection following CT26 challenge. This vaccination strategy using a single non-protective mimotope
also resulted in a significant increase in tumor-free survival. Despite having fewer AH1-specific cells at the time
of tumor challenge, we show that these AH1-specific cells have increased affinity for AH1 tetramer and
increased functional avidity against AH1. Additionally, we show that AH1-specific cells generated with this
vaccination strategy secrete multiple cytokines upon peptide stimulation and produce more IFN- on a per-cell
basis than cells elicited by mimotope vaccination alone. These data suggest that using mimotope vaccines in
conjunction with native peptide antigen may improve the quality of the cross-reactive T cells and enhance antitumor immunity.
P17 - TNFR:TNFR-Ligand Interactions Drive CD8+ T Cell Clonal Competition
1
2
**MATTHEW A. BURCHILL , Philippa Marrack , and Ross M. Kedl
1
2
2
National Jewish Health, Denver, CO; Integrated Department of Immunology, University of Colorado Denver, Howard
Hughes Medical Institute, Denver, CO
The ability to respond to self and foreign antigens requires a network of signals between antigen-presenting
cells and responding T lymphocytes. In our studies, we demonstrate that the ability to access the TNFR-ligand
CD70 confers a competitive advantage to CD8+ T cells in response to foreign antigen. Specifically, adoptive
transfer studies have demonstrated that CD8+ T cells sufficient for CD27 are able to suppress the expansion of
endogenous antigen-specific CD8+ T cells to a greater extent than CD27-deficient CD8+ T cells. This difference
is a direct consequence of access to CD70 on antigen presenting cells such that in the presence of CD70
deficient antigen presenting cells both CD27-sufficient and CD27-deficient cells are equally competitive. To
dissect the mechanism by which access to CD70 on antigen presenting cells regulates the competitive ability of
CD8+ T cells we utilized in vitro and in vivo assays. These assays demonstrated that dendritic cell surface
expression of CD70 is reduced in the presence of CD27+ T cells, but not CD27- T cells. Furthermore,
preliminary studies indicate that upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the
surface of CD8+ T cells and captured by CD70-expressing dendritic cells. These data indicate that CD8+ T cells
of the same specificity can compete against one another by directly restricting access to TNFR-ligands
expressed by antigen presenting cells. Preliminary experiments using two-photon microscopy have
demonstrated that CD27-sufficient OTI T cells have a slower mean velocity than CD27-deficient OTI T cells
shortly after immunization. Current studies are directed at dissecting if the differences in velocity observed are
due to differential contact durations between T cells and antigen presenting cells. Collectively, our data
demonstrate that the mechanism of competition between antigen-specific CD8+ T cells is through the regulation
of interactions between CD27 and its ligand CD70.
P18 - Ongoing Priming of Melanoma/Melanocyte-Specific CD8 T Cells Occurs in Hosts with Vitiligo
KATELYN T. BYRNE and Mary Jo Turk
Dartmouth College, Lebanon, NH
Vitiligo, or the autoimmune destruction of melanocytes, has long been correlated with improved survival in
melanoma patients. While vitiligo has often been disregarded as a side effect of robust anti-melanoma T cell
responses, recent data from our laboratory show that antigen provided by melanocyte destruction is actually
required for long-lived CD8 T cell memory against melanoma-expressed self-antigens. However, the possibility
that autoimmune melanocyte destruction itself may prime CD8 T cells that can cross-react against melanoma
has not been investigated. Previously, we reported vitiligo development in 60% of mice that are depleted of
regulatory CD4 T cells while bearing established B16 melanoma tumors. To probe the immunogenicity of vitiligo,
we adoptively transferred naïve melanocyte antigen (gp100)-specific pmel CD8 T cells into actively
depigmenting hosts. We find that CD8 T cell activation occurs in an antigen-specific manner; pmel cells
upregulate CD44 and undergo several rounds of division only in hosts with vitiligo. Furthermore, although the
vitiligo-primed CD8 T cells do not produce IFN-, the cells do produce Granzyme B and express LAMP-1.
Additionally, vitiligo-primed CD8 T cells express the skin-homing marker P selectin ligand. Interestingly,
although vitiligo-primed CD8 T cells appear strongly activated, the cells are unable to form robust memory
populations in the presence of CD4 T regulatory cells. Ongoing studies aim to investigate the anti-melanoma
capabilities of vitiligo-primed CD8 T cells. These studies will further enhance our understanding of the direct
relationship between tumor immunity and autoimmunity, and the contributions of each to protecting the host
against recurrent tumor growth.
P19 - A Molecular Basis for Antigen Specificity and Functional Responses of Natural Killer T Cells
1
2
2
1
1
2
GARTH CAMERON , Onisha Patel , Kwok Wun , Adam Uldrich , Daniel Pellicci , Travis Beddoe , Jamie
2
1
Rossjohn , and Dale Godfrey
1
Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia;
Crystallography Unit, Monash University, Melbourne, Australia
2
The Protein
Natural Killer T (NKT) cells respond to a range of CD1d-restricted antigens despite maintaining a restricted TCR
repertoire. The prototypic NKT cell ligand, -galactosylceramide (-GalCer) has been shown to provide antitumor effects in various cancer models. However, several other glycolipid antigens are emerging as potential
alternatives such as -C-galactosylceramide (-C-GalCer) and -mannosylceramide (-ManCer). There is
strong evidence that structurally distinct glycolipids can elicit different responses, although the molecular basis
for antigen discrimination by the NKT cell TCR receptor is unclear. Utilizing a range of structurally distinct
glycolipid analogues, we have attempted to dissect the molecular basis for antigen discrimination by combining
functional, affinity, and structural data. We have demonstrated that modifications in the composition of the
glycosyl headgroup (3’-deoxy--GalCer, 4’-deoxy--GalCer, and -GlcCer) or modification of the glycosidic
linkage (-C-GalCer) directly impacted TCR interactions, which in turn affected the NKT cell cytokine response,
with ligand potency broadly correlating to the t1/2 life of the interaction. Acyl chain modification (C20:2) did not
affect the interaction, but did impact on ligand potency in terms of NKT cell proliferation, suggesting an effect on
antigen processing and presentation. Truncation of the phytosphingosine chain (OCH) resulted in an induced fit
mode of TCR binding, which reduced TCR affinity. It has also been demonstrated that certain glycolipid antigens
preferentially expanded subsets of NKT cells based on V expression. Glycolipids such as 4’ deoxy--GalCer
and -GlcCer showed a bias toward NKT cells that expressed V7, whereas OCH preferentially expanded those
expressing V8, which suggests a role for TCR- chain in discrimination between different glycolipid antigens.
P20 - DNA Vaccination with Alpha-Enolase Significantly Delays Tumor Progression in a GEM Model of
Pancreatic Cancer
1,2
1,2
1,2
1
3
PAOLA CAPPELLO , Simona Rolla , Roberta Curto , Robert Chiarle , Federica Cavallo , Mirella
1,2
1,2
Giovarelli , and Francesco Novelli
1
2
Center for Experimental Research and Medical Studies (CERMS), San Giovanni Battista Hospital, and Department of
3
Medicine and Experimental Oncology, University of Torino, Torino, Italy; Molecular Biotechnology Center, University of
Torino, Turin, Italy
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid
progression, invasiveness, and resistance to treatment. We have used SERological Proteome Analysis
(SERPA) to define -enolase as a PDAC-associated antigen and evaluated its ability to elicit specific CD4+ and
CD8+ T cell reactivity. The majority of PDAC patient sera contains IgG to -enolase, an enzyme of the glycolitic
cascade that also acts as a plasminogen receptor, and that is overexpressed in PDAC compared to normal
pancreas and chronic pancreatitis. Alpha-enolase-pulsed dendritic cells specifically stimulate healthy autologous
T cells to proliferate, secrete IFN- and lyse PDAC cells. In vivo, -enolase-specific T cells inhibited the growth
of PDAC cells in immunodeficient mice. Taken as a whole these data indicate that -enolase induces an
integrated humoral and cellular response in PDAC patients and could elicit specific T cell reactivity in vivo.
By using genetically engineered mice (GEM) developing spontaneously PDAC, we investigated the potential of
-enolase as a therapeutic vaccine. In GEM vaccinated with a vector expressing cDNA for human -enolase, a
specific -enolase immune response improves anti-tumor responses and survival. The immune response is
associated with an increased level of anti--enolase-specific IgG, an augmented frequency of -enolase-specific
Th1/Th17 cells, and a decrease of suppressor cells. The ability of α-enolase DNA vaccine to delay pancreatic
cancer progression has major implications for setting new immunotherapies in the treatment of PDAC.
P21 - Targeting TGFβRII in Oncology: A Potent Immunomodulatory Antibody with Various Effects on
Cancer Progression
KYLA DRISCOLL CARROLL, Zhaojing Zhong, Desiree Nugent, Xiaohong Xu, and John Haurum
Imclone Systems/Eli Lilly & Co., New York, NY
Signaling via the TGF pathway plays diverse roles in tumor progression, directly and indirectly driving tumor
cell growth and pathogenesis. Direct pro-tumor effects, which occur in TGF receptor-expressing tumor cells,
include autocrine mitogen production, epithelial to mesenchymal transition (EMT), invasion, migration, and prometastatic cytokine production. Indirect pro-tumor effects, which occur in stromal cells, include
immunosuppression and angiogenesis. Due to the pleiotropic effects of the TGF pathway in cancer, we set out
to disrupt signaling using a monoclonal antibody (mAb) that blocks the ectodomain of TGFRII, in order to inhibit
receptor-mediated signaling in target cells. Here we describe a high affinity, fully human anti-TGFRII mAb
along with its murine surrogate. We observed in vivo efficacy in various human and murine tumor models
treated with anti-TGFRII as a monotherapy. In addition, anti-TGFRII therapy functioned at least additively with
the cytotoxic cyclophosphamide. Using immune competent murine models of aggressive disease, we have
shown that the major indirect mechanism of action of our antibody involves enhanced anti-tumor immunity.
Specifically, using an immune-depletion strategy, we have shown that depletion of CD8+ cytotoxic T
lymphocytes (CTL) eliminated the ability of anti-TGFRII to inhibit primary, but not metastatic, tumor growth.
Conversely, depletion of NK cells eliminated the ability of anti-TGFRII to inhibit metastasis while having little
effect on inhibition of primary tumor growth. Analysis of NK cells and CTL ex vivo showed that treatment with
anti-TGFRII significantly induced killing activity by these two populations of cells. In addition, the Th1 cytokine
response marked by IFN secretion by NK cells and CTL was increased in anti-TGFRII treated animals.
Therefore, antibody treatment significantly induced anti-tumor immunity in vivo. Finally, the circulating
immunosuppressive T regulatory (Treg) and MDSC populations were reduced in anti-TGFRII treated animals.
In vitro, treatment with the anti-TGFRII antibody inhibited TGF-induced conversion of naïve T cells into Treg
cells and Treg cell-mediated inhibition of T cell proliferation. Collectively, these data demonstrate that selective
blockade of TGFRII with a neutralizing antibody suppresses primary tumor growth and metastasis through both
direct and indirect attenuation of TGFβ signaling. The results of these studies provide compelling data
supporting the utilization of a neutralizing anti-TGFRII antibody as a novel therapeutic strategy for the
treatment of TGF-dependent tumors.
P22 - IL-2 and IL-7 Drive IL-17 Secreting CD8 Cells (Tc17) Toward IFN-Production In Vitro
CHRISTINA M. CECCATO, Nicholas Durham, Christopher Nirschl, Angela Alme, Xiayou Pan, Ada Tam, and
Charles G. Drake
Johns Hopkins School of Medicine, Baltimore, MD
Inflammation plays an important role in both infection and cancer, especially in the context of the tumor
microenvironment. Many groups have shown that CD4+ T cells activated in the presence of inflammatory
cytokines and TGF- secrete IL-17 (Th17). There is conflicting evidence about the role of Th17 cells in a cancer
setting and whether these cells have pro- or anti-tumor effects. We and others showed that CD8+ T cells
cultured in the presence of IL-6 and TGF- acquired the capacity to secrete IL-17 (Tc17s) and lyse specific
targets in vivo but not in vitro. Others have shown that in a melanoma model, Tc17s exhibit an anti-tumor
response. The mechanism behind this anti-tumor response is poorly understood. We have demonstrated in vivo
plasticity of the Tc17 phenotype by showing that adoptively transferred Tc17s convert from an IL-17 to an IFN-
secreting phenotype but are maintained in high numbers. The mechanism to drive this conversion from an IL-17
secreting phenotype to an IFN- phenotype is not well understood. It is our hypothesis that there is a connection
between conversion and an effective anti-tumor response. In this report, we show that antigen-specific, sorted
IL-17+IFN-- (pure Tc17) cells that are re-cultured in vitro can convert to an IFN- secreting phenotype. This
conversion occurs not in the presence of Tc1 skewing cytokines but rather in the presence of IL-2 or IL-7. These
data suggest the Tc17 phenotype can be converted with common gamma chain cytokines (IL-2 or IL-7).
Although the molecular mechanisms behind plasticity have yet to be elucidated, our data provide insight into the
activity and nature of these cells that could be important in clinical scenarios such as chronic infectious diseases
or adoptive T cell immunotherapy.
P23 - Autoantibodies as Immunobiomarkers in Lung Cancer and Their Use in Early Cancer Diagnosis
1
2
2
3
4
CAROLINE CHAPMAN , Graham Healey , Andrea Murray , Peter Boyle Laura Peek , Geoffrey Hamilton2
1
Fairley , and John F.R. Robertson
1
2
3
Division of Surgery, The University of Nottingham, Nottingham, UK; Oncimmune Ltd, Nottingham, UK; iPRI, Lyon,
4
France; Oncimmune USA LLC, DeSoto, Kansas
TM
Background: The EarlyCDT -Lung test, which measures circulating autoantibodies (AAbs) to tumorassociated antigens (TAAs), has been made available to clinicians as an aid to early detection of lung cancer in
a high risk population. Improvements in the specificity of such a test for lung cancer would not only improve its
cost-effectiveness, but also decrease the number of individuals who would suffer anxiety associated with a false
positive test result.
Patients and Methods: Optimization set: Samples from 235 patients with newly diagnosed lung cancer (68%
with early-stage disease) and matched controls were measured for the presence of AAbs to nine TAAs (p53,
NY-ESO-1, CAGE, GBU4-5, Annexin I, SOX2, MAGE A4, and HuD). The sensitivity and specificity of the
original six (p53, NY-ESO-1, CAGE, GBU4-5, Annexin I, SOX2) and the new seven (p53, NY-ESO-1, CAGE,
GBU4-5, SOX2, MAGE A4, HuD) AAb panels were compared in this optimization set, as well as an independent
clinical sample set, comprising two consecutive series of 776 and 689 high-risk individuals who had also been
sent for a CT scan by their physician due to these concerns.
Results: Optimization set: The original panel of six AAb assays gave an adjusted sensitivity and specificity of
39%/90% while the panel of seven AAb assays resulted in an adjusted sensitivity and specificity of 41%/93%.
For a lung cancer incidence in a high-risk population of 2.4% this improvement would result in a positive
predictive value (PPV) of 1 in 8 and an overall accuracy of the test of 91.6%.
Analysis of the two different panels in the clinical sample sets confirmed that the seven-antigen panel
maintained the sensitivity of the test, but also resulted in an increase in its specificity from 82% (for the 6 AAb
panel) to 91% (with the 7 AAb panel).
Conclusion: The EarlyCDT-Lung test can be used as part of the armamentarium of tests, available to the
clinician, to aid the diagnosis of early-stage lung cancer. The change from a six- to seven-AAb assay improved
the overall specificity of the test, thereby increasing its clinical usefulness.
P24 - Interleukin-10 Signaling in Regulatory T Cells is Required for Suppression of Th17 Cell-Mediated
Inflammation
1
1
2
1
3
**ASHUTOSH CHAUDHRY , Robert M. Samstein , Piper Treuting , Yuqiong Liang , Marina C. Pils , Jan4
4
5
5
6
Michael Heinrich , Robert S. Jack , F. Thomas Wunderlich , Jens C. Bruning , Werner Muller , and Alexander Y.
1
Rudensky
1
Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY;
3
Department of Comparative Medicine, University of Washington, Seattle, WA; Helmholtz-Center for Infection Research,
4
5
Braunschweig, Germany; Department of Immunology, University of Greifswald, Greifswald, Germany; Max Planck Institute
for Neurological Research Cologne, Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in
Aging-Associated Diseases (CECAD), Center of Molecular Medicine Cologne (CMMC), Center for Endocrinology, Diabetes,
6
and Preventative Medicine (CEDP), University Hospital Cologne, Cologne, Germany; Faculty of Life Sciences, University of
Manchester, Manchester, UK
2
+
Effector CD4 T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the
corresponding type of inflammatory response. Engagement of cytokine receptors by cognate ligands results in
the activation of STAT family proteins. STATs are latent cytoplasmic transcription factors that undergo receptor
signaling-induced tyrosine phosphorylation followed by dimerization and nuclear translocation resulting in
expression of target genes. Regulatory T (Treg) cells integrate environmental cues to suppress particular types
of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation,
is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory
interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the
ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in
selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3deficient Treg cells, while loss of IL-6 receptor and IL-23 receptor did not trigger spontaneous intestinal
inflammation. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative
regulatory circuits operating in immune effector cells.
P25 - ATM Modulates RAG-Mediated Monoallelic Looping of the 3’ End of the Tcra Locus to Control
Accessibility and Cleavage
1
1
1,2,3,4
1
5
6
**JULIE CHAUMEIL , Kristen Johnson , Mariann Micsinai
, Joy M-H Wang , Yanhong Ji , Elphege Nora ,
4
7
8,9
1
Yuval Kluger , Craig H. Bassing , David G. Schatz , and Jane A. Skok
1
2
Department of Pathology, New York University School of Medicine, New York, NY; New York University Center for Health
3
4
Informatics and Bioinformatics and NYU Cancer Institute, New York, NY; Department of Pathology and Yale Cancer
5
Center, Yale University School of Medicine, New Haven, CT; Department of Immunology and Microbiology, School of
6
7
Medicine Xian Jiaotong University, China; Institut Curie, CNRS UMR3215, INSERM U934, Paris, France; Department of
Pathology and Laboratory Medicine, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, and
8
Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA; Department
9
of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT
Tight regulation of antigen receptor rearrangement is required to preserve genome integrity and prevent the
occurrence of translocations that lead to leukemia and lymphoma. Recombination is regulated at multiple levels
to restrict cleavage by the recombinase enzyme RAG1/RAG2 to the appropriate lineage and developmental
stage. In T cells, Tcrd and Tcra recombination is complicated because the two loci share chromosomal location,
are not stringently subjected to allelic exclusion, and recombine at different stages. Furthermore, Tcra can
undergo numerous rounds of recombination. In the face of this complexity, maintaining genome integrity at the
Tcra/d locus might represent a major challenge. Recent studies indicate that RAG1 binds to active chromatin
and localizes to the J segments at the 3’ end of each antigen receptor locus in rearranging cells. Here we show
that in double positive T cells RAG1 increases/stabilizes the formation of large monoallelic loops that separate
the 3’ end of the Tcra locus from the bulk of the chromosome territory where the 5’ end remains embedded.
Looping of one Tcra allele facilitates pairing with the second allele, enabling cross-talk during recombination.
Despite pairing, monoallelic looping coincides with monoallelic RAG-mediated cleavage at the Tcra 3’ end. The
introduction of a break on one allele induces ATM-dependent repositioning of the second allele to repressive
pericentromeric heterochromatin, with inhibition of looping and cleavage. These data support a model in which
looping and pairing facilitate focal binding of RAG and regulate monoallelic cleavage in recombination centers.
P26 - ERM Proteins Play a Role in T Cell Migration and Adhesion
*EMILY J. CHEN, Meredith H. Shaffer, and Janis K. Burkhardt
University of Pennsylvania and Children’s Hospital of Philadelphia, Philadelphia, PA
T cells circulate between the blood and lymph nodes in search of cognate antigen. This process is dependent
on their ability to form a protruding leading edge and a constricted tail-like structure termed the uropod. Cell
polarity is a crucial characteristic for T cells to migrate through blood vessels, adhere to endothelium,
extravasate into lymph nodes, and migrate within lymph nodes. Members of the ezrin, radixin, and moesin
(ERM) family of actin-binding proteins are important regulators of polarity in many cell types. We therefore
analyzed the role of these proteins in regulating T cell shape and migration.
T cells express two ERM family members, ezrin and moesin. Both ezrin and moesin localize to the uropod of
migrating T cells. To test the function of these proteins in migrating T cells, we purified CD4+ T cells from
conditional ezrin knockout mice and suppressed moesin in these cells using siRNA. Ezrin- and moesin-deficient
T cells exhibited defective binding to fibronectin, and formed a uropod half as efficiently as wild type control cells
in response to fibronectin-coated surfaces. Double-deficient cells were also impaired in their ability to migrate
toward a chemokine gradient in vitro and to home to lymph nodes in vivo. Video analysis of migrating T cells
revealed that these cells exhibit altered morphology during migration on integrin ligands. Taken together, these
results show that ezrin and moesin are important to generate polarized T cells capable of normal integrindependent adhesion and efficient movement in vitro and in vivo.
P27 - Optimal Conditions Required for Influenza A Infection Enhanced Cross-Priming of CD8+ T Cells
Specific to Cell-Associated Antigens
Joe Wei, Jason Waithman, Kun Xiao, and WEISAN CHEN
T Cell Laboratory, Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Austin Health, Melbourne, Australia
+
Dendritic cells can take up exogenous tumor antigens and present their antigenic epitopes to CD8 T cells, a
process called cross-presentation. Cross-presentation is especially important in anti-tumor immunity because
tumor cells, although carrying tumor antigens, do not activate naïve T cells efficiently due to a lack of costimulatory molecules. Our group has recently shown that influenza A virus infection of allogeneic cells lead to
+
enhanced cross-priming of CD8 T cells specific to cellular antigens. To develop this into a potential vaccine
strategy, in this study, we have systematically investigated the numbers of allogeneic cells infected by IAV, IAV
doses and their infectious activity, the length of in vitro infection, and other associated factors. We have defined
the optimal immune enhancing conditions, and we have also shown in vivo that such enhanced cross-priming
did lead to enhanced tumor protection. The knowledge should be useful for developing more robust cancer
vaccines in the future.
P28 - Phenotype and the T Cell Receptor Repertoire of Colorectal Cancer Tumor-Infiltrating
Lymphocytes
**JEFFREY CHOU, Christie L. Mortales, and Edus H. Warren
Fred Hutchinson Cancer Research Center, Seattle, WA
+
+
Increased density of tumor infiltrating T lymphocytes (TILs), particularly CD8 and CD45RO cells, in primary
colorectal cancer (CRC) has been correlated with superior prognosis even in patients with advanced or
metastatic disease. It has been difficult to establish whether CRC TILs comprise a tumor-reactive adaptive
immune response or are nonspecifically attracted to the tumor. We reasoned that analyzing the diversity of α T
cells in primary CRC tumors or liver metastases might provide insight into this question. Human CRC primary
tumors with adjacent uninvolved colon or CRC liver metastases with adjacent uninvolved liver were obtained
and digested to single cell suspension. In order to study TIL interaction with autologous CRC tumors in vivo,
-/NOD/SCID IL2R (NSG) mice were implanted with CRC cells for growth of CRC xenografts, which were then
expanded and injected into secondary and tertiary NSG mouse recipients. Histologically, the xenografts closely
resembled the primary tumor in morphology and by immunohistochemistry staining and retained expression of
major histocompatibility complex (MHC) class I, suggesting they would be an appropriate model for future
studies with autologous TILs. T cells obtained from CRC tumor or normal tissue digests have been assessed by
+
+
flow cytometry for CD4, CD8, CD45RA, CD45RO, and CD62L. The majority of CD4 and CD8 T cells are
+
CD45RO /CD45RA /CD62L suggestive of an effector phenotype. T cells were also analyzed by spectratyping or
high-throughput sequencing of their T cell receptor (TCR)  chain complementarity-determining region 3 (CDR3)
repertoire. An oligoclonal TCR repertoire was identified in 3 non-contiguous CRC liver metastases that had been
pre-treated with radiofrequency ablation prior to resection. PCR amplification, cloning, and sequencing of 
chain CDR3 revealed TCR restriction to a single sequence in one V family in all 3 CRC liver metastases. The
same TCR V CDR3 sequence could also be detected in the polyclonal repertoire of adjacent liver. The TCR
repertoire from CRC primary tumor TILs and adjacent colon tissue T cells were also analyzed and compared by
high-throughput sequencing of  chain CDR3. Identified dominant CDR3 sequences in TILs may lead to
identification of T cell clones reactive against CRC tumor antigens and future experiments where they are
evaluated for interaction against autologous xenografts in NSG mice.
P29 - The NLRP3 Inflammasome is Required for NKT Cell-Mediated Immune Responses
1,2
3,#
4
5
*MELVYN T.C. CHOW , Jürg Tschopp , Dale I. Godfrey , Andreas Möller , Mark J. Smyth
1*
Paget
1
1,2*
, Christophe
2
Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Australia; Department of Pathology,
3
4
University of Melbourne, Parkville, Australia; Biochemistry, University of Lausanne, Epalinges, Switzerland; Department of
5
Microbiology and Immunology, University of Melbourne, Parkville, Australia; Cancer Genomics Program, Peter MacCallum
#
Cancer Centre, East Melbourne, Australia; *These authors contributed equally to this work; We dedicate this work to the
late Jürg Tschopp
The NLRP3 inflammasome is emerging as a critical regulator of both the innate and adaptive immune response.
It functions as a platform that induces the activation of caspase-1, in turn triggering the maturation of IL-1 and
IL-18. IL-1 and IL-18 have been demonstrated to potentiate NKT cell-mediated immune responses, but the
functional role of the NLRP3 inflammasome has not been fully addressed. Here, we utilized gene-targeted mice
to assess the role of the NLRP3 inflammasome in NKT cell-mediated immune responses activated by galactosylceramide (-GalCer). We demonstrated that NLRP3 inflammasome-deficient mice exhibited reduced
responsiveness to -GalCer. IFN- production by splenic and hepatic NK cells was greatly reduced in -GalCertreated NLRP3 inflammasome-deficient mice. Our current data suggest that the NLRP3 inflammasome might
act as a bridge for cross-talk between NKT cells and other immune cells.
P30 - Using Transposon Mutagenesis to Identify Genes Regulating B Cell Immune Tolerance
**ANTHONY BYRON COOPER, Bao Duong, Colleen Doyle Cooper, and David Nemazee
The Scripps Research Institute, La Jolla, CA
Immune tolerance is a basic feature of the immune system that must be maintained in order to prevent
unwanted self-reactivity and pathology. Autoreactive B cells are regulated in several ways, including genetic
reprogramming by receptor editing, clonal deletion by apoptosis, and functional inactivation (anergy), involving
down-tuning of antigen receptor signaling. Dysregulation in these processes can promote autoimmunity and
cancer. Moreover, when recognized by lymphocytes, antigen itself can be a potent mitogen driving rapid cell
proliferation or regulating survival and homeostasis. In this work we take advantage of an insertional
mutagenesis approach to identify genes involved in the regulation of immune tolerance.
Our lab has created an in vivo model for negative selection of polyclonal B cell populations using superantigens
against components of the B cell receptor. These superantigens provide a nearly complete negative selection of
all B cells providing an ideal screening system for random mutagenesis. Using the Sleeping Beauty-T2/Onc
gene-trap transposon system and a superantigen reactive to IgM we have begun to identify insertional
mutations that allow individual B cells to escape immune tolerance and survive in the periphery. Sequencing
results have indentified insertions in genes important to apotosis, cell growth, and B cell development, as well as
several genes of unknown function. Work has now begun to target candidate genes for “knock-down” with
shRNA in an immortalized hematopoietic stem cell system.
P31 - NKG2D-Mediated NK Cell/Myeloid Cell Cross-Talk in Patients with Glioma
COURTNEY A. CRANE, Carly Hoffman, Seunggu Han, Brian Ahn, Lewis Lanier, and Andrew Parsa
University of California, San Francisco, San Francisco, CA
Immune surveillance by Natural Killer (NK) cells is critical to eliminating tumor cells and preventing
establishment of a solid tumor. Depleting NK cells in mouse models promotes tumor growth, and patients with
decreased NK cell function are more susceptible to cancer and have a poorer prognosis following diagnosis.
One major mechanism of NK cell activation is through the NKG2D receptor, which can occur when transformed
cells increase expression of one or more of the eight known NKG2D ligands. We have new data to suggest that
NKG2D ligand expression extends to circulating myeloid cells and impairs anti-tumor immune responses
mediated by NK cells. Here we show that soluble proteins secreted by tumor cells induce the expression of
NKG2D ligands on circulating myeloid cells in patients with malignant glioma. In these patients, NKG2D ligand
expression on myeloid cells is independent of steroid treatment, surgical intervention, and chemotherapy.
Transwell assays and dialysis indicate that myeloid cell expression of NKG2D ligands is a consequence of
soluble proteins produced by tumor cells. NKG2D ligand expression on circulating myeloid cells is sufficient to
induce inflammatory responses by activated NK cells. Based on these data, we propose that tumor-derived
soluble factors induce NKG2D ligand expression on circulating and tumor-infiltrating myeloid cells, causing
activated NK cells to preferentially attack myeloid cells instead of NKG2D ligand-expressing tumor cells. As
decoy targets, NKG2D ligand-expressing myeloid cells may promote immune escape of tumor cells in patients
with malignant glioma.
P32 -Tim-3 Expression and Function in Natural Killer Cells in Metastatic Melanoma
1,2,3
INÊS PIRES DA SILVA
1
Bhardwaj
1
1
1
4
1
, Sonia Jimenez-Baranda , Anne Gallois , Vijay K. Kuchroo , Iman Osman , and Nina
2
New York University Langone Medical Center, Cancer Institute, New York, NY; Instituto Português de Oncologia de Lisboa
3
4
Francisco Gentil, Lisboa, Portugal; Programme for Advanced Medical Education; Center for Neurologic Diseases, Harvard
Medical School, Boston, MA
Background: Tim-3 (T cell immunoglobulin domain and mucin domain 3) is a type-I glycoprotein receptor with
opposing roles in innate and adaptive immunity. It synergizes with TLR signaling to induce inflammation in DCs,
while it negatively regulates the Th1-cell responses by interacting with galectin-9. Furthermore, Tim-3 has been
implicated in T cell exhaustion in chronic infectious diseases and some tumors, as metastatic melanoma.
Natural killer (NK) cells, innate immune cells, eliminate tumor cells through cytotoxicity and enhance adaptive
immunity through the production of IFN. They constitutively express Tim-3. Moreover, it has been described
that some tumor cells (including melanoma) express/secrete galectin-9 (Tim-3 ligand). However, nothing is
known about the role of Tim-3 in NK cells, particularly in the presence of galectin-9, in healthy and in melanoma
context.
Aims: (i) Characterize NK cells from melanoma and healthy donors (HD, MD) according to Tim-3 expression;
(ii) Correlate Tim-3 expression with NK cells function; (iii) Study the effect of galectin-9 on NK cells expressing
Tim-3.
Methods: Purified NK cells from PBMCs of 20 MD and 40 HD were characterized according to the expression
of Tim-3 by flow cytometry. NK cell cytotoxicity was evaluated using K562 cells as target cells, and measuring
Lamp-1 expression on NK cell surface after 4 hours. Gmel galectin-9+ and Gmel galectin-9- melanoma cells
were sorted and used as target cells.
Results: MD NKs express higher levels of Tim-3 comparing to HD NKs (p<0,05). Tim-3 expression does not
negatively affect cytotoxicity. In fact, the cells expressing Tim-3 are the ones expressing Lamp-1, either in MD or
HD. More important, we observe a decrease in cytotoxicity when the target cells express galectin-9 (Gmel
galectin-9+) compared to Gmel galectin-9-.
Conclusion: MD NKs express higher levels of Tim-3 compared to HD NKs however the presence of Tim-3 in
the membrane is not inhibitory. Furthermore, melanoma cells expressing galectin-9 down modulate NK cell
cytotoxicity. Therefore, the expression of galectin-9 may be a possible mechanism of tumor evasion of immune
surveillance.
P33 - Genetic Conflicts Drive Rapid Evolution of a Family of Antiviral Immunity Proteins
1
**MATTHEW D. DAUGHERTY and Harmit S. Malik
1
1,2
2
Fred Hutchinson Cancer Research Center, Howard Hughes Medical Institute, Seattle, WA
Greater than 10% of all cancers worldwide are attributable to viral infection. The innate immune system serves
as the first line of defense by detecting and neutralizing viruses before they have a chance to establish a
persistent infection that may lead to cancer. Taken from a viral perspective, these host-encoded antiviral
defenses represent potent barriers to viral replication and, therefore, to be evolutionarily successful, viruses
must find ways to evade them. Indeed, many viruses encode specific factors that allow them to directly
antagonize host proteins, permitting those viruses to avoid host neutralization and maintain a persistent
infection. The identification and characterization of molecular conflicts between host defenses and viral
antagonists is thus highly relevant for understanding human immunity and susceptibility to viruses. I am using
evolutionary signatures of rapid, adaptive evolution of immunity genes to identify host proteins that are likely to
be directly antagonized by viruses. In particular, I am focused on the IFIT (Interferon-induced with
tetratricopeptide repeat) gene family, a highly interferon-induced set of broadly acting antiviral factors that are
important for host defense against many viruses, including several oncoviruses. My work has revealed that
several IFIT family members are very rapidly evolving under positive selection, consistent with them being
antagonized by viral factors. Indeed, I have now discovered a viral factor that specifically interacts with one IFIT
protein, and have shown that evolution of the IFIT gene results in differences in susceptibility to binding. Studies
to dissect the mechanism and functional implications of this host-virus interaction are under way.
P34 - Ectodomain Shedding of the NKG2D Ligand MULT1 Regulates Sensitivity to NK Cells
**WEIWEN DENG, Timothy J. Nice, and David H. Raulet
Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California, Berkeley, Berkeley, CA
Natural Killer group 2D (NKG2D) is an activating receptor expressed by NK cells and some T cells. It recognizes
a diverse family of ligands, including the Rae-1 family, H60, and Mult1 in the mouse and the MICA and ULBP
families in humans. Engagement of NKG2D with its ligands triggers the cytotoxic activity of NK cells toward
target cells expressing ligands. NKG2D ligands show a restricted expression in normal tissues, but they are
frequently upregulated in cellular stress, such as tumorigenesis and infection. Some human NKG2D ligands
such as MICA and MICB can be shed from tumor cells and have been detected in sera from patients with
various types of cancer. Here we report that the mouse NKG2D ligand MULT1 protein is shed from the surface
of cells and that the resulting soluble MULT1 can still bind to NKG2D. Shedding of MULT1 was blocked by the
general matrix metalloproteinase (MMP) inhibitor BB94. Soluble MULT1 was detected in supernatants from
tumor cell lines and in the sera of E-Myc transgenic mice with B lymphomas. Unlike what is reported for soluble
MICA, exposure of NK cells to soluble MULT1 did not cause downregulation of surface NKG2D in vitro. In
contrast, exposure to cell bound MULT1 did cause NKG2D downregulation. Interestingly and unexpectedly,
tumor cells transduced to secrete soluble MULT1 were partially rejected in syngeneic mice, in an NKG2Ddependent fashion, suggesting a role for soluble NKG2D in promoting tumor rejection. Our study represents the
first instance of NKG2D ligand shedding in the mouse, and provides an important animal model to define the
physiological role of shed NKG2D ligands, and the circumstances in which shedding occurs.
P35 - Persistent Tumor Clearance After Transfer of IL-12-Primed CTL into Lymphodepleted Hosts
1
2
1
2
3
3
C. MARCELA DIAZ , Osama Naga , Abdel-Aziz A. Zidan , Mohamed L. Salem , Maria Pallin , Anita Parmigiani ,
4
1
1
2
1
Gail Walker , Eric Wieder , Krishna Komanduri , David J. Cole , Alberto J. Montero , and Mathias G.
3
Lichtenheld
1
Department of Medicine, Division of Hematology/Oncology, Sylvester Cancer Center, University of Miami, Miami, FL;
3
Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; Department of
4
Microbiology and Immunology, University of Miami, Miami FL; Biostatistics and Bioinformatics Core, Sylvester Cancer
Center, University of Miami, Miami FL
2
Adoptive T cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive
costs associated with current technology required for culture and expansion of tumor-reactive T cells, the need
for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of
adoptively transferred T cells remain significant impediments for its clinical implementation. Here we report a
simplified combinatorial approach that involves short activation of CD8+ T cells in the presence of IL-12 followed
by adoptive transfer into tumor-bearing animals after a single injection of cyclophosphamide. This approach
resulted in complete eradication of B16 melanoma, and the establishment of long-term immunological memory
capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique
because they simultaneously exhibited traits for cytotoxic effector function and central memory-like, homing, and
senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional
memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory
stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T
cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which—upon
transfer into lymphopenic hosts—are sufficient to eradicate tumors and generate life-long tumor-specific
memory.
P36 - TRP1 Transnuclear Mice: The Role of TCR Affinity in Anti-Tumor CD8 T Cell Responses
1
2
3
STEPHANIE DOUGAN , Michael Dougan , Esteban Celis , and Hidde Ploegh
1
2
1
3
Whitehead Institute for Biomedical Research, Cambridge, MA; Massachusetts General Hospital, Boston, MA; Moffitt
Cancer Center, Tampa, FL
Somatic cell nuclear transfer (SCNT) allows the efficient generation of mice that will serve as a source of T cells
of defined specificity. By harvesting primary T cells from animals that are at the peak of an anti-tumor response,
and by transfer of the nucleus from such antigen-specific T cells into an enucleated oocyte, embryonic stem
cells that harbor the TCR rearrangements of that T cell may be obtained and used for the construction of cloned
mice. These animals contain T cells of the appropriate tumor specificity, have no genetic alterations other than
the physiological TCR rearrangements and are the closest approximation of the tumor-specific CTLs that
contribute to tumor resolution in vivo. Importantly, the generation of transnuclear mice is rapid, requiring
approximately 6 weeks from T cell harvest to obtaining chimeric animals.
We have generated two independent lines of transnuclear mice from CD8 T cells recognizing the endogenous
melanoma antigen TRP1. These two lines, while having the same specificity, differ by 100-fold in their affinity for
high
low
antigen. Activated CD8 T cells from either TRP1
or TRP1 mouse lines have anti-tumor activity in vivo and
can slow the progression of B16 melanoma, with a median survival increase 4.5 days (control = 16 days;
high
low
high
TRP1
= 21 days; TRP1 = 20.5 days). Naïve TRP1
CD8 T cells confer a 2-day increase in median
low
high
low
survival time, while naïve TRP1 CD8 T cells have negligible effect on tumor growth. TRP1
and TRP1
cells can be found among the tumor-infiltrating lymphocytes and in the tumor-draining lymph nodes of B16
low
bearing mice. TRP1 cells can be found in greater number among tumor-infiltrating lymphocytes, suggesting
that increased proliferation or infiltration can compensate for lower affinity. Thus, high affinity CD8 T cells can
provide moderate tumor protection in the naïve or activated state. Naïve low affinity CD8 T cells do not affect
tumor growth; however, upon activation, low affinity CD8 T cells perform equally as well as high affinity CD8 T
cells with respect to tumor growth and overall survival.
P37 - MFG-E8 Blockade Potentiates GVAX in a Breast Tumor Model
DOBRIN DRAGANOV
Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA
Milk fat globule-EGF factor 8 protein (MFG-E8) is an important mediator of the tolerogenic functions of GM-CSF
in vivo and has the ability to abrogate the anti-tumor potential of GM-CSF-secreting vaccines (GVAX). The RGE
mutant, a dominant-negative form of MFG-E8, antagonizes the tolerogenic functions of wild type MFG-E8
through suppression of phosphatidylserine-dependent phagocytosis of apoptotic cells, inhibition of TGF
production, and by augmenting the secretion of pro-inflammatory cytokines. The RGE mutant protein is known
to potentiate GVAX in a B16 melanoma model, via a mechanism that involves preferential expansion of effector
+
+
+
CD8 T cells over CD4 Foxp3 Treg cells. B16 melanoma cells, however, do not express the wild type MFG-E8
protein, which is frequently overexpressed in various solid and hematological malignancies. Here, we
demonstrate that the RGE mutant provides therapeutic benefit even in the context of a tumor vaccine that
expresses high levels of endogenous wild type MFG-E8. RGE-mediated tumor protection correlates with modest
augmentation of systemic anti-tumor humoral responses, as well as with similarly enhanced cellular responses
at the tumor challenge site, as demonstrated by the increased ratio of Teffectors to Tregs. Our results suggest
that while RGE-modified vaccines have augmented therapeutic potential even in the context of MFG-E8-positive
tumors, they suffer from very limited capacity to impact Treg frequencies and functions at the distant tumor
locations. Adequate treatment of such cancers might, therefore, require alternative strategies that target MFGE8 systemically and at the tumor site, where high levels of MFG-E8, produced locally by tumor or infiltrating
myeloid cells, represent an additional barrier to effective anti-tumor immune responses.
P38 - Novel FACS-Based Characterization of Inflammatory Lung Phenotypes in Patients with Lung
Cancer
*MUBING DUAN
1
1,2,4
1,3
1
3
2
, D. Steinfort , W.C.C. Li , L. Irving , G. P. Anderson , and M.L. Hibbs
2
4
Ludwig Institute for Cancer Research, Parkville, Australia; University of Melbourne, Melbourne, Australia,
4
Melbourne Hospital, Melbourne, Australia, Monash University, Melbourne, Australia
3
Royal
Recent years have seen a growing awareness of the link between chronic inflammation and cancer
tumorigenesis. In innate immunity, chronic inflammation is now thought to promote tumorigenesis through (i) the
establishment of a stromal microenvironment favoring tumor growth and progression by tumor-associated
macrophages (TAMs), and (ii) circulating myeloid-derived suppressor cells that suppress anti-tumor immune
surveillance mediated by cytotoxic T lymphocytes. The role of these two features in the development and
progression of primary lung carcinoma, however, remains relatively unknown. This is chiefly due to a difficulty in
studying lung macrophage phenotypes, whose highly autofluorescent properties hamper their characterization
by fluorescence-activated cell sorting (FACS).
We have recently developed a novel approach of characterizing inflammatory lung macrophages in mice using
FACS. This work has allowed us to profile alveolar macrophages in animal models of acute and chronic lung
+
inflammation, and to identify a pathological Mac-1 alveolar macrophage subpopulation associated with the
development of type II alveolar epithelial cell hyperproliferation and lung fibrosis. We are now translating this
approach to the characterization of alveolar macrophage phenotypes associated with human lung cancer
lesions. Here, we report both its feasibility and the inflammatory BAL profile of patients with lung cancer ±
chronic obstructive pulmonary disorder (COPD) versus sarcoidosis.
P39 - Tumor-Associated Neutrophils Have Anti-Tumor Potential in Prostate Cancer
Shuai Tang, Janet A. Tooze, and PURNIMA DUBEY
Wake Forest School of Medicine, Winston-Salem, NC
A significant number of patients develop castration-resistant prostate cancer, for which there is no cure. Our
goal is to characterize the effector and inhibitory mechanisms that modulate prostate cancer progression. We
have previously shown that CD8+ effector function is transiently increased following androgen ablation in the
prostate-specific Pten knockout mouse model. We evaluated the role of myeloid cells in modulating CD8+ T cell
function in this model. We observed a heavy Gr-1+ cell infiltrate in the primary tumors which was markedly
increased early after castration. Phenotypic profiling of tumor-infiltrating Gr-1+ cells from intact and castrated
mice showed uniform CD11b+Ly6C+Ly6G+ marker expression and neutrophil morphology.
To determine the functional importance of these tumor-associated neutrophils (TANs), we depleted neutrophils
in non-castrated mice using anti-Ly6G antibody. Surprisingly, in contrast to most previous reports where TANs
have pro-tumor function, we found that depletion of TANs slightly increased tumor burden. In addition, the
number of regulatory T cells (Tregs) was elevated in the tumor after neutrophil depletion, and the proportion of
functional CD8+ T cells was unchanged.
In contrast, concomitant castration and neutrophil depletion did not alter tumor weight 2.5 weeks post-treatment.
Although the total number of CD8+ T cells in the tumor increased, the proportion of functional CD8+ T cells was
unchanged compared to castration alone. A striking increase in Treg numbers was also observed.
Using quantitative real-time PCR, we detected high levels of the inflammatory cytokine TNF- by TANs before
and after castration. IL-4R expression was also detected after castration suggesting an alternatively activated
phenotype. However, expression of iNOS or arginase, two signature enzymes produced by granulocytic
myeloid-derived suppressor cells, was not detected. In addition, the TANs were able to cross-present antigen
following immunization with an MHC class I-restricted tumor antigen. Taken together, these data suggest that
the TANs in Pten-/- prostate tumors have anti-tumor function before and after castration, and may modulate
both CD8+ effector T cells and Tregs within the tumor. Our long-term goal is to understand how TANs control
effector and inhibitory arms of the immune system in order to improve outcomes following immunotherapy.
P40 - The Human Leukocyte Antigen-Restricted Glioblastoma Peptidome is an In Vivo Source of TumorAssociated Antigens
1
2
3
3
4
2
VALERIE DUTOIT , Christel Herold-Mende , Norbert Hilf , Oliver Schoor , Philipp Beckhove , Judith Bucher ,
2
3
3
3
2
Katharina Dorsch , Sylvia Flohr , Jens Fritsche , Peter Lewandrowski , Jennifer Lohr , Hans-Georg
5
5
3
3
3
1
Rammensee , Stefan Stevanovic , Claudia Trautwein , Verona Vass , Steffen Walter , Paul R Walker , Toni
3
3
1
Weinschenk , Harpreet Singh-Jasuja , and Pierre-Yves Dietrich
1
2
Center of Oncology, University of Geneva and Geneva University Hospitals, Geneva, Switzerland; University of
3
4
Heidelberg, Germany; immatics biotechnologies GmbH, Tubingen, Germany; The German Cancer Research Center,
5
Heidelberg, Germany; Institute for Cell Biology, University of Tubingen, Germany
T lymphocytes distinguish healthy from diseased cells by monitoring the changes in the peptides presented by
MHC molecules at the cell surface (the cell peptidome). The identity of tumor-associated peptidomes remains
unknown, although their knowledge could bring valuable information for a better understanding of
tumorigenesis, but also to explore its relationships with the immune system. Here, we eluted peptides from the
surface of HLA-A2+ glioblastoma (GBM) samples using liquid chromatography and mass spectrometry, resulting
in the uncovering of the first naturally presented HLA-A2 peptidome. In a second step, we identified peptides
associated with the tumor component of the cells using comparative mRNA microarrays. Out of this, we
selected 10 peptides with high potential for immunotherapy. Tumor-associated expression of these 10 novel
antigens was validated at the mRNA and protein levels and peptide presentation at the cell surface was
quantified, demonstrating that these antigens are highly expressed in tumor cells but not or at very low levels on
normal CNS and non-CNS tissues. In order to confirm the choice of the selected peptides for immunotherapy,
we investigated the presence and function of specific T cells in the blood of HLA-A2+ patients with GBM. Our
results suggest that a repertoire of antigen-specific T cells is available and functional in patients with GBM, with
specific T cells able to kill antigen-expressing tumor cells in vitro. Finally, we were able to detect T cells specific
for one of the antigens at the tumor site in one patient, demonstrating for the first time that glioma is able to
spontaneously induce an antigen-specific immune response in the brain microenvironment. Altogether, we show
that uncovering tumor peptidomes allows for the identification of tumor-associated peptides that have the
required properties to be used for immunotherapeutic purposes and suggest expanding this approach to other
tumor entities.
P41 - Visualizing Acute Negative Selection in the Thymus
**IVAN DZHAGALOV and Ellen Robey
University of California, Berkeley, CA
Negative selection is one of the most important guardians of self-tolerance; when it fails autoimmune diseases
develop. It has been studied mostly through analysis of dissociated cells ignoring the fact that thymocytes
develop and are selected in a three-dimensional environment. We devised a system that allowed us to follow a
synchronous wave of thymocytes undergoing negative selection in their native environment in real time. We
found that upon recognition of its cognate antigen, thymocytes immediately slow down and remain relatively
immotile for hours. Despite the fast and robust activation, it takes 3-4 hours for the first signs of apoptosis to
appear. Significant cell loss becomes obvious after 6-8 hours. We visualized the cell death and clearance and
found an important role of LysM-expressing cells in the phagocytosis of dying thymocytes. Our studies
established a timeline of negative selection and will allow us to assess the effect of various factors such as
dose, distribution and nature of antigen, type of antigen presenting cell, and type of responding cell on the
process.
P42 - Induction of CD8 T Cell Responses Restricted to Multiple HLA Class I Alleles in a Cancer Patient
by Immunization with a 20-Mer NY-ESO-1f (NY-ESO-1 91-110) Peptide
1, 2
3
2
4
2
SHINGO EIKAWA , Kazuhiro Kakimi , Midori Isobe , Kiyotaka Kuzushima , Yoshihiro Ohue , Kazuhiro
1
5
1
6
2
5
Ikeuchi , Akiko Uenaka , Heiichiro Udono , Lloyd J. Old , Mikio Oka , and Eiichi Nakayama
1
2
Okayama University Graduate School of Medicine, Okayama, Japan; Kawasaki Medical School, Kurashiki, Okayama,
3
4
5
Japan; Medinet, University of Tokyo Hospital, Tokyo, Japan; Aichi Cancer Center, Nagoya, Japan; Kawasaki University of
6
Medical Welfare, Kurashiki, Okayama, Japan; Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer
Center, New York, NY
Objectives: Analysis of immune responses of NY-ESO-1 protein-vaccinated patients revealed that there are
two dominant regions recognized by either CD4 or CD8 T cells, one of which is also dominantly recognized by
antibody. Based on these findings, we conducted a clinical trial using NY-ESO-1f peptide (NY-ESO-1 91-110)
derived from one of the dominant regions as a vaccine. In this study, we investigated CD8 T cell responses in
NY-ESO-1f-peptide vaccinated patients.
Methods: CD8 T cell response3 in patients TK-f01 (HLA-A*24:02, B*35:01, 52:01, C*03:03, 12:02) and TK-f05
(HLA-A*02:06, B*39:02, 40:02, C*03:04, 07:02) were investigated. Uptake of NY-ESO-1f peptide by APC for
antigen presentation was examined using immature dendritic cells. HLA restriction in bulk CD8 T cell recognition
of NY-ESO-1 was determined by IFN- capture assay using a panel of EBV-B cells as APC and CD8 T cell
frequency was analyzed. The frequency of CD8 T cells recognizing tumor cells was investigated with TK-f01
PBMCs obtained at day 0 and day 85 (after the 4th vaccination). Clonal analysis of CD8 T cells was performed.
We established f peptide-specific CD8 T cell clones and identified epitopes. Cytotoxicity of CD8 T cell clones
was assessed by aCella-TOX.
Results and Discussion: A robust CD8 T cell response was elicited after NY-ESO-1f peptide vaccination in the
patients. The long 20-mer NY-ESO-1f peptide was internalized and CD8 T cell epitopes were presented on
APCs. The clonal analysis showed that the multiple HLA class I-restricted CD8 T cell responses were induced
against the NY-ESO-1f peptide in TK-f01. The CD8 T cells recognizing tumor cells were induced at high
frequency after NY-ESO-1f peptide vaccination. CD8 T cell clones showed antigen-specific cytotoxicity. These
results indicated that NY-ESO-1f peptide consisting of 20 amino acids included multiple CD8 T cell epitopes and
was strongly immunogenic.
P43 - NLRP6 Regulates Colonic Microbial Ecology and Risk for Colitis
**ERAN ELINAV, Till Strowig, and Richard Flavell
Yale University School of Medicine, New Haven, CT
The intestinal mucosal compartment is a complex environment, in which the host comes into intimate contact
with a diverse microflora ecosystem that contains trillions of bacteria, viruses, fungi, and parasites, separated
from the host inner self by only a single layer of epithelial cells. The complex interactions between the host
epithelial layer, mucosal immune system, and microflora are increasingly recognized to play crucial roles in
many immune and metabolic processes including immune system development and function, metabolic
regulation, and the induction of tolerance, while alterations in this cross-talk is linked to a multitude of
pathologies. Germline-encoded receptors have been suggested to play central roles in regulation of this
complex host-microflora balance. Nod-like receptors (NLR) are cytoplasmatic proteins that are shown to play
important roles in the innate antimicrobial response. Some NLRs are capable of forming inflammasomes, multiprotein complexes that function as sensors of endogenous or exogenous damage-associated molecular
patterns. We recently demonstrated that NLRP6 is highly expressed in the gastrointestinal tract. Deficiency of
NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota
characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. ASCand NLRP6-deficient mice are characterized by spontaneous inflammation and exacerbation of dextran sodium
sulfate (DSS) colitis. In this talk we will discuss new findings implicating NLRP6 and other NLR proteins and
their interactions with other innate sensing pathways in the regulation of the intestinal steady state as well as
damage response, and demonstrate that compartmentalized NLR and inflammasome function is involved in
recognition of intestinal damage of various causes. Alterations in this function may be associated with distinct
effects on the innate and adaptive mucosal immune response.
P44 - Control of Innate Immune Responses to Nucleic Acid Ligands by Endosomal Sorting of Toll-Like
Receptors
**ALEX ENGEL and Greg Barton
University of California, Berkeley, Berkeley, CA
Intracellular Toll-like receptors (TLRs) recognize nucleic acid structures derived from viruses, bacteria,
parasites, host debris, and certain adjuvant mixtures and activate immune responses by innate immune cells
and B cells. These TLRs, TLR3, 7 and 9, sense their respective nucleic acid ligands in membrane
compartments of the endolysosomal system. Ligand routing, receptor trafficking, and signaling adaptor
recruitment to the nucleic acid sensing compartments control the amplitude and quality of cytokine output in
macrophages and dendritic cells. To understand how cells of the innate immune system establish this sensing
compartment we investigated the trafficking of TLR9, a nucleic acid sensing TLR that preferentially responds to
bacterial and viral DNA. DNA is sensed in an acidified compartment by an N-terminally processed form of TLR9.
We tested TLR9 cytoplasmic lysine mutants for signaling and trafficking defects. Substitution of a conserved
lysine (K932) with arginine strongly attenuated TLR9 signaling. Arginine substitution of the two remaining
cytoplasmic lysines further exacerbated the TLR9-K932R signaling defect. Ubiquitin is conjugated to TLR9 in a
post-ER compartment, and this ubiquitination is dependent on the TLR9 cytoplasmic lysines. Intriguingly,
substitution of the cytoplasmic lysines also reduces the efficiency of TLR9 processing, suggesting that these
lysines are important for TLR9 trafficking. Fractionation of macrophage cell contents on density gradients
separated unprocessed and processed forms of TLR9. By tracking various endosome subtype-specific
compartmental markers, we hope to more precisely define where TLR9 is modified, binds ligand, and recruits
signaling adaptors. Understanding the range of compartments where TLR9 is localized and how responsive
TLR9 is when localized to these compartments will yield important insights into how the cell uses endosomal
trafficking to interpret the presence and class of pathogen and respond accordingly.
P45 - Engagement of ICOS Pathway Synergizes with CTLA-4 Blockade as Potent Cancer Immunotherapy
1,2
2
XIAOZHOU FAN , Sergio A. Quezada , and James P. Allison
1
1,2
2
Weill Cornell Graduate School of Medical Sciences, New York, NY; Ludwig Center for Cancer Immunotherapy, Howard
Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY
Blockade of CTLA-4 has demonstrated potent anti-tumor efficacy in both animal models and cancer patients.
We identified ICOS as a T cell costimulatory pathway that could synergize with CD28/CTLA-4 manipulation.
Tumor cells engineered to express the ligand of ICOS (ICOSL), when administered as irradiated cellular vaccine
together with anti-CTLA-4 treatment, moved the balance between effector and regulatory T cells in the tumor by
enriching CD8+ and CD4+ Foxp3- T cells and enhanced proinflammatory cytokine production in CD4+ Foxp3- T
cells and cytolytic activity of CD8+ T cells, resulting in significant protection against multiple types of mouse
tumors. This study established a pivotal role of ICOS in anti-tumor immunity and implicated promising potential
of combining CTLA-4 blockade and ICOS engagement in the clinic.
P46 - Conserved Non-Coding DNA Sequence Element 3 (CNS3) in Foxp3 Locus Stochastically
Determines Foxp3 Induction Efficiency
1
2
3
1
1
**YONGQIANG FENG , Ye Zheng , Steven Josefowicz , Joris van der Veeken , and Alexander Rudensky
1
2
Department of Immunology, Sloan-Kettering Institute, HHMI, New York, NY; Salk Institute for Biological Studies, La Jolla,
3
CA; The Rockefeller University, New York, NY
Immune homeostasis relies on the precise control of regulatory T cells (T reg) that suppress excessive immune
response. Transcription factor Foxp3 plays a central role in T reg cell fate determination and function, therefore,
understanding Foxp3 transcriptional regulation is critical in elucidation of T reg development and function. Without
introducing global effects, our group took the approach to analyze the roles of CNS elements in Foxp3 locus and
showed that these elements play distinct roles in T reg development and maintenance. Further examination of
their roles will provide a unique approach to address the fundamental questions in T reg development and
function. Here we focus on how CNS3 influences T reg development, TCR repertoire, and the immunological
functions. We found that mice lacking CNS3 dramatically reduce thymic and induced peripheral T reg frequency.
Our group previously proposed that TCR signaling acts on CNS3. In order to discriminate whether CNS3
functions as a sensor for TCR signal strength or stochastically promotes Foxp3 induction, we measured T reg
induction efficiency in vitro by titrating TCR signaling. In the absence of CNS3, induction of T reg always showed
similar ratio of lower efficiency, suggesting that CNS3 stochastically promotes Foxp3 induction. This model is
further supported by two lines of additional evidence. First, in the CNS3 mutant and wild type chimeras, both
CNS3 null and wild type Treg cells showed identical expansion potential, TCR target Nur77, and Foxp3 target
gene expression, suggesting that the TCR repertoire carried by CNS3-null Treg cells are similar to the wild type
counterparts. Second, TCR beta chain usage is identical between wild type and CNS3-null Treg cells. In order to
trace the stage of T cell development and factors acting on CNS3, we extended our published data that
monomethylation on histone H3 at lysine 4 (H3K4me1), an epigenetic marker for enhancers, on CNS3 was
identified in T but not B cells. Surprisingly, we found that CNS3 is marked by H3K4me1 at as early as double
negative 1 (DN1) stage where T cell fate has not been determined, indicating that CNS3 is poised even before T
cell fate commitment.
P47 - Previous Exposure to Ionizing Radiation Decreases Hematopoietic Progenitor Cell Self-Renewal
and Increases Selection for Activated Notch1
1
2
2
*COURTNEY FLEENOR , Andriy Marusyk , Vadym Zaberezhnyy , and James DeGregori
1
1,2
2
Integrated Department of Immunology, University of Colorado and National Jewish Health, Denver, CO; University of
Colorado Anschutz Medical Campus, Aurora, CO
Ionizing radiation (IR) exposure has been associated with increased cancer incidence, particularly leukemias,
for over 100 years, but the mechanism underlying this relationship remains poorly understood. IR-induced
carcinogenesis is conventionally attributed to the direct generation of oncogenic mutations. Our lab has
developed an evolutionary-based model for cancer development, Adaptive Oncogenesis. We propose that cells
in young, healthy individuals are highly adapted to their niches, limiting selective pressure for trait-altering
mutations. Environmental insults, such as IR, alter cells as well as their niche in such a way as to reduce the
fitness of the cellular population (defined as a measure of the ability of a cell to pass its particular
epigenotype/genotype on to subsequent cell generations), creating room for improvement. This fitness reduction
increases selective pressure for adaptive mutations that provide an increase in cellular fitness relative to
neighboring cells competing for the same niche. We demonstrate that hematopoietic progenitor cells (HPC) from
P
previously irradiated but homeostatically restored (IR ) mice have decreased fitness relative to control nonirradiated (Ctrl) HPC, as indicated by competitive and limiting dilution bone marrow transplant assays. We find
P
that IR HPC cultured ex vivo display decreased maintenance and expansion relative to Ctrl, and that these
maintenance defects can be reversed by activated Notch1 signaling (leading to selection for Notch1 activation).
Notch1 is a transmembrane receptor integral to hematopoietic stem cell maintenance, and activating Notch1
mutations are found in over half of human T cell acute lymphoblastic leukemia patient samples and are also
found in T cell leukemias of mice exposed to IR. Our data support a model whereby previous exposure to IR
results in specific, reproducible, somatically heritable, and reversible defects in HPC that select for adaptive
oncogenic mutations, such as Notch1, that repair or circumvent the defect, increasing the selective advantage of
the mutant HPC relative to neighboring HPC and thereby driving leukemogenesis.
P48 - Regulation of Connective Tissue Growth Factor by the microRNA Cluster miR-17~92 in
Glioblastoma
1
2
*JAMIE L. FOX , Michael Dews , and Andrei Thomas-Tikhonenko
1
1,2
2
University of Pennsylvania, Philadelphia, PA; Children’s Hospital of Philadelphia, Philadelphia, PA
Connective tissue growth factor (CTGF or CCN2) is a member of the CCN family of secreted proteins that plays
a controversial role in cancer. Depending on tumor type and model system, CTGF has been shown to have both
positive and negative effects on tumor cell migration, metastasis, and angiogenesis. CTGF is overexpressed in
primary glioblastoma multiforme (GBM) tumors compared to normal brain tissue, and its forced overexpression
in GBM cell lines has been shown to increase in vitro cell migration, proliferation, and resistance to
chemotherapeutic drugs, as well as augment tumor size and vascularization. In contrast, a recent study
contends that CTGF has a tumor suppressive role in GBM, which is abrogated by direct suppression via the
miR-17~92 cluster of microRNAs. We and others have recently shown that miR-17~92 represses the TGF-
signaling pathway, which seems critical for GBM malignancy. Since CTGF is activated by the TGF pathway,
we hypothesized that an alternative indirect regulation of CTGF expression by the miR-17~92 cluster may be
involved in GBM tumorigenesis. In the current study, we have determined that miR-18a, one of six members of
the miR-17~92 cluster, inhibits basal and TGF-induced CTGF mRNA and protein levels in A-172 GBM cell line,
both directly and indirectly. Our preliminary data indicate that CTGF and TGF coactivate each other by way of
a positive feedback loop, and that direct inhibition of CTGF by miR-18a via direct 3’UTR binding results in
decreased CTGF protein expression without affecting mRNA turnover. This in turn leads to an attenuation of
downstream TGF signaling, including CTGF itself. Currently, we are investigating the functional effects of
CTGF overexpression or inhibition in in vivo models of GBM to establish its role in angiogenesis and tumor
growth.
P49 - Ex Vivo Characterization and Isolation of Rare Memory B Cells with Antigen Tetramers
1
BETTINA FRANZ Kenneth F. May Jr.
1
3
1,2
1,2
Glenn Dranoff , and Kai Wucherpfennig
1,3
2
Dana-Farber Cancer Institute, Boston, MA; Brigham and Women’s Hospital and Harvard Medical School, Boston, MA;
Program in Immunology, Harvard Medical School, Boston, MA
Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral
blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of
memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated
fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen.
Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies
ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric
antigen labeling demonstrated that a substantial fraction of the B cell repertoire can be missed when monomeric
antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell
sorted tetramer(+) B cells with single-cell RT-PCR of the B cell receptor. All antibodies bound to tetanus antigen
with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell
and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and
sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.
P50 - Effect of ADP on Dendritic Cell and T Cell Responses
K. NICOLE GRAVES, Kenneth A. West, and Robert Liwski
Dalhousie University, Halifax, Nova Scotia, Canada
To evade immune clearance, tumors secrete many factors to create an immunosuppressive or tolerant
environment. Specifically, tumors produce adenosine triphosphate (ATP) and its derivatives adenosine
diphosphate (ADP) and adenosine. While the inhibitory activities of adenosine have previously been described,
ADP functions through distinct purinergic receptors (P2Y1, P2Y12, and P2Y13) and may therefore have a
different mechanism of action. This study investigated the effect of ADP on anti-tumor dendritic cell (DC) and T
cell responses. Together, these cells can help shape adaptive immune responses against tumors. Bone
marrow-derived DCs were isolated from C57BL/6 mice and were found to express P2Y1, P2Y12, and P2Y13 by
RT-PCR. When cultured in the presence of ADP or stable analogue ADPS, DC surface expression of MHC
class II and co-stimulatory molecules CD40 and CD86 was unchanged. Likewise, when DCs were pre-treated
with ADP or ADPS, there was no change in their ability to activate naïve CD4+ T cells. However, when naïve
CD4+ T cells, which express P2Y1, were activated by anti-CD3, anti-CD28 coated beads in the presence of
ADP or ADPS, activation and proliferation were significantly decreased. This correlated with a significant
reduction in IL-2 secretion and CD25 surface expression. Interestingly, proliferation could not be rescued by the
addition of exogenous IL-2 or activated T cell supernatant, indicating a possible defect in the IL-2 receptor
signaling pathway. ADP treatment of CD4+ T cells appeared to cause a decrease in phosphorylation of ERK
and Akt. Conversely, when activated in a similar manner, proliferation of CD8+ T cells was not affected by the
addition of ADP or ADPS. However, treated CD8+ T cells had a significant reduction in IFN- secretion,
suggesting that ADP may cause polarization away from a type 1 response. Surprisingly, cytotoxic T lymphocyte
activity was unaltered. This study demonstrated that ADP can inhibit CD4+ T cell responses specifically by
reducing activation, proliferation, and IL-2 production, and can also strongly inhibit IFN- secretion by CD8+ T
cells. Future investigation into the immunosuppressive properties of ADP and possible antagonism could lead to
the development of a novel anti-tumor therapeutic treatment.
P51 - New mRNA Vaccines which Couple Peptide Presentation to Dendritic Cell Activation Induce a
Potent Anti-Tumor CTL Response In Vivo
1,2
1,3
1,3
1,2
GIDEON GROSS , Gal Cafri , Esther Tzehova , Alon Margalit , and Lea Eizenbach
1
2
3
3
MIGAL, Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel; Weizmann Institute of Science, Rehovot, Israel
Ex vivo-activated DCs loaded with tumor antigens are beginning to show clinical efficacy in cancer
immunotherapy. Recently we have created a membrane-anchored derivative of 2 microglobulin (2m), the
invariant MHC class I light chain, as a novel platform for genetic CTL-inducing vaccines. By linking antigenic
peptides to the N-terminus of the 2m scaffold and the cytosolic domain of TLR4 or TLR2 to the C-terminus we
were able to simultaneously endow the polypeptide products with potent peptide presentation and cell activation
capacities when expressed in different mouse APC lines in vitro (Cafri et al. Int. Imm. 2011).
In the present work we evaluated the functional properties of such constructs in mRNA-transfected BM-derived
mouse DCs, both ex vivo and in vivo. We show that MHC class I presentation of the linked peptide by
transfected DCs is clearly higher than that exhibited by the same DCs loaded with a saturating concentration of
the synthetic peptide. The same mRNAs, but not mRNA encoding an inert membranal anchor instead of the
TLR moiety, induced IL-12 production at levels comparable to LPS, and the same was true for additional
cytokines and chemokines. In vivo, mRNA-transfected mouse DCs were markedly superior to peptide-loaded,
LPS-activated DCs in inducing peptide-specific CTLs. Moreover, mice pre-immunized with the former DCs, but
not the latter, were fully protected against lung metastases upon challenge with a highly tumorigenic melanoma
cell line. Comparative experiments evaluating immunotherapy of pre-established tumors are now under way and
are beginning to show higher efficacy of these bifunctional mRNA vaccines.
P52 - Endocytic Receptors Differentially Confer Cross-Ppresentation Ability to Non-professional APCs
**JEFF GROTZKE and Peter Cresswell
Yale University, New Haven, CT
+
Cross-presentation is a major mechanism by which CD8 T cells can be alerted to the presence of bacterial,
viral, and tumor antigens. While the general pathways by which antigens are cross-presented have been
elucidated, the molecular mechanisms that facilitate cross-presentation are incompletely understood. One such
poorly defined area is the role that endocytic receptors play in determining whether an internalized antigen gets
cross-presented. To examine this, 293T cells were stably transduced with mannose receptor (MR), DC-specific
ICAM-3 grabbing non-integrin (DC-SIGN), scavenger receptor A1 (SRA1), macrophage receptor with
collagenous structure (MARCO), and lectin-like oxidized LDL receptor-1 (LOX-1). Interestingly, after incubation
with ovalbumin (OVA)-conjugated beads, only cells expressing SRA1 and DC-SIGN were able to stimulate the
OVA-specific B3Z hybridoma. Cross-presentation ability did not correlate with the ability to phagocytose beads,
as similar levels of bead uptake were seen between cross-presenting and non-cross-presenting receptors.
Internalization by the expressed receptor was required, as incubation with receptor-specific competitive
inhibitors blocked cross-presentation. Furthermore, the cross-presentation pathway utilized by 293T cells was
similar to that used by DC, as it required phagocytosis, retrotranslocation to the cytosol, and proteasomal
degradation. To determine whether the cytoplasmic tail of SRA1 is required for cross-presentation, the SRA1 tail
was replaced with the tail from MARCO, resulting in a loss of cross-presentation ability. Furthermore,
serine/threonine phosphorylation may be required for SRA1-mediated cross-presentation, as mutation of
conserved serine and threonine residues in the SRA1 tail results in reduced cross-presentation. These data
demonstrate that the ability to internalize antigens is insufficient to mediate cross-presentation, and that
receptor-specific events can influence the fate of phagocytosed antigens at the level of cross-presentation. The
identification of factors induced by receptor engagement leading to cross-presentation is an area of intense
investigation.
P53 - Structural Studies on the Lymphocyte Calcium Channel Pore-Forming Protein ORAI1
**APARNA GUDLUR, Anjana Rao, and Patrick Hogan
La Jolla Institute for Allergy and Immunology, La Jolla, CA
Calcium signaling is crucial for proper functioning of the immune system, and dysregulated calcium signaling is
implicated in certain cancers. Detection of foreign antigens by immune-cell receptors causes depletion of
intracellular calcium stores and activates a protein STIM1 that senses calcium concentration inside the cells.
STIM1 initiates store-operated calcium entry (SOCE) through the plasma membrane-located calcium channel
Orai1. We are interested in solving the X-ray crystal structure of the Orai1 calcium channel in order to
understand its molecular architecture, selectivity for calcium, and overall regulation of calcium signaling by this
calcium channel.
Overexpression constructs of human Orai1, a human homolog hOrai2, and the drosophila ortholog dOrai have
been overexpressed in the yeast Pichia pastoris. Detergent-based solubilization of Orai proteins and
subsequent purification processes have been standardized. Extensive screening was performed to identify
detergent and buffer conditions in which Orai proteins are monodispersed and stable. Preliminary experiments
indicate that Orai solubilized in the most stabilizing detergent can form oligomers of homogenous size.
Detergent-solubilized Orai also retains its ability to interact with its known physiological binding partners STIM
and calmodulin.
In order to grow good quality crystals of Orai that would yield high-resolution X-ray diffraction data, large-scale
crystallization trials are being conducted using various lipids in the physiologically relevant context of the lipid
bilayer. Various soluble and membrane protein tags for Orai that are expected to aid in the crystallization
process are being explored. Crystallization of Orai in complex with specific antibodies and physiological binding
proteins is also being initiated. Preliminary crystallization trials of pure detergent-solubilized Orai yield crystallike ‘spherulites’ that show promising X-ray diffraction patterns.
P54 - CTCF Binding Elements Mediate Control of V(D)J Recombination
**CHUNGUANG GUO, Hye Suk Yoon, Andrew Franklin, Suvi Jain, Anja Ebert, Claudia Bossen, Christian
Vettermann, Jamie G. Bates, Mark S. Schlissel, Cornelis Murre, Meinrad Busslinger, Cosmas C. Giallourakis,
and Frederick W. Alt
Howard Hughes Medical Institute, The Children’s Hospital, The Immune Disease Institute; Department of Genetics, Harvard
Medical School, Boston, MA
Immunoglobulin heavy chain (IgH) variable region exons are assembled from V H, D and JH gene segments in
developing B lymphocytes. Within the 2.7 megabase (Mb) mouse IgH locus, V(D)J recombination is regulated
to ensure specific and diverse antibody repertoires. Herein, we report a key IgH V(D)J recombination regulatory
region, termed InterGenic Control Region-1 (IGCR1), that lies between the VH and D clusters. Functionally,
IGCR1 employs CTCF looping/insulator factor binding elements and, correspondingly, mediates IgH loops
containing distant enhancers. IGCR1 promotes normal B cell development and balances antibody repertoires by
inhibiting transcription and rearrangement of D H-proximal VHs and promoting rearrangement of distal VHs.
IGCR1 maintains ordered and lineage-specific VH(D)JH recombination, respectively, by suppressing VH joining to
Ds not joined to JHs and VH to DJH joins in thymocytes. IGCR1 also is required to allow feedback regulation and
allelic exclusion of proximal VH to DJH recombination. Our studies elucidate a long-sought IgH V(D)J
recombination control region and implicate a new role for the generally expressed CTCF protein.
P55 - Proliferating Cell Nuclear Antigen (PCNA) is a Novel Inhibitory Ligand for NKp44 Receptor
1
1
1
2
1
UZI HADAD , Benyamin Rosental , Michael Brusilovsky , Kerry S. Campbell , Alex Braiman , and Angel
1
Porgador
1
2
The Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer Sheva, Israel;
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA
Natural killer (NK) cells selectively kill tumor or virus-infected cells. This activity is delicately balanced by
activating and inhibitory signals from a repertoire of NK cell surface receptors that bind to their ligands. Natural
Cytotoxicity Receptors (NCRs) are a family of three receptors: NKp46, NKp44, and NKp30. Their expression is
predominantly restricted to NK cells, and they have the ability to activate NK cells in the absence of additional
stimuli. It is well documented that certain tumor cells express several ligands for NCRs though only a few
cellular ligands have been identified so far. Our group showed, in several studies, that all NCRs bind to different
Heparan-Sulfate / Heparin epitopes, and that this interaction is important for the recognition of tumor target cells
by the NK cells. Other studies identified BAT3 and B7-H6 as cellular ligands for NKp30.
Proliferating Cell Nuclear Antigen (PCNA) is a nuclear/cytoplamic protein that is overexpressed in hyperplasia
and cancer cells. Our study identified PCNA as novel inhibitory ligand for NKp44. We found that upon incubation
with NK cells, target PCNA translocates to the immunological synapse, interacts directly with NKp44, and
inhibits different NK functions, including cytokine secretion and cytotoxicity. Since the early research of NKp44,
it is known to contain an ITIM sequence in its cytoplasmic tail. However, previous studies failed to delineate its
activity. Our study demonstrates that the inhibitory effect of PCNA-NKp44 interaction is mediated through the
ITIM component of NKp44.
Our research indicates that PCNA could serve as a potential target for NK based immunotherapy for cancer.
P56 - Persistent Antigen at Vaccination Sites Induces CD8+ T Cell Sequestration, Dysfunction, and
Deletion
YARED HAILEMICHAEL, Zhimin Dai, Nina Jaffarzad, Yang Ye, Miguel A. Medina, Xue-Fei Huang, Stephanie
M. Dorta-Estremera, Nathaniel R. Greeley, Giovanni Nitti, Zhi Qiang Wang, Wencai Ma, Brian Rabinovich,
1
Nahum Puebla-Osorio, Kimberly S. Schluns, Richard E. Davis, Patrick Hwu, and Willem W. Overwijk
The University of Texas M.D. Anderson Cancer Center, Houston, TX
To understand why cancer vaccine-induced T cells often fail to shrink tumors, we studied immune responses in
mice vaccinated with gp100 peptide emulsified in incomplete Freund’s adjuvant (IFA), commonly used in clinical
cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in
tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells underwent antigen-driven
apoptosis, resulting in systemic tolerance. Treatment with anti-CD40 antibody, TLR7 agonist, and interleukin-2
reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation
shifted T cell localization towards tumors, inducing superior anti-tumor activity. Short-lived formulation also
reduced systemic T cell exhaustion and promoted memory formation. Persisting peptide/IFA vaccine depots,
currently used to vaccinate cancer patients, can induce specific T cell sequestration at vaccination sites followed
by exhaustion and deletion; short-lived depot formulations may overcome these limitations and result in greater
therapeutic efficacy of peptide-based cancer vaccines.
P57 - Macrophages are More Potent Immune Suppressors than Myeloid-Derived Suppressor Cells in
Mice Bearing 4T1 Metastatic Mammary Carcinomas
MELISA J. HAMILTON-VALENSKY, Judit Banáth, Nancy E. LePard, Gerald Krystal, and Kevin L. Bennewith
British Columbia Cancer Agency Research Centre, Vancouver, British Columbia, Canada.
Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSCs) and macrophages
(Ms), play pivotal roles in the promotion of primary tumor growth and are emerging as possible players in
metastasis. However, little is known about the relative immunosuppressive potencies of different myeloid cell
types or whether the strength of immune suppression correlates with the functions of these cells in tumor growth
or metastasis. To address these questions we isolated different populations of MDSCs and Ms from mice
bearing metastatic 4T1 murine mammary tumors and compared their ability to suppress T cell proliferation in
vitro. Ms were more potent suppressors of T cell proliferation than MDSCs on a per cell basis in each tissue we
investigated, including the spleen and the lung, which is a key metastatic target. Moreover, they exerted their
suppressive effects via different mechanisms. MDSCs suppressed T cells via a contact-independent mechanism
that could be blocked by catalase, suggestive of a role for extracellular ROS production. On the other hand, M
suppression was found to be contact-dependent and reversed by N-acetyl-cysteine (NAC), but not cysteine
supplementation, indicating a role for intracellular ROS production. Next, we investigated the roles of Ms and
MDSCs in promoting tumor growth and metastasis in vivo. All-trans retinoic acid (ATRA) induces immature cells
to terminally differentiate and is used to treat some forms of acute myeloid leukemia. We implanted 4T1 tumorbearing mice with ATRA pellets to promote the differentiation of MDSCs to Ms and DCs. As expected, mice
treated with ATRA had decreased MDSCs and increased Ms in their lungs. Interestingly, although ATRA
treatment did not alter the rate of primary tumor growth, mice treated with ATRA displayed significantly more
lung metastases than untreated or placebo-treated mice. We repeated these experiments using the 4T07
murine mammary tumor model and found ATRA treatment had an even more pronounced effect on increasing
metastatic growth. Taken together, these findings suggest that Ms may support metastasis to a greater extent
than MDSCs, which is consistent with our finding that Ms are more immunosuppressive than MDSCs on a per
cell basis.
P58 - Regulatory Mechanism of IgG Sialylation in Immune Responses
**JIN-HWAN HAN and Jeffrey V. Ravetch
Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY
A potent immunomodulatory molecule, immunoglobulin G (IgG), is a glycoprotein that carries an N-glycan on its
constant region (Fc). It was demonstrated that, in inflammatory conditions, the disease-associated IgGs have
decreased terminal sialic acid and galactose residues on their Fc glycan. Despite increasing experimental and
clinical evidence supporting such a notion, the exact regulatory mechanism of IgG sialylation is completely
unknown. We show here that IgG sialylation is differentially regulated by different modes of immune responses.
We also discovered that fully functional follicular helper T (T FH) cell help is required for the production of
asialylated antigen-specific IgG, suggesting that germinal center reaction plays a crucial role in the suppression
of IgG sialylation. Our findings can potentially lead to a novel vaccine strategy that enhances IgG-mediated
effector function against tumor cells and infectious agents by targeting TFH cells.
P59 - Adjuvant Breast Cancer Vaccine Improves Overall Survival of Breast Cancer Patients
JONATHAN F. HEAD and Robert L. Elliott
Elliott-Elliott-Head Breast Cancer Research and Treatment Center, Baton Rouge, LA
Background: Breast cancer patients were vaccinated in the adjuvant setting with a whole cell vaccine,
containing autologous and allogeneic breast cancer cells, to evaluate the effect on host lymphocyte immunity
and overall survival.
Material and Methods: We began preparing whole cell preparations for a breast cancer vaccine clinical trial in
1995. Stage I and II breast cancer patients had host lymphocyte immunity against tumor-associated antigens
evaluated before standard therapy. Those patients with depressed immunity determined by a lymphocyte
blastogenesis assay (LBA) were offered the whole cell vaccine. Patients were given six intradermal injections
(three weekly followed by three monthly). Ten weeks after the last injection the LBA was repeated.
Results: Thirty-seven patients were vaccinated in the adjuvant setting with the whole cell vaccine, containing
autologous and allogeneic breast cancer cells. Some patients experienced slight pain and swelling at the
injection site. There were no severe toxicities and the vaccine was well tolerated. There has been a ten year
follow-up for all vaccinated patients. The survival data of the vaccinated patients with depressed immunity (79%
survival at 10 years) compared to a historic control of unvaccinated patients with depressed immunity to their
tumor-associated antigens (59% survival at 10 years) demonstrates an improvement in overall survival of the
vaccinated patients.
Conclusions: This study confirms the importance of maintaining good host lymphocyte immunity after
completion of standard therapy, and validates cancer immunotherapy in the adjuvant setting.
P60 - The Cancer/Testis Antigens CABYR A/B and CABYR C are Expressed in a Subset of Colorectal
Cancers and Hold Promise as Targets for Specific Immunotherapy
1
3
2
1
2
H.M.C. Shantha Kumara , Otavia L. Caballero , Tao Su , Xiaohong Yan , Aqeel Ahmed , CHANDANA S.H.
1
1
1
4
2
1
HERATH , Vesna Cekic , Daniel D. Kirchoff , Andrew J. Simpson , Carlos Cordon-Cardo , Richard L. Whelan
1
2
St. Luke Roosevelt Hospital, New York, NY; Herbert Irving Comprehensive Cancer Center, Columbia University, New York,
3
4
NY; Ludwig Institute for Cancer Research, Johns Hopkins University Baltimore, MD; Ludwig Institute for Cancer Research,
New York, NY
Introduction: Human CABYR is a testis-specific polymorphic calcium binding tyrosine phosphorylation
regulative protein isolated from spermatozoa. CABYR is expressed in the human germ line but not in adult
human tissues, thus, it is considered a cancer-testis (CT) protein The expression patterns of CABYR genes in
colorectal cancer (CRC) are not well characterized. The aim of this study is to evaluate the expression of
CABYR a/b and c in CRC tumors and to determine if they hold promise as vaccine targets.
Method: Consenting CRC patients (pts) who underwent elective resection for whom tumor samples were
available were included in the study. Tissues were OCT embedded and stored at -80°C until analysis. CABYR
a/b and c expression was analyzed by quantitative PCR (QPCR) using the SYBR-Green platform. Tumor and
normal tissue expression levels were determined and compared, as were tumor and testis CABYR expression
levels. Tumors with expression levels of 0.1% or more than the testis were considered positive. Expression of
CABYR a/b and c in a set of normal human tissues was analyzed by RT-PCR.
Results: A total of 47 paired CRC and normal tissue specimens were studied. The percent of pts with a relative
expression ratio of malignant to normal tissues (MN ratio) over 1 was 70% for CABYR a/b and 72% for CABYR
c. The percent with both an MN ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR a/b
and 25.5% for CABYR c. Except for very low expression of CABYR a/b in the brain, CABYR a/b or c was not
expressed in normal tissues.
Conclusion: RT-PCR analysis confirmed the frequent overexpression of CABYR a/b and c in most CRC tumors
compared to adjacent normal tissues. In 23-26% of tumors expression was more than 0.1% of the expression
level of testis. A larger and more diverse group of tumors (Stage 1-4) needs to be assessed to determine if
CABYR expression correlates with T, N, or final tumor stage. Evaluation of blood for anti-CABYR antibodies is
also needed. These results support CABYR as a potential therapeutic vaccine candidate in CRC.
P61 - Potent Anti-Tumor Strategies Based on OX40 Engagement
1
1
1
1
DANIEL HIRSCHHORN-CYMERMAN , Gabrielle A. Rizzuto , Taha Merghoub , Adam D. Cohen , Francesca
1
1
2
1,3,4
1,3,4
Avogadri , Alexander M. Lesokhin , Andrew D. Weinberg , Alan N. Houghton , and Jedd D. Wolchok
1
2
Memorial Sloan-Kettering Cancer Center, New York, NY; Robert W. Franz Cancer Center, Earle A. Chiles Research
3
4
Institute, Providence Portland Medical Center, Portland; Weill Medical College, Cornell University, New York, NY; Graduate
School of Medical Sciences of Cornell University, New York, NY
Expansion and recruitment of CD4+ Foxp3+ regulatory T cells (Tregs) are mechanisms employed by growing
tumors in order to evade immune elimination. In addition to expansion of effector CD8+ T cells, successful
therapeutic interventions may require reduction of Tregs within the tumor microenvironment. Here we report that
the combined use of the alkylating agent cyclophosphamide (CTX) and an agonist antibody targeting the costimulatory receptor OX40 (OX86) provides potent anti-tumor immunity capable of regressing established,
poorly immunogenic B16 melanoma tumors. CTX administration resulted in tumor antigen release, which
following OX86 treatment significantly enhanced the anti-tumor T cell response. We demonstrated that an
important cellular target of the combination therapy are Tregs. Paradoxically, the combination therapy led to an
expansion of Tregs in the periphery. In the tumor, however, the combination therapy induced a profound Treg
depletion that was accompanied by an influx of effector CD8 T cells leading to a favorable T effector/Treg ratio.
Closer examination revealed that diminished intratumoral Treg levels resulted from hyperactivation and Tregspecific apoptosis. Thus, we propose that CTX and OX40 engagement represents a novel and rational chemoimmunotherapy.
P62 - Dynamic Capture of Tumor Propagation and Associated Immune Responses in CNS
Microenvironment
1
1
1
1
1
1
ALEX Y. HUANG , Jay T. Myers , Agne Petrosiute , Deborah Barkauskas , Jennifer Tsai , Kathryn Davis , Y.
1
2
2
Othman , Justin D. Lathia , and Jeremy N. Rich
1
2
Case Western Reserve University School of Medicine; Cleveland Clinic Foundation, Lerner Research Institute, Cleveland,
OH
Ex vivo experimental systems often lack the appropriate conditions to fully capture the complex intercellular
communication between tumor cells and surrounding tissues – a critical feature in understanding cancer
development and immune evasion. In vivo imaging modality such as bioluminescence lacks the resolution
necessary to discern subtle structural differences and heterogeneity in the tumor niche. Microscopic
examination of fixed specimens is devoid of the 3-dimensional context or sequential evolution of tumor
progression within the same host. Recent insights into in vivo tumor formation and associated immune
responses have come from studies involving the use of intravital 2-photon laser scanning microscopy (2P-LSM),
a technique that allows deep visualization (>300um) with single-cell resolution (<1um), thus enabling direct
observation of cellular behavior in intact tissues at a suitable dynamic spatial-time resolution. Our laboratory
studies the role of tumor microenvironment in shaping immune repertoire towards tolerance and develops
strategies to modify tumor microenvironment to enhance anti-tumor immunity. One such example is our study of
+
CNS tumors such as glioblastoma multiforme (GBM), which contain a cellular hierarchy with a CD133 subpopulation representing self-renewing and tumorigenic GBM stem cells (GSCs). In a xeno-transplant model, a
single GSC was capable of tumor initiation in the mouse brain. To directly test the relative tumorigenic potential
+
of GSCs (CD133 ) and non-GSCs (CD133 ), we inoculated paired tumor populations from the same primary
GBM tumor cells and monitored tumor competition by serial 2P-LSM through implanted cranial windows. Serial
2P-LSM imaging shows that after 35 days, in vivo GBM formation was driven exclusively by GSCs but not nonGSCs. In order to interrogate CNS tumor-associated immune responses, we inoculated syngeneic mouse
glioma tumors into immunocompetent C57BL/6 mice. Using this and a CNS-inflammatory model, the
experimental autoimmune encephalomyelitis, we have begun to undercover the role of perivascular antigenpresenting cells and microglia in guiding the recruitment of CNS-bound lymphocytes. Our data provide the first
direct functional evidence that CSCs are responsible for tumor propagation in GBM, and represent an in vivo
experimental platform to monitor immunotherapeutic interventions.
P63 - Structural and Functional Analysis of RAR-related Orphan Receptor Gamma
**WENDY HUANG, Jun R. Huh, Jawara Allen, and Dan R. Littman
Skirball Institute; Howard Hughes Medical Institute, New York University Langone Medical Center, New York, NY
Tumor pathogenesis is associated with chronic inflammation. Recent studies have highlighted important roles of
interleukin 17 (IL-17)-secreting T-helper (Th17) lymphocytes in multiple chronic inflammatory diseases, including
multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, asthma, and tumor development. Retinoidrelated orphan receptor-gamma (RORt) has been shown to be a key transcription factor critical for promoting
Th17 cell differentiation and production of IL-17. However, the molecular mechanisms and upstream signaling
pathways that regulate the function of RORt are poorly characterized. We performed evolutionary conservation,
surface exposure, and protein interactome analysis to identify post-translational modifications and important
protein co-regulators of RORt. We now show that component of the proteasome and nuclear pore complexes,
as well as the phosphorylation status of serine residues on RORt, play important roles in modulating protein
stability and transcriptional activities of RORt. Studies to dissect the functional implications of these regulatory
events in Th17-modulating inflammatory conditions are under way.
P64 - ITAM Signaling Negatively Regulates Dendritic Cell Function via SOCS-1
1,2
2
1,2
**HIROAKI ITO , Laura Lau , and Jessica A. Hamerman .
1
2
Benaroya Research Institute at Virginia Mason, Seattle, WA and Department of Immunology, University of Washington,
Seattle, WA
Dendritic cells (DCs) play a crucial role in both the innate and adaptive immune system. DCs use several
receptor systems to recognize pathogens, including Toll-like receptors (TLR). TLR signaling in DCs promotes
the presentation of antigens derived from pathogens and the production of proinflammatory cytokines,
chemokines, and type I Interferons (IFNs). We have found that two immunoreceptor tyrosine-based activation
motif (ITAM) adaptors (DAP12 and FcR) negatively regulate TLR responses in DCs, however, the precise
mechanism by which ITAM signaling suppresses TLR responses remains to be elucidated. Here, we show that
aberrant SOCS-1 expression was found in ITAM signaling-deficient DCs. SOCS-1 is a well-described
suppressor for TLR signaling, as well as cytokine signaling, including signaling from the IFN- and IFN-
receptors. We found decreased SOCS-1 expression in DAP12/FcR-deficient BM-derived DCs (BMDCs) in
comparison with WT BMDCs before and after TLR stimulation at both mRNA and protein levels. MHC class II
expression on DAP12/FcR-deficient BMDCs was higher than that of WT BMDCs even under steady state
conditions, similar to SOCS-1 deficient DCs. Consistent with the decrease of SOCS-1 expression in
DAP12/FcR-deficient BMDCs, STAT-1 phosphorylation upon IFN- stimulation was increased in DAP12/FcRdeficient BMDCs in comparison with WT BMDCs. These results suggest that ITAM signaling positively regulates
SOCS-1 expression to prevent DC activation in the steady state. Because miR-155 can degrade SOCS-1
mRNA, we investigated whether miR-155 expression was dysregulated in DAP12/FcR-deficient BMDCs.
Indeed, ITAM-deficient BMDCs had increased miR-155 compared to WT BMDCs, suggesting one mechanism
for reduced SOCS-1 in these cells. In conclusion, these results suggest that ITAM signaling regulates the
balance between SOCS-1 and miR-155 expression to maintain appropriate DC responses in the face of
pathogen challenge.
P65 - MerTK Inhibition in Tumor-Associated Macrophages as a Potential Therapeutic Target in Breast
Cancer
*KRISTEN M. JACOBSEN and Douglas K. Graham
University of Colorado Denver Anschutz Medical Campus, Aurora, CO
Tumor-associated macrophages (TAMs) can have pro-tumorigenic effects that promote angiogenesis,
lymphangiogenesis, tumor proliferation, and metastasis. An increased number of TAMs has also been
associated with poor prognosis in a number of solid tumors, such as breast, lung, and prostate cancers,
gliomas, and lymphomas. Previous studies have shown that reeducating or depleting the pro-tumor TAM
population can lead to a reduction in tumor growth and metastasis. The MerTK receptor tyrosine kinase is
differentially expressed during macrophage development and polarization. MerTK has also been shown to play
a role in normal macrophage function, such as apoptotic cell clearance and cytokine production. Preliminary
studies have suggested MerTK expression in the tumor microenvironment or on infiltrating hematopoietic cells
promotes tumor growth in a mouse model of mammary gland carcinoma. In order to investigate the role of
MerTK expression in the tumor microenvironment, the MMTV-PyMT model was utilized, in which expression of
the polyoma middle T oncoprotein is driven by the mouse mammary tumor virus promoter resulting in
spontaneous development of mammary gland tumors. Females expressing the MMTV-PyMT transgene were
+/+
-/lethally irradiated and transplanted with MerTK or MerTK bone marrow, and mammary tumor growth was
-/measured using MRI. The primary tumor burden of mice transplanted with MerTK bone marrow was reduced
+/+
50% compared to mice transplanted with MerTK bone marrow. Interestingly, the differential expression of
MerTK in the transplanted bone marrow cells did not seem to affect the macrophages’ ability to migrate and
infiltrate the mammary gland tumors, suggesting the effect maybe through functional differences between the
+/+
-/MerTK and MerTK macrophages in the tumor microenvironment. Therefore, future studies need to be
conducted in order to elucidate the mechanism by which lack of MerTK expression in the tumor
microenvironment leads to a reduction in tumor growth in this model. However, our results suggest that MerTK
inhibition in tumor-associated macrophages may be a novel therapeutic for solid tumors by targeting both the
tumor cells and the tumor microenvironment.
P66 - Identifying Disease Susceptibility Loci by Expression QTL Analysis
**KIRK D.C. JENSEN, Musa Hassan, Kenneth Hu, Erwan Boedec, and Jeroen P.J. Saeij
Massachusetts Institute of Technology, Department of Biology, Cambridge, MA
Disease outcome is significantly influenced by one’s genetic background. Here we explore the genetic basis for
differences in host susceptibility to infection with Toxoplasma, an orally acquired parasite that currently infects 2
billion people worldwide and can cause severe disease in animals and man. Over twenty years ago, McLeod
and colleagues reported the survival of thirty recombinant inbred (RI) mouse strains, which are derived from
crosses between susceptible C57BL/6 and resistant A/J mice, following oral challenge with Toxoplasma and
approximated that five genetic loci controlled differences in survival between the strains. The genetic map of
each of these RI strains is now available and of the five susceptibility loci that were originally postulated, four
can now be positioned to genetic regions on mouse chromosomes 1, 9, 11, and 15.
To identify candidate host genes within these regions, we explored the transcriptional response of macrophages
following Toxoplasma infection or inflammatory stimuli that might occur during oral infection. The macrophage
cell type was chosen because it elicits many of the host’s immunoregulatory and toxoplasmacidal mechanisms
following infection. Bone marrow derived macrophages from each RI mouse strain were generated and their
transcriptome was determined by RNA sequencing after Toxoplasma infection or TNF + IFN stimulation. The
gene expression of each gene was considered a quantitative trait (expression QTL) that was subsequently
mapped to mouse genetic loci, and both trans- and cis-regulation of gene expression was observed. Results
from these screens will be discussed, including ongoing investigation to determine potential gene candidates in
mice that control disease outcome.
P67 - Trypanosoma cruzi as an Effective Cancer Antigen Delivery Vector
1,2
1,2
1,2
1
CAROLINE JUNQUEIRA , Bruno Galvão-Filho , Luara I. Santos , Santuza M. R. Teixeira , Flávia G.
2
3
1
4
4
4
Rodrigues , Wanderson D. DaRocha , Egler Chiari , Achim A. Jungbluth , Gerd Ritter , Sacha Gnjatic , Lloyd J.
4
1,2,5
Old , and Ricardo T. Gazzinelli
1
Departamento de Bioquímica e Imunologia and Departamento de Parasitologia, Instituto de Ciências Biológicas,
2
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Laboratório de Imunopatologia, Centro de Pesquisas
3
René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil; Departamento de Bioquímica e Biologia Molecular,
4
Universidade Federal do Paraná, Curitiba, MG, Brazil; Ludwig Institute for Cancer Research, New York Branch at Memorial
5
Sloan–Kettering Cancer Center, New York, NY; Division of Infectious Disease and Immunology, University of
Massachusetts Medical School, Worcester, MA
One of the main challenges in cancer research is the development of vaccines that induce effective and longlived protective immunity against tumors. Significant progress has been made in identifying members of the
Cancer/testis Antigen (CTA) family as potential vaccine candidates. However, it remains to be developed an
ideal form for antigen delivery that induces robust and sustainable antigen-specific T cell responses, and in
+
particular of CD8 T lymphocytes. Here we report the use of a recombinant non-pathogenic clone of
Trypanosoma cruzi as a vaccine vector to induce vigorous and long-term T cell-mediated immunity. The
rationale for using the highly attenuated T. cruzi clone was: (i) the ability of the parasite to persist in host tissues
and, therefore, to induce a long-term antigen-specific immune response; (ii) the existence of intrinsic parasite
agonists for Toll-Like Receptors (TLRs) and consequent induction of highly polarized Th1 responses; and (iii)
the parasite replication in the host cell cytoplasm, leading to direct antigen presentation through the endogenous
+
pathway and consequent induction of antigen-specific CD8 T cells. Importantly, we found that parasites
expressing a CTA (NY-ESO-1) were able to elicit solid protection against melanoma in a mouse model.
Furthermore, in a therapeutic protocol, the parasites expressing NY-ESO-1 delayed the rate of tumor
development in mice. We conclude that the T. cruzi vector is highly efficient in inducing T cell-mediated
immunity and protection against cancer cells. More broadly, this strategy could be used to elicit long-term T cellmediated immunity and used for prophylaxis or therapy of chronic infectious diseases.
Supported by: The Atlantic Philanthropies/LICR; CNPq; FAPEMIG; CAPES.
P68 - Activation of Dendritic Cells During Chronic Viral Infection Improves Virus-specific T Cell
Responses
**ALICE O. KAMPHORST, Andreas Wieland, Pablo Penaloza-MacMaster, Cullen LaClair, and Rafi Ahmed
Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA
Chronic infections are established when the immune system fails to eliminate a pathogen. A hallmark of chronic
infections is T cell exhaustion: the antigen-specific T cells that persist lack effector functions. T cell exhaustion
was first described in lymphocytic choriomeningitis virus (LCMV) infection and later shown to occur in many
persistent infections, such as HIV and hepatitis B and C virus infections, as well as during cancer. Exhausted T
cells express several inhibitory receptors such as Programmed Death (PD)-1, that attenuates T cell receptor
signaling. Sustained inhibitory signaling on the T cells contributes to dysfunction maintenance. Control of
chronic infections can be achieved by reverting the exhausted state of antigen-specific lymphocytes, for
example through blockade of the PD-1/PD-L1 pathway. However, there remains a need to develop further
strategies that boost T cell responses to clear chronic infections and tumors. Conventional dendritic cells (DCs)
are professional antigen presenting cells necessary to initiate immune responses by linking innate to adaptive
immunity. The primary function of DCs is antigen presentation to T cells, while modulating T cell activation with
cytokines and co-stimulatory molecules. To determine how DCs can contribute to functional restoration of
exhausted CD8 T cells we used agonistic anti-CD40 antibodies to activate DCs during LCMV chronic infection.
Upon anti-CD40 treatment we observed an increase in virus-specific T cell numbers, as well as effector function
(IFN- production). However, this effect was not sustained and virus load could not be controlled. We have
found that blockade of the PD-1/PD-L1 pathway does not lead to DC activation. Therefore, we are currently
addressing whether a combination therapy with anti-CD40 and anti-PD-L1 might have a synergistic effect to
rescue exhausted CD8 T cells and promote viral clearance during stringent LCMV chronic infection. Agonistic
anti-CD40 antibodies can also activate other cell types besides DCs. It will also be important to determine if the
rescue of exhausted CD8 T cells mediated by anti-CD40 treatment is a direct result of DC activation and
whether B cells have a role.
P69 - The Activation-Induced Cytidine Deaminase (AID) Targets DNA Less Efficiently in Stable
Nucleosomes
**PRASHANT KODGIRE and Ursula Storb
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL
The humoral arm of adaptive immunity is characterized by a large and diverse population of antibodies
generated by V(D)J recombination, followed by random mutations in the antigen binding region of the antibody
gene by a process of somatic hypermutation (SHM), which creates further diversity as well as enhances
antibody affinity more than 1000-fold. The somatic hypermutation of antibody genes is initiated by the cytidine
deaminase AID creating cytidine (C) to uridine (U) mutations, starting after ~100-200 bp from the promoter and
extending for about 2 Kbp. During SHM these ‘U’s are repaired in error-prone fashion, using translesion DNA
polymerases leading to mutations at and near the ‘U’. The process of SHM is linked to transcription initiation.
We have postulated that AID, which initiates SHM, gets associated with the transcription complex at or near the
promoter and deaminates ‘C’s, while it travels with the RNA polymerase. Transcription occurs in the context of
chromatin which likely modifies the targeting of AID. In this study we are trying to obtain a better insight into how
AID acts in the antibody genes, especially understanding whether targeting of AID is affected by the positioning
of nucleosomes in the Ig genes. We have introduced two copies of a very strong nucleosome positioning
sequence into the variable region of an Ig gene in mutating B cells and are looking for effects on the SHM
pattern. Our results indicate that positioning of nucleosomes in the IgL gene significantly affects the accessibility
of AID to Ig genes and SHM pattern. We postulate that, unlike RNA polymerase, AID has a limited capacity to
negotiate stable nucleosomes. Our experiments are important for determining how the varied repertoire of
antibody genes is created with the potential to react against any foreign antigenic substance, including tumor
cell antigens.
P70 - B Cells Generated Using Soluble Multimeric CD40L Can be Used to Expand Antigen-Specific CD8+
T Cells and CD4+CD25+ Regulatory T Cells
RICHARD S. KORNBLUTH and Mariusz Stempniak
Multimeric Biotherapeutics, Inc., La Jolla, CA
Human B cells proliferate massively ex vivo when treated with a CD40 stimulus and IL-4. Such “CD40-B cells”
are known to be strong antigen-presenting cells (APCs) that can replace dendritic cells (DCs) for many
applications. However, the production of CD40-B cells generally requires co-culture with cells lines that express
membrane CD40L, since this form of CD40L supplies a many-trimer stimulus needed to cluster CD40 receptors
and thereby activate the B cells. This added level of complexity has compromised the practicality of using CD40B cells instead of DCs for generating anti-tumor CD8+ T cells. To circumvent this problem, we tested a 4-trimer,
multimeric soluble form of CD40L produced by fusing the body of surfactant protein D with the extracellular
domain of CD40L to make SP-D-CD40L (UltraCD40L™). This protein was produced by CHO cells and is stable
o
for several weeks at room temperature and >5 months at 4 C. To grow CD40-B cells, CD19+ or CD20+ cells
were immunomagnetically selected from 0.5-15 ml of venous blood and cultured with 10-20% CHO cell
supernatant plus IL-4. About 100-fold expansion of B cells occurred in two weeks and the resulting cells were
>95% positive for CD19, CD80, and CD86. These growing CD40-B cells can be cryopreserved and then thawed
for further expansion many months later. To use these CD40-B cells as APCs for CD8+ T cells, they were
treated with mitomycin C and pulsed with the NLV peptide, a prototypic antigen from CMV pp65 that is an
immunodominant epitope for HLA-A2.1 donors. Using these cells as APCs for either PBMCs or purified CD8+ T
cells, antigen-specific CD8+ T cells were generated that were recognized by NLV/A2.1- tetramer staining. Under
other culture conditions, CD40-B cells were used to expand CD4+CD25+ regulatory T cells (Tregs). Thus, the
use of SP-D-CD40L to grow B cells provides a simple means of generating proliferating APCs that could
eventually supplant the need for nonproliferating dendritic cells in adoptive cellular therapy (ACT)-based
treatment protocols.
P71 - Infusion of an OX40 Agonist Increases CD4+ and CD8+ Memory T Cell Proliferation in Cancer
Patients: Immunologic and Clinical Assessment of a Phase I Trial
MAGDALENA KOVACSOVICS-BANKOWSKI, Edwin B. Walker, Lana Chisholm, Kevin Floyd, Walter J. Urba,
Brendan Curti, Andrew Weinberg
Providence Cancer Center, Providence Portland Medical Center, Portland, OR
OX40, a member of the TNFR superfamily, is a potent co-stimulatory molecule expressed on the surface of
activated T lymphocytes. OX40 engagement increases T cell proliferation, effector function, and survival.
Preclinical studies have shown that OX40 agonists increase anti-tumor immunity and improve tumor-free
survival. These preclinical results have prompted the initiation of phase I clinical trial using a mouse anti-human
OX40 agonistic antibody injected into patients with stage IV cancer. The anti-OX40 antibody was administered
on Day 1, 3, and 5 at 0.1, 0.4, and 2 mg/kg (10 patients/dose). The antibody was well tolerated and there was
evidence of tumor shrinkage in 11/30 patients treated. Immune monitoring analysis, over a 2 month period after
OX40 Ab administration, was performed on PBL by flow cytometry, using a 10-color antibody panel detecting
CD3, CD4, CD8, CD95, CD28, CD25, CD127, CCR7, Foxp3, and Ki-67. Administration of anti-OX40
+
neg
+
monoclonal antibody increased the proliferation of peripheral T cells, both CD4 /Foxp3 and CD8 T cells, with
+
pos
no significant increase in CD4 /Foxp3 regulatory T cells. Anti-OX40 not only triggered T cell proliferation, but
+
also changed the activation phenotype of the CD8 T cells as measured by increased co-expression of CD38
and HLA-DR on the surface of cycling cells. Finally, there was an association between increased proliferation of
+
neg
+
CD4 /FoxP3 and CD8 T cells and lack of disease progression in anti-OX40 treated patients. These results
show that the administration of an anti-OX40 antibody into cancers patients was well tolerated and caused
tumor shrinkage in some patients. It also greatly increased proliferation of a subset of T cells, defining an OX40specific “immunologic signature” that could potentially be used as a biomarker to predict clinical outcome.
P72 - Expression of B7-H3 and B7x on TRAMP-C2 Tumor Cells Leads to Increased Formation of Tumor
Nodules in the Lung
**KATHARINA KREYMBORG, Rebecca Waitz and James P. Allison
Howard Hughes Medical Institute, Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, NY
Although many tumors express antigens that can be recognized by the host’s T cells, spontaneous immunemediated rejection of cancer very rarely occurs. The lack of positive costimulatory ligands or the presence of
inhibitory molecules likely contributes to this phenomenon. One of the most extensively studied inhibitory
receptors is CTLA-4, which is expressed by T cells and binds to B7-1 and B7-2 on antigen presenting cells. In
vivo blockade of CTLA-4 enhances tumor-specific T cell responses in several mouse models, and blocking
CTLA-4 in melanoma patients results in clinical responses and an improved overall survival.
Lately, several additional costimulatory pathways and molecules have been identified, among them B7x and B7H3, which belong to the B7 family of coreceptors. B7x seems to negatively regulate the immune response
whereas B7-H3 has been implicated as both a stimulator as well as an inhibitor of T cells, possibly depending
on the type of responder cell or utilization of multiple receptors with different properties. Interestingly,
overexpression of both B7x and B7-H3 has been observed on a variety of tumors and is associated with
advanced tumor stage and poor clinical outcome. Thus, tumors might use these molecules to locally manipulate
or impede tumor-specific immune responses.
To study the impact of tumor-expressed B7x and B7-H3 on tumor growth as well as on anti-tumor immune
responses, we engineered transplantable prostate tumor cells (TRAMP-C2) to overexpress B7x or B7-H3 and
intravenously injected them into C57BL/6 wt mice. The expression of B7x as well as B7-H3 on TRAMP-C2 cells
caused greatly increased formation of tumor nodules in the lung at day 31 post injection compared to control
TRAMP-C2 cells. This might imply that B7x or B7-H3 expressed on the tumor itself could inhibit tumor-specific
immune responses.
P73 - Single-Cell Tracking of PU.1 and Bcl11b Regulation During Myeloid and Lymphoid Development
**HAO YUAN KUEH, Michael Elowitz, and Ellen Rothenberg
California Institute of Technology, Pasadena, CA
Haematopoietic stem cells utilize circuits of interacting transcription factors to control cell fate decision making
during differentiation. Deregulation of transcription factor levels in these circuits can lead to differentiation arrest
and leukemic transformation. While recent experiments are yielding insights into the topology and function of
these transcription factor circuits, it remains unknown how they mediate fate decision making on a single-cell
level. In this study, we used flow cytometry and timelapse microscopy to track the regulation of two
haematopoietic transcription factors PU-1 and Bcl11b in single progenitor cells. PU.1 is crucial for the
development of multiple haematopoietic fates; it is initially expressed in stem cells and up-regulated during
myeloid development, but down-regulated during lymphoid development. Aberrant regulation of PU.1 levels can
cause oncogenic transformation in a number of different haematopoietic lineages. Bcl11b is specifically required
for T cell fate commitment, and is sharply up-regulated during T cell development. It is suspected to be a tumor
suppressor gene in developing T cell progenitors. To facilitate single-cell measurements, we utilized an existing
transgenic PU.1-GFP reporter strain (Nutt et al., 2005), and also generated transgenic Bcl11b fluorescent
reporter mouse strains using gene-targeting. We found that lymphoid/myeloid bipotent progenitors express
PU.1-GFP at a uniform intermediate level. Consistent with known results, they decrease PU.1-GFP levels as
they undergo B- or T- cell development; increase PU.1-GFP levels as they undergo macrophage or dendritic cell
differentiation; and maintain similar PU.1-GFP levels as they turn on Gr1, a granulocyte marker. By following
PU.1-GFP levels in single cell lineages, we found that the activity of the PU.1 promoter was already maximal in
the earliest progenitors. Further PU.1-GFP level increases occurred not through additional increases in
promoter activity, but by cell cycle lengthening, which allows for additional protein accumulation. PU.1 promoter
activity was markedly reduced in cells that decreased their PU.1-GFP levels, as well as those that maintained
constant intermediate PU.1-GFP levels; once again, differences in PU.1-GFP levels between these two
populations derived primarily from differences in the cell cycle length, not from differences in promoter activity.
Our results uncover an unexpected but important role for the cell cycle in controlling cellular PU.1 levels, and
suggest that dynamic cell cycle control and transcription regulation act in concert to control the levels of
regulatory genes during haematopoietic development.
P74 - Impact of Topical Imiquimod on Immune Responses of Colorectal Cancer Patients Treated with the
Multi-Peptide Vaccine IMA910 and GM-CSF
1
1
1
1
2
3
SABRINA KUTTRUFF , Steffen Walter , Andrea Mayer , Jörg Ludwig , Frank Mayer , Erika Hitre , Bernhard
1
1
1
1
1
1
Rössler , Michael Cannarile , Dominik Maurer , Verona Vass ; Juha Lindner , Nina Pawlowsiki , Claudia
1
1
1
1
1
1
1
Trautwein , Jörn Dengel , Norbert Hilf , Oliver Schoor , Toni Weinschenk , Jürgen Frisch , Carsten Reinhardt ,
1
Harpreet Singh
1
2
3
immatics biotechnologies, Tuebingen, Germany; University of Tuebingen, Tuebingen, Germany; National Institute of
Oncology, Budapest, Hungary
To effectively treat cancer patients with immunotherapy, vaccines have to overcome tolerance and
immunosuppression by combination with powerful immunomodulators. Recently, the efficacy of immunotherapy
has been demonstrated in several settings. However, the selection of an adequate adjuvant for peptide vaccines
still remains difficult. In the past, only a limited number of studies directly compared two different adjuvants in a
single clinical trial, and patient numbers were often restricted.
IMA910 is a peptide-based vaccine consisting of 10 HLA-A*02 binding and 3 long HLA-DR binding tumorassociated peptides (TUMAP), which were presented on colorectal tumors and were overexpressed in
comparison to healthy tissues. Additionally, IMA910 contains a hepatitis B virus core-antigen marker peptide of
+
known immunogenicity. The multi-center clinical trial IMA910-101 enrolled HLA-A*02 colorectal cancer (CRC)
patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. Patients were
infused with a single low dose of cyclophosphamide (300mg/m2) and repeatedly immunized intradermally (up to
16 vaccinations) with IMA910 in combination with GM-CSF (cohort 1; n=66) or IMA910 with GM-CSF plus
topically applied imiquimod (cohort 2; n=26) as an adjuvant. Before and post vaccination patients were analyzed
for CD8 and CD4 T cell responses to IMA910 HLA-A*02- and HLA-DR-restricted peptides. Immunomonitoring
was performed by HLA-multimer assay and intra-cellular cytokine (ICS) assay for CD8 T cell responses and by
ICS assay for CD4 T cell responses. Tumor status of patients was monitored repeatedly by CT/MRI, and
corresponding tumor scans were reviewed centrally for assessment of disease control rate (DCR) and time to
progression (TTP).
Here, we show that IMA910 vaccination was safe and well tolerated. IMA910 overall was immunogenic in 75/80
(94%) evaluable patients. A moderate but significant effect of imiquimod treatment on the number of immune
responses to IMA910 peptides as detected by the ICS assay was observed. Finally, we demonstrate
significantly increased DCR and TTP of class I multi-TUMAP responders in comparison to patients with one or
no TUMAP responses.
P75 - Distinct Immune Profiles of Murine Solid Tumor Models Determine Optimal Immunotherapy
MELISSA G. LECHNER, Christopher Roque, Keegan Barry-Holson, and Alan L. Epstein
USC Keck School of Medicine, Los Angeles, California
Immunotherapy is a promising mode of cancer therapy since it can utilize the inherent antigen specificity,
systemic trafficking, and immune memory of the host. In order to develop successful immunotherapies, murine
tumor models are important preclinical tools that shed light upon strategies by which cancers achieve immune
escape and allow in vivo testing of relevant treatments. This study evaluated the hypothesis that the dominant
immune escape mechanisms employed by different murine tumor models varied significantly and that
identification of these strategies could be used to select optimal immunotherapy regimens to treat each unique
tumor. Six frequently used tumor models [BALB/c syngeneic: 4TI breast, CT26, MAD109, RENCA; C57BL/6:
Lewis (LLC) lung, B16 melanoma] were examined. Characterization of each tumor model’s immune profile was
performed using quantitative analysis of immune-related gene expression, immunohistochemistry, and FACS
analysis of tumor, tumor-draining lymph node (TDLN), and spleen for immune effector and suppressor cell
populations. Furthermore, tumor vasculature and indicators of antigen expression were evaluated on frozen
sections for each model. From these analyses, a distinct immune profile for each tumor model was generated,
including classification by the major immune suppressor cell population present [regulatory T cell (Treg),
myeloid-derived suppressor cell (MDSC), or both] and designation as poorly immunogenic or immune
stimulatory. As an unexpected finding, major differences in the compartmental localization of MDSC and Treg
populations were observed across models. Mature, activated dendritic cells (DCs) were generally decreased in
the BALB/c tumor models compared with strain-matched controls, with the notable exception of CT26. By
contrast, activated DCs were significantly increased in both the C57BL/6 tumor models. Some models were
notable for strong tumor infiltration by PMN, activated T cells, and antigen presenting cells, such as RENCA,
whereas B16 was poorly immunogenic and largely devoid of such immune infiltrate. Based upon the observed
immune signatures, immunotherapeutic protocols were tested to counter the evasive mechanisms seen in
several of the examined tumor models. This approach yielded successful therapy of MDSC-dominant RENCA
and Treg-dominant CT26, and MDSC- and Treg-influenced 4T1 tumor models. These studies characterize the
major mechanisms of immune escape in six well studied murine tumor models and demonstrate that prior
identification of the immune signature can facilitate selection of effective immunotherapies for treatment.
+
P76 - High Density of Tumor-Infiltrating Foxp3 Regulatory T Cells of Primary Tumor or Lymph Node
Metastasis is Associated with Favorable Outcome in Gastric Cancer
1
2
1
3
4
HEE EUN LEE , You Jeong Lee , Min A. Kim , Hye Seung Lee , Byung Lan Lee , Woo Ho Kim
1
1,4
2
Seoul National University Hospital, Seoul, Korea; Center for Immunology, University of Minnesota, Minneapolis, MN;
4
Seoul National University Bundang Hospital, Seongnam, Korea; Seoul National University College of Medicine, Seoul,
Korea
3
+
Background: It has been reported that the frequency of Foxp3 regulatory T cells (Tregs) among tumorinfiltrating lymphocytes is increased in cancer-bearing patients. We designed the study to determine the
+
prognostic significance of Foxp3 Tregs in gastric cancer.
+
Methods: Tissue microarray and immunohistochemistry were used to assess the densities of Foxp3 T regs in
primary tumor tissue (n=216) and unmatched metastatic tumor tissue of regional lymph nodes (n=149) from
+
+
gastric cancer patients. Numbers of Foxp3 Tregs were counted using NIH ImageJ software. Foxp3 Treg
densities were evaluated for correlation with clinicopathologic characteristics and overall survival.
+
Results: With respect to Foxp3 Tregs of primary tumor (Foxp3P), the low density was associated with
advanced T stage, lymph node metastasis, and distant metastasis (p = 0.003, 0.015, and <0.001, respectively,
ANOVA). Foxp3P density was found to be an independent predictor of lymph node metastasis by multivariate
analysis with odds ratios (95% CI) of 0.348 (0.165-0.734). Kaplan-Meier survival analysis revealed that the high
density group of Foxp3P had longer survival time than the low density group (p = 0.001, log-rank test). In
multivariate analysis, Foxp3P density remained an independent prognostic factor with hazard ratio (95% CI) of
+
0.651 (0.435-0.975). Meanwhile, the density of Foxp3 Tregs of lymph node metastasis (Foxp3LN) was not
correlated with T stage of the original tumor, lymph node metastasis, or distant metastasis. However, it was
found that Foxp3LN density was an independent prognostic indicator: high density of Foxp3LN was significantly
associated with improved survival (hazard ratio (95% CI) = 0.532 (0.356-0.795)) in multivariate analysis.
+
Conclusions: Foxp3 Treg densities in not only primary tumor, but also metastatic tumor of regional lymph
nodes, were significantly associated with patient survival in gastric cancer. As our finding that high density of
+
Foxp3 Tregs was related to favorable prognosis contrasts with several previous studies, further investigation
will be needed.
P77 - X-Ray Crystallographic, Biochemical, and SAXS Studies of the CSN Provide Insight into the PCIPCI Interaction on the COP9 Signalosome
**JUNG-HOON LEE and Hao Wu
Structural Biology Program, Department of Biochemistry, Weill Medical College of Cornell University, New York, NY
The CSN (COP9 signalosome) is a conserved eight-subunit complex that functions in the ubiquitin-proteasome
pathway. Previous studies showed that the CSN regulates the activity of cullin-RING ligase (CRL) families of
ubiquitin E3 complexes by deneddylation activity. Furthermore, the CSN is known as a controller in the DNAdamage response, cell-cycle control, and gene expression. Very recent observations indicate that the important
roles of the CSN in the regulation of NF-B (nuclear factor kappaB) in innate immunity, as well as T cell
activation and maturation. For example, the CSN1 subunit of the COP9 signalosome recruits the inositol 1,3,4trisphoaphate 5/6-kinase (5/6-kinase) to phosphorylate IkappaBalpha (IB), which regulates the TNF-induced
NF-B activation. Based upon the emerging functional importance of the CSN, we first determined the atomic
structure of eukaryotic CSN1 at 2.7 angstrom resolution by multi-wavelength anomalous diffraction (MAD)
method and further investigated the protein-protein interaction mediated by the N- or C-terminal PCI
(proteasome, COP9, eIF3) domains of the CSN subunits (e.g., CSN1, CSN2, CSN7 etc) within the COP9
signalosome using in vitro binding studies, mutagenesis, and SAXS (small angle X-ray scattering).
P78 - Identification of BCP-20(FBOX39) as Cancer/Testis Antigen from Colon Cancer Patients by SEREX
1,
2,
2
1
Myung-Ha Song , Jong-Cheon Ha , Sang-Mok Lee , Yeong-Min Park , and SANG-YULL LEE
1
2
1
School of Medicine, Pusan National University, Beomeo-ri, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, Korea;
Biocurepharm Co., Ltd, Jeonmin-Dong, Yuseong-Ku, Daejeon-city, Korea
Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential
CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by
SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through
BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens
were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases
(30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including
breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20
antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were
three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher
titers than those of healthy donors. These data suggest that the BCP-20 gene is new CT antigen and may be
useful for diagnosis and immunotherapy.
P79 - TAP-independent Presentation of the Melanoma Vaccine Candidate Epitope gp100209-217 Requires
no gp100 Sequences Outside the Core Peptide and is Sensitive to Cytosolic TPP2 Degradation
1
**RALF LEONHARDT* , Nathalie Vigneron*
1,2,3
2,3
1
, Benoît Van den Eynde , and Peter Cresswell
1
2
3
Yale University School of Medicine, New Haven, CT; Ludwig Institute for Cancer Research, Brussels Branch; Cellular
Genetics Unit, Institute of Cellular Pathology, Université Catholique de Louvain, Brussels, Belgium; *These authors
contributed equally to this work
gp100209-217 is a promising anti-melanoma peptide vaccine that corresponds to an immunodominant, HLAA*0201-restricted tumor epitope. For immune evasion many tumors downregulate, sometimes completely, the
expression of the TAP transporter, a crucial factor involved in MHC class I-mediated antigen presentation.
Surprisingly, we find that HLA-A*0201-mediated presentation of gp100209-217 is remarkably independent of TAP
and thus the peptide likely uses an unconventional pathway to access MHC class I molecules. Presentation
does not seem to involve proteolysis in one of the diverse protease-rich compartments that gp100 traverses on
its way to melanosomes. Neither is melanosomal targeting or ER-export of gp100 required, nor is even cotranslational insertion of the protein into the ER membrane necessary. However, presentation of the epitope
does depend on proteasome activity. Interestingly, we find that cytosolic tripeptidyl peptidase 2 (TPP2)
efficiently counteracts TAP-independent presentation of gp100209-217 suggesting that tumors might use this
protease for immune evasion. Strikingly, no sequences outside the core peptide region within gp100 are
required for TAP-independent access of the ligand to HLA-A*0201. We conclude that a transporter system
besides TAP must exist in the cell, which can partially compensate for loss of TAP function and mediate access
of critical tumor-specific antigenic peptides to HLA-A*0201. Our ongoing work attempts to identify this
transporter.
P80 - Monocytic CCR2+ Myeloid-Derived Suppressor Cells Promote Immune Escape by Limiting
Activated CD8 T Cell Infiltration Into the Tumor Microenvironment
1,2
4
1
1
ALEXANDER LESOKHIN , Tobias M. Hohl , Taha Merghoub , Daniel Hirschhorn-Cymerman , Francesca
1
5
1
1,2,3
1,2,3
Avogadri , Gabrielle A. Rizzuto , Eric G. Pamer , Alan N. Houghton , Jedd D. Wolchok
1
2
Memorial Sloan-Kettering Cancer Center, New York, NY; Weill Medical College, Cornell University, New York, NY;
4
Graduate School of Medical Sciences of Cornell University, New York, NY; Vaccine and Infectious Disease Division, Fred
5
Hutchinson Cancer Research Center, Seattle, Washington; University of California, San Francisco Medical Center, San
Francisco, California
3
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor
progression in a process driven by soluble factors such as granulocyte-macrophage colony stimulating factor
(GM-CSF). These cells contribute to the suppressive nature of the tumor microenvironment and interfere with
the functions of cytotoxic anti-tumor T effector cells. To date, MDSC heterogeneity has presented a barrier to
studying the properties of individual MDSC constituents in vivo.
Herein, we find that GM-CSF, a cytokine that promotes the numeric and functional development of monocytes,
granulocytes, and dendritic cells, and is frequently used as a vaccine adjuvant, is also critical for the expansion
of a monocyte-derived MDSC population characterized by the expression of CD11b and the chemokine receptor
CCR2. We demonstrate that these cells mediate T cell suppression in a contact-dependent fashion and via the
function of Arginase and inducible nitric oxide synthase, consistent with known MDSC functions. CD11b+CCR2
negative cells do not have suppressive capability despite also being expanded numerically by the actions of
GM-CSF. Utilizing a toxin-mediated ablation strategy that targets CCR2-expressing cells, we demonstrate that
monocytic MDSCs regulate activated CD8 T cell entry into the tumor site in vivo, thereby limiting the efficacy of
immunotherapy.
Our results extend observations on the dual role of GM-CSF in both stimulation and suppression of tumor
immunity and suggest therapeutic targeting of monocytic MDSCs could enhance the outcomes of
immunotherapy.
P81 - MicroRNA Signature in T Lymphocytes from Healthy Donors
1,2
1,2
2
JULIO CESAR CETRULO LORENZI , Dalila Luciola Zannete , Patricia Viana Bonini de Palma , Carla Martins
1,2
3
3
1,2
Kaneto , Takemasa Tsuji , Sacha Gnjatic , Wilson Araύjo Silva Jr.
1
2
Medical School of Ribeirão Preto, University of São Paulo, São Paulo, Brazil; National Institute of Science and Technology
3
in Stem Cell and Cell Therapy, Center for Cell Therapy and Regional Blood Center, Ribeirão Preto, Brazil; Ludwig Institute
for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer Center, NY
MicroRNAs (miRNAs) are a class of endogenous, small regulatory non-coding RNAs that repress gene
expression post-transcriptionally by affecting miRNA stability or translation in a sequence-specific manner.
These molecules play central roles in a wide range of biological processes, including cell proliferation,
differentiation, and apoptosis. Several miRNAs have been described in different processes such as regulation,
maturation, and differentiation of T lymphocytes, which play a major role in the immune response. In this study
we sought to determine the global expression of miRNAs in nine subpopulations of T lymphocytes from healthy
donors. To achieve this goal we obtained PBMC cells derived from healthy donors, then we used magnetic
beads to rescue only pure CD4+ or CD8+ cells. Next, we used a flow cytometry cell sorter in order to isolate
Naïve, Central memory, Effector memory, Effector RA memory, and regulatory T Cells from CD4+ and CD8+
populations. From each population we determined the expression level of 866 human miRNAs using Agilent
miRNA microarray technology. From these analyses we emphasize the presence of 58 miRNA co-expressed in
all CD8 T populations and 13 miRNA co-expressed by all CD4 T populations. We also notice that each
lymphocyte subpopulation has a specific miRNA expression. From these uniquely expressed miRNAs we were
able to draw an miRNA expression signature from each lymphocyte subpopulation. These findings support the
idea that miRNA expression profiles could be used as genetic signatures for healthy T lymphocyte
supbopulations. This T lymphocyte miRNA expression signature from healthy donors will be important to
establish differences in miRNA signatures found in T cell-based diseases and in patients with autoimmune
diseases and cancer.
Financial Support: FAPESP (2010-00310-1), INCTC and FUNDHERP
P82 - AdCD40L Gene Therapy Inhibits Recruitment of Myeloid-Derived Suppressor Cells (MDSC) to the
Tumor Micro Milieu and Suppresses Tumor Growth
Lina Liljenfeldt, Lothar Dieterich, Fredrik Eriksson, Anna Dimberg, and ANGELICA LOSKOG
Science for Life Laboratory, Uppsala University, Sweden
Immunotherapy such as adoptive transfer of T cells is hampered by the accumulation of suppressor cells such
as myeloid-derived suppressor cells (MDSCs) in the tumor milieu. Preconditioning the patients with
cyclophosphamide and/or irradiation reduces the number of suppressor cells, which is beneficial for treatment
outcome. However, preconditioning is harsh and limits the number of patients. New strategies to overcome
immune escape mechanisms that terminate T cell responses are needed. CD40L is a potent stimulator of tumor
immunity. Adenovector CD40L (AdCD40L) has shown promising results in experimental models and in man
(phase I-II trials). We hypothesize that AdCD40L may affect MDSC because of its broad range as an immune
activator. Herein, we determined the level of GR1+CD11b+ MDSCs in tumors and spleens from mice treated
with AdCD40L gene therapy and found that MDSCs were significantly reduced in AdCD40L-treated tumors. To
determine the effect of CD40L on MDSCs they were co-cultured with tumors +/- AdCD40L. Tumors promoted
survival of MDSCs but the presence of CD40L did not alter the viability or phenotype of MDSCs. However,
MDSCs were increased in the cocultures with splenocytes and tumors, suggesting that the tumor promotes
generation of MDSCs. The generation of MDSCs was blocked if the tumor expressed CD40L. Instead, DCs
were detected and the lymphocytes expanded vigorously. In a migration assay, CD40L-expressing tumors had
less attractive capacity on MDSCs. In conclusion, AdCD40L blocks the generation and migration of MDSCs to
the tumor. CD40L gene therapy may be of interest as a sensitizer to T cell therapy.
P83 - Changes in Signaling State Induced by Epithelial-Mesenchymal Transition (EMT)
1
2
3
3
2
1
**HAIHUI LU , Karl Clauser , Ailan Guo , Roberto Polakiewicz , Steven A. Carr , and Robert A. Weinberg
1
2
3
Whitehead Institute for Biomedical Research Cambridge, MA; Broad Institute, Cambridge, MA; Cell Signaling Technology,
Danvers, MA
The great majority (80%) of human solid tumors are carcinomas that originate from various epithelial cell types
throughout the body. In order for carcinoma cells to migrate and invade adjacent cell layers, they must lose cellcell adhesion and acquire motility. The resulting cellular motility shares many similarities with the extensive cell
migration and tissue rearrangements that occur during the epithelial-mesenchymal transition (EMT), a highly
conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal
cells. During the process of EMT and invasion, the tumor cells remodel the microenvironment, and they respond
to various stimuli from the changing microenvironment. Several master transcriptional regulators, including
Twist, Snail, and Slug have been identified that induce EMT, and the expression of these factors has been
correlated with poor prognosis in various types of carcinomas. Overexpression of these transcription factors in
immortalized human mammary epithelial cells (HMLE) induces changes in morphology, cell adhesion, motility
and invasion, typical of the EMT process occurring in vivo. We examined differentially expressed membraneassociated proteins as well as the phosphorylation status of signaling proteins during EMT using SILAC-based
quantitative proteomics profiling. We have observed changes in cell adhesion, cell-cell junction, as well as
growth factor signaling pathways. Cells that have undergone EMT downregulated tight junction components,
and increased collagen and septin secretion. During EMT, MET and RON signaling are attenuated whereas Axl,
PDGFR, and FGFR signaling are increased. Activation of Axl, PDGFR, and FGFR has been indicated in
aggressive tumor progression, consistent with the metastasis-promoting property of EMT. The changes in the
signaling state show close similarity to those observed in the EMT process using human NSCLC lines indicating
the ubiquitous reprogramming by EMT in normal or transformed cells, as well as in different tissues.
P84 - Defining Novel Targets of Immunotherapy for Adult T Cell Leukemia/Lymphoma
1
1
2
3
2
1
YUKA MAEDA , Hiroyoshi Nishikawa , Takashi Ishida , Sacha Gnjatic , Fumiko Mori , Daisuke Sugiyama ,
2
3
2
1
Asahi Ito , Lloyd J. Old , Ryuzo Ueda , and Shimon Sakaguchi
1
2
Experimental Immunology IFReC, Osaka University, Osaka, Japan; Nagoya City University Graduate School of Medical
3
Science, Nagoya, Japan; Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer
Center, New York, NY
Adult T cell leukemia/lymphoma (ATLL) is a unique disease entity caused by human T lymphotropic virus type 1
(HTLV-1). HTLV-1 infection is endemic in southwestern Japan, Africa, the Caribbean Islands, and South
America, and 10-20 million people worldwide are infected with HTLV-1. Approximately 60% of ATLL cells harbor
leukemic cells expressing Foxp3, a key transcription factor for CD25+CD4+ regulatory T cells (Tregs), resulting
in a severe immunodeficiency in ATLL patients. The prognosis for ATLL patients, even though receiving
intensive chemotherapy, remains poor and the mean survival time of patients with aggressive ATLL is less than
1 year. It is required therefore to develop more effective strategies to treat ATLL. Cancer/testis (CT) antigens, of
which more than 100 have been identified, are now under clinical investigation, and immunization with these
antigens elicits humoral and cellular immune responses in cancer patients, raising the possibility that these
antigens could be targets of ATLL immunotherapy. Here, we examined 11 CT antigen (NY-ESO-1, MAGE-A1,
MAGE-A3, MAGE-A4, MAGE-A10, CT-7, CT-10, SSX-1, SSX-2, SSX-4, and SCP-1) expression in primary
tumor cells from 57 individual ATLL patients. Among them, CT antigens such as MAGE-A3, MAGE-A4, and NYESO-1 were highly expressed in ATLL cells (32.7%, 63.6%, 61.8%, respectively) in contrast with other types of
lymphomas or leukemia. Expression of NY-ESO-1 and pan-MAGE protein also was confirmed by
immunohistochemistry. Humoral immune responses against NY-ESO-1 (11.4%, 5/43) and MAGE gene family
+
(4.5%, 2/43) were developed in ATLL patients. NY-ESO-1-specific CD8 T cells were detected in 5 out of 9
+
patients. More importantly, NY-ESO-1-specific CD8 T cells recognized an autologous ATLL cell line and
produced cytokines. These data suggest that cancer/testis antigens could be promising targets of
immunotherapy for ATLL.
P85 - Characterization of a Human Promyelocytic-Like Population Responsible for the Immune
Suppression Mediated by Myeloid-derived Suppressor Cells
1
1
1
2
1
SUSANNA MANDRUZZATO , Samantha Solito , Erika Falisi , Claudia Marcela Diaz-Montero , Laura Pinton ,
1
2
3
Paola Zanovello , Alberto J. Montero , and Vincenzo Bronte
1
3
2
University of Padova, Padova, Italy; Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL;
Immunology Section, Verona University Hospital and Department of Pathology, Verona, Italy
One of the mechanisms of immune tolerance induced by the cancer is linked to the expansion of myeloidderived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells that have been
shown to accumulate in the blood, lymph nodes, bone marrow, and tumor sites in patients and experimental
animals with neoplasia, and are capable of inhibiting both adaptive and innate immunity.
We recently demonstrated that the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs
from precursors present in human bone marrow (BM) and that, analogously to tumor-induced MDSCs, BMMDSCs consist of a heterogeneous population of myeloid cells, able to suppress activation of alloactivated and
mitogen-activated T lymphocytes. To find out whether suppressive activity of BM-MDSCs was either shared by
a number of immature subsets or limited to a specific differentiation stage, we isolated different myeloid fractions
from fresh BM and in vitro-cultured BM-MDSCs. When we tested the immunoregulatory activity of the sorted
subsets, we observed that the entire suppressive activity of BM-MDSCs was contained within a single subset of
immature cells with morphology and phenotype resembling promyelocytes, which were able not only to block
lymphocyte proliferation, but also to affect IFN- production and induce T cell apoptosis.
When we further investigated the relationship between T cell activation and BM-MDSC-mediated suppression,
we found out that the promyelocytic-like population was able to proliferate and maintain its immature phenotype
when co-cultured with activated T lymphocytes; conversely, the same myeloid subset showed a diminished
proliferative index and differentiated to more mature myeloid cells when co-cultured with resting T lymphocytes.
In the blood of breast and colorectal cancer patients we could clearly identify an immature myeloid population
resembling in vitro-generated BM-MDSCs. Our data suggest that circulating MDSC levels, phenotypically similar
to those described in human BM experiments, are clinically relevant and: (i) increase over time in patients with
progressive disease; (ii) correlate with an established prognostic marker (i.e., circulating tumor cells) in
advanced breast cancer; and (iii) their persistently high or increasing levels following chemotherapy are
associated with poorer survival.
P86 - Peritumoral Administration of CD40 Agonist Antibody Delivers Low Systemic Antibody
Concentrations with Anti-Tumor Effects on Local and Distant Tumors
1
2
1
1
1
Linda Sandin , Peter Ellmark , Anna Orlova , Vladimir Tolmachev , Thomas H. Tötterman* , SARA M.
1
MANGSBO
1
2
Uppsala University, Uppsala, Sweden; Lund University, Uppsala, Sweden; *These authors contributed equally to this work
Anti-CD40 therapy results in promising anti-tumor effects in experimental animal models. Antibody has per
default been delivered i.v. with resulting dose-escalating toxicity in humans. Toxicity can also be measured by
liver damage in mice receiving high-dose anti-CD40. To circumvent this, we and others are aiming at delivery of
antibody in close vicinity of the tumor with drainage to tumor-draining lymph nodes where naïve and tumor
antigen-experienced cells can receive optimal co-stimulation by professional antigen presenting cells.
Our data indicate that anti-CD40 can be injected close to the tumor at a low dose with sustained anti-tumor
effects compared to systemic delivery. Effects on distant tumors were registered, which can be due to a
systemic spread of the antibody, recirculation of locally activated immune cells, or both.
Our ELISA results demonstrate that i.v. injection of anti-CD40 results in higher levels of systemic circulating
antibody than peritumoral delivery. As ELISA was estimated as too insensitive we performed a radiolabeled antiCD40 antibody biodistribution study. Locally injected antibody reaches circulation as well as lymphatic organs
such as spleen and lymph nodes within a few hours after injection. Accumulation of specific as opposed to
control antibody is noted in immune cell rich organs. Our experimental bladder cancer model reveals that
animals cured of tumor are immune against tumor rechallenge, and spleen cells of immune mice can transfer
tumor protection.
Our data complement previous findings by Herbert-Fransen et. al. (CCR 2011) who showed that virally induced
tumors were cured using a single dose of anti-CD40 in slow-release oil formulation. Our data indicate that a low
local dose of anti-CD40 leading to plasma antibody levels measurable only by radiolabeling has a good
therapeutic effect on a non-viral tumor. This anti-tumor effect is obtained at a dose associated with limited
toxicity.
P87 - High Affinity Tumor- and Minor Histocompatibility Antigen-Specific TCR-Redirected T Cells for the
Immunotherapy of Prostate Cancer
1,#
TERESA MANZO , Rodrigo Hess Michelini
1,*
1,*
Matteo Bellone , and Anna Mondino
1,#
, Veronica Basso
1
1,#
5
6
, Massimo Freschi , Ton Schumacher ,
Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy;
#
*
Amsterdam, Netherlands; co-first; co-last
6
Netherlands Cancer Institute,
We have demonstrated that the concomitance of minor histocompatibility (H) antigen- and high-affinity tumorspecific T cell responses found in allogeneic hematopoietic transplant recipients causes the eradication of
autochthonous prostate mouse adenocarcinoma (Hess Michelini, 2010). As in most cases donors might lack
high affinity T cells specific for tumor-associated antigens, we have investigated the possibility to supplement
donor T cells with sizeable frequencies of high affinity tumor-specific TCR-transferred lymphocytes. Furthermore,
as graft-versus-host disease (GVHD) is the major complication of allotransplantation, we also investigated
whether combining tumor- and minor H antigen-restricted TCR-transferred T cells might confer graft versus
tumor without evoking GVHD. We report that while minor H antigen- or high affinity tumor-specific TCR+
redirected CD8 T cells per se are insufficient in causing disease eradication, their concomitant infusion
promotes tumor regression. TCR-transferred cells promptly respond to tumor-specific vaccines and promote
tumor infiltration and eradication. Post-transplant vaccination is critical for therapeutic efficacy since it favors
accumulation of tumor-specific CD8+ IFN-+ effector cells and the infiltration of the tumor mass by CD3+
lymphocytes. Likewise, the infusion of tumor-specific TCR-transferred T cells together with a limited number of
minor H antigen-specific TCR-engineered T cells reveal therapeutically effective, in the absence of signs of
GVHD. Thus, together these data support the value of combining TCR-transferred T cells specific for tumor and
minor H antigens for the immunotherapy of prostate cancer.
P88 - BTB-POZ Transcription Factors Recruit the E3 Ubiquitin Ligase Cullin 3 to Initiate Lymphoid
Effector Programs
1
1
1
1
1
**REBECCA MATHEW , Michael P. Seiler , Seth T. Scanlon , Michael G. Constantinides , Clara Bertozzi-Villa ,
2
1,3
Jeffrey D. Singer , and Albert Bendelac
1
3
Committee on Immunology, Department of Pathology, The Howard Hughes Medical Institute, University of Chicago,
2
Chicago, IL; Department of Biology, Portland State University, Portland, OR
The differentiation of several T and B cell effector programs in the immune system is directed by signature
transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-POZ transcription
factor directing the innate-like effector program of NKT thymocytes was prominently associated with cullin 3
(Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate
binding. PLZF induced the transport of Cul3 from the cytosol to the nucleus where the two proteins were
associated within a chromatin associated/modifier protein complex. Furthermore, PLZF expression in
thymocytes resulted in selective changes of ubiquitination of multiple components of this complex. Cul3 was
also found associated with another BTB-POZ transcription factor, Bcl6, which directs the B cell germinal center
and the T follicular helper programs. Conditional deletion in mice demonstrated an absolute, cell-intrinsic
requirement of Cul3 for the development of NKT cells, but opposite effects in germinal center B cell and follicular
helper T cell responses, which were abrogated and exaggerated, respectively. In addition, innate-like B1 B cells
and marginal zone B cells were severely reduced. In contrast, the Th1, Th2, and Th17 differentiation programs
and T-independent B cell responses were unaffected. We conclude that distinct lineage-specific BTB-POZ
transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin
associated/modifier complex. We propose that this novel function is essential to direct the differentiation of
several T and B lymphocyte effector programs, and may also be involved in the oncogenic role of PLZF and
Bcl6 in leukemias and lymphomas.
P89 - Antigen Immunoselection as a Mechanism of Cancer Immunoediting
1,2
1
1
HIROKAZU MATSUSHITA , Matthew D. Vesely , Ravindra Uppaluri , and Robert D. Schreiber
1
1
2
Washington University School of Medicine, St. Louis, MO; Medinet, The University of Tokyo Hospital, Tokyo, Japan
Cancer immunoediting is the process wherein the immune system not only protects against tumor development
but also promotes outgrowth of tumors with reduced immunogenicity. Although we and others identified several
key immune components that participate in this process, we know very little about the targets of cancer
immunoediting. In this study we used a highly immunogenic methylcholanthrene induced sarcoma cell line
(d42m1) to ask whether tumor antigens can be immunoediting targets. Like most unedited MCA sarcomas,
d42m1 cells form tumors when transplanted into immunodeficient mice but are rejected in naïve syngeneic wild
type (WT) mice. We identified two antigens of d42m1 by expression cloning: a point mutant of spectrin-2 and a
point mutant of M-phase phosphoprotein 8 (MPP8). In WT mice, d42m1 occasionally produces escape variants
lacking mutant spectrin-2 but maintaining expression of mutant MPP8 that can form tumors in naïve WT mice.
Enforced expression of mutant but not WT spectin-2 into escape variant cell lines converted them into
regressors. Analysis of the parental d42m1 cell line revealed that only 80% of d42m1 clones expressed mutant
spectrin-2 whereas 100% expressed mutant MPP8. Only spectrin-2-expressing d42m1 clones were rejected
in WT mice. Thus, mutant spectrin-2 is not only the major rejection antigen of d42m1 sarcoma cells but is the
target of a cancer immunoediting immunoselection process that facilitates tumor escape from immune control.
P90 - Vaccination with Recombinant Canarypox Expressing NY-ESO-1/TRICOM Generates High Avidity
Multifunctional CD8+ and CD4+ T Cells in Ovarian Cancer Patients
1
2
3
2
1
JUNKO MATSUZAKI , Rachel Sabado , Sacha Gnjatic , Stephanie Blank , Shashikant Lele , Paulette
1
1
2
2
1
Mhawech-Fauceglia , Nefertiti DuPont , Franco Muggia , Rose Marie Holman , Francine Siedlecki , Juliet
2
3
3
3
1
2*
1*
Escalon , Erika Ritter , Gerd Ritter , Lloyd J. Old , Protul Shrikant , Nina Bhardwaj , Kunle Odunsi
1
2
Roswell Park Cancer Institute, Buffalo, NY; Cancer Institute, New York University, School of Medicine, New York, NY;
Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY; *These authors contributed
equally to this work
3
Poxviruses are strong stimulators of the cellular arm of the immune system, and are thus attractive vectors for
use in immunotherapeutic strategies of stimulating T cell responses to tumor antigens. Preclinical and clinical
studies have demonstrated enhanced T cell priming and expansion when TRICOM (TRIad of COstimulatory
Molecules, B7.1, ICAM-1, and LFA-3) is incorporated into poxviruses. In this study, we conducted a phase I
clinical trial (Protocol LUD2007-005) wherein we proposed three strategies: (i) the use of canarypox vector
(ALVAC) to enhance presentation of NY-ESO-1 to the immune system; (ii) the use of TRICOM to enhance T cell
+
+
responses (second signal), which could preferentially induce and expand high avidity CD8 and CD4 T cells;
and (iii) the use of GM-CSF to enhance the efficacy of APCs. Twelve ovarian cancer patients with NY-ESO-1expressing tumors and minimal disease burden received ALVAC(2)-NY-ESO-1(M)/TRICOM at a dose of 0.5 mL
7
containing ≥ 1x 10 CCID50/mL of the ALVAC virus via subcutaneous administration monthly for 6 months. GMCSF (Sargramostim, Leukine®) was administered subcutaneously as a vaccine adjuvant at a dose of 100
mcg/day for 4 days (days 1-4) starting on the day of ALVAC(2)-NY-ESO-1(M)/TRICOM administration. No
vaccine-related adverse events were noted in the study. ALVAC(2)-NY-ESO-1(M)/TRICOM vaccination elicited
NY-ESO-1-specific immune responses in all patients. 10 baseline seronegative patients developed antibodies
+
following 1-4 vaccinations. There was induction of CD4 T cell responses following vaccination in all patients;
+
+
and CD8 T cell responses were detected in 7/10 (70%) of patients without detectable CD8 T cells at baseline.
+
+
+
Moreover, the vaccine elicited multicytokine producing CD4 and CD8 T cells, skewed CD4 T cells towards
Th1 polarization, and generated T cells with higher TCR avidity compared with preexisting cells. Together, these
results support the use of ALVAC(2)-NY-ESO-1(M)/TRICOM vaccine in future combinatorial vaccine strategies
for human ovarian cancer.
P91 - The Role of Actin Cytoskeleton in Regulating the Immunological Synapse-Kinapse Balance
**VIVEKA MAYYA and Michael L. Dustin
Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY
The functioning of the immune system relies on the appropriate coordination between migratory behavior and
intercellular interactions of constituent cell types. The effector T cells form an organized, radially symmetric, and
sessile junction called the 'Immunological Synapse' (IS) with antigen presenting cells (APCs) to direct the
immune response. However, T cells, especially naïve T cells, can also readily convert to a mobile junction called
'Immunological Kinapse' (IK) by symmetry breaking of the IS. We have established a simple experimental
system to study IS-IK transitions in human naïve CD8 T cells on cover-glass coated with anti-CD3 and ICAM1
(Figure 2). There is considerable heterogeneity in the responses of naïve CD8 T cells, in that some stay sessile
for the duration of imaging; some become mobile almost immediately after spreading on the immobilized
ligands; some others break symmetry at various timepoints and become motile; and finally some cells also show
multiple inter-conversions between IS and IK. We have observed that inhibition of PKC using #C20 inhibitor
completely arrests the cells. Further, pharmacological manipulation of myosinII activity suggests that an optimal
level of myosinII is important for naïve cell motility during antigenic signaling. We are currently using machine
learning approaches to comprehensively characterize the complex motility behavior and capture the
heterogeneity in motility behavior by mathematical description. We are also in the process of establishing
monolayer APC system to study naïve cell motility under more native conditions. This will also allow us to
perform long-term imaging and study the impact the IS-IK balance on CD8 T cell responses.
P92 - Tipping the Scale: Identifying Small Molecule Inhibitors to Promote Myxoma Virus-mediated
Oncolysis and Overcome Antiviral Immunity in Brain Tumor Initiating Cells
1
1
1
1
2
1
BRIENNE MCKENZIE , Franz J. Zemp , Xueqing Lun , Aru Narendran , Grant McFadden , Ebba Kurz , Peter
3
Forsyth
1
3
2
Southern Alberta Cancer Research Institute, University of Calgary, Alberta, Canada; University of Florida, Gainesfille, FL;
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
Background: Glioblastoma multiforme (GBM) is a highly aggressive brain cancer, with an average survival of
12 months post-diagnosis. Current standard-of-care treatment regimens are essentially palliative, highlighting
the need for novel therapeutic approaches. Oncolytic virus (OV) therapy is a promising experimental treatment
that uses replication-competent viruses to selectively infect tumor cells. The anti-tumor efficacy of OVs relies on
two mechanisms: (i) direct tumor cell oncolysis by virus; and (ii) virus-mediated stimulation of anti-tumor immune
responses. Both mechanisms require productive, efficient viral replication in the tumor. Our lab has
demonstrated that a single intra-cranial injection of oncolytic virus Myxoma (MyxV) is sufficient to cure GBMbearing xenografts. However, we have shown that brain tumor initiating cells (BTICs), which are highly
tumorigenic, patient-derived cells that recapitulate key characteristics of human GBMs more accurately than
traditional cell lines, are not cured with MyxV monotherapy. The molecular mechanism of BTICs’ enhanced
antiviral resistance is unknown. We have shown that resistance is partially overcome when MyxV is combined
with mTOR inhibitor Rapamycin in vivo, but cures are not obtained.
Hypothesis: MyxV replication and killing can be enhanced using small molecule inhibitors identified via highthroughput screen.
Results: We used a comprehensive panel of 75 compounds with preclinical/clinical anti-cancer efficacy to
screen for pharmaco-viral synergy, measured by BTIC death in vitro. Several synergistic compounds, each
targeting different pathways, were identified (Chou-Talaly method). The mechanisms of synergy in vitro
(enhanced viral replication, initiation of apoptosis, inhibition of antiviral pathways, etc.) are being determined. In
vivo experiments utilizing combination therapies in BTIC xenografts are under way. In addition to mechanisms
identified in vitro, mechanisms of synergy unique to the in vivo system are also being investigated, including
altered recruitment of tumor-associated macrophage/microglia, altered M1>M2 polarization, and altered cytokine
expression by tumor and/or stromal cells. We intend to characterize combination therapies and mechanisms of
synergy in syngeneic mouse BTIC models in the future.
Significance: Our findings offer an effective avenue to develop efficacious combination therapies that maximize
the anti-tumor efficacy of MyxV.
P93 - Mechanism of Dendritic Cell-Mediated Transfer of HIV-1 to CD4+ T Lymphocytes
1
2
**MICKAEL M. MENAGER , Nicolas Manel , and Dan R Littman
1
1
2
Skirball Institute of Biomolecular Medicine, NYU Langone Medical Center, New York, NY; Institut Curie, Paris, France
Despite progress in unraveling the mechanism of HIV replication cycle in human cells, we have limited
understanding of how the virus is transmitted in vivo, how it causes immunodeficiency, and why immune
responses are generally ineffective in neutralizing it. In vitro studies suggest that while dendritic cells (DCs) are
largely resistant to HIV infection, they are able to bind and transfer intact infectious HIV to CD4+ T cells, a
process known as “trans-enhancement”. The mechanism of trans-enhancement has been the subject of some
controversy. This was, however, recently reconciled by the observation that HIV is internalized in a non-classical
structure, termed the “invaginated pocket”, accessible to solvent, but topologically distinct from the cell
membrane (Yu et al., 2008). Currently, the genes involved in the various steps of trans-enhancement (capture;
invagination; release) are not known. As the formation of the “pocket” implies membrane recruitment and
movement, we are performing an shRNA screen of proteins involved in vesicular and membrane trafficking, in
order to identify key components of this process.
We anticipate that this screen will provide important insight into how HIV exploits DCs to better infect CD4+ T
cells. Performing the screen in primary cells is challenging, but we have made progress in establishing a robust
platform, and have started to implement it with shRNA virus obtained from the Broad Institute. Using Vpxcontaining SIV virus-like particles, we are now able to achieve highly efficient transduction of Monocyte Derived
Dendritic Cells (MDDCs), in 96 well plates, and to obtain good knock down of gene expression with minimal
experimental variation in trans-enhancement between replicates. Once we identify MDDC genes required for
efficient trans-enhancement, we will next proceed to determine if they play a role in capture, internalization, or
transfer of HIV-1 to the DC-T cell synapse. We will carry out confocal and electron microscopy analysis
combined with co-immunoprecipitation experiments to follow the subcellular distribution and interactions of HIV1 with the protein products. In combination with the laboratory’s recently published results (Manel et al., 2010)
on DC sensing of HIV, this approach could provide new tools and new targets for the design of therapies that
limit viral replication or boost innate immune responses that control HIV dissemination.
P94 - RNA Exosome Complex Targets AID to Transcribed Duplex DNA Substrates During CSR
**FEILONG MENG, Caitlyn Wasserman, and Frederick W. Alt
Program in Cellular and Molecular Medicine and Immune Disease Institute, Children’s Hospital Boston, Harvard Medical
School, Boston, MA
Activation Induced cytidine Deaminase (AID) initiates Immunoglobulin (Ig) heavy chain (IgH) class switch
recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating
cytidines on template and non-template strands of transcribed DNA substrates. The RNA exosome, a cellular
RNA processing/degradation complex, targets AID to both DNA strands in vitro, particularly when the template
DNA strand is hybridized to a nascent RNA transcript. Although the RNA core complex alone can promote
robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in
vitro, the catalytic subunits are also required for optimal CSR. Our findings implicate non-coding RNA
surveillance machinery as playing a crucial role in generating antibody diversity, and support a model in which
non-coding RNA, potentially generated by pausing PolII, functions at the IgH locus during CSR.
P95 - STAT1-independent Cytokine Responses in the Immunosurveillance of Tumours
1,2
1
1
2
1
1
*NICOLE L. MESSINA , Kellie M. Banks , B.P. Martin , Adam Uldrich , Ailsa J. Christiansen , Mark J. Smyth ,
1,2
1,2
Christopher J.P. Clarke , and Ricky W. Johnstone
1
Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia;
Melbourne, Parkville, Victoria, Australia
2
University of
STAT1 is involved in mediating anti-viral responses and immunomodulatory functions induced by numerous
-/cytokines. STAT1 mice have major defects in IFN responses and are exquisitely sensitive to pathogenic
infection and tumor growth, however, IFNs are still able to partially influence biological function in these mice.
The aim of this project is to elucidate the role of STAT1-independent signalling pathways in anti-tumor
-/-/-/responses. Tumor cell lines derived from MCA treated STAT1 mice were transplanted into STAT1 , RAG2cg ,
-/and RAG1 mice. These tumors grew progressively in the immunocompromised mice, moreover, absence of
+
-/-/perforin or CD8 and NK cells also resulted in accelerated STAT1 tumor progression and STAT1 tumor cells
were found to be highly susceptible to direct killing by NK cells in a perforin dependant manner. In contrast, their
growth was attenuated in wild type animals suggesting that STAT1 plays an important role in anti-tumor immune
responses to MCA-induced sarcomas, but tumor cell-intrinsic STAT1 expression is not essential for these
responses. To elucidate the role of STAT1-independent IFN responses in immune-mediated rejection of these
-/-/-/-/-/tumors, IFNR1 , IFN , and IFNR mice were challenged with the STAT1 tumor lines. IFNR1 mice were
-/highly susceptible to growth of STAT1 tumors suggesting that type I IFNs mediate the host response to
-/-/STAT1 tumors without directly affecting the tumor. STAT1 tumors also grew progressively in both strains of
mice with defects in the IFN- signalling. Furthermore in vitro studies showed that IFN could induce MHC class
-/-/I upregulation in wild type and STAT1 reconstituted STAT1 tumors but not STAT1 tumors. These data
suggest that type II IFNs act on the host to facilitate the anti-tumor response in a STAT1-deficient setting. These
studies are consistent with a role for STAT1-independent IFN signalling in host cells to mediate rejection of
-/MCA induced sarcomas. The anti-tumor effects of other cytokines on the STAT1 tumors have yet to be
determined and may still impart a role for STAT1-independent signalling in anti-tumor responses.
P96 - Structural Basis of Membrane Bending by the N-BAR Protein Endophilin
**CARSTEN MIM and Vinzenz Unger
Northwestern University, Evanston, IL
Control of the events at the membrane by proteins is a vital but insufficiently explored property of all cells. In
particular, immune cells must regulate membrane homeostasis (e.g., after secretion of antibodies) and the flow
of information across the membrane (e.g., antigen recognition). Endocytosis is the common cellular process to
achieve this, since it controls the amount of membrane and receptors on the surface. Thus endocytotic proteins
need to be able to bind and bend membranes.
Functioning as key players in cellular regulation of membrane curvature, BAR-domain proteins bend bilayers
and recruit specific interaction partners through mechanisms that are only partially understood. Using electron
cryomicroscopy, we here present reconstructions of full-length endophilin and its isolated N-terminal BAR
domain in their membrane-bound state. The reconstructions reveal surface lattices that are fundamentally
different from those observed for the F-BAR family of proteins. Endophilin lattices lack extensive lateral
interactions and expose large areas of membrane surface for access by downstream interaction partners.
Lattice cohesion is achieved through interactions between endophilin’s amphiphatic, N-terminal helices that are
oriented parallel to the tube axis. This design is independent of changes in membrane curvature, which are
accommodated through changes in the number of subunits around the tubular surface. Lastly, endophilin’s SH3
domains form dimers above the BAR scaffold in some but not all cases. This suggests that the spatial
presentation of SH3-domains rather than affinity governs the recruitment of downstream interaction partners.
P97 - Specifitiy of Tumor Antigen-Specific Regulatory T Cell Suppression
*MATTHEW M. MOAKE, Gang Zhou, and Hyam I. Levitsky
Johns Hopkins School of Medicine, Baltimore, MD
Cells within a tumor display heterogeneity in their antigenic expression. In this setting, it is established that
effector T cells (Teffs) demonstrate functional specificity, enacting immunologic rejection of those cells expressing
their cognate antigen and leaving neighboring cells largely unharmed. Regulatory T cells (T regs) are generally
considered to have a negative impact upon tumor prognosis, and are thought to suppress immunologic tumor
rejection. Whether this suppression by T regs follows the same antigenic specificity as tumor rejection by T effs has
not been established and has important implications. In these studies, we sought to examine the functional
specificity of Tregs as it pertains to the suppression of tumor rejection, and in doing so to further examine the key
players involved and mechanisms of action used by T regs.
To explore this question, we have established a mixed, dual-antigen lymphoma model using the BalbC mouse
A20 lymphoma expressing the model antigens Ovalbumin (OVA) or Hemagluttinin (HA), along with the use of
the Luciferase (luci) enzyme to allow direct in vivo monitoring of one of the two tumors in the mix. Mice
challenged in this way developed a heterogenous tumor mass with both OVA and HA expressing tumors in the
same histologic location. Subsequent adoptive transfer of in vitro-generated Teffs cells recognizing either the
OVA or HA antigen resulted in selective clearance of the corresponding tumor with continued growth of the
other. When in vitro-generated OVA- or HA-specific Tregs were added, we saw suppression of tumor rejection
only when the Tregs and Teffs shared specificity. This demonstrates that Tregs display functional specificity in the
same manner as do Teffs.
Based on these observations, we are now undertaking three lines of further inquiry. First, we are expanding our
lymphoma model to examine the ability of antigen-specific Tregs to suppress more complicated immune
responses, including a polyclonal treatment system. Second, we are examining the role of MHC class II on the
tumor versus host antigen presenting cells as it pertains to the suppressive function of T regs. Finally, we are
working to explore the mechanisms T regs use to enact suppression upon Teffs. Once complete, we hope to have a
better understanding of how T regs are able to suppress tumor rejection with the goal of abrogating this
suppression to allow for improved immunologic rejection of tumors.
P98 - Mechanisms of Antigen Presentation by Dendritic Cells
**NGOZI R. MONU and E. Sergio Trombetta
New York University Langone Medical Center, New York, NY
The goal of our studies is to better understand the mechanisms of antigen presentation in order to optimize
vaccine development. Recent findings from our lab indicate that lysosomal proteolysis in dendritic cells (DCs) is
optimized for limited degradation of antigens favoring the rescue of immunogenic peptides for loading onto MHC
molecules. This led to the hypothesis that limiting antigen digestibility could enhance antigen presentation on
both MHC class I and MHC class II.
To test this hypothesis we designed model antigens encoding selected T cell epitopes integrated in the
backbone of recombinant proteins of different susceptibility to lysosomal proteolysis. We use these model
antigens to immunize mice with the goal of studying their immunogenicity in vivo. The capacity of each model
antigen to generate T cell responses is determined by measuring: 1) the activation and expansion of antigenspecific (CD8 or CD4) T cells after vaccination, using peptide-MHC tetramers as a physiological measure of
their capacity to elicit T cell responses; 2) the development of CTL effector function, using in vivo killing assays,
to further substantiate the functionality of primed CD8 T cells; 3) the differentiation of T cells into long-lived
memory cells, using re-challenge protocols in vivo; and 4) the serum IgG responses, as a further measure of
antigen presentation on MHC class II to prime CD4 T cells.
We have produced and characterized some of these model antigens. Current in vitro and in vivo analysis of
antigen presentation of different model antigens based on MBP suggest that, contrary to expectations, easily
degraded antigens are equally (and sometimes more) immunogenic for the induction of CD8 T cell responses
compared to equivalent stable forms of the same antigen (less susceptible to lysosomal proteolysis). In addition,
preliminary experiments show that limiting digestibility does not enhance antigen presentation on MHC class II,
regardless of epitope. We are in the process of verifying these results by analyzing the requirements for priming
CD4 T cell responses in vivo, other T cell effector functions such as cytokine secretion, as well as the
requirements for inducing antibody production under similar immunization conditions.
P99 - Structural Insights into NF-B and Co-Activator CBP Interaction
1
2
2
**SULAKSHANA P. MUKHERJEE , Maria A. Martinez-Yamout , Peter E. Wright , and Gourisankar Ghosh
1
1
2
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA; Department of Molecular
Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA
Transcription factor NF-B is known for its role in immune responses to infection. It binds to its cognate B site
to initiate the transcription of various genes which are in turn involved in various cellular processes. However,
the activity of NF-B is regulated by interactions with components of the general transcription machinery.
General transcription co-activator CBP/p300 is one such component, which interacts with RelA, one of the most
potent members of NF-B family. CBP/p300 interacts with RelA in a bipartite manner; the CBP(TAZ1) domain
interacts with the RelA activation domain 2 (TA2), whereas the CBP(KIX) domain interacts with the DNA-binding
RelA rel homology region (RHR). The latter interaction requires phosphorylation at Ser-276 of RelA
(pS276RelA). Here we depict the structural model of RelA(TA2):CBP(TAZ1) complex determined by NMR
spectroscopy. The RelA(TA2) domain is intrinsically disordered which attains a folded conformation with three
helices on binding to CBP(TAZ1). The structural model provides the first insights into the RelA:CBP complex.
P100 - IDO Drives Tumor-Promoting, Pathogenic Inflammation
1
1
1
1
1
ALEXANDER J. MULLER , Courtney Smith , Mee-Young Chang , James DuHadaway , Laura Mandik-Nayak ,
1
2
2
2
George Prendergast , Katherine Parker , Daniel Beury , Suzanne Ostrand-Rosenberg
1
2
Lankenau Institute for Medical Research, Wynnewood, PA; University of Maryland Baltimore County, Baltimore, MD
Implication of the tryptophan-catabolizing enzyme IDO (indoleamine 2,3-dioxygenase) as a mediator of acquired
immune tolerance in the reproductively essential process of shielding the ‘foreign’ fetus from maternal immunity
has been suggested a means by which tumors might escape immune surveillance. However, in the context of
the classical DMBA/TPA skin carcinogenesis model, we found IDO to be induced by the inflammatory tumorpromoting process itself, independent of tumor immunoediting. Furthermore, loss of IDO did not exacerbate the
severity of inflammation as might be expected if it were simply immunosuppressive. Rather, IDO appears to be
integrally involved in shaping the inflammatory environment to support tumor outgrowth. Intriguingly, in patients
with rheumatoid arthritis or lupus, elevated tryptophan degradation has been correlated with disease activity,
suggesting the possibility that IDO might also play a role in promoting inflammatory, autoimmune pathologies.
Consistent with this idea, in a spontaneous mouse model of arthritis, we found IDO elevated at the onset of
disease, while treatment with the IDO inhibitor 1-methyl tryptophan significantly reduced disease severity. These
findings add to a growing body of evidence suggesting that IDO is a key element shaping the pathogenic nature
of the chronic inflammatory environment. In current studies, we have found that IDO-deficient mice are resistant
to both KRAS-induced lung adenocarcinomas and pulmonary breast carcinoma metastases. Elevation of the
inflammatory cytokine IL-6 was associated with tumor outgrowth in the lungs in both models but was greatly
attenuated with the loss of IDO, consistent with the in vitro demonstration that IDO activity markedly potentiates
IL-6 production. MDSCs (myeloid-derived suppressor cells) exhibited reduced T cell suppressive activity when
isolated from tumor-bearing, IDO-deficient animals that could be rescued by ectopic production of IL-6 in the
tumor. IL-6 production could likewise reverse the pulmonary metastasis resistance exhibited by IDO-deficient
mice. Together, these findings genetically validate IDO as a therapeutic target in the settings of pulmonary
cancer and metastasis and establish the importance of IDO as a driver of IL-6 production and MDSC function.
More generally, our findings provide support for the emerging concept of IDO as a prototypical, integrative
immune modifier that links inflammation and immune escape to foster the establishment of a pathogenic, tumorpromoting environment.
P101 - HLA Class I Expression and/or NY-ESO-1 Expression by Cancer Cells are Requisite for Beneficial
Anti-Tumor Effect of CD8+ Tumor Infiltrating Lymphocytes in Invasive Epithelial Ovarian Cancer: A
Comparative Study Between the US and Japanese Cancer
1
1
1
1
2
TAKESHI NAGAI , Eiichi Sato , Toshitaka Nagao , Keiichi Isaka , Kunle Odunsi
1
2
Tokyo Medical University, Tokyo, Japan; Roswell Park Cancer Institute, Buffalo, NY
We have previously reported that frequent infiltration of CD8 positive tumor infiltrating lymphocytes (TILs) is
associated with patients’ longer survival in epithelial ovarian cancer (EOC). However, EOCs include varied
histological subtypes and their biological behavior is quite different among histological subtypes. In this study,
we performed immunohistochemical and clinico-pathological analyses on larger series of EOC. Specimens were
extensively obtained from the US and Japanese patients, and the significance of histological difference and
clinical stage was assessed with careful deliberation. Two handred and thirty one cases of EOC specimen (118
cases from Roswell Park Cancer Institute and 113 cases from Tokyo Medical University) were
immunohistochemically analyzed for CD8+ TIL infiltration, CD68+ macrophage infiltration, and HLA class I and
NY-ESO-1 expression by cancer cells. Statistical examination was performed with clinico-pathological factors. In
the US series, frequent CD8+ TIL infiltration was favorably prognostic in serous carcinoma, but was not
significantly prognostic in other histological subtypes. In serous carcinoma from the US, CD8+ TIL infiltration
was favorable in subgroups with marked CD68+ macrophage infiltration, higher HLA class I expression by
cancer cells, or NY-ESO-1 positive cases. On the other hand, in Japanese serous carcinoma, such prognostic
significance of CD8+ TIL was not confirmed statistically. This was probably because the Japanese serous
carcinoma series included larger number of earlier stage disease; Stage I disease accounts for 15% of serous
carcinoma of the Japanese series while such earlier stage accounted for none of the US series. Actually, when
Stage III cancers were sampled from the Japanese series, frequent CD8+ TIL was favorably prognostic with
statistical significance. In human sporadic EOCs, it seems that effective immune reaction can arise in cancers of
advanced stage, and it requires expression of HLA class I and some antigens by cancer cells. HLA class I and
immunogenic antigen expression should be carefully considered for the assessment of the immune reaction and
it would also be a significant factor for the induction of immunotherapy.
P102 - Expansion of Donor Anti-Tumor T Cells for CLL Adoptive Immunotherapy
MASAYASU NAITO, Ursula Hainz, Kristen E. Stevenson, Tetsuro Sasada, Donna Neuberg, Gordon J. Freeman,
Catherine J. Wu
Dana-Farber Cancer Institute, Boston, MA
Infusion of mature donor CD8+ T cells with specificity against tumor cells following allogeneic stem cell
transplantation can provide an effective treatment against chronic lymphocytic leukemia (CLL). CLL cells,
however, are weak antigen presenting cells (APCs), and only poorly stimulate antigen-specific T cell expansion.
To overcome this barrier, we evaluated a novel formulation of human recombinant CD40L, a molecule that is
known to enhance the immunostimulatory capacity of normal and malignant B cells. This CD40L (CD40L-Tri)
was designed with the extracellular domain of CD40L connected by a long flexible linker to a leucine zipper for
trimerization and an octahistidine motif for purification. We compared the immunostimulatory activity of CD40LTri with a conventional murine fibroblast cell line (tCD40L/NIH3T3). CD40L-Tri significantly: (1) expanded B cells
over 14 days (p<0.05); (2) increased expression of the costimulatory molecules CD80, CD83, and CD86 to
levels comparable to tCD40L/NIH3T3 (p<0.05); and (3) stimulated the expansion of allogeneic CD4+ and CD8+
T cells, at similar levels to tCD40L/NIH3T3 cells. Finally, CD40L-Tri-activated B cells could present pulsed
peptide to specifically expand CD8+ T cells specific for the influenza peptide M1 from a normal volunteer. These
results confirmed that CD40L-Tri can expand T cells without the risk of xenoantigen exposure incurred through
the use of tCD40L/NIH3T3.
Our ongoing studies explore the use of CD40L-Tri-activated CLL cells to expand HLA-matched donor T cells
with specificity for recipient CLL. We have successfully generated donor CD8+ T cells with specific reactivity
against recipient CLL cells but not allo-antigens in 3 of 4 patients following weekly repeated stimulation against
CD40L-Tri-activated CLLs in the presence of supportive cytokines. These results suggest a potentially effective
strategy for adoptive T cell therapy after transplant in patients with CLL.
P103 - Structural and Biochemical Elucidation of the Membrane-Proximal Events in Toll-Like Receptor
Signaling
**JOHANNA NAPETSCHNIG, Ermelinda Damko, and Hao Wu
Weill Medical College of Cornell University, Department of Biochemistry, New York, NY
In the constant challenge for survival, the innate immune system represents the first defense against the threat
of invasion by microorganisms. Activation of the Toll-like receptor (TLR) pathway by invading pathogens results
in intracellular signaling cascades and ultimately in the development of appropriate immune and inflammatory
responses. After TLR activation the first intracellular event in TLR/IL-1R signaling is the association of MyD88
with TLR. The intracellular TIR domain of the receptor and the TIR domain of adapter proteins govern this
membrane-proximal interaction. Although many TIR domain structures have been reported, no structural
information about any TIR complex is available to date. After the recruitment of MyD88 by the TLR receptor,
MyD88 in turn recruits the IRAK proteins to form the Myddosome, which ultimately results in the activation of
downstream signaling proteins. The Myddosome complexes are regulated dynamically during signaling, but the
exact nature of their regulation is poorly understood.
We are now using an approach integrating biochemistry and structural biology to focus on deciphering the
regulation and structure of these membrane-proximal events. We plan to shed light on the molecular principle of
signal transduction via the intracellular TIR domain of the TLR receptor in mammals, as well as to reveal the
mechanism involved in Myddosome feedback inhibition. We have co-expressed multiple TIR domains to
facilitate formation of stable complexes and are using previously successfully assembled DD complexes as a
scaffold to support TIR complex assembly. Assembled complexes are then biochemically and biophysically
analyzed.
P104 - A Model System to Investigate Antibody Bipolar Bridging Mediated by gE-gI, a Herpes Virus Fc
Receptor
1
3
**BLAISE NDJAMEN , Alex Farley , and Pamela J. Bjorkman
1
2
2
3
California Institute of Technology, Pasadena, CA; Howard Hughes Medical Institute, Pasadena, CA; Massachusetts
Institute of Technology, Cambridge, MA
The Herpes Simplex Virus 1 (HSV-1) expresses two membrane glycoproteins (gE and gI) that bind the Fc
region of host immunoglobulin G (IgG), which allows HSV-infected cells to escape recognition by Fc-dependent
immune cells. HSV-1 gE-gI can further undermine the immune system by participating in antibody bipolar
bridging, a process by which the antigen-binding fragments (Fabs) of the IgG bind a viral antigen while the Fc
binds to gE-gI. Although there is evidence of gE-gI/IgG/viral antigen complex formation, the fate of this complex
is still unknown.
In this study, we engineered an in vitro system to determine whether the gE-gI/IgG/viral antigen complex is
internalized and targeted for degradation in host cells. This system involves HeLa cells transiently expressing
HSV-1 gE-gI and HSV-1 gD, normally localized at the cell surface of HSV virions and infected cells, fused to
Cerulean fluorescent protein. Taking advantage of the fact that HSV-1 gE-gI binds human IgG (hIgG), but not
mouse IgG (mIgG), we constructed two forms of the monoclonal anti-gD antibody: one in which the Fabs were
fused to a hIgG1 Fc (anti-gDhFc), which can bind to gE-gI via its Fc and to gD via its Fabs, and one in which the
Fabs were fused to an mIgG2a Fc (anti-gDmFc), which can bind to gD but not to gE-gI. The 2G12hFc, an hIgG
against an irrelevant HIV-1 antigen was used as a control antibody that binds gE-gI but not gD. Pulse-chase
experiments analyzed by immunofluorescence confocal microscopy showed that cells expressing gE-gI
internalized hIgG and gD but not mIgG and gD, at pH 7.4 but not pH 6.0, correlating with pH-dependent gE-gIIgG interactions. gE-gI and the internalized IgG were mostly located in distinct intracellular compartments,
suggesting that HSV-1 gE-gI and IgG dissociate at low pH. These results suggest that gE-gI plays an active role
in clearing the infected cell surface of both host IgG and viral antigens, providing HSV-1 with a mechanism to
evade IgG-mediated immune responses. Knowledge gained from this study will shed light on viral Fc receptor
functions and Herpes virus immune evasion strategies, which are relevant to cancer and cancer therapies.
P105 – Anti-TIM-3 Antibody Promotes T Cell IFN-γ-Mediated Anti-Tumor Immunity and Suppresses
Established Tumors
1,2
1
3
3
1,2
*SHIN FOONG NGIOW , Bianca von Scheidt , Hisaya Akiba , Hideo Yagita , Michele W. L. Teng , Mark J.
1,2
Smyth
1
2
Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, Australia;
3
University of Melbourne, Parkville, Victoria, Australia; Juntendo University School of Medicine, Tokyo, Japan
New insights into the regulation of T cell activation and proliferation have led to the identification of checkpoint
proteins that modulate T cell reactivity. Monoclonal antibody (mAb) immunotherapies reactive with CTLA-4 or
PD-1 have shown promising therapeutic outcomes in mice and humans, highlighting the application of T cell
checkpoint inhibitors as one of the most promising new immunotherapeutic approaches. T cell immunoglobulin
and mucin domain 3 (TIM-3) is one of many similar inhibitory molecules that are gaining attention as targets in
tumor immunotherapy. Here we report an extensive characterization of the therapeutic activity and mechanism
of action of an anti-mouse TIM-3 mAb against experimental and carcinogen-induced tumors. We specifically
+
+
define the mechanism of anti-tumor action of anti-TIM3 requiring IFN--producing CD8 T cells and CD4 T cells.
Anti-TIM-3 displayed modest prophylactic and therapeutic activity against a small fraction of carcinogen-induced
sarcomas, but comparative and combination studies of anti-TIM3 with anti-CTLA-4 and/or anti-PD-1 against
experimental and carcinogen-induced tumors suggested that these agents might be well-tolerated and very
effective in combination.
P106 - PD-1 and LAG-3 Work Synergistically to Prevent Anti-Tumor Immunity
CHRISTOPHER J. NIRSCHL, Monica Goldberg, Nicholas Durham, Christina Ceccato, George Netto, Alcides
Chaux, Charles G. Drake
Johns Hopkins School of Medicine, Baltimore, MD
Immunotherapies of cancer represent a careful balance between anti-tumor responses and autoimmune-like
symptoms, highlighting the abuse of peripheral tolerance mechanisms by progressing tumors. Within immune
checkpoint molecules there is a wide spectrum of immunological influence, ranging from “on/off” switches such
as CTLA-4, to more subtle controls such as PD-1 or LAG-3. Yet, the combinatorial effects of these more subtle
molecules have not yet been thoroughly investigated in a wide range of model systems. In our studies we have
found that mice lacking both of these checkpoint proteins have a severely attenuated lifespan that is not
-/-/reflected in either single knockout. Pdcd-1 Lag-3 mice also showed intense, organ-specific immune infiltration
in the heart, lungs, and liver, while organs such as the kidney, thymus, and colon showed little to no disruption.
-/-/When transferred into a model of self-tolerance (137), Pdcd-1 Lag-3 CD8 T cells showed increased ability to
-/-/-/escape deletion over either single knockout. Moreover, Pdcd-1 Lag-3 and Pdcd-1 CD8 T cells showed
-/similarly elevated levels of in vivo CTL activity, while Lag-3 CD8 T cells were comparable to controls. After
being transferred into host developing endogenous prostate tumors (ProTRAMP), both single knockouts showed
-/-/increased CTL capability in vivo. However, the Pdcd-1 Lag-3 CD8 T cells had greater in vivo CTL activity than
either single knockout, suggesting a synergistic role for these molecules in preventing anti-tumor immunity.
Lastly, hosts bearing equivalent size SA1N tumors were treated with PD-1, LAG-3, or both. While the PD-1
treatment resulted in slowed tumor growth, the LAG-3 treatment showed no benefit over isotype controls.
However, dual blockade treatment resulted in complete tumor rejection, with no harvestable tumor remaining 13
days after treatment. In addition, antibody treated mice from all groups showed no detectable immune infiltration
in any of the previously described organs. Together, these data describe a synergistic role for the checkpoint
proteins PD-1 and LAG-3 in preventing anti-tumor immunity, and suggest that combinatorial antibody blockades
provide a promising avenue for future clinical immunotherapy.
P107 - Dacarbazine Before Peptide Vaccination Activates AKT Signalling Pathway and Functional
Activity of Melan-A-specific T Cell Clones
2
1
1
1
1
1
Ornella Franzese , Belinda Palermo , Cosmo Di Donna , Duilia Del Bello , Novella Gualtieri , Carmen Nuzzo ,
3
4
1
1
Enrico Proietti , Caterina Catricalà , Virginia Ferraresi , PAOLA NISTICÒ
1
2
3
Regina Elena National Cancer Institute, Rome, Italy; University of Tor Vergata, Rome, Italy; Istituto Superiore di Sanità,
4
Rome, Italy; S. Gallicano Dermatological Institute, Rome, Italy
The administration of dacarbazine (DTIC) one day before peptide (Melan-A and gp100)-vaccination in disease+
free HLA-A2 melanoma patients increases the number of peptide-specific effector-memory CD8 lymphocytes.
The TCR repertoire diversity of Melan-A CD8+ clones is broadened while maintaining high avidity in long
surviving patients treated with DTIC before vaccination, whereas patients treated with vaccine alone showed a
reduction of the TCR repertoire diversity and a decline of tumor lytic activity. To identify the mechanisms
underlying the DTIC-elicited functional effects and TCR repertoire enlargement, we analyzed CD8+ Melan-Aand gp100-specific T cell clones isolated before and at different times after treatment. The induction and
maintenance of the long lasting CD8+ Melan-A T cell clone phenotype and functional activity were analyzed in
relationship to the AKT signaling and telomerase activity which are reported to be related to the expression of
CD28/CD27 costimulatory molecules and to the replicative potential of memory T cells. In Melan-A-specific
CD8+ clones telomerase activation correlates with the AKT phosphorylation, but not with CD28 and CD27
expression. Differently in gp100-specific clones, AKT and telomerase activation correlate with CD28/CD27
expression. The identification of extracellular stimuli responsible for DTIC-mediated activation of the AKT
signaling pathway is currently under investigation.
P108 - Characterization of the Human Splenic Littoral Cell, a Rapidly Evolving Constituent of Red Pulp
and the Chief Source of SIRPin Primate Spleen
1,5
2,5
4,5
2,5
2,5
**JAVIER G. OGEMBO , Dan Milner , Keith Mansfield , Scott J. Rodig , Jeffery L. Kutok , Geraldine S.
2,5
3,5
1,5
Pinkus , Anthony John Iafrate , and Joyce Diane Fingeroth
1
2
Division of Infectious Diseases, Beth Israel Deaconess Medical Center; Department of Pathology, Brigham and Women’s
3
4
5
Hospital; Department of Pathology Massachusetts General Hospital; New England Primate Research Center; and Harvard
Medical School, Boston MA
Asplenic individuals are profoundly compromised not only in their ability to destroy many infectious agents, but
also in their increased risk of death from autoimmune disease, certain tumors, and ischemic heart disease.
Enhanced mortality is attributed to lack of phagocyte populations sequestered in spleen that efficiently engulf
and destroy appropriate targets, though related cells are found at other sites in man. To investigate whether a
unique population(s) regulates red blood cell (RBC) and pathogen clearance as well as filtration of altered self,
we focused on a little-characterized cell that dominates the splenic red pulp of man and closely related primates,
the venous sinus lining or littoral cell (LC). High expression of the formin, FHOD1, outlines the LC population.
Though endothelial-like in distribution, LCs also express several macrophage-associated proteins, the RBC
antigen DARC, and T cell co-receptor CD8/, though they lack lineage markers CD34 and CD45. Strikingly,
signal regulatory protein alpha (SIRP), expression in human spleen concentrates on the LC, consistent with a
+
key role in RBC turnover, as well as elimination versus release of infected or altered self. Human LCs (SIRP ,
+
+
FHOD , CD8/ , CD34 , CD45 ) comprise a highly specialized barrier cell population that has emerged late in
the course of evolution accompanied by CD8 expression. Unique to higher primates (Hominidae), LCs may
provide the ultimate filtration function(s) of human spleen.
P109 - Correlation of Antibody and T Cell Responses Against XAGE-1b in NSCLC Patients
1
1,2
1,2
1
2
1
YOSHIHIRO OHUE , Shingo Eikawa , Yu Mizote , Hirofumi Matsumoto , Nami Okazaki , Midori Isobe , Akiko
3
1
3
Uenaka , Mikio Oka , and Eiichi Nakayama
1
2
Kawasaki Medical School, Kurashiki, Okayama, Japan; Okayama University Graduate School of Medicine, Dentistry and
3
Pharmaceutical Sciences, Okayama, Japan; Faculty of Health and Welfare, Kawasaki University of Medical Welfare,
Kurashiki, Okayama, Japan
Background: XAGE-1b is a cancer/testis antigen identified in lung cancer using autologous patient serum by
SEREX. The expression of XAGE-1b is observed frequently in lung adenocarcinoma and rarely in some other
tumors. Recently, we showed spontaneous T cell response against XAGE-1b in XAGE-1b-antibody positive
non-small cell lung cancer (NSCLC) patients. In this study, we analyzed the frequency of XAGE-1b-reactive
CD4 and CD8 T cells in NSCLC patients showing high, intermediate, and low antibody responses.
Materials and Methods: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical
School Hospital between 2009 and 2011. Antibody response was determined for XAGE-1b protein by ELISA
using O.D. values at 1:900 serum dilution and classified as high ≥ 3.0, 3.0 > intermediate ≥ 1.0 and 1.0 > low.
CD4 and CD8 T cell responses against XAGE-1b were examined by IFN-γ ELISA using 12- or 16-mer XAGE1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein in antibody-positive patients. Cytotoxicity
was analyzed determining GAPDH release by aCellaTox kit.
Results: The number of XAGE-1b antibody-positives was 29 (9.0%) of 323 NSCLC patients. Within those, the
patients showing high, intermediate, and low antibody response was 6, 11, and 12, respectively. The frequency
of XAGE-1b-reactive CD4 T cells in patients showing high, intermediate, and low antibody response was 5.5
±2.1 x 10-5, 1.5 ±0.7 x 10-5, and < 1.1 x 10-5, respectively. The frequency of XAGE-1b-reactive CD8 T cells
was 6.9 ±1.6 x 10-6, 4.6 ±1.6 x 10-6 and < 1.1 x 10-6, respectively. The ratio of the CD8 T cells recognizing
XAGE-1b-expressing tumor to the XAGE-1b-peptide reactive CD8 T cells in patients showing high and
intermediate antibody responses was 60.0% and 14.3%, respectively.
Conclusion: Our findings indicate correlation of antibody response
P110 - Dualistic Responses of Carcinomas to Single and Multiple Immune Reactants Over a Narrow
Range
**OLIVER M.T. PEARCE, Paula C. Soto, Patrick Secrest, Renee Chow, Nissi Varki, Jack Bui, and Ajit Varki
University of California, San Diego, La Jolla, CA
Explorations into the modulation of cancer progression by the immune system have a long history. In the 1950s
Burnett and Thomas coined the term ‘immunosurveillance’ [1] to indicate that the immune system is constantly
recognizing nascently transformed cells and eliminating them. More recently, the term ‘immunoediting’ was
coined by Schreiber and colleagues [2], denoting a three-step mechanism that better explains how the immune
system can be both protective and tumor sculpting. On the other hand much data indicates that chronic immune
reactions can promote tumor progression. This apparent inconsistency can be reconciled by Prehn’s suggestion
[3, 4], that the immune system can either facilitate or limit tumor progression, in a hormetic curve that depends
on the relative level of immune reactants. We have directly addressed the Prehn hypothesis using a human-like
murine model with antibodies directed against a human-specific tumor antigen, analyzing the growth of tumors
exposed to varying doses of tumor-specific antibodies. We found that tumor growth could be first stimulated and
then inhibited in an immune response curve (IRC) over a linear and remarkably narrow dose range.
Furthermore, when the antibodies with lower affinity were used, the IRC was shifted appropriately to the right.
Meanwhile we also used an independent model of Siglec-E null mice to mimic the human-like down-regulation
of inhibitory innate immune leukocyte Siglecs. Tumors grown in such Siglec-E null mice showed increased
growth, apparently due to an enhanced innate immune reaction. Finally, we combined both inflammatory
enhancing effects (namely the Siglec-E deficiency and antibodies) which shifted the IRC to the left. This work
affirms the Prehn hypothesis and directly demonstrates that the immune system plays a dualistic role in tumor
progression, depending on the relative levels of one or more different immune reactants, over a narrow range.
These results also have practical relevance to the prevention of human cancer and to the design of anti-cancer
immunotherapeutics.
[1] Burnett, F. M. Br. Med. Bull. 20: 154-158, 1964.
[2] Dunn, G. P. et al. Nat. Immunol. 3: 991-998, 2002.
[3] Prehn, R. T. Science. 176:170-171, 1972.
[4] Prehn, R.T., and Prehn, L.M. 222:341-356, 2008.
+
+
P111 - Imaging the Response of Melanoma-Specific CD4 and CD8 T Cells to CTLA-4 Blockade In Vivo
TSVETELINA PENTCHEVA-HOANG and James P. Allison
Memorial Sloan-Kettering Cancer Center, New York, NY
Real-time imaging of tumor-specific immune responses can provide information about the cellular dynamics in
lymph nodes and tumors, as well as how cell behaviors change as a result of various immunotherapies. We
have recently developed a 2 photon imaging system that allows us to track GFP-positive tumor-specific T cells
+
+
(PMEL, TRP1, or polyclonal CD4 and CD8 ) in tdTomato-expressing B16 tumors and tumor-draining lymph
+
+
nodes (TDLNs). The average velocities of CD8 PMEL and CD4 TRP1 T cells are lower in tumors compared to
+
+
those in TDLNs, consistent with tumor antigen recognition. The velocities of polyclonal CD4 and CD8 T cells
appear comparable in tumors and TDLNs. When anti-CTLA-4 antibodies are injected intraperitoneally in live
animals, the velocities of PMEL T cells in TDLN but not in tumors increase, regardless of initial treatment.
Injection of control hamster immunoglobulin has no effect on PMEL velocities in TDLNs, suggesting that CTLA-4
might be contributing to T cell stopping in TDLNs. Similar experiments with TRP1 and polyclonal T cells indicate
that CTLA-4 blockade does not change their velocities, possibly because of differences in TCR avidity. Under
the same conditions, MHC class II antibodies seem to increase TRP1 velocities slightly but significantly,
consistent with a reversal of the TCR-mediated STOP signal. Preliminary experiments with Foxp3-GFP mice
suggest that it is possible to track endogenous T regulatory cells, and their velocities in TDLNs are lower than
those of effector T cells. Future studies will address the behavior of multiple cell populations in the same tumorbearing mouse using actin-CFP and CD11c-YFP animals.
P112 - Antigen Dose Regulated Changes in CD8-Dependent T Cell Receptor Binding
*KARLO PERICA, Joan Bieler, Malar Durai, Kapil Gupta, Jonathan Schneck
Johns Hopkins School of Medicine, Baltimore, MD
The T cell receptor (TCR) is characterized by high specificity and sensitivity but low affinity for cognate antigen.
In addition, it has become apparent that individual T cells can regulate their overall avidity for antigen through
persistent changes in membrane organization. Using biophysical analysis of soluble Major Histocompatability
Complex (MHC) dimer binding, our laboratory showed that TCRs are pre-clustered several days after activation,
leading to enhanced binding of low dose antigen. This stands in contrast to observations from other groups
describing a loss of soluble MHC binding after stimulation that is not due to TCR internalization or decreased
surface TCR expression.
Here, we show for the first time that the dose of antigen stimulation drives activated T cell binding behavior.
High antigen dose in vitro-stimulated T cells are rendered transiently incapable of binding soluble MHC dimer at
Day 4, beginning recovery by Day 7. Conversely, low-dose stimulated cells display TCR clustering and
enhanced binding. The enhanced and defective binding behaviors cannot be explained by expression of TCR,
CD8, or other surface receptors.
We also show that this defect is confined to “CD8-dependent” soluble MHC binding, and can be reversed by
inhibition of surface sialylation but not N-glycosylation. Furthermore, we provide initial evidence that defective
cells segregate CD8 and TCR in distinct membrane domains. Thus, we suggest a model in which T cells
integrate antigen dose to regulate future binding behavior through a glycosylation-dependent mechanism.
Glycosylation in turn may regulate TCR and CD8 surface organization, which controls binding of CD8dependent antigen.
P113 - Immunological Studies with Mouse Bcr-abl-Transformed 12B1 Cells
MARTINA PETRÁČKOVÁ, Vincent Lučanský, Monika Krmenčíková, Eva Sobotková, Martina Dušková, Libor
Staněk, Vladimír Vonka
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
In our laboratory, we are focused on the development of therapeutic vaccines against chronic myeloid leukemia
(CML), using as a model system mouse bcr-abl-transformed cells, namely the cell lines designated B210 and
12B1 cells. Both these cells are of early B cell lineage and induce leukemia after i.v. inoculation; the 12B1 cells
also induce solid lymphoma-like tumors after s.c. inoculation. Our present research is primarily directed to the
development of vaccines based on tumor cells gene-engineered to secrete immunostimulating cytokines and to
the DNA vaccines. In the present series of experiments we isolated and tested gene-modified 12B1 cells
secreting IL-2 and GM-CSF. The properties of these cells markedly differed. The pathogenicity of the IL-2
producing sublines was reduced and most of the animals, which did not develop tumors after administration of
these cells, were found resistant to the challenge with parental cells. Analysis of the tumor population revealed
genetic instability of the IL-2 secreting cells. On the other hand, GM-CSF secreting cells were more pathogenic
than the parental 12B1 cells and, in addition to leukemia and solid tumors, they also induced extensive
pathological changes in several organs. At variance with the IL-2 secreting cells they were genetically stable in
vivo. When analyzing the effects of cell clones markedly differing in the production of GM-CSF, it became
apparent that the pathogenicity was directly related to the extent of the cytokine production. On the other hand,
when used in the form of inactivated vaccines, the most immunogenic were the high GM-CSF producer cells. In
another set of experiments we induced immunity in mice with a DNA vaccine containing the whole bcr-abl gene
and then challenged the immunized animals with 12B1 cells. To determine the role of CD4+, CD8+, and NK
cells in the immunity developed, these cell populations were separately deleted by monoclonal antibodies.
Surprisingly, CD4+ cells were the dominant population involved in the immune response. Experiments are under
way to analyze these observations and their significance for immunity against bcr-abl-transformed cells.
This work was supported by grant NS 10634-3/2009 from IGA MZ ČR and the research project MZOUHKT2005.
P114 - Candida albicans Infection of Caenorhabditis elegans Induces Antifungal Immune Defenses
1,2
1,2
**READ PUKKILA-WORLEY , Frederick M. Ausubel , Eleftherios Mylonakis
1
1,2
3
Massachusetts General Hospital, Boston, MA; Harvard Medical School, Boston, MA
Candida albicans yeast cells are found in the intestine of most humans, yet this opportunist can invade host
tissues and cause life-threatening infections in susceptible individuals. To better understand the host factors that
underlie susceptibility to candidiasis, we developed a new model to study antifungal innate immunity. We
demonstrate that the yeast form of C. albicans establishes an intestinal infection in Caenorhabditis elegans,
whereas heat-killed yeast are avirulent. Genome-wide, transcription-profiling analysis of C. elegans infected with
C. albicans yeast showed that exposure to C. albicans stimulated a rapid host response involving 313 genes
(124 upregulated and 189 downregulated, ~1.6% of the genome) many of which encode antimicrobial, secreted,
or detoxification proteins. Interestingly, the host genes affected by C. albicans exposure overlapped only to a
small extent with the distinct transcriptional responses to the pathogenic bacteria Pseudomonas aeruginosa or
Staphylococcus aureus, indicating that there is a high degree of immune specificity toward different bacterial
species and C. albicans. Furthermore, genes induced by P. aeruginosa and S. aureus were strongly overrepresented among the genes downregulated during C. albicans infection, suggesting that in response to fungal
pathogens, nematodes selectively repress the transcription of antibacterial immune effectors. A similar
phenomenon is well known in the plant immune response, but has not been described previously in metazoans.
Finally, 56% of the genes induced by live C. albicans were also upregulated by heat-killed yeast. These data
suggest that a large part of the transcriptional response to C. albicans is mediated through “pattern recognition,”
an ancient immune surveillance mechanism able to detect conserved microbial molecules (so-called pathogenassociated molecular patterns or PAMPs). This study provides new information on the evolution and regulation
of the innate immune response to divergent pathogens and demonstrates that nematodes selectively mount
specific antifungal defenses at the expense of antibacterial responses.
P115 - Tumor-Associated Myeloid Cells can be Activated In Vitro and In Vivo to Mediate Anti-tumor
Effects
ALEXANDER RAKHMILEVICH, Mark Baldeshwiler, Tyler Van de Voort, Mildred Felder, Richard Yang, Nickolas
Kalogriopoulos, David Koslov, and Paul Sondel
University of Wisconsin-Madison, Madison WI
Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs.
These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy.
In this study we tested the possibility of activating tumor-associated myeloid cells to mediate anti-tumor effects.
Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM)
had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and
lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC correlated with the
+
+
influx of CD11b Gr-1 myeloid cells to the peritoneal cavity. Non-adherent PEC were responsible for most of the
+
+
NO production in TBM, whereas in naïve mice NO was mainly secreted by adherent CD11b F4/80
+
+
+
macrophages. Sorted CD11b Gr-1 monocytic and CD11b Gr-1 granulocytic PEC from TBM had a reduced
ability to secrete NO following in vitro stimulation (compared to naïve PEC), but effectively suppressed
proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with
anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction of local tumor burden, and
prolongation of mouse survival. Taken together, the results indicate that in tumor-bearing hosts, tumorassociated myeloid cells can be activated to mediate anti-tumor effects.
P116 - Analysis of Antigen Uptake and B Cell Activation in Peyer’s Patches
**ANDREA REBOLDI and Jason G.Cyster
Department of Microbiology and Immunology, University of California, San Francisco, and Howard Hughes Medical Institute,
San Francisco, CA
An essential step in humoral immune responses is for B cells to contact the antigen, and the sites of encounter
have only recently been visualized in lymph nodes. However, whether a similar mechanism takes place in other
secondary lymphoid organs is not known yet. Peyer’s patches (PPs) are part of gut-associated lymphoid tissue
(GALT) and they’re positioned close to the intestinal lumen where they play an essential role in the mucosal
immune response. Immunoglobulin A production takes place in PPs and represents a key process in protecting
intestinal mucosal surfaces against invasion by pathogenic microorganisms and confining commensal bacteria
in the intestinal lumen. We’re trying to elucidate how antigen can gain access to B cells in PPs, since these are
the principal sites of induction of high and low affinity IgA, which protects intestinal mucosal surfaces against
invasion by pathogenic microorganisms and confines commensal bacteria in the intestinal lumen. Our lab has
recently shown that Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a key regulator in
positioning B cells in lymphoid organs. Moreover, the oxysterol 7,25-OHC has been identified as the ligand for
EBI2 in regulating B cells trafficking after an immune challenge. Since it has been suggested that oxysterols can
affect IgA production, we’re currently investigating how the axis EBI2-7,25-OHC impacts on B cell positioning
and class switch specificity in Peyer’s patches.
P117 - Phase I/II Study of Resiquimod as an Adjuvant for NY-ESO-1 Protein in Patients with Melanoma
1
3
1
1
1
RACHEL LUBONG SABADO , Sacha Gnjatic , Crystal Cruz , Isabelita Vengco , Angelica Angiulli , Farbod
2
2
1
1
1
1
Darvishian , Luis Chiriboga , Rose Marie Holman , Juliet Escalon , Caroline Sorlie , Crystal Escano , Ethel
1
1
1
2
3
3
3
Yepes , Dunbar Sharpe , Patrick Ott , Achim Jungbluth , Linda Pan , Ralph Venhaus , Llyod Old , Anna
1
1,2
Pavlick , and Nina Bhardwaj
1
2
3
NYU Cancer Institute, New York, NY; New York University School of Medicine, New York, NY; Ludwig Institute for Cancer
Research, New York, NY
Background: The TLR 7/8 agonist Resiquimod has been shown to induce local activation of immune cells,
production of cytokines, and antigen presentation by dendritic cells, features desirable for cancer vaccine
adjuvants. In this study, we evaluated the safety and immunogenicity of vaccination with NY-ESO-1 protein
emulsified in Montanide ISA-51 VG when given with or without Resiquimod in surgically resected stage IIB-IV
melanoma patients.
Methods: This is a two-part study design. Part I represents an open-label dose-escalation with Resiquimod
using 2 cohorts treated with 100ug NY-ESO-1 protein emulsified in 1.25mL Montanide (day1) followed by topical
application of 1000mg of the 0.2% Resiquimod gel on days 1 and 3 for cohort-1 (N=3) or days 1, 3, and 5 for
cohort-2 (N=3). The cycles were repeated every 3 weeks, total of 4 cycles. In Part II, patients were randomized
to receive 100ug NY-ESO-1 protein emulsified in 1.25mL Montanide (day1) followed by topical application of
placebo gel (Arm-A; N=8) or 1000mg of 0.2% Resiquimod gel (Arm-B; N=12) using the dosing regimen
established in Part I. Blood samples were collected at baseline, one week after each cycle of vaccination, and at
the follow-up visit for the assessment of NY-ESO-1-specific humoral and cellular immune responses.
Results: The vaccines were generally well tolerated, with no grade 4 adverse events or study-related deaths.
The most common toxicities were mild to moderate and included local injection site reactions (granuloma,
puritis, induration) and systemic flu-like symptoms. Evaluation of immune responses induced by the vaccine
showed the induction of both humoral and cellular immune responses against NY-ESO-1. 24/26 patients
developed NY-ESO-1 antibodies, some starting after the second vaccine, and a substantial number developing
5
titers of >10 . Preliminary analysis of T cell responses shows induction of NY-ESO-1-specific CD4+ and CD8+ T
cell responses.
Conclusion: This study demonstrates the safety and immunogenicity of Resiquimod as an adjuvant for NYESO-1 protein vaccination. Ongoing analysis of immune responses will provide further information on the quality
of immune responses induced by the vaccine.
P118 - Bacteria-Induced Gap Junctions in Tumors Favor Antigen Cross-Presentation and Anti-Tumor
Immunity
1
1
3
1
1
2
FABIANA SACCHERI , Chiara Pozzi , Francesca Avogadri , Sara Barozzi , Mario Faretta , Paola Fusi , and
1
Maria Rescigno
1
2
3
European Institute of Oncology, Milan, Italy; University of Milano-Bicocca, Milan, Italy; Memorial Sloan-Kettering Cancer
Center, New York, NY
Antigen presenting dendritic cells (DCs) trigger the activation of cytotoxic CD8 T cells that target and eliminate
cells with the antigen on their surface. Although DCs usually pick up and process antigens themselves, they can
also receive peptide antigens from other cells via gap junctions. We demonstrate here that infection with
Salmonella can induce, in both human and murine melanoma cells, the up-regulation of connexin 43 (Cx43), a
ubiquitous protein that forms gap junctions and that is normally lost during melanoma progression. Bacteriatreated melanoma cells can establish functional gap junctions with adjacent DCs. After bacterial infection, these
gap junctions transferred preprocessed antigenic peptides from the tumor cells to the DCs, which then
presented those peptides on their surface. These peptides activated cytotoxic T cells against the tumor antigen,
which could control the growth of distant uninfected tumors. Melanoma cells in which Cx43 had been silenced,
when infected in vivo with bacteria, failed to elicit a cytotoxic anti-tumor response, indicating that this Cx43
mechanism is the principal one used in vivo for the generation of anti-tumor responses. The Cx43-dependent
cross-presentation pathway is more effective than standard protocols of DC loading (peptide, tumor lysates, or
apoptotic bodies) for generating DC-based tumor vaccines that both inhibit existing tumors and prevent tumor
establishment. In conclusion, we exploited an antimicrobial response present in tumor cells to activate cytotoxic
CD8 T cells specific for tumor-generated peptides that could directly recognize and kill tumor cells. (Saccheri et
al., Sci TM 2010). The ultimate goal is to bring the protocol into the clinic. Experiments with human sample show
encouraging results that are consistent with the ones obtained in the murine model. These achievements
opened the path to the experimentation on patients.
P119 - Cholesterol Transporter ABCG1 as a Novel Mediator of Anti-Tumor Immunity
DUYGU SAG, Caglar Cekic, Joel Linden, and Catherine C. Hedrick
La Jolla Institute for Allergy and Immunology, La Jolla, CA
ATP Binding Cassette Transporter G1 (ABCG1) is a member of the ABC transporter family that effluxes
cholesterol from cells to HDL particles for reverse cholesterol transport. Reverse cholesterol transport is the only
path for elimination of cholesterol from the body. ABCG1 is expressed in many cell types including myeloid cells,
lymphocytes and endothelial cells. ABCG1 has recently been shown to modulate macrophage and lymphocyte
immune responses. In this study, we investigated the role of ABCG1 in tumor progression. MB49 bladder
-/+/+
carcinoma or B16 melanoma cells were injected subcutaneously into ABCG1 or ABCG1 control mice. Both
groups of mice were fed with either Western diet containing 0.2% cholesterol or chow diet containing no
-/cholesterol starting a week before injection of tumor cells. ABCG1 mice fed a Western diet showed
+/+
dramatically reduced (80%) tumor growth compared to ABCG1
control mice. We did not observe any
-/differences in tumor size between groups on chow diet. ABCG1 mice fed Western diet showed also diminished
+/+
lung metastasis of B16 melanoma. Moreover, we found that chimeric ABCG1 control mice reconstituted with
-/+/+
ABCG1 bone marrow had significantly diminished tumor growth compared to ABCG1 mice reconstituted with
+/+
ABCG1 bone marrow, demonstrating that the impact of ABCG1 deficiency on tumor growth is immunemediated. Analysis of the cells of the tumor microenvironment by flow cytometry revealed that reduced tumor
-/size in ABCG1 mice on Western diet was associated with a significant increase in the frequency of NK cells
and decrease in the frequency of macrophages and T regulatory cells. Furthermore, we found that mixed
+/+
-/-/chimeric mice containing ABCG1 and ABCG1 bone marrow (mixed at a 1:1 ratio) possessed fewer ABCG1
+/+
macrophages in spleen, and these macrophages are more apoptotic compared to ABCG1 macrophages.
These results identify the cholesterol transporter ABCG1 as a novel mediator of anti-tumor immunity.
Understanding how ABCG1 impacts anti-tumor immune responses could lead to production of entirely new
therapeutic approaches for cancer immunotherapy. Studies to determine the mechanisms leading to impaired
tumor growth as a result of ABCG1 deficiency are underway and will provide novel insights as to how ABCG1
impacts tumor immunity.
P120 - Identifying Factors Influencing Immunogenicity by Screening Mutated Forms of Wilms Tumor 1
1
2
1
*ASHLEY SAINT FLEUR , David Spence , and Hyam I. Levitsky
1
2
Johns Hopkins School of Medicine, Baltimore, MD; University of Delaware, Newark, DE
Wilms Tumor 1 (WT1) is a tumor associated antigen overexpressed in many cancers. As WT1 is also a self
antigen present at low levels in some normal cell populations, active immununotherapy targeting WT1 is
constrained by mechanisms of immune tolerance. By randomly mutating the wild type WT1 cDNA by errorprone PCR, an unbiased library of mutations is created. Mutations in nucleic acid result in altered codons, which
may lead to amino acid substitution or premature translation termination. Proteins produced from these mutants
may: (a) yield novel epitopes that serve as altered peptide ligands, (b) have increased turnover augmenting the
density of peptide/MHC complexes, or (c) enter alternate degradation pathways revealing cryptic epitopes, now
immunogenic.
The overall objective of this project is to identify those factors that influence immunogenicity using WT1 as a
model antigen. We have created a mutant mouse WT1 cDNA library and we are establishing an in vitro assay
capable of detecting immunogenic mutants. We will screen the mutant library by transfecting antigen presenting
cells with pools of mutants and quantifying the stimulation of polyclonal T cells from mice vaccinated with wild
type WT1. Pools that score positive will be subcloned. We will validate the immunogenicity of the identified
clones by vaccination in tumor protection experiments and identify the impact of specific immunogenic mutations
on antigen processing and presentation. Vaccines utilizing such mutant cDNA may have the potential to break
tolerance and improve immune responses to antigens that are normally seen as self antigens, or that only elicit
a weak immune response.
P121 - Trypanosoma cruzi Expressing NY-ESO-1 Induces Anti-tumor Immunity Associated with the
+ +
+
Generation of Memory CD8 T gzmB
1,2
1,2
2
2
LUARA ISABELA SANTOS , Bruno Galvão-Filho , Lis R. Antonelli , Caroline Junqueira , and Ricardo Tostes
1,2
Gazzinelli
1
2
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Rene Rachou Research Center, FIOCRUZ, Belo
Horizonte, MG, Brazil
The NY-ESO-1 is a highly immunogenic protein, member of the family Cancer/testis Antigens (CTA) and a
strong candidate for therapeutic and prophylactic vaccines against cancer. While multiple clinical trials
demonstrate the development of measurable immune responses anti-NY-ESO-1 only a minority of patients
experienced clinical benefit, such as tumor regression. Mice immunization with the attenuated Trypanosoma
cruzi strain expressing the NY-ESO-1 (CL14-NY-ESO-1) is able to prevent in vivo the growth of a melanoma cell
line (B16) expressing NY-ESO-1. Here, we evaluated the induction of cellular immune response induced by
vaccination with CL14-NY-ESO-1 and the therapeutic effect of this live vaccine. Mice immunized with transgenic
+
parasite were evaluated for cytokine production and phenotype of CD8 T cells. We observed that transgenic
parasites were able to induce the generation of effector and memory effector CD8+ T cells. When compared to
one dose, the homologous prime-boost protocol with transgenic parasites was more efficient in inducing the
generation of granzime B+ CD8+ T cells. We observed a persistent production of IFN gamma and IL-2
production after stimulation with recombinant NY-ESO-1. Specific NY-ES0-1-CD8T cells also were generated
and capable to produce IFN gamma after stimulation. In the therapeutic protocol mice were challenged with
melanoma cell line B16F10 expressing NY-ESO-1 and the tumor growth scored by measuring perpendicular
diameters. Five doses of anti-CTLA-4 were administrated every 3 days starting at day 4 after tumor challenge
and the parasites were inoculated three times every 5 days starting at day 3 after tumor challenge. The results
show that anti-CTLA-4 when combined with parasite expressing NY-ESO-1 delays the growth of highly
tumorigenic murine melanoma. This study shows that the transgenic parasite induces high levels of effector
+ +
+
memory CD8 T gzmB T cells and when combined with the neutralization of CTLA-4 is able to control
melanoma growth.
Financial support: LICR, INCTV, CNPq and FAPEMIG
P122 - GITR Ligation During Tumor Immunotherapy Induces Foxp3 Loss and Regulatory T Cell
Instability
DAVID SCHAER, Campbell Bryson, Roger Pellegrini, Nicole Malandro, Taha Merghoub, Alan Houghton, and
Jedd Wolchok
Memorial Sloan-Kettering Cancer Center, New York, NY
In vivo GITR ligation has been shown to augment T cell-mediated anti-tumor immunity, yet the underlying
mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not
been fully elucidated. We have previously demonstrated that in vivo GITR ligation by agonist antibody (DTA-1)
modulates Tregs directly during induction of murine melanoma tumor immunity. DTA-1 causes a ~50%
reduction of intra-tumor Tregs as a consequence of both impaired Treg tumor trafficking and intra-tumor down
modulation of Treg Foxp3 expression. Foxp3 loss was absent in IgG-treated tumors and in DTA-1 treated
TDLN, demonstrating a tumor-specific effect. Inflammatory conditions of tumor growth and lymphopenic
environments appear to make Tregs particularly susceptible to GITR ligation induced instability.
As this immunotherapeutic approach is translated into the clinic it is important to better understand its
mechanism(s) of action. Investigating changes in the periphery, we have found that the effect on Treg stability
also extends to the Tregs in the blood. Immediately after therapy, there is a 30-40% reduction of Tregs in the
PBMCs, which lasts for at least seven days. At the same time, there are no changes to Tregs in spleen or tumor
draining lymph node. Using putative natural and induced Treg markers Helios and Neuropilin-1, we found that in
the PBMCs, DTA-1 affected both Treg subsets. While Tregs were uniformly Helios and Neuropilin-1 high in the
tumor of control treated mice, there was a marked drop in Helios and Neuropilin-1 staining of the remaining
Tregs in DTA-1 treated tumors. This suggests that GITR ligation modulates all intra-tumor Tregs, and not just
those which have lost foxp3. Current research is investigating how incorporation of GITR signaling causes Tregs
to lose Foxp3 and if the level of IL-2 signaling affects Treg vulnerability to GITR induced instability.
Recently, GITR ligation has entered phase one clinical trials for cancer immunotherapy. Understanding the
exact cause of the Treg phenotypic changes associated with GITR ligation will contribute to defining biomarkers,
which may elucidate the effects of therapy in human patients.
Supported by: NIH K12 CA120121-01, CA056821, P01CA59350, P01CA33049, Swim Across America, John D. Proctor
Foundation, Goodwin Commonwealth Foundation, Lita Annenberg Hazen Foundation, and Experimental Therapeutics
Center of MSKCC
P123 - Epigenetic Memory: Tolerant T Cells Cannot Escape Their Fate
1,2
2
**ANDREA SCHIETINGER , Jeff Delrow , and Philip D. Greenberg
1
1,2
2
University of Washington, Seattle, WA; Fred Hutchinson Cancer Research Center, Seattle, WA
A hallmark of tolerant, self antigen-specific CD8 T cells is the failure to proliferate in response to antigen, but the
regulatory mechanisms underlying the proliferative block are less clear. Using an in vivo mouse model of
peripheral CD8 T cell tolerance, we show that tolerant T cells proliferate and become functional under
lymphopenic conditions even in a tolerogenic environment. However, T cell rescue is only transient, with
tolerance re-imposed upon repletion of the lymphoid compartment even in hosts lacking the tolerogen. Such
reacquisition of tolerance in non-tolerogenic hosts challenges the prevailing paradigm that continuous exposure
of T cells to self-antigen is required to maintain tolerance. Instead, genome-wide mRNA and microRNA profiling
revealed that tolerant T cells harbor a tolerance-specific gene signature resulting in functional
unresponsiveness, which can be temporarily overridden under lymphopenic conditions leading to rescue of
tolerant T cells, but is eventually reasserted. These findings imply that tolerance is dictated by “epigenetic
memory”, suggesting that permanent rescue of tolerant T cells will require strategies that lead to erasure of
tolerance-specific epigenetic memory.
P124 - Non Coding RNAs in the Development and Function of T Cell Lineages
**MACLEAN SELLARS, Andrew Sczesnak, Maria Ciofani, Mark M.W. Chong, and Dan R. Littman
The Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, New
York University School of Medicine, New York, NY
Large non-coding RNA transcripts (lncRNA) are widely expressed in mammalian cells. They are thought to play
crucial roles in regulating diverse processes, ranging from proliferation and metastisis, to stem cell pluripotency
and differentiation. Whether lncRNAs control the differentiation and function of the immune system is unclear.
To begin to answer this question, we have performed RNA-sequencing and constructed ChIP-seq based
chromatin state maps to identify transcribed regions in developing thymocytes and polarized T helper cells.
Combining these approaches, we have identified hundreds of lncRNAs, including many that are differentially
expressed during differentiation into mature CD4+ helper and CD8+ cytotoxic T cells, or correlate with the
expression of critical cytokines. We are currently testing candidate lncRNAs to determine their roles in T cell
development and effector function.
P125 - Targeting High-Fidelity and Error-Prone Repair Pathways During Somatic Hypermutation
**RASHU BHARGAVA SETH and David G. Schatz
Yale Medical School, New Haven, CT
Somatic hypermutation (SHM) is a B cell-specific genetic process that causes point mutations within the variable
regions of the immunoglobulin (Ig) loci, and underlies the process of Ig affinity maturation. This mutagenic
process is initiated at the Ig loci by the enzymatic activity of activation-induced cytosine deaminase (AID), which
deaminates cytosine residues in the DNA to uracils. This is followed by the recruitment of error-prone base
excision repair (BER) and/or mis-match repair (MMR) machineries that introduce mutations at or near the uracil
residue. The error-prone nature of BER and MMR in resolving uracil residues created by AID at the Ig loci is in
contrast to the high-fidelity repair these processes achieve while resolving uracil residues that arise in the
genome spontaneously or as a result of mis-incorporation during replication.
A study published by Dr. David Schatz’s lab has revealed two key features of SHM regulation in mice: (1.) In B
cells, AID does not target genes as specifically as previously believed. Many transcribed non-Ig genes are ‘hit’
by AID e.g., Bcl6, Myc, Cd83, Ocab, etc.; (2.) Most of the AID-generated mutations in these non-Ig genes are
resolved by high-fidelity repair, which protects them from accumulating mutations that could lead to genomic
instabilities and disease conditions like cancer. The aim of my project is to understand the molecular
mechanisms that regulate the gene-specific differential recruitment of high-fidelity and error-prone repair
pathways to AID-generated mutations. To achieve this goal, efforts are underway to develop a human cell
culture model that will allow the study of high-fidelity and error-prone repair pathways during SHM. Further,
experiments are being conducted to test a model that the cell-cycle stage at which different genes are
expressed or deaminated by AID determines the type of repair pathway that will be recruited. Details of the
experimental design being employed in this project and preliminary data will be discussed at the meeting.
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P126 - CTLA-4-Blockade Prevents Accumulation of Vaccine-Induced Tumor Infiltrating CD4 Foxp3 and
IL-10 Producing Regulatory T Cells
TYLER R. SIMPSON, Sergio A. Quezada, Karl S. Peggs, Jalena Vider, Ronald Blasberg, and James P. Allison
Ludwig Center for Cancer Immunotherapy, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New
York, NY
Blockade of the T cell coinhibitory receptor CTLA-4 has shown promise in the treatment of late stage metastatic
melanoma and other malignancies in mouse models of cancer as well in the clinic. Using the transplantable
B16-BL6 model of melanoma, we have previously shown that treatment with blocking -CTLA-4 monoclonal
antibodies in combination with a GM-CSF-secreting B16-BL6 tumor cell vaccine (GVAX) induces potent tumor
rejection associated with activation of the tumor vasculature and an increased intra-tumor effector
+
+
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CD8 /CD4 Foxp3 regulatory T cell ratio. Despite our general understanding of the mechanisms underpinning
CTLA-4 blockade in inducing anti-tumor T cell activity, the independent contribution of different immune cell
+
subsets and particularly the role of tumor reactive CD4 T cells remains unstudied. We studied the endogenous
+
systemic and tumor infiltrating CD4 T cell compartment as well as the function of tumor reactive TCR
+
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transgenic CD4 T cells (CD4 Trp1) specific to the melanoma differentiation antigen Trp1. We found that
+
depletion of CD4 T cells ablates GVAX+-CTLA-4 mediated tumor rejection, indicating that the CD4+
compartment is necessary for tumor protection. Surprisingly, whereas GVAX alone induced rapid expansion and
+
+
+
infiltration of CD4 Trp1 cells into tumor and vaccine sites, a great percentage of those cells were CD4 Foxp3
+
+
and capable of secreting IL-10. Remarkably, incorporation of -CTLA-4 prevented accumulation of CD4 Foxp3
T cells in the tumor and vaccine sites and reduced, on a per cell basis, the production of IL-10 whilst increasing
+
IFN- production. In combination with GVAX and -CTLA-4 treatment, the transferred CD4 Trp1 cells enhanced
+
protection against B16-BL6, suggesting that supplementing CD4 T cell help can enhance anti-tumor immunity.
+
Together these data demonstrate that tumor specific CD4 T cell responses play a necessary role and are
shaped by CTLA-4 blockade in cancer immunotherapy. Lastly, the data illustrate how cellular vaccinations
strategies can induce immune-regulatory pathways detrimental to the patient but that can be prevented through
CTLA-4 blockade.
P127 - Vaccination with Listeria-Mage-b and Curcumin is Highly Effective Against Metastatic Breast
Cancer
MANISHA SINGH, Ilyssa Ramos, Denise Asafu-Adjei, Wilber Quispe-Tintaya, Dinesh Chandra, Arthee
Jahangir, Bharat B Aggarwal, and Claudia Gravekamp
Albert Einstein College of Medicine, Bronx, NY
Controlling metastatic cancer, including breast cancer, is one of the biggest challenges in cancer therapy. It has
been shown in mice and humans that vaccines can have a favorable effect on metastases, but that vaccine
efficacy is strongly reduced by immune suppression in the tumor microenvironment. Interleukin (IL)-6 is one of
such immune suppressive cytokines that is highly produced by metastatic breast and other cancers. In a
previous study we found high levels of IL-6 produced by breast tumor cells of an aggressive metastatic breast
tumor model 4T1, expressing tumor-associated antigen (TAA) Mage-b. This IL-6 strongly reduced T cell
responses to Mage-b, but elimination of IL-6 using anti-IL-6 antibodies restored T cell responses to Mage-b in
vitro. Based on these results we hypothesized that reduction of IL-6 will improve vaccine efficacy against
metastatic breast cancer through improved T cell responses. To prove this hypothesis, we combined vaccination
of Listeria-Mage-b with Curcumin. Curcumin, an Indian spice, is known for reducing IL-6. One preventive
followed by two therapeutic immunizations completely eradicated the metastatic breast cancer in the 4T1 model,
which was not possible with Listeria-Mage-b or Curcumin alone. The complete eradication by Listeria-Mage-b
and Curcumin correlated with improved T cell responses to Mage-b compared to Listeria-Mage-b alone. Also
various therapeutic immunization strategies were tested. One therapy involved Listeria-Mage-b alternated with
Curcumin administrations after tumor development, which reduced the number of metastases by 80% and
growth of primary tumors by 78%, compared to the saline group. Since Curcumin is part of the food consumed
by many people all over the world, we tested Curcumin given before tumor development followed by ListeriaMage-b vaccinations after tumor development. This resulted in reduction of metastases by 96% and in tumor
growth by 56%. The results of this study strongly suggest that Curcumin improves vaccine efficacy of ListeriaMage-b through improved T cell responses to Mage-b. This is a highly promising approach and may be
applicable to other IL-6-producing metastatic cancers as well.
P128 - The Functional Role of Killer Cell Lectin-Like Receptor G1 (KLRG1) Expression by Foxp3+
Regulatory T Cells (Treg)
*KATE SMIGIEL and Dan Campbell
University of Washington and Benaroya Research Institutue, Seattle, WA
The T cell-mediated immune response relies on numerous cosignaling molecules to fine-tune the processes of
priming, expansion, contraction and tolerance. Similar to the co-inhibitory molecules PD-1 and BTLA, KLRG1
contains a cytoplasmic ITIM and has been shown to trigger inhibitory signaling pathways in CD8+ T cells and
NK cells. As KLRG1 is expressed by a substantial proportion of Foxp3+ Treg, I have hypothesized that this
expression functions to negatively regulate their activity. For CD8+ T cells, KLRG1 expression is associated with
terminally differentiated short-lived effector cells that have limited memory potential.
For Foxp3+ cells, I have found KLRG1 expression to also be associated with markers of activation and function,
including CD44, ICOS and CTLA-4. Furthermore, KLRG1 expression on Treg is associated with expression of
the peripheral tissue homing molecules CCR4, CCR6 and P-selectin ligand. As the physiological ligand for
KLRG1 is Epithelial (E)-cadherin, it was striking to discover that KLRG1-expressing Treg constitute between 40
and 70% of the total Treg in tissues such as the gut and skin, where levels of E-cadherin are highest.
To directly test the function of KLRG1 in the homing, activation and expansion of peripheral tissue Treg,
KLRG1-deficient mice were used to create mixed bone marrow chimeric animals. In these 50/50 WT/KO
chimeric mice, there is significant skewing of the Treg population towards KO-derived cells only in the peripheral
tissues (skin/gut) previously mentioned as having enriched frequencies of KLRG1-expressing Treg. This implies
that KLRG1 signaling, presumably via the E-cadherin present in these tissues, limits the competitive fitness of
wild-type Treg during homeostatic reconstitution. To further address the function of KLRG1 ligation in these
tissues, studies are underway to compare the ability of KLRG1-deficient Treg to control T cell-mediated colitis
and DNFB-mediated skin contact hypersensitivity, which are models of inflammation that have been shown to
be affected by E-cadherin levels or by the expression of molecules that compete with KLRG1 for E-cadherin
binding.
P129 - Regulation of Tr1 Differentiation and IL-10 Secretion by CD55 Costimulation of Naïve T Cells
IAN SPENDLOVE, Ruhcha Sutavani, and Lindy Durrant
The University of Nottingham, City Hospital, Nottingham, UK
The immune system exists as a balance of systems controlling pro and anti-inflammatory events. At the cellular
level there are Th1, Th2, Th17, amongst other pro-inflammatory cell types and nTreg, Th3 and Tr1 antiinflammatory cells. There is growing evidence that differentiation, from naïve to effector cells, by these
populations is governed by the activation status of the APC, cytokine milieu but also by a growing number of
costimulatory molecules. The influence of the classical costimulator (CD28) is modulated by cytokines leading to
Th1 and Th2 differentiation. Recently a number of papers have implicated the role of other costimulators in
moderating the effects of CD28 and promoting a ‘switch’ to a regulatory phenotype. We have previously shown
that engagement of CD55 by CD97, a 7-TM receptor, results in costimulation of human T cells in a similar way
+
to CD28. When purified naïve (CD45RA ) human Tcells were costimulated via CD55 or CD28 the result was
proliferation and survival of these populations. However, analysis of the cytokines produced showed a Th1
lo
phenotype for CD28 costimulated cells (IFN-, TNF-, IL-4 ) but a predominant, IL-10 producing, Tr1
Hi
lo
phenotype for the CD55 costimulated cells (IL-10 , IL-4 , IFN- ). Analysis showed only a small population of
cells producing IL-10 (0.5%-1.0% in 10 donors). Kinetic analysis showed that 24 hour stimulation of freshly
isolated naïve T cells with either PMA-ionomycin or CD55 costimulation failed to induce IL-10 production.
+
However at day three following CD55 costimulation IL-10 was detectible and the number of IL-10 cells had
increased at 5 days. In contrast IL-10 remained undetectable in CD28 costimulated cultures. Restimulation of
these cultures following 5 days rest demonstrated that CD55 costimulation had a predominant effect on IL-10
secretion, unlike CD28. Similarly MACS enriched, IL-10 secreting cells maintained the Tr1 phenotype but the IL10 depleted cells reverted to Th1. The Tr1 supernatants were able to inhibit naïve cell stimulation in an IL-10
dependent manner. This work highlights a novel costimulator (CD55) of naïve T cells that promotes the
differentiation and IL-10 production in a discrete population of Tr1 cells. Understanding the regulation of these
cells will help in reversing the anti-inflammatory imbalance in the tumor environment.
P130 - Efforts Toward Small Molecule Antibody-Recruiting Therapeutics for Cancer Treatment
1
1
1
1
1
DAVID A. SPIEGEL , Eugene F. Douglass , Kelly J. Fitzgerald , Charles E. Jakobsche , Patrick J. McEnaney ,
2
1
Ryan P. Murelli , and Andrew X. Zhang
1
2
Yale University, New Haven, CT; Brooklyn College, CUNY, Brooklyn, NY
Antibody-based therapeutics have become critical instruments in treating diseases ranging from rheumatoid
arthritis to cancer in recent years. However, antibodies and other therapeutic proteins are limited in therapeutic
applications by their chemical structures: because they are peptide-based, they require intravenous
administration, are often highly immunogenic or allergenic, and treatment regimens are often very costly.
Here we describe recent research efforts in our laboratories toward the design, chemical synthesis, and
biological characterization of small molecule antibody recruiting therapeutics against prostate cancer,
Staphylococcus aureus, and the human immunodeficiency virus (HIV). These are bifunctional small molecules
designed to redirect antibodies already present in the human bloodstream to the surfaces of pathogenic cells,
such as cancer cells, bacteria, and virus particles. The ternary complex formed between these agents,
endogenous antibodies, and target cells will lead to immune-mediated pathogen destruction. In theory, this
strategy would exploit many of the advantages of biologics, while circumventing the disadvantages, by
capitalizing on the chemical properties of small molecules (e.g., high oral bioavailability, facile synthesis, and
low cost).
It is our hope that this small molecule-based strategy will serve as starting point toward entirely novel
therapeutic approaches to a wide range of disease states.
P131 - Deciphering the Molecular Zipcodes Required for Effector CD8 T Cell Entry into Tumors at
Distinct Sites
NITHYA SRINIVASAN, Andrew R. Ferguson, Elizabeth D. Thompson, and Victor H. Engelhard
Carter Center for Immunology Research, University of Virginia, Charlottesville, VA
Immunization with dendritic cells (DC) via different routes targets distinct lymphoid organs (LO) and induces
CD8 T cell effectors that express different adhesion molecules and chemokine receptors. We aimed to decipher
which of these homing receptors are necessary for effector CD8 T cell infiltration into tumors at distinct sites.
Using IV DC immunization route, CD8 effectors activated in mediastinal LN and spleen were > 90% α4β1
+
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integrin , 60% CXCR3 , and 35-50% P- Selectin Ligand (PSL) . These cells infiltrated both subcutaneous (sc)
and intraperitoneal (ip) tumors. Interestingly, while PSL and E selectin ligand (ESL) are implicated in T cell
migration into inflamed skin, blocking P- or E-Selectin on sc tumor vasculature had no effect on infiltration.
However, sc tumor entry was dramatically inhibited by blocking 41 integrin. In support of a role for chemokine
receptors, CXCR3-/- transgenic T cells also showed reduced sc tumor entry. Tumor location influences homing
requirements of naïve CD8 T cells. For instance, blocking 41 integrin on naïve cells inhibits sc but not ip
tumor entry, correlating with significantly higher expression of its ligand (VCAM-1) on sc tumors. In contrast,
despite lower VCAM-1 expression, 41 integrin remained essential for ip tumor infiltration. This correlated with
a significantly greater number and per cell levels of 41 integrin on effectors compared to naïve cells. Similar
-/to sc tumors, infiltration into ip tumors did not depend on ESL and PSL but was impaired in CXCR3 T cells.
Therefore, in the absence of ESL and no apparent role for PSL, rolling and firm adhesion mediated by 41
-/integrin, coupled with chemokine signaling through CXCR3 are necessary for infiltration of IV primed effectors.
It is, therefore, important to decipher whether molecular requirements for tumor entry at specific sites vary for
distinct effector populations activated in other LOs such as skin or gut.
Funded by NIH grant R01 CA78400.
P132 - A Role for ATM in Antigen Receptor Allelic Exclusion
*NATALIE STEINEL and Craig Bassing
University of Pennsylvania, Philadelphia, PA
The majority of lymphocytes rearranges and expresses antigen receptors originating from a single allele, a
phenomenon known as allelic exclusion. Feedback inhibition, a major mechanism contributing to allelic
exclusion, involves the assembly and expression of an antigen receptor from one allele which suppresses
further V-to-(D)J rearrangements on the homologous allele. For feedback inhibition to successfully mediate
allelic exclusion asynchronous rearrangement of homologous alleles is required. Recently we showed that the
Ataxia Telangiectasia mutated (ATM) protein kinase prevents bi-allelic RAG cleavage at the Ig locus in G1
arrested abl-transformed pre-B cells. Since RAG DNA cleavage activates the ATM kinase, these data imply that
ATM may initiate signals which repress additional V(D)J recombination events and enforce asynchronous
rearrangement of homologous alleles. To investigate whether ATM enforces allelic exclusion in vivo, we
examined ATM mouse lymphocytes by FACS for Ig, IgH, or TCR allelic inclusion. Analysis of mice
heterozygous for an engineered allotypic difference at the Ig locus revealed increased Ig allelic inclusion in
ATM deficient B lymphocytes compared to wild type controls. Similarly, mice expressing allotypically marked IgH
alleles showed increased IgH allelic inclusion in the absence of ATM. Investigation of TCR V expression by
FACS revealed a slight increase in TCR allelic inclusion in ATM-deficient  T cells compared to wild type
controls. Consistent with this result, ATM  T cells showed both increased bi-allelic V-to-DJ
rearrangements, as well as increased usage of the 3’ D2J2 cluster compared to wild-type cells, suggesting
that ATM may inhibit secondary V-to-DJ rearrangements both in cis and in trans. Deletion of cyclin D3,
which drives G0 phase lymphocytes into the cell cycle, further increased TCR allelic inclusion in ATM mice,
suggesting that the ability of cyclin D3 to drive cells through G1 phase may cooperate with ATM signals to inhibit
bi-allelic V rearrangements. Additionally, ATM mice containing a pre-assembled DJ complex that reduces
TCR recombination events and increases V rearrangement frequencies also showed further increased TCR
allelic inclusion in ATM mice. Based on these data, we hypothesize that ATM promotes antigen receptor allelic
exclusion in lymphocytes by enforcing asynchronous V-to-(D)J recombination of homologous antigen receptor
allele to ensure efficient feedback inhibition. In this context, allelic exclusion may have arisen from pressure to
maintain genomic stability and suppress transforming lesions during V(D)J recombination.
P133 - A High-Throughput Engineering and In Vivo Selection System for Establishing Optimal Affinities
of T Cell Receptors for Anti-Cancer Responses
1
1
2
3
3
2
JENNIFER D. STONE , Adam Chervin , Carolina Soto , Boris Engels , Hans Schreiber , Ed Roy , and David M.
1
Kranz
1
2
3
University of Illinois at Urbana-Champaign, Urbana, IL; University of Illinois at Urbana-Champaign, Urbana, IL; The
University of Chicago, Chicago, IL
Adoptive T cell therapies have shown significant promise in the treatment of cancer. Further advantages have
been realized by the introduction of tumor antigen-specific T cell receptor (TCR) genes into T cells activated ex
vivo. This approach could, in principle, overcome central tolerance mechanisms that prevent efficient responses
by the endogenous T cell repertoire. To date, however, the rules for binding affinity that govern a TCR’s ability to
+
+
optimally recruit CD8 and CD4 T cells to a cancer are unknown. Investigation of these issues is complicated
by the need to test each TCR for efficacy on a one-by-one basis. To more rapidly query the impact of diverse
TCR binding properties, we developed a high-throughput platform for examining T cell targeting of cancer by
constructing libraries of structurally related TCRs with a wide range of affinities for a model tumor epitope. The
library was characterized via T cell display in vitro, and the variants with the best binding to the soluble target
+
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epitope were identified. Administration of CD8 or CD4 T cells transduced with this library mixture of TCRs
provided effective T cell therapy of cancer in a mouse model. High-throughput sequencing of individual TCR
transcripts present in mouse tumors and tissues allowed rapid determination of those TCR sequences which
were selected in vivo for optimal T cell infiltration into tumors and peripheral T cell persistence. Our results
provide insight into the relationship between TCR binding and adoptive T cell effectiveness against tumors, and
they can guide improvements to adoptive T cell strategies.
P134 - Overcoming Obstacles of T Cell Immunotherapy of Cancer: Testing Strategies to Improve T Cell
Function in Clinically Relevant Mouse Tumor Models by Targeting Negative Regulators
1
1
1
1
3
**INGUNN M. STROMNES , Carla Fowler , Christina M. Georgopolos , Xiaoxia Tan , Joseph N. Blattman ,
1,2
1,2
Sunil R. Hingorani , and Philip D. Greenberg
1
2
University of Washington, Seattle, WA; Fred Hutchinson Cancer Research Center, Seattle, WA
Adoptive immunotherapy is a promising treatment modality for patients with cancer. However, there are many
obstacles to success, such as the low avidity of self/tumor-reactive T cells and the failure of transferred T cells
to persist and function in an immunosuppressive tumor environment. We initially evaluated if abrogating the
negative regulator of lymphocyte function, Src homology region 2 domain-containing tyrosine phosphatase-1
(SHP-1), in tumor-reactive, effector T cells influenced efficacy of adoptive therapy in a mouse model of
disseminated leukemia. A conditional knockout of SHP-1 in mature, tumor-specific transgenic T cells (TCRgag)
was developed to analyze cell intrinsic effects of SHP-1 ablation on T cell therapeutic activity. Abrogation of
SHP-1 in in vitro-expanded TCRgag cells significantly enhanced the therapeutic efficacy of adoptive therapy.
-/SHP-1 effector T cells had improved cytolytic activity, enhanced functional avidity and significantly greater
short-term accumulation after transfer into mice with disseminated leukemia. Despite the enhanced short-term
accumulation, SHP-1 ablation did not impact the ability of donor T cells to form long-lived memory with
functional capacity. We are currently evaluating such strategies in a spontaneous mouse tumor model of
pancreatic ductal adenocarcinoma (PDA), which faithfully recapitulates the human disease. Two antigens are
being targeted in the PDA model: a candidate tumor antigen, Mesothelin, which is naturally overexpressed in
PDA and in our mouse model; and a model antigen expressed as a transgene in the tumor. We have cloned
Mesothelin-specific T cells with a range of avidities to determine the TCR affinity that has the best therapeutic
efficacy without toxicity. This model presents additional obstacles to leukemia, including adequate T cell access
to the tumor. We aim to determine how the properties of target antigen (tumor antigen with malignant properties
vs. a normal self-antigen), the tumor-reactive T cell (T cell receptor affinity, expression of negative regulators)
and the tumor microenvironment (vascularity of tumors, immunosuppressive cells) influence the therapeutic
outcome of adoptive immunotherapy. Strategies deemed promising in our mouse models will be adapted for
clinical trials by our group in patients with cancer.
P135 - Effects of Corticosteroids on Tumor Immunity Induced by CTLA-4 Blockade Depends on Dose
and Timing
1
1
2
2
1
3
DAISUKE SUGIYAMA , Yuka Maeda , Jianda Yuan , Takuma Kato , Shimon Sakaguchi , Hiroshi Shiku , Lloyd
2
2
1
J. Old , Jedd D. Wolchok and Hiroyoshi Nishikawa ,
1
2
Immunology Frontier Research Center, Osaka University, Osaka Japan; Ludwig Institute for Cancer Research New York
3
Branch, Memorial Sloan-Kettering Cancer Center, New York, NY; Mie University Graduate School of Medicine, Mie, Japan
+
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CTLA-4 blockade activates/augments CD4 and CD8 tumor-specific T cell responses and results in tumor
rejection. However, immune-related adverse events (irAEs), such as enterocolitis and dermatitis, occurring in
some patients having anti-CTLA-4 mAb therapy suggest that tissue-specific inflammation may accompany antitumor responses. As a consequence, patients may receive immunosuppressive drugs, such as corticosteroids
to control the adverse events. While the immunosuppressive effect of corticosteroids is well recognized, a
surprising observation from human clinical trials using CTLA-4 blocking antibodies is that the use of
corticosteroids or other immune suppressing medications to treat adverse events does not interfere with the
anti-tumor effect. Few details of the mechanism underlying this observation are known. Here, we addressed the
impact of corticosteroids on tumor immunity induced by anti-CTLA-4 mAb using mouse model. We inoculated a
murine fiblosarcoma cell line, CMS5a stably transfected with NY-ESO-1 (CMS5a-NY-ESO-1) into BALB/c mice,
and anti-CTLA-4 mAb was administered 3 days after tumor inoculation. Anti-CTLA-4 mAb treatment induced >
80% tumor regressions. When mice treated with anti-CTLA-4 mAb were injected with corticosteroids [high dose
(100 mg/kg) or low dose (1 mg/kg)] on the day of anti-CTLA-4 mAb treatment, tumor regression was inhibited by
+
+
either dose. In these mice, we also observed diminished NY-ESO-1-specific CD4 and CD8 T cell responses.
When corticosteroid injection was started 7 days after anti-CTLA-4 mAb treatment, tumor-recurrence was
observed with high dose corticosteroids (100 mg/kg), but not low dose (1 mg/kg). The low dose steroid is
allometrically scaled to represent the dose used in humans. Notably, when steroid treatment was delayed until
complete tumor regression (14 days after anti-CTLA-4 mAb treatment) was observed, tumor recurrence was not
observed in either high dose (100 mg/kg) or low dose (1 mg/kg) corticosteroid groups. We propose that dose
and timing are important considerations for the use of immunosuppressive drugs, such as corticosteroids, to
control irAEs induced by CTLA-4 blockade.
P136 - Genome-Wide Mapping of RAG1 and RAG2 Protein Binding
1
2
2
2
1
**GRACE TENG , Wolfgang Resch , Arito Yamane , Rafael Casellas , and David Schatz
1
2
Yale University School of Medicine, New Haven, CT; Genomic Integrity and Immunity, NIAMS, National Institutes of
Health, Bethesda, MD
V(D)J recombination assembles the antigen-binding domains of antigen receptor genes from arrays of V, D, and
J gene segments. The RAG1 and RAG2 proteins drive this process of somatic DNA rearrangement, catalyzing
double-stranded DNA breakage at recombination signal sequences (RSSs) in the antigen receptor loci. These
breaks are repaired in a manner that combines one V, one D, and one J segment into a functional exon. The
generation of these programmed DNA breaks, though central to lymphocyte development, has been linked to
several lymphoid malignancies. Thus, it is important to understand the mechanisms that target the RAG proteins
either to antigen receptor genes, or to ectopic sites elsewhere in the genome.
We previously showed that the RAG proteins localize to discrete “recombination centers” positioned over J gene
segments in the antigen receptor loci. We have now expanded our studies to include the genome-wide
localization patterns of RAG1 and RAG2 in the B- and T-lymphocyte lineages using ChIP-seq. These data
illustrate, in much higher resolution, that RAG1 and RAG2 accumulate in peaks that span the RSSs of gene
segments that are primed for, or have undergone, V(D)J recombination. We also find a surprising abundance of
RAG1 and RAG2 binding sites outside of the antigen receptor genes. The RAG proteins are bound to several
thousand other locations in the genome, largely in conjunction with peaks of tri-methylated histone H3 lysine 4
(a marker of transcriptional activation). We are currently analyzing the ChIP-seq data to understand: (1) the
common features of ectopic RAG binding sites (chromatin signatures, primary sequence motifs, etc.); (2) the
degree of cooperation and interdependence between RAG1 and RAG2 for binding to genomic targets; and (3)
the contribution of peripheral regulatory domains of the RAG proteins to their targeting.
P137 - Modulation of Notch1 and Notch3 Signaling Through the Negative Regulatory Regions
1
2
2
**KITTICHOAT TIYANONT , Thomas E. Wales , John R. Engen , and Stephen C. Blacklow
1
1
2
Dana Farber Cancer Institute - Harvard Medical School, Boston, MA; Northeastern University, Boston, MA
The Notch signaling pathway is a key regulator of normal development in the hematolymphoid system and adult
tissue homeostasis. Proper regulation of Notch activity is crucial, as mutations that trigger aberrant Notch signal
lead to developmental defects and to several diseases including cancer. In human T cell acute lymphoblastic
leukemia (T-ALL), for example, activating mutations are frequently found in the negative regulatory region
(NRR) of the Notch1 receptor. In the absence of ligand stimulation, the normal Notch remains silent. The T-ALLassociated mutations within the NRR bypass autoinhibition, allowing ligand-independent signaling. In its
autoinhibited state, the NRR consists of three LIN12/Notch repeats (LNR-A, LNR-B, and LNR-C) wrapping
around the globular heterodimerization domain (HD). Signals are initiated when ligand binding induces
proteolysis at a site called S2 within the HD domain. We used hydrogen exchange in conjunction with mass
spectrometry (HX-MS) to investigate the activation dynamics and the inhibition of the human Notch1 and Notch3
NRRs. To activate the NRRs, a calcium chelator EDTA was used for Notch1, and an agonist antibody A13 was
used for Notch3. In both cases, the results suggest that the NRR activation may take place by a sequential
peeling of LNR-A and LNR-B away from the HD domain, resulting in the exposure of the S2 cleavage site. The
NRR activation can be circumvented using an inhibitory antibody WC629 against Notch1, and an antibody A4
against Notch3. The results show that both Notch1 and Notch3 inhibitory antibodies bind the corresponding
NRRs at the LNR-A and HD domains, locking the NRRs in the autoinhibited state. These studies will help us
gain a better understanding of the activation step in Notch signaling and serve as a foundation for the
development and evaluation of mechanism-based modulators for the treatment of T-ALL and other diseases
associated with dysregulation in Notch signaling.
P138 - Variable Input Stimulation: Using Engineering Approaches to Interrogate Dynamics and
Feedback in T Cell Signaling
1
1,2
**JARED TOETTCHER , Orion Weiner , and Wendell Lim
1
2
1,3
3
University of California San Francisco, San Francisco, CA; Cardiovascular Research Institute, San Francisco, CA; Howard
Hughes Medical Institute, San Francisco, CA
Our project aims to use engineering approaches to address a central question in cancer immunology: how do T
cells sense and process activating signals that vary in space and time? A recently developed technology—the
Phy/PIF light-inducible protein-protein interaction—makes this line of investigation possible by providing a
precision instrument for delivering custom intracellular signaling inputs. To address this question we must (i)
develop experimental and computational systems to deliver precisely controlled light inputs to individual live
cells and (ii) ‘wire in’ the Phy/PIF optogenetic module to control activity at various T cell signaling nodes.
Using light as a quantitative intracellular input faces many challenges. Individual cells differ in expression of
optogenetic components, signaling pathways’ light-to-activity relationship can be complex and nonlinear, and
cells can activate feedback loops that change these activity relationships over time. To overcome these
challenges, we implemented a feedback control strategy to use real-time measurements of pathway activation
to tune input light levels on the fly. We have used this strategy to automatically identify light inputs leading to
user-defined time courses of Phy/PIF binding, such as linear or exponential ramps of activity. Our controller is
also able to compensate for cell-to-cell variability and drive a tight distribution of PIF levels across a population
of cells.
A central hypothesis of this project is that we can use light to recruit cytoplasmic regulators of T cell signaling to
the plasma membrane to specifically activate T cell signaling nodes. To control T cell signaling with light, we
have constructed a library of genetic constructs encoding fluorescent light-gated T cell signaling proteins. One
early success has been our development of light control over PI3K activity, an important signal in T cell
activation and immune cell migration. Spatially localized recruitment of a fluorescent, PIF-tagged subunit of PI3K
was capable of driving PIP3 accumulation in live cells. This activity is also compatible with our feedback
controller. We can quantitatively clamp PIP3 levels despite cellular changes in PI3K activity induced by inhibitors
or drugs. Finally, preliminary data indicates that light-gated PI3K activity on the cell boundary can induce
protrusion, demonstrating that PI3K activity is sufficient for polarization.
+
P139 - Post-Transcriptional Regulation of CD8 T Cell Response
1
1
2
**SARA TRIFARI , Matthew Pipkin , Hozefa Bandukwala , and Anjana Rao
1
1
2
La Jolla Institute for Allergy and Immunology, San Diego, CA; Immune Disease Institute, Harvard Medical School, Boston,
MA
+
Differentiation of naïve CD8 T cells into effector and memory cytotoxic T lymphocytes (CTLs) during bacterial or
viral infections is governed by multiple parameters, which include the frequency of antigen (Ag)-specific
precursors, Ag availability, IL-2 signals, and inflammation. Recently our group showed that strong IL-2 signals
+
are sufficient to promote differentiation of activated CD8 T cells into fully competent CTLs.
Much less is known about the post-transcriptional regulation of effector molecules in CTLs. Recently,
+
DICER−deficient CD8 T cells have been shown to have an altered pattern of survival/migration in vivo. We
have found that acute deletion of DICER in developing CTLs leads to increased expression of lytic molecules,
as well as to cytokine dysregulation. As DICER deletion substantially impairs miRNA processing, these data
+
suggest that one or more microRNAs may normally contribute to restrain effector molecule expression in CD8 T
cells, and conditions may exist, in vivo, in which this inhibition needs to be relieved. The observation that DICER
+
knock down in human CD8 T cells substantially recapitulates the phenotype of DICER−deficient mouse T cells
suggests that an miRNA−regulated post−transcriptional pathway of CTL differentiation may be conserved
between human and mouse. The role of individual miRNA in regulating the expression levels of lytic molecules
in CTLs is being investigated by a systematic overexpression approach. In parallel, a quantitative proteomic
approach will be implemented to investigate the impact of DICER deficiency on global protein levels in CTLs.
P140 - A Human-Derived Monoclonal Antibody Against NY-ESO-1 Improves the Efficacy of
Chemotherapy
1
1
2
2
2
Anurag Gupta , Natko Nuber , Christoph Esslinger , Mareike Wittenbrink , Martin Treder , Alexandro
1
3
3
3
1
3
Landshammer , Marcus Kelly , Sacha Gnatic , Erika Ritter , Lotta von Boehmer , Takuro Noguchi , Hiroyoshi
4
5
3
3
1
1
Nishikawa , Hiroshi Shiku , Lloyd Old , Gerd Ritter , Alexander Knuth , and MARIES VAN DEN BROEK
1
2
3
University Hospital Zurich, Zurich, Switzerland; CT-Atlantic, Schlieren Switzerland; Ludwig Institute for Cancer Research
4
New York Branch, Memorial Sloan-Kettering Cancer Center, New York, NY; Immunology Frontier Research Center, Osaka
5
University, Osaka, Japan; Mie University Graduate School of Medicine, Mie, Japan.
Purpose: We investigated whether antibodies against intracellular tumor-associated antigens support tumorspecific immunity when administered together with a treatment that destroys the tumor. We propose that
released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic
cells.
Experimental Design: We cloned the first human monoclonal antibody against NY-ESO-1, an immunogenic
intracellular cancer-testis antigen. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates
crosspresentation of an NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells and whether this
results in the maturation of dendritic cells in vitro. We investigated the clinical efficacy of 12D7 in combination
with 5-fluorouracil using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1.
Results: Human dendritic cells that were incubated with NY-ESO-1 : 12D7 immune complexes efficiently
stimulated NY-ESO-1157-165/HLA-A2 specific human CD8 T cells to produce interferon-, whereas NY-ESO-1
alone didn’t. Furthermore, the incubation of dendritic cells with NY-ESO-1 : 12D7 immune complexes resulted in
the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil
plus 12D7 was significantly more effective than treatment with 5-fluorouracil alone.
Conclusions: We propose antibody-facilitated T cell induction in cancer (AFTIC) as a novel concept in
immunotherapy. AFTIC is based on the application of monoclonal antibodies against tumor-associated antigens
plus a treatment that promotes the local release of those antigens resulting in immune complex formation, which
induces concomitant maturation of DCs, AFTIC supports boosting as well as de novo activation of tumor+
specific CD8 T cells at the relevant anatomic location.
P141 - Local Immunoregulation in Clear Cell Renal Cell Carcinoma
Stefanie Dannenmann, Julia Thielicke, Martina Stöckli, Giovanni Sais, Peter Schraml, Maurizio Provenzano,
Holger Moch, Alexander Knuth, and MARIES VAN DEN BROEK
University Hospital Zurich, Zurich, Switzerland
The prognosis of patients with clear cell renal cell carcinoma (ccRCC) is poor as 30% of newly discovered
tumors are already in a metastatic stage and non-surgical treatment options are still not satisfactory. RCCs are
thought to be immunogenic tumors as they are frequently infiltrated by immune cells and in a proportion of the
patients clinical responses were seen after immunostimulation with IFN- and IL-2. It is becoming increasingly
clear that the tumor microenvironment delivers suppressive signals to the immune system, which impair the
effector function of tumor-specific T cells and we therefore tried to identify an immunological signature within the
tumor with prognostic value. We performed retrospective analysis of paraffin biopsies from 60 ccRCC patients
with a known course of disease. Using immunohistochemistry and quantitative real-time PCR, we analyzed 37
different immunological parameters including cell subsets, surface markers and molecules. We found no
correlation between the number of infiltrating immune cells (CD45+) and tumor stage or patient survival. High
levels of FoxP3 and CD68 correlated significantly with decreased survival time, whereas high levels of iNOS
mRNA correlated significantly with increased survival time. Thus, the coexistence of high levels of FoxP3 and
CD68 in tumors seems to constitute an unfavourable immunological signature. FoxP3 is a transcription factor
expressed by regulatory T cells, and an association between increased infiltration of regulatory T cells and
reduced survival was already reported for different cancer types including breast, colon and pancreatic cancer.
+
CD68 is expressed by macrophages and there is evidence from preclinical models that CD68 macrophages on
the one hand promote metastasis and on the other hand are involved in local immunosuppression. The latter is
low
mainly mediated by iNOS M2 macrophages. We are currently performing experiments with fresh ccRCC
biopsies to further identify the role of macrophages in local immunosuppression.
P142 - -Irradiation Enhances Immunogenicity of Cancer Cells by Increasing the Expression of CancerTestis Antigens In vitro and In vivo
1*
1
3
1
5
1
Anu Sharma , Beata Bode , Roland H. Wenger , Kuno Lehmann , Alessandro A. Sartori , Holger Moch ,
1
1*
1*
Alexander Knuth , Lotta von Boehmer , and MARIES VAN DEN BROEK
1
2
University Hospital Zurich, Zurich, Switzerland; University of Zurich, Zurich, Switzerland; *These authors contributed
equally to this work
-Irradiation is an effective treatment modality for cancer. There is accumulating evidence that radiotherapy
supports tumor-specific immunity. It was described that irradiation induces de novo protein synthesis and
enhances antigen presentation, we therefore investigated whether -irradiation results in increased or de novo
expression of cancer-testis (CT) antigens and MHC class I (MHC-I), thus allowing efficient immunological
control. This is especially relevant because the expression of CT-antigens and MHC-I on tumor cells is often
heterogeneous.
We found that in vitro irradiation of cancer cell lines and of fresh tumor biopsies induced a higher or de novo
expression of different CT-antigens and a higher expression of MHC-I in a time- and dose-dependent fashion. In
+
vitro irradiated, but not unirradiated, cells were recognized by NY-ESO-1157-165/HLA-A2-specific CD8 T cells,
which underscores the increased susceptibility of tumor cells to immunological control upon irradiation. The
analysis of paired biopsies taken from a cohort of sarcoma patients before and after radiotherapy confirmed our
findings and, in addition showed that irradiation resulted in higher infiltration by lymphocytes. Other forms of
stress including hypoxia, hyperthermia, and genotoxic stress did not have an impact on the expression of CTantigens or MHC-I.
Our findings suggest that -radiation enhances the immunogenicity of tumors. We therefore propose that
combining radiotherapy with treatments that support tumor-specific immunity may result in increased therapeutic
efficacy.
P143 - Structure and Multimerization Properties of a T Cell Activity Modulating Signal Molecule B7H3
1
2
2,3
**VLADIMIR VIGDOROVICH , Udupi A. Ramagopal , Steven C. Almo , and Stanley G. Nathenson
1,4
Albert Einstein College of Medicine, Bronx, NY
Mammalian immune system is responsible for defending the host against externally introduced infectious
agents, as well as against internally arising malignancies. The ability to target and eradicate infected and
diseased cells is controlled through complex systems of molecular recognition and signaling events.
Regulation of T cell activity is a tightly controlled process involving many molecular signals. Some of the key
ligands in this system belong to the B7 family of proteins. B7H3 is a recently identified member of this family and
has been implicated in inhibition of T cell activity in peripheral tissues, acting through an unknown receptor.
Importantly, B7H3 expression has been repeatedly reported to be highly upregulated in various tumors, and this
condition was strongly correlated with poor clinical outcomes.
In order to contribute to the understanding of the molecular organization and function of B7H3, we determined
its atomic structure using X-ray crystallography. This structure identifies surface residues that may facilitate its
binding to the receptor on T cells. In addition, we discovered that B7H3 forms an unusual dimer in the crystal
and in solution, and used mutagenesis to identify residues that play a role in the dimer formation. Subsequent
studies using the mutants may reveal the role that dimerization plays in B7H3 function as well as the location of
its receptor-binding site.
P144 - Regulation of Immunosuppressive Adenosine 2a Receptor (A2aR) Signaling in CD8 T Cells
1
2
ISAAH VINCENT , Joel Linden , and Victor H. Engelhard
1
1
2
Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA; La Jolla Institute for Allergy and
Immunology, La Jolla, CA
Adenosine is generated in sites of tissue damage, hypoxia, and necrotic cell death, including tumors, and is
immunosupressive. Adenosine signaling via the A2aR on CD8 T cells diminishes their ability to control tumor.
Although in vitro treatment with an A2aR agonist decreased the number of naïve CD8 T cells that were activated
by anti-CD3, and the number of differentiated effector CD8 T cells that released IFN- and TNF-,
subpopulations remained that activated or produced cytokines at levels equivalent to untreated cells. The
activity of these subpopulations was blocked by treatment with forskolin, to active adenylate cyclase, or
rolipram, to block phosphodiesterase 4, suggesting that their insensitivity was due to inadequate signaling
through the A2aR. Sensitive and insensitive CD8 T effector subpopulations were apparent in B16 tumor bearing
mice, whereas all effectors in vaccinia-infected mice were insensitive. In vitro, IL-12 in combination with antiCD3 rendered all CD8 T effectors insensitive to an A2aR agonist. These IL-12 differentiated CD8 T effectors
were less sensitive to rolipram and forskolin, as well as the cAMP mimetic Sp-8-bromo-cAMPS. Thus, IL-12
treatment results in an additional manifestation of insensitivity downstream of cAMP. These results indicate that
the sensitivity of CD8 T cells to A2aR signaling is regulated at two levels: cAMP accumulation and sensitivity to
cAMP action. While the immunological context of vaccinia infection and IL-12 enhances insensitivity, CD8 T
cells that differentiate in a tumor-baring mouse remain sensitive. This work has uncovered a previously
unappreaciated complexity in the regulation of A2aR signaling. Enhancing the development of CD8 T cells that
are insensitive to immunosuppressive A2aR signaling is an attractive approach to making qualitative
improvements in the immune response to tumors.
Support: NIH R01 CA78400, R01 HL37942 and F31 CA130117
P145 - How Does Human CD4 Engage pMHC II and T Cell Receptor? A First Detailed Look at the Ternary
Complex by Directed Evolution and X-ray Crystallography
**XIN XIANG WANG, Yili Li, Yiyuan Yin, and Roy A. Mariuzza
University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD
The T cell receptor (TCR)CD3 complex is composed of a diverse TCR heterodimer associated with the
invariant CD3 dimers CD3, CD3, and CD3. The TCR mediates peptide-MHC(pMHC) recognition, while
the associated CD3 molecules transduce activation signals to the T cell. In addition to CD3, T cell activation
requires the co-receptors CD4 or CD8, which bind MHC class II or class I molecules, respectively. Whereas
much is known about downstream T cell signaling pathways, the mechanisms whereby TCR engagement by
peptide-MHC initiates signaling remain a mystery. Key to solving this problem is establishing the molecular
architecture of the TCR-MHC-CD4 and TCR-CD3 complexes. However, all previous attempts to crystallize
these multiprotein assemblies for X-ray crystallographic analysis have been frustrated by the low affinities of
MHC-CD4 and TCR-CD3 interactions. We have employed a powerful new approach, in vitro directed evolution
by yeast surface display (YSD), to increase the affinity of human CD4 for HLA-DR1. A CD4 mutant (Q40Y,
T45W) bound HLA-DR1 with KD = 8.8 M, compared to >400 M for wild-type CD4. This higher affinity CD4
variant has allowed us to determine the detailed structure of human CD4 in complex with HLA-DR1 to 2.4 Å
resolution. The structure revealed how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules
by targeting invariant residues in their 2 and 2 domains. More importantly, this human CD4 variant has
facilitated formation of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a
myelin-derived self-peptide bound to HLA-DR4, and CD4. Detailed structural analysis of this complex, in
conjunction with previous mutagenesis data, has enabled us to understand how the CD4 co-receptor engages
pMHC on TCR for T cell signaling, specifically how pMHC-TCR binding is coupled with T cell activation.
Similarly, the directed evolution approach is being taken to investigate the CD3-TCR interaction.
P146 - DPX-Survivac: An Enhanced Peptide Vaccine for Ovarian Cancer Targeting Survivin
1
3
1
1,2
1,2
1
Olga Hrytsenko , Neil Berinstein , Marianne Stanford , Mohan Karkada , GENEVIEVE WEIR , Rita Nigam ,
1
1
Lisa MacDonald , and Marc Mansour
1
2
Immunovaccine Inc., Halifax, Nova Scotia, Canada; Dept. Microbiology & Immunology, Dalhousie University, Halifax, Nova
3
Scotia, Canada; Sunnybrook Research Institute, Toronto, Ontario, Canada
Survivin is a tumor associated antigen that is overexpressed by many types of cancers and is essential to their
growth and survival. Survivin is unique in that it is a ‘crossroads signalling molecule’ and has been implicated in
the majority of the 10 hallmarks of cancer, making it an ideal target for immunotherapy. Survivin is a particularly
good target for ovarian cancer since it is overexpressed in more than 80% of all ovarian cancers, greater than
NY-ESO or WT-1. Analysis of 285 ovarian tumor samples from GEO DataSet GSE 9891 revealed that survivin
is equally overexpressed in cancer cells of ovarian, fallopian, and peritoneal origin. High levels of survivin
expression are strongly associated with malignant tumor type and with poor histological grade. Survivin
expression is increased in all ovarian cancer subtypes without apparent differences amongst them. The vaccine
contains a mixture of survivin MHC class I restricted peptide antigens that have been selected for their superior
immunogenicity across multiple HLA restrictions. The mixture includes modified peptide mimetics with enhanced
immunogenicity, as well as naturally strong antigenic sequences. The antigens were previously evaluated in a
phase I clinical trial in advanced solid tumors and were proven safe. We plan to enhance the immunogenicity of
TM
the antigens by formulating the peptides in DepoVax
(DPX), a novel liposome-in-oil adjuvanted depot
formulation that increases the potency of peptide-based cancer vaccines. Immunovaccine Inc. recently obtained
FDA approval for a phase I/II clinical trial of DPX-Survivac in ovarian cancer patients. The trial design
incorporates a strategy to limit immune suppression by T regulatory cells throughout the vaccination period. The
trial is expected to begin enrolment in the Fall of 2011.
P147 - Plexin-A4 Signaling Plays a Key Role in the Development of Intestinal Inflammation
**HAITAO WEN, Justin E. Wilson, and Jenny P-Y. Ting
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC
Plexins and semaphorins are ligand-receptors that serve as guidance molecules in the nervous system, and
their roles in immunity are emerging. Plexins are similar to the toll-like receptors (TLR) in their evolutionary
conservation from flies to mammals. Previously we showed a novel intersection of plexin-A4 with TLRs. Plexin-/A4-deficient (Plxna4 ) cells exhibit defective inflammatory cytokine response upon activation by a spectrum of
TLR agonists and bacteria. Mechanistically, plexin-A4 is required for activation of the small guanosine
triphosphate hydrolase (GTPase), ras-related Rac1, upon stimulation with TLR agonists. Rac1 activation is
accompanied by JNK and NFB activation culminating in TLR-induced binding of NFB and AP-1 to the
-/promoters of pan-cytokines. Physiologically, Plxna4 mice are resistant to TLR agonist-induced inflammation
and polymicrobial peritonitis caused by cecal ligation and puncture (CLP). More recently we have been studying
the role and mechanism of plexin-A4 in intestinal inflammation. We found that plexin-A4 exacerbates the
disease severity and colon inflammation in dextran sodium sulfate (DSS)-induced colitis and DSS-/azoxymethane (AOM)-induced colitis-associated cancer (CAC). Plxna4 mice exhibit less disease outcome
compared to WT controls in both colitis and CAC models, including less severity in body weight loss and
shortened colon length, and improved histological scores of tissue damage. Meanwhile, colon tissues from
-/Plxna4 mice contain lower amounts of inflammatory cytokines such as IL-1, IL-6 and TNF- than those from
WT mice. These findings expand the role of plexins to intestinal inflammation and suggest plexin-A4 as a new
intervention point for treating inflammation-related diseases such as colitis and CAC.
P148 - Sipuleucel-T Therapy Enhances T Cell Activation In Vivo
JOHNNA WESLEY, J. Trager, N. Perdue, L.Y. Kuan, F. Stewart, and N. Sheikh
Dendreon Corporation, Seattle, WA
Background: Sipuleucel-T is an autologous cellular immunotherapy approved for the treatment of
asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). Sipuleucel-T is
manufactured from peripheral blood mononuclear cells cultured ex vivo with PA2024, a recombinant fusion
protein of prostatic acid phosphatase linked to granulocyte-macrophage colony-stimulating factor. This abstract
details the activated T cell (T act) and regulatory T cell (T reg) profiles during the manufacture of sipuleucel-T from
the Phase 2 ProACT trial in men with mCRPC.
Materials and Methods: Sipuleucel-T treatment comprises 3 infusions, each prepared from a fresh
+
+
+
+
+
leukapheresis at 2-week intervals. Treg cells (CD4 CD25 Foxp3 CD127 ) and CD4 and CD8 Tact (Foxp3 ,
CD25, CD45RA, CD134 [OX40], CD137 [4-1BB], and CD278 [ICOS] expression) were assessed in pre-culture
(indicative of in vivo status) and post-culture (final product for infusion) using flow cytometry. Cytokines were
analyzed in the culture supernatants collected during manufacture using multiplex MesoScale Discovery
technology.
Results: The frequency of in vivo Treg cells was not altered during the course of treatment. The median percent
of Treg cells increased modestly post-culture at the second and third infusions compared to the first infusion, but
remained within the normal range of healthy individuals. Post-culture we observed an increase in the percent of
+
+
CD4 CD25 Foxp3 T cells with increases detected at the second and third infusions. Modulation of CD45RA
+
+
expression on CD4 and CD8 T cells pre- and post-culture was also consistent with T cell activation. An
+
+
+
+
+
+
increase in the percent of CD134 and CD137 CD4 and CD8 T cells post-culture and of CD278 CD4 T cells
was observed after the first infusion of sipuleucel-T. Activation-associated cytokines (e.g., IFNγ, IL-2, and TNFα)
predominated in culture supernatants, with a minimal level of the T reg-associated immunosuppressive cytokine,
IL-10, produced.
Conclusions: These data demonstrate that sipuleucel-T treatment results in an increase in T act cells without
inducing a similar increase in T reg cells over the course of therapy. Increases in T act cells at the second and third
infusions exhibit a prime-boost profile. The cytokine profile during manufacture indicates that T reg cell
functionality is not enhanced. Collectively, these data indicate that sipuleucel-T may alter the Tact/Treg ratio in a
direction favoring therapeutic efficacy.
P149 - Durable Protection from Intravaginal HIV-1 Transmission to Humanized Mice Using CD4 AptamersiRNA Chimeras as a New Topical Microbicide Candidate
1,2
3
2
2
2
3
3
3
3
*LEE A. WHEELER , V. Vrbanac , R. Trifonova , E Basar , X Zu , E Seung , M Deruaz , T Dudek , T Allen , A
3
3
2,4
2
Luster , AM Tager , D. Dykxhoorn , and J. Lieberman
1
2
Harvard Medical School, Boston, MA; Immune Disease Institute & Program in Cellular & Molecular Medicine, Children's
3
3
Hospital Boston, Boston, MA; University of Miami Medical School, Miami, FL; Massachussetts General Hospital, Boston,
4
MA; University of Miami School of Medicine, Miami, FL
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive
strategy is a topical vaginal microbicide. We previously showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs)
specifically suppress gene expression in CD4+ T cells and macrophages in vitro and in polarized cervicovaginal
tissue explants. CD4-AsiCs that knockdown HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue
explants. We hypothesized that CD4-AsiCs against HIV genes or the CCR5 co-receptor, when applied
intravaginally prior to infection, could effectively inhibit viral transmission to humanized mice.
We first demonstrated that topically applied CD4-AsiCs silenced target gene expression in the CD4+ cells
resident in the vaginal mucosa of humanized mice, without activating lymphocytes or stimulating innate
immunity. Importantly, the knockdown effect persisted for up to 15 days post-treatment. When applied up to a
week prior to viral challenge, CD4-AsiCs that knockdown essential host factors such as CCR5 effectively
reduced HIV transmission, whereas CD4-AsiCs targeting HIV genes such as gag and vif reduced transmission,
though somewhat less effectively, when applied either 2d before or 4h after viral challenge. Administering a
mixture of CD4-AsiCs targeting HIV genes and CCR5 was the most effective approach, fully blocking vaginal
HIV transmission to the mice.
P150 - Enhancement of CTL and Th1 Responses by Vaccination with “Smart” Nanocarriers for Antigens
and Oligonucleotide Adjuvants
**JOHN T. WILSON, Salka Keller, Anthony J. Convertine, Chen-Chang Lee, and Patrick S. Stayton
University of Washington, Seattle, WA
The clinical potential of cancer vaccines remains largely unrealized due to inadequate stimulation and
+
+
persistence of tumor antigen-specific CD8 cytotoxic T lymphocytes (CTL) and CD4 Type 1 helper (Th1) T
cells. We postulate that cancer vaccines that actively promote antigen cross-presentation and concurrently
augment the immunostimulatory potency of antigen presenting cells provide a strategy for synergistically
enhancing anti-tumor immunity. Toward this end, we have developed a new class of vaccine delivery vehicle
based on pH-responsive, “smart” polymers designed to promote cytosolic delivery of biologic cargo. Diblock
copolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and
comprised a pH-responsive, endosomolytic, terpolymer block containing dimethylaminoethyl methacrylate
(DMAEMA), propylacrylic acid and butyl methacrylate, and a polycationic DMAEMA-rich block doped with thiolreactive pyridyl disulfide groups to allow for electrostatic complexation of oligonucleotides as well as reversible
conjugation of thiol-bearing peptides and proteins. Polymer chains self-assembled into 20-40 nm micelles,
demonstrated potent pH-dependent membrane destabilizing activity, and facilitated simultaneous packaging of
oligonucleotides (siRNA and CpG ODN) and a thiolated model protein antigen, ovalbumin (Ova). Conjugation of
Ova to pH-responsive polymers significantly enhanced MHC class I presentation by a dendritic cell line (DC2.4)
in an in vitro co-culture assay, an effect inhibited by chloroquine and lactacystin, demonstrating a dependence
on both endosomal acidification and protoesomal processing in polymer-mediated cross-presentation.
Intradermal vaccination of C57BL/6 mice with polymer-Ova conjugates elicited a significantly higher Ova+
+
specific CD8 IFN response as measured by intracellular cytokine staining and ELISPOT compared to mice
vaccinated with free Ova or Ova delivered with CpG ODN 1826 (CpG) as an adjuvant. To demonstrate the
potential for dual delivery of antigen and an oligonucleotide adjuvant from a single carrier platform, mice were
immunized with polymer-Ova conjugates electrostatically complexed with CpG. Immunization with such dual+
+
+
+
delivery vehicles for Ova and CpG markedly enhanced both CD8 IFN and CD4 IFN (Th1) responses relative
to immunization with polymer-Ova conjugates or Ova administered with free CpG, suggesting a synergistic
response associated with delivery of antigen and adjuvant on pH-responsive nanocarriers. Collectively, these
investigations demonstrate the potential of “smart” polymers as a multimodal and tunable drug delivery platform
for improving the clinical efficacy of cancer vaccines through enhancement of CTL and Th1 responses.
P151 - The Ubiquitin-Like Protein, ISG15, is a Novel Tumor-Associated Antigen for Cancer
Immunotherapy
LAURENCE WOOD, Zhen-Kun Pan, Matthew Seavey, Geetha Muthukumaran, and Yvonne Paterson
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
The recent announcement of the first FDA approved therapeutic vaccine for prostate cancer, Provenge, is a
watershed moment for the field of tumor immunotherapy. However, while Provenge provides a powerful tool to
clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic
strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in
development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results
with HER2 vaccines appear promising in terms of efficacy, however, the lack of HER2 overexpression by a
majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy
suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified
ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein
regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast
cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen
receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis
and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these
challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with
immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions
in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated
antigen for cancer immunotherapy.
P152 - Study of Pallbearer, an E3-Ubiquitin Ligase that Regulates Phagocytosis of Apoptotic Cells in
Drosophila
**HUI XIAO and Nathalie C. Franc
The Scripps Research Institute, La Jolla, CA
The swift removal of cells that are dying via genetically programmed cell death (apoptosis) by specialized cells,
such as macrophages, is a critical event in the shaping of tissues during the development of all multicellular
organisms, from worm to man. Defects in the removal of dying cells in a process known as phagocytosis or
clearance of apoptotic cells can contribute to the development of inflammatory autoimmune disorders, such as
systemic lupus erythematosus and neurodegenerative diseases, such as Alzheimer’s. Despite tremendous
research efforts in the past decades, little is known still about the molecular mechanisms controlling the
clearance of apoptotic cells, largely owing to the redundancy and complexity of this process in mammalian
systems. We have established the fruit fly, Drosophila, as a suitable genetic model system in which to study
phagocytosis. In a quest to genetically dissect the molecular mechanisms of apoptotic cell clearance, we have
identified pallbearer (pall), a gene encoding a novel F-Box protein. F-box proteins form multi-molecular
Skp/Cullin/F-Box (SCF) complexes that act as ubiquitin ligases, thereby targeting other modified proteins to
degradation via a pathway known as the ubiquitin-proteasome pathway. Identifying its substrates is important to
further our understanding of how the ubiquitin-proteasome promotes phagocytosis. Mutant macrophages for pall
are small in size and poorly phagocytic. We have identified the ribosomal protein RpS6 as a putative substrate
for PALL by Co-IP and tandem Mass Spectrometry. RpS6 plays a role in immune cells, as RpS6 mutants have
melanotic tumors, the sign of an aberrant immune response, as well as enlarged macrophages, the opposite
phenotype of pall mutants. We also found RpS6 as a gene that enhances AC clearance in a genome-wide RNAi
screen in S2 cells and confirmed this finding by using new RpS6-specific siRNAs. We are assessing the role of
RpS6 in phagocytosis and testing its genetic interaction with pall. In addition, by immunostaining with HA
antibody and transfection of PALL:GFP fusion protein in S2 cells, we have found a regulation of expression and
subcellular localization of PALL in response to apoptotic cells. We are studying the subcellular localization of
PALL, and its relevance for the function of PALL in AC clearance.
P153 - Mechanism of Immune Regulation by Zap-70/Syk Tyrosine Kinase Family
**QINGRONG YAN and John Kuriyan
University of California, Berkeley, Berkeley, CA
The T cell receptor (TCR) signaling cascade, which is crucial for functional immune responses, is tightly
regulated by protein tyrosine kinase ZAP-70. A regulatory segment that includes a tandem SH2 domain controls
the catalytic activity of ZAP-70. A recent crystal structure of full length ZAP-70 reveals a “linker-kinase
sandwich” intra-molecular interface formed by the tandem SH2 domain and kinase domain is important to
stabilize the inhibitory architecture of ZAP-70. Here we utilized an in vitro FRET based approach to measure the
catalytic activity of ZAP-70 on its physiological substrate LAT. Full length ZAP-70 exhibited a very low catalytic
activity towards LAT. Release of the inhibitory architecture of ZAP-70 by a doubly phosphorylated-ITAM peptide
or introduction of a point mutation slightly increased ZAP-70 kinase activity. However, these increased activities
were not comparable with the catalytic activity of ZAP-70 kinase domain alone. Notably, even the presence of a
linker peptide alone at the N-terminus of ZAP-70 kinase domain, which connects the tandem SH2 domain and
kinase domain, inhibited ZAP-70 kinase activity. Phosphorylation of ZAP-70 by Lck or Src fully restored the
inhibited ZAP-70 activity. Taken together, these results suggest that an intermediate inhibited state of ZAP-70 is
present after ZAP-70 is recruited to the TCR receptor complexes that contain the phosphorylated ITAM motifs,
then ZAP-70 will be further activated by Src family tyrosine kinases.
P154 - Non-Competitive Inhibitors of MALT1 Identified by High Throughput Screening
1
2
1
2
**CHENGHUA YANG , Lorena Fontan Gabas , Chao Zheng , Ari M. Melnick , and Hao Wu
1
1
2
Department of Biochemistry, Department of Medicine, Weill Medical College of Cornell University, New York, NY
Diffuse large B cell lymphoma (DLBCL) is the most common type of adult lymphoma in humans. Among
DLBCLs, the activated B cell (ABC) subtype is the most aggressive and clinically challenging. ABC-DLBCLs are
generally characterized by constitutive NF-κB signaling through T/B cell antigen-receptor signaling pathways.
Emerging data have implicated that CARMA1/BCL10/MALT1 complex has a pivotal role in canonical and noncanonical NF-κB activation. MALT1 is a paracaspase enzyme that functions at least in part by cleaving and
inactivating the negative NF-B regulator A20. It has been demonstrated that constitutive proteolytic activity of
MALT1 plays a critical role in ABC-DLBCL cell survival, and inhibition of MALT1 enzymatic activity by a peptide
inhibitor is lethal to ABC-DLBCL cells, suggesting MALT1 could be a bona fide therapeutic target for ABCDLBCLs. Here we performed a small molecule high throughput screening for MALT1 inhibitors. 324 hits were
identified among ~50,000 compounds in the primary screen. A total of 27 compounds with IC50 at micromolar
range were picked for in vivo studies. Two lead compounds, which inhibit MALT1 activity non-competitively,
showed the blockage of A20 cleavage and significant suppression effect on ABC-DLBCL cell growth. Three
analogue compounds were identified in further analogue compound screening that showed better ABC-DLBCL
cell growth inhibition effect. The mechanism of how the inhibitors block the proteolytic activity of MALT1 will be
determined by X-ray crystallography. The efficacy of the top leads in ABC-DLBCL in vivo will also be tested in
mice.
+
P155 - Investigating the Role of Asymmetric Cell Division in Fate Determination of CD8 T Cells
*MOHAMMED YASSIN, Edwin Hawkins, Jane Oliaro, Richard Tothill, Mandy Ludford-Menting, Raz Shimoni,
Sarah Russell, and Patrick Humbert
Peter MacCallum Cancer Centre, Melbourne, Australia
Our lab and others have recently shown the importance of asymmetric cell division (ACD) in hematopoietic cell
function and fate, and preliminary data suggests that it may even impact anti-tumor immunity. The process of
fate determination in hematopoietic cells has been studied at the population level for decades. Here we propose
a single cell approach to study this process and understand the molecular players in setting up asymmetry and
determining subsequent fate. To achieve this, we have developed a time-lapse microscopy system to study the
+
proliferation and differentiation of naïve CD8 T cells at a single cell level. We are also in the process of
+
performing single cell RNAseq analysis on the daughters of naïve CD8 T cells in order to elucidate the
differential expression and segregation of mRNA and microRNA in the daughter cells. Our project will provide
+
the first systematic analysis of molecular differences between the progeny of a naïve CD8 T cell and elucidate
a possible role for ACD in the process of fate determination in hematopoietic cell.
®
P156 - RNActive Vaccines Provide Strong Anti-Tumor Effect, Especially in Combination with antiCTLA4 Therapy or Radiation
1
1
1
1
2
KAI ZANZINGER , Regina Heidenreich , Mariola Fotin-Mleczek , Christina Lorenz , Stephan Huber , and Karl1
Josef Kallen
1
2
CureVac GmbH, Tübingen, Germany; University Hospital Tübingen, Tübingen, Germany
Based on modified and protamine-formulated messenger RNA, two-component RNActive vaccines combine two
principle activities: high antigen expression and immune stimulation mediated by TLR7. That they do not need
any additional adjuvant to be active and can be administered via single intradermal injection supports their
unique potency. RNActive vaccines induce innate as well as balanced and long-lasting adaptive immune
response and provide strong anti-tumor effect. To increase the therapeutic effect, potentially allowing for the
control of large established tumors, we combined RNActive vaccines with two different standard therapies.
E.G7-OVA bearing mice were treated therapeutically with either OVA- RNActive alone or in combination with
anti-CTLA4 antibody. Combination therapy resulted in a strong, synergistic anti-tumor effect, yielding a higher
frequency of mice with complete tumor rejection. Remarkably, the complete responders were protected against
rechallenge with parental ovalbumin-negative EL-4 tumors, indicating an antigen spreading in this group.
Interestingly, following combination therapy, tumors which escaped the control of the immune system exhibited
strongly reduced ovalbumin expression. Next we tested whether combination of RNActive vaccines with
radiotherapy can inhibit the growth of established, low immunogenic and radiation-insensitive, Lewis lung
carcinoma (LLC) tumors. Immunotherapy alone, targeting two tumor/self-antigens, was not effective in inducing
tumor protection. High dose radiation of the tumors induced only transient growth stagnation. However,
combined radioimmunotherapy dramatically improved anti-tumor efficacy and supported surveillance of large
tumors in treated mice. To understand the cellular and mechanistic base for the synergistic effect, we conducted
a comprehensive flow cytometric analysis of LLC tumor tissues one week after treatment began. Although at
this time point the tumor volume was still comparable in all mice, we observed dramatic changes in the cellular
composition of the tumor tissue. Tumors from mice receiving radiation and combination therapy were highly
infiltrated by immune cells; the frequency of cells associated with adaptive immunity was particularly increased
in mice treated with combination therapy. These findings highlight that combination of RNActive vaccines with
standard cancer therapies, such as antibody therapy or radiation, creates highly synergistic anti-tumor effects,
which may have the potential to improve long-term survival in cancer patients.
P157 - Barriers to Oncolytic Myxoma Virus Therapy in Syngeneic Orthografts of Glioma: Independent of
Adaptive Immunity, Interferon and Microglia… What’s Left?
1
1
1
2
FRANZ J. ZEMP , Brienne McKenzie , Xueqing Lun , Grant McFadden , Peter Forsyth
1
2
1,3
3
University of Calgary, Calgary, Alberta; University of Florida, Gainsville, Florida; Moffitt Cancer Center, Tampa, Florida
Initial trials using oncolytic virotherapy (OV) for malignant gliomas (MGs) have demonstrated their safety, yet
failed to show significant clinical responses. Myxoma virus (MYXV) has produced tremendous results in mouse
orthotopic xenografts, in some instances ‘curing’ animals with a single intracranial injection. However, when
translating these studies into syngeneic orthografts in rodent glioma models, we fail to see any treatment
efficacy with MYXV alone. Our studies utilize primary glioma lines derived from a spontaneous murine glioma
+/+/model (NF1 TP53 mice). These grafts accurately recapitulate the genetic and histologic appearance of
human MGs in C57Bl/6 mice. Several of these lines display exquisite susceptibility to MYXV replication and
oncolysis in vitro, yet fail to show any replication or efficacy in vivo, with complete viral clearance occurring
within ~5 days post-treatment. Rapid viral elimination suggests involvement of CNS innate immune responses,
-/and this was confirmed using immunodeficient RAG1 mice. We have further demonstrated that resistance is
independent of Type-I Interferon. Flow cytometry data demonstrate that these tumors contain resident microglial
and macrophage populations. Ablation or inactivation of microglia using CD11b::DTR mice or minocyclin found
that we can cause an initial increase in viral replication, but overall rate of viral elimination and survival remains
unchanged with these treatments. Taken collectively, these results suggest that there remains an anti-viral
component either already present or immediately produced/recruited in the tumor that mediates viral clearance.
Assessment of the tumor microenvironment using multiplex ELISA has identified several candidate molecules
that could potentially be involved in the anti-viral state of the tumor, and current studies determining their roles
are underway. Further, studies directed at ablating tumor-resident and recruited macrophages using chlondrate
-/-/lipososmes and CCR2 /CXCR3 deficient mice will determine whether these cell types are involved. These
studies will allow us to understand the barriers to OV therapy for MGs, and allow us to direct the genetic and/or
pharmacological manipulation of the tumor microenvironment to maximize this therapy’s potential.
P158 - The Role of the Diacylglycerol Pathway in Regulatory T Cell Differentiation
*TAO ZOU
Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
Regulatory T cells, (Treg)s, are crucial for the maintenance of immune homeostasis and self-tolerance. A
population of natural Tregs develops in the thymus along with other T cell subsets. The current model of Treg
lineage commitment includes roles for cytokines and co-stimulatory molecules while offering a dominant role for
the T cell receptor (TCR) in this cell fate decision. However, the specific signaling pathways downstream of the
TCR that orchestrate Treg differentiation have not been clearly defined.
Two major second messengers that are generated upon TCR engagement are diacylglycerol (DAG) and
2+
intracellular calcium (Ca ). Each of these second messengers activates multiple signaling pathways that
together lead to the activation of the T cell. Cellular levels of DAG are regulated positively by TCR signals and
negatively by DAG kinases (DGK), which metabolizes DAG to phosphatidic acid. Using mice deficient in one
isoform of DGK, DGK zeta, we examine the role of the DAG pathway in Treg differentiation. Our preliminary
data implicate DGK zeta as a regulator of this cell fate decision, although the precise mechanisms that mediate
this process remain undefined.
P159 - Expression of Soluble TGF-β Receptor II by Recombinant Vaccinia Virus Enhances E7 Specific
Immunotherapy of HPV16 Tumors
KAMILA ZURKOVA, K. Babiarova, L. Kutinova, J. Krystofova, P. Hainz, and S. Nemeckova
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
Transforming growth factor  (TGF-) is pleiotropic cytokine with regulatory roles in many processes. It
regulates functions of T and NK cells, dendritic cells and macrophages. TGF- produced by tumor cells
downregulates immune responses to tumor antigens and allows the tumor to evade immune surveillance. High
risk human papilloma viruses (HPV) have been identified as etiological agents of anogenital tract carcinomas
and other tumors. One of the mechanisms involved in their tumorogenicity is based on the ability of viral early
E7 protein to upregulate TGF- expression in transformed cells.
In our experimental model, we tried to eradicate tumors induced by s.c. inoculation of TC-1 cells expressing
HPV16 E6/E7 antigens onto mice back. We enhanced therapeutic effect of rVACV strain Praha 13 (P13)
expressing HPV16-E7 tumor antigen in a highly immunogenic form SigE7LAMP with co-expression of soluble
TGF- receptor II (sTRII). We used extracellular sTRII domain or chimeric sTRII fused to IgG-Fc portion
tagged with Jun fragment (sTRII-Fc-Jun) which should have higher biological activity due to easier
dimerization.
Soluble receptors were expressed either under control of early H5 promoter or synthetic E/L promoter which is a
potent inducer of strong late phase expression. Both types of sTRII produced in rVACVs infected cells were
secreted in properly glycosylated form and partially remained associated with cells as underglycosylated
precursor. Higher sTRII/sTRII-Fc-Jun levels were present in cytoplasm of cells infected by P13-H5-sTRII or
P13-H5-sTRII-Fc-Jun at 20 h.p.i. However, the amount of soluble receptors released into medium of all
rVACVs infected cells was equal in later intervals.
Therapeutic effect of immunization was examined in animals carrying TC-1 tumors. Regression of tumors
occurred only in animals simultaneously immunized with SigE7LAMP and treated with soluble TRII mainly with
P13-H5-sTRII-SigE7LAMP recombinant, whereas none of the chimeric sTRII-Fc-Jun expressing viruses
increased therapeutic potential of rVACV. The co-expression of either sTRII or sTRII-Fc-Jun did not
+
significantly enhance the response of HPV16-E7 specific CD8 T cells. Moreover, reduction of tumor growth by
P13-H5-sTRII-SigE7LAMP was not caused even by changed multiplication of the virus.
This work was supported by grants P304/10/1511, NS 10660-3/2009 of the Grant Agency of CR and MZ0UHKT2005 of the
Ministry of Health of CR.
Sanket Acharya
Harvard University
617-800-4755
acharya@fas.harvard.edu
James Arnold
University of Cambridge
+44 00 1223 336814
jna24@cam.ac.uk
Diego Acosta-Alvear
University of California, San Francisco
415-476-5676
Diego.Acosta-alvear@ucsf.edu
Patrick Baeuerle
Micromet, Inc.
+49-89-895277601
patrick.baeuerle@micromet-inc.com
Sepideh Afshar
Eli Lilly & Co.
858-638-8500
s.afshar@lilly.com
Sanjay Bagaria
Mayo Clinic
904-953-2523
bagaria.sanjay@mayo.edu
James Akin
Harvard University
617-575-9071
jakin@fas.harvard.edu
Malina Bakowski
University of California, San Diego
858-246-0882
mbakowski@ucsd.edu
Steven Albelda
Perelman School of Medicine, University of
Pennsylvania
215-573-9933
albelda@mail.med.upenn.edu
Yael Barach
Albert Einstein College of Medicine
718-430-4154
yael.barach@phd.einstein.yu.edu
Diana Alvarez Arias
Dana-Farber Cancer Institute
617-632-4618
Diana_AlvarezArias@dfci.harvard.edu
Rupesh Amin
Fred Hutchinson Cancer Research Center
510-579-4859
ramin@fhcrc.org
Ana Anderson
Brigham and Women’s Hospital and Harvard
Medical School
617-525-5850
aanderson@rics.bwh.harvard.edu
Dan-Oscar Antson
Ludwig Institute for Cancer Research
212-450-1546
dantson@licr.org
Nicole Aqui
University of Pennsylvania School of Medicine
215-746-1195
aqui@mail.med.upenn.edu
Charlotte Ariyan
Memorial Sloan-Kettering Cancer Center
2126396280
ariyanc@mskcc.org
Myron Arlen
Neogenix Oncology
516 4821200
MArlenMD@msn.com
Michael Becker
MD Becker Partners LLC
267-756-7094
michael@mdbpartners.com
Peter Beemiller
University of California, San Francisco
415-514-3250
peter.beemiller@ucsf.edu
Marcelo Behar
University of California, San Diego
760-815-3045
mbehar@ucsd.edu
Danielle Bello
Memorial Sloan-Kettering Cancer Center
2126396280
bellod@mskcc.org
Nicole Belmar
Abbott Biotherapeutics
650-454-2041
nicole.belmar@abbott.com
Pavel Belousov
Moscow State University
+74959391445
belous_p@rambler.ru
Alexey Berezhnoy
University of Miami
3052431045
aberezhnoy@med.miami.edu
Tessa Bergsbaken
University of Washington School of Medicine
206-685-3613
bergst@u.washington.edu
Sadna Budhu
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
budhus@mskcc.org
Stephane Bertolini
Ludwig Institute for Cancer Research
2154501587
sbertolini@licr.org
Jonathan Buhrman
University of Colorado-Denver, National Jewish
Health
303-398-1320
jonathan.buhrman@ucdenver.edu
Nina Bhardwaj
New York University Medical Center
212-263-5814
nina.bhardwaj@nyumc.org
Adam Blaisdell
New York University School of Medicine
2122630406
adam.blaisdell@nyumc.org
Julie Magarian Blander
Mount Sinai School of Medicine
(212)659-9407
julie.blander@mssm.edu
Eric Borges
Boehringer Ingelheim RCV GmbH & Co KG
+43180105
sylvia.schoen@boehringer-ingelheim.com
Walter Braga
UNIFESP
55 11 55764240
wmoises@uol.com.br
Kenneth Brasel
Dendreon Corporation
206-219-7831
kbrasel@dendreon.com
Erica Braverman
Howard Hughes Medical Institute and National
Institutes of Health
516-695-9313
bravermane@hhmi.org
Matthew Burchill
National Jewish Medical and Research Center
303-398-1332
burchillm@njc.org
Elena Burova
Regeneron Pharmaceuticals
914-345-7675
elena.burova@regeneron.com
Kate Byrne
Dartmouth College
603-653-3688
katelyn.t.byrne@dartmouth.edu
Margaret Callahan
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
callaham@mskcc.org
Garth Cameron
The University of Melbourne
0411281003
g.cameron6@pgrad.unimelb.edu.au
John Campbell
Militenyi Biotech Ltd.
+447739819897
johnc@miltenyibiotec.co.uk
Valerie Campello-Iddison
Bristol-Myers Squibb
609-252-4641
valerie.campelloiddison@bms.com
Javier Briones
Hospital Santa Creu I Sant Pau
34935677887
jbriones@santpau.cat
Paola Cappello
Universita degli Studi di Torino
+39 0116335737
paola.cappello@unito.it
Vincenzo Bronte
Verona University
00-39-049-8215897
vincenzo.bronte@univr.it
Kyla Carroll
ImClone Systems, Inc.
646-638-5008
kyla.carroll@imclone.com
Matthew Buckwalter
Institut Pasteur
3144389502
mbuckwal@pasteur.fr
Amy-Jo Casbon
University of California, San Francisco
476-4758
amyjo.casbon@ucsf.edu
Michael Cavnar
Memorial Sloan-Kettering Cancer Center
646-888-3210
cavnarm@mskcc.org
Yongwon Choi
University of Pennsylvania School of Medicine
215-746-6404
ychoi3@mail.med.upenn.edu
Jonathan Cebon
Ludwig Institute for Cancer Research,
Melbourne
+614 19376794
jonathan.cebon@ludwig.edu.au
Jeffrey Chou
Fred Hutchinson Cancer Research Center
206-667-7456
jchou2@uw.edu
Christina Ceccato
Johns Hopkins University College of Medicine
410-502-9778
cceccat1@jhmi.edu
Michela Cesco Gaspere
ICGEB, Trieste
+39.040.3757310
cesco@icgeb.org
Caroline Chapman
University of Nottingham
44 115 8231825
caroline.chapman@nottingham.ac.uk
Ashutosh Chaudhry
Memorial Sloan-Kettering Cancer Center
646-888-3160
chaudha1@mskcc.org
Julie Chaumeil
New York University School of Medicine
1 212 263 0503
julie.chaumeil@nyumc.org
Melvyn Chow
Peter MacCallum Cancer Centre
+61396561238
melvyn.chow@petermac.org
Ramon Chua
Ludwig Institute for Cancer Research, NYB
646-888-2340
chuar@mskcc.org
Gisele Colleoni
Federal University of São Paulo
55-11-5539-6151
gcolleoni@unifesp.br
Anthony Cooper
The Scripps Research Institute
858-784-9545
abcooper@scripps.edu
Emily Corse
Memorial Sloan-Kettering Cancer Center
6468882350
corsee@mskcc.org
Nabil Chehab
ImClone Systems, Inc.
646-747-3705
nabil.chehab@imclone.com
George Coukos
Ovarian Cancer Research Center, University of
Pennsylvania School of Medicine
215-662-3316
gcks@mail.med.upenn.edu
Emily Chen
University of Pennsylvania School of Medicine
61394963700
weisan.chen@ludwig.edu.au
Courtney Crane
University of California, San Francisco
415-672-5614
courtney.crane@ucsf.edu
Weisan Chen
Ludwig Institute for Cancer Research,
Melbourne
215-628-4061
emilyjc@mail.med.upenn.edu
Michael Curran
Memorial Sloan-Kettering Cancer Center
6468882348
curranm@mskcc.org
Rita Chiu
Ludwig Institute for Cancer Research, NYB
(646) 888-2200
chiur@mskcc.org
Helen Cho
Pfizer Inc.
858 526 4748
helen.k.cho@pfizer.com
Janet Dally
MD Becker Partners LLC
267-756-7094
janet@mdbpartners.com
Tao Dao
Memorial Sloan-Kettering Cancer Center
646-888-2206
t-dao@ski.mskcc.org
Matthew Daugherty
Fred Hutchinson Cancer Research Center
206-667-4512
mdaugher@fhcrc.org
Jacqueline Doody
ImClone Systems, Inc.
6466385131
jacqueline.doody@imclone.com
Thomas Davis
Celldex Therapeutics, Inc.
908-454-7120
scalon@celldextherapeutics.com
Stephanie Dougan
Whitehead Institute for Biomedical Research
(617)780-9935
dougan@wi.mit.edu
Carissa Dege
University of Colorado - Anschutz Medical
Campus, National Jewish Health
303-398-1315
carissa.dege@ucdenver.edu
Peter Dowling
VA Medical Center, East Orange, NJ
973-676-1000
dowlinpc@umdnj.edu
Amy Degnim
Mayo Clinic
507-284-4499
degnim.amy@mayo.edu
Ronald DeMatteo
Memorial Sloan-Kettering Cancer Center
212.639.3976
dematter@mskcc.org
Dobrin Draganov
Harvard Medical School
617-935-6213
draganov@fas.harvard.edu
Charles Drake
Johns Hopkins University School of Medicine
(410) 502-7523
cdrake@jhmi.edu
Weiwen Deng
University of California, Berkeley
510-642-9522
wwdeng@berkeley.edu
Erika Duan
Ludwig Institute for Cancer Research,
Melbourne
61 08 99030714
Erika.duan@ludwig.edu.au
C. Marcela Diaz
University of Miami
3052435771
cdiaz1@med.miami.edu
Purnima Dubey
Wake Forest School of Medicine
336-716-7078
pdubey@wakehealth.edu
Catherine Diefenbach
New York University Medical Center
212 731-5670
catherine.diefenbach@nyumc.org
Lindy Durrant
University of Nottingham
01158231863
lindy.durrant@nottingham.ac.uk
Mustafa Diken
TRON, University of Mainz
+49 6131 178165
mustafa.diken@tron-mainz.de
Valerie Dutoit
University of Geneva
+41223729882
valerie.dutoit@hcuge.ch
Wendy Diller
The Pink Sheet
212-633-3182
w.diller@elsevier.com
Ivan Dzhagalov
University of California, Berkeley
510-643-6957
ild@berkeley.edu
Alena Donda
University of Lausanne
++41 216925857
alena.donda@unil.ch
Shingo Eikawa
Okayama University
+81-86-235-7192
opera_house0908@yahoo.co.jp
Zhiwan Dong
Memorial Sloan-Kettering Cancer Center
6468883157
dongz@mskcc.org
Knut Elbers
Boehringer-Ingelheim Venture Fund
+49-6132-77-0
knut.elbers@boehringer-ingelheim.com
Eran Elinav
Yale University School of Medicine
203-737-2216
eelinav@gmail.com
Bettina Franz
Dana-Farber Cancer Institute
617-632-3931
bettina_franz@dfci.harvard.edu
Alex Engel
University of California, Berkeley
510-642-2043
alexcengel@gmail.com
Zvi Fridlender
Hadassah - Hebrew University
972-2-6777794
fridlender@hadassah.org.il
Victor Engelhard
University of Virginia Health System
434-924-2423
vhe@virginia.edu
Denise Frosina
Ludwig Institute for Cancer Research, NYB
(646) 888-2200
frosinad@mskcc.org
Alan Epstein
University of Southern California, Keck School of
Medicine
323-442-1172
aepstein@usc.edu
Tihui Fu
MD Anderson Cancer Center
713-745-2369
futihui@yahoo.com
Zelig Eshhar
Weizmann Institute of Science
+972 89343965
zelig.eshhar@weizmann.ac.il
Jitka Fucikova
Univerzita Karlova v Praze (Charles University in
Prague)
+420733568377
jitka.fucikova@lfmotol.cuni.cz
Xiaozhou Fan
Memorial Sloan-Kettering Cancer Center
646-888-2349
fanx@mskcc.org
Etienne Gagnon
Université de Montréal
514-343-6702
etienne.gagnon@umontreal.ca
Yongqiang Feng
Memorial Sloan-Kettering Cancer Center
646-888-3168
fengy2@mskcc.org
Humilidad Gallardo
Memorial Sloan-Kettering Cancer Center
646-888-2585
h-gallardo@ski.mskcc.org
Luis Ferran
Ludwig Institute for Cancer Research, NYB
(646) 888-2200
ferranl@mskcc.org
Hormas Ghadially
MedImmune Ltd.
0044 1223898194
ghadiallyh@medimmune.com
Courtney Fleenor
University of Colorado, Anschutz Medical
Campus
303-724-3233
courtney.fleenor@ucdenver.edu
Sacha Gnjatic
Ludwig Institute for Cancer Research, Memorial
Sloan-Kettering Cancer Center
(646) 888-2339
gnjatics@mskcc.org
Peter Forsyth
H. Lee Moffitt Cancer Center
813-745-3063
lacetta.cline@moffitt.org
Wayne Godfrey
BN ImmunoTherapeutics Inc.
6506814653
wayne.godfrey@bn-it.com
Jamie Fox
University of Pennsylvania
215-272-4569
jlynne@mail.med.upenn.edu
Gisela Gonzalez
Center of Molecular Immunology
+53-7-217645
gisela@cim.sld.cu
Ruth Franklin
Weill Cornell Graduate School
(646) 888-2761
raj2006@med.cornell.edu
Girija Goyal
Harvard University
617-953-2360
girija_goyal@dfci.harvard.edu
Claudia Gravekamp
Albert Einstein College of Medicine
718-430-4048
claudia.gravekamp@einstein.yu.edu
Chunguang Guo
Immune Disease Institute, Inc.
617-713-8550
cguo@idi.harvard.edu
Nicole Graves
Dalhousie University
902 494-5132
kngraves@dal.ca
Uzi Hadad
Ben-Gurion University of the Negev
972-8-6477276
uzihad@bgu.ac.il
Philip Greenberg
Fred Hutchinson Cancer Research Center
206-543-8306
pgreen@u.washington.edu
Yared Hailemichael
University of Texas MD Anderson Cancer
Center
713-792-1931
yhailemi@mdanderson.org
Jonathan Greer
Memorial Sloan-Kettering Cancer Center
646-888-3210
greerj@mskcc.org
Howard Grey
La Jolla Institute for Allergy and Immunology
858 752-6568
hgrey@liai.org
Veronika Groh
Thomas Spies, Fred Hutchinson Cancer
Research Center
206-667-6940
vgroh@fhcrc.org
William Haining
Dana-Farber Cancer Institute
(617) 632-5293
nicholas_haining@dfci.harvard.edu
Melisa Hamilton
BC Cancer Research Centre
604-675-8000
mhamilto@bccrc.ca
Jin-Hwan Han
The Rockefeller University
212-327-7323
jhan@rockefeller.edu
Gideon Gross
Tel-Hai College, MIGAL
972-4-8181701
gidi@migal.org.il
Simon Harrison
Peter MacCallum Cancer Insititute
+613 96561701
simon.harrison@petermac.org
Jeffrey Grotzke
Yale University School of Medicine
203-737-2452
jeffrey.grotzke@yale.edu
Jonathan Head
The Elliott-Elliott-Head Breast Cancer Research
& Treatment Center
225-755-3070
jhead@eehbreastca.com
Aparna Gudlur
La Jolla Institute for Allergy and Immunology
858-952-7178
aparna@liai.org
Jennifer Guerriero
Dana-Farber Cancer Institute
617-582-9759
Jennifer_Guerriero@dfci.harvard.edu
Jose Guevara
Loyola University
708 327 3155
jaguevara@lumc.edu
Xuemei Guo
Imclone Systems
646-895-3848
xuemei.guo@imclone.com
Kevin Heller
Bristol-Myers Squibb
2125238735
cmudiyanselage@chpnet.org
Chandana Herath
St. Luke Roosevelt Hospital Center
609-252-6727
kevin.heller@bms.com
Jonathan Hill
Tempero Pharmaceuticals
617-374-8830
jonathan.a.hill@gsk.com
Atsushi Hinohara
Kyowa Hakko Kirin Co., Ltd.
81-55-989-3603
atsushi.hinohara@kyowa-kirin.co.jp
Daniel Hirschhorn-Cymerman
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
hirschhd@mskcc.org
Hiroaki Ito
Benaroya Research Institute at Virginia Mason
206-583-6525
hito@benaroyaresearch.org
Tsvetelina Hoang
Memorial Sloan-Kettering Cancer Center
646-888-2350
hoangt@mskcc.org
Tyler Jacks
Massachusetts Institute of Technology
(617) 253-8511
tjacks@mit.edu
F. Stephen Hodi
Dana-Farber Cancer Institute
617-632-5053
stephen_hodi@dfci.harvard.edu
Kristen Jacobsen
University of Colorado
303-724-0680
kristen.jacobsen@ucdenver.edu
Rikke Holmgaard
Memorial Sloan-Kettering Cancer Center
+1-646-888-2349
HolmgaaR@mskcc.org
Stephen Jameson
University of Minnesota, Minneapolis
612-625-1496
james024@umn.edu
Megan Holz
Ludwig Institute for Cancer Research, NYB
(646) 888-2200
holzm@mskcc.org
Robert Jenq
Memorial Sloan-Kettering Cancer Center
6468882317
Jenqr@mskcc.org
Axel Hoos
Bristol-Myers Squibb
203-677-5640
axel.hoos@bms.com
Kirk Jensen
Massachusetts Institute of Technology
617-324-5133
kirkj@mit.edu
Karoline Hosiawa
Regeneron Pharmaceuticals
914-345-7823
karoline.hosiawa@regeneron.com
Hyungjun Jeon
Albert Einstein College of Medicine
9172855462
neokaze77@gmail.com
Sakie Hosoya-Ohmura
University of Michigan Medical School
734-615-7248
hsakie@umich.edu
Carl June
Abramson Cancer Center, University of
Pennsylvania
(215) 573-5745
cjune@mail.med.upenn.edu
Alex Huang
Case Western Reserve University
617-726-8156
yhuang@steele.mgh.harvard.edu
Wendy Jia Men Huang
New York University Medical Center
2163681271
alex.y.huang@case.edu
Yuhui Huang
Massachusetts General Hospital
212-263-7520
wendyjiamenhuang@gmail.com
Ella Ioffe
Regeneron Pharmaceuticals
914-345-7955
ella.ioffe@regeneron.com
Achim Jungbluth
Ludwig Institute for Cancer Research, NYB
646-888-2340
jungblua@mskcc.org
Richard Junghans
Roger Williams Medical Center
401-456-2507
rjunghans@rwmc.org
Markus Junker
University of Wuerzburg
0049-931-201-25807
markusjunker@gmx.net
Caroline Junqueira
Federal University of Minas Gerais - FIOCRUZ
55 31 34092634
carolinejunqueira@cpqrr.fiocruz.br
Lothar Kaiser
Dendrimun GmbH
+496021920605
dr.kaiser@dendrimun.com
Prashant Kodgire
University of Chicago
773-702-4500
pkodgire@bsd.uchicago.edu
Xiaoqiang Kang
ImClone Systems
646-638-5009
xiaoqiang.kang@imclone.com
Nikita Kolhatkar
University of Washington
206-543-1010
nkolhat@uw.edu
Andreas Katopodis
Novartis
+41 79 3674550
andreas.katopodis@novartis.com
Richard S. Kornbluth
Multimeric Biotherapeutics, Inc.
619-846-8603
rkornbluth@multimericbio.com
Steven Katz
Roger Williams Cancer Center
401-456-2484
skatz@chartercare.org
Magdalena Kovacsovics
Providence Portland Medical Center
503 2153190
Magdalena.kovacsovics@providence.org
Ross Kedl
University of Colorado
303-270-2061
ross.kedl@ucdenver.edu
Linda Kozick
Bristol-Myers Squibb
609-252-5188
linda.kozick@bms.com
Bridget Keenan
Johns Hopkins School of Medicine
410-614-1772
bkeenan3@jhmi.edu
Katharina Kreymborg
Memorial Sloan-Kettering Cancer Center
646-888-2349
kreymbok@mskcc.org
Andrew Kent
Mount Sinai School of Medicine
(212)659-9348
andrew.kent@mssm.edu
Ada Kruisbeek
DC Prime BV
+3120 59 85601
buller@tto.vu.nl
Khashayarsha Khazaie
Robert H. Lurie Comprehensive Cancer Center,
Northwestern University
312-503-1901
khazaie@northwestern.edu
Hao Yuan Kueh
California Institute of Technology
626-395-4915
kueh@caltech.edu
Kien Khuu-Duong
Dendreon Corporation
206.829.1534
kkhuu-duong@dendreon.com
Teresa Kim
Memorial Sloan-Kettering Cancer Center
646-888-3210
kimt1@mskcc.org
Shigehisa Kitano
Memorial Sloan-Kettering Cancer Center
646-888-2343
kitanos@mskcc.org
Alexander Knuth
University Hospital Zurich
011 41 44 255 9779
alexander.knuth@usz.ch
Sudha Kumari
New York University Medical Center
212-263-3208
sudha.kumari@nyumc.org
Sabrina Kuttruff
Immatics Biotechnologies GmbH
+49 7071 5397
thiercy@immatics.com
Samuel Lam
Washington University School of Medicine
3148149763
sklam@go.wustl.edu
Christoph Lampert
Roche Glycart AG
0041447556169
christoph.lampert@roche.com
Quy Le
University of Washington School of Medicine
206-435-4859
quyl@u.washington.edu
Ronald Levy
Stanford University School of Medicine
650-725-6423
levy@stanford.edu
Melissa Lechner
University of Southern California, Keck School of
Medicine
323-442-1172
lechner@usc.edu
Philippe Lewalle
Université Libre de Bruxelles (ULB)
32 2 541 37 27
philippe.lewalle@bordet.be
Hee Eun Lee
Seoul National University Hospital
82-2-2072-0342
heleemd@gmail.com
Jung-Hoon Lee
Weill Medical College of Cornell University
212-746-6458
jul2020@med.cornell.edu
Sang Yull Lee
Pusan National University
82-51-510-8084
sangyull@pusan.ac.kr
Wan-Ling Lee
Washington University School of Medicine
314-362-2004
wlee@pathology.wustl.edu
Yu-Jung Lee
University of Minnesota, Minneapolis
415-502-6638
youjeong77@gmail.com
Nikoletta Lendvai
Memorial Sloan-Kettering Cancer Center
2126393368
lendvain@mskcc.org
Ralf Leonhardt
Yale University School of Medicine
203-785-5042
Ralf.Leonhardt@yale.edu
Alex Lesokhin
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
lesokhia@mskcc.org
Ross Levine
Memorial Sloan-Kettering Cancer Center
646-888-2796
leviner@mskcc.org
Hyam Levitsky
Johns Hopkins University School of Medicine
410-614-0552
hy@jhmi.edu
Xintong Li
VA Medical Center, East Orange, NJ
973-676-1000
xint.li@gmail.com
Yanxia Li
ImClone Systems, Inc.
6468952767
yanxia.li@imclone.com
Weiping Li
VA Medical Center, East Orange, NJ
973-676-1000
wpli54@hotmail.com
Wei Liang
City of Hope Cancer Center
6263215419
wliang@coh.org
Lauren Licata
Roger Williams Medical Center
914 450 4587
lauren.licata@CharterCARE.org
Mathias Lichtenheld
University of Miami
3052435771
mlichten@med.miami.edu
Cailian Liu
Memorial Sloan-Kettering Cancer Center
6468882323
liuc3@mskcc.org
Albert Lo
University of Pennsylvania
215-898-3904
loalbert@mail.med.upenn.edu
Hans Loibner
APEIRON Biologics AG
+4318656577
hans.loibner@apeiron-biologics.com
Julio Lorenzi
São Paulo University
1632897895
julioclorenzi@gmail.com
Angelica Loskog
Uppsala University
+46186119181
angelica.loskog@igp.uu.se
Susanna Mandruzzato
University of Padova
+390498215898
susanna.mandruzzato@unipd.it
Israel Lowy
Regeneron Pharmaceuticals
914.785.3278
israel.lowy@regeneron.com
Sara Mangsbo
Uppsala University
+46186119183
sara.mangsbo@igp.uu.se
Haihui Lu
Whitehead Institute for Biomedical Research
973-676-1000
ludeer113@gmail.com
Alberto Mantovani
Istituto Clinico Humanitas IRCCS, University of
Milan
011-39-2-8224244
alberto.mantovani@humanitas.it
Wei Lu
VA Medical Center, East Orange, NJ
626-475-6160
hlu@wi.mit.edu
Immanuel Luescher
Ludwig Institute for Cancer Research, University
of Lausanne
+41 21 692 5988
immanuel.luescher@unil.ch
Teresa Manzo
San Raffaele Scientific Institute
+39-02-26434800
manzo.teresa@hsr.it
Patricia Marinello
Bristol-Myers Squibb
203-677-5246
patricia.marinello@bms.com
Jacqueline Lypowy
ImClone Systems, Inc.
917 606 4109
jacqueline.lypowy@imclone.com
Rebecca Mathew
University of Illinois at Chicago
773-702-9196
rmathew@uchicago.edu
Jean-Pierre Mach
Universite de Lausanne
0041216925733
Jean-Pierre.Mach@unil.ch
Hirokazu Matsushita
The University of Tokyo Hospital
+81-3-5805-3161
hmatsu924@m.u-tokyo.ac.jp
Yasuhiro Maeda
VA Medical Center, East Orange, NJ
81-6-6879-4963
yuka-m@ifrec.osaka-u.ac.jp
Junko Matsuzaki
Roswell Park Cancer Institute
716-845-4374
junko.matsuzaki@roswellpark.org
Yuka Maeda
Osaka University, Immunology Frontier
Research Center
973-676-1000
Yasuhiro.Maeda@va.gov
Viveka Mayya
New York University Medical Center
212-263-3208
viveka.mayya@med.nyu.edu
Tak Mak
Campbell Family Institute for Breast Cancer
Research, Princess Margaret Hospital,
University of Toronto
+1-416-946-4501
tmak@uhnresearch.ca
Nicole Malandro
Memorial Sloan-Kettering Cancer Center
646-888-2322
malandrn@mskcc.org
Brienne McKenzie
University of Calgary
403 826-8321
mckenzba@ucalgary.ca
Thomas Meagher
Dendreon
2062746781
cmeagher@dendreon.com
Lydie Meheus
Reliable Cancer Therapies
3222684816
lydie.meheus@reliablecancertherapies.com
Mickael Menager
New York University Medical Center
646-469-5465
mickael.menager@med.nyu.edu
Alexander Muller
Lankenau Institute for Medical Research
484-476-8034
mullera@mlhs.org
Feilong Meng
Children's Hospital Boston
617-919-2608
meng@idi.harvard.edu
Judith Murphy
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
murphyj1@mskcc.org
Taha Merghoub
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
merghout@mskcc.org
Anne Murray
Ludwig Institute for Cancer Research, NYB
(646) 888-2200
murraya1@mskcc.org
Nicole Messina
Peter MacCallum Cancer Insititute
+61396561238
nicole.messina@petermac.org
Cristina Musselli
Agenus Inc
7816744711
dimitra.dariotis-diaz@agenusbio.com
Jean Michaud
GlaxoSmithKline Biologicals
450-978-4564
jean.z.michaud@gskbio.com
Takeshi Nagai
Tokyo Medical University
+81-3-3342-6111
t-nagai@tokyo-med.ac.jp
Carsten Mim
Northwestern University
847.491.3741
c-mim@northwestern.edu
Yasuhiro Nagata
National Nagasaki Medical Center
81-957-52-3121
nagatay1961@mac.com
Matthew Moake
Johns Hopkins University School of Medicine
410-614-3606
moake@jhmi.edu
Masayasu Naito
Dana-Farber Cancer Institute
617-632-3351
Masayasu_Naito@DFCI.HARVARD.EDU
Alberto Montero
University of Miami
3052435771
amontero2@med.miami.edu
Johanna Napetschnig
Weill Medical College of Cornell University
646-346-3515
jon2011@med.cornell.edu
Ngozi Monu
New York University Medical Center
212-263-9277
Ngozi.Monu@nyumc.org
Carl Nathan
Weill Medical College of Cornell University
212-746-6505
cnathan@med.cornell.edu
Duane Moogk
NYU School of Medicine
2122639276
duane.moogk@nyumc.org
Blaise Ndjamen
California Institute of Technology
626-395-8351
ndjamen@caltech.edu
Adele Mount
CSL Limited
(+613) 93892409
adele.mount@csl.com.au
Shin Foong Ngiow
Peter MacCallum Cancer Insititute
+61396561238
shinfoong.ngiow@petermac.org
Sulakshana Mukherjee
University of California, San Diego
1-858-822-0470
spmukherjee@ucsd.edu
Christopher Nirschl
Johns Hopkins University School of Medicine
410-502-9778
cnirsch1@jhmi.edu
Hiroyoshi Nishikawa
Osaka University
81-6-6879-4963
nisihiro@ifrec.osaka-u.ac.jp
Youmna Othman
Case Western Reserve University
2168446063
Youmna.Othman@uhhospitals.org
Paola Nistico
Regina Elena Cancer Institute
+390652662539
nistico@ifo.it
Willem Overwijk
The University of Texas M.D. Anderson Cancer
Center
(713) 792-2921
woverwijk@mdanderson.org
Daniel Nixon
Western Regional Medical Center
843 469 1190
dnixonun@aol.com
Takuro Noguchi
Ludwig Institute for Cancer Research, NYB
646-888-2343
takuro_noguchi@hotmail.com
Francesco Novelli
University of Turin
0116334463
franco.novelli@unito.it
Marianna Nuti
Universita de Roma 'La Sapienza'
+39 06 49973029
marianna.nuti@uniroma1.it
Kunle Odunsi
Roswell Park Cancer Institute
716-845-8376
kunle.odunsi@roswellpark.org
Javier Ogembo
Beth Israel Deaconess Medical Center
617-735-2522
jogembo@bidmc.harvard.edu
Yoshihiro Ohue
Kawasaki Medical School
+81-86-462-1111
yoshipoohsan@hotmail.com
Mikio Oka
Kawasaki Medical School
+81-86-432-1111
moom@med.kawasaki-m.ac.jp
Karyn O'Neil
Centyrex/J&J
610-240-8126
koneil@its.jnj.com
Lisa Osborne
University of Pennsylvania School of Medicine
215-898-6268
losborne@mail.med.upenn.edu
Dipa Patel
ImClone Systems, Inc.
2244756546
neela.patel@abbott.com
Neela Patel
Abbott Laboratories
646-638-5136
dipa@nycmail.com
Oliver Pearce
University of California, San Diego
858-405-7533
opearce@ucsd.edu
Virginia Pedicord
Memorial Sloan-Kettering Cancer Center
646 8882350
vap2003@med.cornell.edu
David Peng
Canaccord Genuity
2128493923
dpeng@canaccordgenuity.com
Karlo Perica
Johns Hopkins University School of Medicine
801-598-0369
karlo@jhmi.edu
Robert Petit
Advaxis, Inc.
609-452-9813
petit@advaxis.com
Martina Petrácková
Institute of Hematology and Blood Transfusion
+420221977391
martina.petrackova@uhkt.cz
Agne Petrosiute
Case Western Reserve University
2168443345
agne.petrosiute@uhhospitals.org
Kristen Picha
Johnson & Johnson
6106516286
KPicha@its.jnj.com
Jeffery Pollard
Center for the Study of Reproductive Biology
and Women’s Health, Albert Einstein College of
Medicine
718-430-2090
jeffrey.pollard@einstein.yu.edu
Michael Postow
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
postowm@mskcc.com
Gerd Ritter
Ludwig Institute for Cancer Research, NYB
646-888-2339
rittere@mskcc.org
Bruce Robinson
University of Western Australia, Sir Charles
Gairdner Hospital
61-8-9346-2098
bwsrobin@cyllene.uwa.edu.au
Read Pukkila-Worley
Massachusetts General Hospital
617-726-3812
rpukkilaworley@partners.org
Pedro Romero
Ludwig Institute for Cancer Research, University
of Lausanne
41 21 314 01 98
pedro.romero@unil.ch
Ellen Puré
Wistar Institute
215-898-1570
pure@wistar.org
Ronald Rooke
Transgene SA
33.3.88.27.92.31
rooke@transgene.fr
Sonia Quaratino
Merck KGaA
49 6151 72
sonia.quaratino@merck.de
Steven Rosenberg
National Cancer Institute
301-496-4164
steven_rosenberg@NIH.gov
Allen Radin
Regeneron Pharmaceuticals
9143457361
allen.radin@regeneron.com
Redouane Rouas
Institut Jules Bordet, Université Libre de
Bruxelles
003225413727
mrouas@ulb.ac.be
Alexander Rakhmilevich
University of Wisconsin-Madison
608-263-5193
rakhmil@humonc.wisc.edu
Jeffrey Ravetch
The Rockefeller University
212-327-7321
ravetch@rockefeller.edu
Andrea Reboldi
University of California, San Francisco
+41 91 820 0300
andrea.reboldi@ucsf.edu
Tiffany Reddin
Celgene Cellular Therapeutics
732-564-3510
treddin@celgene.com
Xiaodi Ren
Imclone Systems, Inc.
9178435223
xiaodi.ren@imclone.com
Erika Ritter
Ludwig Institute for Cancer Research, NYB
646-888-2340
ritterg@mskcc.org
Marcus Ruscetti
University of California, Los Angeles
240-344-0817
mar8e@ucla.edu
Rachel Sabado
New York University Medical Center
2122638046
rachel.sabado@nyumc.org
Fabiana Saccheri
European Institute of Oncology
0039-0294375091
fabiana.saccheri@ifom-ieo-campus.it
Duygu Sag
La Jolla Institute for Allergy and Immunology
8582287588
dsag@liai.org
Ashley Saint Fleur
Johns Hopkins University School of Medicine
4106143606
saint@jhmi.edu
Andres Salazar
Oncovir, Inc.
202-3421726
asalazar@starpower.net
Luara Santos
Universidade Federal de Minas Gerais
55 31 34092634
luaraisabela@gmail.com
Eiichi Sato
Tokyo Medical University
81-3-3342-6111
sato-e@tokyo-med.ac.jp
David Schaer
Memorial Sloan-Kettering Cancer Center
(646) 888-2323
schaerd@mskcc.org
Andrea Schietinger
University of Washington School of Medicine
206-910-0462
aschieti@u.washington.edu
Christine Sedrak
Ludwig Institute for Cancer Research, NYB
646-888-2339
sedrakc@mskcc.org
MacLean Sellars
New York University Medical Center
(212) 263-6921
MacLean.Sellars@med.nyu.edu
Alejandro Sepulveda
Memorial Sloan-Kettering Cancer Center and
Howard Hughes Medical Institute
6468882348
sepulvem@mskcc.org
Inês Pires da Silva
New York University Langone Medical Center Cancer Institute
404-727-9301
akampho@emory.edu
Tyler Simpson
Memorial Sloan-Kettering Cancer Center
6468882348
simpsont@mskcc.org
Manisha Singh
Albert Einstein College of Medicine
718-430-4067
manisharaj@gmail.com
Gaurav Singh
Memorial Sloan-Kettering Cancer Center
646-888-2348
singhg@mskcc.org
Jaafar Sleiman Haidar
Imclone Systems/ Eli Lilly
646 358 5087
Jaafar.Haidar@imclone.com
Craig Slingluff
University of Virginia Health System
434-924-1730
cls8h@virginia.edu
Mark Sliwkowski
Genentech, Inc.
650-225-1000
sliwkowski.mark@gene.com
Rashu Seth
Yale University School of Medicine
203-785-3247
rashu.seth@yale.edu
Kate Smigiel
University of Washington School of Medicine
206-543-1010
kssmigiel@gmail.com
Padmanee Sharma
University of Texas M.D. Anderson Cancer
Center
713-792-2830
padsharma@mdanderson.org
Mark Smyth
Peter MacCallum Cancer Insititute
61-3-9656-3728
mark.smyth@petermac.org
Seung-Uon Shin
University of Miami
305-243-3668
sshin@med.miami.edu
Andrew Sikora
Mount Sinai School of Medicine
646 315 5841
agsikora@gmail.com
Alice Silva
Emory University
212-263-6727
Ines.Silva@nyumc.org
Paul Sondel
The University of Wisconsin
608-263-9069
pmsondel@facstaff.wisc.edu
Eric Sorenson
Memorial Sloan-Kettering Cancer Center
6468883210
sorensoe@mskcc.org
Ian Spendlove
University of Nottingham
+4 115 8231857
ian.spendlove@nottingham.ac.uk
David Spiegel
Yale University School of Medicine
203-432-8697
david.spiegel@yale.edu
Sumit Subudhi
Memorial Sloan-Kettering Cancer Center
646-888-2348
subudhis@mskcc.org
Nithya Srinivasan
University of Virginia Health System
434 924 1706
ns4ed@virginia.edu
Daisuke Sugiyama
Osaka University, Immunology Frontier
Research Center
+81-6-6879-4963
sugiyama@ifrec.osaka-u.ac.jp
Bhaskar Srivastava
Yale University School of Medicine
203-737-2423
bhaskar.srivastava@yale.edu
Subhashini Srivatsan
Washington University School of Medicine
6179351954
ssrivats@go.wustl.edu
Blaze Stancampiano
Boehringer Ingelheim GmbH
+1 (203) 798-4964
blaze.stancampiano@boehringer-ingelheim.com
Shannon Steinberg
Dartmouth College
6036533688
Shannon.Steinberg@dartmouth.edu
Natalie Steinel
University of Pennsylvania
512-413-4119
nsteinel@mail.med.upenn.edu
Susan Stern
Department of Veterans Affairs
202-443-5703
susan.stern@va.gov
Jennifer Stone
University of Illinois at Urbana=Champaign
217-244-2823
jstone@illinois.edu
Bhavani Stout
Celgene Cellular Therapeutics
908-673-9000
bstout@celgene.com
Ingunn Stromnes
University of Washington School of Medicine
206-605-5157
ims@u.washington.edu
Edward Stuart
CT Atlantic
+49 (0)170 53 711
edward.stuart@ct-atlantic.com
Sapna Tandon
Memorial Sloan-Kettering Cancer Center
6468882114
tandons@mskcc.org
Justin Taylor
University of Minnesota, Minneapolis
612-626-1188
tayl0611@umn.edu
Ryan Teague
Saint Louis University School of Medicine
314-977-8871
rteague@slu.edu
Grace Teng
Yale University School of Medicine
203-785-3247
grace.teng@yale.edu
Gavin Thurston
Regeneron Pharmaceuticals
914-345-7575
gavin.thurston@regeneron.com
Kittichoat Tiyanont
Dana-Farber Cancer Institute
617-525-4418
ktiyanont@partners.org
Jared Toettcher
University of California, San Francisco
415-502-8133
jared.toettcher@ucsf.edu
Brian Tomkowicz
Johnson & Johnson
6106516000
Btomkowicz@its.jnj.com
Thomas Totterman
Uppsala University
+46 18 611 4184
thomas.totterman@igp.uu.se
Sinhan Tran
Ludwig Institute for Cancer Research
212-450-1541
stran@licr.org
Martin Treder
CT Atlantic
+41447319406
martin.treder@ct-atlantic.com
Jannine Villella
Winthrop-University Hospital
516-663-2018
jvillella@winthrop.org
Sara Trifari
La Jolla Institute for Allergy and Immunology
650-892-8629
strifari@liai.org
Isaah Vincent
University of Virginia
4349241706
isv3v@virginia.edu
Cornelia Trimble
Johns Hopkins University School of Medicine
410-502-0512
ctrimbl@jhmi.edu
Lotta von Boehmer
University Hospital Zurich
044 255 9779
Lotta.vonBoehmer@usz.ch
Angeliki Tsagaratou
La Jolla Institute for Allergy and Immunology
858-752-6500
tsagaratou@liai.org
Silvia von Mensdorff-Pouilly
Glycotope
+3170 3259603
s.vonmensdorff@vumc.nl
Takemasa Tsuji
Ludwig Institute for Cancer Research, NYB
646-888-2343
tsujit@mskcc.org
Qi Wang
University of California, Berkeley
607-342-4890
qwang@berkeley.edu
Alisa Tubbs
Ventana - Roche
520.396.0389
alisa.tubbs@ventana.roche.com
Li-Hsien Wang
Regeneron Pharmaceuticals
914-345-7972
li-hsien.wang@regeneron.com
Mary Jo Turk
Dartmouth Medical School
603-653-3549
mary.jo.turk@dartmouth.edu
Xin Xiang Wang
University of Maryland Biotechnology Institute
240-314-6228
wangxin@umbi.umd.edu
Alice Tzeng
M.I.T.
337-255-6099
atzeng@mit.edu
Tomoko Watanabe
BioWa, Inc.
609-580-7503
watanabe.tomoko@biowa.com
Fred Valentine
New York University School of Medicine
212-263-6401
fred.valentine@nyumc.org
Joyce Wei
Memorial Sloan-Kettering Cancer Center
646 888 2348
weij@mskcc.org
Maries van den Broek
University Hospital Zurich
+41445563134
maries@van-den-broek.ch
Genevieve Weir
ImmunoVaccine, Inc.
19024215735
gweir@imvaccine.com
Matthew Vesely
Washington University School of Medicine
3143628746
veselym@msnotes.wustl.edu
Irving Weissman
Stanford University School of Medicine
650-723-6520
irv@stanford.edu
Vladimir Vigdorovich
Albert Einstein College of Medicine
718-430-2226
vladimir.vigdorovich@einstein.yu.edu
Haitao Wen
University of North Carolina at Chapel Hill
919-966-2662
haiwen@med.unc.edu
Chien-Huan Weng
MSKCC
6468882348
chw2019@med.cornell.edu
Qingrong Yan
University of California, Berkeley
510-643-1710
yanqr510@gmail.com
Johnna Wesley
Dendreon Corporation
2062197921
jwesley@dendreon.com
Chenghua Yang
Weill Medical College of Cornell University
212-746-6458
chhyang@gmail.com
Lee Wheeler
Harvard University
617-771-4941
lee_wheeler@hms.harvard.edu
Mohammed Yassin
Peter MacCallum Cancer Centre
+61396561238
mohammed.yassin@petermac.org
Clarence Williams
Ludwig Institute for Cancer Research, NYB
646-888-2343
william1@mskcc.org
Tangsheng Yi
University of California, San Francisco
415-502-6638
tangsheng.yi@ucsf.edu
John Wilson
University of Washington School of Medicine
206-685-0163
wilsonjt@u.washington.edu
Beatrice Yin
Ludwig Institute for Cancer Research, NYB
646-888-2343
yinb@mskcc.org
Jedd Wolchok
Memorial Sloan-Kettering Cancer Center
(646) 888-2395
wolchokj@MSKCC.ORG
Jianda Yuan
Memorial Sloan-Kettering Cancer Center
646-888-3073
yuanj@mskcc.org
Laurence Wood
University of Pennsylvania School of Medicine
215-898-2145
woodla@mail.med.upenn.edu
Dmitriy Zamarin
Memorial Sloan-Kettering Cancer Center
646-888-2350
zamarind@mskcc.org
Catherine Wu
Dana-Farber Cancer Institute
617-632-5943
cwu@partners.org
Xingxing Zang
Albert Einstein College of Medicine
718-430-4155
xing-xing.zang@einstein.yu.edu
Lan Wu
ImClone Systems, Inc.
646-336-7858
lan.wu@imclone.com
Kai Zanzinger
CureVac GmbH
+49(0)7071920530
kw@curevac.com
Hui Xiao
The Scripps Research Institute
858-784-7660
huixiao@scripps.edu
Franz Zemp
Southern Alberta Cancer Reserach Institute,
University of Calgary
403-220-8693
fjzemp@ucalgary.ca
Ming-Hong Xie
Oncomed
650-225-8281
minghong.xie@oncomed.com
Xiaohong Xu
ImClone Systems, Inc.
9175898128
xiaohong.xu@imclone.com
Shan Zeng
MSKCC
646-8883209
zengs@mskcc.org
Ben Zeskind
Immuneering Corporation
617 6943782
bzeskind@immuneering.com
Yan Zhang
Weill Medical College of Cornell University
212-7464423
yaz2006@med.cornell.edu
Ying Zheng
Johns Hopkins University School of Medicine
412-478-7857
yzheng25@jhmi.edu
Samuel Zhang
Bristol-Myers Squibb
609-252-3303
samuel.zhang@bms.com
Tao Zou
University of Pennsylvania School of Medicine
215-410-0240
taozou@mail.med.upenn.edu
Yi Zhang
ImClone Systems, Inc.
6466386384
yi.zhang@imclone.com
Kamila Zurkova
Institute of Hematology and Blood Transfusion
+420221977364
zurkova@uhkt.cz
Ruihua Zhao
Albert Einstein College of Medicine
718-430-4154
rzhao1@einstein.yu.edu
About the Cancer Research Institute
The Cancer Research Institute (CRI), established in 1953, is the world’s only nonprofit
organization dedicated exclusively to transforming cancer patient care by advancing
scientific efforts to develop new and effective immune system-based strategies to
prevent, treat, and cure cancer.
Guided by a world-renowned Scientific Advisory Council that includes three Nobel
laureates and thirty members of the National Academy of Sciences, CRI has invested
more than $200 million in support of research conducted by immunologists and tumor
immunologists at the world’s leading medical centers and universities, and has
contributed to many of the key scientific advances that have led to the recent
explosion of interest in the potential for immunotherapy to change the face of cancer
treatment.
To accelerate the pace of progress in the field, CRI convenes and coordinates global
collaborations among academics, industry scientists and decision makers, regulatory
representatives, and health research associations focused on discovery, development,
and refinement of new cancer immunotherapies. A founding visionary and scientific
leader in tumor immunology, CRI is helping to shape the emerging field of immunooncology, and is ushering in a new era of medical progress to bring more treatment
options to cancer patients sooner.
The Cancer Research Institute has one of the lowest overhead expense ratios among
nonprofit organizations, with more than 85 percent of its resources on average going
directly to the support of its science, medical, and research programs. CRI meets or
exceeds all 20 standards of the Better Business Bureau Wise Giving Alliance, the
most comprehensive U.S. charity evaluation service, and has earned the GuideStar
Exchange Seal, indicating CRI’s commitment to transparency of organizational
information to the public. CRI has also received an 'A' grade for fiscal disclosure and
efficiency from the American Institute of Philanthropy, as well as top accolades from
other charity watchdog organizations.
For more information, visit http://www.cancerresearch.org.
National Headquarters · One Exchange Plaza · 55 Broadway, Suite 1802 · New York, NY 10006
Phone 212-688-7515 · Fax 212-832-9376 · Web site www.cancerresearch.org
cancer immunotherapy consortium
2012 scientific Colloquium
immune signatures in the tumor
and beyond: toward predictive and
prognostic markers
April 19-21, 2012
gaylord convention center • national harbor, md
Further information about the meeting will be available soon at
www.cancerresearch.org/colloquium 2012