LIVER SINUSOIDAL ENDOTHELIAL CELLS ENDOCYTOSED ALLOGENEIC CELLS TOLERIZE T CELL RECOGNIZING ALLO-ANTIGENS THROUGH INDIRECT RECOGNITION BY FAS-FAS LIGAND PATHWAY.

Abstract

P953 Although it has been well known that portal injection (PI) of donor cells leads to tolerance to allografts, the mechanism has not been fully understood. The difficulty in determining the precise mechanisms may be due to a difference between tolerizing mechanisms in T cells with direct allospecificity and those with indirect allospecificity. In the present study, we have demonstrated that PI of class II-deficient donor splenocytes led to indefinite acceptance of subsequently transplanted donor-type heart allografts. This finding indicates that PI of donor cells efficiently induces tolerance at least among T cells with indirect allospecificity. PKH-26-labeled B6 splenocytes (30x106 cells/mouse) that were treated with 30-Gy irradiation were injected via the portal vein into Balb/c mice, so that host antigen presenting cells that had taken up the injected splenocytes could be identified as cells that have PKH-26 labeling. At 12 hours after PI, approximately 60% of host liver sinusoidal endothelial cells (LSECs) cells had taken up PKH-26-labeled splenocytes. FCM analyses revealed that naive LSECs were MHC class II+, CD40+, CD80+ and CD86+ phenotypes that are consistent with those of antigen presenting cells (APCs). In contrast, LSECs capturing allogeneic splenocytes lost CD40 expression and inductively expressed Fas-ligand on their surface. To test tolerizing capacity of host LSECs capturing allogeneic splenocytes toward T cells with indirect allospecificity, we examined the effect of antigen presentation by LSECs to naive T cells on the responsiveness of those T cells to subsequent antigen presentation by professional APCs. Balb/c mouse splenocytes first underwent transmigration across the LSECs from Balb/c mice that had been treated with PI of either naïve or irradiated allogeneic B6 splenocytes, enabling direct interaction between T cells and LSECs. The transmigrated T cells, which were predominantly CD4+ T cells, were subsequently stimulated with splenic APCs from Balb/c mice that had been stimulated with intravenous injection of B6 splenocytes. The proliferative response of CD4+ T cells that had transmigrated across the LSECs from mice that had been treated with PI of irradiated splenocytes was significantly reduced compared with that of LSECs from untreated mice. As far as we know, these are the first demonstration that indirect antigen presentation by LSECs contributes to alloreactive T cell tolerance induced by portal injection of donor splenocytes.