Journal: Cellular and Molecular Immunology

Article Title: Cell-associated galectin 9 interacts with cytotoxic T cells confers resistance to tumor killing in nasopharyngeal carcinoma through autophagy activation

doi: 10.1038/s41423-024-01253-8

Figure Lengend Snippet: High G9 in tumors affects T-cell cytolytic activities. A Mono-(1) Poly- (>1) functional profile of gated CD8 response in G9-knockdown or overexpressed cocultures Pie charts representing the mono-poly-functional profile of gated CD8 response (left) in G9-knockdown or overexpressed cocultures. Size of each pie segment corresponds to the frequency of the corresponding cytokines (color-coded). Arcs depict cytokine makeup within pie slice. Bar graph (right) indicating the percentage of monofunctional profile studied (pie slice in light blue). Representative granzyme B (GzB) staining on cytospun cocultured CTL shown at the side. B Representative zebra plot (left) and quantitative plot (right) of CD107 + CD8 + and GzB + CD107 + CD8 + cells, respectively. C Schematic representation of the workflow for the co-culture system using an anti-EpCAM antibody to distinguish CD8 + T cells. Sorted cells were then cytospun on the side for GzB staining. Pie chart of GzB percentages with the corresponding representative fluorescence staining images. Scale bar: 20 μm. D Representative staining CD8 (turquoise), granzyme B (GzB; pink), and nuclei (DAPI, blue) in the tumor region of NPC tissues presented with PanCK+G9+ ( n = 3) and PanCK+G9− ( n = 3). The illustration depicts the distance of a cell (PanCK+G9+ and PanCK+G9−) to another cell type (CD8+GzB + ), defined as the distance between the reference cell (RC) and its nearest neighbor cell (NC) of another cell type. Histogram lines represent CD8+GzB+ paired with PanCK+G9+ cells within 300 µm, with shading indicating an effective distance of 15 µm. The percentage of NC at specified distances relative to RC is shown on the right. E A schematic showing palmitoylation modification allows G9 to translocate onto the cell surface (left column). Representative confocal fluorescence images of E-cadherin (red) and G9 (green) showing cells with (NPC43OE6-Palm; bottom) and without (NPC43OE6; middle) palmitoylation. NPC43Vector as control. The middle column, line profiles quantifying fluorescence signals from E-cadherin (red) and G9 (green) along the yellow lines in the representative image. Scale bar: 10 μm. The right column, sorted cells were then cytospun on the side for GzB staining with corresponding pie chart of GzB percentages. Scale bar: 5 μm. F Schematic illustration of tumor cells loaded with exogenous, activated GzB protein (10 μg) with pore-forming protein streptolysin-O. Endogenous GB puncta Green masks and magnified insets of representative regions staining (left) and quantitative data on GzB puncta in target NPC cells. Scale bar: 50 μm. G Schematic representation of GranToxiLux assay. Representative flow cytometry analysis of granzyme B activity (FITC + ) into the TFL4+ TarTC upon cocultures (left) and quantitative data on percentage of death TarTC (GzB-incorporated target cells) (right). The data were pooled from three ( n = 3) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (one-way ANOVA test for ( A – C ), ( E – G ); two-tailed unpaired Student’s t -test for ( D )). Data points are shown ± SEM

Article Snippet: DAPI was stained before imaging on ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices).

Techniques: Functional Assay, Knockdown, Staining, Co-Culture Assay, Fluorescence, Modification, Control, Flow Cytometry, Activity Assay, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: Cell-associated galectin 9 interacts with cytotoxic T cells confers resistance to tumor killing in nasopharyngeal carcinoma through autophagy activation

doi: 10.1038/s41423-024-01253-8

Figure Lengend Snippet: G9 control on the necrotic and autophagic machinery is conserved in NPC patients. A Experimental setup: antiCD3/28 activated pre-expanded CD8 + T cells (Ex-CD8 + T cells) cocultured with TarTC. NPC cells with or without transduced with tandem RFP-GFP-LC3B and then independently treated with specific autophagy inhibitors: N-acetylcysteine = NAC (6 uM); chloroquine = CQ (60 uM), pepstatin A = pep (10ug/ml), at indicated time point. Autophagic processes were analyzed through different assays. B Western blot analysis on autophagy biomarkers at 6 h cocultures. C RFP-GFP-LC3B dual reporter assay to analyze the blockade of autophagy flux. D Quantification of autophagosomes and autolysosomes in cell soma. Total number of puncta per region of interest was normalized by number of nuclei. 3 region-of-interest were counted per condition. E Representative images of G9-knockdowned or overexpressed NPC tumor cells transfected with the tandem RFP-GFP-LC3B plasmids, following by coculturing with Ex-CD8 + T cells with or without autophagy inhibitors. Cells were then fixed with 4% PFA followed by confocal microscopy. The right parts are enlarged from the boxed areas in the left parts. Autophagosomes denote yellow punta and autolysosomes red punta. F Autophagosomes in cocultured TarTC. CTL cocultured with TarTC for 6 h was stained with autophagy-specific green dye and analyzed by microplate reader. Data presented as green signal normalized with blue DAPI signal. G Western blot analysis on biomarkers for necrosis and immune checkpoint inhibitor at 6 h cocultures. H Western blot analysis on biomarkers for necrosis at 6 and 24 h cocultures. I The stacked bar chart represents the percentage (%) of death-related proteins that were upregulated (pink), and downregulated (green) and proteins not overlapped in each treatment group (white). The secondary y-axis (up) shows the ratio (orange dots connected by a line) of proteins from the dataset that map to the autophagy pathway divided by the total number of proteins that map to the programmed cell death (PCD; apoptosis) and non-PCD (necrosis) pathway. Quantitative Gartner-Aldman plot (right) of expression ratio changes of proteins detected in necrosis pathway (GO0070266). J Viability assay, K colony formation assay, and L soft agar colony formation assay (left bar plot: area intensity; right lollipop graphical plot: volume intensity) were employed to determine the tumorigenicity in the presence of indicated autophagy inhibitors. M Representative image of the tumor regions, which were selected for G9+, RIPK1+, and Beclin+ cell quantification. Normal tissues ( n = 5; region-of-interest ROI n = 34), tumor tissues ( n = 9; ROI = 21). Scale bar: 50 μm. N Estimation plot on G9 intensity and ( O ) bar plot on immunoreactivity on RIPK1 and Beclin in tumors with high G9 and low G9 expression. * P < 0.05, ** P < 0.01 and *** P < 0.001 (one-way ANOVA for ( D , F , J , K , and L ) and two-tailed unpaired Student’s t -test for ( I ))

Article Snippet: DAPI was stained before imaging on ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices).

Techniques: Control, Transduction, Western Blot, Reporter Assay, Transfection, Confocal Microscopy, Staining, Expressing, Viability Assay, Colony Assay, Soft Agar Assay, Two Tailed Test