Biased t cell receptor v gene usage in rheumatoid arthritis. oligoclonal expansion of t cells expressing vα2 genes in synovial fluid but not in peripheral blood

Objective. To analyze the T cell receptor (TCR) variable (V) region gene usage in the rheumatoid joint. Methods. Monoclonal antibodies (MAb) were used to determine the prevalence of selected V elements on T cells in synovial fluid (SF) from rheumatoid arthritis (RA) patients and in peripheral blood (PB) from RA patients and normal controls. V_α2-positive PB and SF T cells from 1 patient were cloned by immediated limiting-dilution and analyzed by restriction mapping. Results. In 9 of 14 RA patients, SF was enriched in at least 1 of the selected V elements, compared with PB. TCR genes of the V_α2 family were the most frequently overrepresented in the SF (4 patients). The expanded V_α2-positive cells were oligoclonal in SF but heterogeneic in PB. Conclusion. Our data showing biased and clonally restricted TCR elements in the rheumatoid joint indicate major histocompatibility complex–restricted antigen recognition, rather than a “superantigen,” in the pathogenesis of RA.     

Emergence of oligoclonal t cell populations following therapeutic t cell depletion in rheumatoid arthritis

Objective. To examine the compartment of CD4+ T cells in patients with rheumatoid arthritis (RA) who have developed persistent lymphopenia following antibody-mediated T cell depletion and to investigate why T cell depletion is of limited therapeutic efficacy. Methods. Circulating T lymphocytes from 10 patients with seropositive RA treated with the monoclonal antibody (MAb) CAMPATH-1H were longitudinally monitored by fluorescence-activated cell sorter analysis with MAb. To assess the molecular diversity of repopulating T cells, random samples of T cell clones from the peripheral blood of 3 patients were analyzed by sequencing the T cell receptor (TCR) β chains. At the time of recurring disease, the synovial tissue was examined by immunohistochemistry, and the repertoires of peripheral and synovial tissue T cells were compared by TCR β-chain sequencing and by semiquantitative hybridization with oligonucleotides specific for the V–D–Jβ junctional region of selected clones. Results. The reconstitution of the peripheral T cell compartment was very slow. A mean CD4+ T cell count of 105/μl was reached 34 weeks following MAb treatment. After treatment, the percentage of CD4+ T cells with the CD45RO+ phenotype was significantly increased (P = 0.001), indicating the expansion of antigen-primed memory T cells. TCR β-chain sequences revealed a marked restriction in the diversity of repopulating T cells with the emergence of dominant clonotypes. Despite the low counts of peripheral CD4+ T cells, the synovial tissue was infiltrated by CD4+ T cells to a similar extent as that in RA patients not treated with MAb. Selected clonotypes that had emerged in the peripheral blood compartment dominated the repertoire of tissue-infiltrating T cells in the synovium. Conclusion. In patients with RA, T cell depletion induces a long-term imbalance in T cell homeostasis. Clonal proliferation of CD4+ T cells severely restricts the diversity of available T cell specificities and results in the emergence of dominant clonotypes, which accumulate in the synovial tissue despite peripheral lymphopenia.     

Mycobacteria and human heat shock protein—specific cytotoxic t lymphocytes in rheumatoid synovial inflammation

Objective. To study the cytotoxic capacity of mycobacteria-specific T lymphocyte lines and clones from sites of inflammation in patients with rheumatoid arthritis (RA). We also studied antigen specificity, surface phenotype, expression of T cell receptors (TCR), and HLA restriction. Methods. Autologous macrophages (Mπ) from the synovial membrane (SM), synovial fluid (SF), or peripheral blood (PB) were used as target cells in cytotoxicity assays. Results. All SM and SF cell lines tested thus far have shown specific lysis of the autologous Mπ from SM or PB that had been pulsed with BCG (bacillus Calmette-Guerin), but no cytotoxicity when the targets were pulsed with irrelevant antigens such as tetanus toxoid and Chlamydia. Both CD4+ and CD8+ cells were shown to be involved in the specific cytolysis. The majority of the cytotoxic T lymphocyte (CTL) lines were TCRα/β+ cells. However, both TCRα/β+ and TCRγ/δ+ clones (TCR δ1+) from one RA patient showed antigen-specific lysis. Antigen-specific recognition by a number of CTL lines and clones generated from SF and SM was restricted by HLA—DR molecules. Two Mycobacterium bovis 65-kd heat shock protein (HSP)–specific TCRα/β+ SF T cell clones also lysed Mπ that had been pulsed with a recombinant human 65-kd HSP. Conclusion. Joint inflammation and destruction might be partly attributable to a cross-reaction of mycobacteria-induced cytotoxic T cells with self HSP.     

Increased usage of vβ2 and vβ6 in rheumatoid synovial fluid t cells

Objective. To determine if the T cell antigen receptor V_β usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). Methods. Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V_β gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V_β primers with a constant region C_β primer, and 2 C_α primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. Results. There were consistent differences between the V_β gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA–DR haplotype. In all synovial specimens, V_β2 was increased relative to the peripheral blood, while V_β13.1 and V_β13.2 were decreased. V_β6 and V_β21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V_β bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V_β gene usage of separated CD4+ and CD8+ synovial T cells showed that V_β2 and V_β6 were more highly expressed on CD4 cells. Conclusion. Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V_β gene usage compared with PB T cells. V_β2 and V_β6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V_β bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.     

Selection of T cell receptor Vβ elements by HLA-DR determinants predisposing to Rheumatoid Arthritis

Objective. Rheumatoid arthritis (RA) is genetically linked to a sequence motif encoding for the middle portion of the α-helical loop, which is adjacent to the antigen-binding groove of the HLA-DR molecule. The disease-associated element might be involved in binding the antigen or in interacting with the T cell receptor (TCR). To investigate the contribution of the disease-associated element in T cell recognition events, we studied structural requirements in the interaction of T cell clones with HLA-DR determinants. Methods. T cell clones restricted to disease-associated HLA-DR determinants were established by allogeneic stimulation of HLA–DRB1*0401+ or *0401–responders with HLA–DRB1*0404/8+ stimulators. Allele-specific primer sets were used to identify the Vβ gene segment expressed by individual clones. Sequence analysis was applied to study the diversity of the TCR β-chain junctional regions. Results. The repertoire of TCR Vβ elements was strongly biased toward the usage of Vβ6. HLA–DRB1*0401+ and *0401– donors preferentially recruited Vβ6+ T cells to recognize the disease-associated HLA–DR determinant. Sequence data revealed that the Vβ6.6/7 and Vβ6.8/9 subtypes of the Vβ6 multigene family were overrepresented. The TCR β chains were characterized by a high degree of junctional diversity, supporting the view that a multitude of peptide–DR complexes were recognized and that the preferential use of Vβ6 was dictated by the TCR β chain–DRβ1 chain contact. Conclusion. T cells reactive with the disease-associated HLA–DR structure are nonrandomly selected. The HLA-DR component predisposing to RA might define molecular requirements that restrict the TCR–HLA interaction. Thus, the phenomenon of HLA association in RA might reflect a genetic control of T cell recognition, through the selection of the TCR repertoire.     

Restricted cytokine expression in rheumatoid arthritis

Objective. To determine the cytokine profile of the phenotypically activated T cell in rheumatoid arthritis (RA) synovium. Methods. Interleukin-2 (IL-2), IL-2 receptor (IL-2R), IL-6, IL-4, and interferon-γ (IFNγ) gene expression was examined in T cells from freshly isolated synovial fluids (SF) and synovial tissues (ST) from patients with RA. Estimates of baseline expression were determined using unstimulated peripheral blood (PB) T cells from healthy individuals. The corresponding positive controls were phytohemagglutinin-activated tonsil T cells. Results. In studies of paired PB and SF T cell samples from 17 RA patients, IL-2 messenger RNA (mRNA) levels in only 1 PB and 3 SF samples were more than 2 standard deviations above the mean of levels in unstimulated PB from healthy donors. Similarly, only 5 PB and 7 SF samples exhibited elevated IL-2R mRNA levels. IFNγ gene expression was not detected in any of the paired RA PB or SF samples. Fractionated T cells from 12 RA ST were screened with similar results: Only 1 of 12 samples exhibited IL-2 mRNA levels more than 2 standard deviations above levels in baseline controls. IL-2R mRNA levels were low or not detected, and IFNγ mRNA was absent. Subsequent studies showed that IL-4 and IL-6 gene expression levels were also low in RA tissues compared with tonsil T cell–positive controls. Conclusion. These data provide evidence for restricted cytokine expression in the T cell population in RA tissues.     

Clonal expansion of mycobacterial heat-shock protein–reactive T lymphocytes in the synovial fluid and blood of rheumatoid arthritis patients

Objective. To examine the reactivity pattern and T cell receptor (TCR) characteristics of mycobacterial heat-shock protein 65 (hsp65)-reactive T cells generated from paired synovial fluid (SF) and peripheral blood (PB) samples obtained from rheumatoid arthritis (RA) patients and from healthy subjects. Methods. The reactivity pattern of hsp65-reactive T cell clones generated under limiting-dilution conditions was analyzed in ³H-thymidine incorporation assays. The TCR variable regions of these hsp65-reactive T cells were characterized by polymerase chain reaction with TCR AV- and BV-specific primers and by DNA sequence analysis of the third complementarity-determining region (CDR3). Results. The hsp65-reactive T cells derived both from RA patients and controls preferentially recognized the 1–170 and 303–540 regions of hsp65 and did not cross-react with human hsp60. The hsp65-reactive T cell clones derived from RA patients displayed a restricted TCR AV and BV gene usage, which can be attributed to the limited clonal origin(s) of the independent T cell clones, as evidenced by CDR3 sequence analysis. These clonally expanded T cells were found in both PB and SF and in different inflamed joints of RA patients. Conclusion. Our study suggests that there is in vivo clonal activation and expansion of mycobacterial hsp65-reactive T cells in patients with RA.     

HLA and T cell receptor β-chain DNA polymorphisms identify a distinct subset of patients with pauciarticular-onset juvenile rheumatoid arthritis

Objective. To evaluate and extend upon a reported association of a T cell receptor (TCR) V _β coding region polymorphism with pauciarticular-onset juvenile rheumatoid arthritis (JRA). Methods. TCR V _β6.1 genotypes and haplotypes in JRA and control groups were determined by DNA amplification. Results. Haplotypes of the V _β6.1 gene which encode a nonfunctional form of V _β6.1 were significantly associated with pauciarticular JRA in patients possessing the HLA–DQA1*0101 allele (P = 0.0073). Conclusion. A TCR V _β gene segment in the vicinity of V _β6.1, possibly V _β6.1, is apparently involved in the pathogenesis of pauciarticular-onset JRA in DQA1*0101-positive individuals.     

Cd8high+ (CD57+) T cells in patients with rheumatoid arthritis

Objective. To investigate the development and T cell receptor (TCR) usage of CD8+, CD57+ T cells in rheumatoid arthritis (RA) patients. Methods. Three-color flow cytometry using monoclonal antibodies (MAb) to CD8, CD57 and different TCR V_β gene products. Results. The proportion of CD8+ T cells expressing CD57 (CD57/CD8) was significantly higher in RA patients compared with age-matched controls. Expanded TCR V_β populations were more frequent, and were found in both RA patient—derived CD8^high+(CD57+) and CD8+, CD57– populations. TCR V_β5+ and TCR V_β13+ expansions were present at high frequency (5 of 26 and 7 of 26, respectively). TCR V_β expansions in CD8^high+(CD57+) lymphocytes from RA patients were significantly larger than those in age-matched controls (expansion index 2.38 ± 0.28, n = 41 and 1.63 ± 0.09, n = 32, respectively), and were stable over time. Conclusion. RA leads to an increase in the frequency of expanded CD8+ T cell subsets expressing selected TCR, due to expansion of TCR V_β+ populations in CD8^high+(CD57+) T cells. Their restricted TCR usage suggests potential specificity for RA antigens and, therefore, a potential role in the pathogenesis of RA.     

Evaluation of TCR Vβ subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vβ repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vβ subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated (⁶⁰Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34⁺ cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vβ repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vβ usage was exhibited in the bone marrow of mice, which included TCR Vβ 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vβ subfamilies (Vβ9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vβ subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15–20 days. Oligoclonal expression or oligoclonal trend of some TCR Vβ subfamilies (Vβ10, 11 and Vβ2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vβ11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vβ subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34⁺ cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

Restricted junctional usage of T cell receptor Vβ2 and Vβ13 genes,which are overrepresented on infiltrating T cells in the lips of patients with sjögren's syndrome

Objective. To analyze the clonality of T cell receptor (TCR) Vβ2—and Vβ13—positive T cells, which are predominantly expressed in the lips of patients with Sjögren's syndrome (SS). Methods. The junctional sequences of complementary DNA clones encoding TCR Vβ2 and Vβ13 genes were determined by the polymerase chain reaction. Forty-one Vβ2 and 45 Vβ13 clones established from the lips of 3 SS patients were sequenced. Results. The Vβ2/Jβ2.3 pair was enriched in 2 of the 3 patients (44% and 46% of the clones, respectively), and the Vβ13/Jβ2.1 sequence was dominant in 2 of the 3 (23% and 45%). These pairs were not used preferentially in peripheral blood lymphocytes from the same patients. Conclusion. Infiltrating Vβ2- and Vβ13-positive T cells from the lips of all 3 patients with SS were polyclonal, but the junctional usage of cells from 2 lip samples was restricted, compared with cells from peripheral blood. This suggests that not all expanded cells from the lips of SS patients are stimulated by superantigens.     

T Cell Influence on Superantigen-Induced Arthritis in MRL-lpr/lpr Mice

Objective. To define the influence of the T cell receptor (TCR) and the lpr autoimmune gene on the induction and progression of superantigen-induced arthritis in V _β8 transgenic MRL-lpr/lpr mice. Methods. The time to onset and the extent of synovial hyperplasia after the induction of arthritis by intraarticular injection of staphylococcal enterotoxin B (SEB) were compared in mice having T cells that bear the V _β8 transgene alone (V _β8 TCR transgenic MRL-+/+), the lpr gene without the V _β8 gene (nontransgenic MRL-lpr/lpr), both the V _β8 gene and the lpr gene (V _β8 transgenic MRL-lpr/lpr), or neither gene (nontransgenic MRL-+/+). Synovial hyperplasia was compared in SEB-injected V _β8 transgenic MRL-lpr/lpr mice after treatment with cyclosporin A (CSA), anti-V _β8 and anti-CD4 monoclonal antibodies, and in V _β8 transgenic MRL-lpr/lpr mice after injection of a non—V _β8-reactive superantigen, staphylococcal enterotoxin A (SEA). Results. At day 30, increased synovial cells were observed in all SEB-treated mice, but the increase was greatest in the V _β8 transgenic MRL-lpr/lpr mice. T cell involvement was indicated by the inability of either heat-denatured SEB or SEA to induce severe arthritis, the reduction in the severity of the arthritis on systemic treatment with CSA or anti-V _β8, and the correlation of synovial hyperplasia with in vitro SEB reactivity of T cells. Conclusion. These observations suggest that super-antigens can induce chronic arthritis and that the induction and progression of the arthritis requires an underlying T cell defect in anergy induction in addition to exposure to the superantigen.     

The programmed death 1/programmed death ligand 1 inhibitory pathway is up‐regulated in rheumatoid synovium and regulates peripheral T cell responses in human and murine arthritis

Objective

T cells play a major role in the pathogenesis of rheumatoid arthritis (RA). The programmed death 1 (PD‐1)/programmed death ligand 1 (PDL‐1) pathway is involved in peripheral tolerance through inhibition of T cells at the level of synovial tissue. The aim of this study was to examine the role of PD‐1/PDL‐1 in the regulation of human and murine RA.

Methods

In synovial tissue and synovial fluid (SF) mononuclear cells from patients with RA, expression of PD‐1/PDL‐1 was examined by immunohistochemistry and flow cytometry, while PD‐1 function was assessed in RA peripheral blood (PB) T cells after stimulation of the cells with anti‐CD3 and PDL‐1.Fc to crosslink PD‐1. Collagen‐induced arthritis (CIA) was induced in PD‐1^−/− C57BL/6 mice, and recombinant PDL‐1.Fc was injected intraperitoneally to activate PD‐1 in vivo.

Results

RA synovium and RA SF were enriched with PD‐1+ T cells (mean ± SEM 24 ± 5% versus 4 ± 1% in osteoarthritis samples; P = 0.003) and enriched with PDL‐1+ monocyte/macrophages. PD‐1 crosslinking inhibited both T cell proliferation and production of interferon‐γ (IFNγ) in RA patients; PB T cells incubated with RA SF, as well as SF T cells from patients with active RA, exhibited reduced PD‐1–mediated inhibition of T cell proliferation at suboptimal, but not optimal, concentrations of PDL‐1.Fc. PD‐1^−/− mice demonstrated increased incidence of CIA (73% versus 36% in wild‐type mice; P < 0.05) and greater severity of CIA (mean maximum arthritis score 5.0 versus 2.3 in wild‐type mice; P = 0.040), and this was associated with enhanced T cell proliferation and increased production of cytokines (IFNγ and interleukin‐17) in response to type II collagen. PDL‐1.Fc treatment ameliorated the severity of CIA and reduced T cell responses.

Conclusion

The negative costimulatory PD‐1/PDL‐1 pathway regulates peripheral T cell responses in both human and murine RA. PD‐1/PDL‐1 in rheumatoid synovium may represent an additional target for immunomodulatory therapy in RA.