Effects of Immunosuppressive Drugs on Hematopoietic Engraftment after Simultaneous Infusion of Apoptotic Cells and Bone Marrow Cells.

We have recently showed in a murine bone marrow transplantation (BMT) model that apoptotic cell infusion simultaneously with a bone marrow (BM) graft favors hematopoietic engraftment, and induces a long term tolerance restricted to BM cell allo-antigens. This effect is observed whatever the origin of apoptotic cells and does not lead to the development of autoimmunity or GvHD. This favorable effect on engraftment was obtained without immunosuppressive (IS) drugs. In human BMT, IS treatment is always required. The aim of this study was to investigate the interactions between apoptotic cells and IS drugs, especially since some of them might interfere with other strategies of tolerance induction. The roles of ciclosporin A (CSA), mycophenolate mofetil (MMF), sirolimus (Rapa) and LF150195 (LF), a deoxyspergualin analog, were tested. Recipient Balb/c mice (H2^d) were sublethally irradiated (6 Gy) 1 day before receiving 10⁶ BM cells with or without apoptotic cells (5x10⁶ γ-irradiated splenocytes) from donor FvB mice (H2^q). Drugs were injected IP: CSA 50mg/kg/d from day 0 (d0) to d14, MMF 20mg/kg/d from d0 to d14, Rapa 1.5mg/kg/d from d0 to d14, and LF 0.625mg/kg/d from d0 to d10. Engraftment was assessed by flow cytometry analysis in peripheral blood. Skin grafts from BM donor mice were performed >70 days after BMT, to assess the influence of IS drugs on our tolerance induction protocol. CSA treatment didn’t favor engraftment in mice receiving BM alone (22% engraftment vs 20% without IS drug, p=NS), and induced only transient chimerism (<d70). CSA inhibited the effect of apoptotic cells on engraftment (13% engraftment with BM, apoptotic cells and CSA, vs 41% with BM and apoptotic cells without IS drug, p<0.05). MMF didn’t promote engraftment in mice receiving BM alone (11% engraftment vs 20%, p=NS), and didn’t have any effect when associated with apoptotic cell infusion (30% engraftment vs 41%, p=NS). Rapa was very effective in permitting engraftment of BM alone (93% engraftment vs 20%, p<0.05), and when associated with apoptotic cells, Rapa achieved a stable and long term chimerism in all grafted mice. LF increased engraftment after infusion of BM cells alone (40% engraftment vs 20%, p<0.05), and after BM and apoptotic cell infusion, 93 % of mice engrafted (p<0.05). Engraftment was stable in all mice (up to 600 days post-BMT), except for the group receiving CSA and BM alone. Skin grafts were tolerated in all chimeric mice (up to 400 days after skin graft), and were rejected in the same way by non chimeric mice (either because there wasn’t any BM engraftment, or because chimerism was lost at the time of skin graft) and control naive mice. This study confirms that IS drugs can interact with tolerance induction protocols, and particularly with our model of cell-based therapy using IV apoptotic cell infusion to promote BM engraftment. CSA shouldn’t be associated with apoptotic cells, as it seems to antagonize the favoring effect on engraftment. MMF could be used, since it doesn’t hamper the action of apoptotic cells. Rapa is highly effective even without apoptotic cells, and doesn’t interfere with their action. LF seems to be the most interesting drug in our model, because its effects are synergic with apoptotic cells in promoting engraftment. In clinical settings, Rapa and MMF can be used in addition to apoptotic cell infusion to favor engraftment after reduced intensity conditioning regimens.