Tolerance induction through megadose bone marrow transplantation with two-signal blockade

Background. Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. Materials and methods. Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2 × 10⁷ cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2 × 10⁸ cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vβ5 or Vβ11 T cells and intrathymic presence of donor MHC class II⁺ cells. Induction of chimerism-based tolerance to skin allograft was further determined. Results. High levels (∼23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (∼10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. Conclusions. The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.