Analysis of T-cell Receptor Usage in Activated T-cell

Journal of Autoimmunity (1989) 2, l-13
Analysis of T-cell Receptor Usage in Activated
T-cell Clones frorn Hashirnoto's Thyroiditis and
Graves'Disease
Marie Lipoldovar*t Marco Londeirf Beatrix GrubeckLoebenstein,f Marc Feldmannf and MichaelJ. Owen*
* Imperial Cancer Research
Fund Laboratories, St Bartholomeut's Hospital, Dominion
House' Bartholomew close, London ECI A 7BE;
lThe charing cross sunley
Research Cenne, Lurgan Aaenue, Hammersmith, London W6 gLW, UK
Rearrangements to the T-cell receptor (TcR)
B and y gene loci were studied
in T cells derived from the thyroid glands of a patient with Hashimoto's
(HT) and another with Graves' (GD) autoimmune
thyroiditis. The cells
studied were freshly isolated mononuclear cells, T-celi lines grown in the
presence of anti-cD3 and.lL-2 and r-cell clones. Numerous different rearrangernents to the constant regions of rcRp and rcRy and in the variable
gene region of rcRB were observed. These findings indicate that the T-cell
response in autoimrnune thyroiditis is multiclonal and may have implications for the epitopes recognized by autoreactive T cells and for the
mechanisms of the disease.
To function effectively, the immune system must be able to distinguish self from
non-self. The mechanism(s) that mediate this process are not yet fully understood.
Various ones have been implicated, such as the involvement of suppressor cells,
tolerance induction and idiotypic regulation (reviewed in
Il]).
The major structure which discriminates between T cells of various specificitiesis
the T-cell receptor (TcR) for antigen. Studies over the past 3 years have identified
the TcR on the great majority of T cells as a disulphide-linked o,p heterodimeric
glycoprotein, in close associationon the membrane with the signal transducing CD3
complex [2]. It would be predicted that T-cell recepror usage should be related to
the mechanisms of discrimination of self from non-self, and recently Kappler and
colleagueshave produced evidence that this is so. Thus, in certain mouse strains.
tOn leave from Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovo
namesti 2, 16637 Praha 6-Dejvice, Czechoslovakia.
0896-8411/89/010001
+ 13$03.00/0
@ 1989 Academic Press Limited
M. Lipoldova
et al.
whilst there are immature thymocytes expressing the VB l7a gene segment) these
cells do not mature into T cells. A clear correlation was found with the expression
of I-E suggesting that Vp 17a was involved in I-E recognition and that Vp 17a
suppressionwas related to the processofselfrecognition [3].
In autoimmune disease, there is a failure of the mechanisms preventing self
recognition. This has been demonstrated at the T-cell level in Graves' disease,
where there is a high frequency of T cells recognizing thyroid cell surface antigens
in association with HLA Class II antigens [4]. Graves' diseaseand Hashimoto's
thyroiditis are two autoimmune diseasesof the thyroid which have distinct endocrine
sequelae,with the former being associatedwith hyperthyroidism whilst the latter,
due to destruction of the thyroid follicular cells, leads to hypothyroidism. In both
conditions, the thyroid is infiltrated by T lymphocytes many of which are activated,
as judged by the expressionof the IL-2 receptor and Class II antigens. The maiority
of the cells, including the thyroid follicular cells, expressClass II antigens. Based on
these observations, it was proposed that the pathogenesisofthese diseasesinvolved
the chronic restimulation of activated T cells by epithelial antigen-presenting cells
whose Class II expressionwas maintained by lymphokines produced by the activated
T cells [5]. Several aspectsof this hypothesis have been substantiated, with IFNy
being shown to induce Class II antigen expression on thyroid epithelial cells [6],
which can then act as antigen-presenting cells in several systems [7], including
autoreactive T-cell clones [4].
However, little is known about why these diseasesoccur in certain individuals.
Both diseasesare LILA-D region associated,as are most autoimmune diseases[8],
but the remaining components of the aetiology are not understood (reviewed in [9]).
We have investigated the nature of the T-cell receptors on the activated T cells in
Graves' and Hashimoto's thyroiditis in order to determine whether the TcR may
contribute to causing these diseases.In this study TcRB gene rearrangement was
investigated using a variety of probes. These experiments failed to show a simple
predominant pattern of rearrangement in T cells from Hashimoto's or Graves'
thyroids and indicate significant heterogeneity in these cells. These results have
implications both for the epitopes recognized on the thyroid membrane and for
TcR repertoire usage.
Materials
and methods
Patients
Intrathyroidal T lymphocytes from a patient with Hashimoto's thyroiditis (HT)
(female, aged 39 years) and from another with Graves' disease(GD) (female, aged
46 years) were used. Purification and culture of thyroid-infiltrating lymphocytes
were performed as follows: surgically removed thyroid tissue was dispersed with
collagenase(Cooper Biomedical Collagenase, Type IV, 5 pg/ml, in RPMI 1640
medium containing l5o/o fetal calf serum, Gibco) for 3h and pipetted through a
200 p mesh. The red blood cells were then lysed with an ammonium chloride buffer.
Infiltrating mononuclear cells were separated from thyroid follicular cells on
the basis of 16h adherence and purified over Ficoll gradient centrifugation
(Lymphoprep, Nyegard, Oslo, Norway). Inztiao activated T cells were selectedfrom
T-cell receptor usage in autoimmune
thyroiditis
this population by the expressionof functional IL-2 receptors, by one week's culture
in RPMI supplemented with 10o/ohuman serum and recombinant interleukin-2
(IL-2,0.025 pg/ml; Sandoz, Vienna, Austria, or Ajinomoto, Japan). The T-cell
lines were established by culturing these cells in the presence of CD3 antibody
(OKT3, 0.03 pg/ml; American Type Culture Collection, ATCC, Rockville,
Maryland, USA) together with irradiated (3,000 rad) autologous peripheral blood
mononuclear cells as feeders andrL-2 (0.025 pg/ml). This technique supplies nonspecific stimuli and allows the growth of T cells while retaining their specificity [10,
11]. After 3 weeksof culture, T-cell cloneswere prepared from the lines. Cloning and
screening of the clones for phenotype (T /T8) and thyroid autoantigen recognition
(specific proliferation in response to autologous thyroid follicular cells and thyroglobulin) was performed as previously describedfl2,l3]. A detailed description of
the clones is given elsewhere[12,l4].
Southern blot analysis
DNA from 5 x 105cells was prepared in agaroseblocks as described by Williams [ 15] .
Briefly, the cells were immobilized in low gelling temperature agarose,protein was
digested by incubation for 2 h at 50'c with proteinase K, and the products of
digestion were removed by washing with l0 mla Tris-cl pH 8.0 and I mrvrEDTA at
4"C. DNA was then digested in agarosewith one of the restriction enzymesBam HI,
EcoRI or Hind IIL The fragments were separatedby electrophoresisthrough 0.g,o/o
agarose, transferred to Amersham Hybond N nylon membrane and hybridized for
16 h at 65'c with 32Plabelled probes (specific activity 2-5 x 108dpm/pg) in 0.45 m
Nacl,0.045 msodiumcitrate, 10 x Denhardt's,0.1% sDS,5o/odextransulphateand
50 pg/ml salmon sperm DNA. Blots were washed twice for 30 min at 65'c with 0.3 m
Nacl, 0.03 rr.rsodium citrate, 0. I % sDS and once for l5 min at 65'c with d.ots rvr
Nacl, 0.0015 l' sodium citrate, 0. I % sDS and exposedat - 70'c on Kodak xAR-5
film [6].
The probes used detected cPl and cp2 sequences[17], TcRy sequences(a gift
fromDrT. Mak), vB8 (agiftfromDrT. Rabbitts) [18],vp2 andvpl l (gifts fromDr
D. Loh) [l9], and Vp5 which was subcloned from a TcR cDNA from the cell line
HPB-ALL [20].
Northern blot analysis
Total RNA from l-2 x l06cells prepared by the guanidinium cscl method
[21] was
electrophoresed on lo/o agarose [22], transferred to Hybond N and hybridized for
l6 h at 42"c witt. 32Plabelled probes (specific activity 2-5 x r08dpm/pg) in 0.75 m
Nacl, 0.075 trl sodium citrare, 50o/oformamide, 0.05 lt sodium phosphate pH 6.4,
1 x Denhardt's, 0. I o/osD s, 50 pg/ml salmon sperm DNA. Blots were washed twice
for 30 min at 50'C with 0.075 l.r NaCl, 0.0075rvrsodium citrate, 0.1% SDS.
Results
TcRp gene rearrangementsin Hashimoto's thyroiditis
In order to estimate whether one T-cell clone dominates the immune response to
Hashimoto's thyroiditis or whether the response is polyclonal, T-cell receptor rearrangementswere examined in freshly isolated thyroid mononuclear cells and in the
M. Lipoldova
et al.
f
PPf
]r o < - N
i
v N * .)
'
iiiiP*.|-i-i;
€€€€
s $+ x
E
rxxx;8838
VY--LL'LM
::II
:r
kb
'",ry
{4S:
-23.1
3.te',S1
,tu,,... ..
, ... :,;,,,,,,r.
xr
&&
-9.4
-6.5
-4.3
*a*
*
;t
*
Figure 1. Rearrangements to the CB 1 and Cp 2 loci in T-cell clones from HT. DNA from the B-celi line
was digestedwiththe
BRl8 and fromclones HTCX25-24,HTCX25-4I,I{TCX25-42andHTCX25-54
restriction enzyme EcoRI (RI) orHind III (H3) and examined by Southern blot analysisusing a CB probe.
corresponding T-cell lines grown for one month in the presenceof IL-2 and subsequently CD3 antibody. Severalrestriction enzymeswere used in these experiments.
Thus, EcoRI and Hind III were used to detect rearrangementsto the CB 1 and Cp 2
loci respectively, whereas Bam Hl was used to detect allelic rearrangements. Only
ttre 23 kb germline band was detected by Southern blotting analysis of Bam Hldigested DNA from freshly isolated thyroid mononuclear cells, whereas a smear of
rearrangementswas detected in Bam H1-digested DNA from the T-cell line grown
for one month in the presenceof IL-2 and anti-CD3 (data not shown). An additional
difuse band of about 20 kb was also detected.This band was not, however, exclusive
to Hashimoto's thyroiditis since it was also observed in other T-cell populations
grown in IL-2, such as phytohemaglutinin stimulated T blasts and T cells stimulated
with an anti-clonotypic T-cell receptor antibody 3D6, and may reflect an'average'of
many rearrangement events detected with Bam H1.
These experiments using Bam Hl failed to provide evidence for a predominant
population of T cells with rearrangementscharacteristicfor Hashimoto's thyroiditis.
Similar experiments, in which rearrangements to Cp I and CB 2 were studied
separatelyusing EcoRI and Hind III, gave similar results (data not shown).
In order to study further the TcRp rearrangementsin Hashimoto's thyroiditis, l2
T-cell cloneswere studied. A representativeblot from these experiments is shown in
Figure I and the results from the entire set of clonesare summarized in Table 1. Each
clone exhibited a different rearrangement pattern. For example, as shown in Figure
l, in clone }ITCX25-24 one Cp I gene was deleted and the second rearranged,
whereas in clone }JTCX25-41 both Cp I genes were deleted. In clone HTCX25-42
both Cp I geneswere rearranged and in clone HTCX25-54 one Cp I gene was in the
germline configuration and the second was rearranged. The CB 2 locus also showed a
different pattern of rearrangement in these clones. Thus, in clone HTCX25-24 the
T-cell receptor
usage in autoimrnune
thyroiditis
Table l. CB gene anangements in thyroid-derived T-cell clones
from a patient with
H ashimoto's thvroiditis
HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX
25-7t 25-13 25-t9 25*66 25_76 25_82 25_15 25__242541 2542 25_54
.f4
BR18 T4
T4
T8
T8
T4
T4
T8
T4
T4
CBl
CF2
GDDDDGRRDDRR
GRDDDRRRRDGG
GRRGGRGRRG
GRGGGGGRGG
The phenotypes (T4 or T8-positive) ofthe clones used are presented under their names.
Rearrangements (G: germline, D : deleted, R: rearranged) to the cp 1 and cB 2 loci are shown.
cB 2 gene(s)were in a germline configuration, whereas in clones lHTCX25-41 both
cB 2 genes were rearranged. clone HTCK25-42 exhibited one cB 2 gene in the
germline configuration and a deleted allele. In clone HT CX2-54C8 2 gene(s)were in
the germline configuration. There was no correlation between the phenotype (T4+
or T8+) and the rearrangement pattern of the respective clones (Table l). Taken
together, these data indicate that at the clonal level there is no predominant TcRp
gene rearrangement associatedwith Hashimoto's thyroiditis. The clones used were
of independent origin. This conclusion is confirmed by the results of Southern
blotting analysisusing a TcRy geneprobe in which multiple different rearrangement
patterns were also found (Figure 2).
Eighteen vB subfamilies have been described for the human TcRB locus
123).twe
have studied the rearrangement and use of four vB subfamilies, vp 2, v g 5,VB g and
VB I I which together constitute at least 25o/o@s estimated by the number of bands
hybridizing on southern blots) of human vp genes and are dispersed over about
400kb of DNA [23]. Sequenceshybridizing with a vB probe can in principle be
either rearranged in T-cell DNA, indicating non-productive or productive rearrangement to the TcRB constant region, or deleted, indicating usage of a more 5,
VB gene (Figure 3).
The results of Southern blotting analysis using the Vp probes are summarized in
Table2. Representativeblots are shown in Figure 3. No deletions or rearrangements
were observed using a vB 5 probe, consistent with this subfamily being the most
distant from the CB locus of the four subfamilies studied
[23]. Co-deletions in parts
of the vp 8 and vp I I subfamilies in clones HTCX25-19, HTCX25-g4HTCx2566,HTCX25-76, and HTCX25-15, probably reflect linkage disequilibrium between
the two vB families. A linkage disequilibrium between a 2 kb fragment of the vp g
subfamily and a25 kb fragment of the vp ll subfamily, and a 23 kb band from VB g
and22 kb band from vB I I has been describ ed,[2al.Thus, the patient from whom the
present clones have been derived is probably heterozygous for these differently
linked fragments.
Rearranged bands which were coincident with bands hybri dizing to a cB probe
were observed in clone HTCX25-82 using vB 2 and VB g probes. To assesswhether
M. Lipoldova
et al,
fi)
\
co
N
N
I
lr)
N
x
(-)
F
T
m
Nrn
N-
llro
|r):1
;ax;
*
a)
r
v
-
T!
kb
23,t 9.4 -
43-
2?'-
2 . O-
ffi
Figure 2. Rearrangements to the TcRy locus in HT T-cell clones. DNA from BRlS and from clones
HTaXZ5-22 and HTCX25-15 was digested with the restriction enzyme Bam HI (B) or Hind III (H3),
electrophoresed and hybridized with a TcR^y probe.
one of these rearrangementswas productive, total RNA was isolated from the clones
HTCX25-15, HTCX25-41, and HTCX25-82, and probed for vB expression.The
results of this experiment are shown in Figure 4. Using aV92 probe a 1.3kb
transcript was detected in RNA from clone HTCX25-82 but not in the other clones
analysed. When this blot was hybridized with a CB probe, 1.3 kb transcripts were
detected in all samples, although the weakest hybridization was in the HTCX-82
track.
in Grqaes' disease
TcRp rearcangements
Six clones from a patient with Graves'thyroiditis (GT) were studied. Phenotypes
and thyroid autoantigen specificity are listed in Table 3. Three of the six clones
proliferated in responseto autologous thyroid follicular cells, suggesting specificity
to one or several not yet defined membrane-bound autoantigens. None of the six
clones reacted to thyroglobulin. Figure 5 shows the allelic TcRB rearrangements in
theseclones.As was the casefor HT, no predominant rearrangementswere detected,
demonstrating that all the clones were of independent origin. The rearrangements
involving Vp 2 and Vp 5 were also studied. No deletions or rearrangements were
found using a Vp 5 probe. After hybridizing with aYB 2 probe the T-cell clone 14.4
showed rearranged bands and clones 814.9 and Bl4.l2 showed deletions in this
subfamily. However no predominant use of these subfamilies was detected. T-cell
T-cell receptor usage in autoimmune
N
O
I
(O
(o
O
F
I
N(o(o
ol.-(ou)
Illl
tr)|r)Ir)La)
NNNN
SgSo
t.-6Y
|r) | lJr-l ) l r ) | ( )
N
NC\JN
)< X X X m
llll
tr)|r)lr)'A
NNNN
X
xXx
Yi-Yl,
OQO=
FFI_ft
IITco
thyroiditis
KO
()tr)tr)r?
NNNN
xxx><m
999e=
co
t
m
TT
N@(oo,
@f-(o;
tltL
XXXX
TTM
.ffi
KD
"
,'
23.1-
?3.t-
)_ .
r'*
Ii
6 . 5-
fin
KO
2 3 . 1-
I
l
;.l;
9 . 4-
co
E
co
OOOO
FFFF
I:fI:T
9.4-
94-
6 . 5-
:
4 . 3- f
;
la
a"
*
6.5-
l
4.3 -
*'lw
+i
iI
i
I
.
t#.
l
i , ,.-"'
t"..,.1,,-..-.-j
t
M'rs
&.d.' ,,
B
Figure 3. Southern blot analysis of four Vp subfamilies in HT T-cell clones. DNA from clones
HTCX25-82, HTCX25-76' HTCX25-66, HTCX25-19 and BRl8 was digested with Bam Hl and hybridized in sequencewith (A) vB 2, (B) vB 5, (c) vB 8 and (D) vB l l probei. After each hybridization the
blot was stripped and checked for absenceof a signal before rehybridizing.
receptor rearrangements did not distinguish thyroid autoreactive and non-specific
clones and the three autoreactive clones did not show a common rearrangement
parrern.
Discussion
There is considerable evidence that T cells are intimately involved in both human
autoimmune diseasesand animal models. The chronic phaseor perpetuation of many
human autoimmune diseases,such as thyroiditis or insulin-dependent diabetes,
depends on the HLA Class II-expressing target tissues, and autoreactive T-helper
cells. These cells in turn produce mediators which further induce tissue Class II
expressionand initiate the inflammation and destruction of the target tissues .
[5]
Evidence to support this concept has come from a variety of sources. First,
immunohistological analysishas shown that most autoimmune diseases(e.g. Graves'
and Hashimoto's thyroiditis, diabetes, rheumatoid arthritis) over-express HLA
Class II in the tissues [e.g. 9, 25]. Histological and functional analysis shows that
there are many activated T-cells infiltrating the tissues in these diseases
[4]. These
cells may be cloned, and in Graves' diseaseit has been shown that many of these are
restimulated by HLA Class Il-expressing thyrocytes [4]. products of activated T
cells such as IFN1 and tumour necrosis factor or lymphotoxin are involved in the
inductionof ClassII intissue cellsinztilzo[6,26f.
In animal models, the role of T cells has been demonstrated even more directly. T
cells and T-cell clonesmay transfer a variety of diseases,such as experimental allergic
r-
T-cell receptor usage in autoimmune
Nrtr)
OV
thyroiditis
s+u'
lll
lr)lr)Lr)
()tr,l;
aNNN
f-NXXX
FCO-JOOO=
]
/)COFFFE
I-O-Ir=m
@NNN
r'-Nxxx
co
E
c0
FCOJOOC)
f(,MFF
-IO-TII
v;:
t:.7:',
a.t
- 28S
- 28S
-18s
iRt::i
&
:5
5
t
-, ,
A
B
Figure 4' Expression of Vp 2 and Cp transcripts. Total cellular RNA from BR18 (5 x 106cells), T-cell
clones HTCX25-15, HTCX25 -4l,}{TCX25-82 (106cells), peripheral blood lymphocytes grown in the
p r e s e n c e o fI L - 2 ( 5 x 1 0 6 c e l l s ) a n dI p g p o l y ( A ) + R N A f r o m T - c e l l l i n e s H U T T 8 a n d H S B 2 w e r e u s e d f o r
Northern hybridization analysis. The RNA was hybridized with a VB 2 probe (A), stripped and rehybridized with a CB probe (B). Exposure time in Figure 4A was 10 d at - 70'C. In Figure 4BBRI8, HTCk2515' HTCX25-41 and HTCX25-82 tracks were exposed overnight at -70'C and peripheral blood
lymphocytes, HUT78 and HSB2 tracks for 2 h at room temDerarure.
Table 3. Phenotype and thyroid autoantigen reactiaity of thyroid-derizted T-cell clones
from a patient with Graaes' disease
Clone No:
Phenotype
(T4 : helper/T8 : cytotoxic suppressor)
autologous thyroid
follicular cells
thyroid autoanrigen
reactivity
thyroglobulin
B 1 4 . 18 1 4 . 3 8 1 4 . 4 8 1 4 . 5 B 1 4 . 1 0 B L 4 . t 2
T8
T4
T4
T4
T4
T4
+++
cells recognize. Some of the GD clones recognizethyrocytes, but the lack of residual
thyrocytes makes it impossible to ascertain if this is the casein HT. However, the
expressionof IL-2 receptor implies very recent activation (within the last week [30]),
and thus these clones are clearly relevant to the diseaseprocess. It is conceivable
that a single dominant clone may be responsible for the autoimmune diseaseprocess
- I8S
10
M. Lipoldova
et al,
Noo.o
sd-d-vs-.;
commmm$6
KD
4 . 3-
2'3-
''':i:"'"' ''i
2.O_,
.,
Figure 5. Rearrangements to the CB I and Cp 2 loci in T-cell clones from GD. DNA was digested with
Bam Hl and hybridized after electrophoresiswith a Cp probe.
as proposed by Burnet [31] . However, the heterogeneity of autoantibodies detected
in GD and HT would seem to render unlikely the idea that a single helper T-cell
clone could be responsible,on the basisof linked recognition of T and B cell epitopes
[32]. The results presented here demonstrate that no predominant F gene rearrangement is found in infiltrating cells, autoreactive T-cell lines and clones in either
Flashimoto's or Graves' thyroiditis. In the latter diseaseit was possible to ascertain
that these were autoreactive T cells (Table 3) and even these were heterogeneousin
their rearrangement pattern, with the sizesof the observed rearrangementsimplying
different Vp usage. Productive TcRB gene rearrangements are generated in two
stages,namely DB to lB and VB to DFIp. In principle, different rearrangements,as
detected by Southern hybridization analysis,could be generatedby the use ofdifferent Vp and/or JB segments.Although none of the four Vp families studied here are
preferentially utilized in clones from HT, it cannot be ruled out that another VB
subfamily is used by these clones without the use of additional VB probes or direct
DNA cloning. However, the variation in sizeof the observedrearrangementsstrongly
suggestsdifferent Vp usagein these clones in addition to any differential JB usage.
The results presented here provide the first molecular data about T-cell receptors
in HT and GD obtained by studying autoimmune clones derived from the activated
T-cell pool. Similar experiments in other autoimmune diseaseshave given conflicting results. Thus, in human multiple sclerosis(MS) IL-2 dependent clones derived
from active MS plaques and blood, two of seven clones from blood and one of six
clones from brain exhibited the same rearrangement pattern [33]. In thymus and
peripheral lymph nodes of /pr/lpr andglrlglr mice which develop a diseasesimilar to
systemic lupus erythematosis, non-clonal rearrangementsof TcRp were found [34].
In T-cell populations grown from synovial specimensfrom patients with rheumatoid
arthritis, distinct B gene rearrangements were detected in 13 of 14 cultures,
suggestinga limited number of T-cell specificities [35].
T-cell receptor usage in autoimrnune
thyroiditis
11
Analysis of a variety of model systems has indicated that clones recognizing a
defined antigen and restricted to a defined MHC allele generally use a restricted
number of Vo and/or VB genes. In the immune response to pigeon cytochrome c,
Fink er al. have shown that in five TcRs studied, TcR gene usage correlated with
epitope specificity. Thus three different receptors using closely related Vo and vp
genes were identified with VB usage, correlating with MHC specificity of the TcR
[36]. Consistent with this finding was the transfer of MHC specificity by rransfection
of a TcRB gene from a TcR recognizing moth cytochrome c plus IEb [32]. In addition
lTinoto et al. demonstratedthat there was predominant use of a Vo gene in severalTcell clones recognizing pigeon cytochrome c [38]. Also, dominance of one TcR has
been found in the responseto a simple determinant TNP [39]. However, in another
study different specificities of two TcRs with the same vo and vB regions were
obtained by usage of separate diversity and joining regions [40]. Taken together,
these results indicate that T-cell clones recognizing the sameor very similar epitope
tend to utilize a severelyrestricted number of V regions. The epitopes recognized by
autoreactive T cells on thyroid tissue are unknown. However, the observed heterogeneity of VB expression suggeststhat multiple epitopes are recognized as foreign.
Alternatively epitope recognition may indeed be restricted but may be associated
primarily with Vo (or Ju) expression. The extended nature of the Jo locus makes
analysis of gene rearrangement using Southern blotting generally uninformative.
Detailed sequenceanalysiswill be required to analysefurther both Vo and VB usage
in these clones.
Recently, such an analysis was described with murine T-cell clones recognizing
peptide l-9 of myelin basic protein in PL/J and (PL/J x SJL) mice [41]. A very
restricted usage of TcR Va, vB and J segments was nored. Sequence analysis of
the HT and GD clones described here will determine whether a difference in the
heterogeneity of the T-cell response indeed exists between these two autoimmune
diseases.
Acknowledgernents
we thank Drs T. Mak, c. Terhorst, T. Rabbitts and D. Loh for gifts of probes and
Ms Celia Joseph for typing this manuscript. This study was supported by ICRF
(M.L., M.J.O.), the liTellcome Trust, Juvenile Diabetes Foundation and British
Council (M.L., B.G-L and M.F).
References
1. Nossal, G. J. v. 1983. cellular mechanisms of immunologic tolerance. Ann. Rez,t.
Immunol. lz33-62
Meuer, S. C., K. A. Fitzgerald, R. E. Flussey, J. C. Hodgon, S. F. Schlossman, and
E. L. Reinheru. 1983. Clonotypic structures involved in antigen-specific human T-cell
function. J. Exp. Med. 157: 705-7 19
3 . Kappler, J. II(/., N. Roehm, and P. Marrack. 1987. T-cell tolerance by clonal elimination
in the thymus. Cell 49:273-280
4 . Londei, M., G. F. Bottazzo, and M. Feldmann. 1985. Human T-cell clones from auroimmune thyroid glands: specific recognition of autologous thyroid cells. Science 2282
85-89
t2
M. Lipoldova
et al.
5. Bottazzo, F. G., R. Pujol-Borrell, T. Flanafusa,and M. Feldmann. 1983.Role of aberrant
HLA-DR expression and antigen presentation in endocrine autoimmunity. Lancet 2z
1115-1119
6. Todd, I., R. Pujol-Borrell, L. J. Hammond, G. F. Bortazzo, and M. Feldmann. 1985.
Interferon-1 induces HLA-DR expression by thyroid epithelium. Clin. Exp. Immunol.
6L2265-273
7. Londei, M., J. R. Lamb, G. F. Bottazzo, and M. Feldmann. 1985.Epithelial cells expressing aberrant MHC Class II determinants can present antigen to cloned human T cells.
Nature 3l2z 639-64I
8. Immunol.Redews.1983.Vol. 70.
perspective. Ann. Rezt.
9. Smith, H. R. and A. D. Steinberg. 1983. Autoimmunity-a
Immunol. lt 175-2lO
10. Padula, S. J., M. K. Pollard, E. G. Lingenheld, and R. B" Clark. 1985. Maintenance of
antigen specificity by human interleukin-2-dependent T-cell lines, use of antigen presenting cells and OKT3 antibody in the absenceof antigen. J. Clin. Invesr. T5:788-797
I l. Londei, M., B. Grubeck-Loebenstein, P. deBerardinis, C. Greenall, and M. Feldmann.
1988. Efficient propagarion and cloning of human T cells in the absence of antigen using
OKT3, IL-2 and antigen-presenting cells. S cand.J. Immunol. 17z 3546
12. Londei, M., B. Grubeck-Loebenstein, C. Greenall, M. Turner, and M. Feldmann. 1987.
Comparison of cellular infiltrate of Hashimoto's thyroiditis in aiao and after in ztito cell
growth. Acta Endouinol. ( Kbh) 115 (suppl 281): 86-89
13. Grubeck-Loebenstein, B., M. Londei, C. Greenall, K. Pirich, H. Kassal, \$[' lfaldhausl,
and M. Feldmann. 1988. The pathogenic relevanceof HLA-Class II expressing thyroid
follicular cells in non-toxic goitre and in Graves' disease.J. Clin. Inaest.81: 1608-1614
14. Grubeck-Loebenstein, B., M. Turner, M. Londei, K. Pirich, rW. \(/aldhausl, and M.
Feldmann, The production of cytokines by thyroid derived helper T-cell clones in
patients with Graves' disease. (In preparation)
15. \Tilliams, G. T. 1987. High efficiency preparation of DNA from limiting quantities of
eukaryotic cells for hybridization analysis. Gene 53212l'126
16. Feinberg, A. O. and B. Vogelstein. 1984. A technique for radiolabeling DNA restriction
endonucleasefragments to high specific activity. Anal. Biochem. 137t266-267
17. Collins, M. K. L., A. M. Kissonerghis,M. J. Dunne, C. J. ril7atson,P.W.J.Rigby, and
M. J. Owen. 1985. Transcripts from an aberrantly rearranged human T-cell receptor Bchain gene. EMBO J. 4z l2ll-1215
18. Sims, J. E., A. Tunnacliffe, Itr7.J. Smith, and T. H. Rabbitts. 1984. Complexity of
human T-cell antigen receptor p-chain constant- and variable-region genes. Nature 3l2z
54t-545
19. Tillinghast, J. P., M. A. Behlke, and D. Y. Loh. 1986. Structure and diversity of the
human T-cell receptor p-chain variable region genes.Science233:879-883
20. Yoshikai, T., D. Antoniou, S. P. Clark, Y. Yanagi, R. Sangster, P. van den Elsen, C.
Terhorst, and T. $7. Mak. 1984. Sequenceand expression of transcripts of the human
T-cell receptor p-chain gerres.Nature 3l2z 52L-524
21. Chirgwin, J., A. Przybyla, R. MacDonald, and tJ7.Rutter. 1979. Isolation of biologically
active ribonucleic acid from sources enriched in ribonucl ease.Biochemisty 18:5294-5299
22. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
23. La| E., P. Concannon, and L. Hood. 1988. Conserved organisation of the human and
murine T-cell receptor p-gene families. Nature 33lt 54i-546
24. Concannon, P., R. A. Gattis, and L. E. Hood. 1987. Human T-cell receptor Vp gene
polymorphism.J. Exp.Med. 165z1130-1 140
25. Hanafusa, T., R.Pujol-Borrell, L. Chiovato, R. C. G. Russell, D. Doniach, and G. F.
antigen on thyrocytes in Graves'
Bottazzo. 1983. Aberrant expression of HLA-DR
disease:relevancefor autoimmunity. Lancet 2: I I I 1-1 I I 5.
26. Pujol-Borrell, R., I. Todd, M. Doshi, G. F. Bottazzo, R. Sutton, D. Gray, G. R. Adolf,
andM.Feldmann. lgsT.HLAClassllinductioninhumanisletcellsbyinterferon-yplus
tumour necrosis factor or lymphotoxin. N ature 3262304-306
T-cell receptor usage in autoimmune
thyroiditis
13
27. Holmdahl, R., L. Klareskog, K. Rubin, E. Larsson, and H. !(rigzell. r9g5. scand.
J.
Immunol.22:295-306
28. Hayosh, N. S. and R. H. Swinborg. 1987. Autoimmune effector cells: inhibition of adoptive transfer of autoimmune encephalomyelitis with a monoclonal antibody specific for
interleukin-2 receptors.J. Immunol. l3gz 3771-3775
29. Todd, I., R' Pujol-Borrell, L. J. Hammond, J.M.McNally, M. Feldmann, and
G. F.
Bottazzo. 1986. Enhancement of thyrocyte HLA Class II eipression by thyroid stimulating hormon e. Clin. Exp. Immunol. 69: 524-531
30. Boitard c., S. Michie, P. serrurier, G. Butcher, A. Larkius, and H. o. McDevitt. 19g5.
In aizto prevention of thyroid and pancreatic auroimmunity in the BB rat by antibody ro
classllmajorhistocomparibilirycomprexgeneproducts. proc.Narl.Acad.sci.usAtz:
6627-6631
31. Burnet, F. M. 1959. The clonal serection Theory of Acquired Immunity. cambridge
University Press, London.
32. Mitchison, N. A. 1983. The rise and fall of the anrigen bridge. ln: Immunoregulation.
N. Fabris, E. Garaci, J. Hadden and N. A. Michison, eds. plenum press, N& york.
pp.3543
33. Haffer, D. A., A. D. Duby, D. Benjamin, J. G. seidman, and H.
J. !7einer. 19g6.
Neurology 36 (suppl l)t314
34. Mowrfiz, J. D., K. Huppi, J. F. Mushinski, M. F. Seldin, and A. D. Steinberg. 19g6.Fed.
Proc. 4527 12
35. Stamenkovic, I., M. Ste-gagno,K. A. !7right, S. M.Krane, E. p. Amento, R. B.
Colvin,
R. J. Duquesnoy, and J. T. Kumick. rggg. clonar dominance among T-lymphocyti
infiltrates in arthritis. Proc. Natl. Acad. Sci. USAg5z 1179_11g3
36. Fink, J. P., L. A. Matis, D. L. McElligott, M. Bookman, and S. M. Hedrick. 19g6.
Correlations between T-cell specificity and the structure of the antigen receptor. Nature
321:219-226
37. Saito, T. and R' N. Germain. 1987. Predictable acquisition of a new MHC recognition
specificity following expression of a transfected T-ceil receptor
B-chain gene. NatureJ2gz
256-259
38. \winoto, A., J. L. IJrban, N. c. Lan, J. Goverman, L. Hood, and D. Hansburg. 19g6.
Predominant use of a Vc, gene segment in mouse T-cell receptors for cytochr-ome
C.
Nature 324:679-682
39. Hochgeschwender, u., H. G. Simon, H. U. lfeltien, F. Bartels, A. Becker, and
J. T.
Epplen. 1987. Dominance of one T-cerl receptor in the H2Kb/TNF responseNa trri
326
307-309
40. Rupp, F., J. Brecher, M.A. Giedlin, T. Mosmann, R. M. Zinkernager, H. Hengartner,
and N. Joho. 1986. T-cell antigen receptors with identical variable rigions but d-itrereni
diversity and joining region gene segments have distinct specificities but cross-reacrive
idiotypes. Proc. Natl. Acad. Sci. USAg4:2lg-223
41. Acha-orbea, H., D. J. Mitchell, L. Timmermann, D. c. vraith, G. S. Tausch,
M.K.
'Waldor,
S. S. Zamvil, H. O. McDevitt, and L. Steinman. 1988. Limited heterogeneity of
T-cell receptors from lymphocytes mediating autoimmune encephalomyeliiis
allows
specific immune intervention. Ceil 54:263-273