TCR-BETA SPECTRATYPING IN RA - EVIDENCE OF CLONAL EXPANSIONS IN PERIPHERAL-BLOOD LYMPHOCYTES

Objective-To compare the TCR beta repertoire of peripheral blood CD8 e nriched (CD8+) and depleted (CD8-) T cells in rheumatoid arthritis (RA ) patients and controls using CDR3 length analysis (spectratyping). Me thods-CD8+ and CD8- T cells were separated from 14 RA patients and 12 controls, using magnetic beads coated with anti-CDS monoclonal antibod ies. cDNA was prepared as the template for amplification with 22 V bet a-C beta primer pairs. The products were resolved by electrophoresis i n an ABI373 sequencer using GENESCAN software. Expansions were identif ied as dominant CDR3 lengths, where the area underlying the correspond ing peak exceeded the sum of the areas of the two adjacent peaks. This method was validated by sequencing 10 samples displaying dominant pea ks. The expansion frequencies in RA patients and controls were compare d using the chi(2) test statistic Results-Dominant peaks were evident in several V beta 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 sample s exhibiting dominant peaks revealed an unequivocal clonal expansion i n nine (90%). Conclusions-RA patients exhibited a significantly increa sed 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-inflammato ry cytokines or a defect of T cell regulation that predates the onset of RA and may itself predipose to disease.