The presence of donor-reactive CD4 T cells in early-phase liver-induced tolerance in rats: Analysis using donor passenger leukocytes from the recipient

In previous studies, we showed that the recipient spleen 12 h after liver transplantation included highly immunogenic donor passenger leukocytes (PLs) in the nonrejector rat strain combination (Lewis: LEW→Wistar Furth: WF). Furthermore, we noted that giving anti-CD4 mAb to WF recipients at the time of liver grafting prolonged the liver damage due to primary rejection reaction, suggesting that CD4 T-cell regulation but not the deletional mechanism played a dominant role during the induction phase of tolerance in this combination. We proposed that in vitro CD4 T-cell response to the donor PLs from the recipient lymphoid organ may be observed in the early phase of tolerance induction. We initially observed donor-specific acceptance by 10 days after liver transplantation from the result of secondary heart transplantation, and showed that donor (LEW) PLs in recipient WF spleen 12 h after grafting were able to prime cytotoxic CD8 T cells in vivo. We then isolated functional donor PLs from the recipient WF spleen and established in vitro proliferation assay. When the enriched donor PLs were used as stimulator cells in the proliferation assay, a powerful alloimmunostimulatory ability was observed. Moreover, a potent response of recipient splenocytes to the PLs was observed on postoperative day 14. From these observations, we confirmed that clonal deletion of donor-reactive CD4 T cells did not participate in early phase of tolerance induction in this setting.