Fig 2: T/NK cell clusters in NPC.a t-SNE plot showing 10 clusters of 17,263 T/NK cells (indicated by colors). b t-SNE plot, color coding for the expression of the marker genes (gray to red) for the indicated cell subtypes. c Average expression of selected T cell function-associated genes of naïve markers, inhibitory receptors, cytokines and effector molecules, co-stimulatory molecules, and Treg markers in each cell cluster. d Potential developmental trajectory of CD4+ T cells (n = 5694) inferred by analysis with Monocle 2. Arrows show the increasing directions of certain CD4+ T cell properties annotated with the signatures shown in e. e Traceplots of (left) CD4+ T cell activation signature along activation component and (right) terminal differentiation signature along terminal differentiation component for the CD4+ T cells. Cells are projected along the component, with the blue line indicating the moving average of the expression of signatures (a sliding window of length equal to 5% of the total number of CD4+ T cells was used), and the shaded area displaying SEM. Signatures used are presented in Supplementary information, Table S9. f Potential developmental trajectory of CD8+ T cells (n = 6975) inferred by analysis with Monocle 2. Arrows show the increasing directions of certain CD8+ T cell properties annotated with the signatures shown in g. g Traceplots (as in e) of (left) CD8+ T cell activation signature along activation component and (right) terminal differentiation signature along terminal differentiation component for the CD8+ T cells. Signatures used are presented in Supplementary information, Table S9. h Spearman correlation between the activity of CD8+ T cells (n = 6975), as measured by average granzyme expression (GZMA, GZMB and GZMH), and the expression of CD8+ T cell-specific genes. Genes encoding known immune checkpoint molecules are highlighted in blue. CD4+ Tconv, conventional CD4+ T cell; CD8+ Tdys, dysfunctional CD8+ T cell; NK, natural killer. i Heatmap showing the activity of TFs in each T/NK cell subtype. The TF activity is scored using AUCell. j Peripheral CD8+ T cells and NK cells were transfected with negative control (NC), EOMES-specific, RUNX3-specific, or XBP1-specific siRNAs, followed by immunoblot analysis to determine protein expression of Eomes, Runx3, or XBP1. ß-actin is the loading control. The experiments were repeated independently for three times with similar results. k Left, representative histograms depicting the expression of GZMB and perforin on peripheral CD8+ T cells transfected with NC and EOMES-specific (n = 6 donors, n = 5 independent experiments), RUNX3-specific (n = 6 donors, n = 6 independent experiments), and XBP1-specific siRNAs (n = 6 donors, n = 5 independent experiments). Right, percentage of GZMB+ or perforin+ cells in siNC and siEOMES (n = 6), siRUNX3 (n = 6), and siXBP1 (n = 6) CD8+ T cells. l Left, representative histograms depicting the expression of GZMB and perforin on peripheral NK cells transfected with NC and EOMES-specific (n = 6 donors, n = 5 independent experiments), RUNX3-specific (n = 6 donors, n = 5 independent experiments), and XBP1-specific siRNAs (n = 6 donors, n = 5 independent experiments). Right, percentage of GZMB+ or perforin+ cells in siNC and siEOMES (n = 6), siRUNX3 (n = 6), and siXBP1 (n = 6) NK cells. *P < 0.05, **P < 0.01 (paired Student’s t-test).