title: 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.
abstract: 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 × 10(6) 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.
authors.affiliation: 1 Centre for Kidney Research, The Children's Hospital at Westmead, University of Sydney, NSW, Australia. 2 Centre for Transplant and Renal Research, Westmead Millennium Institute for Medical Research, University of Sydney, NSW, Australia. 3 Children's Cancer Research Unit, The Children's Hospital at Westmead, University of Sydney, NSW, Australia. 4 Centre for Medical and Molecular Bioscience, University of Wollongong, NSW, Australia. 5 Australia Microsurgical Consultants, Melbourne, VIC, Australia. 6 Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, University of Sydney, NSW, Australia. 7 Oncology Research Unit, School of Medical Science, University of New South Wales, NSW Australia.
authors.forename: Min
authors.initials: M
authors.lastname: Hu
authors.forename: Belinda
authors.initials: B
authors.lastname: Kramer
authors.forename: Geoff Y
authors.initials: GY
authors.lastname: Zhang
authors.forename: Yuan Min
authors.initials: YM
authors.lastname: Wang
authors.forename: Debbie
authors.initials: D
authors.lastname: Watson
authors.forename: Brian
authors.initials: B
authors.lastname: Howden
authors.forename: Geoff
authors.initials: G
authors.lastname: McCowage
authors.forename: Ian E
authors.initials: IE
authors.lastname: Alexander
authors.forename: Peter
authors.initials: P
authors.lastname: Gunning
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Alexander
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D016026:Bone Marrow Transplantation; D002330:Carmustine / Q000494:pharmacology*; D015254:DNA Modification Methylases / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004195:Disease Models, Animal; D006086:Graft vs Host Disease / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism*; D016027:Heart Transplantation; D007108:Immune Tolerance / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D016038:Skin Transplantation; D018183:Transplantation Chimera; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C528331:MGMT protein, mouse; D045643:DNA Repair Enzymes; D002330:Carmustine