MHC ALLOPEPTIDE REACTIVE T CELL CLONES GENERATED FROM HUMAN RENAL ALLOGRAFT RECIPIENTS: INDIRECT ALLORECOGNITION IN LONG TERM ENGRAFTMENT

Abstract

9 T cells obtained from patients with chronic allograft dysfunction are primed to recognize and respond to donor-specific MHC allopeptides in vitro. Class II MHC allopeptide-specific Th1 clones generated from rejecting animals exhibit a restricted TCR Vβ repertoire and transfer delayed type hypersensitivity responses in vivo indicating, the important role they play in the genesis of rejection. In order to evaluate the role of T cells primed by donor specific MHC peptides in chronic allograft dysfunction, T cell lines and clones were generated from peripheral blood lymphocytes (PBLs) of human renal allograft recipients, all of whom had been transplanted for more than one year. All patients were maintained on the same immunosuppressive protocol. Nine patients mismatched for MHC class II specificities for which peptides were available were studied. The patients were divided into 2 groups; 1. chronic allograft dysfunction (n=4) and 2. stable graft function (n=5). T cell lines were generated from PBLs by repeated stimulation with the relevant mismatched class II MHC allopeptide presented by self antigen-presenting cells. The mean proliferative response of T cells lines from "rejecting" patients was significantly greater than that of the lines from "stable" patients; (10743.0±1834.2 versus 1175.4±127.3 cpm, p<0.0001). The production of cytokines generated in response to the allopeptides was evaluated by ELISA and RNAse protection assays. Cell lines from the "rejecting" group produced the Th1 cytokine IFN-γ but not the Th2 cytokine IL-4 in culture supernatants. One of the cell lines produced TGF-β. In contrast, the "stable" group produced IL-4 but not IFN-γ in response to re-challenge with the allopeptides. These data were confirmed by RNAse protection assay. T cell clones were then generated by limiting dilution and were phenotyped using flow cytometric analysis. T cell clones from both groups were all found to be CD4+. RT-PCR transcript analysis with specific human TCR Vβ primers showed that the clones expressed a restricted TCR Vβ repertoire with some overlaps. The mean proliferative response of the "rejecting" T cell clones was also significantly greater than that of the "stable" clones; (20352.0±2678.7 versus 2873.2±348.1 cpm, p<0.0001). Interestingly, without exception, analysis of 49 T cell clones showed that all the clones from rejecting patients produced IFN-γ but not IL-4, while clones from stable patients produced IL-4 but not IFN-γ. This is the first demonstration, at the clonal level, that chronic allograft rejection in humans is associated with the presence of CD4+ MHC allopeptide-specific T cell clones that express a Th1 phenotype. In contrast, MHC allopeptide-specific T cell clones generated in the absence of rejection are associated with a Th2 phenotype that may be protective. These data provide an interesting insight into the potential mechanisms underlying chronic allograft dysfunction and more importantly suggest an innovative approach to immune surveillance.