Rorgt⁺ ILC3s nonredundantly control the homeostasis of Sirpα⁺CD4⁺Esam⁺ cDC2s. (A) Representative immunofluorescence image of WT splenic cDCs and ILCs. Spleen sections were stained with the indicated markers allowing identification of the different cDC and ILC subsets. CD11c⁺ voxels were used to mask cDC-specific markers as shown in the middle panel. ILC subsets were identified based on the expression of Eomes, Gata3, NK1.1, and Rorgt. Note also the presence, in the right panel, of Eomes⁺NK1.1⁻ cells that most likely represent regulatory T cells. n = 3 spleens. Scale bars, 200 µm for low-magnification images, 100 µm for insets. (B) Schematic representation of cDC and ILC subset distribution in the spleen shown in A. (C) Spatial frequency distribution of WT cDCs around Rorgt⁺ ILC3s for the spleen shown in A. The numbers on the plot reflect the percentage of cDCs from each subset present in a 25-µm radius from Rorgt⁺ ILC3s. Bottom: Summary of median nearest neighbor distance for the three spleens analyzed. Repeated-measures ANOVA with Tukey’s multiple comparisons post-test. (D) Probability of observing a cDC of a given subset as a function of spatial distance from Rorgt⁺ ILC3s in the spleen shown in A. The observed probability is shown in bold. The result of randomly relabeling Rorgt⁺ ILC3s across the ILC repertoire is show in gray (n = 39). The upper and lower boundary values for those simulations are depicted by the dotted lines. (E) Representative flow cytometry plots and quantification of splenic cDCs in Rorc^+/+, Rorc^GFP/+, and Rorc^GFP/GFP mice. n = 7 Rorc^+/+ mice, 8 Rorc^GFP/+ mice, and 8 Rorc^GFP/GFP mice; two independent experiments; two-way ANOVA with Sidak’s multiple comparisons post-test. (F) Representative flow cytometry plots and quantification of splenic cDCs in Rag2^−/− x Rorc^+/+, Rag2^−/− x Rorc^GFP/+ and Rag2^−/− x Rorc^GFP/GFP mice. n = 10 Rag2^−/− x Rorc^+/+ mice, 11 Rag2^−/− x Rorc^GFP/+ mice, and 7 Rag2^−/− x Rorc^GFP/GFP mice; three independent experiments; two-way ANOVA with Sidak’s multiple comparisons post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.