Fig. 2: PD-1 is required for epidermal and dermal T_RM cell formation and specifies T_RM cell fate.

a, Schematic showing the transfer of equal mixes of 1 × 10⁵ each of PD-1^+/+ CD45.2⁺Thy1.2⁺ C57BL/6J OT-I cells with PD-1^−/− CD45.2⁺Thy1.1⁺ OT-I cells or PD-1^+/+ CD45.2⁺Thy1.2⁺ OT-I cells with PD-1^+/+ CD45.2⁺Thy1.1⁺ (sex-matched littermate controls of PD-1^−/− CD45.2⁺Thy1.1⁺) OT-I cells into CD45.1 Thy1.2 C57BL/6J mice at day −1, followed by VACV-OVAss at day 0 and quantification of OT-I cells in the skin at days 10, 21 and 42 after infection (left) and quantification of the total number of PD-1^+/+ or PD-1^−/− CD3⁺CD8⁺Va2⁺CD69⁺CD103^+/− OT-I T_RM cells isolated from skin at days 10, 21 and 42 after infection (right). Data were pooled from three independent experiments (n = 14) at day 10, six independent experiments (n = 30) at day 21 and four independent experiments (n = 20) at day 42 for the PD-1^+/+ + PD-1^−/− mixes and from two independent experiments (n = 9) at day 10, one independent experiment (n = 4) at day 21 and three independent experiments (n = 12) at day 42 for PD-1^+/+ + PD-1^+/+ (littermate control) mixes. Statistical significance between the number of PD-1^+/+ OT-I and PD-1^−/− OT-I cells was estimated using a linear mixed-effects model with group by time interaction and replication as fixed effects and a random intercept for each mouse; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. b, Representative flow cytometry plots showing skin PD-1^+/+ or PD-1^−/− CD8⁺Va2⁺ OT-I cells from the mixed transfers as in at days 10, 21 and 42. c, Quantification of ear skin PD-1^+/+ and PD-1^−/− CD3⁺CD8⁺Va2⁺CD69⁺CD103^+/− OT-I T_RM cells in mice transferred with an equal mix of PD-1^+/+ and PD-1^−/− OT-I cells as in at day 42 after infection; ***P ≤ 0.001. d, Schematic showing the transfer of an equal mix of PD-1^+/+ CD45.2 Thy1.2⁺ and PD-1^−/− CD45.2⁺Thy1.1⁺ OT-I cells at day −1 into C57BL/6J mice, followed by VACV-OVAss at day 0, administration of BrdU by intraperitoneal injection daily at days 5–10 and isolation of T cells from ear skin at day 10 after infection (top) and representative flow cytometry plots and quantification (bottom) of the percentage of skin BrdU⁺ cells among PD-1^+/+ or PD-1^−/− cells among ear skin Va2⁺CD69⁺CD103^+/− OT-I T_RM cells (bottom; n = 18, data were pooled from three independent experiments). Bars and error bars show mean ± s.e.m. Statistics were estimated using a linear mixed-effects model with group by treatment interaction as fixed effects and a random intercept for each mouse; ****P ≤ 0.0001. e, Schematic showing the transfer of tdTomato⁺ OT-I cells into CD11c–eYFP recipient mice at day −1, followed by VACV-OVAss at day 0, intraperitoneal anti-PD-1 or isotype treatment on days 0, 3, 6 and 9 after infection, isolation of ear tissue at days 10–14 after infection (top) and representative immunofluorescence images showing tdTomato⁺ OT-I cells (white arrows), CD11c–eYFP⁺ DCs and DAPI nuclear staining in skin cross-sections from CD11c–eYFP mice at days 10–14 after infection (bottom). Dashed lines indicate the dermal–epidermal border. f, Quantification of tdTomato⁺ OT-I cells isolated from epidermal sheets of isotype- or anti-PD-1-treated C57BL/6J mice as in e on days 10–14 after infection; isotype, n = 12; anti-PD-1, n = 14. Data were pooled from three independent experiments. Statistics were estimated using a linear mixed-effects model with group by treatment interaction as fixed effects and a random intercept for each mouse. Bars and error bars show mean ± s.e.m.; *P ≤ 0.05. g, Schematic showing the transfer of tdTomato⁺ or Thy1.1⁺ C57BL/6J OT-I cells into Thy1.2⁺ C57BL/6J mice at day −1, followed by VACV-OVAss immunization at day 0, treatment with inhibitory anti-PD-1 or isotype by intraperitoneal injection on days 0, 3, 6 and 9 after infection, treatment with FTY720 by intraperitoneal injection every 2 days starting at day 35, challenge with topical PBS or SIINFEKL on the right (R) or left (L) depilated flank sites of each mouse at day 42, isolation of PBS- and SIINFEKL-treated flank skin at day 49 (left) and quantification of epidermal CD69⁺CD103⁺ T_RM cells and dermal CD69⁺CD103⁻ T_RM cells from anti-PD-1- and isotype-treated mice at day 49 after infection (right). Data were pooled from four independent experiments; isotype, n = 13; anti-PD-1, n = 18. Statistics were estimated using a linear mixed-effects model with group and by treatment challenge as fixed effects and a random intercept for each mouse; **P ≤ 0.01; ***P ≤ 0.001.