Purpose: We have formerly achieved functional tolerance in fully MHC miamatched rat facial allotransplantation model under cyclosporine-A (CsA) monotherapy. In this study we tested the effect of donor bone marrow transplantation (DBMT) under short-term alpha-betaTCRmAb and CsA 7-day protocol on development of chimerism, allograft survival, and presence of regulatory T-cells in face allograft model across MHC barrier.

Methods: Thirty six hemiface allotransplantation were performed between LBN(RT1^l+n) donors and LEW(RT1^l) recipients in 6 groups (6 rats each). Controls: Group 1 isograft and Group 2 allograft without treatment, Group 3 received intraosseous DBMT only. Groups 4, 5 and 6 received alpha-betaTCR/CsA 7-day therapy. Additionally Groups 5 and 6 were augmented with intraosseous DBMT of 35x10⁶, and 100x10⁶ of bone marrow cells (BMC) respectively. Before transplantation BMC were stained with PKH dye to evaluate engraftment and migratory process of donor BMC.

Flow cytometry assessed immunodepletion of T–lymphocytes, donor-specific chimerism for MHC class I (RT1ⁿ) antigens and presence of regulatory T-cells CD4+/CD25+. Grading of graft rejection was assessed by H+E staining.

Results: Isograft controls survived indefinitely, whereas controls without immunosuppression rejected transplanted allograft within 5 to 8 days. Group 3 treated with only DBMT accepted transplant up to 13 days. Median survival time (MST) of facial allograft (Group 4) under alpha-betaTCR/CsA therapy was 35 days. In both Groups 5 and 6 augmented with donor BMC MST was 48 days. However, the longest survival time in Groups receiving 35x10⁶ or 100x10⁶ BMC was 465 and 498 days respectively. In long-term survaivals split tolerance was found as presented by acceptance of skin and rejection of hair components.

PKH-positive cells of donor origin were present within lymphoid organs and skin of recipients. In rejected allografts T-cell chimerism declined <1%, and B-cell chimerism was at 4.5% of CD45RA/RT1ⁿ. In contrast, in long-term survival animals T-cell chimerism was 2%-4% (RT1ⁿ) during follow-up period and B-cell chimerism reached 12.4% CD45RA/RT1ⁿ at day 465 post-transplant. At day 7 post-transplant, regulatory CD4+/CD25+ T-cells initially were detected at 1.8%, however in long-term survivals reached 4.0%, whereas rejected allografts reveled 2.2% of CD4+/CD25+ T-cells at the time of euthanasia.

Conclusion: Long-term face allograft survival was achieved under 7-day alpha/beta-TCRmAb/CsA protocol augmented with DBMT without chronic immunosuppression. Long-term allograft acceptance was supported by development of regulatory T-cells and maintenance of donor T-cell and B-cell chimerism.