scRNA-seq reveals heterogeneity in thymic DC2. (A) scRNA-seq of CD11c⁺ and CD11b⁺ FACS-sorted cells from thymus of 7-wk-old C57BL/6 mice. Cells were bioinformatically filtered to include only clusters expressing Flt3, Csf1r, and Csf3r. UMAP plots show the analysis of 8,514 transcriptome events, identifying 9 major clusters, marked by color-coded lines. (B) Violin plots displaying normalized expression of signature genes associated with cell clusters defined in A. (C) Representative flow cytometry gating strategy for identifying the cell populations defined in A and B. Cells were pre-gated as shown in Fig. S1 D. The gating strategy identifies granulocytes (Ly6G/SiglecF⁺CD11b⁺), pDCs (SiglecH⁺), macrophages (Ly6C⁻CD64⁺), monocytes (Ly6C⁺CD11b⁺), aDC1 (CD11c⁺MHCII⁺CCR7⁺XCR1⁺), aDC2 (CD11c⁺MHCII⁺CCR7⁺SIRPα⁺), DC1 (CD11c⁺MHCII⁺ XCR1⁺), DC2 (CD11c⁺MHCII⁺SIRPα⁺CD11b⁺), and CX3CR1⁺ DC2 (CD11c⁺MHCII⁺SIRPα⁺CD11b^LowCX3CR1⁺). (D) Frequency of thymic myeloid cell populations among total CD11c⁺ and CD11b⁺ cells in the thymus of C57BL/6 mice from birth (0 days old) to 105-day-old mice; n = 2–7 mice from two independent experiments. (E) Total numbers of cells per thymus in 7-wk-old C57BL/6 mice; n = 3 mice. (F) Representative gating strategy for thymic CD11c⁺MHCII⁺ SIRPα⁺ cells. The graph represents the percentage distribution of individual thymic subpopulations in 7-wk-old C57BL/6 mice; n = 2 mice from two independent experiments. Data are shown as mean ± SD.