Abstract
The recognized diversity of innate lymphoid cells (ILCs) is rapidly expanding. Three ILC classes have emerged, ILC1, ILC2 and ILC3, with ILC1 and ILC3 including several subsets. The classification of some subsets is unclear, and it remains controversial whether natural killer (NK) cells and ILC1 cells are distinct cell types. To address these issues, we analyzed gene expression in ILCs and NK cells from mouse small intestine, spleen and liver, as part of the Immunological Genome Project. The results showed unique gene-expression patterns for some ILCs and overlapping patterns for ILC1 cells and NK cells, whereas other ILC subsets remained indistinguishable. We identified a transcriptional program shared by small intestine ILCs and a core ILC signature. We revealed and discuss transcripts that suggest previously unknown functions and developmental paths for ILCs.
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Acknowledgements
We thank our colleagues in the ImmGen consortium, especially C. Benoist and L. Lanier, for input and discussion; the core ImmGen team, K. Rothamel and A. Rhodes, for contributions and technical assistance; M. Artyomov, G. Krishnan and J. Siegel for computational assistance; D. Sojka for discussion; E. Lantelme and D. Brinja for sorting assistance; P. Wang for microscopy assistance; and eBioscience and Affymetrix for support of the ImmGen Project. Supported by the US National Institutes of Health (R24AI072073 to the ImmGen Consortium; 1U01AI095542, R01DE021255 and R21CA16719 to the Colonna laboratory; MSTP T32 GM07200 to M.L.R.; and Infectious Disease Training Grant T32 AI 7172-34 to V.S.C.).
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M.L.R. analyzed data; A.F., M.L.R., J.S.L. and Y.W. sorted cell subsets; M.L.R., A.F. and V.S.C. performed follow-up experiments and analyzed data; S.G. maintained mice; S.K.D. provided critical reagents; M.L.R., A.F., S.G. and M.C. designed studies; M.L.R. and M.C. wrote the paper; and the ImmGen Consortium contributed to the experimental design and data collection.
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Integrated supplementary information
Supplementary Figure 1 Characteristics of sorted ILC and NK cell populations.
(a) Gating strategy for siLP ILC1, NK, NKp46+ ILC3, and NKp46− LTi-like ILC3 subsets after gating on live CD45+ and CD3−CD19− cells in C57BL/6 mice or RorγteGFP/+ reporter mice. ILCs are gated on intracellularly stained Rorγt+ cells (left) or RorγteGFP+ cells (right). For microarray analysis, the gating strategy as shown on the right was used. (b,c) Histograms of NK1.1 (b) and CD45 (c) expression from sorted siLP subsets used in array generation (n = 5 pooled mice each). (d) Two-dimensional views of PCA. Data are representative of at least ten experiments (a) or five independent experiments (b,c).
Supplementary Figure 2 Validation of ILC core signature.
(a) Extracellular and intracellular staining of TCRδ on (or in) ILCs and NK cells, continued from Figure 6. Numbers in outlined areas indicate percent TCRδ+ cells (outline colors (red) match those in keys above plots). (b) Representative flow plots of percentage of CXCR6+ cells from CXCR6eGFP/+ reporter mice. siLP NKp46− LTi-like ILC3, siLP ILC1, siLP NK, and siLP NKp46+ ILC3 percentages were analyzed after fixation of cells. Color-coded gates demonstrate percentage of indicated subsets that are CXCR6+ (n = three or four mice per genotype per tissue). Data are representative of two independent experiments (a) or three independent experiments (b).
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Supplementary Text and Figures
Supplementary Figures 1 and 2 (PDF 1153 kb)
Supplementary Table 1
Unique transcripts of individual ILC subsets (XLSX 50 kb)
Supplementary Table 2
Shared transcripts between siILC subsets (XLSX 73 kb)
Supplementary Table 3
Transcripts differentially expressed among ILC3 subsets (XLSX 60 kb)
Supplementary Table 4
Transcripts differentially expressed between NK cells and ILC1 cells in a single tissue (XLSX 84 kb)
Supplementary Table 5
Transcripts differentially expressed between NK cells and ILC1 cells in two tissues (XLSX 48 kb)
Supplementary Table 6
Core ILC1 and NK cell signatures (XLSX 54 kb)
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Robinette, M., Fuchs, A., Cortez, V. et al. Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat Immunol 16, 306–317 (2015). https://doi.org/10.1038/ni.3094
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DOI: https://doi.org/10.1038/ni.3094
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