Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: M1-like macrophages secrete CXCL16 and support CXCR6 + CD8 + T-cell recruitment but are progressively lost during PCa progression. ( A ) UMAP plot of tumor-infiltrating myeloid cells from PCa tissues, identifying five distinct clusters, including an IL1B + macrophage subset. ( B ) Dot plot showing average expression and detection frequency of selected marker genes across macrophage and dendritic cell (DC) clusters. ( C ) Violin plots illustrating the expression of key pro-inflammatory ( IL1B , TLR2 , CD86 ), anti-inflammatory ( CD163 , MRC1 ), and chemokine ( CXCL16 ) genes across myeloid subsets. ( D ) AUCell-based quantification of M1 and M2 gene signatures across clusters; IL1B + macrophages exhibit the highest M1 signature score. Kruskal-Wallis test, ****p<0.0001. ( E ) CellChat network visualizing outgoing macrophage-derived signals to CD8 + T-cell subsets; IL1B + macrophages prominently interact with CXCR6 + TEff-like CD8 + T cells. ( F ) Bubble plot visualizing the results of ligand–receptor interaction analysis; CXCL16–CXCR6 axis ranks among the strongest predicted signals. ( G ) Gating strategy for the identification of murine bone marrow-derived macrophages (BMDMs) induced with M-CSF. ( H ) Flow cytometry of BMDMs polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by CD80 and CD206 expression. ( I ) Confocal images of THP-1-derived macrophages stained for CD68 after PMA induction. ( J ) Flow cytometry of THP-1-derived macrophages polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by MHC-II and CD206 expression. ( K ) Immunoblots showing higher CXCL16 levels in M1-polarized BMDMs compared with their M2 counterparts. ( L ) ELISA quantification of secreted CXCL16 in the supernatants of M1-polarized and M2-polarized THP-1-derived macrophages. Mann-Whitney U test, **p<0.01. ( M ) Immunoblot analysis demonstrating elevated levels of CXCL16 in M1-polarized THP-1-derived macrophages compared with M2-polarized cells. (N, O). A total of 5×10⁶ TRAMP-C1 cells suspended in 100 µL PBS were subcutaneously implanted into the right flank of 5–6-week-old male WT C57BL/6J mice (n=5 per group). Tumors were harvested at day 35 (early stage) and day 49 (advanced stage) post-inoculation. Flow cytometric analysis of TAMs revealed a significant reduction in the ratio of MHCII + CD206⁻ (M1-like) to MHCII⁻ CD206 + (M2-like) macrophages during tumor progression. Mann-Whitney U test, **p<0.01. ( P ) Multiplex immunohistochemistry of human PCa tissues (GS=3+4 vs GS=5+5) demonstrated spatial proximity between CXCL16 + M1-like macrophages (HLA-DRA + ) and CXCR6 + CD8 + T cells in lower-grade (GS=3+4) tumors, which was largely diminished in high-grade (GS=5+5) lesions. Black arrows indicate matched regions across serial tissue sections. Scale bars: upper panels, 100 µm; lower panels, 40 µm. AUCell, area under the recovery curve; GS, Gleason Score; M-CSF, macrophage colony-stimulating factor; PCa, prostate cancer; PBS, phosphate-buffered saline; TAMs, tumor-associated macrophages.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Multiplex Assay, Immunohistochemistry, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: 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; Pten flox/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 –log 10 (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.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Activity Assay, Cell Culture, Activation Assay, Western Blot, Flow Cytometry, Derivative Assay, RNA Sequencing, Transformation Assay, Quantitative Proteomics, Labeling, Multiplex Assay, Immunofluorescence, Single Cell