To characterize circulating T cell subpopulations in B chronic lymphocytic leukemia patients, TCR V alpha and V beta gene-segment use was analyzed by PCR using a panel of V gene-segment subfamily-specific oligonucleotide primers (V alpha 1-29/V beta 1-24). Virtually all V alpha and V beta subfamily specificities were expressed in these patients (nine stage A and four stage C), and the mean values obtained for each specificity were similar to those of a group of 13 healthy donors. Nonetheless, individual analysis revealed that unique V alpha or V beta gene-segment transcripts were overrepresented in patients compared with the control group. Overrepresentation of some TCR V beta chains was also detected by cytofluorometric analysis using a panel of 18 anti-V beta-specific mAbs. To further characterize these T cell subpopulations, we sequenced five different V beta-C beta PCR products in two selected stage A patients and found highly predominant recurrent transcripts in each of the five V beta specificities (50% to 100% of the analyzed sequences with identical V(D)J regions). These results were confirmed on bulk cDNA (i.e., without cloning) and extended to other V beta specificities (up to nine clonal expansions of 24 V beta specificities in one patient) and two other patients using a PCR-based method that determines V(D)J junction size patterns. Finally, it was observed that a V beta 19+ T cell subpopulation was clonally expanded in one patient to up to 30% of circulating T cells. This V beta 19+ CD8+ T cell clone was shown to specifically recognize the autologous tumor cells in vitro, as determined in cytokine release assays. Together, these results support the view that multiple expansions of unique T cell clones may derive in vivo from B chronic lymphocytic leukemia tumor-associated Ag stimulation.

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