DONOR PBMC INDUCED TOLERANCE IS ASSOCIATED WITH THE EMERGENCE OF ANERGIC T CELLS FROM THE THYMUS FOLLOWED BY GENERATION OF REGULATORY T CELLS IN THE GRAFT.

O110 We have previously demonstrated that pretransplant infusion in Lewis (Lw) rats of donor Brown-Norway (BN) peripheral blood mononuclear leukocytes (PBMC) under appropriate immunomodulating conditions promotes donor-specific tolerance to a BN kidney allograft, a phenomenon that was dependent on the migration of donor PBMC into the recipient thymus. T cells isolated from lymph nodes (LN) from long term survival rats had anti-donor specific hyporesponsiveness. To investigate the mechanisms involved in tolerance induction, we examined anti-donor alloreactivity in T cells isolated from thymus and LN 20 days after donor cell infusion (pretransplant). In addition, alloreactivity of T cells from recipient LN and kidney grafts was investigated 5, 30 and 60 days after transplantation. In MLR the anti-donor response of pretransplant CD4+ thymocytes (SI: 10.5±1.9) and LN cells (SI: 4.4±0.9) from rats receiving BN PBMC was lower (p <0.05) than that of cells from naïve animals (thymocytes: SI: 17.5±2.0; LN: SI: 15.0±2.0), whereas MLR response against third-party WF antigens was normal. The hyporesponsiveness of CD4+ thymocytes and LN cells against BN antigens was partially but not completely reversed by addition of IL-2 (50U/ml), indicating that both a partial clonal deletion and the formation of anergic T cells had occurred. Neither CD4+ thymocytes nor LN cells taken pretransplant were capable to down-regulate the anti-donor alloreactivity of naïve LN cells in co-culture MLR, excluding the presence of regulatory cells at this stage. Early posttransplant (day 5) LN alloreactivity against donor stimulators did not differ from that of LN from naïve rats, however donor specific hyporesponsiveness was again found in LN cells at day 30 and 60 post-transplant. From day 30 LN cells had a marked regulatory effect, as shown by potent suppression of a naïve anti-BN MLR in a co-culture system (30 day: SI: 7.9±0.6, 60 day: SI: 5.5±0.9, p< 0.05 vs. naïve co-culture: SI: 12.8±2.0). A high % of CD4+ T cells was found in the graft at 5 days (11.7±0.7%, p <0.05 vs. untreated rats acutely rejecting their grafts: 6±1.6%) and 60 days post-transplant (17.2±4.3%, p 0.05 vs rejecting). Interestingly, graft CD4+ cells isolated from BN-PBMC infused rats were hyporesponsive to BN stimulators in MLR (SI 5 days: 3.2±1.0, SI 60 days:2.2±0.5, p< 0.05 vs. naïve CD4+: SI: 10.6±3.2) and in co-culture assays they markedly suppressed a naïve MLR against donor antigens (SI 5 days:1.6±0.4, SI 60 days: 5.5±1.0 p< 0.05 vs. naïve co-culture) indicating that graft CD4+ T-cells had potent regulatory activity. Additional co-culture MLR revealed that the immunoregulatory activity in LN (60 days) and in graft CD4+ cells (5 days) was confined in the CD4+CD25+ subset. Based on these findings we hypothesize that tolerance induction by pretransplant infusion of donor PBMC is a multistep process: an induction thymic-dependent phase leading to the generation of anergic T cells, followed by a maintenance phase characterized by the appearance of regulatory T cells first in the graft and then in peripheral LN.