Extended Data Fig. 1: PD-1 is rapidly expressed on activated T cells but retained on TRM after VACV-OVAss.

a. Human skin and b. PBMCs showing the relative distribution of NK, CD8+ and CD4 + T cell clusters by CyTOF analysis. c. Quantification of cell percent derived from skin phenograph or louvain clusters. Green lines represent 80% skin specific clusters while blue lines are clusters that are less than 80% skin specific. d. Schematic showing Thy1.1 C57BL/6J OT-1 cells were transferred into Thy1.2 C57BL/6J mice at day −1 followed by VACV-OVAss immunization on day 0 and early harvest kinetics (top). PD-1 expression on OT-I cells isolated from the draining LN (dLN), non-draining LNs, and spleen at 0, 48, 72 and 120 hours after mice received 2 × 106 viral pfu on ear by VACV-OVAss. Flow cytometry of data from one individual mouse is shown per time point (bottom). e. Representative flow cytometry of Vα2 + Vβ5 + CD8+ populations 0, 48, 72 and 120 hours post-infection. f. Day 42 representative flow cytometry gating showing percent of cells with PD-1 cell surface expression using anti-PD-1 clone: RMP1-30 or g. using anti-PD-1 clone: 29 F.1A12. Quantification of percent fraction of PD-1 expressing skin TRM relative to LN and spleen TCM or TEM populations at 6 weeks using anti-PD-1 clone 29 F.1A12 (left). Each dot represents individual mice. h. Schematic showing Thy1.2 + OT-I cell were transferred into Thy1.1 + C57BL/6J mice at day −1, followed by VACV-OVAss (4 × 106 viral pfu on ear and tail) on day 0, and skin, LN and spleen harvest at 1 to 6 weeks post-infection (left). PD-1 geometric mean fluorescence intensity (MFI) on skin, spleen, and LN CD8 + OT-I cell subsets between 1 to 6 weeks post infection (middle). CD69 and CD103 cell-surface expression was used to gate four quadrants of CD8 + OT-I cells in skin. TRM were gated from Va2 + CD8+Thy1.2+ donor cells, and defined as CD69 + CD103+ (epidermal) or CD69 + CD103- (dermal). TCM and TEM were identified by KLRG1-/CD62L+ and KLRG1-/CD62L- gating respectively, while TEffector were defined by KLRG1 + CD62L +/− in LN and spleen (right). i. Schematic of OT-I Thy1.1 transfer into Thy1.2 recipients with immunization as in h. but with harvest of flank, ear, and tail skin at day 10–12 post-infection (top). PD-1 representative histograms of epidermal CD69 + CD103+ or dermal CD69 + CD103- skin OT-I TRM of ear, flank, and tail skin were concatenated from 5 mice per site (bottom). j. % PD-1 positive cells in flank, ear, and tail dermal and epidermal TRM, with mean fluorescence intensity (MFI) of PD-1+ populations. An unpaired T-test was performed for g. Bars and error bars show mean +/- SEM. +p ≤ 0.1 * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.