De novo T_RM generation upon secondary vaccination in previously unperturbed sites. (A and B) Mice received GFP⁺ OT-I T cells (A) or barcode-labeled OT-I T cells (B) and were subjected to DNA vaccination on the right hind leg, whereas the other hind leg remained unperturbed. (A) Analysis of T_RM frequencies in vaccinated (right leg) and nonvaccinated (left leg) skin sites >60 d after vaccination. (B) More than 60 d after primary vaccination, the nonvaccinated (left leg) skin site was subjected to DNA vaccination. More than 60 d after secondary vaccination, the primary and secondary vaccinated skin sites were harvested, and GFP⁺ T_RM at the two sites were quantified. Number of barcode-labeled T_RM detected at the primary and secondary skin vaccination site of nine mice. *, P < 0.05, Wilcoxon signed-rank test. Dots represent individual mice.