Tfh cells, in part via IL-6 dependent production of IL-21, are specialized providers of B cell help. Extending previous work in which we showed that signaling through C5aR expressed on T cells and antigen presenting cells (APCs) enhances alloreactive effector T cell proliferation, differentiation and expansion while inhibiting regulatory T cells (Treg), we assessed the impact of these signals on Tfh induction and function. When we stimulated WT and C5aR-/- CD4 T cells with anti-CD3/CD28, recombinant IL-6, and blocking anti-IFNg, anti-TGFb and anti-IL4 antibodies, we observed less IL-21 produced by C5aR-/- CD4 T cells (p=0.016 vs control). Because IL-6 signals via Stat3, we tested whether C5aR deficiency/blockade altered IL-6 induced Stat3 phosphorylation. These experiments showed decreased IL-6-induced pStat3 in C5aR-/- CD4 T cells vs. controls (p<0.05 at 15 and 30 min), and showed a dose dependent decrease in pStat3 when WT CD4 T cells were stimulated with IL-6 plus a specific C5aR antagonist (p<0.05). C5aR deficiency/blockade did not alter IL-10-induced Stat3 phosphorylation suggesting specificity for an IL-6/C5aR interaction. Expression of IL-6R and gp130 (IL-6 co-receptor) were similar on WT and C5aR-/- T cells. Together the results imply that C5aR expression enhances IL-6R signaling on T cells. To test effects of C5aR on Tfh cell induction and function in vivo we employed a Tfh-cell-dependent, lupus-like model of chronic graft versus host disease (cGVHD) that results in glomerulopathy: we adoptively transferred equal numbers of B6 WT or C5aR-/- CD4 T cells into (BALB/c x B6) F1 hosts. Two weeks later we observed that the recipients of C5aR-/- cells contained reduced numbers of donor derived Tfh cells (p=0.08) and recipient germinal center B cells (p=0.02) as well as decreased anti-dsDNA antibodies (p=0.04) vs. WT controls. The numbers of donor derived Foxp3+ follicular Treg were not different between groups. In separate experiments in which we followed animals (n=4-5/group) for 3 months post transfer we observed less hematuria and decreased histological evidence of glomerulopathy in recipients of C5aR-/- CD4 cells. Overall our data provide new mechanistic insight by showing that T cell expressed C5aR regulates IL-6 dependent induction and function of Tfh cells. The findings support the need for testing the efficacy of targeting C5aR to prevent allosensitization in the clinic.

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