CD8⁺ T cells in REL samples have lower cytotoxic potential. (A) Differential TF activity analysis using SCENIC. Heat map indicating log2 odds ratios; only significantly enriched TFs are colored (Fisher exact test false discovery rate < 0.05) (left). Characterization of target genes based on known gene sets (right). Assignment of target genes to known functions was performed using publicly available gene sets (supplemental Methods). The colored bars represent the fraction of target genes per TF, which belong to the different gene sets (IFN, IFN response; Activation, Immune cell activation; TNF, TNF signaling). (B) CD8⁺ T gene regulatory network (GRN) of exemplary differentially active TFs (TBX21, REL, and FOS) and their target DEGs. (C) UMAP highlighting the T-cell clusters that belong to the CD8⁺ effector memory (EM) cells and were used for the differential expression analysis. (D) Heat map depicting scaled expression (z-score) across all 6 samples of 235 DEGs (CR, 144 genes; REL, 91 genes) in CD8⁺ EM clusters. Analysis was performed using the MAST algorithm (log2FC > 0.5; adjusted P value [p.adj] < 0.05 after Bonferroni correction). (E) Gene ontology and hallmark enrichment analysis on the DEGs from D. Terms were selected from the top-enriched terms. Full list provided in supplemental Table 8. (F) UMAP indicating the normalized gene expression of ADGRG1/GPR56. (G) GPR56 and CD27 expression across pseudotime of CD8⁺ cells, split per condition. The bar plots below indicate the percentages of GPR56⁺ and CD27⁺ cells in REL and CR samples when considering all CD8⁺ clusters. (H) Percentage of GPR56 positive cells in the indicated fractions determined by flow cytometry in the same 6 samples used for scRNA-seq. (Top left cartoon) Gating strategy used to identify naive, central memory (T_CM), EM (T_EM), and CD45RA⁺ EM (T_EMRA) cells using CCR7 and CD45RA. P values were calculated using Student t test. (I) Volcano plot illustrating the DEGs between GPR56⁺ (purple) and GPR56^– (orange) CD8⁺ T_EM cells of patients in CR. The y-axis represents P value after Bonferroni correction (p.adj), and points were colored according to absolute log2FC > 0.5 and P.adj < .05 (purple/orange). (J) Box plots illustrating the percentage of GZMB⁺ (top) and PRF1⁺ (bottom) T cells in the GPR56⁺ and GPR56⁻ fractions assessed by intracellular flow cytometry. Connected points indicate fractions originating from the same sample. P value was calculated using paired Wilcoxon test, n = 10.