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

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 x10(8) fully allogeneic bone marrow cells (BMC) of Buffalo (BUF; RT1(b )) rats were intrathymically injected into Wistar Furth (WF; RT1(u)) r ats at the time of BUF cardiac allografting with short-course therapy of antilymphocyte serum (ALS) and FK506 in our experimental model. Res ults. Allogeneic IT injection of BUF BMC with ALS and FK506 indefinite ly prolonged graft survival (mean survival time >210 days) in all WF r ats. On day 130 after grafting, tolerant WF rats accepted donor BUF sk in grafts (>120 days) but not third-party Lewis skin grafts. In contro l 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 re action (MLR), spleen T cells of tolerant rats at 1 month after graftin g 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 1:4,926, and frequency of IL- a-producing T helper cell precursors (fThp) of 1:23,925, compared with naive rats (1:2,158 and 1:4,266, respectively). By 6 months after gra fting, 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 1:2,842 and of th e (fThp) of 1:5,630, 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 (int erferon-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokines was detecte d in the early phase; thereafter, expression was completely inhibited, except for interferon-gamma in the chronic phase. Conclusions. Perfec t donor-specific tolerance was obtained by IT injection of donor BMC a t the time of transplantation, while alloimmune responses were maintai ned at levels similar to those of naive rats.