Adoptive transfer of CD4+Foxp3+ regulatory T cells (Treg) has potential for promoting transplantation tolerance but is limited by the scarcity of thymic-derived Treg and the functional instability of peripherally generated (induced) iTreg. Prior studies have shown that immune cell derived C3a and C5a enhance effector T cell immunity, but effects on iTreg generation and stability are not known.
We generated alloantigen-reactive iTreg (allo-iTreg) using naÏve CD4+ T cells from B6 WT or C3a/C5a-receptor deficient (C3aR-/-C5aR-/-) Foxp3GFP reporter mice plus Balb/c DCs and TGFb. Flow sorted Foxp3-GFP+ allo-iTreg were a) restimulated in vitro and by transfer into allogeneic hosts, b) tested for stability (Foxp3GFP, cytokines by flow cytometry), and c) analyzed for methylation of the Foxp3 promoter. Suppressive function was assessed in vitro using proliferation assays and in vivo using acute allogeneic graft vs. host disease models. Effects of C3aR/C5aR signaling on human iTreg induction and function were tested in vitro in response to recombinant C3a/C5a, siRNA knockdown of decay accelerating factor (augments local C3a/C5a production) and blocking C3aR/C5aR with antagonists.
C3a/C5a receptor deficiency or blockade enhanced iTreg conversion (>50%, p<0.05) and correlated with less AKT-dependent Foxo1 phosphorylation, a signaling pathway known to be attenuated in Treg. Murine WT and C3aR-/-C5aR-/- iTreg were functionally equivalent, but WT allo-iTreg lost Foxp3 expression and secreted IFNg and TNFa upon restimulation in a secondary MLR or following transfer into allogeneic hosts. C3aR-/-C5aR-/- more stably expressed Foxp3 and resisted Th1 conversion both in vitro (p=.0.01, 3 exp) and in vivo (p<0.05, n=6, 3 exp). The enhanced stability of C3aR-/-C5aR-/- allo-iTreg was associated with reduced Foxp3 promoter methylation. Adoptive transfer of C3aR-/-C5aR-/- allo-iTreg delayed acute GVHD compared to WT allo-iTreg (p<0.05, n=8/grp). C3a and C5a prevented TGFb-dependent human iTreg generation while C3aR antagonism rescued Foxp3 expression and maintained suppressive function. Our data support the novel conclusion that immune cell derived C3a/C5a prevent iTreg induction and negatively affect iTreg stability in favor of Th1 immunity. Our findings suggest that C3aR/C5aR blockade may be leveraged to promote stable iTreg-dependent transplant tolerance.
To cite this abstract in AMA style:Touw Wvander, Paolo C, Wing K, Peter H. Blocking C3a/C5a-Receptor Signaling Promotes Induction and Stability of Murine and Human Alloreactive Regulatory T Cells [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/blocking-c3ac5a-receptor-signaling-promotes-induction-and-stability-of-murine-and-human-alloreactive-regulatory-t-cells/. Accessed April 22, 2021.
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