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CDR3 Spectratyping Analysis of the TCR Repertoire in Myasthenia Gravis¹

Yoh Matsumoto^2,*, Hidenori Matsuo^,, Hiroshi Sakuma^*, Il-Kwon Park^*, Yukiko Tsukada^*, Kuniko Kohyama^*, Takayuki Kondo, Satoshi Kotorii and Noritoshi Shibuya^,

^* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan; Division of Clinical Research and Department of Neurology, National Hospital Organization, Nagasaki Medical Center of Neurology, and Department of Clinical Neurosciences, Nagasaki University Graduate School of Biomedical Sciences, Kawatana, Nagasaki, Japan

	Abstract

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Because myasthenia gravis (MG) is an autoimmune disease mediated by Abs specific for the acetylcholine receptor, helper T cells play a role in Ab production. In this study, we have performed large-scale cross-sectional and longitudinal TCR studies by CDR3 spectratyping using PBL and thymus tissues from MG patients. We found that there was no preferential usage of any particular TCR -chains that was identical among MG patients. However, the longitudinal study clearly demonstrated that one or more TCR V expansions persisted frequently in MG patients. Importantly, persistent TCR expansions correlated with clinical severity and high anti-acetylcholine receptor Ab titer. Finally, examinations of T cells expressing CXCR5, i.e., follicular B-helper T cells, revealed that spectratype expansions in MG patients were detected mainly in the CD4⁺ CXCR5⁺ T cell populations, whereas CD8⁺ T cells were the major source of clonal expansion in healthy subjects. These findings suggest that persistent clonal expansions of T cells in MG patients are associated with the development and maintenance of MG. Close examination of pathogenic T cells in MG provides useful information to elucidate the pathogenesis and to estimate the disease status.

	Introduction

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Myasthenia gravis (MG)³ is an autoimmune disorder characterized by weakness and fatigability of skeletal muscles caused by Abs against the acetylcholine receptor (AChR). The Abs bind to AChR at the postsynaptic membrane, leading to receptor destruction and disturbance in neuromuscular transmission (1). Although it is clear that Abs against AChR in most patients are final effectors, T cells play an important role in helping the Ab production in MG. Another characteristic feature of MG is abnormalities in the thymus. Lymphofollicular hyperplasia of the thymic medulla occurs in 65% and thymoma is found in 15% of MG patients (2). Furthermore, it was demonstrated that AChR, a key molecule for the development of MG, is expressed in the thymus (3) and that thymi showing hyperplasia or with thymoma generate more T cells, including AChR-specific T cells (4, 5, 6). On the basis of these findings, it is generally believed that anti-AChR Abs are generated with the help of T cells in the thymus of MG patients. Thus, characterization of the AChR-specific T cells, especially their TCR usage, is essential for the elucidation of the pathomechanisms of MG and for the development of specific immunotherapy for the disease.

To date, analysis of the TCR usage by pathogenic T cells in MG patients has revealed extremely controversial results. Cohen-Kaminsky and colleagues (7) showed that V5.1-positive T cells in the thymus were slightly increased in MG patients compared with control subjects (8). In contrast, other groups observed oligoclonal expansion of V4, V6, V12, or V13 in MG patients (9, 10, 11). Moreover, Infante et al. (12) found a diverse TCR repertoire with no preferential usage of particular TCRs in MG patients. Although the precise reasons for these discrepancies remain unclear, they may reflect the difference in the materials used and the methods for the TCR analysis. In a previous study of multiple sclerosis (MS) patients by CDR3 spectratyping analysis (13), we have demonstrated persistent or transient oligoclonal expansions of V5.2 in the PBL and/or cerebrospinal fluid T cells in >70% of the patients studied longitudinally but only in 30% of MS patients in a cross-sectional study. These results strongly suggest that a longitudinal study is essential for TCR repertoire analyses.

In this study, we have performed both cross-sectional and longitudinal studies using PBL and thymus tissues from MG patients and PBL from healthy subjects. It revealed no preferential usage of TCRs that recurred in MG patients in the cross-sectional study. However, persistent oligoclonal expansions of Vs, which differed from patient to patient, were detected in the longitudinal study. Importantly, they correlated with clinical severity and high anti-AChR Ab titer. These findings suggest that activated and clonally expanded T cells detected by CDR3 spectratyping are associated with the development of MG and provide useful information to elucidate its pathogenesis and to develop tailor-made immunotherapy.

	Materials and Methods

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Patients and healthy subjects

Thirty-eight MG patients, 8 males and 30 females (mean age, 57.9 ± 15.5 years; range, 25–80 years), attending the Departments of Neurology, Nagasaki Medical Center of Neurology, or Nagasaki University Hospital were included in the present study. All subjects’ consent was obtained, and the study was approved by the Institute Review Board. The diagnosis of MG was made on the basis of the clinical presentation and confirmed by laboratory examinations, such as repetitive nerve stimulation tests, anti-AChR, and computerized tomography images of mediastinum. For clinical assessment of the severity of the disease, the Myasthenia Gravis Foundation of America clinical classification (from class I: purely ocular disease to class V: defined by intubation) (14) and MG-activities of daily living (15) were used. Muscle-specific tyrosine kinase-seropositive MG patients were not included in this study. Forty-four healthy volunteers, 21 males and 23 females (mean age, 40.1 ± 17.6 years; range, 22–88 years) who had no episodes of common cold or influenza infection within the previous month were examined as controls. Ten milliliters of heparinized blood was drawn from patients and control subjects, and PBL was isolated using the density gradient method. Thymus tissues (n = 9) of MG patients were obtained by thymectomy. Six of them showed hyperplasia and three were uninvolved thymus adjacent to a thymoma.

Cross-sectional and longitudinal studies

The cross-sectional study was performed as described above. When the same person was examined more than once, the results of the first examination were included in the cross-sectional study. The longitudinal study was performed on 22 MG patients and 17 healthy subjects, and the results obtained from patients and subjects with sufficient follow-up periods were selected for analysis. The spectratype expansion pattern obtained in the longitudinal study was classified into the three categories: 1) heterogeneous, where no particular spectratype expansion was observed in multiple examinations in which the normal pattern was included frequently; 2) persistent, where spectratype expansion of particular Vs were constantly observed during the observation period; and 3) intermediate, where a particular spectratype expansion was occasionally observed.

cDNA synthesis and PCR amplification

RNA was extracted from PBL using RNAzol B (Biotecx Laboratories). cDNA was then synthesized by reverse transcription using ReverTra Ace (Toyoba) and amplified in a thermal cycler (PerkinElmer) using primer pairs for TCR. Primers for V1-24 were the same as those used in a previous study (16). C primer was labeled with Cy-5 or rhodamine or was left unlabeled.

CDR3 spectratyping

CDR3 spectratyping was performed as described previously (17). cDNA was amplified with V-specific and rhodamine-labeled C primers, and undiluted or diluted PCR products were added to an equal volume of formamide/dye loading buffer and heated at 94°C for 2 min. Two microliters of the samples was applied to a 6% acrylamide sequencing gel. Gels were run at 30 W for 3 h 30 min at 50°C. Then, the fluorescence-labeled DNA profile on the gel was directly recorded using an FMBIO fluorescence image analyser (Hitachi). Spectratype expansion was evaluated by visual inspection and density analysis of the image using software attached to the fluorescence image analyzer as shown in Fig. 1, C and D, and graded into two categories: 1)"Oligoclonal pattern," appearing as an increase of the density and thickness of one band within a normal spectratype profile (distortion of the Gaussian distribution) (18); and 2) "Monoclonal" one, with a marked increase of the density and thickness of one band with faint or no additional spectratypes (18). We tested for contaminations by the reagents used in PCR every 10 PCR analyses by doing PCR without templates; if present, all reagents used and results obtained during the period were discarded.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. CDR3 spectratyping profiles of PBL showing the normal spectratype pattern (A) in a healthy subject and spectratype expansion (B) in a MG patient. In the normal pattern, each V shows a Gaussian distribution without any spectratype expansion (A). In contrast, this MG patient exhibits expansion of several spectratypes indicated by arrows (B). The profiles of V4, V8, and V20 spectratypes of a healthy subject (A) and V3, V8, and V14 spectratypes of a MG patient (B) revealed by the density analysis are shown in C and D, respectively.

cDNA isolated from spectratypes of interest on the acrylamide gel was reamplified with V and unlabeled C primers to remove the fluorescence attached to the primer used for CDR3 spectratyping. This process was essential for cloning. To avoid biased amplification of a particular TCR clone, PCR was performed for only five cycles. Then, PCR products were ligated into pT-Adv vector and cloned using the AdvanTAge PCR Cloning Kit (Clontech Laboratories) according to the manufacturer’s instructions. The plasmid DNA was then sequenced using Cy5-labeled C primer and Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit on an ALFexpress DNA sequencer (Pharmacia Biotech). CDR3 length is defined as a region starting from an amino acid residue after the CASS sequence of most V segments and ending before the GXG box in the J region, as described previously (19).

Isolation and TCR analysis of CXCR5-positive T cells

Because the majority of CXCR5-expressing cells were CD4⁺ T and B cells (20), we decided to examine CD4⁺, CD8⁺, and CD4⁺CXCR5⁺ T cells. The number of CD8⁺CXCR5⁺ T cells was too small for the analysis. CD4⁺CXCR5⁺ T cells were isolated using an AutoMACS (Miltenyi Biotec) following the manufacturer’s instructions. In brief, we removed adherent cells and some B cells from MG and control PBL by negative selection with 1F5 (anti-CD20) plus LM2/1.6.11 (anti-Mac-1) followed by anti-mouse IgG-microbeads (Miltenyi Biotec). The cells were then positively (the CD8⁺ T cell population) or negatively (the CD4⁺ T cell population) selected with OKT8. Finally, CD4⁺ T cells were further separated into CD4⁺CXCR5⁺ and CD4⁺CXCR5^– T cells with anti-CXCR5 mAb (DakoCytomation) and anti-mouse IgG-microbeads. Three populations, CD4⁺ T, CD8⁺ T, and CD4⁺CXCR5⁺ T cells, were subjected to the TCR analysis. We determined the purity of isolated cells by flow cytometry after incubation of the cells with FITC-labeled anti-mouse IgG and confirmed that each population was >90% pure.

	Results

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Cross-sectional study of MG patients and healthy individuals by CDR3 spectratyping

We first performed a cross-sectional study of 38 MG patients and 44 healthy subjects to characterize the TCR repertoire in MG. Of 38 patients, six cases of the ocular type, 15 cases of MG with thymoma, 13 cases of the early onset, and two cases of the late onset were included. Our previous studies on neuroimmunological disorders revealed that a particular type of V TCR, i.e., V5.2, showed clonal expansion more frequently in MS (13), whereas there was no preferential usage of TCR in Guillain-Barré syndrome (21). When multiple examinations were performed on the same individuals, the results obtained in the first examination were included in the cross-sectional study. Representative spectratyping profiles are shown in Fig. 1 and the results summarized in Table I. Nearly half of controls (46.5%) showed a normal spectratype pattern (Fig. 1A). The density analysis revealed unskewed Gaussian distributions (Fig. 1C). In contrast, only a quarter of the MG patients showed the normal pattern (p < 0.05) (Table I). Remaining control and MG samples contained one or more expanded spectratypes (arrows in Fig. 1B). The distorted Gaussian distributions (V3 and V8 in Fig. 1D) and the monoclonal pattern (V14 in Fig. 1D) were detected in the density analysis. In healthy subjects, V9 spectratype expansions seemed to be more frequent compared with other Vs, but there was no significant difference. In MG patients, 29 cases (77.3%) showed the expansion of one or more spectratypes (Table I). Although these were more frequent in the MG patients than in healthy subjects, they did not show preferential usage of a common V, unlike MS.

View this table: [in this window] [in a new window]   Table I. The frequency of V expansion in healthy subjects and patients with MGa

View larger version (34K): [in this window] [in a new window]   FIGURE 2. CDR3 spectratyping of the thymus tissue. All six thymus tissues examined showed the normal spectratyping pattern without any expansion.

We have currently performed longitudinal studies on 22 MG patients and 17 healthy subjects. Among them, we selected 16 MG and 11 control cases; we were able to examine MG patients for more than 2 years and healthy subjects for 1 year (Table II and Table IV). Representative profiles obtained from MG patients are illustrated in Fig. 3, and all of the results are summarized in Table II. Longitudinal studies of oligoclonal expansions of TCR revealed three behavior patterns. 1) In persistent type, expansions were detected in all samples, as illustrated in Fig. 3 and Table II. In Fig. 3, V9 and V11 expansion persisted over 1 year and 5 mo. Nine of 16 patients showed this type of spectratype expansion during the observation periods (patients 4, 5, 7, 8, 10, 11, 12, 13, and 15 in Table II). 2) Heterogeneous type ranged from a normal pattern to one of short-lived expansions (patients 1, 2, 3, 14, and 16 in Table II). 3) In intermediate type, a particular spectratype expansion was intermittently observed (patients 6 and 9 in Table II).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Longitudinal studies of a MG patient showing the persistent pattern. Multiple examinations revealed that V9 and V11 expansions (A–C) indicated by arrows persisted over 1.5 years. Spectratype expansions other than V9 and V11 are not marked. This patient corresponds to Patient 8 in Table II.

CDR3 sequences of TCR clones derived from expanded spectratypes in MG

In a series of previous studies in MS (13) and its animal models (17, 22), we demonstrated that persistently expanded spectratypes represent expansions of particular TCR clones. To test whether that also applies in MG, we did a similar analysis using PBL from patient 7 (Table II), which showed V11 expansion over 2 years in 4 of 5 examinations. The expanded spectratypes were cut from the gel, and cDNA was extracted, cloned, and sequenced. As clearly demonstrated in Table III, we found a TCR clone with the CASS-ATPNEQFF-GPG sequence in the CDR3 throughout the 2 years. Evidently, it represents an expansion of a single T cell clone.

View this table: [in this window] [in a new window]   Table III. Amino acid and nucleotide sequences of the CDR3 of V11 TCRs extracted from patient 7a

We performed similar longitudinal studies in elderly healthy subjects who occasionally show clonal T cell expansion (23, 24, 25) to look for differences from those in our MG patients. As can be seen in Table IV, persistent spectratype expansions were found in subjects in their seventies and transient ones in their fifties and sixties. These TCR expansions are thought to occur due to exogenous Ag stimulation/infections and to reduce the T cell diversity (26). Interestingly, one volunteer (37 years old) residing in the community had a persistent cough when first tested (not included in Table IV). As shown in Fig. 4A, there were V2, 3, 6, 14, 20, and 24 expansions at that time point. Three months later when the cough had disappeared, the number of expanded spectratypes had reduced markedly (Fig. 4B). This change strongly suggests that spectratype expansions in middle-aged to elderly subjects can be elicited by exogenous Ag stimulation and may be short-lived.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. CDR3 spectratyping profile of one of control subject during persistent cough (A) and after it subsided (B). A volunteer had persistent cough when he was first examined by CDR3 spectratyping. As shown in A, there were V2, -3, -6, -14, -20, and -24 expansions at that time point. Three months later when the symptoms had disappeared, expanded spectratypes were markedly reduced (B).

These longitudinal studies on total T cells may indicate persistent TCR clonal expansions in autoimmune responses in MG patients. However, some persistent clonal expansions in healthy subjects were indistinguishable from those found in MG patients. To pursue that further, we spectratyped CD4⁺CXCR5⁺ T cells isolated from healthy subjects and MG patients (Table VI). Because these T cells reportedly act as helpers for follicular B cells (20), they might play an essential role in anti-AChR Ab production and in thymic hyperplasia, a classic sample of lymphoid neogenesis in autoimmune disorders. As shown in Table VI, the spectratype expansions found in three healthy subjects were mainly restricted to CD8⁺ T cells, as reported previously (26, 27), whereas in 2 of 3 cases CD4⁺ and CD4⁺CXCR5⁺ T cells showed normal spectratype patterns. Characteristically, in most MG patients, spectratype expansions found in the CD4⁺ T cell population were also detected in CD4⁺CXCR5⁺ T cells. These findings clearly demonstrate that the clonal expansions of T cells in MG patients are different from those in healthy subjects.

	Discussion

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Most studies used either FACS analysis with anti-TCR mAbs or CDR3 spectratyping. By FACS analysis using PBL or thymic cells, V5.1- (7), V12- (9), and V13- (11) positive T cells were reported to be significantly increased in MG patients. In our experience, however, this method is not sufficiently sensitive for TCR analysis of pathogenic T cells in autoimmune diseases. In EAE induced in Lewis rats by immunization with myelin basic protein, several lines of evidence indicate that encephalitogenic T cells mainly use V8.2. Clonal expansion of this cell type was detectable by CDR3 spectratyping in the lymphoid organ at the very early stages of the disease (32). In sharp contrast, by flow cytometry, this expansion could not be detected in the lymphoid organ throughout EAE (33). V8.2 expansion was only detectable at the peak of EAE in the CNS, where V8.2-positive cells accounted for >20% (33). This is mainly because the numbers of circulating encephalitogenic T cells are too small for detection by FACS analysis. So far, two CDR3 spectratyping studies have focused on the TCR repertoire in MG. Navneetham et al. reported that V4 and V6 were used significantly more in MG patients than in controls (10). In contrast, Infante et al. (12) found no significant difference in the TCR repertoire between MG patients and control subjects. The inconsistencies between these two reports and between these reports and our results may be largely attributable to the number of patients and controls and to the presence or absence of the longitudinal examination. In previous studies, the number of cases for the analysis was too small, and each case was examined only once.

We now summarize intriguing findings from the large-scale cross-sectional and longitudinal analyses of PBL and thymus taken from MG patients and healthy subjects by CDR3 spectratyping. First, TCR spectratype expansions were more frequently found in MG patients than healthy subjects (p < 0.05), though without preferential usage of any particular TCR -chains. Second, the longitudinal study clearly demonstrated persistent TCR V expansions more frequently in the patients with severe MG. Although persistent spectratype expansions varied from patient to patient, they correlated with severe clinical status and high anti-AChR Ab titers. Finally, examination of CXCR5⁺ T cells may help to focus on pathogenic Th cells in MG. In MG patients, spectratype expansions were found in CD4⁺ and CD4⁺CXCR5⁺ T cells, whereas spectratype expansion seen in healthy subjects was observed in CD8⁺ T cells. We have tried to exclude the possibility that preceding infection occurred in MG patients may induce spectratype expansion unrelated to the MG pathogenesis as follows. For this purpose, we have checked all of the patients subjected for the longitudinal study whether they have clinical signs and laboratory data suggesting infection. All of the patients did not have fever and increased white blood cells at the time of spectratype examinations. Only patient 12 at the second examination exhibited slightly increased C-reactive protein. However, it is unlikely that this would modulate the results of CDR3 spectratyping, because the results at the second examination were almost the same as those obtained in other examinations. Taken together, the present study strongly suggests that T cells showing persistent clonal expansion are involved in the development and maintenance of MG.

It should be noted that all nine thymus tissues, including six hyperplasmic thymi, exhibited normal spectratype patterns. Although the thymus has previously been implicated as a possible site of the disease origin (75% of patients have thymic abnormalities) (1), its role in MG is still a mystery (29). Our findings from CDR3 spectratyping suggest either that the thymus includes no clonally expanded pathogenic T cells or that their frequency is too low to detect even using this method. Importantly, >30% of MG patients do not show clinical improvement after thymectomy (34). Collectively, it is possible that the thymus plays a role in exacerbation of the disease in some, but not all, MG patients. In this regard, recent progress in elucidating the mechanism of lymphoid neogenesis provides useful information for the understanding of the relationship between thymic abnormalities and MG. As reviewed by Weyand et al. (35), ectopic germinal centers are formed in several organs in the presence of autoantigens, CXCL13-producing tissue cells, follicular dendritic cells, and B and T cells. Although it remains to be elucidated whether all of these factors are present in the thymus of MG patients, it is possible because AChRs are found in the thymic myoid cells (36).

In a series of reports, Berrih-Aknin and colleagues (7, 37) have insisted that V5.1-positive T cells are pathogenic in MG, and that circulating anti-V5.1 Abs may regulate the excess of pathogenic V5.1-positive T cells (8). This conclusion was drawn on the basis that by FACS analysis, V5.1-positive T cells were slightly but significantly increased in number in the thymus of MG patients. However, this finding was not confirmed by other groups using flow cytometry and CDR3 spectratyping. Similarly, we have not observed significant V5.1 clonal expansion in this study. Taking all these findings into consideration, it can be said that the conclusion by Berrih-Aknin et al. (7, 37) remains unestablished.

In summary, we were able to identify disease-associated and clonally expanded T cells in patients with MG by CDR3 spectratyping. Close examination of pathogenic T cells in MG provides useful information to elucidate the pathogenesis and to estimate the disease status.

	Acknowledgments

 
We are grateful to Dr. Masakatsu Motomura (First Department of Internal Medicine, Nagasaki University, School of Medicine, Nagasaki, Japan) for providing information about MG patients.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported in part by Grants-in-Aid from the Ministry of Education, Japan and from the Neuroimmunological Disorders Research Committee of the Ministry of Health, Labor and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Yoh Matsumoto, Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6 Fuchu, Tokyo 183-8526, Japan. E-mail address: matyoh{at}tmin.ac.jp

³ Abbreviations used in this paper: MG, myasthenia gravis; AchR, acetylcholine receptor; MS, multiple sclerosis.

Received for publication October 27, 2005. Accepted for publication February 2, 2006.

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