Mechanism of portal venous tolerant long-term MHC Class I L^d-specific skin graft survival in transgenic 2CF1 mice

Background: Administration of alloantigen via the portal vein (PV) in non-transgenic animals has been shown to promote immunologic tolerance and enhance transplant allograft survival. The underlying mechanisms remain unclear. In 2C×dm2 F1 (2CF1) transgenic mice, the monoclonal antibody, 1B2, identifies specific 2C TCR transgenic CD8+ T cells that are cytotoxic against Class I MHC L^d. In these mice, the specific response by these cells to L^d+ skin grafts after PV administration of L^d+ antigen was determined. Materials and methods: Saline (control) or allogeneic C57BL/6×BALB/C F1 (CB6F1) spleen cells (25×10⁶), which differ from 2CF1 only at L^d, were injected PV into 2CF1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these 2CF1 mice. Skin graft rejection was defined as > 50% loss of the graft. Parallel experiments were performed in non-transgenic littermates [B6F1 (C57BL/6×dm2)]. FACS analysis of 2CF1 peripheral blood for 1B2+, CD4+ , and CD8+ T cells was performed 2 days before PV injection (9 days prior to skin grafting), 5 days after PV injection (2 days prior to skin grafting), and 7, 14, 21, 28, and 60 days after skin grafting. FACS analysis of naive, saline control, and CB6F1 PV-treated 2CF1 thymocytes was also performed. Responsiveness of saline (control)-treated and PV-treated 2CF1 splenocytes was measured by in vitro cytotoxic T lymphocyte (CTL). Results: All CB6F1 skin grafts were rejected in < 14 days by PV saline controls. However, a single PV injection of donor L^d+ CB6F1 cells was sufficient to induce indefinite CB6F1 (L^d+) skin allograft survival in 100% of non-transgenic B6F1 and transgenic 2CF1 (anti-L^d) TCR transgenic recipients. FACS analysis of 1B2+ T cells demonstrated that PV injection of donor antigen followed by a CB6F1 skin graft led to a 70% decrease in peripheral donor-reactive 1B2+ CD8+ T cells by day 7, while central thymocytes were unchanged. CTL of 2CF1 splenocytes following PV CB6F1 demonstrated that they were hyporesponsive to L^d compared to saline-treated 2CF1 splenocytes. Despite recovery of peripheral CD8+ T cells to near normal levels by 60 days post-transplantation, skin graft survival persisted indefinitely. Conclusions: Administration of specific PV antigen results in exquisite long-term L^d+ skin allograft acceptance. This tolerance induction is related to a significant peripheral deletion of donor-reactive 1B2+ CD8+ transgenic T cells and anergy of the residual T cells.