Fig. 3. A subset of RORγt⁺T-bet⁺CD4⁺ ILC3 emerges during embryonic development.

(a-d) Flow cytometric analysis of embryonic T-bet ZsGreen reporter mice. a, Representative gating of E14.5 small intestine (SI) for identification of T-bet⁺RORγt^- ILC1 (grey shaded gate) and RORγt⁺ ILC3 subsets based on expression of CD4 and T-bet ZsGreen. (b) Representative histograms of marker expression by ILC1/NK or ILC3 subsets from E14.5 SI. (c) Representative plots of T-bet ZsGreen or NKp46 and CD4 expression by RORγt⁺ ILC3 isolated from SI at different indicated embryonic and postnatal time-points. (d) Quantification of frequency of designated RORγt⁺ ILC3 subsets over time in ontogeny. Graphs depict data as mean ± SEM, E14.5 n=4 (pooled samples) examined over 4 independent experiments, E16.5 n=2 (pooled samples) examined over 2 independent experiments, E18.5 n=10 (individual samples) examined over 3 independent experiments, d1 n=8 (individual samples) examined over 2 independent experiments, 4 w n=7 examined over 2 independent experiments. (e) Representative SI gating on T-bet⁺ RORγt-protein^- ILC1 and identification of NK1.1⁺RORγt-fate map(FM)⁺ ex-ILC3 in one day old Rorc(gt)^cre/wt × R26^eYFP newborn mice. Quantification of frequencies and absolute numbers of ex-ILC3 as mean ± SEM, n=6 examined over 2 independent experiments. (f) Representative SI gating on RORγt-protein⁺CD4⁺ LTi cells and identification of T-bet^-protein^-T-bet^-FM⁺ ILC3 in one day old Tbx21^cre/wt × R26^eYFP newborn mice. Quantification of frequencies and absolute numbers of T-bet-FM⁺ LTi cells as mean ± SEM, n=5 examined over 2 independent experiments. Detailed statistics are available in source data.