H2-Aa deficiency increased cDC2 and altered their transcriptional program to promote cross-presentation. (A) Representative flow cytometry plots of the indicated cDC populations in spleens of WT and H2-Aa^cit/cit mice. Cells were gated on Lin− CD45⁺ Ly6C− CD11c+. (B) The total number of cDC2 in spleens of WT and H2-Aa^cit/cit mice. (C) MHC-I and CD80 mean fluorescence intensity (MFI) on cDC2. (D and E) Frequency (D) and MHC-I MFI (E) of cDC2 among tumor-infiltrating lymphocytes isolated from B16F10 melanomas collected on day 11 after B16F10 inoculation into the flank of mice. (F) Immunoblot analysis of Nlrc5 in lysates of panDC enriched from spleens of WT and H2-Aa^cit/cit(cit/cit) mice. α-tubulin was used as a loading control. (G) Uniform Manifold Approximation and Projection (UMAP) clustering of scRNA-seq data from splenic DC sorted from 3 naïve H2-Aa^cit/cit mice (right) and 3 naïve WT littermates (left), showing 10 color-coded clusters at a resolution of 0.2. (H) Proportion of each cell cluster identified in G. (I) KEGG pathway enrichment analysis of genes significantly increased in H2-Aa^cit/cit relative to WT Ccr2+ cDC2 (adjusted P ≤ 0.05, n = 410). One-sided hypergeometric test was used to determine the statistical significance of enrichment. (J) Volcano plot showing differentially expressed genes in WT versus H2-Aa^cit/cit(cit) Ccr2+ cDC2 (adjusted P ≤ 0.05, n = 854). Shaded areas contain genes with Log₂Fold change (FC) > 0.4 and Log₂FC less than −0.4. Data points represent individual mice with four mice per group (B–E). Data are representative of one experiment (G–J) or two independent experiments (A–F). WT littermates were used as controls (A–H). Error bars indicate SD (B–E). P values were determined by Student’s t test (B–E). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F4.