Induction of stable long-term mixed hematopoietic chimerism following nonmyeloablative conditioning with T cell-depleting antibodies, cyclophosphamide, and thymic irradiation leads to donor-specific in vitro and in vivo tolerance

Abstract

Background: Successful transplantation of solid organs relies on long-term immunosuppression for the prevention of graft rejection. Donor-specific tolerance without the need for continuous immunosuppression can be observed after allogeneic BMT. However, its routine use for tolerance induction has been precluded so far by the high conditioning-related toxicity of standard BMT regimens. Our laboratory has recently established a cyclophosphamide (CTX) plus thymic irradiation (TI)-based nonmyeloablative conditioning protocol for the treatment of hematologic malignancies. We have recently described the successful clinical application of this approach for the induction of donor-specific tolerance in a patient receiving a living-related kidney transplant, which resulted in graft acceptance without long-term immunosuppression. The aim of this study was to evaluate the induction and maintenance of host-versus-graft tolerance following this CTX-plus-TI-based regimen in a mouse model.

Methods: Induction of mixed hematopoietic chimerism and development of donor-specific tolerance following the CTX-based nonmyeloablative conditioning regimen (200 mg/kg CTX, in vivo T-cell depletion [anti-CD4 monoclonal antibody (MoAb) GK1.5 and anti-CD8 MoAb 2.43], and 7 Gy TI) was studied in the fully major histocompatibility complex (MHC)-mismatched B10.A (H2a)-->B6 (H2b) strain combination.

Results: The conditioning regimen allowed allogeneic bone marrow engraftment and persistent (>30 weeks) mixed lymphohematopoietic chimerism in almost all recipients. TI was essential to allow engraftment and development of tolerance, which was evident in all lasting chimeras. Compared to animals receiving a similar TBI-based conditioning regimen, overall levels of chimerism were significantly lower in the CTX-plus-TI-conditioned animals. However, donor-specific tolerance in vitro and in vivo was evident in CTX-plus-TI-conditioned chimeras. Tolerance was associated with the presence of donor-type MHC class II+ cells in the thymus and deletion of donor-reactive cells, as determined by Mtv-8 and Mtv-9 superantigen-mediated deletion of Vbeta11+ and Vbeta5/1.2+ T cells.

Conclusion: Engraftment, long-term chimerism, and induction of donor-specific tolerance can be achieved using a nonmyeloablative CTX-based conditioning regimen in fully MHC-mismatched BMT recipients without the induction of GVHD.