Single-cell analysis of tumor-infiltrating T cells. (A) Experimental design. NSG mice were injected IV with 2.5 × 10⁵ NY-ESO-1⁺ Raji cells, followed by IV injection of 3 × 10⁶NY-ESO-1 TCR⁺ CD161⁺ cells on day 6 (n = 5 mice per treatment group) and treatment with either CD161 mAb or isotype control. On day 13, cells were isolated from bone marrow of both hind limbs, and fluorescence-activated cell sorted on live CD3⁺ T cells (human CD45⁺, human CD19^–, ZsGreen^–, and mouse CD45^–). Cells were analyzed by scRNA-seq using the 10X Genomics platform. (B) Identification of CD4 and CD8 T-cell clusters across all samples (n = 5 mice per treatment cohort). (C) Differentially expressed transcripts in CD8 (left) and CD4 (right) T cells in CD161 mAb–treated vs isotype–treated mice. Only transcripts expressed in >10% CD4 or CD8 T cells with an adjusted P value < .05 are shown (supplemental Tables 1 and 2). (D) Distribution of granzyme A (GZMA), granulysin (GNLY), and granzyme B (GZMB) expression in isotype control (top) vs CD161 mAb (bottom) treatment groups. (E-F) Violin plots showing expression of GZMA, GNLY, and GZMB in CD4⁺ (E) and CD8⁺ (F) T-cell clusters. Values trimmed at 2% and 98% for plotting. (G) Distribution of C-X-C motif chemokine receptor 6 (CXCR6) and integrin subunit alpha E (ITGAE) expression in isotype control (top) vs CD161 mAb (bottom) treatment groups. (H-I) Violin plots showing differential expression of tissue-residency markers (CXCR6, ITGA1, CD69, and ITGAE) in CD4 (H) and CD8 (I) T cells in CD161 vs isotype control treatment groups. Data from 1 scRNA-seq experiment with hashtagged cells from 5 mice per treatment group. Two-sided unpaired Wilcoxon rank sum test, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. UMAP, uniform manifold approximation and projection.