Human PD1 ⁺ TIGIT ⁺ CD8 T cells in blood and tissues are clonally related and express effector molecules. (A) UMAP of scRNA-seq data derived from FACS-sorted CD8 T cell populations of human peripheral blood, skin, and fat of one donor (“Donor RT2”). Left, cells color-coded based on tissue of origin. Middle, expression of gene signature (PDCD1, TOX, IL10, IFNG, AREG, and TIGIT). Right, cells clustered in 20 groups. (B) Annotation of clusters using signature genes shown in (B) and labeled in UMAP. Sort info and quality control (QC) in Fig. S2; experimental repeat with second donor (“Donor RT1”) in Fig. S2. (C) TCRs derived from fat (left) and skin (right) CD8 T cells in effector cell clusters 6, 7, 8, and 16 (fat: 8,158 cells, skin: 4,573 cells) were highlighted in yellow (fat) or blue (skin) and displayed in all other clusters. (D) Clonality of clusters (top, white) or tracking of fat (middle, orange) or skin (bottom, blue) CD8 T cells in blood-based clusters of the same donor. The percentage indicates the fraction of detected clones among the total clones for the donor, with the total number of clones shown above. Each slice represents a clonotype with the angle representing its fraction among all cells in the respective cluster. (E) DEGs between TCR-identical cells in clusters 6, 7, 8, 16 and 0, 2, 3, 5, 9, 11, 14, 18. Several genes highlighted in red and labeled, P_adj values <2⁻¹⁰⁰⁰ capped at 2⁻¹⁰⁰⁰. Values >3 capped at 3. (F) Gene expression of NR4A1, NR4A2, CD69, TOX, XCL1, XCL2, AREG, TNF, IFNG, GZMB, CCL3, PDCD1, and CCL4 in CD8 T cells from donor RT2. CDR3 sequences are listed in Table S4. (G) Expression of intracellular TOX in PD1⁺TIGIT⁺ CD8 T cells from human blood, fat, skin, and liver tissue. All data are derived from two or more independent experiments with the indicated number of human donors.