*Purpose: Activation, differentiation and expansion of alloreactive CD8⁺ T cells, the dominant effectors that mediate murine heart allograft rejection, requires allorecognition, costimulation, and cytokine-initiated signals. While previous work showed that alloreactive CD4⁺ T cell immunity entails immune cell-produced and locally activated complement, whether and how C3a receptor 1 (C3aR1) signaling impacts transplant outcomes and the mechanisms linking C3aR1 to alloreactive CD8⁺ T cell activation/expansion remain unclear.

*Methods: To test whether preventing C3aR1 signaling interacts with calcineurin inhibition to prolong allograft survival, we transplanted groups of WT B6 mice recipients with BALB/c hearts and treated the recipients with tacrolimus ± a pharmacological C3aR1 antagonist (C3aR1-A) administered via subcutaneous osmotic pump.

*Results: Tacrolimus administration prolonged graft survival in WT recipients from a median survival time (MST) of 7 to 14 days (p<0.05), but remarkably synergized with the blockade of C3aR1 to prolong allograft survival to a MST of 20 days (p<0.05 vs. tacrolimus only). To test the effects of the tacrolimus plus C3aR1-A on the donor-reactive T cell responses, we transplanted groups of WT mice, treated them with tacrolimus ± C3aR1-A, and sacrificed them on day 10 post-transplant (all grafts beating) to perform ex vivo immune analyses. We observed 2-fold lower numbers of IFNγ-producing, TNFα-producing, and CD107+ (a marker of cytotoxic degranulation) splenic CD8+ T cells in the day 10 post-transplant tacrolimus+C3aR1-A treated recipients vs. tacrolimus treated controls (p<0.05 for each, n=4/group). When we analyzed CD8+ T cells within the graft-infiltrating lymphocytes (GILs) on day 10 post-transplant, we observed fewer IFNγ+ and fewer CD107+ CD8+ T cells in the tacrolimus-treated recipients in which C3aR1 signaling was pharmacologically impeded. Reciprocal adoptive transfer studies of WT or C3aR1-deficient CD8+ T cells into WT or C3aR1-deficient hosts revealed that C3aR1 expressed on CD8+ T-cells drives mTOR- and T-bet dependent T cell differentiation and that C3aR1 expressed on APCs alters costimulatory molecule expression and innate cytokine production.

*Conclusions: Together the data discern multiple effects of C3aR1 on alloreactive T cell immunity and identify C3aR1 as a testable therapeutic target for future studies aimed at improving human transplant outcomes.

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