Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein–directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.
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We are grateful to the Ares staff, genotyping facility and flow cytometry core for their technical assistance. This study was supported by grants from the UK Medical Research Council (U105178805 to J.L.B., M.W.D.H., M.G., H.E.J., M.D., R.B., A.C.) and the Wellcome Trust (100963/Z/13/Z to A.C.F.F., J.A.W., P.A.C., S.K., A.L.). A.C.H.S. was supported by a Croucher Cambridge International Scholarship.
The authors declare no competing interests.
Peer review information Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended Data Fig. 1 Characterization of DN1 embryonic thymus populations using 5xpolychromILC mice.
(a) Flow-cytometry analysis of the indicated cell surface markers or transcription factors in the E13.5, E14.5, E15.5, E17.5 and E19.5 embryonic thymus of 5xpolychromILC mice. (b) Flow-cytometric analysis of cKit and CD44 expression in DN1-Id2–Bcl11b–, DN1-Id2+Bcl11b+, DN2 and DN3 cells in the E15.5 embryonic thymus of 5xpolychromILC mice. (c) Principal component analysis (PCA) of RNA-seq data from indicated thymic cell populations (Fig. 2a) at E19.5 (n = 3). (d) Heatmap of genes from bulk RNA-seq analysis selected from single-cell gene expression analysis data (Fig. 1c). (e) Relative gene expression (from bulk RNA-seq analysis) of Zbtb16 and Pdcd1, in different embryonic thymus populations at E15.5 and E19.5 (n=3). Mean ± s.e.m; two-way ANOVA with Tukey post-hoc test. (f) Flow-cytometry analysis of RORγt-Katushka, Bcl11b-tdtomato, GATA-3-hCD2, RORα-Teal and Id2-BFP expression in the double negative (DN) and double positive (DP) cell subsets from E17.5 embryonic thymus. (g) Flow-cytometry analysis of RORγt-Katushka, Bcl11b-tdtomato, GATA-3-hCD2, RORα-Teal and Id2-BFP expression in ETP and ILC2p (lin-ST2+IL7Ra+) from E17.5 embryonic thymus.
(a) Flow-cytometry analysis of Bcl11b-tdtomato, GATA-3-hCD2, RORα-Teal and Id2-BFP in IL-7Rα+ST2+ ILC2p during embryonic thymus development. (b) Flow-cytometry analysis of ST2 and NK1.1 expression in the DN1 population in E16.6 embryonic thymus from wildtype (WT), Il33+/- or Il33-/- mice.
(a) Representative flow-cytometry gating strategy for the purification of ETP cells. (b) Representative flow-cytometry analysis of GFP, CD44 and ICOS expression by cells generated in vitro after co-culture of ETPs, transduced with empty or RORα overexpressing vector, with OP9-DL1 stromal cells in the presence of growth factors (IL-7 and Flt3-L). GFP+ cells represent the positively transduced cells. (c) Flow-cytometric analysis of Bcl11b-Tom, Id2-BFP, RORα-TEAL and GATA-3-hCD2 expression in pro-T cells (ICOS-CD44-) and ILC2p (ICOS+CD44+) generated in vitro after co-culture of ETPs purified from 5xpolychromILC mice with OP9-DL1 stromal cells in the presence of growth factors (IL-7 and Flt3-L). (d) Western blot analysis from HEK cells transiently transfected with overexpressing constructs (pcDNA3) for RORα-FLAG-T2A (62 kD), RORα-FLAG (58 kD) or GATA-3-HA-T2A (48 kD), immunoprecipitated with anti-T2A or anti-FLAG antibody and detected using anti-FLAG or anti-T2A antibody, respectively. Data are representative of 2 independent experiments.
(a) Representative ATAC-seq tracks for thymic ETP, DN2, DN3, NKp and ILC2p, and binding profiles of RORα-T2A and GATA-3-T2A in ILC2 purified from lymph nodes of Rorateal/teal, Gata3hCD2TR/+ or wild type mice, and expanded in vitro with IL-7 and IL-33, around the Arntl and Clock loci. Tracks shown are representative of three independent experiments. (b) Representative ATAC-seq tracks for thymic ETP, DN2, DN3, NKp and ILC2p, and binding profiles of RORα-T2A and GATA-3-T2A in ILC2 (as in Fig. S4a) around the Il1rl1, Il2ra and Rora loci.
a) Gene expression (RPKM from bulk RNA-seq analysis) (top panel) and tSNE plots (log2 expression from single cell analysis) (lower panel) showing Arg1, Il13, Icos and Il1rl1, in different embryonic thymus populations. Data represent mean ± s.e.m. (n=3 biologically independent samples). (b) Representative ATAC-seq tracks for thymic ETP, DN2, DN3, NKp and ILC2p, and binding profiles of RORα-T2A and GATA-3-T2A in ILC2 purified from lymph nodes of Rorateal/teal, Gata3hCD2TR/+ or wild type mice, and expanded in vitro with IL-7 and IL-33, around the type-2 cytokine locus. Tracks shown are representative of three independent experiments.
(a) Representative ATAC-seq tracks for thymic ETP, DN2, DN3, NKp and ILC2p around the Bcl11b locus. Tracks shown are representative of three independent experiments. (b) Representative ATAC-seq tracks for thymic ETP, DN2, DN3, NKp and ILC2p and binding profiles of RORα-T2A and GATA-3-T2A in ILC2 in ILC2 purified from lymph nodes of Rorateal/teal, Gata3hCD2TR/+ or wild type mice, and expanded in vitro with IL-7 and IL-33 around the Id2 and Nfil3 locus. Tracks shown are representative of three independent experiments. (c) A luciferase assay shows the activity (relative to empty vector) of a DNA fragment containing the RORα-binding site from the Id2-associated -143 kb regulatory region, in the presence of increasing concentrations (as indicated) of RORα, GATA-3 or both. Data are representative of three independent experiments; mean ± s.e.m.; two-way ANOVA with Tukey post-hoc test.
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Ferreira, A.C.F., Szeto, A.C.H., Heycock, M.W.D. et al. RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus. Nat Immunol 22, 166–178 (2021). https://doi.org/10.1038/s41590-020-00833-w
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