Donor-specific tolerance by perioperative intrathymic injection of bone marrow cells in the rat cardiac allograft model: use of FK506 can shorten the necessary duration of pretransplant intrathymic conditioning.

ABSTRACT

BACKGROUND: Many strategies of tolerance induction by intrathymic (IT) injection of donor alloantigens have been reported to date; however, the timing of IT injection is usually 1-3 weeks before transplantation. METHODS: To apply IT injection to cadaveric organ transplantation, 1 x 10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1b) rats were intrathymically injected into Wistar Furth (WF; RT1u) rats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model. RESULTS: Allogeneic IT injection of BUF BMC with ALS and FK506 indefinitely prolonged graft survival (mean survival time > 210 days) in all WF rats. On day 130 after grafting, tolerant WF rats accepted donor BUF skin grafts (> 120 days) but not third-party Lewis skin grafts. In control groups, syngeneic IT injection of WF BMC or intravenous injection of donor BUF BMC in combination with ALS/FK506 therapy failed to induce tolerance. In vivo testing was performed during induction (1 month) or during maintenance (6 months of tolerance. In the mixed lymphocyte reaction (MLR), spleen T cells of tolerant rats at 1 month after grafting displayed hyporesponsiveness after stimulation with donor cells. The addition of interleukin (IL)-2 to MLR culture did not restore T-cell responsiveness. Tolerant rats had a significantly decreased frequency of T cytotoxic cell precursors (fTcp) of 14,926, and frequency of IL-2-producing T helper cell precursors (fThp) of 123,925, compared with naive rats (1 2,158 and 14,266, respectively). By 6 months after grafting, however, the anti-donor MLR proliferative responses of tolerant rats had been restored to the levels of naive splenic T cells. These tolerant rats displayed restoration of the (fTcp) of 12,842 and of the (fThp) of 15,630, which were comparable frequencies of naive rats. Suppressor T cells did not contribute in this model. In cardiac grafts of tolerant rats induced by IT injection, expression of both Th1 (interferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detected in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase. CONCLUSIONS: Perfect donor-specific tolerance was obtained by IT injection of donor BMC at the time of transplantation, while alloimmune responses were maintained at levels similar to those of naive rats.