The terminal differentiation of Sirpα⁺CD4⁺Esam⁺ cDC2s requires ILCs. (A) Representative immunofluorescence image of WT splenic cDCs. Spleen sections were stained with the indicated markers, allowing identification of the major anatomical regions and cDC subsets. CD11c⁺ voxels were used to mask cDC-specific markers and to create the surface renderings that are plotted on top of anatomically demarcated regions. Esam staining demarcated vessels and the marginal zone, but expression on DCs was too low for accurate quantification. n = 4 spleens. Scale bars, 200 µm for low-magnification images, 100 µm for insets. (B) Representative flow cytometry plots and quantification of splenic cDCs in WT, Rag2^−/−, and Rag2^−/− x γc^−/− mice. n = 12 WT mice, 8 Rag2^−/− mice, and 7 Rag2^−/− x γc^−/− mice; three independent experiments; two-way ANOVA with Sidak’s multiple comparisons post-test. (C) Representative flow cytometry plots and quantification of splenic cDCs in Rag2^−/− treated with depleting NK1.1 or CD90 antibodies. n = 12 Rag2^−/− + isotype antibody control mice, 11 Rag2^−/− + aNK1.1 mice, and 8 Rag2^−/− + aCD90 mice; at least two independent experiments per treatment regimen; two-way ANOVA with Sidak’s multiple comparisons post-test. (D and E) Number of ILCs (D) and cDCs (E) recovered from the spleens of Rag2^−/− x γc ^−/− mice, 14 d after adoptive transfer. n = 11 Rag2^−/− x γc ^−/− PBS control mice, 13 Rag2^−/− x γc^−/− + ILCs; three independent experiments; Mann–Whitney U test in D; two-way ANOVA with Sidak’s multiple comparisons post-test in E. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.