Cutting edge: Receptors for C3a and C5a modulate stability of alloantigen-reactive induced regulatory T cells

Abstract

CD4(+)Foxp3(+) regulatory T cells (Treg) are critical regulators of immune homeostasis and self-tolerance. Whereas thymic-derived or natural Treg stably express Foxp3, adaptive or induced Treg (iTreg) generated from peripheral CD4 T cells are susceptible to inflammation-induced reversion to pathogenic effector T cells. Building upon our previous observations that T cell-expressed receptors for C3a (C3aR) and C5a (C5aR) drive Th1 maturation, we tested the impact of C3aR/C5aR signaling on induction and stability of alloreactive iTreg. We observed that genetic deficiency or pharmacological blockade of C3aR/C5aR signaling augments murine and human iTreg generation, stabilizes Foxp3 expression, resists iTreg conversion to IFN-γ/TNF-α-producing efffector T cells, and, as a consequence, limits the clinical expression of graft-versus-host disease. Taken together, the findings highlight the expansive role of complement as a crucial modulator of T cell alloimmunity and demonstrate proof-of-concept that targeting C3a/C3aR and C5a/C5aR interactions could facilitate iTreg-mediated tolerance to alloantigens in humans.

FIGURE 1. C3aR/C5aR signaling on CD4⁺ T cells modulates allo-iTreg generation

(A) Representative flow plots (left and middle, 12,000 events shown in each plot) and total number (right) of Foxp3⁺ cells generated from CFSE-labeled CD4+ WT or C3ar1^−/−C5ar1^−/− Tconv. (B) Suppression capacities of WT (solid line) or C3ar1^−/−C5ar1^−/− (dashed line) allo-iTreg. (C) Total Foxp3^GFP+ cells generated as in (A) ± blocking anti-C3a/C5a mAb. (D) Percentages (left/middle) and total numbers (right) of Foxp3⁺ cells generated as in (A) using allogeneic WT or Daf1^−/− DCs. (E) Splenic frequencies of Foxp3^GFP+ (left) and total CD4+ cells (right) 14d after adoptive transfer of naive WT or C3ar1^−/−C5ar1^−/− CD4+ cells into syngeneic rag1^−/− mice. (F) Immunoblot for pFoxo1 and total Foxo1 from stimulated iTreg cell lysates. Results are each representative of 3 independent experiments. Error bars indicate mean ± s.d. *p<0.05

FIGURE 2. Absent C3aR/C5aR signaling enhances allo-iTreg stability

(A) Percentage (left/middle) and number (right) of Foxp3^GFP+ cells from sorted allo-iTreg 5d after restimulation with allogeneic DCs + IL-2. (B) Representative intracellular IFNγ/TNFα cytokine staining of cultures gated on ex-Treg. (C) Percentage (left/middle) and number (right) of Foxp3^GFP+ cells stimulated as in (A) ± C3aR-A/C5aR-A antagonists (Antag). (D–F) Sorted CD45.2 allo-iTregs were injected into lethally irradiated BALB/c mice with B6CD45.1 bone marrow transplant + Tconv. Foxp3 expression in splenic CD45.2 (D) and intracellular IFNγ (E) and TNFα (F) in CD45.2 Foxp3GFP^neg (ex-Treg), and Foxp3GFP⁺ (iTreg) populations are shown. Results are each representative of at least 3 independent experiments. Error bars indicate mean ± s.d. *p<0.05

FIGURE 3. Allo-iTreg deficient in C3aR/C5aR suppress acute GVHD and exhibit enhanced in vivo stability

(A) Weight changes and (B) clinical scores in C.B.-17 scid mice that received CD45.1⁺ Tconv alone (black, n=4), or Tconv + WT (red n=7) or C3ar1^−/−C5ar1^−/− (blue, n=8) allo-iTreg. Normal growth curve in PBS controls (green, n=5). (C) Quantified percentages of Foxp3^GFP+ iTreg among CD45.2⁺ T cells in the spleen and liver of recipients at 4 wk. Results are cumulative data of 3 independent experiments. Error bars indicate mean ± s.e.m. *p<0.05

FIGURE 4. C3aR/C5aR signaling modulates generation and stability of human iTreg

(A) Percentage of Foxp3⁺ CD4+ cells on d5 of human iTreg induction cultures ± C3aR-A or C5aR-A (n=8). *p<0.05. (B) Suppressive capacities of CD25⁺ iTreg generated with C3aR-A (red), C5aR-A (blue) or PBS control (black). (C) Representative flow plot of DAF expression on humans DCs transfected with scrambled-targeted (black) or DAF-targeted (red) siRNA. (D) Plots depicting human Foxp3⁺ CD4+ cells generated with control or DAF^kd DCs, ± C3aR-A (mean % Foxp3+ cells shown, n=4,*p<0.05 vs. other 2 groups). (E) Percentages and (F) total numbers of Foxp3 expressing, CD25-enriched human iTreg restimulated for 3d ± C3aR-A or C5aR-A. Numbers represent means and s.d. of 3 replicates, *p<0.05 vs. control. (G) Weights of NOD scid γc^null mice transferred with human PBMC treated with PBS (black), C5aR-A (red), CTLA4-Ig (blue), or CTLA-Ig + C5aR-A (green), n=4–6 per group *p<0.05 (CTLA4Ig vs. CTLA4Ig+C5aR-A), **p<0.05 vs. PBS and vs. C5aR-A alone. (H) Total numbers of human CD4+ cells (left) or CD4+Foxp3+ cells (middle) in the spleens, and percentages of Foxp3⁺ cells (right) within the human CD4⁺ gate, 6 wk after adoptive transfer (n=3–6/group), *p<0.05.