LIVER SINUSOIDAL ENDOTHELIAL CELLS IN LIVER ALLOGRAFTS INDUCE ALLO-SPECIFIC NONRESPONSIVENESS OF T CELLS ACROSS MHC BARRIERS BY THE MECHANISM INVOLVING FAS-FAS LIGAND PATHWAY THROUGH DIRECT RECOGNITION

Abstract

P936 Although livers transplanted across MHC barriers in mice are normally accepted without recipient immune suppression, underlying mechanisms remain to be clarified in detail. To identify the cell type and mechanism that contributes to induction of such a tolerance state, we established an allogeneic mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay. Hepatic constituent cells (HCs) were isolated from B6 and Balb/c mice as stimulators, and splenocytes were isolated from B6 mice as responders. Irradiated HCs were co-cultured with CFSE-labeled B6 splenocytes. In the allogeneic MHLR, whole HCs did not promote proliferation of allo-reactive T cells. Only when CD105+ cells, which are exclusively liver sinusoidal endothelial cells (LSECs), were depleted from whole HCs by sorting, both allo-reactive CD4+ and CD8+ T cells proliferated markedly in the allogeneic MHLR. Such proliferation of allo-reactive T cells was inhibited by returning LSECs to the MHLR. Physical separation of LSECs from the responder-stimulator cells by using a transwell culture system in the MHLR eliminated LSEC-induced inhibitory effects on allo-reactive T cell proliferation. To detect immunogeneic cells in liver, bone marrow chimeric mice were produced. Hepatocytes, LSECs and etc. in liver were derived from Balb/c mice, but bone marrow-derived cells including dendritic cells were replaced by cells of B6 mice. Even when LSECs were depleted from whole HCs, significant allo-reactive T cell proliferation was not observed in MHLR in the combination of chimeric mouse liver cell stimulators and B6 mouse lymphocyte responders. These results suggest that immunogeneic cells in liver were bone marrow-derived cells. To test the tolerizing capacity of LSECs toward allo-reactive T cells, B6 non-adherent splenocytes that had transmigrated through the monolayer of either B6, Balb/c or SJL/j (third party) LSECs were restimulated with irradiated Balb/c splenocyte stimulators. Non-responsiveness of T cells that had transmigrated through Balb/c LSECs and marked proliferation of T cells that transmigrated through B6 and SJL/j LSECs were observed after the restimulation. These findings indicate that allogeneic LSECs have a capacity to induce allo-reactive T cell tolerance through sufficient cell contact in the context of MHC restriction. To address this tolerizing mechanism, we analyzed the phenotype of the LSECs. Naïve LSECs constitutively expressed FasL. Blocking FasL by mAbs eliminated the tolerizing capacity of the LSECs. Consistently, in allogeneic MHLR using whole HCs (including the LSECs) as stimulators, T cells at the early period of cell division expressed phosphatidylserine, which is expressed on the surface of apoptotic cells. Moreover, the proliferation of T cells that had transmigrated through gld (FasL mutant mice with Balb/c background) LSECs was significantly high compared with that of T cells that had transmigrated through wild Balb/c LSECs after the restimulation. Thus, FasL-induced apoptosis participate in tolerization of allo-reactive T cells by LSECs of liver allografts.