Transcriptional signatures of migratory DCs in distinct draining LNs. B6 mice were immunized in the ear pinnae and front footpad with papain plus EαGFP and the corresponding dLNs were harvested 48 h later. (A) Representative plots showing total EαGFP expression within live cells. (B) Quantification of the number of EαGFP⁺ migratory DCs and EαGFP⁺ gMFI of migratory DCs. (C) Quantification of total EαGFP⁺ cells for the indicated DC subsets. (D–F) EαGFP⁺ DC populations were sorted from dLNs on day 2 for bulk RNA sequencing. DCs were sorted on Live, CD64⁻, Lineage⁻ (CD3, CD19, NK1.1), EαGFP⁺, MHC-II^HI CD11c^Int, XCR1⁻, EpCAM⁻, then sorted into CD11b⁺ CD301b⁺, CD11b⁺ CD301b⁻, and CD11b⁻ CD301b⁻ populations. The same gates were used for EαGFP⁻ DCs in naïve LNs. (D) PCA plot of all samples (left) and papain immunized DC populations (right). (E) Heatmap of the top 100 DEGs between EαGFP⁺ migratory DCs from auricular versus brachial dLNs (FDR < 0.05, 2× fold change expression). (F) Volcano plot of DEGs between antigen-bearing migratory DCs from auricular versus brachial dLNs. Red indicates a FDR < 0.05. Genes with a log fold change >2 are shown. (G and H) EαGFP⁺ migratory DCs were assessed for surface CD80 and CD86 expression by flow cytometry. (G) Representative flow plots for CD80 and CD86 expression on EαGFP⁺ CD301b⁺ cDC2s after papain OVA or CpG OVA immunization and (H) quantification for three indicated DC subsets are shown. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two 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–C, G, and H are representative of three to four independent experiments. D–F are representative of one independent RNA sequencing experiment with n = 3 per group.