Abstract

Different types of effector and memory T lymphocytes are induced and maintained in protective or pathological immune responses. Here we characterized two human CD4+ TH17 helper cell subsets that, in the recently activated state, could be distinguished on the basis of their expression of the anti-inflammatory cytokine IL-10. IL-10+ TH17 cells upregulated a variety of genes encoding immunoregulatory molecules, as well as genes whose expression is characteristic of tissue-resident T cells. In contrast, IL-10 TH17 cells maintained a pro-inflammatory gene-expression profile and upregulated the expression of homing receptors that guide recirculation from tissues to blood. Expression of the transcription factor c-MAF was selectively upregulated in IL-10+ TH17 cells, and it was bound to a large set of enhancer-like regions and modulated the immunoregulatory and tissue-residency program. Our results identify c-MAF as a relevant factor that drives two highly divergent post-activation fates of human TH17 cells and provide a framework with which to investigate the role of these cells in physiology and immunopathology.

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Change history

  • 17 November 2018

    In the version of this article initially published, in the legend to Fig. 1b, the description of the frequency of TH17-IL-10+ clones was incomplete for the first group; this should read as follows: “...13 experiments with clones isolated from CCR6+CCR4+CXCR3 T cells...”. Also, the label along the vertical axis of the bottom right plot in Figure 5b was incomplete; the correct label is ‘IFN-γ+ cells (%)’. Finally, in the first sentence of the final paragraph of the final Results subsection, the description of the regions analyzed was incorrect; that sentence should begin: “DNA motif–enrichment analysis of the subset-specific H3K27ac-positive regions...”. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank S. Monticelli (Institute for Research in Biomedicine, Bellinzona) for reagents and for critical reading of the manuscript; and I. Barozzi for discussion of ChIP-seq analyses. Supported by the European Research Council (grant 323183 PREDICT to F.S.), the Swiss National Science Foundation (grants 149475 and 170213 to F.S.), the US National Institutes of Health (P01 grant NS076410 to H.L.W.) and the Helmut Horten Foundation (F.S. and the Institute for Research in Biomedicine).

Author information

Author notes

    • Dominik Aschenbrenner

    Present address: Translational Gastroenterology Unit, NDM Experimental Medicine, University of Oxford, Oxford, UK

  1. These authors contributed equally: Samuele Notarbartolo and Federica Sallusto.

Affiliations

  1. Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana, Bellinzona, Switzerland

    • Dominik Aschenbrenner
    • , Mathilde Foglierini
    • , David Jarrossay
    • , Antonio Lanzavecchia
    • , Samuele Notarbartolo
    •  & Federica Sallusto
  2. Swiss Institute of Bioinformatics, Lausanne, Switzerland

    • Mathilde Foglierini
  3. Ann Romney Center for Neurologic Diseases and Evergrande Center for Immunologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    • Dan Hu
    • , Howard L. Weiner
    •  & Vijay K. Kuchroo
  4. Institute of Microbiology, ETH Zurich, Zurich, Switzerland

    • Federica Sallusto

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Contributions

D.A. designed and performed experiments, analyzed data and edited the manuscript; M.F. conducted computational data analysis; D.J. performed cell sorting and edited the manuscript; D.H. performed experiments and edited the manuscript; H.L.W., V.K.K. and A.L. provided discussions and edited the manuscript; S.N. designed, performed and supervised experiments, analyzed data and wrote the manuscript; F.S. designed and supervised the study, analyzed data and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Samuele Notarbartolo or Federica Sallusto.

Integrated supplementary information

  1. Supplementary Figure 1 Sorting strategy and cytokine production by subsets of human memory TH17 cells.

    a. Sorting of human memory TH17 cells from PBMCs based on chemokine receptor expression or cytokine secretion. MACS-enriched CD4+ T cells were stained with antibodies to CD45RA, CD25, CCR7, CCR6, CCR4 and CXCR3. CD4+ memory TH17 cells were sorted, after exclusion of CD45RA+CCR7+ naïve T cells (TN) and CD8+ CD14+ CD19+ CD56+ CD25+ cells, as CCR6+CCR4+CXCR3 or, alternatively, as CCR6+CXCR3IL-17A+ (a, lower panels). Dot plots from one donor, representative of more than 10 donors analyzed. b. Cytokine production as assessed by intracellular staining in day 0 (resting) and day 5 (activated) TH17-IL10+ and TH17-IL-10 clone pools after 5 h stimulation with PMA plus ionomycin. Data are represented as mean + 95% c.i., with each dot indicating a T cell clone pool from independent experiments (n ≥ 9) (note: the number of clone pools is paired and the same in TH17-IL-10+ and TH17-IL-10, but can vary for different cytokines (IL-10 and IFN-γ, n = 15; IL-22, n = 13; IL-4, n = 10; GM-CSF, n = 9). *P < 0.05; **P < 0.01; ****P < 0.0001, as determined by ratio paired t test.

  2. Supplementary Figure 2 Gene ontology (GO) analyses of differentially expressed genes and lncRNAs in TH17-IL-10+ and TH17-IL-10 cells.

    Gene ontology (GO) analyses on the biological processes associated to the differentially expressed protein-coding genes (a) or lncRNAs (b) in day 0 (resting) and day 5 (activated) TH17-IL-10+ and TH17-IL-10 cells. GO terms are clustered by semantic similarity and a synthetic description is shown. P-value as determined by MetaCore based on hypergeometric distribution; P-value threshold (vertical line) set at 1 × 10–5.

  3. Supplementary Figure 3 Differential surface marker expression in TH17-IL-10+ and TH17-IL-10 cells.

    a,b. Expression of CTLA4, PD-1, CD25, CD69, CXCR6, CCR7 and FOXP3 as assessed by flow cytometry in day 5 (activated) TH17-IL-10+ and TH17-IL-10 clone pools. Shown are representative histogram plots (a) and cumulative data of clone pools from independent experiments (mean + 95% c.i.; CTLA-4 and PD-1, n = 10; CD25, CXCR6 and CCR7, n = 9; CD69 and FOXP3, n = 7) (b). *P< 0.05; **P< 0.01; ***P < 0.001; ****P < 0.0001, as determined by ratio paired t test. c. Expansion of TH17-IL-10+ and TH17-IL-10 cells in response to stimulation with CD3/CD28 antibodies. TH17-IL-10+ and TH17-IL-10 cells were stimulated with plate-bound CD3/CD28 antibodies for 48 h and their proliferation was measured at the indicated time points. Shown is the average of two independent experiments (mean ± s.e.m.).

  4. Supplementary Figure 4 Gene set enrichment analysis (GSEA) of TH17-IL-10+ and TH17-IL-10 transcriptional signatures in autoimmune diseases.

    Enrichment of TH17-IL-10+ and TH17-IL-10-associated gene signatures in publicly available transcriptional datasets of TH17-mediated autoimmune diseases. Gene set enrichment in ileal biopsies from healthy donors (HD) vs. Crohn’s patients (CD) (a) and in PBMCs and synovial fluid mononuclear cells (SFMCs) from healthy donors (HD) vs. juvenile rheumatoid arthritis patients (RA) (b) are shown. GSEA enrichment results were reported as normalized enrichment score and familywise enrichment (FWER) P value. FWER P values smaller than 0.05 (dashed line) were considered significant. c. c-MAF expression in ileal biopsies from healthy donors (HD), ulcerative colitis (UC) and Crohn’s disease (CD) patients, with histologically graded disease severity, is shown in a box (interquartiles, with a line indicating the median value) and whiskers (min to max values) plot. P-value as determined by Kruskal-Wallis test. Micro. Infl. = microscopic inflammation; Macro. Infl. = macroscopic inflammation.

  5. Supplementary Figure 5 c-MAF binds enhancer-like regions in proximity to genes involved in the immune response.

    a. Position weight matrix (PWM) of the top ranked DNA motives identified by the MEME suite software in the day 5 (activated) TH17-IL-10+ c-MAF ChIP-seq dataset; e-values: MAF (DREME), 5.5 × 10–212; MAF (MEME), 6.6 × 10–175; AP-1, 1.7 × 10–146; NFAT, 3.9 × 10–145; RUNX, 2.6 × 10–81; ETS, 1.1 × 10–57; TCF, 1.1 × 10–20. b. Gene Ontology (GO) analysis of genes associated to c-MAF-bound regions in day 5 (activated) TH17-IL-10+ cells. GO biological processes are ranked (top to bottom) according to their binomial P value. c. Graphical representation of a previously characterized (MARE_2) and a novel (IL10_MAF) c-MAF binding sites at the IL10 locus by the Integrative Genome Viewer (IGV). d. The 1 kb genomic region centered on the novel putative IL10 enhancer (IL10_MAF) was aligned to an isometric genomic region centered on a c-MAF peak localized about 9 kb upstream of Il10 in mouse TH17 cells1. The core enhancer region shown is highly conserved between human and mouse (83% identities) and includes a canonical c-MAF binding site (highlighted in yellow). e. The activation status of the newly identified putative IL10 enhancer was evaluated by quantifying H3K27ac and H3K27me3 levels in day 5 (activated) TH17-IL-10+ and TH17-IL-10 cells by ChIP-qPCR (mean + s.e.m.; n = 2). 1Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289-303 (2012).

  6. Supplementary Figure 6 Examples of c-MAF binding to immunoregulatory and tissue-residency genes loci.

    a-g. Graphical representation of c-MAF binding profiles at the locus of the indicated genes using the Integrative Genome Viewer (IGV).

  7. Supplementary Figure 7 c-MAF is partially sufficient to induce IL-10 expression and to repress proinflammatory gene transcription in TH17-IL-10 cells.

    a. c-MAF isoform a (upper panels) and isoform b (lower panels) ectopic expression in day 5 (activated) TH17-IL-10 cells as measured by intracellular staining; representative plots (left panels) and combined results (right panels) are displayed. b. IL-10 production secondary to c-MAF ectopic expression in the same cells as determined by intracellular staining. Data are represented as mean + 95% c.i. (isoform a: n = 5, isoform b: n = 7). c. Expression of c-MAF and IL-10 in TH17-IL10 cells upon ectopic expression of c-MAF isoform b. Control (empty vector) cells and c-MAF-b (c-MAF isoform b) expressing TH17-IL10 cells were polyclonally stimulated in the presence or absence of IL-27 (25 ng/ml) and c-MAF and IL-10 expression was measured in day 5 (activated) cells by flow cytometry, after 5 h stimulation with PMA + ionomycin (mean + s.e.m.; n = 3). d. Expression of c-MAF-dependent genes in TH17-IL10 cells upon ectopic expression of c-MAF isoform a (left panel, n = 4) or b (right panel, n = 6), as measured by qPCR. Black bars indicate TH17-IL10+-associated genes, grey bars indicate TH17-IL10-associated genes. Shown is the average log2 fold change over empty vector (mean + s.e.m). n.a., not assessed. e,f. Representative dot plots (e) and cumulative data of expression of IFN-γ and IL-22 (f) as assessed by intracellular staining of Day 5-activated TH17-IL10 cells following ectopic expression of c-MAF isoform b or control empty vector (eV). Data are expressed as percentage over empty vector and represent the mean + s.e.m. (n = 7), with each dot indicating a T cell clone pool from independent experiments. **P < 0.01; ***P < 0.001, as determined by ratio paired t test (a-c) and paired t test (f).

  8. Supplementary Figure 8 Potential cooperative and antagonistic binding of transcription factors in TH17-IL-10+ and TH17-IL-10 cells.

    a. c-MAF peaks from day 5 (activated) TH17-IL-10+ cells were ranked according to the fold enrichment over the input, corrected for P-value, and clustered in bins of 1000 peaks (cluster 7 is made of 778 peaks). c-MAF peaks-associated nucleosome-free regions (NFRs) in the same cells were identified from H3K27ac ChIP-seq data and their size was compared to the corresponding NFRs in day 5 (activated) TH17-IL-10 cells. If a NFR was not detected, the size was reported as 0. Each dot in (a) represents a NFR and the white lines indicate median and interquartile ranges. P-values are obtained by Wilcoxon matched-pairs signed rank test. b. Number of total H3K27ac peaks (left panel) identified in day 0 and day 5 TH17-IL-10+ and TH17-IL-10 cells, as assessed by ChIP-seq (MACS P-value ≤ 10 × 10–10, FDR ≤ 5% and fold enrichment ≥ 5). Subset-specific H3K27ac peaks (right panel) were identified, among the total ones, as H3K27ac peaks that were specifically enriched (MACS P-value ≤ 10 × 10–6 and fold enrichment ≥ 3) in the indicated population. Multiple H3K27ac peaks residing within 1 kb were collapsed into a single domain. c. Position weight matrices (PWMs) of DNA enriched motives identified in NFRs associated to recently-activated TH17-IL-10+-specific and TH17-IL-10-specific H3K27ac domains. d. Consensus PWMs of the indicated transcription factors were downloaded from the Hocomoco v.11 database2. Overlapping nucleotides in consensus motifs are highlighted by a light grey box in background e. Gene expression profile of the indicated transcription factors in day 5 TH17-IL-10+ and TH17-IL-10 cells was obtained from RNA-seq data (Fig. 4). 2Kulakovskiy, I.V. et al. HOCOMOCO: towards a complete collection of transcription factor binding models for human and mouse via large-scale ChIP-Seq analysis. Nucleic Acids Res 46, D252-D259 (2018).

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https://doi.org/10.1038/s41590-018-0200-5