Molecular and functional heterogeneity of IL-10-producing CD4+ T cells - PubMed (original) (raw)

doi: 10.1038/s41467-018-07581-4.

Shiwa Soukou 1, Babett Steglich 1 2, Paulo Czarnewski 3, Lilan Zhao 2, Sandra Wende 1, Tanja Bedke 1, Can Ergen 1, Carolin Manthey 1, Theodora Agalioti 2, Maria Geffken 4, Oliver Seiz 1, Sara M Parigi 3, Chiara Sorini 3, Jens Geginat [ 5](#full-view-affiliation-5 "INGM-National Institute of Molecular Genetics "Romeo ed Enrica Invernizzi", 20122, Milan, Italy."), Keishi Fujio 6, Thomas Jacobs 7, Thomas Roesch 8, Jacob R Izbicki 2, Ansgar W Lohse 1, Richard A Flavell 9 10, Christian Krebs 11, Jan-Ake Gustafsson 12, Per Antonson 12, Maria Grazia Roncarolo 13, Eduardo J Villablanca 3, Nicola Gagliani 14 15 16, Samuel Huber 17

Affiliations

Molecular and functional heterogeneity of IL-10-producing CD4+ T cells

Leonie Brockmann et al. Nat Commun. 2018.

Abstract

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Mouse IL-10-producing CD4+ T cells are heterogeneous. a–c IL-10-producing Foxp3neg CD4+ T cells (IL-10pos) were isolated from small intestine or spleen of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice. Cells were transferred into lymphopenic hosts and colitis development was assessed by weight loss (c) and endoscopic colitis score (b) 5 weeks upon transfer (IL-10pos cells small intestine n = 12; IL-10pos cells Spleen n = 11; lines indicate mean ±SEM). Results are cumulative of three independent experiments. A Mann–Whitney U test was used to calculate significance. d _t-_SNE analysis of single cell RNA sequencing of IL-10pos cells (including Foxp3+ cells) isolated from small intestine or spleen of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice. e Bootstrap analysis of HVGs in single cell RNA sequencing data of IL-10pos cells from small intestine and spleen. f Expression of indicated genes in _t-_SNE analysis

Fig. 2

Fig. 2

Splenic IL-10-producing CD4+ T cells contain a regulatory cluster. a Spearman correlation coefficient between each cluster from small intestine and spleen. b Fold change analysis of genes enriched in indicated clusters compared to other clusters that were amongst the 100 most enriched genes. c Expression of indicated genes encoding cytokines and chemokines. d Expression of indicated genes encoding transcription factors. e Expression of indicated genes encoding other factors. f Expression of indicated genes encoding surface receptors

Fig. 3

Fig. 3

CIR identify regulatory IL-10-producing CD4+ T cells. _vi_SNE analysis of IL-10pos Foxp3neg CD4+ T cells. Clustering is based on MFI of PD-1, LAG-3, TIGIT, TIM-3, CD49b, and CCR5. Blue circle indicates co-inhibitory receptor rich (CIR rich) region. a Analysis of cells from small intestine, spleen, and lung of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice (n = 4). Data are representative of two independent experiments. b Analysis of untreated IL-10eGFP Foxp3mRFP double reporter mice (n = 3). Data are representative of three independent experiments. c Analysis of P. berghei infected IL-10eGFP Foxp3mRFP double reporter mice (n = 3). Data are representative of two independent experiments

Fig. 4

Fig. 4

CIR rich CD4+ T cells have a high suppressive capacity. a In vitro suppression of co-inhibitory receptor rich (CIR rich) and co-inhibitory receptor poor (CIR poor) IL-10pos Foxp3neg CD4+ T cells isolated from spleen of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice. Representative histograms of five independent experiments, a paired _T-_test was used to calculate significance. b In vitro suppression of CIR rich and CIR poor IL-10pos Foxp3neg CD4+ T cells isolated from spleen of P. berghei infected IL-10eGFP Foxp3mRFP double reporter mice. Representative histograms of four independent experiments. c In vitro suppression of CIR rich and CIR poor IL-10pos Foxp3neg CD4+ T cells isolated from spleen of aCD3-treated IL-10eGFP Foxp3mRFP reporter mice. TIM-3 and LAG-3 were blocked using blocking antibodies. To block IL-10 receptor signaling responder T cells were isolated from IL-10R dominant negative mice (DN IL-10R Responder). Results are cumulative of three independent experiments. d CIR rich and CIR poor IL-10pos Foxp3neg CD4+ T cells and IL-10neg CD4+ T cells were isolated from spleen of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice. Cells were transferred into lymphopenic hosts and colitis development was assessed by weight loss and endoscopic colitis score 5 weeks upon transfer (IL-10neg n = 6; CIR poor n = 9; CIR rich n = 6; lines indicate mean±SEM). Results are cumulative of three independent experiments. One-way ANOVA (post-test Tukey) was used to calculate significance (*p < 0.05). e CIR rich IL-10-producing Foxp3neg CD4+ T cells, IL-10neg CD4+ T cells and Foxp3+ Treg cells were isolated from spleen of aCD3-treated IL-10eGFP Foxp3mRFP double reporter mice. IL-10pos CIR rich or Foxp3+ Treg cells were co-transferred with IL-10neg CD4+ T cells and colitis development was assessed by weight loss and endoscopic colitis score 5 weeks upon transfer (IL-10neg n = 7; IL-10neg + CIR rich n = 6; IL-10neg + Foxp3+ Treg n = 6; lines indicate mean±SEM). Results are cumulative of three independent experiments. One-way ANOVA (post-test Tukey) was used to calculate significance (*p < 0.05)

Fig. 5

Fig. 5

CIR rich CD4+ T cells have a distinct transcriptional program. a Volcano plots of bulk RNA sequencing data of indicated populations (IL-10pos CIR rich n = 2; IL-10pos CIR poor n = 2; IL-10neg n = 2). b Expression of known TR1 signature genes comparing IL-10pos CIR rich with IL-10neg and IL-10pos CIR rich with IL-10pos CIR poor. c Differentially expressed transcription factors between IL-10pos CIR rich and IL-10pos CIR poor. mRNA expression of indicated genes normalized to Hprt from at least three independent experiments (separated low-high, line represents median). d CD4+ T cells isolated from _Lxrα_-/- CD45.2 and wildtype CD45.2 mice were co-transferred with wildtype CD4+ T cells (CD45.1/2) into lymphopenic hosts. Animals (WT:KO n = 7; WT:WT n = 6) were treated with aCD3 antibodies 5 weeks upon transfer and cells were isolated from small intestine. Results are cumulative of two independent experiments. A Wilcoxon test was used to calculate significance. e Correlation between genes significantly higher expressed in IL-10pos CIR rich versus IL-10pos CIR poor cells and expression pattern of scRNA-seq data

Fig. 6

Fig. 6

Human IL-10-producing CD4+ T cells are heterogeneous. a RNA single cell sequencing data of IL-10pos cells (including Foxp3+ T cells) isolated from PBMCs of healthy donors (n = 3), stimulated with SEB overnight. b _vi_SNE analysis of IL-10pos CD25low CD4+ T cells from PBMCs of healthy donors (n = 8) and healthy colon biopsies (n = 4), stimulated with SEB overnight. Clustering is based on MFI of PD-1, LAG-3, TIGIT, TIM-3, CD49b, and CCR5. Blue circle indicates co-inhibitory receptor rich (CIR rich) region. c In vitro suppression of CIR rich and CIR poor IL-10pos CD25low CD4+ T cells isolated from PBMCs of healthy donors, stimulated with SEB overnight. Representative histograms of five independent experiments, a paired _T-_test was used to calculate significance. d, e Analysis of IL-10pos CD25low CD4+ T cells (d) and expression of CD49b/LAG-3 within those (e) of colon biopsies from IBD patients: UC active n = 22 (inflamed n = 20, blue dots; non-inflamed n = 17, black dots); UC Remission n = 2, violet dots; CD active n = 9 (inflamed n = 5, blue dots; non-inflamed n = 6, black dots); CD remission n = 5, violet dots and healthy controls (n = 18). Cells were stimulated with SEB overnight, One-way ANOVA (multiple comparisons) was used to calculate significance (**p < 0.005)

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