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. 2007 Nov 13;104(46):18169-74.
doi: 10.1073/pnas.0703642104. Epub 2007 Oct 31.

Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells

Jun Wei  1 , Omar Duramad, Olivia A Perng, Steven L Reiner, Yong-Jun Liu, F Xiao-Feng Qin
Affiliations

Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells

Jun Wei et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies have highlighted the importance of peripheral induction of Foxp3-expressing regulatory T cells (Tregs) in the dominant control of immunological tolerance. However, Foxp3(+) Treg differentiation from naïve CD4(+) T cells occurs only under selective conditions, whereas the classical T helper (Th) 1 and 2 effector development often dominate T cell immune responses to antigen stimulation in the periphery. The reason for such disparity remains poorly understood. Here we report that Th1/Th2-polarizing cytokines can potently inhibit Foxp3(+) Treg differentiation from naïve CD4(+) precursors induced by TGF-beta. Furthermore, antigen receptor-primed CD4(+) T cells are resistant to Treg induction because of autocrine production of IFNgamma and/or IL-4, whereas neutralizing IFNgamma and IL-4 not only can potentiate TGF-beta-mediated Foxp3 induction in vitro but can also enhance antigen-specific Foxp3(+) Treg differentiation in vivo. Mechanistically, inhibition of Foxp3(+) Treg development by Th1/Th2-polarizing cytokines involves the activation of Th1/Th2 lineage transcription factors T-bet and GATA-3 through the canonical Stat1-, Stat4-, and Stat6-dependent pathways. Using IFNgamma and IL-4 knockouts and retrovirus-mediated transduction of T-bet and GATA-3, we further demonstrate that enforced expression of the Th1/Th2 lineage-specific transcription factors is sufficient to block Foxp3 induction and Treg differentiation independent of the polarizing/effector cytokines. Thus, our study has unraveled a previously unrecognized mechanism of negative cross-regulation of Foxp3(+) Treg fate choice by Th1/Th2 lineage activities. In addition, these findings also provide an attainable explanation for the general paucity of antigen-triggered de novo generation of Foxp3(+) Tregs in the periphery.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inhibition of Foxp3+ Treg differentiation by IL-12, IFNγ, and IL-4. Naïve CD4+ T cells from WT mice were stimulated by plate-bound anti-CD3 and soluble anti-CD28 (A) or by soluble anti-CD3 and spleen APCs (B) in the presence of 1 ng/ml TGF-β1. Naïve CD4+ T cells from OT-II TCR-transgenic mice were stimulated by OVA323–339 peptide and spleen APCs (C) or by OVA323–339 peptide and bone marrow-derived dendritic cells (D) in the presence of 1 ng/ml TGF-β1. Recombinant IL-12 (10 ng/ml), IFNγ (100 ng/ml), or IL-4 (10 ng/ml) was added to the cultures as indicated. Representative FACS plots of anti-Foxp3 and anti-CD25 double staining (Left) and histograms of anti-Foxp3 single stain (Right) are shown. (E) The cells from A were analyzed by real-time PCR analysis for Foxp3 mRNA expression, and relative expression levels are plotted. (F) Suppressive activities of induced Tregs from A were measured by CFSE-based suppression assay with percentage numbers showing target cells divided more than once (average of two independent experiments). (G) Naïve CD4+ cells from WT mice were stimulated by anti-CD3/soluble anti-CD28 in the presence of TGF-β1 (≈0.1–10 ng/ml) with or without neutralizing mAbs (10 μg/ml) to IFNγ and IL-4. Histograms of Foxp3 expression are shown.
Fig. 2.
Fig. 2.
Neutralizing IFNγ/IL-4 enhances de novo induction of Foxp3+ Tregs in vivo. Rag2−/− DO11.10 CD4+ T cells were adoptively transferred to BALB/c mice, and the recipients were immunized with four conditions: no peptide; 25 μg of OVA323–339 peptide; 25 μg of OVA323–339 peptide plus control Abs; and 25 μg of OVA323–339 peptide plus anti-IFNγ/IL-4-neutralizing Abs. Nine days after immunization the spleen (A–C) and peripheral lymph node (D–F) samples were analyzed for Foxp3 and CD25 expression in the KJ1-26+CD4+ donor T cells. (A and D) Representative FACS plots and histograms showing coexpression of Foxp3 and CD25 and the percentage of Foxp3-expressing KJ1-26+CD4+ donor cells. (B and E) Statistical representation of Foxp3 induction in the donor cells. (C and F) Enumeration of the absolute number of Foxp3+ KJ1-26+CD4+ cells. Each bar represents the mean value from three individual mice per group. *, P > 0.1; **, P < 0.05.
Fig. 3.
Fig. 3.
IFNγ/IL-4 inhibits Foxp3 induction via the canonical signaling pathways required for Th1/Th2 lineage differentiation. Naïve CD4+ T cells from WT mice, Stat1−/−, and T-bet−/− were stimulated by soluble anti-CD3/spleen APC (A) or plate-bound anti-CD3/soluble anti-CD28 (B) in the presence of 1 ng/ml TGF-β1. IFNγ (100 ng/ml) was added to the cultures as indicated. (C) Naïve T cells from WT and Stat6−/− mice were stimulated by plate-bound anti-CD3/soluble anti-CD28 in the presence of 1 ng/ml TGF-β1. IL-4 (10 ng/ml) was added to the cultures as indicated. Representative FACS plots of Foxp3 staining are shown with the percentage numbers averaged from two independent experiments.
Fig. 4.
Fig. 4.
Negative cross-regulation of Foxp3+ Treg development by Th1/Th2 effector cytokines and the lineage-specific transcription factors. (A) Naïve CD4+ cells from WT mice were stimulated in primary cultures (1° stimulation) by anti-CD3/anti-CD28 under different priming conditions: 1, nonpolarizing, no neutralizing mAbs or Th1/Th2-polarizing cytokines; 2, Th1-polarizing plus 10 ng/ml IL-12; 3, Th2-polarizing plus 10 ng/ml IL-4; 4, neutral plus neutralizing mAbs (10 μg/ml) to IFNγ and IL-4. The cells were harvested on day 7 and restimulated by plate-bound anti-CD3 and TGF-β1 in secondary cultures (2° stimulation) with or without neutralizing mAbs (10 μg/ml) to IFNγ and/or IL-4 to induce Foxp3+ Treg. A rat IgG isotype control was included (control Ab). Representative FACS plots of Foxp3 staining are shown (average of two independent experiments). (B) The cells from A were restimulated in the presence of anti-IFNγ/IL-4 to induce Foxp3+ Treg differentiation. Suppressive activities of the induced Tregs were determined by CFSE-based suppression assay (average of two independent experiments). Naïve CD4+ cells from WT (C), IFNγ−/− (D), or IL-4−/− (E) mice were activated under the neutral condition and transduced with an EGFP bicistronic retroviral vector encoding T-bet or GATA-3. Empty vector was included as control (Vector ctrl). The transduced cells (GFP+) were isolated by cell sorting for subsequent experiments. (Top and Middle) Intracellular cytokine staining for IFNγ and IL-4 production. (Bottom) Foxp3 expression induced by anti-CD3 plus TGF-β1 stimulation in secondary culture (average of two independent experiments). (F) The cells from C–E were subjected to real-time PCR analysis for Foxp3 mRNA expression, and relative expression levels are plotted. (G) WT CD4+ T cells were transduced with the mutant variants of T-bet and GATA-3, and Foxp3 induction was as in C. Histograms of Foxp3 expression are shown. T-bet ΔDBD, deletion of the T-box DNA binding domain; T-bet ΔAD, deletion of the C-terminal transcription activation domain; GATA-3 ΔNF, deletion of the N-terminal zinc finger DNA binding domain; GATA-3 ΔND, deletion of the N-terminal activation domain.
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