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 x 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 x BALB/c F1 (CB6F1) spleen cells (25 x 10(6)), 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 x 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 nai;ve, 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.