Activation of conventional DC1 and DC2 requires distinct signals. (A) scRNA-seq of CD11c⁺ and CD11b⁺ FACS-sorted cells from the thymus of 7-wk-old C57BL/6 mice. Cells were bioinformatically filtered to include only clusters expressing Flt3. UMAP plots show the analysis of 6,928 transcriptome events, identifying seven major clusters, marked by color-coded lines. (B) Violin plots displaying the normalized expression of signature genes associated with cell clusters defined in A. (C) Representative flow cytometry gating strategy for identifying thymic DCs (Ly6C⁻CD64⁻CD11c⁺MHCII⁺), DC1 (CCR7⁻XCR1⁺), aDC1 (CCR7⁺XCR1⁺), DC2 (CCR7⁻SIRPα⁺), and aDC2 (CCR7⁺SIRPα⁺). (D) Representative flow cytometry plots showing expression of eYFP by thymic DC populations described in C in Xcr1^iCreRosa26^eYFP (Xcr1^eYFP) mice. Gray cells represent all thymic cells gated as in C; green cells represent eYFP⁺ cells. (E) Frequency of eYFP⁺ cells (gated as in C) in Xcr1^iCreRosa26^eYFP (Xcr1^eYFP) mice; n = 4 mice from three independent experiments. (F) Numbers of thymic DCs (gated as in C) in Batf3^−/− and Zeb2^Δ1+2+3 mice, shown as KO/WT ratio of cell numbers; n = 7–9 mice from three independent experiments. (G) Numbers of thymic DCs (gated as in C) in Ifnar1^−/−Ifnlr1^−/−, Cd40l^−/−, and Tcra^−/− mice, shown as KO/WT ratio of cell numbers; n = 4–13 mice from at least two independent experiments. Data are shown as mean ± SD. Statistical analysis was performed by a one-sample t test and Wilcoxon test with a theoretical mean of 1; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and n.s., not significant.