Clonotype Analysis of Human Alloreactive T Cells: A Novel Approach to Studying Peripheral Tolerance in a Transplant Recipient

Abstract

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vβ and Vα genes and made clonotype-specific probes that allowed 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 rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction 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 possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

Human allograft acceptance is associated with immune regulation

“…The relationship between patients and their graft is likely a dynamic one and may change over time. Should the protective pathway be overridden, for example, by infection or by a sudden loss of T-cell clones that may be essential for the protective pathway (17), the destructive pathway is fully functional and leaves these patients susceptible to acute rejection (discussed in ref. 12).…”

“…He had good graft function for 7 years, and his biopsies also displayed focal infiltrates. However, between 9 and 10 years after transplant, patient J.B. developed a cellular acute rejection episode and lost graft function (12,17). Patient J.B. was tested while on dialysis, waiting for retransplant.…”

Loss of Direct and Maintenance of Indirect Alloresponses in Renal Allograft Recipients: Implications for the Pathogenesis of Chronic Allograft Nephropathy

“…One recent study investigated a live-related recipient who had stopped all immunosuppression. T cells were analyzed by the extremely sensitive method of RT-PCR, looking for clonotypic alloreactive T cells (46). This patient had previously been shown to exhibit donorspecific hyporesponsiveness in functional assays (47).…”

Human CD4+CD25low Adaptive T Regulatory Cells Suppress Delayed-Type Hypersensitivity during Transplant Tolerance

“…Although some tolerant patients may maintain donor-specific hyporesponsiveness throughout their posttransplant course, in others anti-donor DTH and CTL activity may resurface after a long period of quiescence (34,35). This "metastable" feature of allotolerance, which in some cases may result in transplant rejection, creates an opportunity in longitudinal studies for comparisons to be made by the adoptive transfer of putative regulatory cells.…”