Group 2 innate lymphoid cells (ILC2s) are distributed systemically and produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Transcriptional profiling of ILC2s from different tissues, however, grouped ILC2s according to their tissue of origin, even in the setting of combined IL-25-, IL-33-receptor-, and TSLP-receptor-deficiency. Single-cell profiling confirmed a tissue-organizing transcriptome and identified ILC2 subsets expressing distinct activating receptors, including the major subset of skin ILC2s, which were activated preferentially by IL-18. Tissue ILC2 subsets were unaltered in number and expression in germ-free mice, suggesting that endogenous, tissue-derived signals drive the maturation of ILC2 subsets by controlling expression of distinct patterns of activating receptors, thus anticipating tissue-specific perturbations occurring later in life.
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We thank M. Consengco and M. Ji for technical expertise; Z. Wang for cell sorting; A. Barczak, R. Barbeau, and J. Pollack for assistance with RNA-seq; E. Wan for assistance with scRNA-seq; J. Turnbaugh (University of California San Francisco) for providing germ-free mice; and M. Ansel and A. Marson for comments on the manuscript. This work was supported by the National Institutes of Health (AI030663 and HL128903 to R.M.L., AI122702 to J.L., DK101604 to A.B.M., and AI113143 to J.C.N.), Dermatology Foundation (R.R.R.-G.), A.P. Giannini Foundation (R.R.R.-G.), Robert Wood Johnson Foundation (R.R.R.-G.), Swiss National Science Foundation (P2EZP3_162266 and P300PA_171591 to C.S.), Howard Hughes Medical Institute (R.M.L.), and the Sandler Asthma Basic Research Center at the University of California San Francisco (R.M.L).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a-e, Gating strategy for sorting lung (a), skin (b), gut (c, small intestine lamina propia), fat (d, perigonadal white adipose tissue) and bone marrow (e, BM) ILC2s as described in Methods.
a-e, RNA-seq analysis of select transcripts significantly enriched (FDR < 0.01) in R5 + ILC2s from peripheral tissues (lung, gut, fat, skin) versus Yarg + BM ILC2s. a, enzyme / ion channel. b, extracellular / nucleus. c, plasma membrane, d, cytoplasm. e, transporter. Data represent mean normalized read counts (fragments per million mapped reads) from biological replicates (n = 3, BM; n = 5, lung; n = 6, fat, gut, skin).
a-b, Number of ILC2s (Lin–CD45+Thy1.2+CD25+) in fat (a), and bone marrow (b). c-h, Quantitative RT-PCR (qPCR) of Gata3 (c), Arg1 (d), Il1rl1 (e), Il17rb (f), Tnfaip3 (g), and Il18r1 (h) transcript abundance among germ-free (GF) or specific pathogen free (SPF) ILC2s sorted from BM, lung, gut, skin, and fat. Data represent biological replicates (n = 3; mean ± SD) and transcripts are normalized to Rps17. * p < 0.05.
Representative flow cytometry of CD45+Lin– cells from indicated single knockout (KO) or TKO mice on YRS backgrounds. Numbers within the gate represent percentage of activated (IL-5-producing) ILC2s among total ILC2s (Lin–CD45+Thy1.2+CD25+ in lungs, fat, and skin; Lin–CD45+IL17Rb+KLRG1+ in gut). Data are representative of 2 or more independent experiments representing at least 3 mice in each group.
a, Heat map of log2 fold change relative global average for top 1000 genes by variance. b-g, Mean normalized read counts (fragments per million mapped reads) for Gata3 (b), Il7r (c), Il5 (d), Il13 (e), Il17rb (f), and Il18r1 (g) transcripts among WT and TKO ILC2s from BM, lung, fat, gut and skin from biological replicates (n = 3, WT BM, TKO BM, TKO gut; n = 4, TKO lung, TKO fat; n = 5, WT lung, TKO skin; n = 6, WT fat, WT gut, WT skin). * p < 0.05; ** p < 0.005; *** p < 0.0005.
a, tSNE plot representing 35,396 ILC2s sorted from BM (Lin–CD45+Yarg+), lung, fat, gut, and skin (Lin–CD45+Red5+) analyzed by single-cell RNA sequencing (scRNA-seq). b, tSNE plot of graph-based clustering of tissue-resident scRNA-seq reveals distinct intra-tissue ILC2 subsets. c, Heat map of hierarchical clustering of top 100 up-regulated (log2 fold change) genes from intra-tissue clusters shown in (b). Arrows highlights differences in Il1rl1 and Il18r1 between clusters.
Normalized relative expression of Tbx21, Rorc, Il2rg, Arg1, Il13, Thy1, Crlf2, Areg, Klrg1, Il2ra, Il2rb, Itgae, Icos, Cd69, and Cd44 from tissue ILC2s (BM (Lin–CD45+Yarg+), lung, fat, gut, and skin (Lin–CD45+Red5+) by scRNA-seq. Data are representative of 2 independent experiments.
a, b, Number of R5 + ILC2s (Lin–CD45+Thy1.2+Red5+) in skin (a) and lung (b) of WT and IL-18KO mice. Data pooled from 2 individual experiments with n ≥ 10 per group and represented as mean±SD.
Supplementary Figures 1–8
RNA sequencing analysis of tissue ILC2s. Bulk RNA-seq data table related to Figures 1, 4, and Supplementary Figs. 2 and 5.
Primers used for qRT-PCR.
Pipeline code for scRNA-seq Cellranger aggr.
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Ricardo-Gonzalez, R.R., Van Dyken, S.J., Schneider, C. et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat Immunol 19, 1093–1099 (2018). https://doi.org/10.1038/s41590-018-0201-4
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