O271 Aims: We could recently introduce in a rat system less-toxic BMT conditioning using only an anti-CD45 mAb. As severe side effect in fully MHC mismatch setting risk of aplasia was observed and most likely provoked by an atypical GvH reaction on bone marrow level. In this study, unspecific immunosuppression (IS) was tested as addition to anti-CD45 mAb conditioning for establishment of a safe and tolerizing BMT protocol. Methods: The model consisted of LEW.1W (RT1u, RT7a) recipients, LEW.7B (RT1l,RT7b) BM donors, a single injection of anti-CD45 mAb (7.5 mg/kg BW) detecting RT7a as conditioning and orally applicated cyclosporine A (CsA 5 or 15 mg/kg BW) or sirolimus (1 or 2 mg/kg BW) as IS for 14 days. Animals were monitored for clinical status and haematological parameters. They were tested for multilineage chimerism at day 20 and day 100 by FACS. Long-term survivors received skin grafts of BM donor- and 3rd party-type on day 100. Results: Only animals of high-dosage sirolimus (2 mg/kg BW) treated group showed reproducible stable chimerism (9 of 12) compared to all other groups (no IS: 1 of 9, CsA 5: none of 9, CsA 15: 1 of 9, sirolimus 1: 1 of 9). Thereby, initial leukocyte depletion by anti-CD45 mAb as parameter for conditioning extent was highly critical for BMT success. Animals, which did not became stable chimeras, showed transient chimerism but suffered mainly from severe aplasia syndrome and died between days 29 to 70 (no IS: 7 of 9, CsA 5: 9 of 9, CsA 15: 6 of 9, sirolimus 1: 5 of 9, sirolimus 2: 3 of 12). GvHD signs were only seen in high-dosage CsA treated animals (2 of 7). The attempt to stabilize BM engraftment by prolongation of high-dosage CsA treatment (28 days) even aggravated GvHD symptoms (4 of 6) like thymic selection disturbance by thymic irradiation did in high-dosage sirolimus group (4 of 4). Moreover, analysis of persisting anti-CD45 mAb coated thymocytes suggested reduced thymocyte selection progress. Long-term lineage-typing of stable chimeras from high-dosage sirolimus group revealed mainly high-graded mixed chimerism in lymphoid and fully chimerism in myeloid lineages. Stable chimeras tolerated skin grafts only of donor-type (> 100 days), while acutely rejecting 3rd party control grafts like none chimeric survivors rejecting both graft-types. Conclusions: Addition of high-dosage sirolimus to isolated anti-CD45 mAb BMT conditioning reduced significantly risk of fatal aplasia in allogeneic BMT. This is in contrast to CsA, where the combination of intrathymically persisting anti-CD45 mAb and CsA seems to disturb TC selection processes leading to severe GvHD without stabilizing HSC engraftment. Without any clinical signs for GvHD the combination of anti-CD45 mAb and sirolimus in allogeneic BMT is promising for generation of stable mixed chimerism and tolerance induction.