C-Kit-Derived CD11b+ Cells are Critical for Cardiac Allograft Prolongation by Autologous C-Kit+ Progenitor Cells

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Published results show that autologous C-Kit+ cells robustly prolong cardiac allografts in a C-Kit and dose-dependent fashion. To extend our understanding, we determined if the allograft protective effects of C-Kit+ cells were preserved under conditions of immune suppression, what the primary signal for differential engraftment of the allograft and related immune tissues by C-Kit-derived cells was, and the class of C-kit-derived cells responsible for allograft prolongation. Survival studies were performed with 107 or 2 × 106 GFP-tagged C-Kit+ cells as previously published. CTLA4-Ig (200ug RO day 2) and Cyclosporine A (30mg/kg IP days 0-10) were utilized with C-Kit+ cells in co-therapy survival experiments. Differential engraftment and differentiation studies were performed with 107 GFP-tagged C-Kit+ cells on day 1 with harvest for FACS analysis on day 4 post-transplant. Diphtheria toxin was used to in vivo deplete C-Kit inoculum-derived CD11b+ cells in CD11b-DTR C-Kit+ cell treated allograft recipients. In vivo TCR75 T-cell proliferation assays were performed with fluor-labelled TCR75 cells on day 3 post-transplant as were intracellular IFNγ re-stimulation assays. Results demonstrated that Cyclosporine A and CTLA4-Ig did not inhibit the graft promoting effects of C-Kit+ cells with CTLA4-Ig co-therapy nearly abrogating rejection altogether. Results also showed that differential engraftment did not occur under isolated ischemia reperfusion whereas differential engraft did occur under combined alloimmunity/ischemia reperfusion - suggesting that alloimmunity may be an important signal for C-Kit cellular trafficking. Differentiation studies demonstrated that the majority of C-Kit+ inoculum-derived cells were CD11b+ by day 4 post transplantation and predominantly co-expressed myeloid markers. Importantly, CD11b depletion studies demonstrated dramatic abrogation of allograft prolongation when CD11b+ cells derived from the autologous C-Kit+ cellular inoculum were selectively removed in vivo. These results strongly implicate a C-Kit-derived myeloid population as critical for allograft prolongation and demonstrate the potential therapeutic application of autologous C-Kit+ cells as calcineurin inhibitor-sparing agents and possibly as co-therapeutics for durable graft survival.