TCR Vβ Usage of TSH Receptor-specific CD4+T Cells in Graves' Disease Patients and Healthy Humans

Journal of Autoimmunity (1997) 10, 479–489

TCR Vâ Usage of TSH Receptor-specific CD4 + T Cells in Graves’ Disease Patients and Healthy Humans Raghavanpillai Raju1*, Duraiswamy Navaneetham1, Sirid-Aime´e Kellermann1†, Susan L. Freeman2, John C. Morris3, Daniel J. McCormick4 and Bianca M. Conti-Fine5** 1 Department of Biochemistry, University of Minnesota, St Paul, MN 55108, USA 2 Section of Endocrinology, St PaulRamsey Medical Center, 640 Jackson Street, St Paul, MN 55101 and Section of Endocrinology, School of Medicine, University of Minnesota, 516 Delaware Street, Minneapolis, MN 55455, USA 3 Division of Endocrinology and Metabolism, Department of Medicine, Mayo Clinic and Medical School, 200 First Street Southwest, Rochester, MN 55905, USA 4 Department of Biochemistry and Molecular Biology, Mayo Clinic and Medical School, 200 First Street Southwest, Rochester, MN 55905, USA 5 Department of Biochemistry, University of Minnesota, St Paul, MN 55108 and Department of Pharmacology, University of Minnesota, 435 Delaware Street, Minneapolis, MN 55455, USA

Received 12 November 1996 Accepted 12 May 1997

Healthy humans have CD4 + T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4 + cells present in healthy persons. We studied CD4 + cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves’ disease (GD) patients. We used short-term CD4 + cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vâ usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vâ region family, in conjunction with a constant region primer. Six lines preferentially used one Vâ family (42–94%), different for each line. In all lines, three or less Vâ families accounted for approximately 60% or more of the Vâ usage. Different Vâ regions were used by each subject. There was no obvious difference between the Vâ usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4 + cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4 + cells specific for the same autoantigen in healthy subjects. © 1997 Academic Press Limited

Key words: Graves’ disease, thyroid, TCR Vâ usage, autoimmunity

Introduction

TSH receptor (TSHr) and myelin basic protein (MBP), the autoAg involved in myasthenia gravis (MG), Graves’ disease (GD) and multiple sclerosis (MS) respectively—have been demonstrated in healthy humans and rodents [1–4]. The relationship between the pool of those potentially autoimmune, inactive CD4 + cells and the activated pathogenic CD4 + cells involved in autoimmune diseases is not clear, and is it not known whether they share similar properties or whether autoimmune T cells have unique characteristics. Studies on the epitope repertoire recognized by human CD4 + cells on the AChR, the TSHr and MBP did not reveal unique differences between normal and autoimmune subjects [1–4]. However, the TCRs used

T cells recognizing self-components which are target autoantigens (autoAg) in autoimmune diseases—such as the nicotinic acetylcholine receptor (AChR), the Correspondence to: Dr Bianca M. Conti-Fine, Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St Paul, MN 55108. Fax: (612) 625-5780. *Present address: Division of Immunology, Mayo Clinic, Guggeinheim-310, 200-1st Street SW, Rochester, MN 55905. †Present address: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Box 609 UMHC, 420 Delaware Street SE, Minneapolis. **Previously known as Bianca M. Conti-Tronconi. 479 0896-8411/97/050479+11 r25.00/0/au970155

© 1997 Academic Press Limited

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Table 1. Graves’ disease patients and healthy controls used in this study Subject Patients

Controls

Age, Sex

HLA-DR and DQ haplotype*

P-CT P-DB P-DH P-EM

30, 56, 27, 29,

F M F F

DRw12(5),w13(6) DR1 DRw15(2),w17(3) DRw11(5),7

DRw52b,w52c DRw51,w52 DRw52b,w53

DQw5(w1) DQw5(w1) DQw6(w1),w2 DQw2,w7(w3)

C-AS C-HA C-TB C-MK C-PK

33, 30, 27, 46, 29,

M M F F M

DRw15(2),w7 DRw15(2) DRw17(3),8 DR1,w15(2) DRw15(2),4

DRw51,w53 DRw51 DRw51 DRw51 DRw51,w53

DQw6(w1),w2 DQw6(w1) DQw6(w1) DQw5(w1),w6(w1) DQw6(w1),w8(w3)

*As reported in [4].

by potentially autoreactive CD4 + cells in healthy persons and those of CD4 + cells actively engaged in an autoimmune response may differ, and give clues to the mechanism of activation and clonal expansion of the pathogenic autoimmune T cells. Most T cells express TCR that are áâ heterodimers [5]. The variable region of the â-subunit results from somatic rearrangement of germline-encoded V (variable), D (diversity) and J (joining) segments. The human TCR Vâ genes are classified into 24 different families based on the extent of sequence similarity [5, 6]. Several studies have investigated the Vâ repertoire selected during the autoimmune CD4 + responses. In rodents, the pathogenic CD4 + T cells against MBP recognize a limited epitope repertoire, and preferentially use certain Vâ families [7]. Studies on T cells isolated from tissues and blood of autoimmune patients yielded conflicting results [reviewed in 8, 9]. For example, some studies on T cells from affected joints or blood of rheumatoid arthritis patients found clonal dominance and restricted V region usage [10, 11 and references therein], while others found heterogeneity of the T cell clones and of their TCRs [12, 13 and references therein]. Also, studies on the TCR usage in multiple sclerosis yielded inconsistent results [14, 15 and references therein]. T cells inducing anti-DNA autoantibody in lupus [16], and T cells infiltrating affected tissues in Sjogren’s syndrome [17], polymyositis [18] and acute myocarditis and dilated cardiomyopathy, which might have an autoimmune origin [19], have restricted Vâ usage. AChRspecific CD4 + lines propagated from MG patients had restricted TCR Vâ usage, although there was individual heterogeneity in different patients [20, 21]. The goal of the present study was to determine the Vâ usage of the potential autoreactive CD4 + cells commonly present in healthy subjects and of autoimmune CD4 + T cells specific for the same autoAg. CD4 + cells specific for the TSHr are suitable for this purpose, because they are present with high frequency both in healthy subjects and in GD patients, and they recognize a limited number of TSHr epitopes [4 and references therein]. We have previously propagated and characterized [4] TSHr-specific CD4 + cell lines from four GD

patients and five healthy controls, by cycles of stimulation with overlapping synthetic sequences of the TSHr, spanning the putative extracellular domains. Each line recognized one to five TSHr peptides, different for each line. In the present study, we determined the TCR Vâ usage of those lines, using a semi-quantitative reverse transcriptase PCR (RT-PCR) assay.

Materials and Methods Characteristics of the patients CD4 + polyclonal lines were propagated from four GD patients and five healthy controls by cycles of stimulation with the pool of synthetic TSHr sequences described below. Their DR haplotype is reported in Table 1. We also used peripheral blood mononuclear cells (PBMC) and PBMC depleted in CD8 + cells (hereafter referred to as CD8 + -depleted PBMC) from seven other healthy subjects, to assess the TCR Vâ usage by unselected blood T cells.

Ag used The CD4 + lines had been propagated from patients and controls by cycles of stimulation with a pool of 29 overlapping synthetic peptides, corresponding to the sequence [22] of the amino-terminal extracellular domain of the TSHr and of the three extracellular loops (hereafter referred to as the TSHr pool). The peptides overlapped by five amino acid residues and were 20 residues long, except for peptides 376–394 (19 residues) and for two peptides, corresponding to the putative extracellular loops between the second and the third transmembrane segments (21 residues) and the sixth and seventh transmembrane segments (12 residues). The synthesis of the peptides used and their characteristics have been previously described in detail [23]. Amino acid composition was determined for all peptides in a Beckman 6300 automated amino acid analyzer, and yielded results corresponding to the expected theoretical values for most peptides.

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Mass spectrometry analysis was carried out on the peptides whose amino acid composition was not fully satisfactory, as well as on a few other randomly selected peptides, using a Bio-Ion 20R biopolymer mass analyzer (Applied Biosystems): for all peptides tested, one major peak of the expected MW was present.

Patients 20

P-CT

10

10

Isolation of PBMC CD8 + -depleted PBMC

C-AS

10

20

P-DB

C-HA

10

30

cpm × 10

–3

5 cpm × 10–3

PBMC were isolated on a Ficoll-Hypaque density gradient (Pharmacia LKB, Uppsala, Sweden). CD8 + depleted PBMC were prepared as described in [24], washed twice with PBS containing 10% FCS (PBS– FCS) and collected by centrifugation for RNA isolation.

Controls 20

P-DH

20

20

C-TB

10 10

20

P-EM

10

C-MK

Anti-TSHr CD4 + T cell lines 20 hTSHr

0 Cells + APC

5

APC

10

Cells

C-PK

10

hTSHr

Cells + APC

APC

0 Cells

We used nine short-term CD4 + T cell lines specific for TSHr sequences described in detail previously [4]. They are indicated with codes which include the letter P or C for Patient and Control, and two letters that represent the code used for the same subjects in our previous study [4]. The lines had been propagated as follows. PBMC from heparinized blood were cultivated in T cell medium (TCM; RPMI medium, 10% heat inactivated human serum, 2 mM L-glutamine, 100 U/ml penicillin, 50 ìg/ml streptomycin) containing TSHr pool (1 ìg/ml of each peptide) for 1 week. Activated lymphoblasts were isolated on Percoll (Pharmacia) gradients and expanded for 5 days in TCM supplemented with 10% T cell growth factor containing 10 U/ml Interleukin 2 (IL-2) (Lymphocult; Biotest Diagnostics Inc., Dreieich, Germany). CD4 + cell lines enriched in TSHr-specific T cells were obtained by weekly cycles of stimulation with the TSHr pool (1 ìg of each peptide/ml), in the presence of irradiated (4000 rad) autologous or DR-matched (as assessed by oligonucleotide typing) PBMC as antigen presenting cells (APC) for 2 days, following by stimulation for 5 days with IL-2 (Lymphocult, Biotest Diagnostic Inc.: concentration of IL-2, 10 U/ml) [4]. The lines used here had been propagated for only two to three cycles of stimulation, to reduce the risk of biased clonal propagation, introduced by the in vitro manipulation for long periods of time. They were predominantly or exclusively CD3 + , CD4 + , CD8 − [4]. In 3-day proliferation assays [4] the lines responded to the TSHr pool strongly and specifically (Figure 1). We identified previously [4] the individual TSHr peptides recognized in proliferation assays by the CD4 + T cell lines from both GD patients and healthy controls: they are listed in Table 2. Most lines recognized two or more TSHr sequences, and each subject had an individual pattern of peptide recognition. Although some TSHr peptides were recognized by more than one subject, we did not identify any immunodominant sequence region recognized by all or most patients and/or controls.

Figure 1. Response to the TSHr pool of CD4 + T cell lines propagated from GD patients and healthy controls by stimulation with the TSHr pool. The lines were challenged in proliferation assays with TSHr pools (1 ìg/ml of each peptide). The columns represent the average of triplicate cultures ±SD. Note the different scale used for the y-axis of line C-MK. See text for experimental details.

Anti-tetanus toxin (TTX) CD4 + cell lines As a control of the Vâ usage, we used two CD4 + cell lines, propagated from the same control used to establish the line C-PK by stimulation with pools of overlapping synthetic peptides corresponding to two sequence regions of TTX (hereafter referred to as TTX pool 1 and TTX pool 2; residues 1–305 of the TTX light chain, and 476–780 of the TTX heavy chain, see [25]). The length and number of the TTX peptides, their overlap, and the length of the sequence regions they spanned were the same as, or similar to, the TSHr peptides used here and the length of the TSHr sequence they spanned. The lines, described in detail previously [25], had been propagated with the TTX pools 1 and 2, using the procedure described above for the TSHr-specific CD4 + lines, and two to three cycles of Ag/IL-2 expansion. They strongly recognized the TTX molecule and several of the TTX peptides used for their propagation (Table 3).

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Table 2. Sequence specificity of the T cell line used in this study† Line

Peptide sequences recognized and P values‡

Vâ families preferentially used (% usage)

P-CT P-DB P-DH P-EM

76–95* 46–65*** 136–155** 271–290* 121–140* 67–95**** 181–200**** 286–305****

Vâ15 Vâ13 Vâ18 Vâ14

(94%) (58%) Vâ15 (5%) (26%) Vâ12 (24%) (44%) Vâ6 (21%) Vâ20 (10%) Vâ24 (7%)

C-AS C-HA C-TB C-MK C-PK

106–125**** 226–245**** 31–50*** 46–65* 196–215**** 226–245* 46–65* 61–80* 76–95*** 226–245** 256–275**** 46–65** 226–245*** 46–65* 226–245**** 286–305*

Vâ11 (62%) Vâ15 (11%) Vâ21 (35%) Vâ18 (25%) Vâ12 (15%) Vâ16 (11%) Vâ6 (65%) Vâ22 (8%) Vâ11 (6%) Vâ6 (42%) Vâ8 (23%) Vâ8 (28%) Vâ5 (27%) Vâ6 (18%)

†For each line, we report the peptides recognized significantly, as determined by an increase in 3H-thymidine incorporation above the background values, as reported in [4]. ‡As reported in [4]. Asterisks indicate the level of significance of the response: ****P<0.001; ***P<0.005; **P<0.01; *P<0.05.

Table 3. Sequence specificity of the anti-TTX cell lines used in this study* Line

TTX peptide sequences recognized

TTX pool 1

L31–50, L91–110, L151–170, L166–185, L226–245, L241–260, L256–275

TTX pool 2

H491–510, H506–525, H521–540, H536–555, H566–585, H581–600, H611–630, H641–660, H656–675, H671–690, H686–705, H701–720, H716–735, H731–750, H746–765, H761–780

*For each line we report the peptides recognized, as determined by a highly significant increase in 3H-thymidine incorporation above the background values [from reference 25].

cDNA synthesis Total RNA was isolated from the CD4 + T cell lines (1×106 cells), PBMC, or CD8 + depleted PBMC (5×106 cells) using RNAzol B reagent (Tel-Test Inc. Friendswood, TX) [26]. cDNA was prepared from the total RNA using MLV reverse transcriptase (SuperScript RNase H − , Gibco BRL, Gaithersburg, MD) [26].

RT-PCR assay of the TCR Vâ usage The Vâ usage of the lines, of the PBMC and of the CD8 + -depleted PBMC was analysed by a RT-PCR assay reported in detail in [26] that yields simultaneous semi-quantitative determination of the relative usage of the 24 different Vâ-region families in polyclonal T cell populations. It uses primers at the 5′ end of the Vâ regions specific for each known V-region family [described in detail in 27]. Each Vâ primer has comparable efficiency of amplification when 25 PCR cycles or less are used [21, 27]. When used in association with a 3′ end constant region primer, each primer yields a product of defined size [21, 27; see also Figure 2]. Thus, the Vâ-specific primers can be used either individually or as pool (see below), because the resulting products can be identified from their size. To assess the Vâ usage of the CD4 + lines, the 24 different 5′ primers, each representing a specific family of TCR Vâ gene [27], were mixed into eight pools:

each pool comprised three primers yielding products of different sizes (Table 4), and also included the constant region 3′ primer [27]. To assess the Vâ region usage of PBMC and CD8 + -depleted PBMC, each V-region 5′ primer was used individually, in association with the constant region 3′ primer (Figure 2). As a control of the effectiveness of the PCR amplification, we co-amplified cDNA of the CD4 protein, using specific 3′ and 5′ primers [27]. The 5′ CD4 primer and 3′ TCR constant region primer were end-labelled by ã-32P ATP (New England Nuclear, DuPont Company, Wilmington, DE), using T4 polynucleotide kinase (Promega Corporation, Madison, WI). The PCRs were carried out in 10 ìl of 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 200 ìM dNTPs, 1.5 mM MgCl2, 0.5 ìM primers, 10% sucrose with cresol red, 0.25 U of Taq DNA polymerase (PerkinElmer, Norwalk, CT) and 1 ìl cDNA. The reaction was ‘hot started’ using petroleum jelly [28] and carried out in a Perkin Elmer 9600 thermal cycler using the following parameters: 95°C, 30 s; 55°C, 15 s; 72°C, 45 s. We used a number of cycles such that the intensity of the Vâ band products did not reach a plateau. For the CD4 + lines we carried out eight PCRs using the same cDNA template and the different Vâ primer pools. For the PBMC and CD8 + -depleted PBMC we carried out 24 PCRs, one for each individual Vâ primer. 6 ìl of the PCR mixture was made up to 40% in formamide, and the PCR products were resolved on a sequencing gel (6% acrylamide, 7 M urea). The bands were identified by autoradiography, and their intensity was

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483

100

24

23

22

21

20

19

18

17

16

15

14

13

11 12

10

9

8

7

6

5

4

3

2

1

A

B

Vβ usage (%)

80 60 40 20 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 TCR Vβ

Figure 2. TCR Vâ usage of PBMC or CD8 + -depleted PBMC from healthy controls, determined in RT-PCR assays, as described in Materials and Methods. One individual family-specific Vâ primer was used for each reaction, in conjunction with the constant region primer. See text for experimental details. (A) Results obtained with the PBMC of one control. The primers yielded products of the expected size. Comparable results were obtained with cDNA from PBMC or CD8 + -depleted PBMC. The RT-PCR products—which span the V-D-J junctions—may comprise multiple bands separated by three base pairs. This size heterogeneity is especially evident for the primers of Vâ regions yielding products of low MW, that are better resolved by polyacrylamide gel electrophoresis (e.g. Vâ13 and Vâ21, see horizontal dashes). (B) Shows the relative Vâ usage as determined by a densitometric scan of the autoradiogram shown in (A) (j), and the average usage of the different Vâ families in seven healthy subjects (h). Some Vâ families appeared to be used more than others, but even the most used Vâ families (e.g. Vâ2, Vâ4, Vâ5, Vâ13) were always <20% of the overall usage. See text for experimental details.

quantified using an AMBIS densitometer (Ambis, Inc. San Diego, CA). Radiolabelling of the PCR products increases the sensitivity of the reaction, and reduces the number of cycles needed to obtain a good signal. This is important because when using many PCR cycles small differences in the efficiency of amplification between target sequences can lead to large differences in the yield of the PCR products [27 and references therein]. In our assay, the signals generated by the different V regions using our primers were comparable at 25 cycles or less [21, 27]. In this study, we carried out two or more independent RT-PCR experiments using different, low numbers of cycles (15–25 cycles) for the cDNA of each T cell line, PBMC or CD8 + -depleted PBMC cDNAs, to verify that the amplification of the different Vâ regions was consistent and proportional

to the number of cycles. The high resolution and low background of sequencing gels allows detection of PCR products originated from multiple clones, using the same V region but separated by three base pairs (see Results). The autoradiogram can be scanned, and the relative intensity of the bands—which for brief exposures is linear with the amount of radioactivity—can be quantified.

Verification of the identity of the PCR products and sequencing To verify the identity of the PCR products, gel slices of the bands of the appropriate MW seen on an autoradiogram were cut out by superimposing the dried gel with the autoradiogram. Water (100 ìl) was

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Table 4. Vâ primers* present in each of the primer pools used in the RT-PCR assay of the TCR Vâ usage and approximate size of the expected products† Pool A

Pool B

Pool C

Pool D

Pool E

Pool F

Pool G

Pool H

Vâ10 (387) Vâ8 (378) Vâ14 (368) Vâ6 (356) Vâ20 (345) Vâ4 (342) Vâ1 (337) Vâ23 (372) Vâ24 (341) Vâ9 (305) Vâ18 (272) Vâ16 (270) Vâ12 (269) Vâ3 (268) Vâ11 (266) Vâ22 (339) Vâ19 (306) Vâ2 (244) Vâ5 (236) Vâ15 (217) Vâ7 (214) Vâ21 (200) Vâ13 (175) Vâ17 (265) *The primers are described in detail in [27]. †For each pool, the Vâ regions amplified by the specific primers are listed according to the size of the expected products, with the heaviest of them at the top. The size of the expected products (in base pairs), indicated in brackets, was calculated based on one randomly chosen representative gene for each family. The size of the actual products may be slightly different, due to different arrangement at the V-D-J junctions.

added to each slice and heated at 100°C for 15 min under mineral oil. An aliquot (1 ìl) of the eluate was tested for re-amplification of the specific product by PCR, using the proper specific 5′ primer and an internal 3′ constant region primer [26]. The amplified product was resolved on a 1% agarose gel and the PCR products visualized by ethidium bromide staining. We verified the identity of the PCR products by sequencing the DNA contained in some randomly selected bands. The PCR products were cloned into Eco RI cut plasmid Bluescript KS II + . The DH5á strain of E. coli was transformed with the plasmids containing the insert, and cloned. The colonies were tested for presence of the insert by PCR, using specific primers, and the PCR products were directly sequenced. Sequencing was carried out using a Promega fmol y DNA Sequencing System kit (Promega Corporation, Madison, WI).

Results TCR Vâ usage by PBMC and CD8 + -depleted PBMC of healthy controls To compare the Vâ usage of PBMC with that of the CD4 + lines and to verify that all the primers yielded products of the expected size, we performed RT-PCR assays of the Vâ usage using cDNA from PBMC or CD8 + -depleted PBMC from seven healthy controls. For these assays, we used one individual familyspecific Vâ region primer, in conjunction with the constant region primer, for each reaction. Comparable results were obtained with cDNA from PBMC or CD8 + -depleted PBMC. Hereafter, we will refer to the Vâ usage of PBMC and of CD8 + -depleted PBMC collectively as ‘PBMC Vâ usage’. We did not have enough blood to carry out similar experiments using GD patients. However, in a previous study we compared the TCR Vâ usage of PBMC from healthy controls and from MG patients, and we obtained comparable results [21]. PCR products of the expected size were obtained for each family-specific primer, demonstrating that the primers amplified each of the 24 Vâ families tested. Figure 2A reports the results obtained with the PBMC of one of the controls, which are representative of those obtained for all controls. The

signal-to-noise ratio obtained in this experiment is representative of that obtained in all RT-PCR experiments, including those carried out with the CD4 + lines (e.g. Figure 2, top panels). Some families (Vâ14, Vâ15, Vâ19, and Vâ20) yielded a very faint band when PBMC were used. For Vâ14, Vâ15 and Vâ20, this was due to low frequency in the blood of T cells expressing these gene families, not to poor amplification efficiency of the primers, because this (see below) and a previous [21] study found that those Vâ families were preferentially used by some Ag-specific CD4 + T cell lines. We observed for the products of most V regions—which span the V-D-J junctions—multiple bands separated by three base pairs [Figure 2A and Reference 21]. This was especially evident for the primers of Vâ regions yielding products of low MW, that are better resolved by polyacrylamide gel electrophoresis, such as Vâ13 and Vâ21 (identified by horizontal dashes in Figure 2A). This size heterogeneity was always present when PBMC were used, and it is due to the presence of multiple T cell clones that use the same Vâ region but have diversity at the V-D-J junctions. The three base pair spacing is due to selection for having the proper reading frame. The black columns of Figure 2B are the result of a densitometric scan of the autoradiogram shown in Figure 2A. No Vâ region exceeded 25% of the overall usage. The relative usage of the different Vâ families had a similar pattern in all subjects. The white columns in Figure 2B represent the average usage of the different Vâ families by PBMC from seven healthy subjects. Although some Vâ families were used more than others, the average usage of even the most used Vâ families (e.g. Vâ2, Vâ4, Vâ5, Vâ13) was <20% of the overall usage.

TCR Vâ usage of TSHr-specific CD4 + lines For all lines we carried out two or more independent experiments using different, low cycle numbers, that yielded consistent results. Figure 3 reports the results of one experiment for each line from the GD patients (left-hand plots) and the healthy controls (right-hand plots), as indicated, expressed as a percentage of the total Vâ usage (black columns). We include in each plot, for the sake of comparison, the average Vâ usage

TCR Vâ usage in Graves’ disease

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A A B C D E F G H

B A B C D EF G H

P-CT

C-AS

Vβ11

100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0

P-CT

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

P-DB

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

P-DH

Vβ usage (%)

Vβ usage (%)

Vβ15

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

P-EM

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0

C-AS

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

C-HA

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

C-TB

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

C-MK

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

100 C-PK 80 60 40 20 0

TCR Vβ

1 2 3 4 5 6 7 8 9 10 11 1213 14 151617 18 192021 22 2324

TCR Vβ +

Figure 3. TCR Vâ usage of TSHr-specific CD4 lines. Results of one RT-PCR assay experiment for each of the CD4 + lines propagated from the GD patients (left-hand side), and from five healthy subjects (right-hand side), as indicated in each panel. For all lines we show a plot of the relative Vâ usage, determined from densitometric scans of autoradiograms of RT-PCR experiments, as described in Materials and Methods (j). For the sake of comparison, we also indicated in each plot the average Vâ usage observed for the PBMC of seven healthy subjects (h). For lines P-CT and C-AS we also show the autoradiogram from which the corresponding plots were derived. See text for experimental details.

of PBMC from the healthy subjects. For lines P-CT and C-AS we also show the actual autoradiograms from which the plots were derived: they representative of those obtained for all lines. For many lines we carried out further experiments using a high number of cycles, to verify that when using low cycle numbers we did not miss Vâ families used to a low extent.

The lines from both healthy controls and GD patients preferentially used one or a few Vâ gene families. At variance with the Vâ PCR products obtained for the PBMC, that included multiple bands separated by three base pairs, indicative of the presence of multiple T clones, the Vâ regions preferentially used by the TSHr-specific lines consistently yielded products that included only one or two bands,

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even for the Vâ families best resolved in the gel electrophoresis. This finding was specific for the Vâ families preferentially used, since it did not occur for the Vâ regions that were not preferentially used, whose presence is likely to be due to contamination by the irradiated PBMC used as APC: for those Vâ regions, three to five bands were frequently detected (data not shown). For example, for line C-PK, Vâ2, Vâ4, and Vâ13 were not preferentially used, yet they yielded five, three, and four bands respectively; for line P-DH, Vâ2 and Vâ13 were not preferentially used and yielded three and four faint bands, respectively. Table 2 summarizes the Vâ family preferentially used by the lines. The line P-CT used Vâ15 almost exclusively (94%), and only one product band could be detected (Figure 3), in spite of the excellent resolution of the Vâ15 PCR products in our gel system (Figure 2). The highly restricted Vâ usage of this line might reflect its recognition of one peptide only (peptide 76–95). The line P-DB preferentially used Vâ13 (58%, two bands). Vâ15 was also used to an extent that, although small (7%, two bands), was higher than that found for PBMC. This line recognized three peptide sequences. Patient DB is homozygous at the DR/DQ locus, and is homozygous for DR1 (Table 1); thus, he expressed only the â1 DR subunit and only one DR molecule, DR1. The preferential usage of Vâ13 in recognition of different epitopes might be related to the relative homogeneity of the class II molecules expressed by this patient. Line P-DH recognized only one peptide significantly, yet it used preferentially and to a similar extent two Vâ families, Vâ12 (24%) and Vâ18 (26%). For the PCR products of both those Vâ regions only one band could be detected on the gel. Line P-EM recognized three TSHr sequences and primarily used Vâ14 (44%), whose product included only one band. Also Vâ20 and Vâ24 were used more substantially (10%, two bands, and 7%, one band, respectively) than by the PBMC. This line also used Vâ6 to a slightly higher extent than the average use by PBMC (20.5%, three bands). The presence of three PCR product bands for Vâ6, while for all the other Vâ regions preferentially used by all lines only one or two products could be detected, suggests that the Vâ6 usage detected might be due to APC contamination. The five lines obtained from healthy controls all recognized two or more TSHr peptide sequences (Table 2) and preferentially used a limited number of Vâ families, which in most cases appeared related to the number of TSHr sequences recognized by the line (Figure 3 and Table 2). Lines C-AS and C-MK recognized two TSHr peptides and preferentially used two Vâ families: line C-AS used primarily Vâ11 (62%, one band) and to a much lesser extent Vâ15 (11%, one band); line C-MK used primarily Vâ6 (42%, two bands) and Vâ8 (23%, one band). Line C-HA recognized four TSHr peptides and preferentially used four Vâ families, for all of which only one PCR product band was detected: Vâ12 (15%), Vâ16 (11%), Vâ18 (25%) and Vâ21 (35%). Line C-TB recognized four TSHr peptide sequences and preferentially used Vâ6

(65%, two bands); also the usage of Vâ11 (6%, one band) and Vâ22 (8%, one band) was higher than that of PBMC. Three peptides recognized by this line (46–65, 61–80 and 76–95) overlap, and peptide 76–95 is recognized much more strongly than the others [4]. It is possible that the TSHr sequence region 46–95, that comprises those peptides, forms only one or two CD4 + epitopes, possibly in the region(s) of peptide overlap. Line C-PK recognized three peptides, and preferentially used three Vâ families: Vâ5 (27%), Vâ8 (28%), and to a lesser extent Vâ6 (18%). However, as for the P-EM line (see above) the Vâ6 product obtained included three bands, raising the possibility that the Vâ6 products might be due to contamination by APC.

TCR Vâ usage of TTX-specific CD4 + lines We investigated the Vâ usage of two CD4 + lines specific for the exogenous Ag, TTX, propagated from control PK using the procedure employed for the TSHr cell lines, and pools of overlapping synthetic TTX peptides (TTX pool 1 and TTX pool 2) similar to those used for the TSHr sequence. Figure 4 reports the results obtained in one of the two consistent experiments carried out for each line. The plots also report the average Vâ usage observed for the PBMC of healthy subjects. Both lines used several Vâ regions, each to a relatively small extent. None of the Vâ products we detected comprised one band only, and most of them comprised multiple bands, similar to what we found for the Vâ usage of the PBMC. Line TTX pool 1 used Vâ2 to a higher extent than the PBMC. No preferential usage of any region could be detected for line TTX pool 2.

Discussion We report here that the potentially autoreactive antiTSHr CD4 + cells from healthy subjects, similar to the actively autoimmune CD4 + T cells from GD patients, have a limited but individual heterogeneity of the TCR Vâ genes they use. For most anti-TSHr lines, the extent of heterogeneity of the Vâ transcripts correlated with the number of TSHr sequences recognized by the line. This indicates that, as it occurs for the autoimmune anti-AChR CD4 + cells in MG [21], in both GD patients and healthy subjects the CD4 + cells recognizing a given TSHr epitope use one or very few Vâ families, and CD4 + cells of the same subject recognizing different TSHr epitopes use different Vâ families. The present data, although from a limited number of subjects, suggest that CD4 + cells recognizing the same TSHr sequence in different subjects do not use the same Vâ families, because there was no correlation between shared Vâ usage and recognition of the same peptide sequences (Table 2 and Figure 3). For example, peptide 76–95 was recognized by lines P-CT and P-EM, whose Vâ repertoire did not overlap. Peptide 226–245 was recognized by all the lines from

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100 80 60

Vβ usage (%)

40 20 0 100 80 60 40 20 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 TCR Vβ

+

Figure 4. TCR Vâ usage of TTX-specific CD4 lines, obtained from subject PK, using synthetic peptide pools spanning two TTX sequence regions of length comparable to that of the TSHr sequence investigated here. We show a plot of the relative Vâ usage obtained in one of two consistent experiments (j). In each plot we also show the average Vâ usage observed for PBMC of healthy controls (h). See text for experimental details.

healthy subjects, yet no individual Vâ family was used by all lines. Peptide 46–65 was recognized to a moderate extent by four control lines that did not share a preferential usage of one Vâ family. The Vâ15 and the Vâ6 families were preferentially used by more than two lines (three lines for Vâ15 and four for Vâ6), but their usage did not correlate with recognition of a particular peptide sequence. Some lines used a lesser number of Vâ families that the peptides recognized (e.g. lines C-TB and C-DB), suggesting that occasionally the same Vâ gene family might be used for recognition of different epitopes, as may occur in some T cell responses to exogenous Ags or autoAgs [e.g. 21, 29]. A limited TCR repertoire of the anti-TSHr CD4 + cells is supported by the finding that the PCR products obtained for the Vâ families preferentially used by the anti-TSHr lines generally showed one or—less frequently—two bands, consistently with a limited clonal diversity of the CD4 + cells using the same Vâ region. On the other hand, in unselected PBMC the V-region PCR products—which span the V-D-J junctions—generally included multiple bands separated by three base pairs, due to the presence of multiple T cell clones that use the same Vâ region but differ at the V-D-J junctions and/or in the D and J genes used (Figure 2). The number of PCR products detected in our system represents the minimum number of T cell clones in the population studied that used a given Vâ family, and each of the PCR products we observed may well include different species that have the same size. The present results agree well with a model of tolerance postulating that most autoreactive T cells are deleted during the ontogenesis of the immune system, and only a limited pool of potentially autoreactive T

cells survive clonal deletion, that carry a limited Vâ repertoire [30]. The pathogenic CD4 + cells involved in autoimmune diseases are likely to be recruited from that surviving limited pool, since they have similar characteristics of epitope and TCR repertoire as the CD4 + cells specific for the same autoAg present in healthy subjects. An important and closely related question is the relative abundance of autoreactive TSHr-specific CD4 + cells in the blood of GD patients as compared to healthy subjects. To answer that question—which we have not yet addressed in our studies because it requires a different experimental approach, namely precursor frequency studies—will be an essential further step in understanding the relationship between the active autoimmune T cells of GD patients and the quiescent anti-TSHr T cells of healthy subjects. The use of Ag-specific CD4 + lines propagated in vitro has the important caveat that any type of in vitro manipulation of the original population of Ag-specific T cell clones may result in biased clonal propagation, and yield CD4 + lines that are not representative of the original clonal repertoire [25 and references therein]. After propagation for extended periods of time, only clone(s) especially apt to be simulated by the peptides used may survive; furthermore, long-term CD4 + lines may ‘clone’ themselves out. Although we propagated our lines for short periods of time to avoid this risk, the limited Vâ repertoire we observed might have resulted from artefactual selection in vitro, and not reflect the Vâ usage occurring in vivo. The results we obtained with the lines TTX pool 1 and TTX pool 2 partially dispel this concern, because although they were propagated using the same procedure as for the TSHr lines, and using peptide pools similar to that used for the TSHr sequence, both lines used several

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Vâ regions. Line TTX pool 2 did not have any preferential Vâ usage, and line TTX pool 1 used Vâ2 to a higher extent than the PBMC, in addition to several other Vâ families. Also, as for PBMC, the Vâ products obtained with these lines included multiple bands (data not shown) indicating that each Vâ family was used by an oligoclonal or polyclonal T cell population. The TSHr-specific line from the same subject (line C-PK, Figure 3) preferentially used Vâ5, Vâ8, and Vâ6. Given the complex antigenic structure of the TSHr [4, 31, 32], with recognition of many epitopes by CD4 + cells, the restricted Vâ usage by T cells specific for an individual epitope may be obfuscated by the complexity of their epitope repertoire, when unselected populations of autoAg-specific T cells are studied. Thus, the present results may reconcile the disparate findings of several previous studies on the Vá and Vâ usage of autoimmune T cells in GD. Some studies found biased TCR Vâ and/or Vá usage in the T cells infiltrating the thyroid tissue in GD patients [33 and references therein], while others did not [34, 35 and references therein]. Those negative results may be explained by considering that the T cells eluted from the thyroid, in the absence of further selection in vitro for autoAg specificity, probably included variable, large amounts of ‘bystander’ T cells of different, unrelated specificities [8, 36]. Furthermore, different thyroid proteins can be the target of autoimmune responses in GD [36] and each of them might form a variety of epitopes recognized by autoreactive CD4 + cells. Thus, even the truly autoimmune T cells eluted from the thyroid are likely to have a broad variability in clonal composition and TCR repertoire. The present data do not support a role of superAg(s) in GD pathogenesis, but do not suffice to exclude that possibility, since intervention of a superAg limited to one epitope might be sufficient to trigger a complex autoimmune response; spreading of the immune response to other epitopes may occur at later stages of the autoimmune response [37, 38]. Given the limited population of GD patients we studied, and the limited epitope specificity of their anti-TSHr CD4 + cell lines, we cannot rule out that we may have missed the CD4 + populations originally expanded by the action of a superAg, which might have been a very small minority at the time the disease reached the severity observed in our patients. In MS and its experimental model, experimental autoimmune encephalomyelitis, the autoimmune CD4 + response against MBP involves well-defined dominant epitopes, recognized by CD4 + cells that have highly restricted Vâ usage [7]. These features of the T cell response to MBP have led to specific therapeutic approaches, targeted on the TCR Vâ regions used by the pathogenic T cells [7], that have now been implemented in multiple sclerosis patients [39]. The relatively large and different repertoire recognized by the autoimmune CD4 + cells against the TSHr in GD patients, and the finding that different Vâ regions are used for different epitopes, reduce the hopes that the TCR expressed by the anti-TSHr CD4 + cells in GD might be used as a target for specific

immunosuppressive approaches, similar to those developed for MS.

Acknowledgements This study was supported by the NINCDS grant NS 23919 (to B.M.C.-F.).

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