c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies

Abstract

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44 ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1β and IL-23. We also report a role for transforming growth factor-β in promoting the conversion of c-Kit ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.

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Fig. 1: IL-17-producing ILCs activated by C. albicans originate from ILC2s.
Fig. 2: IL-17 production in ILC2s is controlled by the reciprocal regulation of RORγt and GATA-3 activity, and blocked by IL-4.
Fig. 3: c-Kit and CCR6 identify a RORγt + ILC2 subset that can readily transdifferentiate into IL-17-producing cells.
Fig. 4: TGF-β regulates the transition of c-Kit ILC2s into RORγt+ ILC2s.
Fig. 5: scRNAseq analysis identifies skin-homing RORγt+ ILC2s in peripheral blood.
Fig. 6: IL-17-producing ILC3s from psoriatic lesions can convert to ILC2s.

Data availability

Sequence data that support the findings of this study have been deposited in the Gene Expression Omnibus with accession numbers GSE124474, GSE129238 and GSE114396 for bulk RNAseq data (Fig. 3) for ILC subsets, microarray data of human ILC2 subsets cultured under IL-17-polarizing conditions (Fig. 4) and scRNAseq data of human ILC2s (Fig. 5), respectively. The data that support the findings from these studies are available from the corresponding authors upon request.

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Acknowledgements

We thank B. Hooibrink for help with flow cytometry. We thank P.I. Spuls and staff at the Department of Dermatology, Academic Medical Center, Amsterdam, the Netherlands, for providing tissue samples of patients with psoriasis and W.G. van Selms and staff at the Department of Plastic Surgery, St. Lucas Andreas Hospital, Amsterdam, the Netherlands, for healthy human skin tissue samples. We thank the MedImmune Flow Cytometry core, C. Groves, G. Rosignoli, R. Rayanki and R. Grady for the support with flow cytometry and cell sorting; MedImmune Phamacogenomics group, R. Raja, R. Halpin and Y. Lee for the support of genomics experiments; I. Nikaido for the advice on processing RNAseq samples; A. Berlin, A. Copenhaver, R. Dagher, M. Yang, O. Wyatt, H. Tompkins and A. Gonzales for laboratory support; Y.J. Liu for the initial support for this work; J.S. Silver, J. Kearley and S. Rieder for the critical discussion; the LAR staff for maintaining the experimental animals; and S. Farshadi for helping figure preparation.

Author information

J.H.B., Y.O., M.B.M.T. and X.R.R. designed the study, performed experiments, analyzed the data and wrote the manuscript. J. Wang, J. Wu and Y.S. performed single-cell and bulk RNAseq experiments and analyses. M.A.d.R. provided psoriatic tissue. L.K., C.G., S.v.T. and I.R. performed experiments and analyzed data. R.V. and J.K. analyzed microarray data. H.S. and A.A.H. designed the study, analyzed the data and wrote the manuscript. H.S. is supported by an advanced grant of the European Research Council ERC-2013-ADG number 341038.

Correspondence to Hergen Spits or Alison A. Humbles.

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Competing interests

Y.O., X.R.R., J.Wang, Y.S. and A.A.H. are employed by and shareholders of AstraZeneca, and J.Wu and C.G. are employed by Novartis. H.S. is employed part time by AIMM Therapeutics.

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Peer review information: Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 IL-17-producing ILCs activated by C. albicans originate from ILC2s.

(a) Flow cytometory analysis of intracellular IL-17 levels following restimulation with PMA and ionomycin of LinCD127+CD161+ total ILCs (gating as in Fig. 1a) from healthy skin that have been stimulated for 6 days with C. albicans hyphae (left panel), in the presence of autologous irradiated dermal cells (middle panel) or in the presence of both irradiated dermal cells and C. albicans hyphae (right panel). Shown data is representative of 3 independent experiments. (b) Phenotypical characterization of hyphen-stimulated IL-17-secreting ILCs based on the expression of CRTH2 and c-Kit. Data shown (left) are representative of 3 independent experiments with 1 to 2 donors each that is quantified in bar diagram (right). Numbers adjacent to outlined areas indicate percent IL-17A+ cells and in quadrants indicate percent cells throughout. (c). Dermal ILC subsets were sorted as described in Fig. 1a and analysed for IL17A gene expression. Combined data of 3 experiments with independent donors. Relative units (AU) relative to the expression of β-actin. Error bars represent SEM. (d) Flow cytometry phenotyping of surface receptor expression in ILC populations from healthy skin gated as in Fig. 1a. Data are representative of 3 independent experiments with independent donors. (e) Flow cytometry gating strategy for ILC subsets from human peripheral blood, based on the expression of CD127, CD161, lineage markers (Lin) (CD1a, CD3, CD14, CD16, CD19, CD34, CD94, CD123, BDCA2, FcεR1a, TCRαβ, TCRγδ), CRTH2 and c-Kit. Numbers in gates or quadrants indicate percent cell in each. Data are representative of at least 3 independent experiments. (f) Expression of CD62L and CD69 in peripheral blood ILC2s of healthy individuals; light gray-shading, isotype-matched control antibody. Data are representative of at least 3 independent experiments. (g) Isolated ILC2s from dermis and peripheral blood from 2 healthy donors were analysed for IL5 and IL17A mRNA expression by qPCR. Relative units (RU) relative to the expression of β-actin. Bar diagrams show mean ± SEM. (h) IL-1β, IL-23 and TGF-β as detected by ELISA in the supernatant of C. albicans hyphae-stimulated irradiated dermal cells or irradiated dermal cells alone. Accumulative data of 3 experiments with one donor each. Diagrams show mean ± SEM. (i) Flow cytometry analysis of frequencies of ILC2-derived IL-17A+ cells (as in Fig. 1a) supplemented with anti-IL-1β, anti-TGF-β, anti-IL-23 (all 5 µg/ml). Bar graphs is quantification of 3 independent experiments with 1 donor each. 1-way ANOVA followed by Tukey’s test; * P < 0.05. Error bars represent SEM. Numbers adjacent to outlined areas indicate percent IL-17A+ cells. (j) Flow cytometry analysis of intracellular IL-17, IL-22, IFN-γ and IL-5 of blood-derived CRTH2-c-Kit+ ILCs following stimulation with IL-1β, IL-23 and TGF-β and restimulation with PMA and ionomycin. Data are representative of at least 3 independent donors. Number in quadrants and gates indicate percentage of cells in each. (k) Flow cytometry analyzing the expression of CRTH2, c-Kit and NKp44 in ILC2s cultured in the presence of IL-2 only (dotted line) or IL-2, IL-1β, IL-23 and TGF-β (black line) for 7 days; filled grey is isotype-matched control antibody. Data are representative of 4 experiments with independent donors. (l) Flow cytometry analysis of intracellular IL-17A and IL-5 in blood ILC2s cultured with indicated cytokines for 7 days followed by stimulation with PMA and ionomycin. The plots are representative of 3 experiments with independent donors (right). The bar diagram is quantifying frequencies of IL-17+ cells (n = 3, independent donors). Number in quadrants and gates indicate percentage of cells in each. 1-way ANOVA followed by Tukey’s test; * P < 0.05; ** P < 0.01; Error bars represent SEM. (m) Flow cytometry analysis of intracellular IL-17A, IL-13 and IL-5 in blood ILC2s cultured with IL-1β, IL-23 and TGF-β ± IL-33 for 7 days followed by stimulation with PMA and ionomycin. The plots are representative of 3 experiments with independent donors (right). The bar diagram is quantifying frequencies of IL-17+, IL-13+, IL-5+ cells (n = 3, independent donors). Number in quadrants and gates indicate percentage of cells in each. 1-way ANOVA followed by Tukey’s test; ** P < 0.01; *** P < 0.001; NS not significant. Error bars represent SEM.

Supplementary Figure 2 IL-17 production in ILC2s is controlled by the reciprocal regulation of RORγt and GATA-3 activity, and blocked by IL-4.

(a) RORγt and RORα inhibition of promoter activity in transfected cells with RORγ/GAL4 and RORα/GAL4 reporter systems in the presence of various concentration of a ROR γt inhibitor (compound 8h) as judged by luciferase reporter activity. IC50; half minimal inhibitory concentration. RLU; Relative light units. SD; standard deviation. Concentration-response curves were generated and IC50 values were calculated by nonlinear fitting using the GraphPad Prism software package. Representative example of concentration-response curves from two experiments with duplicate readings is shown. (b) RORC mRNA of Th cells under TH17 polarization conditions in the presence of various concentrations of the RORγt inhibitor as quantified by RT-qPCR. Individual data and mean ± SD are depicted. Results are representative of three independent experiments with triplicate readings. (c, d) IL-17 secretion by TH17 (c) and Tc17 cells (d) in the presence of increased concentrations of the RORγt inhibitor examined by ELISA. Concentration-response curves were generated and IC50 values were calculated by nonlinear fitting using the GraphPad Prism software package. Representative examples of concentration-response curves from at least three experiments with triplicate readings are shown. (e) IL17A, IL17F and CCR6 expression in purified human CD4+ T-cells polarized towards Th17 cells and treated at the beginning of the cell culture with various concentrations of the RORγt inhibitor or with DMSO for 72 hours examined by RT-qPCR. Gene expression was normalized to GUSB levels and expressed as arbitrary units. Individual data and mean ± SD are shown. Results are representative of three independent experiments with triplicate readings. (f) IL-17A amount quantified by ELISA in the supernatant of blood-derived ILC2s and ILC3s transduced with a retroviral vector encoding GATA3 wild-type (wt) or an empty vector, sorted as transduced GFP+ cells on day 3 and cultured with either IL-1β, IL-23 and TGF-β and (white) or IL-1β and IL-4 (black) for 7 days. (g) RORC expression following stimulation with IL-1β, IL-23 and TGF-β for 7 days of blood-derived ILC2s that are transduced with a retroviral vector encoding either an empty vector or a GATA3 wt vector. (h) GATA3 expression following stimulation with IL-1β, IL-23 and TGF-β in the presence or absence of a RORγt inhibitor, and restimulation with PMA and ionomycin of blood-derived ILC2s. Arbitrary units (AU) relative to the expression of ACTB. NS, not significant. Bar graphs is quantification of 3 independent experiments with 1 donor each. (i) Flow cytometry analysis of IL-5 and IL-17 production from ILC2-derived IL-17 secreting cells isolated using an IL-17-secretion assay from blood ILC2s cultured with IL-1β, IL-23 and TGF-β for 5 days (see material and methods). IL-17 secreting ILCs were culture with IL-1β and IL-4 or in the presence of IL-1β, IL-23 and TGF-β for 5 days. Intracellular IL-5 and IL-17 was measured after restimulation with PMA and ionomycin. Data are representative of 2 experiments with independent donors (left). Number in quadrants and gates indicate percentage of cells in each. Bar graphs are the quantification of 2 independent experiments with one donor each.

Supplementary Figure 3 c-Kit and CCR6 identify a RORγt+ ILC2 subset which can readily transdifferentiate into IL-17 producing cells.

(a) Flow cytometry analysis of blood ILC2s with CCR6, RORγt and c-Kit, and gating strategy to isolate c-Kit+CCR6+ ILC2s, c-Kit ILC2s, CD56bright NK cells and CRTH2c-Kit+ ILCs, from human peripheral blood. Numbers adjacent to outlined areas indicate percent of each cell. Data are representative of 6 experiments with independent donors. (b) Heatmap display of expression levels of ILC subsets defining genes in c-Kit+CCR6+ ILC2s (n = 8), c-Kit ILC2s (n = 8), CRTH2c-Kit+ ILCs (n = 5) and CD56bright NK cells (n = 10) from blood and ILC1s (n = 5), NKp44+ ILC3s (n = 6) and NKp44 ILC3s (n = 6, all independent donors) from tonsils determined by RT-qPCR.. Subsets of ILCs were isolated as in Supplementary Fig. 3a. Red and blue doted box indicate ILC3- and ILC2- specifying genes, respectively. (c) Bar plots of expression level of selected genes from (b) in blood c-Kit+CCR6+ ILC2s, c-Kit ILC2s, CRTH2c-Kit+ ILCs and CD56bright NK cells. NS, not significant. 1-way ANOVA followed by Tukey’s test; * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P< 0.0001. Error bars represent SEM. (d) Flow cytometry analyses of IL-13 production in naive, uncultured ILCs from blood stimulated with PMA and ionomycin (left) and dot plots for quantification of the frequency of IL-13 producing cells to parental cells (n = 3, independent donors). Numbers adjacent to outlined areas indicate percent IL-13+ cells. (e) Cloning strategy for differentiation of blood-derived ILC2s into ILC3s with IL-2, IL-23, TGF-β and IL-1β in the presence of feeder cells (irradiated allogeneic PBMCs and JY-cells) in 96-well plates. (f) Quantification of the number of c-Kit+CCR6+ ILC2s (n = 8), c-Kit ILC2s (n = 7), CRTH2c-Kit+ ILCs (n = 6) and CD56bright NK cells (n = 6, all independent donors) recovered from one well (among an initial 3 × 102 cells per well plated at day 0) after 7 d of culture with IL-1β, IL-25 or IL-33. 1-way ANOVA followed by Dunnet’s test (vs control condition, eg. no cytokines); ** P < 0.01. Error bars represent SEM. (g) Concentration of IL-5 in supernatants of c-Kit+CCR6+ ILC2s, c-Kit ILC2s (key) plated as in b and cultured for 4 d with IL-1β, IL-25 or IL-33 (n = 4, independent donors). 1-way ANOVA followed by Dunnett’s test (vs control condition, eg. no cytokines); ** P < 0.01, *** P < 0.001 and **** P< 0.0001. Error bars represent SEM.

Supplementary Figure 4 TGF-β regulates the transition of c-Kit ILC2s into RORγt+ ILC2s.

(a) Principle component analysis of global gene expression of c-Kit+ ILC2 and c-Kit ILC2 populations isolated from 3 healthy donors, stimulated with indicated cytokines. c-Kit+ ILC2s (grey) and c-Kit ILC2s (dotted circle) incubated under different cytokine combination (dot shown in different colors as indicated) are displayed in PC1 vs PC2 (top) and in PC1 vs PC3 (bottom) are shown. (b) Venn diagrams comparing differently expressed genes of c-Kit+ ILC2s and c-Kit ILC2s stimulated with indicated cytokines from global gene expression data in (a). Red circle represents gene-expression differences of c-Kit+ ILC2s stimulated with IL-1β vs IL-1β IL-23. The blue circle represents differences of c-Kit ILC2 stimulated with IL-1β vs IL-1β plus IL-23. Differently expressed genes of c-Kit ILC2 stimulated with IL-1β vs IL-23 or IL-1β, IL-23 and TGF-β are shown in the green circle. Numbers in the Venn diagram represent numbers of differently expressed genes. (c) Expression of GATA3, IL17RB, IL1RL1 and CRLF2 determined by RT-qPCR in c-Kit+ ILC2s (n = 8) and c-Kit− ILC2s (n = 6, all independent donors) stimulated with IL-1β or IL-1β TGF-β for 7 days. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P< 0.0001. 1-way ANOVA followed by Tukey’s test. Error bars represent SEM. (d) Flow cytometry analysis of RORγt and GATA-3 expression in c-Kit+ ILC2s, c-Kit ILC2s, CD56bright NK and CRTH2c-Kit+ ILCs stimulated with IL-1β or IL-1β TGF-β for 7 days. Data are representative of 3 independent experiments with individual donors. Numbers adjacent to outlined areas indicate percent of each cell.

Supplementary Figure 5 Single cell RNAseq analysis identifies skin-homing RORγt+ ILC2s in peripheral blood.

(a) Representative differentially expressed genes among all cluster identified. Dot size represents the fraction of cells in the cluster expressing the gene and the color code shows average expression levels of the gene. (b) Overlay visualization of GATA3 and RORA expression on tSNE plots of single cell RNAseq data of ILC populations isolated from peripheral blood from one donor. (c, d) Expression of genes identified as differentially expressed genes between cluster 1 and cluster 2 in single cell RNAseq (c) and ILC2-related genes (d), determined by RT-qPCR in sort purified c-Kit+CCR10+ ILC2s, c-Kit+ ILC2s, c-Kit ILC2s, CD56bright NK and CRTH2c-Kit+ ILCs (all n = 4, independent donors). Data are shown in box-whisker plots. Line and “+” represent median and mean. Whiskers show min to max value. 1-way ANOVA followed by Dunnett’s test; * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P< 0.0001; ns, not significant.

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