Figure Supplementary 4: Skin T_RM cells undergo local proliferation and cell death after rechallenge.

a Experimental schematic. Mice were seeded with effector Thy1.1⁺ gBT-I (T_EFF) cells and treated with DNFB, or were seeded with naïve Thy1.1⁺ gBT-I (T_N) cells and infected with VV-gB. >30d after treatment or infection mice were administered Thy1.1 antibody to deplete Thy1.1⁺ gBT-I T_CIRCM cells. Mice were then challenged with HSV infection or transcutaneous peptide application and treated daily with BrdU i.p. post-challenge. b, c Ki67 expression by b DNFB-lodged skin Thy1.1⁺ gBT-I T_RM cells or c splenic Thy1.1⁺ gBT-I T_CIRCM cells 7d after HSV challenge. Data are representative of 2 experiments with n=2, 3 or 6 in b or n=5 or 6 mice per group in c. **p=0.0072, Kruskal Wallis test with Dunn’s multiple comparisons in b and **p=0.0043, two-tailed Mann Whitney U-test in c. d Proportion of BrdU⁺ or BrdU^- DNFB-induced skin Thy1.1⁺ gBT-I cells expressing CD103 in Thy1.1 antibody treated mice 7d post-HSV challenge. Data are pooled from 3 experiments, n=6 mice. n.s.; non-significant, p=0.2835, two-tailed Mann Whitney U-test. e Mice were seeded with either Thy1.1⁺ gBT-I T_N cells and infected with HSV or Thy1.1⁺ gBT-I T_EFF cells and treated with DNFB. >30 d later mice were treated with Thy1.1 antibody. Shown is proportion of CD103⁺ Thy1.1⁺ gBT-I T_RM cells in DNFB-treated or previously HSV infected skin incorporating BrdU 2d following topical application of gB peptide. Data are pooled from 3 experiments with n=3 or 4 mice per group. n.s.; p=0.5137 and p=0.6070, ***p=0.0001, ****p<0.0001, two-tailed Mann Whitney U-test. f Number of DNFB-lodged Thy1.1⁺ gBT-I T_RM cells in DNFB treated skin (left panel) of Thy1.1 antibody treated mice, and AnnexinV and PI expression by these cells at the indicated time points following HSV infection above the DNFB patch. Data are representative of 1 or 3 experiments with n =3 or 4 mice per time point per experiment. *p=0.0435 (middle panel) or 0.0291 (right panel), Kruskal Wallis test. g AnnexinV and PI staining of DNFB-lodged Thy1.1⁺ gBT-I skin T_RM cells 7d post-HSV infection above the DNFB patch or at steady state. Data are pooled from 3 experiments with n=11 or 13 mice per group. ***p=0.0002, two-tailed Mann Whitney U-test. Bars represent the mean.