Methyl-guanine-methyl-transferase transgenic bone marrow transplantation allows N, N-bis (2-chloroethyl)-nitrosourea driven donor mixed-chimerism without graft-versus-host disease, and with donor-specific allograft tolerance
Background: Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed-chimerism strategies were limited either by loss of long-term mixed chimerism or risk of graft-versus-host disease (GVHD). Selective bone marrow (BM) engraftment using marrow protective strategies are currently reaching clinical use. In this study, we tested the utility of methyl-guanine-methyl-transferase (MGMT)-transgenic-C57BL/6 BM into a major histocompatibility complex mismatched-BALB/c model followed by N,N-bis(2-chloroethyl)-nitrosourea (BCNU) treatment to enhance donor-cell engraftment and then evaluated transplant tolerance induction. Methods: A single-dose of anti-CD8 antibody and busulfan was administered into BALB/c-host-mice at day 1. The BALB/c-mice also received costimulatory blockade through multiple-doses of anti-CD40L antibody. 10 x 106 BM-cells from MGMT-transgenic-mice were transplanted into host BALB/c mice at day 0. The BCNU was administered at 4 time points after BM transplantation (BMT). Heterotopic donor C57BL/6 cardiac allografts were performed at day 243 after BMT. Skin transplantation with third-party CBA, host BALB/c and donor C57BL/6 grafts was performed at day 358 after BMT. Results: The BALB/c-mice showed long-term stable and high-level donor-cell engraftment with MGMT transgenic C57BL/6 BMT after BCNU treatment, demonstrating full reconstitution and donor cardiac-allograft tolerance and no GVHD with expanded donor and host Foxp3+ T regulatory cells. Further, skin grafts from donor, host, and third party showed good immune function with rejection of third-party grafts from all mice and benefit from enhanced chimerism after BCNU with less cell infiltrate and no chronic rejection in the donor skin grafts of BCNU treated mice compared no BCNU treated mice. Conclusions: High-level mixed chimerism without GVHD can be achieved using MGMT transgenic BM in a mixed-chimerism model receiving BCNU across a major histocompatibility complex mismatch. Enhanced mixed chimerism leads to long-term donor-specific allograft tolerance.