Prolonged survival of anti-CD154-(MR1)-treated murine heart transplant recipients is dependent on regulatory T cells (Treg) and MDSC. Based on studies in the tumor literature suggesting C5a receptor (C5aR1) is required for MDSC function, we tested the hypothesis that myeloid cell-expressed C5aR1 is required for MR1-induced allograft survival. We generated C5aR1fl/fl mice crossed with LysMcre-expressing mice and verified absence of C5aR1 specifically on all myeloid cells. We transplanted groups of MR1-treated B6 C5aR1fl/flLysMcreand B6 C5aR1fl/fl controls with BALB/c hearts. While MR1 prolonged graft survival in the C5aR1fl/fl to >60d vs 8d in untreated mice, allografts survived only 30d in the MR1-treated C5aR1fl/flLysMcrerecipients (n=7-10/grp, p<0.05 vs control). We phenotyped intragraft CD11b+ myeloid cells on d6 posttransplant using differential expression of Ly6C/G. We observed all myeloid subsets in controls expressed C5aR1. MR1 induced a Ly6Clo subset expansion that we previously showed was required for prolonged graft survival. In grafts of C5aR1fl/flLysMcre recipients we observed fewer Ly6Clo cells (25.05% vs 40.08%, n=10/grp p<0.05) all of which lacked C5aR1. When we analyzed splenic immune responses on d15 posttransplant we observed higher frequencies of donor reactive CD8+ IFNg-producing T cells in the C5aR1fl/flLysMcre recipients (1.46% vs 0.5%; n=6/grp p<0.05) along with higher ratios of donor reactive CD8/Treg (p<0.05 vs control). To test the impact of myeloid cell C5aR1 on Treg expansion we adoptively transferred tracer numbers of TCR transgenic TEa CD4+ cells into MR1-treated transplant recipients and quantified splenic Foxp3+Tea+ cells on day 7. The assays showed ~40% less Treg expansion in the C5aR1fl/flLysMcre mice (n=4/grp p<0.01). Our prior work showed that the Ly6Clo MDSC derived from Ly6Chi precursors. To test whether C5aR1 impacts this conversion we isolated CD45+CD11b+Ly6Chi bone marrow cells from C5aR1fl/fland C5aR1fl/flLysMcre mice,fluorescently labeled and co-transferred them into MR1-treated WT allograft recipients. Analyses on d 3 showed fewer C5aR1-/- vs WT Ly6Clo cells in the grafts (n=6/grp p<0.02). In translational work we observed that human PBMC obtained 6 mo post kidney transplant contained C5aR1+ CD14+CD16-HLADR-CD11b+CD33+ MDSC. Our results newly demonstrate myeloid cell-expressed C5aR1 modulates MDSC induction/function in MR1-treated murine heart transplant recipients and raise concerns about potential deleterious effects of C5aR1 blockade in transplantation.

CITATION INFORMATION: Llaudo I., Ochando J., Heeger P. C5aR Regulates Myeloid Derived Suppressor Cells (MDSCs) in Transplantation Am J Transplant. 2017;17 (suppl 3).

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