Increased usage of v_β2 and v_β6 in rheumatoid synovial fluid t cells

Abstract

Objective. To determine if the T cell antigen receptor V, 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, 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 , primers with a constant region C, primer, and 2 C, primers that co-amplied 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, gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA-DR haplotype. In all synovial specimens, V, 2 was increased relative to the peripheral blood, while Vj13.1 and V,13.2 were decreased. V& and b 2 1 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 Submitted for publication April 19, 1994; accepted in revised form June 28, 1994. patient. The SF V, 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 , gene usage of separated CD4+ and CD8+ synovial T cells showed that V $ and V ' 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, gene usage compared with PB T cells. V, 2 and V, 6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V, bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.

Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay.

“…Another observation that does not support random clonal expansion is that not all genes analyzed were found to be used by the expanded clonotypes, particularly BV5S1, as also noted by others (5,19), which is normally highly expressed and might account for 5-20% of total TCRBV expression (8,15,33). Moreover, the repeated observation of T cell clones expressing members of particular TCRBV gene families, such as BV1, BV2, BV3, BV6, BV8, and BV12, could be connected to the increased expression of several of these genes identified previously in the synovia of RA patients (2,(5)(6)(7). Clearly, functional in vitro studies with T cell clones and synovial antigens or synthetic peptide libraries will be required to characterize the specificity of the clonotypes expanded in the inflamed joints and to determine the nature of the stimulus.…”

“…Several lines of evidence, including the massive T cell infiltration in inflamed joints and the association with particular MHC class II molecules, clearly implicate autoreactive T cells in the pathogenesis of this disease (1). Previous studies on TCR usage by synovial T cells, although sometimes conflicting, often indicated clonal expansions, either documented by TCR cloning and sequencing or suspected on the basis of altered V gene expression profiles (2)(3)(4)(5)(6)(7). Generally, however, polyclonal T cell repertoires were observed, probably due to large cell populations secondarily attracted to the affected joints by inflammatory factors.…”

Correction of DiGeorge Anomaly with EBV-Induced Lymphoma by Transplantation of Organ-Cultured Thymus and Epstein–Barr-Specific Cytotoxic T Lymphocytes

“…T cell receptor ␤-chain usage in her blood mononuclear cells was measured by a quantitative polymerase chain reaction (10) and the degree of heterogeneity of the T cell repertoire was assessed by amplification of T cells of the BV 22JB1s6 subfamily. The PCR product was cloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA 92008), and colonies were selected randomly and sequenced.…”

HLA-DQB1 * 0201/0302 is associated with severe retinopathy in patients with IDDM

“…The DNAwas mixed with specific amplification primers and the amplified DNAwas electrophoresed in 3 % agarose gels stained with ethidium bromide to control amplification before hybridization. Details have been described elsewhere [18]. Nylon membranes (Schleider and Schuell, Keene, N. H., USA) were prepared as described [18], prehybridized for 30 min and then hybridized for 1 h at 53°C.…”

“…Details have been described elsewhere [18]. Nylon membranes (Schleider and Schuell, Keene, N. H., USA) were prepared as described [18], prehybridized for 30 min and then hybridized for 1 h at 53°C. Stringency washing was performed at 55°C for 15 min and the DIG-labelled (digoxigenin-AP) PCR products were detected by alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany).…”