Fig. 3: PD-1-dependent programming is skin specific and highly enforced during skin T_RM cell formation.

a, Schematic showing mixed adoptive transfer of PD-1^+/+ CD45.1⁺Thy1.2⁺ or CD45.2⁺Thy1.1⁺ and CD45.2⁺Thy1.1⁺ or PD-1^−/− CD45.2⁺Thy1.2⁺ OT-I cells into CD45.2 Thy1.2 C57BL/6J recipients at day −1, followed by VACV-OVAss infection in the ear and tail on day 0 and isolation and sorting of ear skin PD-1^+/+ or PD-1^−/− OT-I cells at day 14 after infection (top) and volcano plot depicting DEGs between skin PD-1^+/+ and PD-1^−/− OT-I cells (log (fold change) ≥ 1.5, FDR ≤ 0.05, P ≤ 0.05) isolated at day 14 after infection and analyzed by RNA-seq (Supplementary Tables 1 and 2); FCH, fold change. b, Gene set enrichment analysis (GSEA) showing the similarities between PD-1-dependent programming between skin PD-1^−/− CD69⁺CD103^+/− T cells and spleen PD-1^−/− T_CM cells isolated at day 14 after activation from mice in public datasets (E-MTAB-1569 (ref. ³³)). For each spleen signature, the number of genes (n) and normalized enrichment score (NES) are annotated; ***P ≤ 0.001; NS, not significant. c, Schematic showing the generation of Pdcd1-WT and Pdcd1-KO transcriptomes by subtracting naive CD8⁺Va2⁺ OT-I cell signatures from skin PD-1^+/+ and PD-1^−/− OT-I T_RM cells signatures, respectively (left), and Venn diagram showing the 226 transcripts (27.7%) specific to Pdcd1-WT transcriptomes (Pdcd1 WT_specific program), 296 transcripts (36.4%) specific to the Pdcd1-KO transcriptome (Pdcd1 KO_specific program) and 291 transcripts (35.7%) shared between the Pdcd1-WT and Pdcd1-KO transcriptomes (shared programs); d.p.i., days postinfection. d, Changes in Pdcd1 WT_specific, Pdcd1 KO_specific and shared gene programs defined as in c in skin T_RM cells at specific time points during days 5–90 after VACV-SIINFEKLss in mice as in a relative to day 0 naive OT-I cells (GSE79805)³⁴. The gene set variation analysis (GSVA) signature activity change between days 5 and 30 (boxed; percent change shown) was calculated using mixed effect models (MEMs; see Methods). e, Shared transcripts (291 genes) that overlap with T_RM cell programming identified across skin, lung and gut T_RM cell programs relative to naive T cells (Supplementary Tables 3–6). Black, unique T_RM transcripts (common to three infections/sites and independent of T_CM/T_EM/naive T (T_N) cells) within the T_RM core¹⁶; red, T_RM transcripts present exclusively in T_RM cells (and not significantly enriched in T activated (T_Act), T_ex or T_M cells (Supplementary Fig. 3e)) and using public datasets from skin, lung and gut infections generating CD8⁺ T cell programs¹⁶. f, Expression of CD127, KLRG1, T-bet and eomesodermin in CD45.2⁺Thy1.2⁺CD8⁺ or CD45.2⁺Thy1.1⁺CD8⁺ OT-I cells isolated at day 10 after VACV-OVAss from the flank skin of C57BL/6J mice after adoptive transfer with an equal mix of PD-1^+/+ and PD-1^−/− OT-I T cells at day −1 and infection with VACV-OVAss at day 0 as in Fig. 2a. g, Scoring of repressed and enforced programs of established exclusive viral T cell state signatures defined previously using public datasets from skin, lung and gut infections generating CD8⁺ T cell programs¹⁶ in Pdcd1-WT and Pdcd1-KO signatures as defined in c; ***P ≤ 0.001.