CHARACTERIZATION OF MHC ALLOPEPTIDE REACTIVE T CELL CLONES FROM HUMAN RENAL ALLOGRAFT RECIPIENTS

Abstract

382 We have recently demonstrated that T cells obtained from patients with chronic renal allograft dysfunction are primed to recognize and respond to specific donor-derived class II MHC allopeptides in vitro. In experimental animal models, class II MHC allopeptide-specific Th1 clones generated from rejecting animals express restricted TCR Vß repertoire and transfer delayed type hypersensitivity responses in vivo. In this study we generated T cell clones from PBMCs of two human renal transplant recipients; the first patient has chronic allograft dysfunction and the second control patient has stable graft function. Patient 1 received a HLA-DR*0101 mismatched living donor renal allograft and currently has chronic allograft dysfunction (creatinine: 2.9mg/dl) with typical histopathological features of chronic rejection. We have previously demonstrated T cell reactivity to peptide (42-62) of HLA-DR*0101 in this patient. Patient 2 received a HLA-DR*1501 mismatched cadaver renal allograft and currently has stable renal function (creatinine: 1.6mg/dl). Allopeptide reactivity was not detectable to any of 5 HLA-DR*1501 allopeptides on each of three separate occasions in this patient. T cell lines were generated from PBMCs of both patients by repeated stimulation with self APC pulsed allopeptide; peptide (42-62) of HLA-DR*0101 was used for patient 1 and peptide(1-21) of HLA-DR*1501 was used for patient 2. T cell clones were then generated by limiting dilution and were phenotyped using flow cytometric analysis. Culture supernatants were analyzed for cytokine production by ELISA, and the TCR Vß expression was characterized by RT-PCR. T cell clones from patient 1 were found to be CD4+ cells which specifically proliferated to peptide (42-62) of HLA-DR*0101 and which produced IL-2 and IFN-γ after re-stimulation with the peptide. Clones from patient 2 proliferated weakly, yet specifically to peptide (1-21) of HLA-DR*1501 and were found to be CD4+ cells which produced IL-4 and IL-10 after re-stimulation with the peptide. RT-PCR transcript analysis with specific human TCR Vß primers showed that the clones from both patients expressed a restricted TCR Vβ repertoire: 5.2, 8, 9, 13.1 and 13.2 for patient 1, and Vß 4, 6, 13.1, 13.1 and 13.1 for patient 2. The 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 (? protective) Th2 phenotype. Expanding these studies and understanding the mechanisms of immune deviation in humans should yield relevant information on the pathogenesis of chronic allograft rejection, the major clinical problem in organ transplantation.