Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease
Jinfeng Yang, Abdulraouf Ramadan, Dawn K. Reichenbach, Michael Loschi, Jilu Zhang, Brad Griesenauer, Hong Liu, Keli L. Hippen, Bruce R Blazar, Sophie Paczesny
Jinfeng Yang, Abdulraouf Ramadan, Dawn K. Reichenbach, Michael Loschi, Jilu Zhang, Brad Griesenauer, Hong Liu, Keli L. Hippen, Bruce R Blazar, Sophie Paczesny
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Research Article Immunology Transplantation

Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease

Abstract

Soluble stimulation–2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3– T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2–/– Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2–/– versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc–/– T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2–producing type 1 and increased IL-4/IL-10–producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc–/– mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33–stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD.

Authors

Jinfeng Yang, Abdulraouf Ramadan, Dawn K. Reichenbach, Michael Loschi, Jilu Zhang, Brad Griesenauer, Hong Liu, Keli L. Hippen, Bruce R Blazar, Sophie Paczesny

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Figure 2

Rorc deficiency alleviates aGVHD severity associated with increased frequencies of functional ST2+ Tregs in the inflamed intestine and restoration of Tcon/Treg balance.

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Rorc deficiency alleviates aGVHD severity associated with increased freq...
(A) Representative plots of Foxp3 expression on CD4+ T cells in gut of naive B6 WT or Rorc–/– mice. n = 3, data are shown as mean ± SEM; unpaired t test, ***P < 0.001. (B) Representative plots of ST2 expression on Tregs in gut of naive B6 WT or Rorc–/– mice, respectively. n = 3, data are shown as mean ± SEM; unpaired t test, *P < 0.05. (C) Clinical score and survival curve of lethally irradiated C3H.SW mice receiving B6 WT TCD BM with WT or Rorc–/– donor T cells. n = 5, data are shown as mean ± SEM; unpaired t test, **P < 0.01 for clinical score; a log-rank test was used for survival analysis,**P < 0.01. (D) sST2 and IFN-γ concentrations in plasma collected from the mice described above at the indicated time points after HCT. n = 3, data are shown as mean ± SEM; unpaired t test, *P < 0.05. (E) Representative plots of Foxp3 expression on CD4+ T cells isolated from the gut of C3H.SW mice receiving WT or Rorc–/– T cells on day 14 after allo-HCT, n = 3, data are shown as mean ± SEM; unpaired t test, *P < 0.05. (F) Representative plots of ST2 expression on Tregs isolated from the gut of C3H.SW mice receiving WT or Rorc–/– T cells on day 14 after allo-HCT. n = 3, data are shown as mean ± SEM; unpaired t test, **P < 0.01. (G) mRNA expression of ST2 on Tregs sorted from the gut of mice in C on day 14 after allo-HCT. n = 3, data are shown as mean ± SEM; unpaired t test, **P < 0.01. (H) Representative plots of ST2, TIGIT, and Helios expression on Foxp3+ Tregs from the gut of C3H.SW mice receiving WT or Rorc–/– T cells on day 14 after HCT. n = 3, data are shown as mean ± SEM; unpaired t test, *P < 0.05, **P < 0.01. (I and J) mRNA expression of ST2 and sST2 by sorted Foxp3GFP– effector T cells isolated from the gut of mice in C on day 14 after HCT. n = 3, data are shown as mean ± SEM; unpaired t test, **P < 0.01, ***P < 0.001. (K and L) Representative plots of IFN-γ, IL-17, IL-4, and IL-10 production by Foxp3GFP– effector T cells from the gut of C3H.SW mice receiving WT or Rorc–/– T cells on day 14 after HCT. n = 3, data are shown as mean ± SEM; unpaired t test, *P < 0.05.