Fig. 1: PD-1 is retained on mouse and human CD8⁺ T_RM cells in the absence of persistent antigen.

a, CyTOF uniform manifold approximation and projection plots showing the relative expression of PD-1 protein on T cells with the relative distribution of NK cells, CD8⁺ T cells and CD4⁺ T cells in human PBMCs versus human skin. After biexponential transformation, each plot is normalized to minimum and maximum intensities, based on downsampled and combined flow cytometry standard (FCS) files of human PBMCs (n = 4, combined with roughly equal weight) and human skin (n = 5, combined with roughly equal weight). b, Representative histogram plot of PD-1 expression on CD4⁺ or CD8⁺ CD69⁺CD103^+/−CD4⁺ T_RM cells or CD69⁺CD103^+/−CD8⁺ T_RM cells isolated from human skin (n = 5, combined) and PBMCs (n = 4, combined). c, Expression of PD-1, CD69, CD45RO, CD103 and CLA on CD4⁺ and CD8⁺ T cells from human skin versus PBMCs as in b. d, Heat map of immune checkpoint, activation and chemokine molecules derived from the CyTOF phenograph or Louvain clusters (shown as median intensity); green, skin-specific clusters; red, common markers of T_RM cells. e, Median PD-1 intensity (number of ions detected by CyTOF) ± s.e.m. for total antigen-experienced CD45RO⁺CD45RA⁻CD8⁺ T cells (CD8⁺ T_RM cells) or antigen-experienced CD45RO⁺CD45RA⁻CD69⁺CD49⁺CD8⁺ T_RM cells (antigen-experienced T cells) across PBMCs (n = 4), skin (n = 5), lung (n = 4), colon (n = 6), tonsil (n = 5), liver (n = 3) and spleen (n = 3). Linear model with heterogenous variance, with site as a fixed effect; **P ≤ 0.01; ***P ≤ 0.001. f, Schematic showing Thy1.2⁺ or Thy1.1⁺ C57BL/6J mice injected intravenously with 2 × 10⁵ Rag1^−/− Thy1.1⁺ or Thy1.2⁺ OT-I T cells (OT-I cells, unless otherwise specified) infected with VACV-OVAss at day 0 followed by identification of donor Thy1.1⁺/Thy1.2⁺Va2⁺CD8⁺ OT-I cells at week 6 after infection with 1 × 10⁶ viral plaque-forming units (p.f.u.) on each ear and 2 × 10⁶ on the tail (top) and representative histogram of PD-1 expression based on staining with antibody clone RMP1-30 relative to isotype on CD69⁺CD103^+/− T_RM cells (skin T_RM cells), spleen and LN KLRG1⁻CD62L⁺ T_CM cells (T_CM cells) and spleen and LN KLRG1⁻CD62L⁻ T_EM cells (T_EM cells) at week 6 after infection (bottom); D, day. g, Percentage of PD-1⁺ cells among CD69⁺CD103^+/–CD8⁺ T_RM, CD127⁺CD62L⁺CD8⁺ or KLRG1⁻CD62L⁺CD8⁺ T_CM and CD127⁺CD62L⁻CD8⁺ or KLRG1⁻CD62L⁻ CD8⁺ T_EM cells isolated at week 6 from the spleen or LN of VACV-OVAss-infected mice as in f. Data were pooled from four independent experiments (n = 12) and were analyzed by unpaired t-test; ****P ≤ 0.0001. h, Real-time PCR of viral load in the tail skin of mice as in f at day 10, 21 or 42 after infection (n = 5 mice per time point); LOD, limit of detection (marked with a horizontal line); W, week. i, Schematic showing transfer of Thy1.2⁺ OT-I cells into Thy1.1 C57BL/6J mice at day –1 before skin scarification with VACV-OVA at day 0 and collection of skin, LN and spleen every week between weeks 1 and 6 after infection (left), representative flow cytometry plots of CD69 versus CD103 on CD8⁺ OT-I cells isolated from ear skin and KLRG1 versus CD62L on CD8⁺ OT-1 cells isolated from the LNs of Thy1.2⁺ C57BL/6J recipient mice at week 2 and week 1 after infection, respectively (middle), and frequency of PD-1⁺ cells in skin Thy1.2⁺Va2⁺CD8⁺PD-1⁺CD8⁺ OT-I cells, including CD69⁻CD103⁺ cells, epidermal CD69⁺CD103⁺ T_RM cells, dermal CD69⁺CD103⁻ T_RM cells and circulatory CD69⁻CD103⁻ T cells and KLRG1^+/−CD62L^+/− cells in LN and spleen, including CD62L⁺KLRG1⁻ T_CM cells, CD62L⁺KLRG1⁺ T_eff cells, CD62L⁻KLRG1⁺ T_eff cells and CD62L⁻KLRG1⁻ T_EM cells at weeks 1 to 6 after infection (right; n = 5–10 mice pooled from two independent experiments). j, Schematic showing tdTomato⁺ OT-I cells transferred to 6- to 8-week-old C57BL/6 mice at day –1, followed by skin scarification with VACV-OVA at day 0 and analysis of T_RM cells in the tail skin at day 657 after infection (top), gating strategy on live CD45⁺CD3⁺CD8⁺ T cells (bottom left) and representative histograms showing the expression of PD-1 on epidermal T_RM cells and dermal T_RM cells at day 657 after infection (bottom right). Histograms were concatenated and calculated from five of six mice with robust detectable PD-1 staining (n = 2–4 mice per group from two independent experiments). k, Percentage of PD-1⁺ cells (left) and PD-1 mean fluorescence intensity (MFI; right) in epidermal CD69⁺CD103⁺ T_RM cells (epidermal T_RM cells) and dermal CD69⁺CD103⁻ OT-I T_RM cells (dermal T_RM cells) isolated at day 657 from mice as in j (n = 5 mice). Bars and error bars show mean ± s.e.m.