LTα₁β₂-expressing Rorgt⁺ ILC3s regulate Sirpα⁺CD4⁺Esam⁺ cDC2 homeostasis. (A) Schematic representation of the computational analysis performed to identify the ligand–receptor pairs potentially involved in intercellular ILC3-Tbet⁺ (Sirpα⁺CD4⁺Esam⁺) cDC2 communication. Differentially expressed (DE) genes between the different cDC subsets were identified; transcriptional data from ILC3s was integrated to infer potential ligands expressed by ILC3s (1), potential receptors expressed by Tbet⁺ cDC2s (2), and the target genes of the identified ligand-receptor pairs among the DE genes in Tbet⁺ cDC2s (3). (B) Hexagonal triwise plot depicting differentially expressed (black) and nondifferentially expressed (gray) genes between XCR1⁺ cDC1s, Tbet⁻ (Sirpα⁺CD4⁻Esam⁻) cDC2s, and Tbet⁺ (Sirpα⁺CD4⁺Esam⁺) cDC2s; each new hexagon reflects a twofold difference from the previous with clipping at 32-fold. Highlighted in green are the genes belonging to the Tbet⁺ (Sirpα⁺CD4⁺Esam⁺) cDC2 gene signature. Highlighted in red are representative genes used throughout the paper to identify the different cDC subsets. The rose plot on the top right corner depicts the percentage of differentially expressed genes in each cellular direction; each circle represents a 5% increase. (C) Top 10 GO terms associated with the Tbet⁺ (Sirpα⁺CD4⁺Esam⁺) cDC2 gene signature. (D) NicheNet analysis of the ILC3-expressed ligands and Tbet⁺ (Sirpα⁺CD4⁺Esam⁺) cDC2-expressed receptors potentially involved in mediating intercellular communication events between the two cell types. The top 20 prioritized upstream ligands based on Tbet⁺ cDC2 gene signature are shown in 1; in 2, the potential receptors associated with each ligand and expressed by Tbet⁺ cDC2s are shown; and in 3, the potential target genes of the identified ligand–receptor pairs are highlighted. (E) Representative flow cytometry histograms and quantification of LTβR expression in splenic XCR1⁺ cDC1s and Sirpα⁺ cDC2s from WT, Rag2^−/−, and Rag2^−/− x γc^−/− mice. n = 4 mice/group; results from one representative experiment of two performed; Kruskal–Wallis test with Dunn’s multiple comparisons post-test. MFI, mean fluorescence intensity. (F) Representative flow cytometry plots and quantification of splenic Sirpα⁺CD4⁺Esam⁺ cDC2s in WT, Rag2^−/− and Rag2^−/− x γc ^−/− mice treated with the antagonist LTβR-Fc decoy receptor. n = 5 control + 5 LTβR-Fc-treated WT mice, 5 control + 5 LTβR-Fc-treated Rag2^−/− mice, and 8 control + 7 LTβR-Fc-treated Rag2^−/− x γc^−/− mice; two independent experiments per genotype; two-way ANOVA with Sidak’s multiple comparisons post-test. (G) Representative flow cytometry plots and quantification of splenic Sirpα⁺CD4⁺Esam⁺ cDC2s in Rag2^−/− x γc ^−/− mice treated with an agonist LTβR antibody. n = 11 Rag2^−/− x γc ^−/− mice treated with control antibody, and 9 Rag2^−/− x γc^−/− mice treated with anti-LTβR; three independent experiments; Mann–Whitney U test. (H) Representative flow cytometry plots and quantification of splenic Sirpα⁺CD4⁺Esam⁺ cDC2s in Ltb^flox/flox control, Ltb^flox/flox x Cd19-Cre, Ltb^flox/flox x Rorc-Cre, and Ltb^flox/flox x Cd19-Cre x Rorc-Cre mice. n = 11 Ltb^flox/flox control, 15 Ltb^flox/flox x Cd19-Cre, 7 Ltb^flox/flox x Rorc-Cre, and 6 Ltb^flox/flox x Cd19-Cre x Rorc-Cre mice; data verified in at least two independent experiments per genotype; Kruskal–Wallis test with Dunn’s multiple comparisons post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.