Tissue signals imprint ILC2 identity with anticipatory function


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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Fig. 1: Steady-state ILC2 activation in multiple peripheral tissues.
Fig. 2: ILC2 distribution and homeostatic function is independent of the microbiota.
Fig. 3: Tissue-resident ILC2s depend on distinct tissue signals.
Fig. 4: Transcriptional heterogeneity of tissue-resident ILC2s.
Fig. 5: Tissue map of ILC2 signals revealed by scRNA-seq.
Fig. 6: IL-18 independently mediates ILC2 subset activation.
Fig. 7: Blunted type 2 skin inflammation in the absence of IL-18.


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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).

Author information




R.R.R.-G. and S.J.V.D. designed and performed experiments, analyzed and interpreted the data, and wrote the manuscript. C.S., J.L., J.C.N., H.-E.L., and A.B.M. contributed to experiments. D.V. analyzed scRNA-seq data, and W.L.E. and D.J.E. provided RNA-seq data analysis and expertise. R.M.L. directed the studies and wrote the manuscript with R.R.R.-G. and S.J.V.D.

Corresponding author

Correspondence to Richard M. Locksley.

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Integrated supplementary information

Supplementary Figure 1 Gating strategy for sorting tissue-resident function-marked ILC2s.

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.

Supplementary Figure 2 RNA-seq of peripheral program gene sets in ILC2s.

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).

Supplementary Figure 3 ILC2 counts and select transcripts in resting SPF versus GF mice.

a-b, Number of ILC2s (LinCD45+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.

Supplementary Figure 4 Steady-state ILC2 reporter expression.

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 (LinCD45+Thy1.2+CD25+ in lungs, fat, and skin; LinCD45+IL17Rb+KLRG1+ in gut). Data are representative of 2 or more independent experiments representing at least 3 mice in each group.

Supplementary Figure 5 Gene expression in ILC2s from multiple tissues.

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.

Supplementary Figure 6 Single-cell transcriptome analysis of tissue-resident ILC2s.

a, tSNE plot representing 35,396 ILC2s sorted from BM (LinCD45+Yarg+), lung, fat, gut, and skin (LinCD45+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.

Supplementary Figure 7 Single-cell RNA-seq reveals gradation in peripheral activation program.

Normalized relative expression of Tbx21, Rorc, Il2rg, Arg1, Il13, Thy1, Crlf2, Areg, Klrg1, Il2ra, Il2rb, Itgae, Icos, Cd69, and Cd44 from tissue ILC2s (BM (LinCD45+Yarg+), lung, fat, gut, and skin (LinCD45+Red5+) by scRNA-seq. Data are representative of 2 independent experiments.

Supplementary Figure 8 Number of R5+ ILC2s in IL-18 KO mice.

a, b, Number of R5 + ILC2s (LinCD45+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 information

Supplementary Figures

Supplementary Figures 1–8

Reporting Summary

Supplementary Table 1

RNA sequencing analysis of tissue ILC2s. Bulk RNA-seq data table related to Figures 1, 4, and Supplementary Figs. 2 and 5.

Supplementary Table 2

Primers used for qRT-PCR.

Supplementary Table 3

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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