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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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
Crotty, S. T follicular helper cell differentiation, function, and roles in disease. Immunity 41, 529–542 (2014).
Victora, G.D. & Nussenzweig, M.C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).
Craft, J.E. Follicular helper T cells in immunity and systemic autoimmunity. Nat. Rev. Rheumatol. 8, 337–347 (2012).
Crotty, S. A brief history of T cell help to B cells. Nat. Rev. Immunol. 15, 185–189 (2015).
Gitlin, A.D., Shulman, Z. & Nussenzweig, M.C. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640 (2014).
Vinuesa, C.G. & Cyster, J.G. How T cells earn the follicular rite of passage. Immunity 35, 671–680 (2011).
Ueno, H., Banchereau, J. & Vinuesa, C.G. Pathophysiology of T follicular helper cells in humans and mice. Nat. Immunol. 16, 142–152 (2015).
Suto, A. et al. Development and characterization of IL-21-producing CD4+ T cells. J. Exp. Med. 205, 1369–1379 (2008).
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).
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).
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).
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).
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).
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).
Nakayamada, S. et al. Early Th1 cell differentiation is marked by a Tfh cell-like transition. Immunity 35, 919–931 (2011).
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).
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).
Gold, E. & Risbridger, G. Activins and activin antagonists in the prostate and prostate cancer. Mol. Cell. Endocrinol. 359, 107–112 (2012).
Muttukrishna, S., Tannetta, D., Groome, N. & Sargent, I. Activin and follistatin in female reproduction. Mol. Cell. Endocrinol. 225, 45–56 (2004).
Munz, B. et al. The roles of activins in repair processes of the skin and the brain. Mol. Cell. Endocrinol. 180, 169–177 (2001).
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).
Aleman-Muench, G.R. & Soldevila, G. When versatility matters: activins/inhibins as key regulators of immunity. Immunol. Cell Biol. 90, 137–148 (2012).
Dalton, S. Signaling networks in human pluripotent stem cells. Curr. Opin. Cell Biol. 25, 241–246 (2013).
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).
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).
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).
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).
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).
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).
Schmitt, N. et al. IL-12 receptor β1 deficiency alters in vivo T follicular helper cell response in humans. Blood 121, 3375–3385 (2013).
Abe, Y., Minegishi, T. & Leung, P.C.K. Activin receptor signaling. Growth Factors 22, 105–110 (2004).
Tsuchida, K. et al. Activin signaling as an emerging target for therapeutic interventions. Cell Commun. Signal. 7, 15 (2009).
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).
Kroenke, M.A. et al. Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation. J. Immunol. 188, 3734–3744 (2012).
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).
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).
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).
Moens, L. & Tangye, S.G. Cytokine-mediated regulation of plasma cell generation: IL-21 takes center stage. Front. Immunol. 5, 65 (2014).
Travis, M.A. & Sheppard, D. TGF-β activation and function in immunity. Annu. Rev. Immunol. 32, 51–82 (2013).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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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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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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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DOI: https://doi.org/10.1038/ni.3494
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