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Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 April 2002, Vol. 99, No. 7, pp. 2499-2504 IMMUNOBIOLOGY Restricted T-cell receptor -chain usage by T cells autoreactive to 2-glycoprotein I in patients with antiphospholipid syndrome Kazue Yoshida, Takahide Arai, Junichi Kaburaki, Yasuo Ikeda, Yutaka Kawakami, and Masataka Kuwana From the Institute for Advanced Medical Research and the Department of Internal Medicine, Keio University School of Medicine, Tokyo; and the Department of Internal Medicine, Tokyo Electric Power Company Hospital, Japan.     Abstract Top Abstract Introduction Patients, materials, and... Results Discussion References We recently identified CD4+ T cells that are autoreactive to 2-glycoprotein I (2GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR)  chains of 2GPI-reactive T cells were examined in 8 2GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant 2GPI. The TCR V segments that expanded oligoclonally after stimulation with 2GPI varied among responders, but the V7 and V8 segments were commonly detected in 6 and 4 2GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of 2GPI-reactive T cells revealed limited diversity, and all V7+ TCRs had an amino acid motif of TGxxN/Q or minor variations. The V8+ TCRs had another motif, PxAxxD/E. Surprisingly, an identical V7+ TCR chain was used by 2GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCR usage between APS patients and healthy responders. Some of the V7+ TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by 2GPI-specific CD4+ T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of V7+ or V8+ T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-2GPI antibody production in response to 2GPI. Our results indicate preferential usage of TCR chains by 2GPI-reactive T cells. These TCR chains can be reasonable targets for TCR-based immunotherapy for patients with APS. (Blood. 2002;99:2499-2504) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Patients, materials, and... Results Discussion References Antiphospholipid syndrome (APS) is characterized by arterial and venous thrombosis and by recurrent fetal loss associated with the presence of antiphospholipid antibody.1 The most common target recognized by the antiphospholipid antibody is 2-glycoprotein I (2GPI),2,3 a plasma glycoprotein that binds various kinds of negatively charged substances, including phospholipids, lipoproteins, and activated platelets.4,5 Accumulated evidence in animal models strongly suggests that anti-2GPI antibodies are directly involved in the pathogenic process of APS.6-8 Although precise mechanisms for the thrombophilia caused by anti-2GPI antibodies remain unclear, one possibility is that these antibodies bind to endothelial cell surfaces by recognizing the adhered 2GPI and induce endothelial cell activation, resulting in the up-regulation of procoagulant and inflammatory processes.9,10 On the other hand, it has recently been shown that anti-2GPI antibodies bind to oxidized, low-density lipoprotein through adhered 2GPI and promote its uptake by macrophages, resulting in the promotion of atherosclerosis.11 We recently identified CD4+ T cells responsive to 2GPI in patients with APS and in some healthy subjects.12 2GPI-reactive CD4+ T cells in patients with APS promoted anti-2GPI antibody production from autologous B cells. Analysis of CD4+ T-cell clones from APS patients specifically responsive to 2GPI revealed that the antigenic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major phospholipid-binding site, is preferentially recognized by 2GPI-specific T cells.13 Helper activity that stimulates B cells to produce anti-2GPI antibodies is mediated through T-cell-derived interleukin (IL)-6 and CD40-CD40 ligand engagement. Because of this essential role of 2GPI-reactive T cells in anti-2GPI antibody production, the elimination or inactivation of pathogenic 2GPI-reactive T cells should inhibit anti-2GPI antibody production and prevent thrombosis and fetal loss in patients with APS. One of the immunologic interventions to induce selective suppression of an autoantigen-specific T-cell response is a strategy targeting the variable regions of T-cell receptors (TCRs) that are preferentially used by autoantigen-reactive T cells.14 This strategy has been effective in suppressing pathogenic autoimmune responses and improving symptoms in animal models for several organ-specific autoimmune diseases.15-18 To apply this selective therapeutic strategy, it is indispensable that the set of TCRs used by autoantigen-reactive T cells be highly restricted, but the TCR chain usage of pathogenic 2GPI-reactive T cells has not been examined to date. In this study, V gene usage and TCR complementarity-determining region 3 (CDR3) sequences of 2GPI-reactive T cells were analyzed in APS patients and healthy responders using polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant 2GPI.     Patients, materials, and methods Top Abstract Introduction Patients, materials, and... Results Discussion References Patients and controls Peripheral blood T cells from 5 Japanese patients with APS were analyzed in this study. They were selected from 12 patients enrolled in our previous study based on the presence of serum anti-2GPI antibody, 2GPI-induced T-cell proliferative response, and availability of blood samples.12 All patients fulfilled the preliminary classification criteria for APS proposed by the International Workshop.19 A diagnosis of primary APS was made in 3 of the patients, and the remaining 2 had secondary APS accompanied by systemic lupus erythematosus. Clinical and serologic characteristics of these patients were described in detail in our previous report.12 At the time of blood examination, all the patients were taking low-dose corticosteroids (less than 10 mg/d) and aspirin. Three healthy persons with 2GPI-induced T-cell proliferative response were selected from 12 volunteers analyzed in the previous study12 and were used as control subjects. All samples were obtained after the patients and control subjects gave their written informed consent, approved by the Keio University Institutional Review Board. HLA class II allele genotyping Genomic DNA was isolated from peripheral blood leukocytes using a standard phenol extraction procedure. HLA-DRB1, DRB4, DQB1, and DPB1 alleles were determined using PCR followed by analysis of restriction fragment-length polymorphisms.20,21 Flow cytometric analysis Two-color cell staining was performed using a phycoerythrin-conjugated monoclonal antibody (mAb) to CD3 (BD PharMingen, San Diego, CA) and an anti-V2, anti-V7, or anti-V8 mAb (Immunotech, Marseilles, France) plus a fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin (Ig) G antibody (Immunotech). Cells were analyzed on a FACSCalibur flow cytometer (BD PharMingen) using the CellQuest software. Antigen preparations GP-F, a recombinant maltose-binding protein (MalBP) fusion protein encoding the entire amino acid sequence of human 2GPI, was prepared and used as an antigen for T-cell stimulation.12 MalBP was also prepared as a control antigen. Cell preparations Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by Lymphoprep (Nycomed Pharma AS, Oslo, Norway) density-gradient centrifugation and were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 50 U/mL penicillin, and 50 µg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. In some experiments, PBMCs were depleted of V2+, V7+, or V8+ cells by mixing with anti-V2, anti-V7, or anti-V8 mAb, respectively, followed by incubation with goat anti-mouse IgG antibody-coupled magnetic beads (Dynal, Oslo, Norway). Bead-bound cells were subsequently removed using a magnetic apparatus. After the depletion treatment, the remaining cells that were positive for these TCR V chains constituted less than 0.1% of the population. GP-F and MalBP-stimulated T cells PBMCs (2 × 106/well) were cultured in 24-well plates with GP-F or MalBP (10 µg/mL). On day 3, 30 U/mL IL-2 was added to the culture. Cells were restimulated with antigen, IL-2 (100 U/mL), and 106 irradiated (40 Gy) autologous PBMCs in fresh medium at day 10. Seven days after the second stimulation, the cells were harvested and stored at 80°C until use. 2GPI-specific CD4+ T-cell clones Six 2GPI-specific CD4+ T-cell clones were established from patients with APS and were reported in detail previously.13 Clones KS3 and KS8 were generated from patient APS3, and clones OM2 and OM7 were generated from patient APS4. The remaining 2 T-cell clones, EY3 and EY8, were established from an another patient with APS whose blood sample was unavailable for this study. KS3, OM2, OM7, EY3, and EY8 were shown to recognize p276-290 in the context of HLA-DRB4*0103 and to induce anti-2GPI antibody production from autologous B cells. Antigenic profile and helper function were not fully analyzed for KS8. Detection of the TCR chains used by 2GPI-reactive T cells Total RNA was extracted from PBMCs, GP-F-stimulated T cells, and MalBP-stimulated T cells using an RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized from the total RNA using Molony murine leukemia virus reverse transcriptase (Takara, Kyoto, Japan) with oligo-dT priming. cDNA was then subjected to PCR amplification using a panel of V (V1-24) region-specific primers in combination with a C region-specific primer.22 PCR products were resolved by electrophoresis on 2% agarose gels and were visualized by staining with ethidium bromide. The intensity of individual V signals was semiquantified by densitometry using Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA). The relative expression of each V product was calculated as a ratio to the intensity of a control C product. TCR V chains, which were expressed in GP-F-stimulated T cells at a level that was at least twice as high as in unstimulated T cells, were selected as candidate TCR V chains used by 2GPI-reactive T cells and were further analyzed by SSCP analysis.23 Specifically, the PCR products in a denaturing solution consisting of 95% formamide and 20 mM EDTA were boiled for 5 minutes and were loaded onto 5% polyacrylamide gels containing 10% glycerol. Gels were run at 30 W constant power for 2.5 hours. After electrophoresis, the gels were stained with a Silver Stain Plus kit (Bio-Rad Laboratories) according to the manufacturer's protocol. Clonally expanded single-strand DNA bands present in GP-F-stimulated T cells, but not in unstimulated or MalBP-stimulated T cells, were regarded as coding for the TCR chains of 2GPI-reactive T cells. Nucleotide sequencing of CDR3 in TCR chains Individual single-strand DNA bands of interest in silver-stained gels were re-amplified with V and C primers using the "bandstab" technique.24 PCR products were ligated into a pGEM-T vector (Promega, Madison, WI) and were transfected into competent DH5 Escherichia coli (Toyobo, Osaka, Japan). Both strands of at least 3 independent colonies were sequenced on an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA) using BigDye Terminator Cycle Sequence Ready Reaction kit (Applied Biosystems). A nucleotide sequence obtained from 2 or more colonies was regarded as that of a TCR chain used by 2GPI-reactive T cells. TCR CDR3 nucleotide sequences of 2GPI-specific T-cell clones were also determined directly from the PCR products as described previously,25 except that an ABI Prism 310 genetic analyzer (Applied Biosystems) was used instead of gel electrophoresis of radiolabeled nucleotides. CDR3 length was defined as the region starting from the amino acid residue after the CASS sequence of most V segments and ending before the GxG box in the J region.26 In vitro production of anti-2GPI antibodies in PBMC cultures In vitro assays to analyze the 2GPI-induced synthesis of anti-2GPI antibodies in PBMC cultures were carried out as described.12 Briefly, PBMCs and V2+, V7+, and V8+ cell-depleted PBMCs were cultured in complete medium with GP-F (10 µg/mL) in the presence of pokeweed mitogen (1 µg/mL) for 10 days. IgG anti-2GPI antibody levels in undiluted culture supernatants were measured by an enzyme-linked immunosorbent assay kit (Yamasa, Choshi, Japan), in which cardiolipin-coated plates were incubated with purified human 2GPI as a cofactor. All cultures were prepared in duplicate, and the results were expressed as the OD450 and were calculated as the mean of the duplicates minus the mean of the blank reference wells, which did not contain sample. Differences in antibody levels between samples with and without the depletion treatment were assessed by Student t test.     Results Top Abstract Introduction Patients, materials, and... Results Discussion References TCR V segments used by 2GPI-reactive T cells The expression of individual V gene segments in unstimulated and GP-F-stimulated T cells was semiquantified by family PCR using a panel of V region-specific primers. Unstimulated T cells expressed many V genes at varying levels, but a limited set of V genes were expressed by GP-F-stimulated T cells. TCR V chains whose expression levels were higher in GP-F-stimulated T cells than in unstimulated T cells were subjected to SSCP analysis. As shown in Figure 1, oligoclonally expanded single-strand DNA bands present in the GP-F-stimulated T cells, but not in the unstimulated or MalBP-stimulated T cells, were identified as DNA encoding TCR of 2GPI-reactive T cells. To verify the initial selection step by densitometry, 12 randomly selected V products that did not increase after GP-F stimulation were examined using SSCP analysis. Results showed that there was no oligoclonal band specifically present in GP-F-stimulated T cells. V gene segments used by 2GPI-reactive T cells and HLA class II alleles in 5 APS patients and 3 healthy responders are summarized in Table 1. Two or more V segments were used by 2GPI-reactive T cells in all subjects except APS2. V segments that were oligoclonally expanded after stimulation with 2GPI varied among subjects, but V7, V8, and V13.1 were detected in 2 or more responders. The most frequently detected V gene segment was V7, which was used in 6 responders including 4 APS patients and 2 healthy donors. V8 and V13.1 were also detected in 4 and 2 responders, respectively. We noted that either V7 or V8 was used by 2GPI-reactive T cells in all 2GPI-responders. There was no apparent difference in the V gene usage of 2GPI-reactive T cells between APS patients and healthy responders, though the number of subjects examined was small. No statistically significant association was found between the V genes used by 2GPI-reactive T cells and HLA class II alleles, but all 4 responders with 2GPI-reactive V8+ T cells were homozygous for DPB1*0501. View larger version (126K): [in this window] [in a new window]   Figure 1. Oligoclonal expansion of TCR after stimulation with 2GPI. PCR-amplified TCR products of unstimulated, GP-F-stimulated, and MalBP-stimulated T cells were fractionated on 5% polyacrylamide-glycerol gels and stained with silver. Oligoclonally expanded single-strand DNA bands present in GP-F-stimulated T cells, but not in unstimulated or MalBP-stimulated T cells, were identified as DNAs encoding TCR chains of 2GPI-reactive T cells.                                View this table: [in this window] [in a new window]   Table 1. HLA class II alleles and TCR V gene segments oligoclonally expanded after stimulation with 2GPI in patients with APS and in healthy responders TCR CDR3 amino acid sequences of 2GPI-reactive T cells SSCP bands for V7, V8, and V13.1 were re-amplified directly from the gels, and their nucleotide sequences were determined after subcloning. The identical sequence was obtained from 2 independent SSCP-derived clones in some cases, and these corresponded to positive and negative DNA strands. We were unable to amplify some of the SSCP bands despite repeated attempts using various PCR conditions, probably because residual silver interfered with the amplification reaction. Deduced amino acid sequences of TCR CDR3 in 2GPI-reactive T cells are summarized in Table 2. Notably, all 7 V7+ TCRs obtained from 6 responders had the amino acid motif TGxxN/Q, or minor variations of it, in their CDR3 sequences. Either V7.1 or V7.2 was rearranged with J1.2, J1.5, or J1.6. The CDR3 length was 12 or 13 amino acids, except in healthy donor HD4. A similar amino acid motif was also detected in 2 V13.1+ TCRs, in which V13.1 was rearranged with J1.2 or 1.5. Surprisingly, an identical CDR3 sequence was detected in V7+ TCRs obtained from 3 APS patientsAPS2, APS4, and APS12. To eliminate possible PCR contamination, the same experiments were repeated in APS2 and APS4 using blood samples obtained at different time points and with entirely new reagents. As a result, V7+ TCR with the identical CDR3 sequence was detected as the TCR -chain of 2GPI-reactive T cells in these patients. Interestingly, the amino acid motif TG was primarily encoded by the germline-encoded D1 segment (5'-acaggg-3'). The TGxxN/Q motif was not detected in 2 V7+ TCRs that oligoclonally expanded after stimulation with MalBP in APS2. In contrast, V8+ TCRs that were derived from 3 responders had the amino acid motif PxAxxD/E in their CDR3. Either V8.1 or V8.2 was rearranged with J2.3 or J2.7, and CDR3 was 9 or 11 amino acids long. The D gene segment could not be definitively identified in V8+ TCRs because of extensive nucleotide deletion. Nearly identical TCR chains were detected from APS patients and healthy responders. For example, V8+ TCRs derived from APS12 and HD4 had the same V8.1-J2.3-C2 gene rearrangement and a nearly identical CDR3 sequence, in which only one amino acid was different.                                View this table: [in this window] [in a new window]   Table 2. V-J-C gene rearrangement, CDR3 amino acid sequence, and CDR3 length in TCR chains used by 2GPI-reactive T cells TCR chains in 2GPI-specific CD4+ T-cell clones TCR chains of 6 2GPI-reactive T-cell clones were analyzed, and the results, in addition to the antigenic specificity and helper activity promoting anti-2GPI antibody production, are summarized in Table 3. The V-J-C rearrangement and the CDR3 sequence of clones KS3 and KS8 generated from APS3 were concordant with those determined by PCR-SSCP analysis in the same patient. T-cell clones OM2 and OM7 generated from APS4 had an identical V7+ TCR chain, which was also detected in 3 APS patients, including APS4, by PCR-SSCP analysis. Furthermore, this particular V7+ TCR was also detected in EY3 and EY8 generated from an APS patient who was unavailable for PCR-SSCP analysis. Identities of the TCR chains detected by the PCR-SSCP analysis and of the 2GPI-specific T-cell clones were further confirmed by SSCP analysis, in which TCR single-strand DNA had the same electrophoretic mobility on gels (results not shown). It was noted that KS3, OM2, OM7, EY3, and EY8, which recognized the immunodominant p276-290 and had helper activity, commonly used the TCR chains with V7 and the TGxxN/Q motif.                                View this table: [in this window] [in a new window]   Table 3. Antigenic specificity, helper activity inducing anti-2GPI antibody production, and TCR chain of 2GPI-specific CD4+ T-cell clones generated from APS patients Effects of V7+ or V8+ T-cell depletion on in vitro anti-2GPI antibody production To evaluate the relative contribution of V7+ and V8+ T cells in anti-2GPI antibody production in APS patients, the effects of V7+ and V8+ T-cell depletion on in vitro anti-2GPI antibody production in PBMC cultures were examined in samples from 3 APS patients. PBMCs depleted of irrelevant V2+ T cells were also examined as a control. As shown in Figure 2, anti-2GPI antibody production was inhibited by the depletion of V7+ T cells, but not by the depletion of V2+ or V8+ T cells, in cultures from patients APS2 and APS3, who had 2GPI-reactive V7+ T cells in circulation. On the other hand, anti-2GPI antibody production was specifically suppressed by the depletion of V8+ T cells from patient APS8, who had V8+ T cells that were responsive to 2GPI. These findings strongly suggest that the 2GPI-reactive T cells with helper activity preferentially use V7 or V8 in patients with APS. However, the proportions of V7+ and V8+ T cells in the peripheral blood T cells were 1.6% to 2.1% and 3.5% to 4.7%, respectively, in 8 responders, and there was no difference in these proportions between APS patients and healthy subjects or between responders with and without 2GPI-reactive T cells with these V segments. View larger version (40K): [in this window] [in a new window]   Figure 2. Effects of V7+ or V8+ T-cell depletion on in vitro anti-2GPI antibody production. V2+, V7+, and V8+ cell-depleted PBMCs and mock-treated PBMCs were cultured with GP-F (10 µg/mL) and pokeweed mitogen (1 µg/mL) for 10 days. Anti-2GPI antibody levels in undiluted culture supernatants were measured by an enzyme-linked immunosorbent assay. Significant inhibition of anti-2GPI antibody production by the depletion treatment is indicated by an asterisk. A representative result from 3 independent experiments is shown.     Discussion Top Abstract Introduction Patients, materials, and... Results Discussion References The current study demonstrates that the TCR chains of 2GPI-reactive T cells are highly conserved in APS patients and healthy responders. In most 2GPI-responders, 2GPI-reactive T cells used TCR chains with a rearranged V7 segment and a TGxxN/Q motif in the CDR3 sequence. Furthermore, the shared V7+ TCR chain was used by 2GPI-reactive T cells in 3 APS patients. We believe that this finding is not attributed to PCR contamination because detection of this particular V7+ TCR was reproducible, and V7+ TCRs of MalBP-reactive T cells detected by the same method used the entirely different CDR3 sequences. On the other hand, V8+ TCRs used by 2GPI-reactive T cells had a different CDR3 sequence, with a PxAxxD/E motif. It is likely that the TCR chains with these characteristics are predominantly used by pathogenic 2GPI-reactive T cells in most patients with APS because (1) V7+ or V8+ TCR was used by 2GPI-reactive T cells in all APS patients examined, (2) V7+ TCRs with the TGxxN/Q motif were also detected in CD4+ T-cell clones known to be specific to 2GPI and can induce anti-2GPI antibody production, and (3) in vitro anti-2GPI antibody production in PBMC cultures was significantly inhibited by the depletion of V7+ or V8+ T cells. 2GPI-reactive T cells using V segments other than V7 and V8 were also detected, but these appeared to be a minor repertoire of 2GPI-reactive T cells or were present only in a small subset of APS patients. CD4+ T-cell clones responsive to p276-290 in the context of DRB4*0103 were frequently generated by patients with APS.13 Five 2GPI-specific T-cell clones with this antigenic specificity (KS3, OM2, OM7, EY3, and EY8) used the V7 segment and the TGxxN/Q motif. Because the CDR3s of TCR and TCR chains provide the principle peptide-binding residues in a TCR molecule, the V7+ TCRs with the TGxxN/Q motif detected by the PCR-SSCP analysis may play a crucial role in recognition of the immunodominant T-cell epitope within p276-290. In addition, V13.1+ TCRs detected in 2 2GPI-responders and 2GPI-specific T-cell clone KS8 also had the TGxxN/Q motif in CDR3. Although the antigenic profile of KS8 was not fully determined, this clone was shown to recognize domain V, which includes p276-290, in an HLA-DR-restricted manner.13 Therefore, it is possible that 2GPI-reactive V7+ and V13.1+ T cells have the same antigenic specificity. In contrast, none of the 2GPI-specific T-cell clones analyzed used V8+ TCRs with the PxAxxD/E motif. V8+ TCR is presumed to recognize another epitope on 2GPI, given that 2GPI-reactive CD4+ T-cell clones were heterogeneous in terms of their antigenic specificity and that T-cell clones recognizing domain I/II in an HLA-DP-restricted manner and those recognizing domain IV in an HLA-DR-restricted manner could be also generated from patients with APS.13 APS patients and healthy responders used similar TCR chains for 2GPI-reactive T cells. This finding is analogous to the response of autoreactive T cells to myelin basic protein (MBP) in patients with multiple sclerosis (MS)27,28 and to topoisomerase I in patients with scleroderma.25 Interestingly, the CDR3 motifs in MBP-specific T cells are shared by MS patients, healthy persons, and an animal model for MS.28 Taken together with our previous finding that T cells responsive to 2GPI are present not only in APS patients but also in some healthy persons,12 the current study further supports the concept that 2GPI-reactive T cells are a component of the normal T-cell repertoire. This hypothesis could be explained by the recognition of cryptic determinants, not generated from a native molecule under normal circumstances,29 in 2GPI-reactive T cells. In fact, 2GPI-specific T-cell clones responded to reduced 2GPI and recombinant 2GPI fragments produced in bacteria, but not to native 2GPI.13 T cells responsive to cryptic self-determinants that escape negative selection in the thymus do not normally encounter the antigenic peptide in the periphery, but they would be activated if cryptic self-peptides were efficiently presented by antigen-presenting cells.30 In this study, PCR-SSCP analysis combined with in vitro stimulation of T cells with 2GPI was used to determine the TCR chains of 2GPI-reactive T cells. In most previous studies, the TCR usage of antigen-specific T cells was determined by analysis using antigen-specific T-cell clones.25,27,28 However, the generation of antigen-specific T-cell clones requires considerable effort and time, and available T-cell clones are potentially biased because T cells with rapid proliferation kinetics tend to be selected. The current study confirmed that the TCR chains detected by PCR-SSCP analysis were concordant with those of 2GPI-specific T-cell clones. Thus, PCR-SSCP analysis combined with in vitro antigenic stimulation of T cells is a powerful tool for analyzing the TCR usage of antigen-specific T cells without generating T-cell clones. Our results indicate the preferential usage of V7 and V8 segments by pathogenic 2GPI-reactive T cells in patients with APS. Although our findings were based on in vitro experiments on a relatively small number of patients, we could propose that the elimination or the inactivation of T cells expressing V7+ or V8+ TCRs may suppress the production of a disease-inducing anti-2GPI antibody. For this purpose, the unique structures of V7 and V8 could be targets of TCR-based immunotherapy, including anti-TCR V mAb administration and vaccination with V peptides or naked DNA encoding V sequences.15-18 Because regulatory T cells recognizing idiotopes on antigen-specific TCRs represent an important peripheral mechanism for controlling autoreactive T cells,31 selective stimulation of an anti-TCR response is a potential strategy for unleashing its powerful immunoregulatory activity. The efficacy of TCR-based immunotherapy was shown in animal models for various autoimmune diseases,15-18 but this strategy in patients was still under active investigation. For example, encephalitogenic T cells to MBP in the peripheral blood and central nervous system plaques of MS patients with DRB1*1501 were highly restricted and expressed similar V5.2+ or V6.1+ TCR chains.32,33 Based on this evidence, a double-blind, placebo-controlled trial to evaluate clinical and immunologic changes after vaccination with the V5.2 CDR2 peptide in patients with progressive MS was performed.34 A strong T-cell response to the V5.2 sequence was induced in 6 of 17 patients who received the V5.2 peptide, but in none of 6 placebo-injected patients. Responders to the V5.2 peptide had a reduced T-cell response to MBP and remained clinically stable without any side effects during 1 year of therapy, whereas nonresponders had increased MBP responses and progressed clinically. Furthermore, in patients with rheumatoid arthritis, vaccination of a combination of peptides corresponding to the CDR2 sequences of V3, V14, and V17, which are predominantly expressed in synovial fluid and tissue,35 diminished disease activity without any side effects.36 TCR vaccination stimulated regulatory T cells that down-regulate pathogenic T cells without necessarily deleting them.37,38 In this regard, in MS patients, V5.2-specific T-helper 2 cells induced by the V5.2 peptide vaccination directly inhibited MBP-reactive T helper 1 cells through the release of IL-10.34 In summary, we have shown that the set of TCR chains of pathogenic 2GPI-reactive T cells in APS patients is highly restricted. TCR-based immunotherapy targeting V7 or V8 is a potential strategy to selectively suppress pathogenic 2GPI-specific T cells, resulting in the inhibition of anti-2GPI antibody production and the prevention of thrombotic events in patients with APS.     Acknowledgments We thank Yuka Okazaki and Kyoko Kimura for their expert technical assistance and Noriko Hattori for helpful discussions.     Footnotes Submitted September 21, 2001; accepted November 20, 2001. Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan; the Keio University Medical Science Fund; and the Japan Intractable Diseases Research Foundation. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; e-mail: kuwanam{at}sc.itc.keio.ac.jp.     References Top Abstract Introduction Patients, materials, and... Results Discussion References 1. Harris EN, Chan JK, Asherson RA, Aber VR, Gharavi AE, Hughes GRV. Thrombosis, recurrent fetal loss, and thrombocytopenia: predictive value of the anticardiolipin antibody test. Arch Intern Med. 1986;146:2153-2156[Abstract/Free Full Text]. 2. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci U S A. 1990;87:4120-4124[Abstract/Free Full Text]. 3. Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. 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Curr Opin Immunol. 1994;6:946-955[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: V. Venturi, H. Y. Chin, D. A. Price, D. C. Douek, and M. P. Davenport The Role of Production Frequency in the Sharing of Simian Immunodeficiency Virus-Specific CD8+ TCRs between Macaques J. Immunol., August 15, 2008; 181(4): 2597 - 2609. [Abstract] [Full Text] [PDF] T. Atsumi New therapeutic targets for antiphospholipid syndrome Blood, December 15, 2007; 110(13): 4141 - 4141. [Full Text] [PDF] Y. Yamaguchi, N. Seta, J. Kaburaki, K. Kobayashi, E. Matsuura, and M. Kuwana Excessive exposure to anionic surfaces maintains autoantibody response to 2-glycoprotein I in patients with antiphospholipid syndrome Blood, December 15, 2007; 110(13): 4312 - 4318. [Abstract] [Full Text] [PDF] X. Tang, I. Maricic, and V. 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