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

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC

doi: 10.3389/fcimb.2021.785166

Figure Lengend Snippet: HLA-DR expression in monocyte. The mean fluorescence intensity (MFI) of HLA-DR was determined in basal CD14pos cells after 20 h of T. cruzi lysate (Tc) stimulation or fenofibrate (Tc + Fen) treatment. Monocytes were selected based on FSC and SSC. After excluding doublets and debris, live cells were selected, monocytes were classified by CD14 positive staining. The mean fluorescence intensity (MFI) of HLA-DR was calculated both in total monocytes (A) . It shows the mean fluorescence intensity (MFI) of CD14pos/HLA-DRpos monocytes in healthy (HI) (B) , asymptomatic (Asy) (C) and chronic Chagas disease (CHD) patients (D) , where each patient is represented by a circle. The results are shown as the mean of the experiments ± SEM. These data were analyzed by fitting a mixed effect model with a Tukey post-hoc test.

Article Snippet: PBMC were stained with LIVE/DEADTM fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience).

Techniques: Expressing, Fluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC

doi: 10.3389/fcimb.2021.785166

Figure Lengend Snippet: HLA-DR expression in T. cruzi stimulated and fenofibrate treated monocyte subpopulations. The mean fluorescence intensity percentage of HLA-DR + cells was determined in PBMC stimulated or not with T. cruzi lysate (Tc) and treated or not with fenofibrate (Tc + Fen) after 20 h, according to CD14 and CD16 expression. It shows the mean fluorescence intensity (MFI) of classical (CD14high/CD16neg) (A) , intermediate (CD14high/CD16pos) (B) and non-classical (CD14low/CD16pos) (C) monocytes with HLA-DR + expression. The results are shown as the mean of the experiments ± SEM. These data were analyzed by fitting a mixed effect model with a Tukey post-hoc test.

Article Snippet: PBMC were stained with LIVE/DEADTM fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience).

Techniques: Expressing, Fluorescence

Journal: JAIDS Journal of Acquired Immune Deficiency Syndromes

Article Title: Granulocytic Myeloid–Derived Suppressor Cells Increased in Early Phases of Primary HIV Infection Depending on TRAIL Plasma Level

doi: 10.1097/qai.0000000000001283

Figure Lengend Snippet: FIGURE 1. MDSC frequency during primary HIV infection. A, Gating strategy used to identify MDSC: in the morphological gate (FSC/SSC) we excluded debris, then we gated Lin2/HLA-DRlow/2 cells. In this gate, we selected CD11b+CD33+ cells (MDSC). The expression of CD14, CD15, CD16, and CD124 is shown on cells selected from the CD11b+CD33+ gate. The expression of CD124, with its correspondent fluorescence minus one control (FMO), is shown as histogram. B, Gr-MDSC frequency in HD (16), PHI (51), and CHI (26). C, Gr-MDSC frequency in PHI grouped after the Fiebig classification, and CHI. Results are shown as box and whiskers. The Mann–Whitney test was applied, and P , 0.05 was considered significant.

Article Snippet: Evaluation of MDSC percentage was accomplished with 0.5 · 106 PBMC stained with anti-Cluster of Differentiation (CD) 15, anti-CD33, anti-Human Leucocyte Antigen-DR (HLA-DR), anti-CD3, anti-CD56, anti-CD19, anti-CD14, anti-CD11b, anti-CD16 (BD Biosciences), and anti-CD124 monoclonal antibodies (mAbs) (R&D system).

Techniques: Infection, Expressing, Control, MANN-WHITNEY