A Minimum Conditioning Protocol towards Transplantation Tolerance in NOD Mice by Mixed Hematopoietic Chimerism

Abstract

Abstract Stable mixed hematopoietic chimerism is a robust method for inducing donor specific tolerance. However, its clinical application is dampened by the toxicity of current recipient conditioning regimens. We previously showed an irradiation-free mixed chimerism protocol in diabetes prone NOD mice is achievable with antibodies to T cells and CD40L together with busulfan and high dose rapamycin. We sought to generate a more clinically feasible chimerism protocol and tested the hypothesis that more efficient recipient T cell depletion would eliminate the need for anti-CD40L (known to cause thromboembolism in humans) and rapamycin. We preconditioned NOD mice with donor specific transfusion from fully mismatched mice (d −10), cyclophosphamide (CYP) (d −8), antibodies against CD90 and/or CD4 + CD8 (d −6, −1, 4, 9, 14), busulfan (d −1) and donor bone marrow transplant (d 0). Flow cytometry was used to detect chimerism. Mixed chimerism was induced in 33/45 NOD mice. Stable chimerism with multilineage donor cells was maintained in 21/33 recipients. Loss of chimerism could be predicted by a lower early level of chimerism at d 4, 9 or 14. Inclusion of αCD90 mAb, busulfan and CYP was critical for chimerism induction. With anti-CD90 17/23 mice became stable chimeras. 5/5 chimeric mice accepted skin from bone marrow donors but rejected skin from MHC-matched and minor antigen mismatched donors. The loss of anti-donor Vβ11+ T cells and anti-recipient Vβ17+ T cells in stable chimeric mice indicated the establishment of chimerism involves clonal deletion. Conclusion A protocol causing rapid and robust recipient T cell depletion generated chimerism without the need for anti-CD40L or rapamycin in tolerance induction resistant NOD mice.