Fig 1: IL-10–STAT3–FOXO1 signaling reprograms CXCR6+CD8+ T cells toward a dysfunctional state. (A) Dot plot showing the expression of IL10RA, STAT3, STAT4, CXCR6, and related markers across CD8+ T cell subsets in scRNA-seq data. (B) IL-10 pathway activity scores across CD8+ T cell clusters. Kruskal-Wallis test, ****p<0.0001. (C, D) Murine splenic CD8+ T cells cultured in a medium containing anti-CD3 (5 µg/mL), anti-CD28 (5 µg/mL), and IL-2 (10 ng/mL) for 48 hours. Following initial activation, cells were maintained in fresh medium supplemented with IL-2 (10 ng/mL) for an additional 7 days. On day 9, cells were treated with 20 ng/mL murine IL-10, IL-15, or STAT3 inhibitor Stattic (2 µM) for 24 hours. The protein expression of STAT3, p-STATS, FOXO1, KLF2, and CXCR6 was assessed by Western blot analysis. (E, F) Flow cytometry of mouse spleen-derived CD8+ T cells shows preferential expression of IL-10R on CXCR6+CD8+ T cells, with upregulation observed following TCR stimulation (anti-CD3/CD28+IL-2, day 10), indicating heightened susceptibility to IL-10-mediated signaling. (G–I) Flow cytometry of human peripheral blood mononuclear cell (PBMC) CD8+ T cells from healthy donors similarly demonstrates enhanced IL-10R expression on CXCR6+CD8+ T cells and its induction on TCR stimulation. (J) PCA of bulk RNA-seq. Prostate tissues from Pb-Cre; Ptenflox/flox (T) and WT mice (n=3/group) were profiled by bulk RNA-seq. PCA separated T (blue) from WT (red) chiefly along PC1 (87.24% variance) and PC2 (5.64%). (K) Sample-to-sample distance heatmap. Distance matrix based on transformed expression values shows tight clustering of biological replicates within genotype and clear segregation between T and WT. (L) Volcano plot. Differential expression analysis between T and WT (cut-offs |log2FC|≥1.5, FDR<0.05). Points are colored by direction (Up=red; Down=blue). Dashed lines indicate thresholds. Il10, Mrc1, Cd163, and Cxcr6 are highlighted in purple; other selected genes are labeled as indicated. Y-axis shows –log10 (adjusted p). (M) Multiplex immunofluorescence (human prostate). Representative fields from human prostate specimens (n=9). Channels: DAPI (nuclei), CD68 (pan-macrophage), HLA-DRA (M1-like macrophage), CD163 (M2-like macrophage), and IL-10. IL-10 signal is enriched in tumor regions and co-localizes with CD68+CD163+ macrophages. Scale bar: 20 µm. DAPI, 4′,6-diamidino-2-phenylindole; FDR, false discovery rate; PCA, principal component analysis; scRNA-seq, single-cell RNA sequencing; WT, wild-type.