Evidence of T cell clonality in the infectious tolerance pathway: Implications toward identification of regulatory T cells

We have shown that a rare population of regulatory CD4+ T cells plays a key role in the acquisition of infectious tolerance in rat sensitized recipien ts of cardiac allografts pretreated with nondepleting anti-CD4 mAb. This st udy was designed to analyze the TCR V beta expression patterns in this tran splantation model. First, we used V beta -specific RT-PCR to show that ther e was no differential usage of TCR V beta genes by T cells mediating reject ion or tolerance. Indeed, graft-infiltrating lymphocytes expressed most of the 22 known rat TCR V beta genes in both recipient groups, suggesting unre stricted TCR V beta repertoire in alloreactive T cells. Then, we applied CD R3 spectrotyping of TCR beta -chain to assess the clonality of T cells at d ifferent anatomic sites. CDR3 size restriction, indicative of the presence of T cell clones, was observed in graft-infiltrating lymphocytes but not in draining lymph nodes or spleen of tolerant hosts. Consisent with the clona l expansion, T cells in tolerated grafts exhibited the memory phenotype at a much higher percentage as compared with peripheral lymphoid organs. Moreo ver, in tolerated graft-infiltrating lymphocytes, the CD3 size restriction occurred in limited V beta gene families, with V beta8.1 and V beta 18 most frequently detected. Hence, T cells at the graft site of tolerant recipien ts contain T cell clones expressing selective V beta genes. This phenotypic characteristics of the tolerogenic GILs may potentially be used as a novel marker to identify operational regulatory T cells in organ allograft recip ients.