TLR signals prevent CD40L-blockade (MR1) induced murine transplant tolerance but mechanisms are incompletely understood. Based on previous findings that immune cell derived complement activation products C3a and C5a enhance effector T cell responses and suppress Treg, similar to observed effects of TLR signals, we tested the hypothesis that TLR signaling and complement activation are linked. Quantitative PCR analysis of CpG (TLR9 agonist)- or LPS (TLR4 agonist)-treated splenic dendritic cells (DC) induced 10-40 fold increases in C3 and factor B (fB) expression at 24h (p<0.05). C3 and fB expression did not change in CpG-treated MyD88-/- DCs or in LPS-treated TRIF-/- DCs indicating the increases depend on canonical signaling pathways. While mixed lymphocytes reactions (MLRs) using WT DCs pretreated with CpG or LPS augmented alloreactive T cell proliferation and expansion (p<0.05 vs. no TLR stimulus), absence of C3 and C5 in the DCs and T cells prevented the effects (p<0.05 vs WT controls). In vitro blockade of C3a/C3aR and C5a/C5aR signaling with neutralizing mABs or genetic deficiency of C3aR/C5aR on responding T cells reduced TLR-induced alloreactive T cell proliferation/expansion by >50% (p<0.05 vs control). C3/C5 deficiency did not alter surface expression of MHCII, CD80 and CD86 on DCs stimulated with CpG at 24h suggesting TLR-induced production of C3a/C5a enhance T cell responsiveness directly through T cell expressed C3aR/C5aR. While CpG-stimulated DCs suppressed in vitro induced, TGFbeta-dependent allo-Treg induction (p<0.05 vs control DC), absence of C3aR/C5aR on the responding T cells prevented the effect. Consistent with previous publications, CpG treatment of WT BALB/c recipients of B6 hearts prevented MR1-induced graft survival (median survival time: CpG 20 d vs control >100 d, p<0.05, n=5 per group). In contrast, allografts survived > 60 d in CpG/MR1-treated C3/C5 deficient, H-2d recipients of B6 allografts (n=5 p<0.05). Grafts survived >100 d in control WT and C3/C5 deficient recipients treated with MR1 alone. Together the data demonstrate a previously unrecognized mechanistic link between TLR signals and immune cell derived complement, and raise the possibility that targeting C3aR/C5aR signaling could overcome potential detrimental effects of pathogens on graft reactive alloimmunity in human transplant recipients.

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