Active eosinophils regulate host defence and immune responses in colitis

Abstract

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies^1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4⁺ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

Fig. 1. A-Eos and B-Eos are two distinct GI-resident eosinophil subsets.

a, Uniform manifold approximation and projection (UMAP) of eosinophil transcriptomes obtained from the BM, blood, spleen, small intestine, stomach and colon of Il5-tg mice (n = 3). b, Eosinophil differentiation trajectory. c, Subset distribution across organs (% of eosinophils). d, Expression of cluster marker genes. A complete list of cluster markers is available in Supplementary Table 1. e, Top, UMAP of Cd80 and Cd274 expression. Bottom, expression levels over pseudotime. f, Top, UMAP of eosinophil proteomic (spectral flow cytometry) profiles isolated from blood, spleen, stomach, colon and small intestine. Bottom, heat map of median surface marker expression across subsets (n = 5, B6J). g, Representative FACS plots of A-Eos (PD-L1⁺CD80⁺) and PD-L1⁻CD80⁻ eosinophils across organs. Numbers indicate percentage of eosinophils. h, Representative immunofluorescence of Siglec-F and CD80 in the mouse colon (n = 3, B6J). Arrows mark Siglec-F⁺CD80⁺ A-Eos (red) and Siglec-F⁺CD80⁻ B-Eos (green). Nuclei stained with DAPI. Scale bar, 20 µm. i, Mean fluorescence intensity (MFI) of CD63, SSC-A and Siglec-F in colonic A-Eos and B-Eos (n = 6, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. j, Left, representative images of cytospinned intestinal A-Eos and B-Eos stained with anti-EPX and DAPI (n = 3, Il5-tg). Scale bar, 10 µm. Right, quantification of EPX staining intensity at cell periphery and centre. Data are mean ± s.d. Two-tailed unpaired Student’s t-test. k, Active-to-basal ratio in luminal versus basal third of colonic crypts (n = 3, B6J). Two-tailed paired Student’s t-test. l, Left, active-to-basal ratio in luminal versus basal third of colonic crypts of healthy human colon cores (n = 5). Two-tailed paired Student’s t-test. Right, active-to-basal ratio in samples from healthy individuals (5 individuals, 9 cores), patients with Crohn’s disease (CD; 5 individuals, 9 cores) and patients with ulcerative colitis (UC; 4 individuals, 8 cores) samples. One-way ANOVA. Data are mean ± s.d. Patient information is provided in Supplementary Table 2. In a,b,e,f, dots represent single cells, coloured by cluster identity.

Fig. 2. A-Eos have antibacterial and immune-regulatory functions.

a–c, A-Eos frequencies in H. pylori-infected (a; stomach, n = 6), C. rodentium-infected (b; colon, n = 5) and DSS-treated (c; colon, n = 8) mice relative to uninfected controls (n = 5–10, B6J). a,b, Data are pooled from two independent experiments. Medians are shown. Two-tailed unpaired Student’s t-test. d, Percentage of eosinophil subsets across organs at steady state and during infection, as assessed by scRNA-seq. e, A-Eos frequencies after conditioning with colon CM. Input: BM-derived (n = 5, B6J), blood (n = 5, Il5-tg) and splenic (n = 5, Il5-tg) eosinophils. Data are mean ± s.d. Two-tailed unpaired Student’s t-test. f, A-Eos frequencies among adoptively transferred CD45.2⁺ eosinophils in colon and spleen of host, 42 h after injection (n = 4, CD45.1). Input A-Eos frequency shown as a reference (spl, splenic eosinophils, n = 2, Il5-tg). Medians are shown. Two-tailed unpaired Student’s t-test. g, Gene expression over common pseudotime at steady state (grey) and during C. rodentium infection (dark red). Dots indicate single cells, coloured by organ (BM, blood and colon). h, C. rodentium (ICC180) viability after exposure to blood, splenic or colonic eosinophils (n = 3, pooled Il5-tg) or conditioned BM-Eos (n = 3, pooled B6J). Technical replicates and medians are shown. Two-tailed unpaired Student’s t-test. i, Expression of MHC-I-restricted antigen processing and presentation signature and IFNγ-regulated genes. Genes used for scores and signatures are listed in Supplementary Table 3. Data are mean ± s.d. Two-sided Wilcoxon test (n = 3, Il5-tg). j,k, Proliferation of anti-CD3 and anti-CD28 (anti-CD3/CD28)-activated, carboxyfluorescein succinimidyl ester (CFSE)-labelled naive CD4⁺ T cells co-cultured with conditioned splenic (j; Spl) or sorted GI (k) A-Eos and B-Eos (n = 7, Il5-tg mice). Medians are shown. One-way ANOVA.

Fig. 3. A-Eos maturation is induced locally by IL-33.

a, Activity of A-Eos-specific regulons across clusters. g, number of genes in regulon. b, Expression of NF-κB signalling components. c, Quantification of pNF-κB p65⁺ cells in colonic A-Eos and B-Eos (n = 3, B6J). Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test. d,e, A-Eos and B-Eos frequencies in antibiotic-treated (d) (n = 16, B6J) and germ-free (GF) (e) (n = 9, B6J) mice relative to controls. SPF, specific pathogen free. d, Data are pooled from two independent experiments. Medians are shown. Two-tailed unpaired Student’s t-test. f, Depleted gene sets in PD-L1⁺CD80⁺ A-Eos (red) and PD-L1⁻CD80⁻ eosinophils (grey), relative to BM stem cells. Kolmogorov–Smirnov test. Dot size indicates gene-set size. Dashed line indicates P = 0.05. g, A-Eos frequencies after conditioning of BM-Eos with IL-33, colon CM and anti-IL-33 (n = 2, pooled B6J). Technical replicates and mean ± s.e.m. are shown. One-way ANOVA. h, Colonic A-Eos and B-Eos frequencies in B6J (n = 21) and Myd88^−/− (n = 15) mice treated with IL-33, relative to untreated controls. Medians are shown. Two-tailed unpaired Student’s t-test. WT, wild type.

Fig. 4. A-Eos co-localize with CD4⁺ T cells in patients with IBD.

a, Top, Venn diagram of significant differentially expressed genes (DEGs) (false discovery rate (FDR) < 0.05, logFC > |2|) in BM-Eos treated with IL-33 and/or IFNγ (n = 4, B6J). All DEGs are listed in Supplementary Table 4. Bottom, expression of subset markers across conditions. Columns are clustered, rows are scaled. CPM, counts per million. b, Proliferation of anti-CD3/CD28-activated, CFSE-labelled naive CD4⁺ T cells co-cultured with BM-Eos conditioned with IL-33 and/or IFNγ (n = 4, B6J). Data are pooled from two independent experiments. Medians are shown. One-way ANOVA. c, A-Eos frequencies in mice treated with IL-33 and/or IFNγ (n = 5, B6J). Medians are shown. One-way ANOVA. d, A-Eos and B-Eos frequencies in DSS-treated B6J (n = 5) and Il33^−/− (n = 4) mice. Medians are shown. Two-tailed unpaired Student’s t-test. e, Frequencies of IFNγ-, IL-17- and TNF-expressing colonic CD4⁺ T cells from mice in d. Medians are shown. Two-tailed unpaired Student’s t-test. f, Left, representative haematoxylin and eosin (H&E)-stained colonic sections of mice in d. Scale bar, 100 µm. Right, colitis score assessed by histopathological examination. Medians are shown. Two-tailed unpaired Student’s t-test. g, Representative molecular cartography images of human ulcerative colitis samples. Nuclei are stained with DAPI; CD4, SIGLEC8 and CD80 RNA molecules are shown in blue, red and yellow, respectively. Scale bar, 200 µm. h, Pairwise proximity score of transcripts across slides. The score indicates the fraction of slides in which the proximity of a pair of transcripts is significantly higher than expected by chance. P values are computed using a permutation test (Methods). T_reg cells, regulatory T cells. i, Mean counts per slide of CD80 and NFKB1 transcripts in the proximity (<10 µm) of SIGLEC8 transcripts spatially associated with CD4 molecules versus SIGLEC8 molecules not associated with CD4 molecules. The central line in the box plot represents the median count per slide, the lower and upper hinge correspond to the first quartiles and the whisker extends from the hinge to the smallest or largest value no further than 1.5 times the interquartile range (IQR) from the hinge. Two-sided paired Wilcoxon test (17 regions of interest (ROIs), n = 4 patients).

Extended Data Fig. 1. scRNA-seq reveals five distinct eosinophil subpopulations.

a, Experimental workflow of scRNA-seq. b, UMAP of all sequenced single-cell transcriptomes passing quality control, clustered and annotated manually based on marker gene expression. c, Distribution of unique molecular identifiers (nUMI, log10 normalized), genes (nGenes, log10 normalized) and mitochondrial gene fraction (mitoRatio, log10 normalized) per cell across samples. d, Expression density of canonical eosinophil marker genes. e, Subset organ distribution. Dashed lines indicate eosinophil subsets from Fig. 1a. f, Significantly enriched (adjusted P < 0.05) GSEA terms across clusters. Kolmogorov–Smirnov test. g, Left: cell-cycle score. Middle: stemness score. Right: granulogenesis score. Data represents mean ± SD. Two-sided Wilcoxon test (n = 3, Il5-tg). h, Expression of cell-cycle genes across eosinophil subsets. Rows are genes and columns are single cells, coloured by scaled expression. i, Expression of mKi67, Epx and S100a6 over pseudotime. j, Receptor gene expression in A-Eos and B-Eos. k, Immune-regulatory score across subsets. Data represents mean ± SD. Two-sided Wilcoxon test (n = 3, Il5-tg). Genes used for scores and signatures are listed in Supplementary Table 3.

Extended Data Fig. 2. PD-L1 and CD80 expression define active eosinophils in the GI tract.

a, GI surface marker gene expression in A-Eos and B-Eos. b, UMAP showing the normalized protein expression intensity of eosinophil surface markers (n = 4, B6J). c, Frequencies of A-Eos as assessed by flow cytometry (n = 4–6, B6J). Data represents mean ± SEM. One-way ANOVA. Data pooled from two independent experiments. d, Mean fluorescence intensity (MFI) of CD9, CD31, CD54 and CD95 across colonic eosinophil subsets. FMO: fluorescence minus one. e, Expression of A-Eos markers, normalized to Gapdh in A-Eos and B-Eos sorted from the small intestine (n = 4, Il5-tg). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. f, Frequencies of CD63⁺, CD9⁺ and CD107a⁺ cells in A-Eos and B-Eos as assessed by flow cytometry (n = 6-7, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. Data pooled from two independent experiments. g, EPX immunofluorescence in sorted blood and spleen eosinophils (n = 3, Il5-tg). Nuclei are stained with DAPI. Scale bar, 10 µm. h, Schematic representation of basal (lower) and luminal (upper) third of the mucosa. i, Representative immunofluorescence images of Siglec-F and CD80 in the mouse colon (n = 3, B6J). Arrows mark Siglec-F⁺CD80⁺ A-Eos (red) and Siglec-F⁺CD80⁻ B-Eos (green). Nuclei are stained with DAPI. Dashed lines delimit the border of luminal and basal third. Scale bar, 20 µm.

Extended Data Fig. 3. PD-L1⁺CD80⁺ A-Eos are specific to the mouse GI and enriched in human IBD.

a, Representative FACS plots of PD-L1⁺CD80⁺ and PD-L1⁻ CD80⁻ eosinophils (n = 3, B6J). Numbers indicate % of eosinophils. b,c, UMAP of eosinophil transcriptomes (shown in Fig. 1a) including those isolated from uterus, lung and adipose tissue (n = 4, Il5-tg). Cells coloured by organ (b) and by cluster (c). d, Subset distribution across organs (% of eosinophils). e, List of shared or unique markers (logFC > 0.5, P adjusted < 0.05) between A-Eos and tissue eosinophils. Non-parametric Wilcoxon rank sum test (FindMarkers function in Seurat). f, Representative FACS plots of PD-L1⁺CD80⁺and PD-L1⁻CD80⁻ eosinophils in HDM- or PBS-treated mice (n = 2, B6J). Numbers indicate % of eosinophils. g, Left: Gating strategy used to identify resident (rEos) and inflammatory (iEos) eosinophils as described by. Right: quantification of PD-L1⁺CD80⁺ and PD-L1⁻CD80⁻ eosinophils in rEos and iEos. Medians are shown. h, MBP and PD-L1 immunofluorescence staining of human tissue microarrays. Representative cores from a healthy individual and a patient with Crohn’s disease are shown (n = 5). Scale bars, 500 µm (core overview) and 10 µm (high magnification insets).

Extended Data Fig. 4. Challenge infection induces a compositional shift toward the A-Eos cluster.

a, Left: Representative FACS plots of the A-Eos and PD-L1⁻ CD80⁻ eosinophils. Numbers indicate % of eosinophils. Right: Absolute counts of A-Eos of mice shown in Fig. 2a–c. Medians are shown. Two-tailed unpaired Student’s t-test. b, Data integration of challenge datasets (dark-red dots, n = 4, Il5-tg). Steady-state dataset (grey) used as a reference. Corresponding steady-state organs shown in black. c, Data integration of DSS dataset (dark-green dots, n = 3, B6J). Steady-state dataset (grey) is used as a reference. Sstate colon shown in black. d, UMAP of integrated (left) and merged (right) steady-state (grey) and challenge (red dots) datasets. e, Left: BM–blood-colon eosinophil Monocle trajectory at steady state and following C. rodentium infection. Each dot represents a single cell coloured by cluster identity. Right: RNA velocities (scvelo) in BM, blood and colon dataset as steady state and during C. rodentium infection. f, Significant DEGs (logFC > 0.5, adjusted P < 0.05) of circulating eosinophils found in the colon vs in the blood of C. rodentium-infected mice. Non-parametric Wilcoxon rank sum test (FindMarkers function in Seurat). g, Single-cell fate probabilities as calculated by CellRank and summarized for each cluster as a pie chart. Arrows represent velocity flow. Cells and pie charts coloured by cluster identity. h, Top: Workflow of in vitro conditioning. Bottom: A-Eos frequencies after conditioning with increasing doses of colon CM. Input: BM-derived (n = 5, B6J), blood (n = 5, Il5-tg) and splenic (n = 5, Il5-tg) eosinophils. Medians are shown. One-way ANOVA. i, Left: EYFP⁺ eosinophil frequencies over time across organs after single tamoxifen pulse in Id2^CreERT2;Rosa^EYFP mice. Data represents mean ± SD. Right: Frequency of A-Eos and B-Eos in colonic EYFP⁺ eosinophils at day 2 and 4 post tamoxifen injection (n = 3, Id2^CreERT2;Rosa^EYFP). Medians are shown. Two-tailed unpaired Student’s t-test. j, Antimicrobial and granulogenesis signature expression in A-Eos. k, Gene expression over common pseudotime at steady state (grey) and upon C. rodentium infection (dark red). Dots indicate single cells, coloured by organ: BM (blue), blood (yellow) and colon (red). l, Edu⁺/Edu⁻ eosinophil ratio in the colon of C. rodentium-infected and control B6J (n = 5) and Il5-tg (n = 3) mice at day 4 post EdU injection. Data represent mean ± SEM. Two-tailed unpaired Student’s t-test. m, Frequencies of eosinophil progenitors (gated as Live CD45⁺CD11b⁺IL5Ra⁺Lin-Sca1⁻CD34⁺) in C. rodentium-infected (n = 17) and control (n = 9) B6J mice. Medians are shown. Data pooled from two independent experiments. Two-tailed unpaired Student’s t-test. n, MFI of CD63 in colonic A-Eos and B-Eos of C. rodentium-infected and control mice (n = 6, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. o, EPX immunofluorescence of sorted A-Eos of C. rodentium-infected and control mice (n = 5, Il5-tg). Nuclei are stained with DAPI. Insets show protrusions. Scale bar, 10 µm.

Extended Data Fig. 5. A-Eos interact with T cells.

a, Ligand–receptor interactions between eosinophils and CD4⁺ T cells (left) or CD8⁺ T cells (right) predicted by CellPhoneDB. Dot size and colour indicate interaction mean. b,c, Representative H&E-stained colonic sections (b) and colitis score (c) in B6J (n = 17) and PHIL (n = 13) mice assessed by histopathological examination; data are pooled from two independent experiments. Medians are shown. Two-tailed unpaired Student’s t-test. Scale bars, 100 µm. d, Frequencies of IFNγ, IL-17 and TNF-expressing colonic CD4⁺ T cells of DSS-treated B6J (n = 17) and PHIL (n = 13) mice. Medians are shown. Two-tailed unpaired Student’s t-test. e, Left: CFSE dilution of T cells co-cultured with BM-derived eosinophils conditioned as indicated and loaded with ovalbumin (OVA) peptide. Right: Representative FACS plots of the CFSE dilution. Numbers indicate % of CFSE dilution (n = 3, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. f, Left: A-Eos (PD-L1⁺CD80⁺) and B-Eos (PD-L1⁻CD80⁻) frequencies in stomach, colon and small intestine of B6J (n = 5) and Il5-tg (n = 5) mice. Medians are shown. Two-tailed unpaired Student’s t-test. Right: Representative FACS plots. Numbers indicate % of eosinophils. g, UMAP of B6J colonic eosinophils (orange) at steady state (n = 6) and during C. rodentium infection (n = 5) integrated in the Il5-tg dataset (grey). Il5-tg colonic eosinophils at steady state and during C. rodentium infection in black. h, Antimicrobial signature, IFNγ-regulated gene signature and antigen processing and presentation via MHC-I in B6J colon and B6J colon + C. rodentium. Data represents mean ± SD. Two-sided Wilcoxon test (n = 3). i, A-Eos frequencies in H. pylori-infected (stomach, n = 5) and C. rodentium-infected (colon, n = 4–7) B6J and Il5-tg mice, relative to uninfected controls. Medians are shown. Two-tailed unpaired Student’s t-test. j,k Absolute counts of A-Eos and B-Eos in colon and small intestine of B6J mice treated with anti-IL-5 (j, n = 5) or anti-CCR3 (k, n = 5) neutralizing antibodies and the respective isotype control. Medians are shown. Two-tailed unpaired Student’s t-test.

Extended Data Fig. 6. A-Eos are induced by NF-κB signalling.

a, Regulon activity across clusters. b, Representative regulons projected on UMAP plot. Cells are coloured by binary regulon activity. c, Pathway activity across clusters according to PROGENy analysis. d, Gene expression relative to Hprt measured by qRT–PCR of eosinophils sorted from the blood (n = 6), spleen (n = 6) and colon (n = 4) of Il5-tg mice. Data represents mean ± SEM. One-way ANOVA. e, Representative images of pNF-κB p65 immunofluorescence staining in colonic eosinophils (n = 3, B6J). Arrows mark A-Eos (Siglec-F⁺ CD80⁺, red) and B-Eos (Siglec-F⁺ CD80⁻, green). Nuclei are stained with DAPI. Scale bar, 20 µm. f, A-Eos frequencies upon conditioning of BM-Eos with colon CM and/or NF-κB inhibitor (n = 5, B6J). Data represents mean ± SD. One-way ANOVA. g, Representative FACS plots of colonic A-Eos (PD-L1⁺CD80⁺) and PD-L1⁻CD80⁻ (B-Eos), relative to Fig 3d. Numbers indicate % of eosinophils. h, Left: Representative FACS plots of colonic A-Eos (PD-L1⁺CD80⁺) and PD-L1⁻CD80⁻ (B-Eos), relative to Fig 3e. Numbers indicate % of eosinophils. Right: MFI of Siglec-F and % CD63 in colonic A-Eos and B-Eos shown in Fig. 3e. Medians are shown. Two-tailed unpaired Student’s t-test. i, Colonic A-Eos and B-Eos frequencies at steady state in B6J (n = 5) Tlr2^−/− (n = 3) and Tlr4^−/− (n = 7) mice. Medians are shown. One-way ANOVA.

Extended Data Fig. 7. IL-33 induces the accumulation of A-Eos in the colon.

a, Experimental workflow of the CRISPR inhibition screen. b, Log10 negative score per gene, as calculated by MAGeCK. Cd80 and Cd274 evidenced in orange. Genes involved in TNF signalling pathway via NF-κB in red, and MAPK signalling pathway in darkred. c, IL-33 concentrations measured by ELISA in colon of DSS-treated mice (n = 12, B6J) and colon and blood of C. rodentium-infected mice (n = 7, B6J), compared to untreated controls (n = 7, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. d, IFNγ, TNF and IL-22 concentrations measured by LEGENDplex in colon (left) and blood (right) of C. rodentium-infected mice (n = 7, B6J), compared to untreated controls (n = 7, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. e, A-Eos (PD-L1⁺CD80⁺) frequencies upon conditioning of BM-Eos with colon CM, IL-22, IL-25, TNF or IL-33 (n = 4, B6J). Data are pooled from two independent experiments. Medians are shown. One-way ANOVA. f, A-Eos frequencies after conditioning with increasing doses of IL-33. Input: BM-derived (n = 5, B6J), blood (n = 5, Il5-tg) and splenic (n = 5, Il5-tg) eosinophils. Medians are shown. One-way ANOVA. g, Western blot of phospho-p38 and phospho-p65 upon conditioning of BM-Eos with colon CM or IL-33 (n = 3, B6J). h, Gene expression normalized to Hprt measured by qRT–PCR of BM-Eos upon conditioning with IL-33 (n = 4, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. i, ST2 expression in BM-Eos upon IL-33 treatment (n = 4, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. j, ST2 expression in colonic A-Eos and B-Eos (n = 5, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t-test. k, ST2 expression across organs (n = 5, B6J). Data represents mean ± SEM. One-way ANOVA. l, A-Eos frequencies upon conditioning of WT (n = 2, pooled B6J) or ST2^−/− (n = 2, pooled) BM-Eos with colon CM or IL-33. Technical replicates and mean ± SEM are shown. Two-tailed unpaired Student’s t-test. m, Left: Representative FACS plots of A-Eos (PD-L1⁺ CD80⁺) and PD-L1⁻CD80⁻ eosinophils in the blood (top) and spleen (bottom). Numbers indicate % of eosinophils. Right: A-Eos frequencies in mice treated with IL-33 (n = 6-7, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. n, A-Eos and B-Eos frequencies in the indicated organs of B6J (n = 7) and Il33^−/− (n = 5) mice at steady state. Medians are shown. Two-tailed unpaired Student’s t-test.

Extended Data Fig. 8. A-Eos co-localize with CD4⁺ T cells in human IBD.

a, Regulon activity in A-Eos across conditions (n = 4, Il5-tg). b, Multidimensional scaling (MDS) plot of bulk RNA-seq samples shown in Fig.4a. c, Heat map of signature gene expression across conditions of samples shown in Fig. 4a. d, A-Eos (PD-L1⁺CD80⁺) frequencies upon treatment of BM-Eos with IL-33 and/or IFNγ. (n = 4, B6J). Data represents mean ± SEM. One-way ANOVA. e, EPX immunofluorescence of A-Eos upon exposure to IFNγ for 90 min. Splenic eosinophils were magnetically enriched (n = 2, Il5-tg), treated overnight with colon CM and A-Eos sorted by flow cytometry. Scale bar, 10 µm. f, Frequencies of PD-L1⁺ and CD80⁺ in colonic eosinophils of WT (n = 6, B6J) and Eo-Cre;Ifngr^fl/fl mice (n = 4) upon C. rodentium infection, relative to uninfected controls (n = 2, B6J). Medians are shown. Two-tailed unpaired Student’s t-test. g, Left: UMAP of single-cell eosinophil transcriptomes isolated from the colon of anti-IFNγR-treated, C. rodentium-infected or control Il5-tg mice (n = 3). Middle: expression of IFNγ target genes. Right: Expression of granule and antimicrobial signatures. Data represents mean ± SD. Two-sided Wilcoxon test. h, Observed vs. expected number of contacts between clusters of SIGLEC8 and CD4 molecules shown per slide. P Values are computed based on a two-sided permutation test (see Methods). i, Proportions of segmented cells expressing SIGLEC8 only (blue) or co-expressing both SIGLEC8 and CD4 (red) across slides. Dotted horizontal line shows mean. j, Mean count per slide of molecules of a given transcript in the proximity (<10 µm) of SIGLEC8 RNA molecules spatially associated with CD4 molecules vs SIGLEC8 molecules not associated with CD4 molecules. The central line in the box plot represents the median count per slide, the lower and upper hinge corresponds to the first quartiles and the whisker extends from the hinge to the smallest or largest value no further than 1.5 x IQR from the hinge. Two-sided paired Wilcoxon test (17 ROIs, n = 4 patients).