An immunoregulatory and tissue-residency program modulated by c-MAF in human TH17 cells

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⁺ T_H17 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⁺ T_H17 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^- T_H17 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⁺ T_H17 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 T_H17 cells and provide a framework with which to investigate the role of these cells in physiology and immunopathology.

Figure 1.. Transient production of IL-10 is a stable feature of a subset of human memory T_H17 cells.

a,b. Production of IL-17 and IL-10 in T_H17 clones analyzed in the resting state (Day 0 and Day 21) and in the recently activated state (Day 5) as measured by intracellular cytokine staining. The clones were divided according to their ability to produce IL-10 on Day 5. Representative staining of a T_H17-IL-10⁺ clone (upper panel) and a T_H17-IL-10^- clone is shown in (a) and data from several T_H17-IL-10⁺ and T_H17-IL-10^- clones representative of more than 15 experiments performed are shown in (b). The percentage of T_H17-IL-10⁺ clones isolated from CCR6⁺CCR4⁺CXCR3^– T cells in 13 experiments performed with different donors was 24.67 ± 3.22 (mean ± s.e.m). A similar frequency of T_H17-IL-10⁺ clones was obtained from 4 experiments performed with different donors in which clones were isolated from IL-17-producing CCR6⁺CXCR3^– T cells (25.6 ± 7.87%, mean ± s.e.m.). c. The capacity of T_H17-IL-10⁺ clones (black symbols) and T_H17-IL-10^- clones (grey symbols) to produce IL-10 was evaluated after repeated rounds of re-stimulation with anti-CD3 and anti-CD28 antibodies, both on Day 0 (square) and on Day 5 (circle); at least 21 days separated two following re-stimulations. Data are representative of 4 independent experiments. In panel c and in all subsequent experiments, the T_H17 cells analyzed were pools of T_H17 clones that comprised ≥ 50% IL-17⁺ cells in the resting state.

Figure 2.. Production of IL-10 by Day 5-activated T_H17 cells requires chromatin remodeling and c-MAF up-regulation.

a. Abundance of histone modifications associated with gene transcription (H3K4me3) or repression (H3K27me3) at the IL10 TSS analyzed by ChIP in Day 0-resting and Day 5-activated T_H17-IL-10⁺ and T_H17-IL-10^- cells. Data are represented as mean + 95% c.i. (n = 6). b. Expression of MAF isoforms a and b was measured in Day 0 and Day 5 T_H17-IL-10⁺ and T_H17-IL-10^- clones by qPCR and normalized to the TATA-box binding protein gene (TBP). Shown are individual values and mean + s.e.m. (isoform a: Day 0, n = 5; Day 0 + 2h and Day 5, n = 6; Day 5 + 2h, n = 7; isoform b, n = 5). c. Expression of c-MAF as detected by intracellular staining in representative Day 0 and Day 5 T_H17-IL-10⁺ and T_H17-IL-10^- cells. d. Mean fluorescence Intensity (MFI) of c-MAF staining in Day 0 and Day 5 IL-10⁺ and IL-10^- T_H17 clones. Data are represented as mean + 95% c.i., with each dot indicating a T cell clone pool (n = 12). e. c-MAF binding to two regions located in the IL10 promoter as determined by ChIP. A developmentally repressed locus (Ctrl_1) and a pericentromeric DNA repeat region (Ctrl_2) were used as negative controls. Data are represented as mean + 95% c.i., with each dot indicating a T cell clone pool (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, as determined by ratio paired t test. Data are representative of at least 3 independent experiments.

Figure 3.. T_H17-IL-10⁻ and T_H17-IL-10⁺ cells differentially polarize monocytes to M1 and M2 macrophages.

CD14⁺ monocytes were sorted from PBMCs and co-cultured with allogeneic Day 5-activated T_H17-IL-10⁺ or T_H17-IL-10^- cells or in medium containing M1- or M2-inducing cytokines. a,b. Expression of M1- and M2-associated surface markers was determined after 24 h culture by flow cytometry (a, representative plots; b, mean + s.e.m.; n = 6). c. Production of TNF and IL-6 by CD14⁺CD4^- monocytes assessed by intracellular staining after 48 h culture (mean + s.e.m.; T_H17, n = 5; M1/M2 medium, n = 2). d. CD14⁺ monocytes were cultured in the presence of supernatants from Day 5 T_H17-IL-10⁺or T_H17- IL-10^- cells or in M1/M2 medium for 7 days and then stimulated with LPS for the indicated time points. RNA was extracted and the expression of M1- and M2-associated genes was measured by qPCR (mean + s.e.m.; T_H17 supernatants, n = 4; M1/M2 medium, n = 3). Genes are ranked according to their differential expression in M1/M2 medium-macrophages stimulated with LPS for 4 hours. *P < 0.05; **P < 0.01; ***P < 0.001, as determined by ratio paired t test. Data are from 3 (a-c) and 2 (d) independent experiments.

Figure 4.. Distinct transcriptional programs in Day 5-activated T_H17-IL-10⁺ and T_H17- IL-10⁻ cells.

a. MA plots of RNA-seq analysis of Day 0-resting and Day 5-activated T_H17- IL-10⁺ and T_H17-IL-10⁻ cells, before or after 2 h stimulation with CD3/CD28 antibodies. Differentially expressed genes are marked in red (fold change ≥ 2) or blue (fold change ≤ 0.5). The genes selected for validation are highlighted by larger dots. b,c. Number of differentially expressed protein-coding genes (b) and lncRNAs (c) in the indicated samples. Data represent the average of two independent experiments. d. qPCR validation of a set of differentially expressed genes in Day 0 (upper panel) and Day 5 (lower panel) T_H17-IL-10⁺ and T_H17-IL-10⁻ cells (mean + s.e.m; n ≥ 3, P-value ≤ 0.05 as determined by paired t test) (⁽¹⁾ P-value = 0.06; ⁽²⁾ fold change ≥ 1.8). In b, c and d, black bars indicate genes more expressed in T_H17-IL-10⁺ cells and grey bars genes more expressed in T_H17-IL-10⁻ cells.

Figure 5.. IL-27 and IL-lβ antagonistically promote the expression of T_H17-IL-10⁺ and T_H17-IL-10^- associated genes.

a,b. T_H17-IL-10⁺ cells were polyclonally activated with or without IL-1β (10 ng/ml) and the expression of representative immunoregulatory, tissue- residency and pro-inflammatory genes was measured on Day 5 at mRNA level by qPCR, after 2 h re-stimulation with CD3/CD28 antibodies (a, mean + s.e.m; n > 3), and at protein level by flow cytometry (b, mean + 95% c.i.; CXCR6, CD25 and CCR7, n = 8; IL-10, IFN-γ and c-MAF, n = 7; CD69 and PD-1, n = 6; CTLA-4, n = 5; BLIMP-1 and FOXP3, n = 4), after 5 h stimulation with PMA+I. c. T_H17-IL-10^- cells were polyclonally activated in presence of IL-27 (25 ng/ml), TGF-β (2 ng/ml) or a combination of the two cytokines and the expression of the indicated proteins was assessed on Day 5 by flow cytometry, after 5 h stimulation with PMA+I (mean + 95% c.i.; n = 6; surface markers TGF-β+IL-27, n = 5). d. T_H17-IL-10⁺ (n = 4) and T_H17-IL-10^- (n = 6) cells were polyclonally activated in the presence of AHR antagonist (CH-223191, 3 μΜ) or agonist (FICZ, 100 nM) and IL-10 production was measured on Day 5 by flow cytometry, following 5 h PMA+I stimulation (mean + s.e.m.). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, as determined by ratio paired t test (a) or ratio paired t test (b-d).

Figure 6.. c-MAF extensively binds bona fide enhancers associated to immune-related genes in T_H17-IL-10+ cells.

a. Number of c-MAF peaks found in Day 0-resting and Day 5- activated T_H17-IL-10⁺ and T_H17-IL-10^- cells, as assessed by ChIP-seq (MACS P-value ≤ 10 × 10⁻¹⁰). b. Pie chart showing the portion of differentially expressed c-MAF-bound genes in Day 0 and Day 5 T_H17-IL-10⁺ cells. The portion of genes associated with T_H17-IL-10⁺ or T_H17-IL-10^- is shown in black and grey, respectively; c-MAF not bound genes are shown in white. c. Position weight matrix (PWM) of DNA motives in T_H17-IL-10⁺ and T_H17-IL-10^- associated genes. d. Genomic distribution of c-MAF peaks; promoters were defined as a −2.5 kb to +1 kb window around gene TSSs. e. ChIP-qPCR analysis of H3K4me1 and H3K4me3 levels on a selected number of c-MAF bound regions in Day 0 and Day 5 T_H17-IL-10⁺ cells (mean + s.e.m.; n = 2). Each region is named according to the associated gene. f. H3K27ac distribution in a 10 kb window around c-MAF peaks, in Day 0 or Day 5 T_H17-IL-10⁺ cells. Data represent the average of two independent experiments.

Figure 7.. c-MAF promotes the immunoregulatory and tissue-residency transcriptional program in Day 5-activated T_H17-IL-10⁺ cells.

a. c-MAF depletion in Day 5-activated T_H17-IL-10⁺ clones by shRNAs. Left panel depicts a representative c-MAF staining in T_H17- IL-10⁺ cells transduced with control shRNA (shLuc) or c-MAF-specific shRNAs (shRNA1). Right panel shows the shRNA-mediated downregulation of c-MAF levels in T_H17-IL-10⁺ clones, as detected by intracellular staining. Two independent c-MAF-targeting shRNAs (shRNAl and shRNA2) were used. Data are represented as mean + 95% c.i., with each dot indicating a T cell clone pool (shLuc and shRNA1 n = 8, shRNA2 n = 7). b. IL-10 expression as determined by qPCR (left panel, mean + s.e.m.) and intracellular cytokine staining (ICCS, right panel, mean + 95% c.i.) in T_H17-IL-10⁺ clones transduced with control shRNA (shLuc) or c-MAF-targeting shRNA1 and shRNA2. Each dot indicates a T cell clone pool (qPCR: shRNA1 n = 5, shRNA2 n = 4, ICCS: shRNA1 n = 7, shRNA2 n = 6). c. MA plots of RNA-seq analysis of Day 5 c-MAF-depleted T_H17-IL-10⁺, after 2 h stimulation with CD3/CD28 antibodies (left panel). Genes associated with T_H17-IL-10⁺ or T_H17-IL-10^- are highlighted in red and blue, respectively. Changes in expression of genes associated with T_H17-IL-10⁺ and T_H17-IL-10^- upon c-MAF depletion are summarized in a box (interquartiles, with a line indicating the median value) and whiskers (min to max values) plot (right panel, mean indicated as a “+”). Data represent the average of two independent experiments. d. Expression of c-MAF-dependent genes in Day 5 c-MAF-depleted T_H17-IL10⁺ cells, as measured by qPCR. Black and grey bars indicate genes associated with T_H17-IL10⁺ or T_H17-IL10^- cells, respectively. Shown is the average log₂ fold change of the two indicated shRNAs compared to the shLuc control (mean + s.e.m.; n = 4). **P < 0.01; ***P < 0.001; ****P < 0.0001, as determined by ratio paired t test (a), paired t test (b) and Wilcoxon matched-pairs signed rank test (c).