FAS LIGAND AND NK CELLS ARE IMPORTANT MEDIATORS OF LATE SKIN GRAFT REJECTION

Abstract

6 We have previously demonstrated prolonged but not indefinite survival of MHC mismatched murine skin allografts with combined perioperative blockade of the CD40 and CD28 costimulatory pathways. The purpose of this study was to determine the role of NK cells, the Fas/Fas ligand, and perforin effector pathways in late skin allograft rejection after combined costimulatory pathway blockade. This will allow the development of strategies to improve the efficacy of this therapy. To determine the effector pathway, three groups of recipients: 1) C57BL/6(B6), 2) B6mn.C3H-Faslgld (mutation in Fas ligand, B6-gld), 3) C57BL/6-Pfptmlsdz (B6-perforin-/-) received BALB/c skin grafts with or without combined CD40/CD28 blockade. The hamster anti-murine CD40L antibody, MR1, and human CTLA4Ig were administered at doses of 500 μg intraperitoneally on postoperative days 0, 2, 4, and 6. In all of the untreated groups, the skin grafts were rejected promptly (MST =8d≈12d). CTLA4Ig and MR1 treatments prolonged the survival in C57BL/6 mice (n=5, MST=40d). The treated B6-perforin-/- group showed mild improved survival (n=5, MST=65d). In contrast, the treated B6-gld group demonstrated excellent survival (n=5, MST>175d). To determine the role of NK cells in late graft loss, C57BL/6 mice received BALB/c skin allografts and treatment in one of three groups: 1) no treatment, 2) CD40/CD28 blockade, 3) CD40/CD28 blockade plus anti-NK antibodies(anti-asialo-GM1 antisera). Polyclonal rabbit anti-asialo-GM1 antisera (50μl) was given on days 0, 4, 8, and 12 post-transplant. In addition, BALB/c skins were transplanted on to C57BL/6 or C57BL/6-Lystbg/+ (beige mouse with impaired NK cell cytotoxicity) with or without CD40/CD28 blockade. In all the untreated groups including the beige mice, the skin grafts were rejected promptly (MST =8d ≈12d). CTLA4Ig and MR1 treatments prolonged the survival in C57BL/6 mice (n=5, MST=40d). The addition of anti-NK antibodies augmented survival (n=5, MST=106d). The treated beige mice also showed marked improved survival (n=7, MST>56d) compared to the treated C57BL/6 group (n=7, MST=28d). The Fas ligand effector pathway and NK cells are important mediators of late skin graft loss after combined costimulatory pathway blockade. These results raise the possibility that NK cells and Fas ligand may be important targets for novel therapies to prevent chronic allograft loss.