Abstract
Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3−/− CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3−/− and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3−/− CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3−/− CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.
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Acknowledgements
We thank the Genome Technology Access Center at the McDonnell Genome Institute at Washington University for RNA-seq, ATAC-seq, CUT&RUN-seq and scRNA-seq. The center is supported in part by the Alvin J. Siteman Cancer Center and Barnes-Jewish Hospital, and the Institute of Clinical and Translational Sciences (ICTS). The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant (P30 CA091842) and the ICTS is funded by the National Institutes of Health’s NCATS Clinical and Translational Science Award (CTSA) program grant (UL1 TR002345). We thank E. Lantelme and D. Brinja and the Pathology and Immunology Flow Cytometry Core for cell sorting. This work was supported by grants UO1 AI095542, RO1 DK126969, RO1 DK132327, RO1 AI134035, RO1 DK124699 and U19 AI142733 (to M. Colonna). K.Y. was supported by the Rheumatology Research Foundation Tobé and Stephen E. Malawista, MD Endowment in Academic Rheumatology, CHRC K12 and K08DK128544.
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K.Y., T.T., R.S., P.F.R., A.U.A., H.I., B.D. and P.L.C. performed experiments and analyzed the data. K.Y., H.I., P.L.C., M. Cella, E.M.O., M.T.B. and M. Colonna designed experiments. H.I. and M.T.B. provided crucial reagents. S.G. maintained the mouse colony. K.Y. and M. Colonna wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 Aiolos does not alter abundance of IELs.
Cell counts of γδ IEL, DN IEL, DP IEL, CD4+ IEL and CD8αβ+ IEL from small intestine in Ikzf3+/+ and Ikzf3−/− mice. Data are representative of two independent experiments (n = 4) and all samples are biological replicates. DN = double negative, DP = double positive (CD4 cytotoxic), Data represent mean ± SD. P values were determined by two-tailed, unpaired Student’s t-test.
Extended Data Fig. 2 NKG2D does not directly induce cytotoxicity in unconventional IELs.
a, Gene expression of NK receptors associated with DAP12 (Klrk1, Klrc2 and Klra8) in γδ IEL and DN IEL from small intestine in Ikzf3+/+ and Ikzf3−/− mice based on RNA-seq. b, γδ IEL and DN IEL from small intestine in Ikzf3+/+ and Ikzf3−/− mice were sorted, and co-cultured with RMA-S or RMA-S overexpressing Rae1γ. Frequency of live target cells are shown. Data showed one experiment in b and all samples are biological replicates. P values in a were calculated by Wald test (DEseq2). WT=Ikzf3 +/+ ; KO=Ikzf3 –/–.
Extended Data Fig. 3 scRNA-seq demonstrate heterogeneity of IELs.
a, UMAP plots of 25,966 cells of Ikzf3+/+ small intestine CD45+ IELs. b, Representative UMAP plots depicting expression of Trac, Cd8a, Tcrg-V7 and Tcrg-V1. c, Heatmap displaying top differentially expressed genes in clusters designated as CD8aa-1, CD8αα-2, CD8αα-3, CD8αα-4, CD8αα-5, CD8αα-6, CD8αβ+, CD4+, pDC, B cells and plasma cells.
Extended Data Fig. 4 CD8αα+ IELs comprise diverse groups of cells.
a, Heatmap displaying top differentially expressed genes in clusters designated as Ikzf3−/−Tcf7−Il2rbhi, Ikzf3+/+Tcf7−Il2rbhi, Tcf7−Il2rblo, Tcf7+, stressed CD8αα, lipidolytic CD8αα, proliferating CD8αα and IFN-stimulated CD8αα. b, Representative UMAP plots depicting expression of Klra4, Klra8, Klrc2, Il2ra, Cd200r1 Cd200r2, Lat, Lat2, Lag3, Cd200r4, Pdcd1, Havcr2, Lair2 and Il7r.
Extended Data Fig. 5 Ikzf3-deficiency enhances responses to IL-15.
a, Heatmap showing differentially expressed cytokine genes in the duodenum and ileum from Ikzf3+/+ mice. b-d, Flow cytometry plots and quantification of NKG2A (b), Ly49A and D (c) and NKG2D (d) on γδ IEL and DN IEL from small intestine in Ikzf3+/+ and Ikzf3−/− mice 72 hours after culturing with either 10 ng/ml or 100 ng/ml of IL-15. Data are representative of two independent experiments and all samples are biological replicates. n=8 in b, c and n=5 (Ikzf3+/+) and 6 (Ikzf3−/−) in d. Data represent mean ± SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test in b, c and d.
Extended Data Fig. 6 Generation and phenotype of Aiolos conditional deficient mice.
a, Diagram of the construct for conditional Ikzf3fl/fl mice showing LoxP sites flanking exon 4 and exon 6. b, Representative histograms show tdTomato expression in γδ IEL and DN IEL, DP IEL, CD4+ IEL, CD8αβ+ IEL, NK cells and B cells from small intestine in E8ICre R26RtdTomato mice. c, Representative image of colon from Ikzf3fl/fl and E8ICreIkzf3fl/fl mice at day 4 post DSS administration in the drinking water. Data are representative of two independent experiments.
Extended Data Fig. 7 Interactions of Aiolos with STAT5 and RUNX members.
a, A pie chart showing the chromosomal location of Aiolos binding regions in the indicated cell types. b, A Venn diagram depicting Aiolos binding peaks in γδ IELs and DN IELs. c, Represenative histogram plots and quantification of pSTAT5 expression in γδ IEL, DN IEL from the small intestine of Ikzf3+/+ and Ikzf3−/− mice. Cells were either not stimulated or stimulated for 15 minutes with IL-15 (100 ng/ml). d, The results of permutation test designed to analyze the significance of overlap of Aiolos with either STAT5, RUNX1 or RUNX3. Evperm: the number of permutation events, Evobs: the number of observed events (overlapped peak counts). Data are representative of two independent experiments and all samples are biological replicates (n = 4 in c). P values were calculated by unpaired, two-tailed Student’s t-test in c and Permutation test in d (ChIPpeakAnno). Data represent mean ± SD. pSTAT5 = phosphorylated STAT5, UTR = untranslated region.
Extended Data Fig. 8 Aiolos shapes a distinct epigenetic landscape.
a, Gene expression of IKZF family members in γδ IEL and DN IEL from the small intestine in Ikzf3+/+ and Ikzf3−/− mice based on RNAseq (see Fig. 2). The expression of Ikzf3 in Ikzf3–/– IEL reflects a portion of the nonfuntional transcript present in the knockout mouse. b, Venn diagrams depicting overlap of Aiolos binding regions, differential H3K27ac peaks and DAR, and the result of permutation test designed to analyze the significance of overlap of differential H3K27ac peaks with DAR in γδ IELs and DN IELs. FDR were calculated by Wald test (DESeq2) in a and P values were calculated Permutation test in b (ChIPpeakAnno). DAR = differential accessible regions.
Supplementary information
Supplementary Tables 1 and 2
Supplementary Table 1 Comprehensive list of DEGs in the indicated cell types, along with their respective associations with DAR, differential H3K27ac, binding of Aiolos, STAT5, RUN1 or RUNX3. ‘+’ indicates positive association, whereas ‘–’ denotes no association. Supplementary Table 2 Quality-control metrics of ATAC-seq and CUT&RUN-seq for Aiolos and H3K27ac.
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Yomogida, K., Trsan, T., Sudan, R. et al. The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes. Nat Immunol 25, 77–87 (2024). https://doi.org/10.1038/s41590-023-01693-w
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DOI: https://doi.org/10.1038/s41590-023-01693-w