Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: Gal9-deficient DCs are impaired in their ability to induce antigen-specific CD4 + T cell proliferation. ( A ) Schematic of human autologous T cell activation assay using WT or KD gal9 DCs and antigen-specific CD4 + [from vaccinated donors with Tetanus toxoid (TT)] or CD8 + (transfected with an mRNA encoding for gp100-TCR) T cells. ( B ) CD4 + T cell proliferation in response to TT or PBS (−TT) presented by WT (gray) or KD gal9 DCs (red). Representative flow cytometry histogram ( Left ) and quantification (n = 4, Right ). ( C ) CD8 + T cell proliferation in response to PBS (−), irrelevant peptide (NY-ESO, Irr), or relevant peptide (gp100, Rel)-treated WT (gray) or KD gal9 DCs (red). Representative flow cytometry histogram ( Left ) and quantification (n = 7, Right ). ( D ) Schematic of murine DC-T cell coculture assay using OVA-treated-WT, gal9 KO ( lgals9 −/− ), or gal9-rescued KO cells ( lgals9 −/− + rgal9) with OT-I or OT-II T cells. ( E and F ) OT-II CD4 + T cell proliferation in response to OVA protein or peptide presented by WT, lgals9 −/− , or lgals9 −/− + rgal9 DCs; representative histograms ( E ) and quantification (n = 4 to 6 mice, F ). ( G ) Division index of OT-II CD4 + T cells shown in ( F ). ( H and I ) OT-I CD8 + T cell proliferation in response to OVA protein or peptide presented by WT, lgals9 −/− , or lgals9 −/− + r-gal9 DCs; representative histograms ( H ) and quantification (n = 4 to 6 mice, I ). ( J ) Division index of OT-I CD8 + T cells shown in ( I ). Data shown as mean ± SEM. Each dot represents an independent donor (human) or mouse. Statistical significance assessed by two-way ANOVA with Šídák’s multiple comparisons test. ns P > 0.05, * P < 0.05, ** P < 0.005.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: Activation Assay, Transfection, Flow Cytometry, Co-culture Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: MHC class I and II–peptide complexes in WT DCs do not differ from gal9 KD DCs. ( A ) Fluorescence intensity quantification of SIINFEKL peptide bound to MHC-I detected in WT and gal9 KO murine DCs after culturing them with 0, 2.5, or 5 mg/mL OVA (or peptide as positive control) at 37 °C or 4 °C for 18 h (n = 4 mice). ( B ) Workflow for MHC-II-bound peptide isolation and analysis by mass spectrometry. Cell pellets from WT and gal9 KD DCs treated with OVA or TT antigens were subjected to mild lysis, followed by overnight incubation with protein G magnetic beads bound to anti-HLA-II antibodies. Peptides bound to MHC-II molecules were eluted from the bead–antibody–HLA–peptide complex using trifluoroacetic acid (TFA), desalted using StageTips, and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS, here: MS). ( C ) Total number of peptides identified in WT (gray) and KD gal9 (red) DCs. Each dot represents a different donor. ( D ) Length distribution (number of amino acid residues) of identified peptides in WT and KD gal9 DCs.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: Fluorescence, Positive Control, Isolation, Mass Spectrometry, Lysis, Incubation, Magnetic Beads, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: Gal9-deficient DCs fail to establish stable interactions with T cells. ( A ) Time-lapse images of superantigen treated-WT or KD gal9 DCs (magenta) cocultured with T cells (green) for the specified time points. White arrows illustrate tracked T cells (t#1-5) migrating toward DCs. ( B ) Migration tracks of individual T cells (t#1-5) in WT or KD gal9 DC–T cell cocultures, plotted across the x-y axis (μm). ( C ) Quantification of the synaptic contact duration (minutes) between T cells and WT (gray) or KD gal9 (red) DCs (n = 4 donors, 10 to 15 cells/donor). ( D ) Quantification of total DC–T cell contacts over time (n = 9 donors). (Scale bar, 10 μm.) Data are presented as mean values ± SEM. Statistical significance assessed by the unpaired t test analysis ( C ) or two-way ANOVA with Šídák’s multiple comparisons test ( D ). * P < 0.05, ** P < 0.005.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: Migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: Gal9 localizes to the IS and interacts with the α and β intracellular domains of HLA-DR. ( A ) Representative immunofluorescence images of superantigen-treated DCs alone ( Top ) or cocultured with T cells (DC synapse, Bottom ), permeabilized and stained against gal9, TCR, and DAPI. Merged and zoomed-in image highlights the localization of gal9 at the DC–T cell interface. Fluorescence intensity depicted as colorized pixels, with grayscale values representing signal intensity. ( B ) Quantification of gal9 fluorescence intensity at the immune synapse or the rear of WT DCs in permeabilized (intracellular) and nonpermeabilized (surface) samples (n = 4 to 6 donors, 6 to 10 cells analyzed per donor). ( C ) Volcano plot depicting gal9-interacting proteins identified by mass-spectrometry following immunoprecipitation with α-gal9 ( Right ) or α-IgG ( Left ). MHC-II (HLA-DRB4) molecule is highlighted in red and gal9 ( lgals9 ) in pink. Dotted gray lines depict the cut-off values; the fold change (x-axis) of 2 and P -value (y-axis) of 0.05. Black dots represent proteins that had fold change >2 and P -value >0.05. ( D ) Immunoprecipitation (IP) of untreated or lactose-treated (35 mM) DCs using α-HLA-DR antibodies or isotype as negative control and resolved and probed with α-gal9 and α-HLA-DR antibodies (n = 3). ( E ) Schematic representation of the NMR experimental design. Gal9 was incubated with HLA-DR α and/or β chain intracellular regions and chemical shift perturbation measurements performed on gal9 amino acids. Lactose (LacNAc) was used as glycan binding control. ( F ) Selected 1 H, 15 N-HSQC NMR spectrum of 15 N-gal9-alone (black) incubated with 1:100 (orange) or 1:200 (purple) of the intracellular domain of HLA-DR β chain. ( G ) Spectra of gal9 alone (black), with 200 equivalents of α and β HLA-DR intracellular domains (orange), and 300 equivalents of lactose (purple). Labels in each crosspeak indicate whether it belongs to gal9 N- or C-ter domains. Arrows indicate the direction of chemical shift perturbations upon peptide addition. All graphs show mean ± SEM. Statistical analysis was conducted by using ordinary one-way ANOVA with Tukey’s multiple comparisons ( B ) or Friedman test ( D ). ns P > 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Fluorescence, Mass Spectrometry, Immunoprecipitation, Negative Control, Incubation, Glycoproteomics, Binding Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: Intracellular gal9 regulates the recruitment of HLA-DR at the immunological synapse and its lateral mobility on the plasma membrane. ( A ) Representative airyscan confocal microscopy of WT or KD gal9 DCs incubated for 2 h with T cells and stained for HLA-DR, gal9, TCR/CD3, and DAPI. Permeabilized (intracellular) and nonpermeabilized (surface) conditions shown. ( B ) Line scans of fluorescence intensity across the synapse to rear (white line in merged composite in ( A ) of DCs depicting HLA-DR (magenta), gal9 (green), and TCR (yellow) signals. ( C and D ) Pearson’s ( C ) and Mander’s ( D ) correlation coefficient quantifying HLA-DR and gal9 colocalization in permeabilized (intracellular) and nonpermeabilized (surface) DCs (n = 4, 10 to 20 cells analyzed/donor). ( E ) Quantification of intracellular HLA-DR intensity at the synapse in WT and KD gal9 DCs cocultured with T cells (n = 4, 10 to 20 cells analyzed/donor). ( F ) Representative FRAP images showing HLA-DR recovery in WT and KD gal9 DCs. Images depict HLA-DR signal and distribution prior to bleaching ( Left ), during the postbleach recovery phase (middle), and at the final postbleach time point ( Right ). The bleached region is indicated by a yellow circle, with time points (in seconds) labeled in the figure. ( G ) Mean FRAP recovery curves of HLA-DR in WT (gray) vs KD gal9 DCs (red), normalized to prebleach intensity. ( H ) Quantification of HLA-DR mobile fractions in WT and KD gal9 DCs (n = 4 donors, 6 to 15 cells analyzed/donor). (Scale bar, 10 μm.) Each dot represents a differenT cell. Statistical analysis was performed by the Mann–Whitney test ( C , D , and H ), one-way ANOVA ( E ), Friedman test ( G ). ns P > 0.05, * P < 0.05, ** P < 0.005, **** P < 0.0001.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: Clinical Proteomics, Membrane, Confocal Microscopy, Incubation, Staining, Fluorescence, Labeling, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

doi: 10.1073/pnas.2501381122

Figure Lengend Snippet: Gal9 loss in DCs impairs tumor rejection in vivo. ( A ) RMA-Muc1 tumor volume over time in WT (gray), lgals9 −/− (red), and cd11c cre Lgals9 fl /fl (green) (n = 14 to 18 mice/genotype). ( B ) Tumor volume at day 14 postinjection in the same mouse cohorts. ( C ) Distribution of animal discomfort levels (%) during tumor progression, assessed per institutional guidelines. Nonmeasurable tumors: Tumors present but too small to be measured with calipers. HEP: Humane end point. All data are shown as mean ± SEM. Statistical analysis was performed by one-way ANOVA with multiple comparisons ( A ), two-way ANOVA with Tukey’s test ( C ), and standard significance thresholds: ns P > 0.05, * P < 0.05, ** P < 0.005.

Article Snippet: For gal9 probing, membranes were blocked in TBS Blocking Buffer (#927-60001, LI-COR) and probed with 1 μg/ml goat α-human gal9 (#AF2045, R&D Systems) overnight at 4 °C.

Techniques: In Vivo