Non-equivalent activation of T cells across skin-draining LNs. (A–D) Mice were transferred with naïve 4get.OT-II cells, injected with papain OVA or CpG OVA in the ear pinnae or front footpad, and dLNs were harvested and assessed by histocytometry or flow cytometry at 48 h. (A and B) Expression of IRF4 and BATF on CD44⁺ OT-II cells after immunization with the indicated adjuvant. (C) Correlation plots of IRF4 gMFI with Gata3 gMFI, BATF gMFI, and frequency of 4get-GFP⁺ cells across different dLNs are shown. (D) Representative histocytometry plots and quantification of the frequency of pS6 and Gata3 expression in IRF4⁺Ki67⁺ OT-II T cells. (E) Mice transferred with naïve 4get.OT-II cells were injected with a fixed amount of papain and an increasing amount of OVA, as indicated, in the ear pinnae or front footpad then dLNs were harvested and assessed by flow cytometry at 48 h. CD44⁺ OT-II cell number, frequency of 4get-GFP⁺ cells, and Gata3, IRF4, and Tbet gMFI expression on CD44⁺ OT-II cells are shown. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two mice per 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). All figures are representative of at least three independent experiments.