Reginald Gorczynski1, Zhiqi Chen, Ismat Khatri, Kai Yu. 1. 1 The Toronto Hospital, University Health Network, Toronto, Canada. 2 Departments of Surgery and Immunology, University of Toronto, Toronto, Canada. 3 Address correspondence to: Reginald Gorczynski, M.D., The Toronto Hospital, University Health Network, 2-805, MaRS Tower, 101 College Street, Toronto, Ontario, Canada M5G1L7.
Abstract
BACKGROUND: CD200 overexpression in transgenic recipients, or by tissue allografts, increases skin and cardiac graft survival in mice receiving rapamycin, in association with increased Foxp3(+)Treg graft infiltration. However, less than 50% of skin grafts persist beyond 25 days. We investigated whether immune ablation using busulphan and cyclophosphamide, followed by autologous marrow reconstitution, would permit long-term survival in a greater percentage of recipients and induce tolerance, allowing for withdrawal of immunosuppressive drugs. METHODS: C57BL/6 wild type (BL/6 WT) or CD200(tg) mice received BALB/c skin grafts with rapamycin (1 mg/kg/36 hr) for 7 days. Thereafter, subgroups received busulphan or cyclophospamide for 6 days, followed by BL/6 marrow transplantation (BMTx), whereas controls were maintained on rapamycin. Beginning 7 days after marrow transplantation, mice receiving BMTx also received rapamycin for a further 7, 14, or 21 days (to a maximum of 42 days after transplantation) after which times, all immunosuppressions were withdrawn. Graft survival was monitored throughout, and mixed leukocyte cultures (MLCs) (using peripheral blood leukocytes) were performed for all groups at 60 days after transplantation. Gene expression was performed in grafts harvested at 80 days. RESULTS: Although all WT mice rejected grafts by 16 days, survival in CD200(tg) was approximately 40% at 60 days, with antigen-specific decreased MLC responses to BALB/c. After BMTx, survival in WT mice was greater than 40% at 60 days, and greater than 90% at 60 days in CD200(tg), with corresponding attenuated MLC responses. Long-term surviving grafts were infiltrated by Tregs of both host and donor marrow origins, as defined using CD45 congeneic donors or recipients. Survival was increased by infusion of an antibody directed to TNFRSF25 on Tregs. CONCLUSION: Autologous BMTx improves graft survival and promotes graft tolerance.
BACKGROUND:CD200 overexpression in transgenic recipients, or by tissue allografts, increases skin and cardiac graft survival in mice receiving rapamycin, in association with increased Foxp3(+)Treg graft infiltration. However, less than 50% of skin grafts persist beyond 25 days. We investigated whether immune ablation using busulphan and cyclophosphamide, followed by autologous marrow reconstitution, would permit long-term survival in a greater percentage of recipients and induce tolerance, allowing for withdrawal of immunosuppressive drugs. METHODS: C57BL/6 wild type (BL/6 WT) or CD200(tg) mice received BALB/c skin grafts with rapamycin (1 mg/kg/36 hr) for 7 days. Thereafter, subgroups received busulphan or cyclophospamide for 6 days, followed by BL/6 marrow transplantation (BMTx), whereas controls were maintained on rapamycin. Beginning 7 days after marrow transplantation, mice receiving BMTx also received rapamycin for a further 7, 14, or 21 days (to a maximum of 42 days after transplantation) after which times, all immunosuppressions were withdrawn. Graft survival was monitored throughout, and mixed leukocyte cultures (MLCs) (using peripheral blood leukocytes) were performed for all groups at 60 days after transplantation. Gene expression was performed in grafts harvested at 80 days. RESULTS: Although all WT mice rejected grafts by 16 days, survival in CD200(tg) was approximately 40% at 60 days, with antigen-specific decreased MLC responses to BALB/c. After BMTx, survival in WT mice was greater than 40% at 60 days, and greater than 90% at 60 days in CD200(tg), with corresponding attenuated MLC responses. Long-term surviving grafts were infiltrated by Tregs of both host and donor marrow origins, as defined using CD45 congeneic donors or recipients. Survival was increased by infusion of an antibody directed to TNFRSF25 on Tregs. CONCLUSION: Autologous BMTx improves graft survival and promotes graft tolerance.
Authors: José Luis Torán; Juan Antonio López; Patricia Gomes-Alves; Susana Aguilar; Carlos Torroja; Marco Trevisan-Herraz; Isabel Moscoso; Maria João Sebastião; Margarida Serra; Catarina Brito; Francisco Miguel Cruz; Juan Carlos Sepúlveda; José Luis Abad; Carlos Galán-Arriola; Borja Ibanez; Fernando Martínez; María Eugenia Fernández; Francisco Fernández-Aviles; Itziar Palacios; Luis R-Borlado; Jesús Vázquez; Paula M Alves; Antonio Bernad Journal: Sci Rep Date: 2019-03-15 Impact factor: 4.379