Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection

Background. Graft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/ granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC, Methods. Expression of perforin, GrB and Fast was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies, Specific inhibitors of the perforin/GrB- and Fast-based pathways were used in Cr-51 release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines, Results. Cells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-Fast polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC, Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas. Conclusions. The perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.