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Activin A programs the differentiation of human TFH cells

Nature Immunology volume 17, pages 976–984 (2016)Cite this article

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An Erratum to this article was published on 20 September 2016

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Abstract

Follicular helper T cells (TFH cells) are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals that regulate the differentiation of human TFH cells. A screen of a human protein library identified activin A as a potent regulator of TFH cell differentiation. Activin A orchestrated the expression of multiple genes associated with the TFH program, independently or in concert with additional signals. TFH cell programming by activin A was antagonized by the cytokine IL-2. Activin A's ability to drive TFH cell differentiation in vitro was conserved in non-human primates but not in mice. Finally, activin-A-induced TFH programming was dependent on signaling via SMAD2 and SMAD3 and was blocked by pharmacological inhibitors.

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Figure 1: High-throughput screening identifying activin A as a potent regulator of human TFH cell differentiation.
Figure 2: INHBA is present in sites relevant for TFH cell differentiation and can be produced by myeloid cells.
Figure 3: Activin A acts in synergy with IL-12 and molds the human TFH gene program.
Figure 4: CD4+ T cells differentiated with activin A acquire functional signature molecules of TFH cells.
Figure 5: Activin A and TGF-β act independently from each other in driving in vitro TFH cell differentiation.
Figure 6: IL-2 antagonizes activin-A-driven TFH cell differentiation.
Figure 7: The role of activin A in in vitro TFH cell differentiation is conserved in CD4+ T cells from non-human primates but not in those from mice.
Figure 8: Activin A activity is mediated by an ALK4–SMAD2-SMAD3 pathway.

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Change history

  • 20 July 2016

    In the version of this article initially published, some of the statistical comparisons in Figures 3f, 3l, 3m, 4a, 4d, 4e, 5g, 6b, 6c and 7d were presented incorrectly in the plots. Also, 'in vitro' was not fully in italics in the abstract, and the author list for reference 36 was incorrect. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Crotty, S. T follicular helper cell differentiation, function, and roles in disease. Immunity 41, 529–542 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Victora, G.D. & Nussenzweig, M.C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Craft, J.E. Follicular helper T cells in immunity and systemic autoimmunity. Nat. Rev. Rheumatol. 8, 337–347 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Crotty, S. A brief history of T cell help to B cells. Nat. Rev. Immunol. 15, 185–189 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gitlin, A.D., Shulman, Z. & Nussenzweig, M.C. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vinuesa, C.G. & Cyster, J.G. How T cells earn the follicular rite of passage. Immunity 35, 671–680 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Ueno, H., Banchereau, J. & Vinuesa, C.G. Pathophysiology of T follicular helper cells in humans and mice. Nat. Immunol. 16, 142–152 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Suto, A. et al. Development and characterization of IL-21-producing CD4+ T cells. J. Exp. Med. 205, 1369–1379 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nurieva, R.I. et al. Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages. Immunity 29, 138–149 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eto, D. et al. IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation. PLoS One 6, e17739 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Choi, Y.S., Eto, D., Yang, J.A., Lao, C. & Crotty, S. Cutting edge: STAT1 is required for IL-6-mediated Bcl6 induction for early follicular helper cell differentiation. J. Immunol. 190, 3049–3053 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Schmitt, N. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Ma, C.S. et al. Early commitment of naïve human CD4+ T cells to the T follicular helper (TFH) cell lineage is induced by IL-12. Immunol. Cell Biol. 87, 590–600 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Schmitt, N. et al. Human dendritic cells induce the differentiation of interleukin-21-producing T follicular helper-like cells through interleukin-12. Immunity 31, 158–169 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nakayamada, S. et al. Early Th1 cell differentiation is marked by a Tfh cell-like transition. Immunity 35, 919–931 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gonzalez, R. et al. Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc. Natl. Acad. Sci. USA 107, 3552–3557 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ray, J.P. et al. Transcription factor STAT3 and type I interferons are corepressive insulators for differentiation of follicular helper and T helper 1 cells. Immunity 40, 367–377 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gold, E. & Risbridger, G. Activins and activin antagonists in the prostate and prostate cancer. Mol. Cell. Endocrinol. 359, 107–112 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Muttukrishna, S., Tannetta, D., Groome, N. & Sargent, I. Activin and follistatin in female reproduction. Mol. Cell. Endocrinol. 225, 45–56 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Munz, B. et al. The roles of activins in repair processes of the skin and the brain. Mol. Cell. Endocrinol. 180, 169–177 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Phillips, D.J., de Kretser, D.M. & Hedger, M.P. Activin and related proteins in inflammation: not just interested bystanders. Cytokine Growth Factor Rev. 20, 153–164 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Aleman-Muench, G.R. & Soldevila, G. When versatility matters: activins/inhibins as key regulators of immunity. Immunol. Cell Biol. 90, 137–148 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Dalton, S. Signaling networks in human pluripotent stem cells. Curr. Opin. Cell Biol. 25, 241–246 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Jones, C.P., Gregory, L.G., Causton, B., Campbell, G.A. & Lloyd, C.M. Activin A and TGF-β promote TH9 cell-mediated pulmonary allergic pathology. J. Allergy Clin. Immunol. 129, 1000–10.e3 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Huber, S. et al. Activin a promotes the TGF-beta-induced conversion of CD4+CD25− T cells into Foxp3+ induced regulatory T cells. J. Immunol. 182, 4633–4640 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Ogawa, K., Funaba, M., Chen, Y. & Tsujimoto, M. Activin A functions as a Th2 cytokine in the promotion of the alternative activation of macrophages. J. Immunol. 177, 6787–6794 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Robson, N.C. et al. Activin-A: a novel dendritic cell-derived cytokine that potently attenuates CD40 ligand-specific cytokine and chemokine production. Blood 111, 2733–2743 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Erämaa, M., Hurme, M., Stenman, U.H. & Ritvos, O. Activin A/erythroid differentiation factor is induced during human monocyte activation. J. Exp. Med. 176, 1449–1452 (1992).

    Article  PubMed  Google Scholar 

  29. Ogawa, K., Funaba, M., Mathews, L.S. & Mizutani, T. Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages. J. Immunol. 165, 2997–3003 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Schmitt, N. et al. IL-12 receptor β1 deficiency alters in vivo T follicular helper cell response in humans. Blood 121, 3375–3385 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Abe, Y., Minegishi, T. & Leung, P.C.K. Activin receptor signaling. Growth Factors 22, 105–110 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Tsuchida, K. et al. Activin signaling as an emerging target for therapeutic interventions. Cell Commun. Signal. 7, 15 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Sáez de Guinoa, J., Barrio, L., Mellado, M. & Carrasco, Y.R. CXCL13/CXCR5 signaling enhances BCR-triggered B-cell activation by shaping cell dynamics. Blood 118, 1560–1569 (2011).

    Article  PubMed  CAS  Google Scholar 

  34. Kroenke, M.A. et al. Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation. J. Immunol. 188, 3734–3744 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Chevalier, N. et al. CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses. J. Immunol. 186, 5556–5568 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Locci, M. et al. Human circulating PD-1+CXCR3−CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity 39, 758–769 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Morita, R. et al. Human blood CXCR5+CD4+ T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 34, 108–121 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Moens, L. & Tangye, S.G. Cytokine-mediated regulation of plasma cell generation: IL-21 takes center stage. Front. Immunol. 5, 65 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Travis, M.A. & Sheppard, D. TGF-β activation and function in immunity. Annu. Rev. Immunol. 32, 51–82 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Oestreich, K.J., Mohn, S.E. & Weinmann, A.S. Molecular mechanisms that control the expression and activity of Bcl-6 in TH1 cells to regulate flexibility with a TFH-like gene profile. Nat. Immunol. 13, 405–411 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Johnston, R.J., Choi, Y.S., Diamond, J.A., Yang, J.A. & Crotty, S. STAT5 is a potent negative regulator of TFH cell differentiation. J. Exp. Med. 209, 243–250 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ray, J.P. et al. The interleukin-2-mTORc1 kinase axis defines the signaling, differentiation, and metabolism of T helper 1 and follicular B helper T cells. Immunity 43, 690–702 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Herbertz, S. et al. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-β signaling pathway. Drug Des. Devel. Ther. 9, 4479–4499 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Shen, M.M. et al. Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies. EMBO J. 13, 1375–1385 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tran, D.Q., Ramsey, H. & Shevach, E.M. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-β dependent but does not confer a regulatory phenotype. Blood 110, 2983–2990 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Marshall, H.D. et al. The transforming growth factor beta signaling pathway is critical for the formation of CD4 T follicular helper cells and isotype-switched antibody responses in the lung mucosa. eLife 4, e04851 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  48. León, B., Bradley, J.E., Lund, F.E., Randall, T.D. & Ballesteros-Tato, A. FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability. Nat. Commun. 5, 3495 (2014).

    Article  PubMed  CAS  Google Scholar 

  49. McCarron, M.J. & Marie, J.C. TGF-β prevents T follicular helper cell accumulation and B cell autoreactivity. J. Clin. Invest. 124, 4375–4386 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bentebibel, S.-E. et al. Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination. Sci. Transl. Med. 5, 176ra32 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank the Protein Sciences Group at Genomics Institute of the Novartis Research Foundation for protein production; G. Silvestri (Emory University) for the peripheral blood mononuclear cell samples from non-human primates; the sequencing core at La Jolla Institute for the generation of RNA-seq data; and the National Disease Resource Interchange for tonsil samples. The contents of the secretomics collection of the Genomics Institute of the Novartis Research Foundation are proprietary. Access to the collection may be considered for further research collaboration agreements on a case-by-case basis. Supported by the US National Institutes of Health (UM1-AI100663 to S.C.) and internal funding from the La Jolla Institute (S.C.) and the Genomics Institute of the Novartis Research Foundation (A.M.).

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Authors and Affiliations

  1. Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Michela Locci, Jennifer E Wu, Fortuna Arumemi & Shane Crotty

  2. Microscopy Core Facility, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Zbigniew Mikulski

  3. Genomics Institute of the Novartis Research Foundation, La Jolla, California, USA

    Carol Dahlberg & Andrew T Miller

  4. Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA.,

    Shane Crotty

  5. Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, California, USA

    Shane Crotty

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  1. Michela Locci
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Contributions

M.L. and J.E.W. performed experiments and analyzed data; F.A. performed SMAD-related experiments; Z.M. generated and analyzed immunofluorescence data; C.D. contributed to optimization of the screen; A.T.M. provided the secretomics library; M.L. and S.C. conceived of and designed the experiments and wrote the paper; and S.C. supervised the study.

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Correspondence to Shane Crotty.

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Integrated supplementary information

Supplementary Figure 1 High-throughput screen for novel inducer of TFH cell differentiation.

(a) Schematic of primary screen. Purified human naïve CD4 T cells were stimulated by anti-CD3/CD28 beads on 384 well plates on day 0. The GNF secretomics recombinant proteins were added at the beginning of the stimulation. Each secretomics protein was tested in duplicate. After 5 d of in vitro culture, cells were evaluated by automated flow cytometry analysis for the expression of Tfh signature markers, including CXCR5 and PD-1.

(b) Overall screen workflow.

(c) Enrichment of CXCR5+ cell induction reported as z-score for each recombinant protein on cells in a. Activin A is shown in red.

Supplementary Figure 2 INBHA expression in human tonsils.

(a) Slide scanner image of INHBA (red), CD3 (green) and Bcl6 (blue) in human tonsils. Image is from one donor representative of six. A magnification of INHBA staining and IgG control staining is shown on the right. Scale bars=100μm.

(b) Confocal image of INHBA (red), CD3 (blue) and CD11c (green) in human tonsil. Image is from one donor representative of two. Rabbit polyclonal IgG and mouse IgG Abs were used as controls for INHBA and CD11c staining, respectively. Scale bars=100μm. White boxes are magnified sections depicted in Fig. 2b.

Supplementary Figure 3 In vitro differentiation of naive CD4+ T cells.

(a) Flow cytometry of naïve CD4+ T cells sorted by flow cytometry and activated by anti-CD3/CD28 beads for 5 d with activin A, with or without IL-12, IL-12 and beads only (–).

(b) Frequency of PD-1+CXCR5+ cells on naïve CD4+ T cells cultured in vitro with anti-CD3/CD28 beads and different doses of activin A for 5 d in the presence of anti-activin A or isotype control mAb (isotype). Dotted lines indicate the average percentages of PD-1+CXCR5+ cells induced by beads only with isotype control mAb.

(c) Flow cytometry of naïve CD4+ T cells cultured in vitro with anti-CD3/CD28 beads and different cytokine combinations for 5 d.

(d) Frequency of PD-1+CXCR5+ cells on cells differentiated as in c. Bars are mean and s.e.m.

In (a-c) data are from 3 or more experiments (n=7 or more).

* P < 0.05 and ** P < 0.01 (two-tailed Wilcoxon matched-pairs signed ranked test).

Supplementary Figure 4 Bcl6 induction by in vitro–differentiated cells.

(a-b) Flow cytometry of intranuclear Bcl6 expression on naïve CD4+ T activated by anti-CD3/CD28 beads for 5 d with activin A, with or without IL-12, IL-12 and beads only (–). One representative donor is shown.

Supplementary Figure 5 Expression of LIF and ITGB7 by tonsil GC TFH cells.

Microarray gene expression values from tonsil GC Tfh cells (CD4+CD45RO+PD-1hiCXCR5hi), Tfh cells (CD4+CD45RO+PD-1loCXCR5lo) and non Tfh cells (CD4+CD45RO+PD-1−CXCR5−). Gene expression data on tonsil CD4+ T cell populations was previously published1. * P < 0.05, ** P < 0.01 (Mann Whitney test).

1. Locci, M. et al. Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity 39, 758–769 (2013).

Supplementary Figure 6 TGF-β induction of the expression of PD-1, CXCR5 and Bcl6.

(a) Flow cytometry of naïve CD4+ T activated by anti-CD3/CD28 beads for 5 d with TGF-β, with or without IL-12, IL-12 and beads only (–). One representative donor is shown.

(b) Frequency of Bcl6 induction by cells differentiated in vitro with TGF-β and TGF-β + IL-12 for 5 d. Average Bcl6 induction from activin A and activin A + IL-12 differentiated cells is shown by the red dotted line.

Data in (a-b) are from 3 independent experiments (n=7). ** P < 0.01 (two-tailed Wilcoxon matched-pairs signed ranked test).

Supplementary Figure 7 Regulation of activin-A-driven TFH cell differentiation by IL-2.

(a) Flow cytometry of naïve CD4+ T activated by anti-CD3/CD28 beads for 5 d with activin A, with or without IL-12, IL-12 and beads only (–) in the presence of anti-IL-2 or isotype mAb. One representative donor is shown.

(b-c) Frequency of PD-1+CXCR5+ (b) and CXCR5+ (c) cell induction on cells in a. Data are cumulative of 3 experiments (n=10). ** P < 0.01 (two-tailed Wilcoxon matched-pairs signed ranked test).

Supplementary Figure 8 Activation of a SMAD-independent pathway downstream activin A.

(a-b) Expression of phosphorylated-MAPK (p-38) and (b) phosphorylated-ERK (p-ERK) by naïve CD4+ T cells (CD4+C45RA+) following stimulation with activin A (red), activin A+ SB 431542 (blue) and in unstimulated cells (US, grey).

(c) Frequency of PD-1+CXCR5+ cell induction by cells differentiated in vitro with activin A+ IL-12 with different doses of Galunisertib or vehicle for 5 d.

(d) RNAseq gene expression on tonsil cell populations were previously generated and described by Gallagher and colleagues2. The graphs show the expression of ACVR1B, ACVR2A and ACRV2B by tonsillar naïve CD4+ cells and GC Tfh (PD-1hiCXCR5hi cells) from 3 or more individual donors. The red dotted line indicates average RPKM of the negative control gene NGFR in naïve CD4+ T cells.

Data in (a-c) are combined from 3 experiments (n=6 or more).

* P < 0.05 and ** P < 0.01 (two-tailed Wilcoxon matched-pairs signed ranked test).

2. Weinstein, J. S. et al. Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes. Blood (2014). doi:10.1182/blood-2014-06-582700

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Locci, M., Wu, J., Arumemi, F. et al. Activin A programs the differentiation of human TFH cells. Nat Immunol 17, 976–984 (2016). https://doi.org/10.1038/ni.3494

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