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* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan;
Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi, Japan; and
Department of Neurology, Kohnan Hospital, Sendai, Miyagi, Japan
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Abstract |
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5.2 were often oligoclonally expanded in peripheral blood of MS patients, but not of healthy subjects. Sequence analysis of the complementarity-determining region 3 region of spectratype-derived TCR clones revealed that the predominant TCR clone was different from patient to patient, but that similar results were obtained in a patient examined at different time points. More importantly, examination of cerebrospinal fluid T cells and longitudinal studies of PBLs from selected patients revealed that V5.2 expansion was detectable in the majority of patients examined. These findings suggest that V5.2 spectratype expansion is associated with the development of MS and that TCR-based immunotherapy can be applicable to MS patients if the TCR activation pattern of each patient is determined at different stages of the disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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17 and, to a lesser extent, V12 were frequently used, while Kotzin et al. (6) found the preferential usage of V5.2 and V6.1 by MBP-specific T cell clones. Later studies failed to confirm the preferential usage of V5.2 by MBP-specific T cell clones (7, 8). Other studies reported that although a particular type of TCR is predominantly used by MBP-specific T cell clones from a single MS patient, it varies from patient to patient, suggesting individual-specific TCR restriction (9, 10). The reasons that the findings obtained were so different remain unclear, but it is partly attributable to the difference in the methods used to establish the MBP-specific T cell lines and clones.
To avoid bias produced during culture, we applied complementarity-determining region 3 (CDR3) spectratyping analysis to identify pathogenic TCRs without culture and succeeded in doing so in several animal models, including experimental autoimmune encephalomyelitis (EAE), experimental autoimmune neuritis, and experimental autoimmune carditis (11, 12, 13, 14, 15). By this method, oligoclonal expansion of pathogenic TCRs could be identified not only using T cells isolated from the target organ, but also using PBL (16). According to Maini et al. (17), oligoclonal expansion of a spectratype (distorted Gaussian distribution) is visible at 1/1000 dilution of a T cell clone in 5 x 106 PBL. Therefore, clonal expansion is recognizable in a large number of nonexpanded T cells. Furthermore, in experimental autoimmune neuritis and experimental autoimmune carditis, pathogenicity of identified TCRs was confirmed by the finding that injection of DNA vaccines encoding TCR Vs selected by spectratyping successfully protects animals from the development of these diseases (14, 15).
In the present study, we analyzed the TCR repertoire of PBL and cerebrospinal fluid (CSF) cells taken from MS patients and healthy subjects by CDR3 spectratyping and determined the CDR3 sequences of TCR clones derived from spectratypes of interest. Although there have been several attempts to analyze the TCR repertoire in MS patients by the same method, they were limited to particular TCR families (18, 19). In this study, we have performed the overall TCR -chain repertoire analysis using PBL and CSF cells and demonstrated that the V5.2 spectratype is expanded more frequently than other Vs in MS patients. These findings suggest that V5.2-positive T cells are associated with the development of MS.
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Materials and Methods |
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A total of 42 MS patients, 5 males and 37 females (mean age of 36.4 ± 10.2 years, range 18–64 years), attending the Departments of Neurology, Tohoku University Hospital and Kohnan Hospital, were subjected to the cross-sectional study. The diagnosis of MS was made on the basis of the criteria proposed by the International Panel (20). The patient group included 35 cases of relapsing-remitting, 2 cases of primary progressive, and 5 cases of secondary progressive types of MS. The intervals between the onset of the disease and the first CDR3 spectratype examination ranged from 6 mo to 15 years. PBL and CSF cells used for the cross-sectional study (results shown in Tables I, II, and V) were taken from patients during relapses before corticosteroid treatment. Patients during remission received no treatment. The presence or absence of the treatment during longitudinal examinations is indicated in Fig. 5. A total of 30 healthy volunteers, 6 males and 24 females (mean age of 38.2 ± 12.1 years, range 19–68 years), who had no episode of common cold or influenza infection within 1 mo, were examined as controls. There was no significant difference in the male:female ratio between the patient and control groups (p = 0.35 by 
2 test). All subjects’ consent was obtained, and the study was approved by the Institute Review Board. Ten milliliters of heparinized blood was drawn from patients and control subjects, and PBL was isolated by the density gradient method. CSF cells were collected from 5 ml of CSF after centrifugation.
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RNA was extracted from PBL and CSF cells using RNAzol B (Biotecx Lab, Houston, TX). cDNA was then synthesized by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan) and amplified in a thermal cycler (PerkinElmer, Norwalk, CT) using primer pairs for TCR. Primers for V1–24 were the same as those used in a previous study (21). C primer was labeled with Cy5 or rhodamine or remained unlabeled.
Determination of the presence of DRB1*1501 allele
The presence or absence of the DRB1*1501 molecules in PBL samples was determined by the protocol proposed in the 11th International Histocompatibility Workshop. cDNA were amplified using a DR-2-specific primer pair (5'-TTCCTGTGGCAGCCTAAGAGG-3' and 5'-CCGCTGCACTGTGGAGCTCT-3'). Then the PCR products were examined for the DR2 allelic subtypes (DRB1*1501, DRB1*15012, DRB1*1601, and DRB1*1602) by dot-blot analysis using three sequence-specific oligonucleotides. Sequence-specific oligonucleotide probes used in this study were as follows: DRB2813 (5'-GTTCCTGGACAGATACTT-3', corresponding to DRB1*1501, DRB1*1502, DRB1*1601, and DRB1*1602), DRB7011 (5'-GACATCCTGGAGCAGGCG-3', corresponding to DRB1*1501 and DRB1*1502), and DRB8603 (5'-AACTACGGGGTTGTGGAG-3', corresponding to DRB1*1501). The probes were labeled with digoxigenin using digoxigenin-tagged dUTP and terminal transferase (Boehringer Mannheim, Tokyo, Japan). Detection of hybridized probes was conducted by the chemiluminescent signal detection system (Boehringer Mannheim).
CDR3 spectratyping
CDR3 spectratyping was performed, as described previously (13). 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 FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan). Spectratype expansion was evaluated by visual inspection and density analysis of the image using a software attached to the fluorescence image analyzer and graded into two categories (see Fig. 3). Oligoclonal pattern of spectratype expansion is referred to as the increase of the density and thickness of a band, keeping other normal spectratype profile (distortion of the Gaussian distribution) (17). Monoclonal type of spectratype expansion is referred to as marked increase of the density and thickness of a band with faint or no additional spectratypes (the monoclonal pattern) (17).
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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 were performed only for five cycles. Then PCR products were ligated into pT-Adv vector and cloned using the AdvanTAge PCR Cloning Kit (Clontech Laboratories, Palo Alto, CA), according to the manufacturer’s instruction. 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, Tokyo, Japan). 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 (22).
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Results |
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CDR3 spectratyping analysis of PBL from healthy individuals
There are several studies showing oligoclonality in the TCR repertoire in healthy individuals (23, 24, 25). Unlike experimental animals, humans are always exposed to T cell-activating agents such as bacteria, viruses, and vaccines. Therefore, it is essential to know the TCR spectratype profile of age-matched healthy subjects to estimate the findings obtained from MS patients. We examined 30 healthy subjects, and the representative results and the summary of overall examinations are shown in Fig. 1 and Table I, respectively. Fig. 1 depicts the normal spectratype pattern in which each spectratype shows a Gaussian distribution without any spectratype expansion. Density analysis of V8 and V12 spectratypes shown in Fig. 1 revealed that this type of spectratype consisted of seven peaks and showed the symmetrical profile with the highest density in the middle (Fig. 3, A and B). We also measured the density of all other spectratypes and obtained essentially the same findings (not shown). One-half of healthy subjects aged in their twenties and thirties showed this pattern, while the rest of this group and the majority of the individuals aged in their forties and fifties possessed one or two spectratype expansions (Table I). There was no predominant V spectratype expansion except V9 expansion, which was detected in four individuals (Tables I and II).
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We made a similar analysis using PBL from MS patients during an active stage or remission (Table I). All the samples shown in this table were taken from patients during the active stage before treatment and those during remission who had no treatment. All patients except four aged in their twenties and thirties during the active stage of the disease showed one or more spectratype expansions (Table I). One patient showed V3, V5.2, and V24 spectratype expansions, as shown in Fig. 2. Density analysis revealed that V3 and V5.2 spectratypes showed the oligoclonal-type expansion in which the peak in the middle increased in density, keeping other normal spectratype profile (Fig. 3, C and D). V24 spectratype expansion was the monoclonal type (Fig. 3E). In the latter case, one spectratype showed marked increase of the density without additional spectratypes. Overall examinations revealed that expanded V spectratypes during the active stage were generally diversified, but that the V5.2 and V24 expansion was relatively frequent compared with others (Table I). Of interest was the spectratype pattern obtained from patients during the remission phase (Table I). The representative results and summary are shown in Fig. 4 and Table I, respectively. Seven of nineteen patients showed the normal pattern without any spectratype expansion, while the remainder had one or two spectratype expansions. In the latter group, only a limited number of Vs was activated, and V5.2 expansion was recognized in 8 of 12 patients (Table I). Because activation of pathogenic T cells bearing a particular type of TCR persists during the remission phase in chronic relapsing EAE (12), V5.2 expansion in MS patients during remission suggests that T cells bearing this phenotype are activated in association with the disease development. The results of longitudinal study of some patients also supported this suggestion (see below). Finally, we determined the frequency of expanded spectratypes in all MS patients and compared it with that of healthy subjects. As shown in Table II, V5.2 expansion was most frequently found and accounted for 31.0% of MS patients. The second group of frequently found Vs was V1, V9, V11, and V24. Frequent expansion of all these Vs except V9 was not observed in healthy subjects. The incidence of V5.2 and 24 expansion in MS patients was significantly increased compared with that in healthy subjects (p = 0.003 and p = 0.034, respectively, by Fisher’s probability test).
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Because V5.2 and V24 spectratype expansion was seen frequently in MS patients, we determined the nucleotide and amino acid sequences of the CDR3 region of TCR clones derived from these spectratypes to see whether there is expansion of particular clones. For controls, we examined normal-looking V5.2 and V9 spectratypes from healthy individuals. A band in the V5.2 spectratype with the same size as that frequently showing expansion in MS patients was cut, and the CDR3 region was sequenced after cloning (Table III). Among 10 TCR clones sequenced, there was no identical sequence indicating that TCR clones in normal spectratypes were quite heterogeneous. Essentially the same results were obtained in the analysis of V9 spectratype (data not shown).
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5.2-derived TCR repertoire became more diverse. These findings suggest that activation and clonal expansion of T cells in some, if not all, patients persist even during the remission phase.
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5.2, V24 showed more heterogeneity (data not shown). In two of three patients, V24 clones possessed the predominant sequence, but the frequency of the clone was very low compared with V5.2. One patient showed complete heterogeneity in the expanded V24 spectratype. Characterization of CSF cells from MS patients
CSF cells were taken from five MS patients (six samples) during the active stage along with PBL and were analyzed by CDR3 spectratyping (Table V). Although V5.2 expansion in PBL was observed in two of five samples taken from five patients, all six CSF samples from the same patients showed oligoclonal expansion of V5.2 spectratype.
Longitudinal studies of selected patients with relapsing-remitting and secondary progressive MS
We also did longitudinal studies on some patients with relapsing-remitting and secondary progressive MS. Seven patients who had been examined for >15 mo were selected for this study (Table VI and Fig. 5). Compared with the cross-sectional study shown in Table II, the proportion of patients bearing HLA-DR2 was high, and six of seven patients possessed HLA-DR2 (five DRB1*1501 and one DRB1*1601). Four patients showed the persistent spectratype expansion of particular Vs (Table VI). The results of the follow-up study of patient YO in Table VI were shown in Fig. 5A. Spectratype examinations were performed seven times during the follow-up period, and V5.2 expansion was found in five examinations (Fig. 5A). In contrast, patient MS in Table VI showed the different characteristic pattern. In the initial two examinations, V5.2 expansion was noted, but V11 expansion became prominent in the subsequent nine examinations (in this study referred to as the switched pattern) (Fig. 5B). Interestingly, V11 expansion was detectable not only at an active stage, but also was found during the remission phase in this patient. One patient (patient FK in Table VI) showed the normalized pattern (Fig. 5C). After the long-lasting remission, the spectratypes showed the normal pattern in multiple examinations. In summary, the longitudinal studies revealed that five of seven patients showed V5.2 expansion during a certain period of, or throughout, the observation period and supported the findings obtained in the cross-sectional study shown in Table II.
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Discussion |
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-chains and probably also 
-chains. Based on this assumption, many studies have tried to identify TCR involved in the process of MS development, but, until now, there is no consensus on the phenotype of TCR used by MS-associated T cells. One of the reasons for this is that the majority of studies were performed using in vitro established neuroantigen-specific T cell lines and clones. Differences in the disease status of MS patients at the time of sampling and the methods for establishing T cell lines and clones may cause variations in the findings.
In the present study, we tried to elucidate the TCR repertoire of MS patients by CDR3 spectratyping analysis. This was mainly based on the findings obtained in our previous studies using animal autoimmune disease models. In rat EAE, only a limited number of spectratypes showed oligoclonal expansion in the spinal cord and PBL throughout the course of rat EAE, whereas irrelevant TCRs became more diverse at later stages of the disease (12, 13). Furthermore, the CDR3 sequence of the majority of TCR clones derived from the EAE-specific spectratype was the same as that of encephalitogenic T cell clones (26, 27, 28). These findings indicate that expanded V spectratypes with the above characteristics well represent TCR of disease-inducing T cells. More importantly, immunotherapy with TCR DNA vaccines targeting Vs selected by spectratyping analysis successfully suppressed the development of autoimmune diseases, indicating that CDR3 spectratyping is a reliable method for identifying pathogenic TCR (14, 15).
Using the same strategy, we analyzed PBL and CSF cells taken from MS patients and healthy individuals and demonstrated that V5.2, but not other V, expansion was recognized frequently in MS patients by several approaches. First, the overall examination using PBL (Table II) demonstrated that the frequency of V5.2 expansion was significantly higher in MS patients than in healthy individuals. Second, all the CSF samples examined showed V5.2 expansion. Third, the longitudinal studies revealed that five of seven patients showed V5.2 expansion during a certain period of, or throughout, the observation period. Interestingly, all these five patients bore DRB1*1501, which was reported to be highly associated with the susceptibility to MS and to be the restriction molecules for MBP-reactive T cell clones established from DR2-positive MS patients (3). Taken together, these findings suggest that activation of V5.2-positive T cells plays a role in the development of MS.
To date, several Vs, including V5.2 (6), V13 (29), and V17 (5), were reported to be frequently used by MBP-specific T cell lines and clones derived from MS patients, but not from healthy individuals (3). However, V13 and V17 expansion was rarely seen in this study. Although the precise reason for this discrepancy is unknown, several factors should be considered. The first factor is the difference in the number of patients examined. In the majority of in vitro studies, T cell lines and clones were established from five or fewer patients, raising the possibility that the nature of the predominant lines and clones varies from one study to another, depending on the patient and culture conditions. Another possibility is that V13 and V17 are used by minor pathogenic T cells. In animal models, immunotherapy with anti-TCR mAb targeting the major encephalitogenic TCR induces the activation of T cells bearing the second pathogenic TCR that was not recognized before the treatment (30). In this regard, the follow-up studies are essential for determination of pathogenic T cells.
It remains poorly understood with regard to the Ag specificity of oligoclonally expanded V5.2-positive T cells. Two pieces of evidence have suggested that these T cells recognize MBP molecules. First, V5.2-positive and MBP-specific T cell clones were frequently established from MS patients, especially from those bearing HLA-DR2 (3). Second, an increase of the proportion of V5.2/5.3-positive cells in PBL and CSF cells in MS patients was detected by FACS analysis. Furthermore, selective in vitro depletion of these T cell subsets resulted in a drastic decrease in the number of MBP-reactive and IFN-
-secreting T cells (31, 32). In addition to these findings, we showed in this study that V5.2 spectratype expansion was equally found in DRB1*1501-positive and -negative patients (Table II). Therefore, the presumed Ag epitope(s) recognized by V5.2-positive T cells would be presented by DRB1*1501 as well as non-DRB1*1501 molecules. Until now, such T cell epitopes presented by different MHC class II molecules were reported as universal T cell epitopes (33, 34). Interestingly, the immunodominant region of human MBP (residues 84–102) is also a universal epitope presented by several HLA-DR molecules, including DRB1*1501 (5, 35, 36). Therefore, V5.2-positive T cells found in the present study could recognize such neuroantigens. However, it should be noted that MBP-reactive T cells would not be restricted to V5.2-positive T cells, as demonstrated previously (10, 29) and suggested in this study. The important point is that the major population of pathogenic T cells should be monitored frequently by appropriate methods.
In the present study, we were able to monitor the TCR repertoire of oligoclonally expanded T cells during the course of MS and demonstrated that V5.2 expansion was more frequently found than other Vs. These findings provide useful information for designing TCR-based immunotherapy.
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Acknowledgments |
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Footnotes |
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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: MS, multiple sclerosis; CDR3, complementarity-determining region 3; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein.
Received for publication July 8, 2002. Accepted for publication February 27, 2003.
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References |
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usage to immunodominant regions of myelin basic protein. Science 248:1016.-chain variable gene use in myelin basic protein-reactive T-cell clones from patients with multiple sclerosis. Proc. Natl. Acad. Sci. USA 88:9161.5.2 in myelin basic protein-specific T cell lines derived from HLA-DR2 positive multiple sclerosis patients and controls. J. Neuroimmunol. 84:7.[Medline]
gene usage by myelin basic protein-specific T-cell clones in multiple sclerosis: predominant genes vary in individuals. Proc. Natl. Acad. Sci. USA 88:2466. chains with different CDR3 motifs by spinal cord T cells in autoimmune encephalomyelitis. J. Neuroimmunol. 96:66.[Medline]
8.2 TCR spectratype in the central nervous system of rats with chronic relapsing EAE. J. Immunol. 161:6993.5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients. Proc. Natl. Acad. Sci. USA 93:12461.5 and V17 clonal diversity in cerebrospinal fluid and peripheral blood lymphocytes of multiple sclerosis patients. Mult. Scler. 4:154. T-cell repertoires are equally complex and are characterized by different levels of steady-state TCR expression. Hum. Immunol. 48:52.[Medline]
and V chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different. J. Exp. Med. 169:27. chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3. J. Exp. Med. 174:1467.13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis. J. Immunol. 163:3530.5.2/5.3 and V6.7 in multiple sclerosis. J. Neuroimmunol. 118:85. (Abstr.).
5.2/5.3+ T cells in multiple sclerosis: results from an MRI-monitored phase II clinical trial. Ann. Neurol. 51:467.[Medline]
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and •, Indicate the examination before and during corticosteroid treatment, respectively. The case shown in A (patient YO in Table VI
in each examination is shown in lower 3–5 rows. A, B, and C, Correspond to patients YO, MS, and FK in Table VI
