Figure Supplementary 6: Multiple challenges generate de novo T_RM cells without displacing pre-existing T_RM cells in skin.

a Experimental schematic related to Fig. 6b. Mice were transferred EGFP⁺ OT-I T_EFF cells i.v. and treated with DNFB to establish a population of (‘old’) T_RM cells. One cohort of mice was left untreated whereas a second cohort was adoptively transferred CD45.1⁺ gBT-I T_N cells followed by i.v. injection of gB-pulsed dendritic cells (DC gB). >20 d following priming mice were boosted with WSN-gB s.c. and left to rest another >30 d before boosting with X31-gB i.n to generate ‘new’ CD45.1⁺ gBT-I skin T_RM cells. DNFB-treated skin was analysed >30 d following final immunization. b Experimental schematic related to Fig. 6c. Mice were transferred CD45.1⁺ gBT-I T_EFF cells and treated with DNFB to establish a population of (‘old’) T_RM cells. Mice were transferred EGFP⁺ OT-I T_N cells then infected with VV-OVA e.c. contralateral to DNFB-treated skin. >30 d following primary VV-OVA infection mice were re-challenged with VV-OVA e.c. at an unrelated site to generate ‘new’ T_RM cells. DNFB-treated skin was analysed >25 d following secondary VV-OVA challenge.