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. 2011 Jun;140(7):2031-43.
doi: 10.1053/j.gastro.2011.03.009. Epub 2011 Mar 17.

Interleukin-12 converts Foxp3+ regulatory T cells to interferon-γ-producing Foxp3+ T cells that inhibit colitis

Ting Feng  1 , Anthony T Cao, Casey T Weaver, Charles O Elson, Yingzi Cong
Affiliations

Interleukin-12 converts Foxp3+ regulatory T cells to interferon-γ-producing Foxp3+ T cells that inhibit colitis

Ting Feng et al. Gastroenterology. 2011 Jun.

Abstract

Background & aims: Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into foxhead box p3 (Foxp3+) interferon (IFN)-γ+ T cells and whether these converted cells retain the ability to inhibit colitis are not clear.

Methods: Foxp3+ Treg cells were generated by culture of naïve CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (Tg) (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor-β. Foxp3GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ-/- mice. Colitis was induced by transfer of naïve CBir1-Tg CD4+ T cells into immunodeficient mice.

Results: Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN-γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity because they suppressed proliferation of naïve T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3+IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells.

Conclusions: IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3+IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells.

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Conflict of interest statement

No authors have conflicting financial interests.

Figures

Figure 1
Figure 1. Naïve CBir1-Tg T cells induce colitis and differentiate into various subsets in TCRβxδ−/− mice
TCRβxδ−/− mice were injected intravenously with PBS, or 1 × 106 naïve CD4+ T cells from CBir1-Tg or OT II mice. (A) Colonic histopathology of the recipients 8 weeks after cell transfer. (B,C) Spleen, MLN, and LP CD4+ T cell cytokine was determined by flow cytometry 4 weeks (B) and 8 weeks (C) after cell transfer. Plot numbers represent the percentage of CD4+ T cells in the respective quadrants (Left). Foxp3 negative or positive T cells were overlaid on CD4+ T cells, and the plot numbers represent the percentage of Foxp3 negative or positive T cells in the respective quadrants (Right). Data are representative of three or more experiments with similar results.
Figure 2
Figure 2. IL-12 and TGF-β are required for induction of Foxp3+IFN-γ+ T cells
CBir1-Tg CD4+ T cells were cultured with CBir1-peptide pulsed-splenic APC with various cytokines or antibody in the absence (A) or presence (B) of IL-2. Five days later, T cell Foxp3 and IFN-γ expression was determined by flow cytometry and analyzed by gating on CD4+ population. (A,B) FACS profiles. One representative of at least three experiments was shown. (C) Bar chart represents aggregate data with mean ± SEM of three experiments.
Figure 3
Figure 3. CBir1-specific Foxp3+ Treg cells convert into IFN-γ+ and IL-17+ T cells in vivo
0.5 × 106 Foxp3GFP + Treg cells were transferred into TCRβxδ−/− mice. (A) Four weeks later, cytokine production of transferred Treg cells was analyzed by flow cytometry by gating on CD4+ cells (Left). Foxp3 negative or positive T cells were overlaid on CD4+ T cells, and the plot numbers represent the percentage of Foxp3 negative or positive T cells in the respective quadrants (Right). (B) Colonic histology of the Treg cell or PBS recipients four weeks after transfer. Data are representative of three experiments.
Figure 4
Figure 4. CBir1-specific Treg cell conversion to effector T cells takes place in the intestine
0.5 × 106 Foxp3GFP+ Treg cells were transferred into TCRβxδ−/− mice. Two (A,B) and six weeks (C,D) later, cytokine production of transferred Treg cells was analyzed by flow cytometry by gating on CD4+ population or CD4+ Foxp3 negative population (A,C). (B,D) Bar chart represents aggregate data with mean ± SEM of three experiments (*P < 0.05; **P < 0.01). Data are representative of two (C,D) or three (A,B) experiments.
Figure 5
Figure 5. IL-12 promotes Foxp3+ Treg cell conversion to IFN-γ-expressing T cells
(A-C) Foxp3GFP+ Treg cells were cultured with CBir1 peptide-pulsed splenic APC. Five days later, T cells Foxp3 expression and IFN-γ production were determined by flow cytometry. Plot numbers represent the percentage of CD4+ population in the respective quadrants (A,C). Supernatant was collected on day 3 of cell culture, and IFN-γ production was determined by ELISA (B). (D) 0.5 × 106 Foxp3GFP+ Treg cells were transferred into TCRβxδ−/− mice. The recipients were injected intraperitoneally with 100 µg of control or anti-IL-12p40 antibodies at the time of cell transfer and weekly thereafter. Four weeks later, cytokine production of transferred Treg cells was analyzed by flow cytometry with CD4+ T cells gated. Data represent two (D) or three (A-C) experiments with similar results.
Figure 6
Figure 6. Th1 cells do not convert to Foxp3+ T cells, and Foxp3+IFN-γ+ T cells develop into IFN-γ+ but not Foxp3+ T cells
(A) IFN-γThy1.1+ Th1 cells from IFN-γThy1.1.CBir1-Tg mice were cultured with CBir1 peptide-pulsed splenic APC. Five days later, T cell Foxp3 and IFN-γ expression was determined by flow cytometry by gating on CD4+ population. (B,C) 0.5 × 106 Th1 cells or PBS were transferred into TCRβxδ−/− mice, and the recipients were sacrificed 10 weeks later. Colonic histopathology was assessed (B), and cytokine production of transferred CD4+ Th1 cells was analyzed by flow cytometry with CD4+ T cells gated (C). (D) 0.25 × 106 CBir1-specific Foxp3GFP−IFN-γThy1.1+, Foxp3GFP+IFN-γThy1.1−, and Foxp3GFP+IFN-γThy1.1+ T cells were transferred into RAG−/− mice. Eight weeks later, the recipients were sacrificed and cytokine production of transferred Treg cells was analyzed by flow cytometry. Plot numbers represent the percentage of CD4+ T cells in the respective quadrants. One of three experiments with similar results was shown.
Figure 7
Figure 7. Foxp3+IFN-γ+ T cells retain regulatory functions
(A) 0.25 × 106 CBir1-Tg Foxp3GFP+IFN-γThy1.1− and Foxp3GFP+IFN-γThy1.1+ T cells were transferred into RAG−/− mice. The recipients were sacrificed and colonic histopathology was analyzed 4 weeks late. (B) 0.2 × 106 CFSE-labeled CD4+ T cells from CD45.1 CBir1-Tg mice were cultured alone, or with 0.2 × 106 CD45.2 CBir1-Tg Foxp3GFP+IFN-γThy1.1− or Foxp3GFP+IFN-γThy1.1+ T cells. T cell proliferation was analyzed by flow cytometry based on the dilution of CFSE intensity by gating on CD4+CD45.1+ T cells. (C) 5 × 104 CD4+ T cells from CBir1-Tg mice were cultured alone, or with 5 × 104 Foxp3GFP+IFN-γThy1.1− or Foxp3GFP+IFN-γThy1.1+ T cells. Tritiated thymidine was added for the last 8 h of a 48-h incubation period for T cell proliferation. Mean CPM of triplicates ± SEM are shown. (D,E) 0.25 × 106 CD45RBhi T cells from CBir1-Tg mice were transferred into RAG−/− mice alone or together with 0.25 × 106 Foxp3GFP+IFN-γThy1.1− or Foxp3GFP+IFN-γThy1.1+ T cells. Four weeks later, the recipients were sacrificed and histopathology was assessed. Combined colon and cecum histological scores (D) and colonic histopathology (E) are shown. ***P < 0.001. Data represent two (D,E) or three (A-C) experiments with similar results.
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