Journal of Autoimmunity (1989) 2, l-13

Analysis of T-cell Receptor Usage in ActivatedT-cell Clones frorn Hashirnoto's Thyroiditis and

Graves'Disease

Marie Lipoldovar*t Marco Londeirf Beatrix Grubeck-Loebenstein,f Marc Feldmannf and MichaelJ. Owen*

* Imperial Cancer Research Fund Laboratories, St Bartholomeut's Hospital, DominionHouse' 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 studiedin T cells derived from the thyroid glands of a patient with Hashimoto's(HT) and another with Graves' (GD) autoimmune thyroiditis. The cellsstudied were freshly isolated mononuclear cells, T-celi lines grown in thepresence of anti-cD3 and.lL-2 and r-cell clones. Numerous different re-arrangernents to the constant regions of rcRp and rcRy and in the variablegene region of rcRB were observed. These findings indicate that the T-cellresponse in autoimrnune thyroiditis is multiclonal and may have impli-cations for the epitopes recognized by autoreactive T cells and for themechanisms of the disease.

To function effectively, the immune system must be able to distinguish self fromnon-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 specificities isthe T-cell receptor (TcR) for antigen. Studies over the past 3 years have identifiedthe TcR on the great majority of T cells as a disulphide-linked o,p heterodimericglycoprotein, in close association on the membrane with the signal transducing CD3complex [2]. It would be predicted that T-cell recepror usage should be related tothe mechanisms of discrimination of self from non-self, and recently Kappler andcolleagues have produced evidence that this is so. Thus, in certain mouse strains.

tOn leave from Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovonamesti 2, 16637 Praha 6-Dejvice, Czechoslovakia.

0896-841 1/89/010001 + 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

suppression was related to the process ofselfrecognition [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 antigensin association with HLA Class II antigens [4]. Graves' disease and Hashimoto's

thyroiditis are two autoimmune diseases of the thyroid which have distinct endocrine

sequelae, with the former being associated with 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 expression of the IL-2 receptor and Class II antigens. The maiority

of the cells, including the thyroid follicular cells, express Class II antigens. Based on

these observations, it was proposed that the pathogenesis ofthese diseases involved

the chronic restimulation of activated T cells by epithelial antigen-presenting cells

whose Class II expression was maintained by lymphokines produced by the activatedT cells [5]. Several aspects of 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], includingautoreactive T-cell clones [4].

However, little is known about why these diseases occur in certain individuals.

Both diseases are 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 inGraves' and Hashimoto's thyroiditis in order to determine whether the TcR may

contribute to causing these diseases. In this study TcRB gene rearrangement wasinvestigated using a variety of probes. These experiments failed to show a simplepredominant 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, aged46 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 selected from

T-cell receptor usage in autoimmune thyroiditis

this population by the expression of functional IL-2 receptors, by one week's culturein RPMI supplemented with 10o/o human serum and recombinant interleukin-2(IL-2,0.025 pg/ml; Sandoz, Vienna, Austria, or Ajinomoto, Japan). The T-celllines 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 bloodmononuclear cells as feeders andrL-2 (0.025 pg/ml). This technique supplies non-specific stimuli and allows the growth of T cells while retaining their specificity [10,11]. After 3 weeks of culture, T-cell clones were prepared from the lines. Cloning andscreening of the clones for phenotype (T /T8) and thyroid autoantigen recognition(specific proliferation in response to autologous thyroid follicular cells and thyro-globulin) was performed as previously describedfl2,l3]. A detailed description ofthe clones is given elsewhere [12,l4].

Southern blot analysis

DNA from 5 x 105 cells was prepared in agarose blocks as described by Williams [ 1 5] .Briefly, the cells were immobilized in low gelling temperature agarose, protein wasdigested by incubation for 2 h at 50'c with proteinase K, and the products ofdigestion were removed by washing with l0 mla Tris-cl pH 8.0 and I mrvr EDTA at4"C. DNA was then digested in agarose with one of the restriction enzymes Bam HI,EcoRI or Hind IIL The fragments were separated by electrophoresis through 0.g,o/oagarose, transferred to Amersham Hybond N nylon membrane and hybridized for16 h at 65'c with 32P labelled probes (specific activity 2-5 x 108 dpm/pg) in 0.45 mNacl,0.045 msodiumcitrate, 10 x Denhardt's,0.1% sDS,5o/o dextransulphateand50 pg/ml salmon sperm DNA. Blots were washed twice for 30 min at 65'c with 0.3 mNacl, 0.03 rr.r sodium citrate, 0. I % sDS and once for l5 min at 65'c with d.ots rvrNacl, 0.001 5 l' sodium citrate, 0. I % sDS and exposed at - 70'c on Kodak xAR-5fi lm [6].

The probes used detected cPl and cp2 sequences [17], TcRy sequences (a giftfromDrT. Mak), vB8 (agiftfromDrT. Rabbitts) [18],vp2 andvpl l (gifts fromDrD. Loh) [l9], and Vp5 which was subcloned from a TcR cDNA from the cell lineHPB-ALL [20].

Northern blot analysis

Total RNA from l-2 x l06cells prepared by the guanidinium cscl method [21] waselectrophoresed on lo/o agarose [22], transferred to Hybond N and hybridized forl6 h at 42"c witt. 32P labelled probes (specific activity 2-5 x r08 dpm/pg) in 0.75 mNacl, 0.075 trl sodium citrare, 50o/o formamide, 0.05 lt sodium phosphate pH 6.4,1 x Denhardt's, 0. I o/o sD s, 50 pg/ml salmon sperm DNA. Blots were washed twicefor 30 min at 50'C with 0.075 l.r NaCl, 0.0075 rvr sodium citrate, 0.1% SDS.

Results

TcRp gene rearrangements in Hashimoto's thyroiditis

In order to estimate whether one T-cell clone dominates the immune response toHashimoto's thyroiditis or whether the response is polyclonal, T-cell receptor re-arrangements were examined in freshly isolated thyroid mononuclear cells and in the

M. Lipoldova et al.

k b- 2 3 . 1

- 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 lineBRl8 and fromclones HTCX25-24,HTCX25-4I,I{TCX25-42andHTCX25-54 was digestedwiththerestriction enzyme EcoRI (RI) orHind III (H3) and examined by Southern blot analysis using a CB probe.

corresponding T-cell lines grown for one month in the presence of IL-2 and subse-quently CD3 antibody. Several restriction enzymes were used in these experiments.Thus, EcoRI and Hind III were used to detect rearrangements to the CB 1 and Cp 2

loci respectively, whereas Bam Hl was used to detect allelic rearrangements. Onlyttre 23 kb germline band was detected by Southern blotting analysis of Bam Hl-digested DNA from freshly isolated thyroid mononuclear cells, whereas a smear ofrearrangements was detected in Bam H1-digested DNA from the T-cell line grown

for one month in the presence of IL-2 and anti-CD3 (data not shown). An additionaldifuse band of about 20 kb was also detected. This band was not, however, exclusiveto Hashimoto's thyroiditis since it was also observed in other T-cell populationsgrown in IL-2, such as phytohemaglutinin stimulated T blasts and T cells stimulatedwith an anti-clonotypic T-cell receptor antibody 3D6, and may reflect an'average'ofmany rearrangement events detected with Bam H1.

These experiments using Bam Hl failed to provide evidence for a predominantpopulation of T cells with rearrangements characteristic for Hashimoto's thyroiditis.Similar experiments, in which rearrangements to Cp I and CB 2 were studiedseparately using EcoRI and Hind III, gave similar results (data not shown).

In order to study further the TcRp rearrangements in Hashimoto's thyroiditis, l2T-cell clones were studied. A representative blot from these experiments is shown inFigure I and the results from the entire set of clones are summarized in Table 1. Eachclone 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 genes were rearranged and in clone HTCX25-54 one Cp I gene was in thegermline configuration and the second was rearranged. The CB 2 locus also showed adifferent pattern of rearrangement in these clones. Thus, in clone HTCX25-24 the

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T-cell receptor usage in autoimrnune thyroiditis

Table l. CB gene anangements in thyroid-derived T-cell clones from a patient withH ashimoto' s thvroiditis

HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX HTCX25-7t 25-13 25-t9 25*66 25_76 25_82 25_15 25__24 2541 2542 25_54

BR18 T4 T4 T8 T8 T4 T4 T8 T4 T4 .f4

C B l

C F 2

G D D D D G R R D D R RG R D D D R R R R D G GG R R G G R G R R GG R G G G G G R G G

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 bothcB 2 genes were rearranged. clone HTCK25-42 exhibited one cB 2 gene in thegermline configuration and a deleted allele. In clone HT CX2-54C8 2 gene(s) were inthe germline configuration. There was no correlation between the phenotype (T4+or T8+) and the rearrangement pattern of the respective clones (Table l). Takentogether, these data indicate that at the clonal level there is no predominant TcRpgene rearrangement associated with Hashimoto's thyroiditis. The clones used wereof independent origin. This conclusion is confirmed by the results of Southernblotting analysis using a TcRy gene probe in which multiple different rearrangementpatterns were also found (Figure 2).

Eighteen vB subfamilies have been described for the human TcRB locus 123).twehave studied the rearrangement and use of four vB subfamilies, vp 2, v g 5,VB g andVB I I which together constitute at least 25o/o @s estimated by the number of bandshybridizing on southern blots) of human vp genes and are dispersed over about400kb of DNA [23]. Sequences hybridizing with a vB probe can in principle beeither rearranged in T-cell DNA, indicating non-productive or productive re-arrangement 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 inTable2. Representative blots are shown in Figure 3. No deletions or rearrangementswere observed using a vB 5 probe, consistent with this subfamily being the mostdistant from the CB locus of the four subfamilies studied [23]. Co-deletions in partsof the vp 8 and vp I I subfamilies in clones HTCX25-19, HTCX25-g4HTCx25-66,HTCX25-76, and HTCX25-15, probably reflect linkage disequilibrium betweenthe two vB families. A linkage disequilibrium between a 2 kb fragment of the vp gsubfamily and a25 kb fragment of the vp ll subfamily, and a 23 kb band from VB gand22 kb band from vB I I has been describ ed,[2al.Thus, the patient from whom thepresent clones have been derived is probably heterozygous for these differentlylinked fragments.

Rearranged bands which were coincident with bands hybri dizing to a cB probewere observed in clone HTCX25-82 using vB 2 and VB g probes. To assess whether

M. Lipoldova et al,

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9 . 4 -

4 3 -

2 ? ' -

2.O -

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 rearrangements was 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.

TcRp rearcangements in Grqaes' disease

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 response to 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

these clones. As was the case for HT, no predominant rearrangements were 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

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Figure 3. Southern blot analysis of four Vp subfamilies in HT T-cell clones. DNA from clonesHTCX25-82, HTCX25-76' HTCX25-66, HTCX25-19 and BRl8 was digested with Bam Hl and hybri-dized in sequence with (A) vB 2, (B) vB 5, (c) vB 8 and (D) vB l l probei. After each hybridization theblot was stripped and checked for absence of a signal before rehybridizing.

receptor rearrangements did not distinguish thyroid autoreactive and non-specificclones and the three autoreactive clones did not show a common rearrangementparrern.

Discussion

There is considerable evidence that T cells are intimately involved in both humanautoimmune diseases and animal models. The chronic phase or perpetuation of manyhuman autoimmune diseases, such as thyroiditis or insulin-dependent diabetes,depends on the HLA Class II-expressing target tissues, and autoreactive T-helpercells. These cells in turn produce mediators which further induce tissue Class IIexpression and initiate the inflammation and destruction of the target tissues [5] .

Evidence to support this concept has come from a variety of sources. First,immunohistological analysis has shown that most autoimmune diseases (e.g. Graves'and Hashimoto's thyroiditis, diabetes, rheumatoid arthritis) over-express HLAClass II in the tissues [e.g. 9, 25]. Histological and functional analysis shows thatthere are many activated T-cells infiltrating the tissues in these diseases [4]. Thesecells may be cloned, and in Graves' disease it has been shown that many of these arerestimulated by HLA Class Il-expressing thyrocytes [4]. products of activated Tcells such as IFN1 and tumour necrosis factor or lymphotoxin are involved in theinductionof Class II intissue cellsinzti lzo[6,26f .

In animal models, the role of T cells has been demonstrated even more directly. Tcells and T-cell clones may transfer a variety of diseases, such as experimental allergic

T-cell receptor usage in autoimmune thyroiditis

cotm

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l r ) l r ) L r )@ N N Nr ' - N x x xF C O J O O C )f ( , M F F- I O - T I I

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( ) t r , l ;a N N Nf - N X X XF C O - J O O O =] / ) C O F F F EI - O - I r = m

T-cell receptor usage in autoimmune thyroiditis

- 28S

- 1 8 s

Figure 4' Expression of Vp 2 and Cp transcripts. Total cellular RNA from BR18 (5 x 106 cells), T-cellclones HTCX25-15, HTCX25 -4l,}{TCX25-82 (106 cells), peripheral blood lymphocytes grown in thepresenceof IL-2(5 x 106cel ls)and I pgpoly(A)+ RNAfromT-cel l l inesHUTT8andHSB2wereusedforNorthern hybridization analysis. The RNA was hybridized with a VB 2 probe (A), stripped and rehybri-dized with a CB probe (B). Exposure time in Figure 4A was 10 d at - 70'C. In Figure 4BBRI8, HTCk25-15' HTCX25-41 and HTCX25-82 tracks were exposed overnight at -70'C and peripheral bloodlymphocytes, HUT78 and HSB2 tracks for 2 h at room temDerarure.

Table 3. Phenotype and thyroid autoantigen reactiaity of thyroid-derizted T-cell clonesfrom a patient with Graaes' disease

Clone No: B14.1 814.3 814.4 814.5 B14.10 BL4. t2

- 28S

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BA

Phenotype(T4 : helper/T8 : cytotoxic suppressor)

autologous thyroidfollicular cells

thyroid autoanrigenreactivity

thyroglobulin

T8 T4 T4 T4+++

T4 T4

cells recognize. Some of the GD clones recognize thyrocytes, but the lack of residualthyrocytes makes it impossible to ascertain if this is the case in HT. However, theexpression of IL-2 receptor implies very recent activation (within the last week [30]),and thus these clones are clearly relevant to the disease process. It is conceivablethat a single dominant clone may be responsible for the autoimmune disease process

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2'3- ' ' ' : i : " ' " ' ' ' i

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Figure 5. Rearrangements to the CB I and Cp 2 loci in T-cell clones from GD. DNA was digested withBam Hl and hybridized after electrophoresis with a Cp probe.

as proposed by Burnet [31] . However, the heterogeneity of autoantibodies detectedin GD and HT would seem to render unlikely the idea that a single helper T-cellclone could be responsible, on the basis of linked recognition of T and B cell epitopes

[32]. The results presented here demonstrate that no predominant F gene rearrange-ment is found in infiltrating cells, autoreactive T-cell lines and clones in eitherFlashimoto's or Graves' thyroiditis. In the latter disease it was possible to ascertainthat these were autoreactive T cells (Table 3) and even these were heterogeneous intheir rearrangement pattern, with the sizes of the observed rearrangements implyingdifferent Vp usage. Productive TcRB gene rearrangements are generated in twostages, namely DB to lB and VB to DFIp. In principle, different rearrangements, asdetected by Southern hybridization analysis, could be generated by the use ofdiffer-ent Vp and/or JB segments. Although none of the four Vp families studied here arepreferentially utilized in clones from HT, it cannot be ruled out that another VBsubfamily is used by these clones without the use of additional VB probes or directDNA cloning. However, the variation in size of the observed rearrangements stronglysuggests different Vp usage in these clones in addition to any differential JB usage.

The results presented here provide the first molecular data about T-cell receptorsin HT and GD obtained by studying autoimmune clones derived from the activatedT-cell pool. Similar experiments in other autoimmune diseases have given conflict-ing results. Thus, in human multiple sclerosis (MS) IL-2 dependent clones derivedfrom active MS plaques and blood, two of seven clones from blood and one of sixclones from brain exhibited the same rearrangement pattern [33]. In thymus andperipheral lymph nodes of /pr/lpr andglrlglr mice which develop a disease similar tosystemic lupus erythematosis, non-clonal rearrangements of TcRp were found [34].In T-cell populations grown from synovial specimens from patients with rheumatoidarthritis, distinct B gene rearrangements were detected in 13 of 14 cultures,suggesting a limited number of T-cell specificities [35].

N o o . os d - d - v s - . ;c o m m m m $ 6

K D

T-cell receptor usage in autoimrnune thyroiditis 1 1

Analysis of a variety of model systems has indicated that clones recognizing adefined antigen and restricted to a defined MHC allele generally use a restrictednumber 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 withepitope specificity. Thus three different receptors using closely related Vo and vpgenes were identified with VB usage, correlating with MHC specificity of the TcR[36]. Consistent with this finding was the transfer of MHC specificity by rransfectionof a TcRB gene from a TcR recognizing moth cytochrome c plus IEb [32]. In additionlTinoto et al. demonstrated that there was predominant use of a Vo gene in several T-cell clones recognizing pigeon cytochrome c [38]. Also, dominance of one TcR hasbeen found in the response to a simple determinant TNP [39]. However, in anotherstudy different specificities of two TcRs with the same vo and vB regions wereobtained by usage of separate diversity and joining regions [40]. Taken together,these results indicate that T-cell clones recognizing the same or very similar epitopetend to utilize a severely restricted number of V regions. The epitopes recognized byautoreactive T cells on thyroid tissue are unknown. However, the observed hetero-geneity of VB expression suggests that multiple epitopes are recognized as foreign.Alternatively epitope recognition may indeed be restricted but may be associatedprimarily with Vo (or Ju) expression. The extended nature of the Jo locus makesanalysis of gene rearrangement using Southern blotting generally uninformative.Detailed sequence analysis will be required to analyse further both Vo and VB usagein these clones.

Recently, such an analysis was described with murine T-cell clones recognizingpeptide l-9 of myelin basic protein in PL/J and (PL/J x SJL) mice [41]. A veryrestricted usage of TcR Va, vB and J segments was nored. Sequence analysis ofthe HT and GD clones described here will determine whether a difference in theheterogeneity of the T-cell response indeed exists between these two autoimmunediseases.

Acknowledgernents

we thank Drs T. Mak, c. Terhorst, T. Rabbitts and D. Loh for gifts of probes andMs Celia Joseph for typing this manuscript. This study was supported by ICRF(M.L., M.J.O.), the liTellcome Trust, Juvenile Diabetes Foundation and BritishCouncil (M.L., B.G-L and M.F).

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