Clonotype analysis of human alloreactive T cells: A novel approach to studying peripheral tolerance in a transplant recipient

The recognition of allo-MHC and associated peptides on the surface of graft -derived APC by host T cells (direct pathway allorecognition) plays an impo rtant role in acute rejection after organ transplantation. However, the sta tus of the direct pathway T cells in stable long term transplants remains u nclear. To detect alloreactive T cell clones in PBL and the allograft durin g the transplant tolerance, we utilized RT-PCR instead of functional assays , which tend to underestimate their in vivo frequencies. We established all oreactive CD4(+) and CD8(+) T cell clones from peripheral blood sampled dur ing the stable tolerance phase of a patient whose graft maintained good fun ction for 9 years, 7 without immunosuppression, We analyzed the sequence of TCR V beta and V alpha genes and made clonotype-specific probes that allow ed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejecti on. We found an unexpectedly high level of donor HLA-specific T cell clonot ype mRNA in peripheral blood during the late tolerance phase. Strong signal s for two CD4(+) clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reductio n in direct pathway T cell clonotype mRNA in peripheral blood and the graft . Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibil ity that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.