TLR stimulation has been shown by others to prevent costimulatory blockade-induced allograft tolerance. Immune cell derived complement activation products C3a and C5a amplify inflammation, enhance effector T cell responses and suppress regulatory T cells (Tregs) analogous to previously reported effects of TLR stimulation, raising the hypothesis that the 2 systems are mechanistically linked. As published, BALB/c hearts transplanted into CpG (TLR9 agonist)-treated B6 mice given MR1 were rejected (d20, n=6) while grafts in MR1 treated controls survived >60 d. In contrast, grafts transplanted into MR1 treated recipients deficient in receptors for C3a and C5a (C3aR-/-C5aR-/-) survived >50d despite CpG (p<0.01, n=6). MR1+CpG treated recipients deficient in C3aR alone rejected their grafts (d25, n=6). Splenic DCs express C3aR/C5aR, and in vitro CpG stimulation of splenic DCs mixed with allo-T cells produced anaphylatoxins. We next treated WT and C3aR-/-C5aR-/- mice with CpG and isolated/analyzed splenic DCs 4 h later. This ex vivo analysis showed that DCs from the C3aR-/-C5aR-/- mice had lower surface expression of class II MHC, CD80, CD86 and higher expression of the co-inhibitory molecule PDL1 vs DCs obtained from CpG treated WT mice (p<0.01). Functionally, CpG-prestimulated WT DCs cocultured with WT T cells augmented allo-T cell responses, while mixed lymphocyte reactions using DCs from CpG-treated C3aR-/-C5aR-/- mice or with C3aR-/-C5aR-/- T cells resulted in 30-60% reduction in allo-T cell proliferation/expansion (p<0.01). To assess links among TLR9 signaling, C3aR/C5aR signaling and Treg stability in vivo we employed ERT2-dTomatoFoxp3-GFP “fate mapping” mice on WT and C3aR-/-C5aR-/- backgrounds so as to identify Foxp3+Tregs (GFP+tomato+) and ex-Tregs (GFPneg tomato+). While CpG treatment to MR1 treated WT recipients of allo-hearts resulted in ~30% conversion of Treg to ex-Treg (Treg instability), the absence of C3aR/C5aR prevented the effects (Treg conversion to ex-Treg was no different from CpG-untreated controls, p<0.001 n=5-8). Together our data support the hypothesis that TLR9-induced resistance to allograft tolerance depends on C3a/C3aR and C5a/C5aR signaling which activates DCs, stimulates effector T cells and induces Treg instability. These findings support the need to test the impact of targeting C3aR/C5aR in human transplant recipients.

CITATION INFORMATION: Sheen J, Heeger P. Toll-Like Receptor (TLR) 9-Induced Resistance to Murine Cardiac Allograft Tolerance Is Dependent Upon C3a and C5a Receptor (C3aR/C5aR) Signaling. Am J Transplant. 2016;16 (suppl 3).

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