TCRbeta spectratyping in RA: evidence of clonal expansions in peripheral blood lymphocytes

Abstract

Objective-To compare the TCR repertoire of peripheral blood CD8 enriched (CD8+) and depleted (CD8−) T cells in rheumatoid arthritis (RA) patients and controls using CDR3 length analysis (spectratyping). Methods-CD8+ and CD8− T cells were separated from 14 RA patients and 12 controls, using magnetic beads coated with anti-CD8 monoclonal antibodies. cDNA was prepared as the template for amplification with 22 V -C primer pairs. The products were resolved by electrophoresis in an ABI373 sequencer using GENES-CAN software. Expansions were identified as dominant CDR3 lengths, where the area underlying the corresponding peak exceeded the sum of the areas of the two adjacent peaks. This method was validated by sequencing 10 samples displaying dominant peaks. The expansion frequencies in RA patients and controls were compared using the 2 test statistic. Results-Dominant peaks were evident in several V families. They were more frequent in RA patients in both the CD8+ subset (RA normalised frequency 10.6; control normalised frequency 8.0; p=0.03) and the CD8− subset (RA normalised frequency 2.9; control normalised frequency 1.5; p=0.02). Sequencing of 10 samples exhibiting dominant peaks revealed an unequivocal clonal expansion in nine (90%). Conclusions-RA patients exhibited a significantly increased frequency of T cell expansions both in the CD8+ and CD8− subsets. This phenomenon may reflect the proliferation of autoreactive cells, a nonspecific expansion of memory T cells in response to pro-inflammatory cytokines or a defect of T cell regulation that predates the onset of RA and may itself predipose to disease. (Ann Rheum Dis 1998;57:319-322) A pivotal role of T cells in the initiation of rheumatoid arthritis (RA) has been inferred from the prominence of CD4+ T cells in the synovium early in disease, the association of RA with particular HLA-DR4 and DR1 antigens, the eYcacy of treatments that deplete or suppress CD4+ cells, and animal models of chronic arthritis. The T cell receptor (TCR) endows this cell with its discriminatory properties. In developing T cells, functional genes for the and chains of the TCR chain are produced by rearranging selected V /J or V /D /J segments and joining them to their respective constant regions. Unlike the CDR1 and CDR2 loops, which are encoded in the germline, the CDR3 loops are determined by the V J or V D J junctional region and diversity is further enhanced by trimming the germline segments and insertion of nongermline encoded nucleotides. The resulting variation in length of the CDR3 loop is the basis for the "spectratyping" technique, in which the CDR3 region is amplified and the distribution of CDR3 lengths can be used as a readout of TCR repertoire.

CD56‐expressing T cells that have features of senescence are expanded in rheumatoid arthritis

“…Oligoclonality was assessed by flow cytometric analysis of cells immunostained with fluorochrome-conjugated antibodies to various TCR AV and BV families/segments (BD Biosciences and Beckman Coulter, Miami, FL) in addition to typical T cell markers. TCR clonality was also assessed by spectratyping procedures (27). In this case, total RNA was prepared from freshly sorted CD3ϩ,CD56ϩ T cells and subjected to reverse transcriptionpolymerase chain reaction experiments for the amplification of the third complementarity-determining region (CDR3) sequences of the indicated TCR BV or AV.…”

Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens

“…For all of these reasons, true clonality in an autoimmune disease is not expected. Indeed, oligoclonality has been reported in studies of experimental autoimmunity in animals [17][18][19] as well as in human rheumatoid arthritis, [22][23][24] systemic lupus erythematosus, 43,44 and IgA nephropathy. 45 Clonal dominance also occurs in viral infection [46][47][48] and in response to tumors and during graft-versus-host disease.…”

“…16 Skewing of the T-cell VB spectrum has also been described for animal models of immunologically mediated diseases such as encephalitis 17,18 and thyroiditis. 19 In humans, expansion of specific CDR3 VB TCR clones has been found in type I diabetes mellitus, 20 thyroiditis, 21 rheumatoid arthritis, [22][23][24] primary biliary cirrhosis, 25 psoriasis, 26,27 or during graft-versus-host disease. 28 TCR VB repertoire analysis has also been performed in patients with AA, 12,13 myelodysplasia (MDS), 29 and paroxysmal nocturnal hemoglobinuria (PNH).…”

Distinct bone marrow findings in T-cell granular lymphocytic leukemia revealed by paraffin section immunoperoxidase stains for CD8, TIA-1, and granzyme B

“…Even evidence of T-cell clonality may not be synonymous with T-cell malignancy because minor T-cell clones can be found in oligoclonal immune reactions. [33][34][35][36][37][38][39] Such clones can be demonstrated in the contracted T-cell repertory of elderly 40,41 or immunosuppressed individuals, 40 and they can be found in blood or bone marrow specimens from patients with autoimmune disorders [42][43][44][45][46] and in other conditions such as myelodysplasia (C.A.H., manuscript in preparation). Thus, it seems that finding a phenotypically distinct lymphocyte population becomes a critical criterion for a T-cell GLL diagnosis.…”