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. 2009 Mar 24;106(12):4793-8.
doi: 10.1073/pnas.0900408106. Epub 2009 Mar 9.

Identification of IL-17-producing FOXP3+ regulatory T cells in humans

Kui Shin Voo  1 , Yui-Hsi Wang, Fabio R Santori, Cesar Boggiano, Yi-Hong Wang, Kazuhiko Arima, Laura Bover, Shino Hanabuchi, Jahan Khalili, Ekaterina Marinova, Biao Zheng, Dan R Littman, Yong-Jun Liu
Affiliations

Identification of IL-17-producing FOXP3+ regulatory T cells in humans

Kui Shin Voo et al. Proc Natl Acad Sci U S A. .

Abstract

IL-17-producing CD4(+) T helper (Th17) cells have recently been defined as a unique subset of proinflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-beta, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORgammat. The maintenance, expansion, and further differentiation of the committed Th17 cells depend on IL-1beta and IL-23. IL-17 was originally found produced by circulating human CD45RO(+) memory T cells. A recent study found that human Th17 memory cells selectively express high levels of CCR6. In this study, we report that human peripheral blood and lymphoid tissue contain a significant number of CD4(+)FOXP3(+) T cells that express CCR6 and have the capacity to produce IL-17 upon activation. These cells coexpress FOXP3 and RORgammat transcription factors. The CD4(+)FOXP3(+)CCR6(+) IL-17-producing cells strongly inhibit the proliferation of CD4(+) responder T cells. CD4(+)CD25(high)-derived T-cell clones express FOXP3, RORgammat, and IL-17 and maintain their suppressive function via a cell-cell contact mechanism. We further show that human CD4(+)FOXP3(+)CCR6(-) regulatory T (Treg) cells differentiate into IL-17 producer cells upon T-cell receptor stimulation in the presence of IL-1beta, IL-2, IL-21, IL-23, and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17(+)FOXP3(+) Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in antimicrobial defense, while controlling autoimmunity and inflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of T-cell subsets that secrete IL-17 and express key factors required for Th17 cell differentiation. (A) ELISA of cytokines in supernatants of T-cell subsets—CD4+CD25lowCD45RA+ (naive), CD4+CD25lowCD45RA− CRTH2− (memory), CD4+CD25high (Treg), and CD4+CRTH2+ (CRTH2)—sorted from PBMCs and stimulated with PMA/ionomycin for 24 h. The purity of each T-cell subset was >95%. (B) Real-time RT-PCR of RORγt, FOXP3, T-bet, and GATA-3 transcripts. Expression level was normalized to GADPH expression level and adjusted to corresponding expression levels in CD4+CD25−CD45RA+ naive T cells. Data are from 5–6 experiments taken from 5–6 different healthy donors. Horizontal bars indicate the median.
Fig. 2.
Fig. 2.
Human tonsil contains a high percentage of FOXP3+IL-17+ CD4+ Treg cells. (A) Intracellular staining for FOXP3 and IL-17A proteins in PMA/ionomycin-stimulated CD4+ T cells isolated from PBL and tonsils. (B) Percentage of IL-17+FOXP3+ T cells among total CD4+FOXP3+ T cells. Arrowhead indicates double-positive for ROR gamma t and FOXP3 expression. A star indicates statistical significance (P < 0.05). (C and D) Immunohistochemistry of FOXP3 and RORγt proteins in human tonsil frozen sections. Horizontal bars indicate the median. (E) IL-17 producer cells are found in the CCR6+CD4+CD25high T-cell fraction. (F) Only CCR6+CD4+CD25high cells secrete abundant IL-17 when stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL). Representative experiments of 2 donors are shown. (G) CCR6+CD4+CD25high T cells suppress CD4+ responder T-cell proliferation. Data are representative of experiments with cells from 2 donors.
Fig. 3.
Fig. 3.
Characterization of PBL-derived FOXP3+IL-17+CD4+ T-cell clones. (A) Intracellular staining for FOXP3 and IL-17A proteins in representative CD4+ T-cell clones derived from CD4+CD25high (FOXP3+IL-17+ and FOXP3+IL-17− lines) or CD4+CD25low T cells (FOXP3−IL-17+ and FOXP3−IL-17− lines). (B) Immunofluorescence microscopy of CD4+ T-cell clones fixed and stained with antibodies specific for human FOXP3 (green) and RORγt (red). DAPI (blue) was used to counterstain the nuclei. (Original magnification: ×400.) (C and D) Western blot analysis for FOXP3 and RORγt expression in T-cell clones as in A. Freshly sorted CD4+CD25lowCD45RA+ naive and CD4+CD24high T cells serve as a negative or positive control for FOXP3, respectively. IL-17− T cells serve as a negative control for RORγt. The β-actin protein serves as a protein loading control. (E) FOXP3+CD4+ T-cell clones suppressed proliferation of conventional CD4+CD25low responder T cells. Carboxyfluorescein succinimidyl ester-labeled CD4+CD25lowCD127+ responder cells were cultured with Treg cells at a ratio of Treg/responder cells (1:2). (F) T-cell suppression requires cell-cell contact. Equal numbers of Treg cells and CD4+ responder cells were used in the transwell experiments. The Treg cells were either cultured together with the responder cells as a positive control for suppression activity or cultured in inner wells. Results represent 1 of 2 independent experiments.
Fig. 4.
Fig. 4.
Th17 cells are absent in thymus but can be derived from CCR6−CD4+ CD25high Treg cells by culturing with IL-1β. (A) T-cell subsets from thymus do not secrete IL-17 upon stimulation with PMA/ionomycin for 24 h. Data are from 3 experiments with 3 donors. (B) IL-1β promotes the differentiation of Th17 cells from CCR6−CD4+CD25high Treg cells. CCR6−CD4+CD25high Treg cells were activated with plate-bound anti-CD3 plus soluble anti-CD28 and then cultured for 15–18 days in the presence of indicated cytokines. Cells were then reactivated with anti-CD3 and anti-CD28 in the absence of cytokine for ELISA assays. Error bars represent SD. (C) IL-2 alone or in combination with IL-6, IL-21, or IL-23 further enhances expansion of Th17 cells from CCR6+CD4+CD25high Treg cells. Data are representative of 3 experiments with different donors.
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References

    1. Fossiez F, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med. 1996;183:2593–2603. - PMC - PubMed
    1. Yao Z, et al. Human IL-17: A novel cytokine derived from T cells. J Immunol. 1995;155:5483–5486. - PubMed
    1. Kawaguchi M, et al. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma. J Immunol. 2001;167:4430–4435. - PubMed
    1. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. - PubMed
    1. Linden A, Laan M, Anderson GP. Neutrophils, interleukin-17A and lung disease. Eur Respir J. 2005;25:159–172. - PubMed

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