The role of complement in solid organ transplantation continues to evolve with our enhanced understanding of complement's regulation of the alloresponse. Prior work has shown that CD4 T-cells and antigen presenting cells (APCs) secrete complement and upregulate expression of C3a receptor (C3aR) and C5a receptor during cognate interactions. As prior studies focused on non-CD8 T-cells, we addressed the role of C3aR signaling in the CD8 T-cell alloresponse. To test the impact of C3aR on alloreactive CD8 T-cells, we transplanted wild type (WT) BALB/c hearts into C3aR-/- or WT B6 recipients. We analyzed donor reactive CD8 T-cells by flow cytometry on post-operative day 6, which revealed ~2 fold fewer donor reactive, interferon gamma producing CD8 T-cells in the C3aR-/- mice vs WT (6% vs 13%, p<0.001). Analogous studies performed in C5aR-/- recipients showed no difference, newly indicating a dominant role for C3aR in modulating alloreactive CD8 T-cells. To differentiate the effects of C3aR expressed on non-CD8 T-cells (extrinsic) vs CD8 T-cells (intrinsic), we adoptively transferred CFSE-labeled WT naïve CD8 T-cells into WT or C3aR-/- B6 mice prior to receiving allogeneic heart transplants. Absence of host C3aR diminished in vivo proliferation of naïve WT CD8 T-cells (13% vs 28%, p<0.01), indicating a CD8 T-cell extrinsic effect of C3aR. Mechanistic studies indicated absence of C3aR lowered APC expression of CD86 and IL12 and reduced CD8 T-cell expression of Tbet, together suggesting C3aR deficiency limits APCs ability to prime CD8 T-cells. To alternatively test the impact of CD8 T-cell intrinsic C3aR on in-vivo CD8 alloresponses, we transferred naïve WT or C3aR-/- CD8 T-cells into WT hosts, followed by allogeneic heart transplants. Proliferation among the transferred C3aR-/- CD8 T-cells was also reduced compared to WT (12% vs 28%, p<0.05), demonstrating a CD8 intrinsic effect of C3aR, independent of CD4 T-cells and APCs. To assess biochemical mechanisms linking C3aR to proliferation in CD8 T-cells, we focused on mTOR signaling and quantified CD8 T-cell expression of phosphorylated S6, an mTOR substrate, by phosphoflow. These assays showed pS6 expression was ~35% lower in the C3aR-/- CD8 T-cells (p<0.01). Overall we newly demonstrate that C3aR signaling crucially regulates alloreactive CD8 T-cell responses through extrinsic and intrinsic mechanisms. The data provide a basis for testing the effects of C3aR blockade on transplant outcomes first in animals and ultimately in humans.

CITATION INFORMATION: Horwitz J., Mathern D., Heeger P. C3a Receptor Regulates the CD8 T-Cell Alloresponse via Intrinsic and Extrinsic Mechanisms Am J Transplant. 2017;17 (suppl 3).

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