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The cytokine TGF-β co-opts signaling via STAT3-STAT4 to promote the differentiation of human TFH cells

Nature Immunology volume 15, pages 856–865 (2014)Cite this article

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Abstract

Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4+ helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-β (TGF-β) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.

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Figure 1: Either TGF-β plus IL-12 or TGF-β plus IL-23 induces the expression of TFH cell–related molecules by human naive helper T cells.
Figure 2: Either TGF-β plus IL-12 or TGF-β plus IL-23 promoted induction of the TFH cell transcriptional signature.
Figure 3: Helper T cells receive signals from TGF-β and IL-12 in the T cell zone.
Figure 4: The combination of TGF-β and IL-12 or of TGF-β plus IL-23 induces the transcriptional signatures of both TFH cells and TH17 cells.
Figure 5: RORγt is expressed by CXCR5+Bcl-6+ helper T cells generated with TGF-β plus IL-12 or with TGF-β plus IL-23.
Figure 6: STAT3 and STAT4 act together with TGF-β in inducing the transcriptional signatures of TFH cells and TH17 cells.
Figure 7: A small fraction of tonsillar TFH cells express Bcl-6 and RORγt.
Figure 8: TGF-β inhibits the expression of IL-21 and ICOS by mouse helper T cells.

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Acknowledgements

We thank E. Trahan, S. Coquery, N. Loof and K. Kayembe for cell sorting; K. Palucka, V. Pascual and Y.-J. Liu for discussions; S. Clayton for confocal imaging; C. Quinn for development of the heat map–generation software; Y. Kanno for advice about chromatin immunoprecipitation; Y.C. Lin for discussions about chromatin-immunoprecipitation data analysis; and M. Kathania for technical help with mouse experiments. Supported by the US National Institutes of Health (U19-AI057234, U19-AI082715 and U19-AI089987), the Alliance for Lupus Research, the Baylor Health Care System (H.U.) and the American Cancer Society (122713-RSG-12-260-01-LIB to K.V.).

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  1. Jacques Banchereau

    Present address: Present address: The Jackson Laboratory for Genomics Medicine, Farmington, Connecticut, USA.,

Authors and Affiliations

  1. Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, Texas, USA

    Nathalie Schmitt, Yang Liu, Salah-Eddine Bentebibel, Indira Munagala, Laure Bourdery, K Venuprasad, Jacques Banchereau & Hideki Ueno

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Contributions

N.S. conceived of and did experiments and analyzed the data; Y.L. and L.B. stained tonsillar tissue; S.-E.B. contributed to the preparation of tonsillar helper T cells; I.M. did NanoString assays; K.V. contributed to experiments with mouse cells; H.U. oversaw and conceived of the entire project and analyzed the data; and N.S., J.B. and H.U. wrote the manuscript.

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Correspondence to Hideki Ueno.

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Integrated supplementary information

Supplementary Figure 1 TGF-β plus IL-12 and TGF-β plus IL-23 induce the expression of TFH cell–related molecules by human naive helper T cells.

(a) Cell viability and cell recovery of human adult blood naive TH cells following 4 days of stimulation with CD3-CD28 mAbs in the presence of the indicated cytokines during the last 3 days. Recovered viable cell numbers are shown in a cell recovery index determined by this formula: recovered cell numbers in the culture with the indicated cytokines / recovered cell numbers in the culture with no cytokine. Each dot represents a result from 10 sets of 4 d-culture experiments. Black bars show the mean value and the blue boxes show the range of ± 1 s.d. (b) Expression of IL-2, IL-21, and ICOS by activated (CFSE-) human naive CFSE-labeled TH cells cultured with no cytokine, IL-12, and IL-12 + IL-4 as in (a). A representative out of 2 experiments. (c) Expression of IL-2, and IL-21 by activated (FSChiSSChi) human naive TH cells cultured with no cytokine, IL-12, IL-12 + TGF-β, and IL-12 + TGF-β + IL-4 as in (a). A representative out of 2 experiments. (d) Expression of CXCR5 and ICOS on activated human naive TH cells stimulated with the indicated cytokines as in (a). Results are from one representative of 13 sets of 4 d-culture experiments.

Supplementary Figure 2 TGF-β plus IL-12 and TGF-β plus IL-23 promote the induction of the TFH cell transcriptional signature.

(a) Isolation of four subsets from ex vivo human tonsillar TH cells according to the expression of CXCR5 and ICOS (gated to CD4+ T cells). (b) Expression of transcription factors by the four tonsillar TH subsets. Data were obtained from TH subsets isolated from 2-3 tonsil samples. (c) Ranking of the differentiation conditions inducing the high ratio of BCL6 to PRDM1 by human adult blood naive TH cells cultured with various cytokines (CK) as in Fig. 2a. The data were normalized to the values in the culture with no cytokines in each experiment. Each dot represents a result from 11 sets of 4 days culture experiments. Black bars show the mean value and blue boxes indicate ± 1 s.d. (d) Ranking of the differentiation conditions inducing the expression of BCL6, BATF, JUN, JUNB, the high ratio of BCL6 to PRDM1, and suppressing the expression of PRDM1 by human cord blood naive TH cells cultured with various cytokines (CK) as in Fig. 2a. Each dot represents a result from 4 sets of 4 days culture experiments.

Supplementary Figure 3 TGF-β plus IL-12 and TGF-β plus IL-23 promote induction of the TFH cell transcriptional signature.

(a) Ranking of the differentiation conditions inducing the expression of MAF and BATF by human adult blood naive TH cells cultured with various cytokines (CK) as in Fig. 1a. Each dot represents a result from 11 sets of 4 days culture experiments. (b) Correlation between BCL6 and MAF, BATF, and PRDM1 expressed by human cord blood naive TH cells cultured as in (a). Pearson R values are indicated. P values were all <0.0001. (c) Two predicted BATF binding sites in the BCL6 gene are shown in the UCSC Genome Browser view. Primer sets for the binding sites #1 and #2 are indicated in the table. (d) Expression of CXCR5, ICOS, IL-21, and Bcl-6 by activated (FSChiSSChi) adult blood naive TH cells stimulated for 4 days with allogeneic CD40L-stimulated DCs in the presence of the indicated amounts of TGF-β. A representative result from 3 independent experiments. Results from 3 independent experiments are shown in panel (e).

Supplementary Figure 4 Expression of BCL6 in cultured cord blood naive helper T cells positively correlates with the expression of AHR, RORA, and RORC.

Correlation between BCL6 and the indicated transcription factors expressed by human cord blood naive TH cells cultured for 3 days with different cytokines as in Fig. 2a. Pearson R values and p values are indicated.

Supplementary Figure 5 RORγt is expressed by CXCR5+Bcl-6+ TH cells generated with TGF-β plus IL-12 or with TGF-β plus IL-23.

(a) The expression of Bcl-6, RORγt, and T-bet by activated (FSChiSSChi) human adult blood naive TH cells cultured with the indicated cytokines for 3 days as in Fig. 1a. A representative of 4 independent experiments. (b) The expression of Bcl-6, RORγt, and T-bet by CXCR5+ TH cells generated by culturing naive TH cells with the indicated cytokines for 3 days. A representative of 4 independent experiments. (c) The expression of Bcl-6 and RORγt by CXCR5+ human adult blood naive TH cells cultured with the indicated cytokines for 3 days as in Fig. 1a. A representative of 3 independent experiments. (d) The expression of Bcl-6, RORγt, and T-bet by activated (FSChiSSChi) human adult blood naive TH cells transfected with either scrambled or RORγt siRNA followed by 2 days of culture with no cytokines or IL-23 + IL-1β + IL-6 + TGF-β. A representative of 3 independent experiments. (e) The expression of IL-17A and IL-21 by activated (FSChiSSChi) cord blood naive TH cells cultured for 3 days with no cytokines or IL-23 + IL-1β + IL-6 + TGF-β. A representative of 3 independent experiments. The frequency of IL-21+ cells among IL-17A+ cells was 38 ± 2% (Mean ± s.d., n=3).

Supplementary Figure 6 A small fraction of GC TFH cells express T-bet but not GATA-3.

(a) Expression of transcription factors by the four tonsillar TH subsets (Mean ± s.e.m, n=3). (b) Human tonsil sections were stained for IgD (indicated by green), CD4 (indicated by blue), and Bcl-6 (indicated by red), and analyzed by confocal microscopy. Scale bar, 100 μm. Representative Bcl-6+ TH cells in GCs are shown in panel (c). Scale bar, 10 μm. (d) The expression of Bcl-6, T-bet, and Gata3 by ex vivo tonsillar CXCR5-ICOS- naive TH cells and CXCR5hiICOShi GC TFH cells. A representative of 4 independent experiments.

Supplementary Figure 7 TGF-β inhibits the expression of IL-21 and ICOS by mouse helper T cells.

(a) Cell viability and cell recovery of mouse naive TH cells following 4 days of stimulation with CD3-CD28 mAbs in the presence of the indicated cytokines during the last 3 days. Recovered viable cell numbers are shown in a cell recovery index determined by this formula: recovered cell numbers in the culture with the indicated cytokines / recovered cell numbers in the culture with no cytokine. Each dot represents a result from 5 sets of 4 d-culture experiments. Black bars show the mean value and the blue boxes show the range of ± 1 s.d. (b) The expression of IL-17, IL-21, CXCR5, and ICOS by activated (FSChiSSChi) mouse naive TH cells cultured for 3 days with the indicated amounts of TGF-β plus IL-12 + IL-6 + IL-21 or plus IL-23 + IL-6 + IL-21 as in (a). A representative of 4 independent experiments. Results from 5 sets of experiments are shown in panel (c). (d) CXCR5 expression by B cells in Spleen and lymph nodes cells analyzed with the anti-mouse CXCR5 mAb used for the phenotyping of mouse TH cells.

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Schmitt, N., Liu, Y., Bentebibel, SE. et al. The cytokine TGF-β co-opts signaling via STAT3-STAT4 to promote the differentiation of human TFH cells. Nat Immunol 15, 856–865 (2014). https://doi.org/10.1038/ni.2947

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