1 Ag-presenting CpG-activated pDCs prime

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regression
Leslie Guéry1, Juan Dubrot1, Carla Lippens1, Dale Brighouse1, Pauline Malinge2, Magali
Irla1,3, Caroline Pot1,4, Walter Reith1, Jean-Marc Waldburger1, and Stéphanie Hugues1,*
1
Department of Pathology and Immunology, University of Geneva Medical School, 1 rue
Michel Servet, 1211, Geneva, Switzerland
2
NovImmune SA, 14 Chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland
3
Centre d’immunology de Marseille Luminy, Université de la Méditerranée, 1300 Marseille,
France
4
Division of Neurology, Department of Clinical Neurosciences, Geneva University Hospitals,
1211 Geneva 14, Switzerland
Running title : Ag-presenting pDCs promote anti-tumor Th17 cells.
Key words: plasmacytoid dendritic cells, antigen presentation, Th17, anti-tumor immunity
Financial support : This work was supported by the Swiss National Science Foundation
(310030-127042) and the European Research Council (pROsPeCT 281365) grants to S.
Hugues.
*
Correspondence to: Stephanie Hugues
Department of Pathology and Immunology, University of Geneva Medical School,
1 rue Michel Servet, 1211, Geneva, Switzerland
Tel.: 00 41 2 23 79 58 93
Fax : 00 41 2 23 79 57 46
Stephanie.hugues@unige.ch
Disclosure : The authors declare no competing financial interests.
Word count : 6145
Figures : 7
Supplemental figures : 4
1
ABSTRACT
Plasmacytoid dendritic cells (pDCs) rapidly and massively produce type I interferon and other
inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T
cell responses.
Moreover, antigen (Ag) presenting pDCs directly regulate T cell
differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or
immunogenic properties. Here we show that CpG-activated pDCs promote efficient Th17
differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on
pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells
infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in
tumors, including tumor-specific CTLs, is altered and results in increased tumor growth.
Importantly, following immunization with tumor Ag and CpG-B, MHCII–restricted Ag
presentation by pDCs promotes the differentiation of anti-tumor Th17 cells that induce
intratumor CTL recruitment, and subsequent regression of established tumors. Our results
highlight a new role for Ag presenting activated pDCs in promoting the development of Th17
cells and impacting on anti-tumor immunity.
2
INTRODUCTION
pDCs exhibit important innate functions during infections, notably by producing large amount
of type I IFN (IFN-I) (1). However, recent evidences directly involved these cells in adaptive
immunity. pDCs up-regulate MHC class II molecules (MHCII) upon inflammation (2) and
function as antigen (Ag) presenting cells (APCs) in vivo in several mouse models of diseases
to induce both T cell-mediated immunity and tolerance. For instance, pDCs promote the
initiation
of
myelin-induced
Th17
responses
and
Experimental
Autoimmune
Encephalomyelitis (EAE) (3). Furthermore, Ag targeting to pDCs via BST-2 in combination
with TLR agonists provide an effective vaccination (4). On the other hand, Treg induction by
pDCs was shown to delay cardiac allograft rejection, dampen asthmatic reactions to inhaled
Ags, and protect against graft versus host disease (5-7). In addition, Ag targeting to pDCs via
Siglec-H inhibits Th cell-dependent autoimmunity (8). We recently formally demonstrated
that MHCII–restricted presentation of myelin Ags by pDCs promotes Treg expansion and
inhibits EAE (9).
The role of pDCs in anti-tumor immunity has been debated. In tumor microenvironnement,
pDCs exhibit a tolerogenic phenotype characterized by low costimulatory molecule
expression and low IFN-I production, and promote tumor growth possibly through Treg
induction (10-13). In contrast, specific Ag-delivery to pDCs using BST2 in combination with
TLR agonists induced protective immunity against tumor growth (4). In humans, intranodal
injections of pDCs activated and loaded with tumor Ag-associated peptides induced antitumor specific T cell responses (14). However, since targeting Ag presentation in pDCs also
increased their production of IFN-I (15), those studies could not determine the contribution of
Ag presentation by pDCs in impacting tumor T cell immunity. In addition, TLR-triggered
pDCs exhibit potent anti-tumor effects. Tumor regression has been correlated for some (ie,
3
R848 and CpG-A), but not all (CpG-B) TLR ligands, with IFN-I production by pDCs (16).
Thus, pDC-mediated anti-tumor potential may rely on other IFN-I independent pDC
functions, possibly via their ability to present tumor Ags.
Here we investigated whether Ag-presenting pDC capacities could be modulated and
exploited to enhance anti-tumor T cell immunity. We show that upon immunization with an
Ag and CpG-B, in vivo Ag-specific Th17 responses are significantly impaired in genetically
modified mice lacking MHCII expression on pDCs. In contrast to what was shown in
conventional DCs (cDCs) (17), MHCII deficiency does not impair innate pDC functions.
Importantly, in mice further challenged with tumors, pDC-primed Th17 cells control tumor
growth by promoting the recruitment of immune cells, including tumor-specific cytotoxic T
lymphocytes (CTLs), into the tumors. In addition, vaccination with an MHCII-restricted
tumor epitope of mice bearing established tumors significantly induce Th17 cells that promote
intra-tumoral CTL recruitment and tumor growth inhibition, in a mechanism dependent on
MHCII expression by pDCs. Thus, CpG-activated pDCs contribute as APCs to the induction
of anti-tumoral Th17 cells that dramatically dampen established tumor growth, a property that
may be of interest in the establishment of anti-tumor immunotherapies.
4
METHODS
Mice
All mice (WT, H2-Aα-/- (18), pIII+IV-/- (19), OT-I (20), OT-II Rag2-/- (21), µMT (22), µMT
pIII+IV-/- (9)) were of pure C57BL/6 background and maintained under SPF conditions. All
animal husbandry and experiments were approved by the animal research committee of the
University of Geneva.
Immunizations and T cell transfer
BM chimeric mice were generated as described (9). Mice were immunized s.c. with OVAII
peptide (ISQAVHAAHAEINEAGR) (2µg) (Polypeptide) in presence of CpG-B 1668
(5µmoles) (Invivogen), and injected i.v. 24h later with OT-II Rag2-/- LN cells labelled with
10µM CFSE (Invitrogen). After 4 days, dLN and ndLN cells were restimulated for 18h with
OVAII (10µg/mL).
In vitro cell differentiation and treatments
pDCs were differentiated from BM with Flt3-L (100ng/mL) (Peprotech) (9), sorted as
CD11cintB220+PDCA-1+ pDCs, using a Moflow Astrios (Beckman Coulter) and treated with
0.3µM CpG-B 1668, 0.3 μM CpG-A 1585 or 1µg/mL Imiquimod (Invivogen). In some
experiments, cells were loaded with 10µg/mL of OVAII peptide.
Flow cytometry
Siglec-H (eBio440c), CD8α (53.6.7), TNF-α (MP6-XT22), Foxp3 (FJK-16s), CD45.1 (A20),
CD3 (145-2C11) and PDCA-1 (eBio927) antibodies were from eBioscience, CD11c (n418),
B220 (RA3-6B2), CD19 (6D5), F4/80 (BM8) and CD11b (M1/70) antibodies from Biolegend
5
and I-Ab (AF6-120.1), CD4 (RM4.5), IL-17 (TC11-18H10) and IFN-γ (XMG1.2) antibodies
from BD. H-2kb SIINFEKL pentamer was purchased from Proimmune.
ELISAs
Cytokine production was assessed in culture supernatants using ELISA Kit from eBioscience
(TNFα, IL-6, IL-17, IL-10 and TGF-β), BD (IFN-γ) or PBL (IFN-α).
Quantitative RT-PCR
Total RNA was isolated and RT-PCR was performed as described (9, 21). Primer sequences
are provided in Supplemental Information.
Dendritic Cell isolation from LN
LN cells were isolated (9, 21), depleted of lymphocytes using CD3 and CD19 antibodies and
magnetic sorting (Miltenyi Biotec). pDCs (CD11cintB220+PDCA-1+) were sorted using a
Moflow Astrios (Beckman Coulter).
Tumor experiments
Mice were immunized s.c. with CpG-B (5µmoles) and OVAII (10µg) either before (7 days) or
after (between 7 and 10 days) tumor cell transplantation (5x105 EG7 thymoma cells, s.c.).
Tumor size was measured with a caliper {L (length) x l (width)}. For TILs analysis, tumors
were digested with collagenase D (1 mg/ml) + DNAse 1 (10 µg/ml) (Roche) and TILs were
enriched using lympholyte M (Cedarlane laboratory) and restimulated for 18h with PMA
(50ng/mL) (Sigma) and ionomycine (1µg/mL) (Sigma). When indicated, mice were depleted
of CD8+ T cells using anti-CD8 mAbs (53-6.72), 100µg i.p. at day 8, 11 and 14 post-tumor
challenge. In some experiments, EG7-tumor bearing mice were adoptively transferred with
6
OVAI-specific CD8+ transgenic T cells (2x106) purified from LN and spleen of OT-I CD45.1
mice using magnetic sorting (Miltenyi Biotec).
In vitro generation of Th17 cells
OT-II naïve T cells were extracted from LN using CD4+ CD62L+ T cells isolation kit
(Miltenyi). Cells were cultured for 3 days on anti-CD3 and anti-CD28 (BioXCell) coated
plates in presence with IL-6 (25 ng/mL) and TGF-β (2.5 ng/mL) (eBioscience). Cells (1.106)
were then injected i.v. into sub-lethally irradiated mice (500Gy).
In vivo killing assay
CFSE labelled splenocytes were loaded (2.5μM CFSE) or not (0.5μM CFSE) with OVAI
peptide (SIINFEKL) (10µg/mL) (Polypeptide) and 5x106 each (ratio 1:1) was injected i.v.
into tumor bearing mice. 20h later, CFSE cells were analyzed in tumor dLN (TdLN) and
ndLN. Specific in vivo killing was calculated as (1-(% CFSE 2.5μM TdLN / % CFSE 0.5μM
TdLN)/(% CFSE 2.5μM ndLN / % CFSE 0.5μM TdLN))*100.
Statistics
Statistical significance was assessed by the Mann-Whitney test or by the Two-Way ANOVA
test with Bonferroni correction for tumor growths using graphpad software. *, p<0.05, **,
p<0.01, *** p<0.001.
7
RESULTS
pDCs induce Th17 cells upon CpG-B activation
We investigated whether the lack of MHCII on pDCs has an impact on Th responses upon
inflammatory antigenic challenge. MHCII expression is regulated by the master regulator
CIITA, itself under the control of cell specific promoters (23) (Supplemental Fig. 1A). MHCII
expression in pDCs strictly relies on pIII, whereas pI is expressed in macrophages and cDCs
(23). We used mice deficient for pIII and pIV of CIITA (pIII+IV-/- mice) (19). Due to deletion
of pIV that is expressed by cortical thymic epithelial cells, pIII+IV-/- mice are devoid of CD4+
T cells (24). Consequently, and as described before (9), we restored the CD4+ T cell
population by generating bone marrow (BM) chimeric mice using irradiated WT recipients.
Furthermore, because pIII is expressed in B cells, we have used BM precursors derived from
B-cell deficient (µMT) mice that were backcrossed with pIII+IV-/- mice. In both cases
(µMT:WT and µMT pIII+IV-/-:WT), BM chimeric mice lack B cells, the only difference
being presence (µMT:WT), or absence (µMT pIII+IV-/-:WT) of MHCII on pDCs
(Supplemental Fig. 1B). In contrast to pDCs, cDCs express normal MHCII levels in µMT:WT
and µMT pIII+IV-/-:WT mice (not shown and (9)). Mice were immunized with OVAII
peptide + CpG-B and transferred with CFSE-labelled OVA specific OT-II CD4+ T cells. T
cells proliferated similarly in draining lymph nodes (dLN), but not in non-dLN (ndLN), of
µMT:WT and µMT pIII+IV-/-:WT mice (Supplemental Fig. 2). The proportion of IFN-γ
producing OT-II cells, as well as the frequency of Foxp3+ OT-II cells in the dLN, was not
affected by the lack of MHCII on pDCs (Fig. 1A-B). Thus, in contrast to our previous study
performed in the context of EAE (9), in which CFA was used as an adjuvant, CpG-activated
pDCs do not promote Treg. The frequency of IL-17 secreting proliferating OT-II cells, as well
as ex vivo IL-17 production, was significantly impaired in dLN of µMT pIII+IV-/-:WT
8
compared to µMT:WT mice (Fig. 1A-C), suggesting that CpG-activated, MHCII-sufficient
pDCs present Ag to CD4+ T cells to promote Th17 responses. Consistently, CpG-B treated,
OVA-loaded pDCs were found competent to induce IL-17 production by OT-II cells in vitro
(Fig. 1D). We further demonstrated that impaired Th17 responses observed in CpG-B context
resulted from the selective abrogation of MHCII expression by pDCs. Indeed, levels of
MHCII expression by other APCs (including CD8+ DCs and macrophages) were rigorously
similar in µMT:WT and µMTpIII+IV:WT chimeric mice, first in steady-state before
immunization, and then equally upregulated 12h after CpG-B treatment (Supplemental Fig.
3). CD11b+ DCs already expressed very high levels of MHCII in naïve condition, and did not
further upregulate MHCII after CpG-B treatment, CD11b+ DCs from µMT:WT and
µMTpIII+IV:WT mice however expressing similar MHCII levels (not shown). Altogether,
our results demonstrated that impaired Ag-specific Th17 observed in µMTpIII+IV:WT
compared to µMT:WT immunized in the context of CpG-B resulted from the selective
abrogation of MHCII expression by pDCs.
Innate pDC functions are not affected by the absence of MHCII
In cDCs, MHCII molecules potentialize TLR-triggered innate functions (17). We tested both
in vitro and in vivo whether pDC innate functions could be affected by the loss of MHCII,
and explain altered Th17 responses in mice lacking MHCII on pDCs. We first analyzed TLRtriggered responses in pDCs derived in vitro from BM of WT, pIII+IV-/- and H2-Aα-/- mice.
Imiquimod and CpG-B treatments induced a significant MHCII upregulation by WT, but not
H2-Aα-/- and pIII+IV-/-, pDCs (Fig. 2A). However, WT, pIII+IV-/- and H2-Aa-/- pDCs
produced equal levels of IL-6, TNF-α and IFN-α in response to both TLR ligands after 6 and
24h (Fig. 2B-C). Intracellular TNF-α staining showed similar results (Fig. 2D). Accordingly,
WT, H2-Aα-/- and pIII+IV-/- pDCs expressed comparable levels of Tnf-α, Il-6, Il-1β and Ifn-α4
9
mRNAs after Imiquimod or CpG-B stimulation (Supplemental Fig. 4A). As described (25),
pDCs produce low IFN-I levels after Imiquimod or CpG-B treatment (Fig.2B, 2C and
Supplemental Fig. 4A). In contrast, expression of IFN-α4 and IFN-β mRNA were similarly
strongly induced after CpG-A treatment in both MHCII-sufficient and MHCII-deficient pDCs
(Supplemental Fig. 4B).
In vivo, LN WT pDCs (Fig. 2E) expressed steady state levels of MHCII whereas pIII+IV-/pDCs were MHCII negative (Fig. 2F). WT and pIII+IV-/- pDCs similarly increased the
expression levels of Il-1β, Il-6, Tnf-α and Ifn-β mRNAs upon CpG-B injection (Fig. 2G),
demonstrating that MHCII deficiency does not impair TLR-mediated pDC innate responses in
vivo. Altogether, our data demonstrate that TLR9-activated pDCs act as APCs to induce Th17
cell differentiation.
Tumor Ag-presentation by CpG-activated pDCs induce Th17 cells that control
subsequent tumor challenge by recruiting immune cells in solid tumors
To determine whether pDC-induced Th17 responses could be exploited in anti-tumor T cell
immunity, we immunized µMT:WT and µMT pIII+IV-/-:WT mice with OVAII + CpG-B and
further challenged mice with OVA-expressing tumors (EG7) in the same flank. Mice lacking
MHCII on pDCs exhibited a significant increase in tumor growth (Fig. 3A), suggesting that
OVAII peptide presentation by CpG-activated pDCs controls tumor growth. Similar results
were observed when BM chimeric mice were immunized with OVA protein instead of OVAII
peptide (Supplemental Fig. 5A), further confirming that pDCs function as bona fide APCs in
our model. In addition, we observed a significant reduction in absolute CD45hi tumor
infiltrating leucocytes (TILs) numbers in µMT pIII+IV-/-:WT compared to µMT:WT mice
(Fig. 3B). Although frequencies of CD4+ T cells, CD8+ T cells, cDCs and pDCs infiltrating
solid tumors were identical whether pDCs express MHCII or not, the dramatic reduction of
10
TILs in absence of MHCII on pDCs led to a significant decrease in absolute numbers of those
cells (Fig. 3C-H).
To investigate whether tumor growth was controlled by Th17 cells primed by CpG-activated
pDCs, we further analyzed effector function of tumor-infiltrating CD4+ T cells. We observed
a significant decrease in frequencies of both IL17+ and IL17+IFN-γ+ CD4+ T cells (Fig. 4A,
4C, 4E) in µMT pIII+IV-/-:WT mice compared to µMT:WT mice. On the contrary, the IFN-γ+
CD4+ T cells (Fig. 4A-4D) and Foxp3+ CD4+ T cell frequencies remained unchanged (Fig.
4B-4F), whereas absolute numbers of all these populations were decreased in uMT pIII+IV-/:WT mice, reflecting the drastic reduction in total TILs (Fig. 4C-F). Consistent with impaired
IL17+ and IL17+IFN-γ+ CD4+ T cell frequencies, the production of IFN-γ and IL-17 by TILs
was significantly decreased in mice lacking MHCII on pDCs (Fig. 4G). In contrast, IL-10 and
TGF-β production were not affected, further confirming that Ag presentation by CpGactivated pDCs did not lead to Treg development (Fig. 4G).
Consistent with a role of tumor-specific Th17 cells in the inhibition of tumor growth, and as
described in the mouse B16 melanoma tumor model (26), we observed a significant reduction
in tumor size (Fig. 4H), as well as increased intratumor IL-17 cytokine levels (Fig. 4I), when
in vitro-differentiated Th17, but not Th0 cells (Supplemental Fig. 6A-B), were adoptively
transferred in mice bearing established tumors.
CpG-activated pDC primed Th17 responses promote CTL recruitment in tumors
Since tumor rejection is mainly mediated by CTLs, we investigated whether tumor-specific
CD8+ T cells were affected by the loss of MHCII expression by pDCs. Although the
percentage of IFNγ+ CD8+ T cells was slightly increased in EG7 tumors from µMT pIII+IV-/:WT compared to µMT:WT, frequencies of OVA-specific (pentamer+) CD8+ T cells were
similar (Fig. 5A-D). Absolute numbers of IFN-γ producing and pentamer+ CD8+ T cells were
11
lower in µMT pIII+IV-/-:WT mice compared to µMT:WT mice (Fig. 5C-D). In dLN,
percentages (Fig. 5E-F) and absolute numbers (not shown) of pentamer CD8+ T cells were
however identical in both groups, suggesting an impaired recruitment of those cells in tumors.
Moreover, CTL cytotoxic activity was similar whether pDCs express MHCII or not in an in
vivo killing assay using OVAI loaded CFSE stained target cells (Fig. 5 G-H). These results
suggest that increased tumor-specific Th17 cells induced by CpG-activated pDCs did not alter
priming of CTL and Th1 cells in dLN but impaired the recruitment of those cells in solid
tumors. Accordingly, total cells isolated from tumor dLN migrated less efficiently toward
EG7 tumor supernatant extracted from µMT pIII+IV-/-:WT compared to µMT:WT chimeras
(Supplemental Fig. 7A), although relative frequencies of LN cell subsets were not affected
(not shown). In addition, the IL17-induced chemokines CCL-2 and CXCL-2, were found
significantly decreased in tumor supernatants of µMT pIII+IV-/-:WT compared to µMT:WT
chimeras (Supplemental Fig. 7B). Altogether, our data demonstrated that CpG-activated
pDCs promoted anti-tumor Th17 cells, intratumoral recruitment of immune cells - including
CTLs - and resulted in tumor growth inhibition.
Ag-presenting functions of CpG-activated pDCs can be exploited for anti-tumor
immunotherapies
To determine whether CpG-activated pDC ability to promote anti-tumor Th17 cells could be
used as a therapeutical strategy, we next transferred CpG-activated OVAII-loaded WT or
pIII+IV-/- pDCs in EG7 tumor-bearing mice. Unfortunately, we could not observe any
regression of tumors from mice injected with MHCII sufficient pDCs (not shown). Similarly,
the vaccination of mice bearing established tumors with OVAII + CpG-B had not beneficial
effect on tumor growth when performed in the flank in which the tumor developed (not
shown), suggesting that pDC ability to prime effector T cells might have been altered by the
12
tumor microenvironment. However, contralateral OVAII + CpG-B vaccination led to a
significant reduction in tumor size (Fig. 6A). In addition, we observed similar results when
tumor-bearing mice were immunized with OVA protein + CpG-B (Supplemental Fig. 5B).
Importantly, tumors from OVAII + CpG-B vaccinated mice exhibited a dramatic increase in
infiltrating CD45hi TILs, with a selective enrichment of Th17 cells (Fig. 6B-6E). In
agreement, IL-17 produced at the vaccination site, and consequently in tumors was
significantly increased (Fig. 6F). These data show that OVAII + CpG-B vaccination of tumorbearing mice promote Th17 cells that induce intra-tumor cell recruitment and tumor growth
inhibition. Consistent with our previous observations, vaccination did not affect tumor
specific CTL frequencies (Fig. 6G-I). However, as a consequence of enhanced cell
infiltration, absolute numbers of Th1, Th17, pentamer+CD8+ and IFN-γ producing CD8+ T
cells were significantly increased (Fig. 6C, 6D, 6G-I).
To determine whether tumor growth inhibition observed following vaccination of tumorbearing mice was dependent on MHCII-mediated Ag presentation by pDCs, we performed
similar experiments in µMT:WT and µMT pIII+IV-/-:WT chimeras. First, tumor growth was
found similar in unvaccinated µMT:WT and µMT pIII+IV-/-:WT mice, demonstrating that,
Ag-presentation by pDCs in tumor microenvironment does not lead to anti-tumor T cell
immunity. However, EG7 tumor bearing µMT:WT chimeras immunized with OVAII+CpG-B
in the contralateral flank exhibited dramatic tumor growth inhibition, whereas, in contrast, no
protective effect was observed in µMT pIII+IV-/-:WT in which pDCs are MHCII deficient
(Fig. 6J). Altogether, these results demonstrate that OVAII+CpG-B mediated tumor
regression, and that this effect was dependent on pDC Ag-presenting functions.
Anti-tumor adaptive immune responses against EG7 tumors are mainly CTL-dependent (2729). To determine whether Th17 mediated control of tumor growth was CTL dependent,
13
CD8+ T cells were depleted in WT tumor-bearing mice vaccinated or not with OVAII + CpGB. As expected, CD8-depletion led to a significant increase in tumor size (Fig. 7A).
Importantly, we observed a total abrogation of OVAII + CpG-B vaccine efficacy in absence of
CD8+ T cells (Fig. 7A), suggesting that OVAII-mediated Th17 cells control tumor growth in a
CD8-dependent manner. Most of the cancer therapies and vaccines developed so far have
been directed to target specific CD8+ T cells (30). Thus, we next wondered whether having a
concomitant anti-tumor Th17 and CD8 T cell responses improve tumor rejection. For that we
adoptively transferred OT-I cells into WT EG7 tumor-bearing mice that we vaccinated or not
with OVAII + CpG-B. We injected sub-optimal OT-I amounts (2x106), which only conferred a
partial and time-limited control of tumor growth when transferred alone (Fig. 7B). OVAII +
CpG-B vaccination increased OT-I recruitment into tumors (Fig. 7C), and consequently
significantly ameliorated and prolonged OT-1 mediated tumor growth inhibition (Fig. 7B).
Accordingly, OVAII + CpG-B vaccination dramatically improved OT-1 mediated tumor
rejection in µMT:WT chimeras (Fig. 7D). In striking contrast, OVAII + CpG-B vaccination
had no effect on tumor growth in OT-I transferred µMT pIII+IV-/-:WT (Fig. 7D), highlighting
the importance of Ag presentation by pDCs in this model.
Altogether, our results demonstrate that anti-tumor vaccination using MHCII-restricted
epitopes in the presence of CpG-B induce tumor antigen presentation by pDCs and
subsequent anti-tumor Th17 responses. pDC-mediated Th17 cells consequently potentialize
CTL-mediated tumor immunotherapies by increasing intra-tumor CTL recruitment.
14
DISCUSSION
pDCs are potent sensors of nucleic acid and become activated to produce large amounts of
IFN-I (31). Those cells, despite low Ag uptake capacity, also function as APCs in several
conditions and, depending on their activation status and the cytokinic environment, promote
either T cell tolerance (3, 4) or effective T cell immunity (5-9). In tumors, it has been
demonstrated that IFN-I display antitumoral effects through anti-proliferative and proapoptotic functions, thus inhibiting tumor cells survival and angiogenesis (32, 33). Moreover,
IFN-I limit tumor progression by enhancing CD8α+ DCs mediated crosspresentation of tumor
Ags and subsequent CTL priming (34, 35). Consequently, IFN-I are used in the treatment of
various cancers (32). However, pDC ability to produce IFN-I is impaired in tumor
microenvironment, therefore converting these cells into tolerogenic pDCs and leading to
immunosuppression in several different cancers (for review, see (33)). In addition, in breast
tumors in particular, pDCs induce Treg expansion in IDO and/or ICOS dependent
mechanisms (10-12). Accordingly, pDC infiltration in tumors has been correlated with poor
clinical outcome (36, 37). Thus, promoting pDC activation in tumor context would restore
IFN-I production, inhibit Treg expansion, and represent an effective approach to induce anti
tumoral immune response. In agreement, CpG-A activated-, IFN-I producing-pDCs injected
directly into tumors initiate an effective and systemic antitumor immunity through the
orchestration of an immune cascade involving the sequential activation of NK cells, cDCs,
and CD8+ T cells (38). Moreover, TLR7 ligands or CpG-B intratumoral injection inhibits
murine mammary tumor growth in a pDC-dependent manner (16). Interestingly however,
protection was linked to restoration of IFN-I occurring after TLR-7 ligand, but not after CpGB, administration. Thus, whereas the role pDC-mediated IFN-I in anti-tumor immunity has
been convincingly demonstrated, it remains unknown whether those cells could directly
15
contribute to anti-tumoral T cell immunity as APCs, and in particular after being activated by
CpG-B.
Here, using mice lacking MHCII expression by pDCs, we show that upon CpG-B activation,
pDCs drive Th17 cell differentiation through MHCII dependent Ag presentation. For
experimental reasons, our mouse model lacks peripheral B cells, which might not be neutral.
However, we compared MHCII+ and MHCII- pDCs in a B-cell deficient background, and
investigated the selective impact of MHCII-mediated Ag presentation by pDCs on CD4+ T
cell responses and tumor growth.
It has been previously demonstrated that TLR triggered pDCs indirectly impact T cell
responses by activating other APCs (1). Our results, together with previous studies, highlight
a direct role for pDCs as APCs in the modulation of T helper activation and outcome. Our
data further establish that, in contrast to what was described for cDCs, innate pDC functions
are not altered by the loss of MHCII. Since MHCII machinery is differentially regulated in
pDCs and cDCs (39, 40), regulation of TLR-triggered inflammatory responses may also be
different in those cells, and, in contrast to cDCs (17), MHCII may not promote TLRsignalling in pDCs. Altogether, our data demonstrate that loss of MHCII expression by pDCs
leads to impaired Th17 responses without affecting pDC innate functions and that CpGactivated pDCs function as efficient pro-Th17 bona fide APCs.
Tissue infiltrating pDC numbers were correlated with the presence of Th17 cells in mucosa
and skin of GvHD patients (41, 42) and in mouse tumors (43, 44). pDC depletion during EAE
leads to impaired encephalitogenic Th17 responses and decreased clinical scores (3). In vitro,
pDCs activated with TLR7 or TLR9 ligands induce Th17 cell differentiation from either naive
or memory T cells (45, 46). In addition, TGF-β-treated pDCs transferred into Collagen
induced arthritis mice promote Th17 cells and exacerbate the disease (47). Depleting antibody
16
experiments demonstrated that production of some cytokines by pDCs, namely TGF-β, IL-6,
IL-1β, IL-23, IFN-α, and TNF-α depending on the studies, were involved in Th17 cell
differentiation (44, 46, 47). We clearly determined that, in addition to pDC-derived cytokines
that are preserved in our model, pDC Ag presenting capacities are implicated in Th17
induction. However, mechanisms accounting for CpG-B activated pDC ability to drive Th17
cells as APCs remain to be elucidated. Future experiments, including careful analysis of
protein expression and dynamic interactions with T cells, will decipher intrinsic pDC
properties which allow them to promote distinct T cell responses (Th1, Th17, Treg)
depending on inflammatory contexts.
Subsequent tumor cell challenge showed that pDCs-primed Th17 cells control tumor growth.
Moreover, our data suggest that pDC-induced Th17 and Th1/17 cells promote inflammation
and intratumor recruitment of immune cells - including Ag-specific CTLs-, resulting in an
increased anti-tumor immunity. Th17 cells implication in tumor immunity remains
controversial. For instance, IL-17 production favors angiogenesis and tumor cell survival (48,
49). Conversely, Th17 might improve tumor rejection by recruiting other immune cells such
as DCs, Th, CTLs and NK cells in the tumor (50-52). Accordingly, the IL17-induced CCL-2
and CXCL-2 chemokines, were decreased in tumor supernatants from mice lacking MHCII
on pDCs. Decrease in CCL-2 and CXCL-2, which induce the recruitment of lymphocytes,
myeloid cells (53-55) and neutrophils (56), respectively, may explain impaired recruitment of
several immune cell populations into tumors in mice lacking MHCII on pDCs. Altogether,
our data demonstrated that MHCII+ pDC-derived, tumor-specific, Th17 cells increase overall
immune tumor infiltrate, including CTLs that would then mediate tumor rejection. Finally,
and in agreement with our results showing a decrease in IFN-γ+IL-17+ Th cells in absence of
MHCII on CpG-activated pDCs, production of IFN-γ by Th17 cells was shown to be essential
for Th17-dependent tumor rejection (26).
17
Tumor microenvironment maintains pDCs in a partially activated phenotype that would
prevent their ability to drive effective anti-tumor T cell responses (10). Accordingly, in tumor
bearing mice, immunization with a tumor Ag in presence of CpG-B in an ipsilateral manner,
or transfer of CpG-B activated and tumor Ag loaded pDCs, failed to inhibit tumor growth.
However, tumor Ag immunization at a distal site leads to induction of tumor-specific Th17
cells, massive recruitment of immune cells in tumors and inhibition of tumor growth.
Importantly, those protective effects are abrogated in mice lacking MHCII on pDCs,
demonstrating requirement of pDC Ag presenting functions for effective tumor regression.
Interestingly, efficacy of therapeutic vaccines was more potent than in a prevention setting.
One probable explanation is that when the vaccine is administrated to tumor-bearing mice,
tumor specific CTLs have already been primed, and as soon as some vaccine-derived Th17
cells are induced, and infiltrate solid tumors to promote intra-tumor recruitment of already
primed endogenous tumor-specific CTLs. In contrast, in the case of preventive vaccine, Th17
cells are primed in absence of any MHCI tumor Ags and consequently attract CTLs only once
they have been generated, days after tumor challenge. OVAII vaccine induced protection is
abrogated in CD8-depleted mice, providing direct evidence that pDC-mediated Th17 induced
tumor rejection relies on tumor-specific CTLs.
Notably, intra-tumoral specific CTL recruitment, as well as CTL-mediated tumor rejection, is
significantly enhanced by concomitant vaccination with MHCII-restricted tumor Ag and
CpG-B. Importantly, this effect is exclusively dependent on tumor Ag presentation by pDCs
to CD4+ T cells and subsequent induction of anti-tumor Th17 responses. Thus, strategies
aiming at enhancing tumor specific CTL priming, such as MHCI tumor epitope vaccines,
could be synergized by promoting CTL recruitment into tumors via induction of pDCdependent tumor specific Th17.
18
Here we demonstrate that Ag-presenting activated pDCs induce potent Ag-specific Th17
cells, suggesting that pDCs could be used not only as inflammatory cytokines producers, but
also as efficient APCs, to improve tumor vaccine efficacy. Those results pave the way for
future manipulation to restore the anti-tumor T cell responses, notably by combining targeting
on both innate and adaptive pDCs functions.
19
Acknowledgments
The authors thank J.P Aubry-Lachainaye for excellent assistance in flow-cytometry, F. V.
Duraes and V. Bochet for help in experiments, N. Page and D. Merkler for providing OT-I
mice, N. Bendriss-Vermare and F. Benvenuti for helpful discussions.
20
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24
FIGURE LEGENDS
Figure 1 : Mice lacking MHCII on pDCs exhibit altered Th17 responses in vivo.
(A-C) µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized in the flank (s.c.) with
CpG-B and OVAII, and CFSE-labelled OT-II Rag2-/- cells were adoptively transferred and
analyzed 4 days later in dLN and ndLN. (A) Representative flow cytometry profiles showing
IL-17+, IFN-γ+ and Foxp3+ cells after gating on CFSE+ CD4+ T cells. (B) Graphs represent
percentages of proliferating T cells expressing IL-17, IFN-γ or Foxp3. (C) IL-17 and IFN-γ
production by T cells in culture supernatants. (B, C) Results show the means and SEM
derived from 7 mice and are representative of 3 independent experiments. (D) BM-pDCs were
incubated or not for 24h with CpG-B and OVAII, washed and co-cultured with OT-II Rag2-/LN cells for 4 days. IL-17 concentration was measured in culture supernatants.
Figure 2 : MHCII deficient pDCs exhibit normal TLR responses in vitro and in vivo.
(A, B, C and D) BM-pDCs from WT, H2-Aα-/- and pIII+IV-/- mice were activated or not with
Imiquimod or CpG-B. (A) Flow cytometry profiles of MHCII expression after 24h. (B, C)
TNF-α, IL-6 and IFN-α production after 6h (B) or 24h (C) of treatment. Results are pooled
from 4-8 mice. (D) Representative flow cytometry profiles of intracellular TNF-α staining
after 6h of treatment. Histograms show percentages of TNF-α+ cells among PDCA-1+ cells.
(A-D) Results are representative of at least three independent experiments. (E-G) WT and
pIII+IV-/- mice were injected in the flank (s.c.) with PBS or CpG-B, and pDCs from dLN were
analyzed 12h later. (E) pDCs were sorted by flow cytometry as CD19-CD11cintB220+PDCA1+ cells. (F) MHCII expression by LN pDCs from PBS injected mice. (G) Tnf-α, Il-6, Il-1b
and Ifn-b mRNA fold increase in sorted pDCs from CpG-B relative to PBS-injected mice.
25
The means and SEM were derived from 4-5 mice and are representative of 3 independent
experiments.
Figure 3 : Tumor Ag-presentation by CpG-activated pDCs controls subsequent tumor
challenge.
(A-H) µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized s.c. with OVAII and
CpG-B. 7 days later, EG7 cells were implanted s.c. in an ipsilateral manner. (A) Tumor
growth was measured every 1-2 days. Results show the mean and SEM derived from 18 mice.
(B-H) At day 8 post-tumor transplantation, TILs were extracted and analyzed. Results show
the mean and SEM derived from 3-6 mice and are representative of 3 independent
experiments. (B) Histograms show absolute numbers of CD45hi TILs. (C-D) Representative
flow cytometry profiles, after gating on CD45hi cells, of CD4+ and CD8+ T cells (C) and cDCs
(CD11c+ cells) or pDCs (SiglecH+ cells) (D). (E-H) Percentages and absolute numbers of
CD4+ T cells (E), CD8+ T cells (F), cDCs (G) and pDCs (H).
Figure 4 : pDCs-primed Th17 cells control tumor growth.
(A-G) µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized s.c. with OVAII and
CpG-B. 7 days later, EG7 cells were implanted s.c. in an ipsilateral manner. At day 8 posttumor transplantation, tumors were extracted and TILs were restimulated 18h with
PMA/Iono. (A) Representative flow cytometry profiles of IL-17 and IFN-γ expression after
gating on CD45hi CD4+ T cells. (B) Representative flow cytometry profiles of CD4 and
Foxp3 expression after gating on CD45hi cells. (C-F) Percentages and absolute numbers of
CD4+ T cells expressing IL-17 (C), IFN-γ (D) IL-17 and IFN-γ (E) or Foxp3 (F). (G) IL-17,
IFN-γ, IL-10 and TGF-β were measured in culture supernatants. (C-G) Results show the mean
and SEM derived from 3-6 mice and are representative of 3 independent experiments. (H-I)
26
EG7 cells were implanted s.c. in WT mice. 7 days later, mice were injected i.v. with OT-II T
cells in vitro polarized into Th0 or Th17 cells. (H) Tumor growth was measured every 1-2
days. (I) TILs were restimulated in vitro with PMA/Iono and IL-17 was quantified in culture
supernatants. (H-I) Results are representative of 3 independent experiments.
Figure 5 : Impaired CTL recruitment in mice lacking MHCII on pDCs.
(A-H) µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized s.c. with OVAII and
CpG-B. 7 days later, EG7 cells were implanted s.c. in an ipsilateral manner. (B-D) At day 8
post-tumor transplantation, TILs were restimulated with PMA/Iono. (A-B) Representative
flow cytometry profiles of CD8 and IFN-γ (A) or CD8 and pent-OVA (B) after gating on
CD45hi cells. (C-D) Percentages and absolute numbers of CD8+ T cells expressing IFN-γ (C)
or the Pentamer OVA (D). (E-F) 8 days post-tumor transplantation, dLN and ndLN were
analyzed. (E) Representative flow cytometry profiles of pent-OVA expression after gating on
CD8+ T cells. (F) Percentages and absolute numbers of CD8+ T cells expressing pent-OVA
(pent+). (G-H) At day 7 post-tumor transplantation, CFSE-labelled target cells were injected
i.v. and dLN were analyzed 20h later. (G) Representative flow cytometry profiles of CFSE.
(H) Histograms show percentages of Ag specific in vivo killing. (A-H) Results show the
mean and SEM derived from 3-6 mice and are representative of 3 independent experiments.
Figure 6 : MHCII-mediated pDC-dependent vaccination with CpG-B + OVAII inhibits
established tumors.
(A-I) EG7 cells were implanted s.c in WT mice. 10 days later, mice were immunized (VAX)
or not (CT) s.c. with OVAII + CpG-B in a contralateral manner. (A) Tumor growth was
measured every 1-2 days. (B-I) At day 5 post-immunization, TILs were analyzed. (B)
Histograms show absolute numbers of CD45hi TILs. (C-E, G-I) Percentages and absolute
27
numbers of CD4+ T cells (C), IFN-γ+ CD4+ T cells (D), IL-17+ CD4+ T cells (E), CD8+ T cells
(G), Pent+ CD8+ T cells (H) and IFN-γ+ CD8+ T cells (I). (F) IL-17 was measured in culture
supernatants. (B-I) Results show the mean and SEM derived from 5 mice and are
representative of 3 independent experiments. (J) EG7 cells were implanted s.c in µMT:WT
and µMT pIII+IV-/-:WT mice. 10 days later, mice were immunized s.c. with OVAII + CpG-B
in a contralateral manner. Tumor growth was measured every 1-2 days. Results show the
mean and SEM derived from 5 mice and are representative of 3 independent experiments.
Figure 7 : MHCII-mediated pDC-dependent beneficial effect of OVAII vaccine to OT-I
transfer in inducing tumor regression.
(A-C) EG7 cells were implanted s.c in WT mice. (A) 8 days later, mice were injected or not
with depleting anti-CD8 mAbs (days 8, 11, 14) and immunized or not in a contralateral
manner with OVAII + CpG-B (day 8). Tumor growth was measured every 1-2 days. Results
show the mean and SEM derived from 6 mice and are representative of 2 independent
experiments. (B, C) 8 days later, mice were transferred or not with OT-I CD45.1 cells (2x106)
and immunized or not in a contralateral manner with OVAII + CpG-B. (B) Tumor growth was
measured every 1-2 days. Results show the mean and SEM derived from 8 mice and are
representative of 2 independent experiments. (C) OT-I cell frequencies (CD45.1+ cells) were
analyzed in tumors 18 days after T cell transfer. Representative flow cytometry profiles, and
histograms represent a pool of 2 experiments with 4 mice / group. (D) EG7 cells were
implanted s.c in µMT:WT and µMT pIII+IV-/-:WT mice. 8 days later, mice were transferred
with OT-I CD45.1 cells (2x106) and immunized or not in a contralateral manner with OVAII +
CpG-B. Tumor growth was measured every 1-2 days. Results show the mean and SEM
derived from 8 mice and are representative of 2 independent experiments.
28
A
8.4
Foxp3
2.3
IL-17
IFN-γ
µMT:WT
11.6
NS
*
2
1
0
2
*
1.5
30
20
1
0.5
10
0
0
μMT:WT
Foxp3+ cells ((%)
0
3
D
IL17 (ng/mL)
5
40
6.4
Foxp3
IL-17
10
C
IFN-γ (ng/mL)
NS
IL-17+ cells ((%)
15
0.9
CFSE
IL-17 (ng
g/mL)
IFN-γ+ cells ((%)
B
12.3
IFN-γ
µMT pIII+IV-/-:WT
CFSE
NS
10
8
6
4
2
0
4
none
OVAII +CpG-B
3
2
1
0
T
T + pDC
μMT pIII+IV-/-:WT
Figure
g
1 : Mice lackingg MHCII on p
pDCs exhibit altered Th17 responses
p
in vivo.
1
0.44
1
0.5
Imi CpG-B
4
2
0
CT
WT
H2-Aα-/pIII+IV-/-
40
20
0
CT
NS
150
PDC
CA-1
SSC
C
CD11c
B220
WT
H2-Aα-/pIII+IV-/-
100
50
0
CT
F
CD19
22.5
22.3
pDC
B220
Imi CpG-B
pDC
WT
pIII+IV-/-
MHCII
18.4
22.6
pIII+IV-/PDCA-1
NS
200
E
cDC
18.9
0.27
Imi CpG-B
250
Imi CpG-B
TNF-α
60
40
NS
30
NS
WT
H2-Aα-/pIII+IV-/-
20
10
0
CT
G
3 Tnf-α
2
1
0
WT
fold incrrease
IL-6 (ng/m
mL)
15
1.5
CpG-B
H2-Aα-/-
80
Imi CpG-B
6
CT
21.7
100
TNF-α+ pDCs
s (%)
1
0 CT
Imi CpG-B
2
0
Imiquimod
None
WT
fold incrrease
CT
2
IFN-α (pg/m
mL)
0
3
IFN-α (pg/mL)
3
1
TNF-α (ng/m
mL)
D
WT
H2-Aα-/pIII+IV-/-
0.39
2
C
CpG-B
MHCII
IL-6 (ng/mL)
TNF-α (ng/mL)
B
Imiquimod
pIII+IV-/-
Imi
5 Il-6
4
3
2
1
0
WT
CpG-B
pIII+IV-/-
8 Il-1β
6
4
2
0
WT
fold incrrease
None
fold incrrease
A
pIII+IV-/-
4 Ifn-β
3
2
1
0
WT pIII+IV-/-
Figure 2 : MHCII deficient pDCs exhibit normal TLR responses in vitro and in vivo.
1
B
A
60
***
40
20
6
8
time (days)
1.05
0
10
μMT pIII+IV-/-:WT
μMT:WT
D
μMT:WT
0.91
CD11c
CD8
8
24.5
10
25
0
4
2
0
NS
*
10
5
0
μMT:WT
H
Freq (%)
6
1
0.5
cDCs
*
3
NS
1.5
50
15
CD8+ T cells
2
0
nb
b (x103)
Freq (%)
*
F
Freq (%)
75
nb ((x103)
Freq (%)
NS
20
8
0 76
0.76
Siglec-H
0
G
4.44
0 98
0.98
26.6
CD4+ T cells
30
μMT pIII+IV-/-:WT
4.53
CD4
E
100
nb (x
x103)
C
4
200
2
1
0
pDCs
1.5
4
NS
1.0
05
0.5
0
nb
b (x103)
0
**
300
µMT:WT
µMT pIII+IV-/-:WT
CD45hhi cell (nb x103 )
tumo
or size (mm2)
80
*
3
2
1
0
μMT pIII+IV-/-:WT
Figure 3 : Tumor Ag-presentation by CpG-activated pDCs controls subsequent tumor challenge.
1
A
μMT:WT
μMT pIII+IV-/-:WT
1.66 10
0.58
79
3.97 85.6
3.79
B
μMT:WT
IL-17
82.5
5
0
E
8
6
4
nb (x102)
0.5
0
**
4
2
**
IFN-γ (ng/mL)
6
6
4
2
0
10
NS
0.2
0
0
μMT pIII+IV-/-:WT
H
I
Th0
Th17
300
Th cell
transfer
200
**
6
*
IL-17 (pg/mL x102)
400
NS
0.4
1
0.5
0
5
0.6
1,5
2
**
0
2
4
μMT:WT
15
0
**
1
10
5
IL-10 (ng/mL)
G8
2
Foxp3+ cells
NS
20
6
**
0
F
0
0
2
0
8
1
3
1
Freq (%)
**
1.5
3
4
2
IL17+ IFN-γ+ cells
IFN-γ+ cells
nb (x103)
10
5
Freq (%)
15
**
NS
nb (x103)
10
**
nb (x103)
Freq (%)
D
IL17+ cells
20
Freq (%)
85.4
TGF-β (ng/mL)
C
IL-17 (ng/mL)
17.5
CD4
IFN-γ
tumor size (mm2)
μMT pIII+IV-/-:WT
14.6
Foxp3
15.4
100
5
4
3
2
1
0
0
0
5
10
15
time (days)
20
Th0
Th17
Figure 4 : pDCs-primed Th17 cells control tumor growth.
1
A
MT:WT
MT pIII+IV-/-:WT
42.1
70.5
57.9
B
MT:WT
CD8
D
IFN-+ CD8+ T cells
91
88.6
*
10
30
20
10
NS
12
8
Freq (%)
Nb (x102)
40
Pent+ CD8+ T cells
6
4
2.5
Nb (x102)
*
50
Freq (%)
11.4
CD8
8
4
2
0
0
MT:WT
*
2
1.5
1
0.5
0
0
μMT:WT
μMT pIII+IV-/-:WT
MT pIII+IV-/-:WT
0.09
0.10
dLN
0.01
Pent
0.01
F
CD8+ pent+ cells (%)
C
E
MT pIII+IV-/-:WT
8.41
Pent
IFN-γ
29.5
0.15
0.10
0.05
0
ndLN
μMT:WT
μMT pIII+IV-/-:WT
CD8
µMT:WT
32.6
67.4
µMT pIII+IV-/-:WT
30.8
69.2
41.1
dLN
39.8
58.9
60.2
CFSE
CFSE
H
Specific in vivo
killing (%)
G
60
40
20
0
ndLN
μMT:WT
μMT pIII+IV-/-:WT
Figure 5 : Intratumoral CTL recruitment is impaired in mice lacking MHCII on pDCs.
1
A
tumor size (mm2)
B
*
CT
VAX
250
4
150
cells (nb x106)
200
*
100
50
d2
5
400
200
2
1
0
IFN-γ+ CD4+ T cells
*
2
1
20
0
1.0
4
0.8
3
0.4
10
5
0.2
0
0
I
IL-17+ CD4+ T cells
*
*
2
1
Freq ((%)
30
nb (x1
103)
3
20
10
F
2
1
IFN-γ+ CD8+ T cells
25
50
20
40
15
10
0
*
0
5
0
40
Pent+ CD8+ T cells
0.6
15
80
0
Freq (%
%)
nb (x10
03)
25
H
*
120
3
0
3
160
4
nb (x103)
10
600
Freq (%
%)
nb (x103)
Freq (%
%)
20
CD8+ T cells
*
800
0
Freq (%
%)
G
CD4+ T cells
30
Freq ((%)
d4
nb (x10
03)
C
0
*
30
20
10
0
*
2.5
IL-17 (pg/mL)
1
nb (x103)
d0
E
2
0
0
D
*
3
2
1.5
CT
**
1
VAX
0.5
0
dLN
tumor
J
tumor size (mm
m2)
120
μMT:WT
μMT:WT VAX
μMT pIII+IV-/-:WT
μMT pIII+IV-/-:WT VAX
90
NS
VAX
60
***
*
30
0
8
10
12
14
16
Figure 6 : MHCII-mediated pDC-dependent vaccination with CpG-B + OVAII inhibits established tumors.
1
A
CT
CT VAX
α CD8
α-CD8
α-CD8 VAX
180
tumor size (mm2)
150
120
NS
***
90
VAX
60
*
30
0
6
8
10
250
tum
mor size (mm2)
12
14
16
Time (days)
B
CpG-B
OT1 + CpG-B
OT1 + CpG-B + OVAII
200
NS
150
**
***
VAX
100
50
0
5
10
15
20
C
CD45.1
OT1
OT1 + VAX
11 4%
11.4%
28 7%
28.7%
CD8
D
tumor size (mm2)
120
CD45.1+ CD8+ ce
ells (%)
Time (days)
40
*
30
20
10
0
OT1 OT1
+
VAX
μMT:WT +OT1
μMT:WT +OT1 +VAX
μMT p
μ
pIII+IV-/-:WT +OT1
μMT pIII+IV-/-:WT +OT1 +VAX
80
**
OT1 +/- VAX
40
0
6
8
10
12
14
Time (days)
Figure 7 : MHCII-mediated pDC-dependent beneficial effect of OVAII vaccine to OT-I transfer in inducing tumor
regression.
1