(A) BALB/c mice were exposed to AFL at different laser settings followed by ID injection of 10 µg OVA right after treatment in A and B or at different time points in C. OVA injection into sham-treated skin served as control. Laser parameter(s) were fixed at 5% in A, 10 mJ/300 Hz in B, and 10 mJ/10%/300 Hz in C. Serum anti-OVA antibody titer was measured 3 weeks after immunization. (D and E) CD4⁺ T cells were purified from DO11.10 mice, stained with CFSE, and adoptively transferred to BALB/c mice followed by ID delivery of 10 μg OVA into AFL- or sham-treated skin or ID delivery of 10 μg OVA in the presence of Alum adjuvant, or left nonimmunized (NI). Draining LNs were harvested 4 days later followed by single-cell suspension preparation, immunostaining, and flow cytometry analysis. Representative dot plots about percentage of KJ1-26⁺ cells in CD4⁺ T cells (D). Percentage of KJ1-26⁺ cells in CD4⁺ T cells of different groups (E). Gating strategies are shown in Supplemental Figure 2A. (F–H) LN cells prepared in D and E were stimulated with OVA_323–339 peptide overnight followed by immunostaining and flow cytometry analysis. Representative dot plots showing percentage of IL-4– and IFN-γ–secreting cells in KJ1-26⁺ CD4⁺ T cells (F). Percentage of IL-4– and IFN-γ–secreting cells in KJ1-26⁺ CD4⁺ T cells are shown in G and H, respectively. Gating strategies are shown in Supplemental Figure 2B. n = 4–8 (A–C), n = 4–6 (D–H). One-way ANOVA with Tukey’s multiple-comparison test was used to compare differences between groups (A and B). One-way ANOVA with Newman Keuls multiple-comparison test was used to compare differences (C, E, G, and H). *, P < 0.05; **, P < 0.01; NS, not significant.