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Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation

Nature Immunology volume 24, pages 42–54 (2023)Cite this article

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Abstract

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

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Fig. 1: Large-scale 3D genome organization in ILCs.
Fig. 2: Differential local 3D structure at key loci correlates with the gene expression programs of each ILC subset.
Fig. 3: Multiple long-distance interactions between the Id2 promoter and LCR1 specifically control ILC2 homeostasis or development.
Fig. 4: Multiple long-distance interactions between the Id2 promoter and LCR1 determine early ILC2 development.
Fig. 5: LCR1 specifically controls group 2 ILC development through Id2 expression regulation.
Fig. 6: LCR1 determines the progression of AAI.

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Data availability

All bulk RNA-seq, ATAC-seq, Hi-C and scRNA-seq datasets have been uploaded to the GEO repository (accession no. GSE191312). The following published datasets were used: GEO GSE11187123, GEO GSE776954, GEO GSE1307759, GEO GSE16954241, GEO GSE14674522 and GEO GSE7942212. Source data are provided with this paper.

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Acknowledgements

The work in this manuscript was supported by funds from CHOP and the NIH grant R01HL136572 (J. H-M.); the Burroughs Welcome Fund, the Chang Zuckerberg Initiative Award, and the NIH grant R01AI168240 (J. H-M. and G.V.); the PEW Charitable Trust (J. H-M. and J.J. T-C.); the NIH grants UC4 DK112217, U01DK11221702A1, R01HL145754, U01DK127768, U01DA052715 (G.V); and F31DK122677 (M.T.J.).

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Author notes

  1. These authors contributed equally: Michaël F. Michieletto, John J. Tello-Cajiao.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michaël F. Michieletto, John J. Tello-Cajiao, Walter K. Mowel, Leonel Joannas, Megan L. Clark, Monica T. Jimenez, Jasmine M. Wright & Jorge Henao-Mejia

  2. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michaël F. Michieletto, John J. Tello-Cajiao, Walter K. Mowel, Aditi Chandra, Sora Yoon, Leonel Joannas, Megan L. Clark, Monica T. Jimenez, Jasmine M. Wright, Patrick Lundgren, Christoph A. Thaiss, Golnaz Vahedi & Jorge Henao-Mejia

  3. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John J. Tello-Cajiao, Aditi Chandra, Sora Yoon & Golnaz Vahedi

  4. Penn Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John J. Tello-Cajiao, Aditi Chandra, Sora Yoon & Golnaz Vahedi

  5. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Patrick Lundgren & Christoph A. Thaiss

  6. Division of Allergy and Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Adam Williams

  7. Division of Protective Immunity, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Jorge Henao-Mejia

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Contributions

These studies were conceptualized by M.F.M., W.K.M., G.V. and J.H.-M. All experiments were designed, performed and analyzed by M.F.M. or W.K.M. with some help from M.L.C., M.T.J. and J.M.W. J.J.T.-C performed all of the bioinformatic analysis with the help of S.Y. for the stripe analysis and of P.L., A.W. and C.A.T. for scRNA-seq analysis. A.C. generated Hi-C, RNA-seq and ATAC-seq libraries and quality controls. L.J. generated the novel mouse strains. J.H.-M. and G.V. supervised the biological and computational parts of this project, respectively. M.F.M., J.J.T.-C. and J.H.-M. wrote the manuscript. G.V. edited the manuscript.

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Correspondence to Golnaz Vahedi or Jorge Henao-Mejia.

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Nature Immunology thanks Shigeo Koyasu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: L.A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Comparison of accessibility between the MNK3 cell line and primary ILC3.

a, Venn diagram representation of the differential chromatin accessibility between ATAC-seq peaks of MNK3 and small intestine (SI) CD4+ ILC3, and SI NCR+ ILC3 cells. Absolute numbers of peaks differentially open (red) or closed (blue) in MNK3, or with similar accessibility (gray) in all samples, are represented. b, View of the chromatin accessibility (ATAC-seq signal) at the Rorc, Il17a, Il17f, Il22 and Il23r loci in small intestine (SI) CD4+ ILC3, SI NCR+ ILC3, and MNK3 cells. c, Venn diagram representation of the differential accessibility analysis results in the region displayed in the right panel. d, View of the chromatin accessibility (ATAC-seq signal) at the Id2 locus in SI CD4+ ILC3, SI NCR+ ILC3, and MNK3 cells. Absolute numbers of peaks differentially open (red) or closed (blue) in MNK3, or with similar accessibility (gray) in all samples, are shown. e, Representative flow cytometry plots showing cell viability and the expression of ILC3-specific markers (IL-22, GM-CSF, CD90.2, RORγt, IL-17 and ID2) by MNK3 cells after stimulation with IL-2, IL-23, and IL-1β. The red line represents activated MNK3 cells and the black line represents unstained MNK3 cells.

Extended Data Fig. 2 Changes in compartment distribution in ILCs are accompanied by changes in expression patterns concordant with euchromatin/heterochromatin state.

a, Fraction of regions in the A or B compartments in common lymphoid progenitors (CLP), group 1 (splenic NK cells), group 2 (bone marrow ILC2s), and group 3 (MNK3) ILC. b, Proportion of regions in CLPs that flipped compartment status in at least one ILC group (brown) or that remained unchanged in all ILC groups (gray). Sidebar indicates the proportion of flipped regions that were in the A (red) or B (blue) compartment at the CLP stage. c, Examples of compartment state distribution in the vicinity of genes with known roles in ILC biology such as Id2, Tcf7, Nfil3, Gata3, Lifr and Tox. Regions in the A and B compartment correspond to positive (red) and negative (blue) values in the PC1 bar, respectively. Each square represents 25kb. d, Percentage of expressed genes among genes within compartments that underwent similar flipping from CLP. The Y-axis represents the different combinations of final compartment states in ILC for flipping regions originally in the status indicated on the left. Parenthetical numbers indicate the total number of genes overlapping the corresponding group of regions. Expressed genes correspond to genes with average RPKM > 1. RPKM: Reads per kilobase of transcripts per million mapped reads. Letter triplets represent the compartment status in group 1, group 2 and group 3 ILC, respectively. e, (left) Heatmap of the compartment status in ILCs of flipping regions that were in the B compartment in CLPs. Regions in the A and B compartment correspond to positive (red) and negative (blue) values in the PC1 bar. (right) Comparison of gene expression levels between group 1 (blue boxes), group 2 (green boxes) and group 3 (red boxes) ILCs of groups of genes undergoing similar compartment flipping from CLP. The Y-axis represents the different combinations of final compartment states in ILC. RPKM: Reads per kilobase of transcripts per million mapped reads. Letter triplets represent the compartment status in group 1, group 2 and group 3 ILC, respectively. Box shows dataset quartiles and whiskers the distribution range. Dots represent outliers as determined by the inter-quartile range. f, (left) Heatmap of the compartment status in ILCs of flipping regions that were in the A compartment in CLP. Regions in the A and B compartment correspond to positive (red) and negative (blue) values in the PC1 bar. (right) Comparison of gene expression levels between group 1 (blue boxes), group 2 (green boxes) and group 3 (red boxes) ILCs of groups of genes undergoing similar compartment flipping from CLP. The Y-axis represents the different combinations of final compartment states in ILC. RPKM: Reads per kilobase of transcripts per million mapped reads. Letter triplets represent the compartment status in group 1, group 2 and group 3 ILC, respectively. Box shows dataset quartiles and whiskers the distribution range. Dots represent outliers as determined by the inter-quartile range. g, Table with the p-values (Mann-Whitney U two-tailed test) for Fig. 1e,f of the pairwise comparison between ILC of expression levels of genes that underwent similar compartment flippings from the A or B compartment in CLP. Letter triplets represent the compartment status in group 1, group 2 and group 3 ILC, respectively.

Extended Data Fig. 3 Local structures of the 3D genome architecture are comparable at the sub-megabase scale and correlate with gene expression in each ILC group.

a, Comparison of the pileup plots computed over conserved vs ILC-specific (left) boundary elements and (right) Topological Associated Domains (TADs) in each group of ILCs. Numbers in the corner indicate average signal strength. b, Boxplot of cross-boundary ratios of TADs for group 1 (blue), group 2 (green) and group 3 (red) ILCs. Box shows dataset quartiles and whiskers the distribution range. Dots represent outliers as determined by the inter-quartile range. c, Boxplot of the size distribution of TADs defined by boundaries detected in each ILC (blue; group 1 ILC, green; group 2 ILC, red; group 3 ILC) and by boundaries conserved across ILCs (white). Box shows dataset quartiles and whiskers the distribution range. Dots represent outliers as determined by the inter-quartile range. d, Expression levels of genes with critical roles in ILC biology in group 1 (blue bars), group 2 (green bars) and group 3 (red bars) ILCs obtained by RNA-sequencing. RPKM: Reads per kilobase of transcripts per million mapped reads. e, Pileup plots of stripes detected in group 1 (left), group 2 (middle) and group 3 ILC (right). Axes in units of mean feature (stripes) size as computed for each group ILC.

Extended Data Fig. 4 3D architecture, chromatin accessibility, and enhancer activity patterns at the Id2 locus in each ILC group.

a, Visualization of the contact heatmap at the Id2 locus in (top) group 1, (middle) group 2, and (bottom) group 3 ILCs. Chromatin accessible regions determined by ATAC-seq (gray track), and H3K27ac deposition (blue track) determined by ChIP-seq (group1 and group 2) or Cut & Run (group 3) in each group of ILCs. Colored arcs represent loops detected in the corresponding contact map (blue; group 1 ILC, green; group 2 ILC, red; group 3 ILC). The location of the Id2 promoter is indicated with an extended vertical dashed line. Dotted and solid lines represent sub-TAD and TAD boundaries, respectively. Arrowheads indicate the position of boundaries conserved across all ILCs. Scale bar in heatmap represents normalized contact frequency.

Extended Data Fig. 5 The locus control region 1 (LCR1), but not LCR2, is specifically required for the development of all tissue resident ILC2s.

a, Representative transcription factor binding motifs enriched in ATAC-seq peaks (peaks from all ILCs pooled) within LCR1 (red section) and LCR2 (blue section). Numbers represent log odds detection scores as computed by HOMER. b, (top) Quantification of absolute numbers of splenic CD4+ T cells, CD8+ T cells, and NK cells in their different maturation states in LCR1+/+ and LCR1−/− mice. (bottom) Quantification of absolute numbers of splenic naive (CD62L+, CD44−) and effector (CD62L−, CD44+) CD4+ and CD8+ T cell populations, as well as CD8+ and CD11b+ dendritic cells. Splenic T cell numbers are one representative experiment that was repeated six times (n = 8 LCR1+/+ and 8 LCR1−/−). Splenic NK cell numbers are a pool of two independent experiments. This experiment was repeated four (n = 9 LCR1+/+ and 9 LCR1−/−). Tregs represents a pool of two independent experiments (n = 9 LCR1+/+ and 9 LCR1−/−) and was repeated three times. Myeloid cell numbers represent one experiment (n = 3 LCR1+/+ and 3 LCR1−/−). Error bars = SEM; and p-values: ns = not significant (Treg panel: Mann-Whitney U two-tailed test; Splenic T cells NK cell and Myeloid cell: Two-way ANOVA with multiple comparison and Bonferroni correction). c, Quantification of absolute numbers of splenic CD4+ T cells, CD8+ T cells, and NK cells in their different maturation state in LCR2+/+ and LCR2−/− mice (n = 4 LCR2+/+ and 4 LCR2−/−). Data represents one experiment that was repeated two times. Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005 (Two-way ANOVA with multiple comparison and Bonferroni correction). d, Representative flow cytometry gating strategy used for the identification of group 1 (NK cell), group 2 (ILC2s), and group 3 (ILC3s) ILC in small intestine lamina propria (siLP) of LCR1+/+ and LCR1−/− animals. e, Quantification of lungs and bone marrow (BM) ILC2s numbers of Arginase1 reporter mouse (Arg1YFP) and Arg1YFP; LCR1−/− mice (Live, CD45+, Lineage−, CD127+, CD90.2+, ST2+, YFP+) at steady state. Data is a pool of two independent experiments and were repeated two times. Each dot represents an individual mouse (n = 7 Arg1YFP; LCR1+/+ and 7 Arg1YFP; LCR1−/−). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005 (Mann-Whitney U two-tailed test). f, Quantification of IL-5 in the serum of LCR1+/+ and LCR1−/− mice at steady state by cytometry bead assay (CBA). Data is representative of two experiments. Each dot represents an individual mouse, (n = 7 LCR1+/+ and 6 LCR1−/−). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05 (Mann-Whitney U two-tailed test). g, (top) Representative flow cytometry gating strategy used for the identification eosinophils in the blood, spleen, and lungs of LCR1+/+ and LCR1−/− mice at steady state and (bottom) their subsequent quantification. Data represents one experiment. Each dot represents an individual mouse, (n = 4 LCR1+/+ and 4 LCR1−/−). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05 (Mann-Whitney U two-tailed test).

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Extended Data Fig. 6 The LCR1 impairs ILC2 development through reduction of Id2 expression and skewing toward ILC1- and ILC3-like ILC progenitors.

a, (left) Expression levels of Il7r and Id2 over the Uniform Manifold Approximation and Projection of the scRNA-seq data set described in Fig. 4a. (right), Violin plot representation of the expression levels of Id2 and Il7r across all identified clusters in Fig.4a. For each cluster (horizontal axis), the expression levels in LCR1+/+ (gray) and LCR1−/− (red) mice is shown. b, Hierarchical representation of similarity between the clusters identified in Fig. 4a. Similarity was measured using complete linkage over the Pearson correlation matrix between clusters. c, Gating strategy used to identify putative ILC3p in the bone marrow of LCR1+/+ and LCR1−/− animals. Lin−, CD127+ cells were pre-gated on Singlets, Live, CD45+ cells. d, Gating strategy for the identification of CLP: Common Lymphoid Progenitors (Live, CD45+, Lin−, CD127+, CD25−, ICOS−, CD135+, α4β7−); α-LP: alpha-Lymphoid Progenitors (Live, CD45+, Lin−, CD127+, ICOS−, CD135+, α4β7+, Id2+, CD25−, CD117+); ChILP: Common helper Innate Lymphoid Progenitors (Live, CD45+, Lin−, CD127+, CD135−, α4β7+, CD25−); ILCp: Innate Lymphoid Cell precursors (Live, CD45+, Lin−, CD127+, ICOS−, CD25−, CD117+, Sca1−, α4β7+, ID2+); ILC2p: group 2 Innate Lymphoid Cell precursors (Live, CD45+, Lin−, CD127+, CD90.2+, ST2+ or CD25+, ICOS+). e, Quantification of ILC2 progenitors in the fetal liver (FL), fetal intestine (FI) or fetal lungs (FLu)of LCR1+/+ and LCR1−/− E15.5 embryos. Data represents the pool of two independent experiments. Each dot represents an individual embryo (numbers: FL: = 21 LCR1+/+ and 20 LCR1−/− ; FI and FLu: 11 LCR1+/+ and 10 LCR1−/−). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005, **** = p ≤ 0.0001 (Mann-Whitney U two-tailed test). f, Volcano plot displaying upregulated (red rectangle) and downregulated (blue rectangle) genes in LCR1+/+ when compared to LCR1−/− cells for clusters in Fig. 5a representing ILCp populations. g, Savitzky-Golay smoothing curve of the normalized expression levels of Tox, Bcl11b, Ets1, Tcf7, and Zbtb16 along the pseudotime axis determined in Fig. 5e. P-value corresponds to Mann-Whitney U test between smoothed signals from LCR1+/+ (black curve) and LCR1−/− (red curve) mice. h, (left) Quantification of ID2 mean fluorescence intensity (MFI) in LCR2+/+ and LCR2−/− bone marrow (BM) CLP, a4b7+ CLP, aLP, ChILP, ILCp, ILC2p gated as shown in Extended Data Fig. 6d. (right) Representative histogram of ID2 expression in LCR2+/+ and LCR2−/− BM. Each dot represents an individual mouse (n = 3 LCR2+/+ and 4 LCR2−/−) and was observed in 2 independent experiments. Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01 (Two-way ANOVA with multiple comparison and Bonferroni correction). i, Representative FACS plots and histograms of GFP expression in wild-type (CD45.1+) or LCR1−/− CD45.2 BM previously transduced with retroviral particles encoding an empty vector expressing the GFP (empty_RV - green) or retroviral particles encoding Id2 and the GFP (Id2_RV – blue), at least eight weeks post engraftment in CD45.1+ hosts. Each dot represents an individual mouse (n = 8 LCR1−/− empty_RV; 12 LCR1−/− Id2_RV). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01 (Two-way ANOVA with multiple comparison and Bonferroni correction).

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Extended Data Fig. 7 Disruption of the 3D architecture of the Id2 locus reduces ILC2 numbers in the lung but does not drastically change other immune cell populations at steady state.

a, Representative flow cytometry gating strategy used for the identification of immune cell populations in digested lungs or in bronchoalveolar lavage fluid (BALF) at steady state or after 16 days of house dust mite (HDM) challenge in LCR1+/+ and LCR1−/− animals. b, Quantification of frequencies among CD45+ cells and absolute numbers of lung parenchyma ILC2s in LCR1+/+ and LCR1−/− mice at steady state, (n = 8 LCR1+/+ and 8 LCR1−/−). Data are representative of one experiment that was repeated five times. Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005 (Mann-Whitney U two-tailed test). c, Quantification of total number of cells of the indicated immune cell populations in Extended Data Fig. 7a in the lung parenchyma of LCR1+/+ and LCR1−/− mice at steady state, (n = 12 LCR1+/+ and 12 LCR1−/−). Data are a pool of two independent experiments, except CD4+ and CD8+ TRM which is a representative experiment. (n = 8 LCR1+/+ and 8 LCR1−/−). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05 (Mann-Whitney U two-tailed test or Two-way ANOVA with multiple comparison and Bonferroni correction).

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Extended Data Fig. 8 Acute intranasal papain and IL-33 challenges in LCR1-deficient mice.

a, Schematic representation of (left) the experimental acute papain or (right) recombinant IL-33 (rIL-33) challenges. Mice were challenged intranasally with either 30mg of papain (in 20mL of PBS) or with 200ng of rIL-33 (in 20ml of PBS) for four consecutive days. Euthanasia was carried out 24 hours after the last challenge. Lung parenchyma ILC2s and eosinophil numbers were assessed by flow cytometry. b, Flow cytometry gating strategy used for the identification of lung parenchyma ILC2s and eosinophils in LCR1+/+ and LCR1−/− mice challenged with papain or LCR1+/+ mice treated with PBS (20mL). Cells were pre-gated on SSC/FSC and Singlets. c, Quantification of ILC2 frequency among CD45+ cells, ILC2 and eosinophil numbers in LCR1+/+ and LCR1−/− mice challenged with papain, or LCR1+/+ mice treated with PBS (20mL). Data represents one experiment with males (green dots; n = 4 LCR1+/+ PBS, 4 LCR1+/+, and 4 LCR1−/− papain – 11.4-12.2-week-old). Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005, **** = p ≤ 0.0001 (One-way ANOVA with multiple comparisons and Bonferroni correction). d, Flow cytometry gating strategy used for the identification of lung parenchyma ILC2s and eosinophils in LCR1+/+ and LCR1−/− mice challenged with rIL-33 (200ng) or LCR1+/+ mice treated with PBS (20mL). Cells were pre-gated on SSC/FSC and Singlets. e, Quantification of ILC2 frequency among CD45+ cells, ILC2 and Eosinophil numbers in LCR1+/+ and LCR1−/− mice challenged with rIL-33 (200ng), or LCR1+/+ mice treated with PBS (20mL). Data is representative of two independent experiments (green dots; n = 3 LCR1+/+ PBS, 3 LCR1+/+ rIL-33, and 4 LCR1−/− rIL-33 – 9.4-9.9-week-old) and was repeated two times. Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.0005, **** = p ≤ 0.0001 (One-way ANOVA with multiple comparisons and Bonferroni correction).

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Extended Data Fig. 9 Disruption of the 3D architecture of the Id2 locus in ILC2 reduces HDM-induced allergic airway inflammation progression.

a, Quantification of NK and NKT cells numbers in the lungs of LCR1+/+ and LCR1−/− mice 16 days after the initial HDM challenge. Data is a pool of two age and sex-matched independent experiments containing males (green dots; n = 5 LCR1+/+ and 5 LCR1−/− – age 12.9-week-old) and females (orange dots, n = 8 LCR1+/+ and 8 LCR1−/− – age 6.9-week-old). The experiments were repeated three times. Error bars = SEM; and p-values: ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01 (Mann-Whitney U two-tailed test). b, Representative histological sections of lungs stained with hematoxylin and eosin (H&E - top) or Periodic Shift Acid (PAS - bottom) of 1 LCR1+/+ mice treated with PBS. The scale represents a length of 500mM and the magnification used is 40x. c, Representative flow cytometry plot (left) and quantification (right) of IL-5+ and IL-13+ producing CD4+ Th2 cells (CD4+, GATA3+) from lung parenchyma. Cells were ex vivo stimulated with PMA and Ionomycin for 4 hours in presence of Golgi inhibitor and identified as Live, CD45+, TCRβ+, CD4+, GATA-3+ cells Data is a pool of two age and sex-matched independent experiments containing males (green dots; n = 5 LCR1+/+ and 5 LCR1−/− – age 12.9-week-old) and females (orange dots, n = 8 LCR1+/+ and 8 LCR1−/− – age 6.9 week-old). The experiment was repeated three times. Error bars = SEM; and p-values: ns = not significant (Mann-Whitney U two-tailed test). d, (top left) Representative flow cytometry plots of IL-13+ and IL-5+ producing naive CD4+ T cells from LCR1+/+ and LCR1−/− mice polarized under Th2 conditions for 6 days. (Bottom left) Proportion of IL-13+, IL-5+ and IL-13+/IL-5+ producing Th2 cells. (Top right), Quantification of the geometric mean fluorescence intensity (gMFI) of GATA-3, and frequency of activated (CD44+) CD4+ Th2 cells at day 6 after polarization. Data is representative of three independent experiments. Each dot represents an individual mouse, (Females; n = 4 LCR1+/+ and 4 LCR1−/−). Error bars = SEM; and p-values: ns = not significant (Mann-Whitney U two-tailed test).

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Supplementary information

Reporting Summary

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Supplementary Tables

Supplementary Table 1. Compartment scores of common lymphoid progenitor, group 1, 2 and 3 ILCs. Each region is annotated with the corresponding set of overlapping genes. Related to Fig. 1 and Extended Data Fig. 1. Supplementary Table 2. RPKM values of group 1, 2 and 3 ILCs bulk RNA-seq data. Related to Figs. 1 and 2 and Extended Data Figs. 1–3. Supplementary Table 3. Cross-boundary ratio of conserved TADs in group 1, 2 and 3 ILCs. Related to Fig. 2. Supplementary Table 4. Coordinates of loops called in group 1, 2 and 3 ILCs. Loops pooled together. Seventh column contains unique identifier for further reference in Supplementary Tables 5–7 and Extended Data Fig. 7. Related to Fig. 3 and Extended Data Fig. 3. Supplementary Tables 5–7. Unique identifiers of loops detected in group 1 (Supplementary Table 5), 2 (Supplementary Table 6) and 3 (Supplementary Table 7) ILCs. Related to Fig. 3 and Extended Data Fig. 3. Supplementary Tables 8–10. Output of the Stripenn algorithm for group 1 (Supplementary Table 8), 2 (Supplementary Table 9) and 3 (Supplementary Table 10) ILCs. Related to Extended Data Fig. 3. Supplementary Table 11. List of motifs identified in LCR1, LCR2 and Rroid regions found by HOMER (knownMotifs.txt file). Related to Extended Data Fig. 5. Supplementary Table 12. List of marker genes of the clusters identified by scRNA-seq. Supplementary Table 13. sgRNA used to generate LCR1−/−, LCR2−/−, Gata3_BS−/−, Rora_BS−/− and Rora_Gata3dKO mice.

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Source Data Fig. 1

Source data for the histograms present in Fig. 1.

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Source data for the histograms present in Fig. 3.

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Source data for the histograms present in Fig. 4.

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Source data for the histograms present in Fig. 5.

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Source data for the histograms present in Fig. 6.

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Source data for the histology images in Fig. 6.

Source Data Extended Data Fig. 5

Source data for the histograms present in Extended Data Fig. 5.

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Source data for the histograms present in Extended Data Fig. 6.

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Source data for the histograms present in Extended Data Fig. 7.

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Source data for the histograms present in Extended Data Fig. 8.

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Source data for the histograms present in Extended Data Fig. 9.

Source Data Extended Data Fig. 9

Source data for the histology images in Extended Data Fig. 9.

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Michieletto, M.F., Tello-Cajiao, J.J., Mowel, W.K. et al. Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation. Nat Immunol 24, 42–54 (2023). https://doi.org/10.1038/s41590-022-01295-y

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