Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice.
Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.
Regenerative Medicine Department, Operational and Undersea Medicine Directorate at the Naval Medical Research Center, Silver Spring, MD 20910-7500, USA.
Khairul
K
Anam
Mihret F
MF
Amare
Stephen R
SR
Zins
Thomas A
TA
Davis
D000818:Animals; D019138:Antibodies, Blocking / Q000008:administration & dosage; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D016026:Bone Marrow Transplantation; D002066:Busulfan / Q000008:administration & dosage; D015704:CD4 Antigens / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology; D002478:Cells, Cultured; D046528:Chimerism; D051858:Forkhead Transcription Factors / Q000096:biosynthesis; D006084:Graft Rejection / Q000276:immunology* / Q000517:prevention & control; D007108:Immune Tolerance; D051379:Mice; D008815:Mice, Inbred Strains; D009713:Nucleotidyltransferases / Q000096:biosynthesis; D016038:Skin Transplantation; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D019172:Transplantation Conditioning
D019138:Antibodies, Blocking; D015704:CD4 Antigens; D016827:CD8 Antigens; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D009713:Nucleotidyltransferases; C057535:lincosaminide O-nucleotidyltransferase; D002066:Busulfan