Immunotherapy with Donor Bone Marrow Supports Facial Allograft Survival by Presence of Regulatory CD4+/CD25+ T-Cell
Aleksandra Klimczak, PhD, Mehmet B. Unal, MD, Yavuz Demir, MD, Maria Siemionow, MD, PhD.
The Cleveland Clinic, Cleveland, OH, USA.
PURPOSE: We have formerly achieved functional tolerance in fully MHC mismatched rat facial allotransplantation model under cyclosporine-A (CsA) monotheraphy. 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 donor 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(RT1l+n) donors and LEW(RT1l) recipients in 6 groups of 6 rats each. Controls: Group 1 isograft and Group 2 allograft recipients 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 (Treg) CD4+/CD25+. Histological 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.