Purpose: We have previously established operational tolerance in fully MHC mismatched rat hemiface allotransplantation model under long-term low dose cyclosporine A (CsA) monotheraphy. Here we evaluated the success of combined CsA and alphabeta-Tcell receptor monoclonal antibody (TCRmAb) 7-day-protocol, augmented with intraosseous donor bone marrow (BM) transplantation on tolerance induction and flap survival. Methods: Twenty-four rats were studied in 6 groups of 4 animals each. Composite hemifacial isotransplantations were performed between LEW(RT1^l) to LEW in Group 1. Allograft transplantations were performed between fully MHC mismatched ACI(RT1^a) donors and LEW(RT1¹) recipients. While Group 2 recipients had no treatment and served as rejection controls, Group 3 rats received TCRmAb 250µg/day, and Group 4 rats received CsA 16mg/kg/day. In Group 5 a combination of TCRmAb and CsA was used, whereas in Group 6 TCRmAb and CsA protocol was augmented with direct intraosseous transplantation of 35-40x10⁶ (BM) cells. Immunosuppression was given for 7 days only, without recipient conditioning. Daily evaluation of clinical signs of rejection like hair loss, erythema, and skin slough were performed. Flow cytometry (FC) was performed to evaluate donor specific chimerism for MHC class-I RTl^a specific antigens in the peripheral blood of recipients at days 7 and 35. Histological grading of skin rejection was evaluated by H&E staining. Results: Isograft controls survived indefinitely, whereas allografts without immunosuppression were rejected within 5-9 days. Rats receiving either TCRmAb or CsA monotherapies rejected their flaps within 14 and 19 days respectively. Treatment with a combination of TCRmAb and CsA extended flap survival up to 42 days, whereas injection of donor BM cells in combination with TCRmAb and CsA protocol further extended flap survival up to 57 days. In hemiface transplants, under αβTCR/CsA protocol, T- cell depletion was significant at day 7 (>95%). FC analysis at day-7 revealed <1% chimerism in TCRmAb recipients, whereas CsA monotherapy recipients showed 6% of CD4/RT^a, 1.6% of CD8/RT^a, and 3.0% of CD11b,c/RT^a donor specific cells. Under combined TCRmAb and CsA protocol, chimerism at day-7 was high and revealed 45.3% of CD4/RT1^a, 27.5% of CD8/RT1^a, and 43.7% of CD11b,c/RT1^a positive cells. These values declined to 9.4%, 3.9%, and 10.0% respectively at day-35 post-transplant. Augmentation of combined TCRmAb and CsA protocol with donor BM transplantation resulted in 51.1% of CD4/RT1^a, 37.5% of CD8/RT1^a, and 54.1% of CD11b,c/RT1^a chimerism at day-7, which declined to 12.1%, 5.5%, and 11.1% respectively at day-35. Histology of rejected skin flaps demonstrated grade 2 and 3 rejection patterns. Conclusion: Significant extension of hemifacial allotransplant survival was achieved across MHC barrier under combined CsA and TCRmAb protocol (p<0.05). Augmentation with BM transplantation further extended allograft survival and correlated with higher levels of donor specific multilineage chimerism.
View Synopsis (.doc format, 85.0 kb)