Transcriptional signatures of naïve and activated cDC2s. (A–D) B6 mice were immunized in the ear pinnae or front footpad with papain or CpG plus EαGFP as indicated and harvested for flow cytometry 48 h later. (A) Total number of EαGFP⁻ cells for the indicated DC subsets. (B) Frequency of total EαGFP⁺ cells of the indicated cell subsets. (C and D) Representative flow plots (C) and quantification (D) showing EαGFP and YAe expression on migratory DCs after immunization with the indicated adjuvant. (E and F) EαGFP⁺ cDC2 populations were sorted from dLNs on day 2 for bulk RNA sequencing as in Fig. 3 D. (E) Heatmap of all DEGs with a FDR < 0.05 and >2× fold change between naïve and papain immunized DCs from auricular and brachial LNs is shown. (F) Volcano plot of DEGs between papain immunized and naïve DCs from auricular and brachial dLNs. Red indicates a FDR < 0.05. Genes with a log fold change >2 are shown. (G) Migratory DCs were assessed for surface PD-L1 and PD-L2 expression 2 days after papain immunization by flow cytometry. Quantification of the frequency of PD-L1⁺ PD-L2⁺ for the indicated DC subsets is shown. (H) GO biological process term enrichment analysis based on all DEGs in EαGFP⁺ CD11b⁺ DC populations between auricular and brachial dLNs. Data shown represents one independent experiment with at least n = 6 independently immunized lymph nodes from three mice per group. Data from multiple pooled experiments are denoted by different symbols within the same group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). A–D and G are representative of three independent experiments. E, F, and H are representative of one independent RNA sequencing experiment with n = 3 per group.