Anti-CD40L (MR1) induced heart transplant tolerance has been recently shown to be dependent on early post-transplant intragraft accumulation of CD11b+Ly6C^LoLy6G^– DC-SIGN expressing Mreg that develop from myeloid precursors via a CSF1-dependent process. The source of the CSF1 and the stimuli required to initiate this process remain unclear. Based on previous publications in the tumor literature linking complement C5a receptor (C5aR) to Mreg function we tested the hypothesis that C5aR signaling is linked to induction of Mreg required for MR1-induced allograft tolerance. We transplanted BALB/c hearts into WT or congenic C5aR-/- B6 recipients +/- MR1 and analyzed intragraft myeloid cell accumulation 5 days later. C5aR was detected on all subsets of the intragraft CD11b+ cells in WT recipients. Whereas the frequency of Ly6C^LoLy6G^– cells increased in WT recipients treated with MR1 compared to untreated controls (45% vs 20%, n=6/group, p<0.05), the frequency of Ly6C^LoLy6G^– cells was the same in MR1 treated and untreated C5aR-/- recipients (30% vs 25%, n=6/group, ns). Whereas BALB/c hearts transplanted into MR1-treated WT recipients survived >60 days grafts, BALB/c hearts transplanted into C5aR-/- recipients underwent delayed rejection (median survival time: 40 days, n=5group, p<0.05), together suggesting that the absence of C5aR prevents MR1-dependent Mreg development required for long term graft survival. To assess mechanisms linking C5aR to Mreg, we cultured WT or C5aR-/- B6 bone marrow (BM) cells (<5% Ly6C^LoLy6G^– myeloid cells at baseline) +/- CSF1+IL4, testing phenotype and function 4 days later. WT and C5aR-/- BM cultures containing CSF1+IL-4 induced ~30% of CD11b+ cells to become Ly6C^LoLy6G^– (<5% in control cultures on day 4). The resultant cells suppressed anti-CD3/CD28 induced T cell proliferation, consistent with induction of Mreg. We next tested whether C3a/C3aR and/or C5a/C5aR signaling on myeloid cells is linked to CSF1 production. Remarkably, C5a (but not C3a) upregulated CSF1 gene expression 3-5 fold over control (p<0.05), and C5a+IL-4 without CSF1 induced differentiation Ly6C^LoLy6G^– Mreg from BM myeloid precursors (p<0.05). These unique findings show for the first time that C5a can induce Mreg generation required for transplant tolerance, in part via stimulating CSF1 production by myeloid cells. As regulatory myeloid cells have been described in human transplant recipients the findings suggest that similar mechanism may be operative in humans.

CITATION INFORMATION: Llaudo I, Conde P, Ochando J, Heeger P. C5a Induces CSF1-Dependent Regulatory Macrophage (Mreg) Development Required for Murine Allograft Tolerance. Am J Transplant. 2016;16 (suppl 3).

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