Extended Data Fig. 4: Transcriptomics supporting selection of PD-1 sufficient T cell in skin.

a. Schematic of mixed adoptive transfer of PD-1 +/+ with PD-1−/− OT-I T cells, VACV-OVAss (4 × 106 viral p.f.u. on ear and tail), and d14 isolation from skin (top). ATAC sequencing of the PD-1 gene locus shows the lack of PD-1 accessibility in naïve T cells and confirms the presence or absence of exons 2 and 3 in PD-1−/− OT-I T cells (bottom). b. Relative enrichment of ECM/GPCR pathway scores comparing donor skin gB-T CD8 + CD103 + TRM isolated from the skin of C57/BL6 mice 30 days post-infection with HSV, versus naive gB-T cells (GSE47045). Pathway enrichment scores were generated using the GSVA ssGSEA algorithm. c. Relative enrichment of ECM/GPCR pathway scores during VACV-SIINFEKL skin OT-I TRM differentiation (GSE79805). Pathway enrichment scores were generated using the GSVA ssGSEA algorithm and normalized by subtracting naïve T cell scores from all samples. d. Scatter plot depicting expression differences of transcripts in PD-1 +/+ vs PD-1−/− T cells in skin and spleen with DEGs in skin (logFC≥1.5, FDR ≤ 0.05, green), spleen (FDR ≤ 0.05, p ≤ 0.05, yellow) or both (purple), and % of all DEGs (n = 328) within each quadrant. Genes that were not differentially expressed by either skin or spleen T cells are depicted as a contour plot (gray). e. Expression of transcription factors (T-bet, Eomes) in PD-1 +/+ OT-I and PD-1−/− OT-I T cells isolated from LN and spleen at day 10, and expression levels of cell surface markers (CD127, KLRG1) on PD-1 +/+ OT-I and PD-1−/− donor OT-I T cells isolated from ear skin of mice on day 21 or 42 post VACV-OVAss. f. Fisher exact test of exclusive T cell differentiation state signatures in Shared, PD-1−/−specific, or WTspecific programs. g. Fisher exact test of non-exclusive (global) CD8 + T cell differentiation state signatures in shared, PD-1−/−specific, or WTspecific programs. Up and down triangles indicate enrichment driven by transcripts either upregulated or downregulated respectively in day 14 skin OT-I relative to naïve controls. Both size and color indicate the level of enrichment.