C5L2 Deficiency Prevents Prolonged Heart Transplant Survival in Mice Treated With Anti-CD40L mAb.

Abstract

C5L2 (GPR77) is a 7 transmembrane spanning receptor that does not signal via G-proteins and binds the complement activation product C5a (and also to its degradation product: C5adesarg). Current concepts are that C5L2 functions as a counter-regulatory receptor, limiting the proinflammatory effects of C5a/C5a receptor (C5aR/CD88) interactions, possibly by internalizing free C5a and thereby acting as a decoy receptor. Recent studies showed that C5a/C5aR interactions enhance alloreactive T effector (Teff) responses and inhibit regulatory T cell (Treg) induction and function but the impact of C5L2 in this context is not known. To address this, we transplanted BALB/c hearts into B6 WT or B6 C5L2-/- recipients +/- anti-CD40L mAb MR1 (250 μg iv at transplant). Untreated WT and C5L2-/- recipients rejected the allografts by day 10 (n=3 mice/group, p=ns). MR1 prolonged survival of heart grafts in WT recipients to a median of 56 days, but only prolonged survival of BALB/c hearts in C5L2-/- to 29 days (n=7-8 mice/group, p<0.01). We repeated the experiment sacrificing recipients 2 weeks post transplantation (allografts beating) to assess donor reactive immunity by IFNγ ELISPOT assay. Spleen cells from WT recipients treated with MR1 contained <10 IFNγ producers/300,000 spleen cells, not different from naïve WT or naïve C5L2-/- animals. In contrast, we observed ˜175 donor reactive IFNγ producers /300,000 spleen cells in C5L2-/- recipients despite treatment with MR1. When we stimulated CFSE labeled WT or C5L2-/- T cells obtained from naïve mice with allogeneic BALB/c APCs we observed similar proliferation (WT 11.3% vs C5L2-/- 11.1% p=ns), indicating that C5L2 does not directly impact alloreactive Teff responses in vitro. In contrast, when we generated iTreg from naïve CD4 cells from WT and C5L2-/- mice, we observed that anti-CD3+TGFβ+IL-2 induced fewer Foxp3+ cells in the absence of C5L2 (57.3% in C5L2-/- vs 82.7 in WT, p<0.05). Together these new results indicate that C5L2 expression is required for prolonged heart transplant survival induced by MR1. The data suggest that C5L2 on naïve CD4 T cells functions by binding immune cell derived C5a and thereby facilitates generation and potentially enhances stability of iTreg in the context of costimulatory blockade.