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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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.).
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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.
(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).
(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.
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).
(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).
(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).
(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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