recombinant human galectin-8  (Galectin Therapeutics)

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Gal-8 was associated with myeloid cell-mediated immune suppression in the tumor microenvironment and binds soluble and membrane LILRB4 (A) Framework diagram of this research. (B) Venn diagram demonstrating Gal-8 with a specific expression pattern. (C) Analysis with the TIDE algorithm shows that LGALS8 plays an important role in T cell dysfunction in the tumor microenvironment. The Z score indicates the interaction term in the Cox proportional hazards model and represents the risk coefficient of LGALS8 expression level and T cell dysfunction. The p value represents the significance of Gal-8 as a risk coefficient. (D) The LGALS8 gene expression value in T cell exclusion signatures calculated with the TIDE algorithm. The association score ( Z score) of T cell exclusion signatures evaluates how LGALS8 associates with immunosuppressive cell types that drive T cell exclusion. (E) The TIMER 2.0 algorithm was used to calculate the MDSC fraction and correlation with LGALS8 expression in the indicated types of tumors from the TCGA dataset. Rho and p values are as shown. (F) ELISA screening of potential immune checkpoint receptors revealed LILRB4 as a Gal-8 interactor. (G) Intracellular localization of LILRB4 and Gal-8 proteins by fluorescence microscopy. Coexpression with LILRB4 colocalized the Gal-8 protein with LILRB4 at the cell membrane, whereas overexpression alone localized the Gal-8 protein within the cytoplasm. Scale bar, 10 μm. (H) Immune blotting of coimmunoprecipitation of FLAG-tagged Gal-8 and hemagglutinin (HA)-tagged LILRB4. (I) BLI assay showing the association-disassociation curve between Gal-8 and LILRB4. The kinetics constants are as follows: kon = 1.29 × 10 5 (1/ms); koff = 1.31 × 10 −1 (1/s); K D = 1.02 μM. See also Figure S1 and Table S1 .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: Membrane, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Over Expression

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Gal-8 binds LILRB4 to induce MDSC expansion (A) Affinity of Fc-tagged Gal-8 protein and HEK293 cell-expressed LILRB4 represented by EC 50 of flow cytometry assay. (B) Schematic illustration of experiment design. (C) Heatmap of the transcriptome sequencing data of CD14 + cells. Each group contains 3 biological replicates. (D) Volcano plot of the transcriptome sequencing data. The analysis was performed based on the false discovery rate q value. The top-ranked genes were strongly correlated with MDSC phenotype and function. (E) GSEA showing RNA sequencing–based monocyte signature evaluated in the context of gene sets representative of immune functions. (F) Flow cytometry assay detecting the percentage of M-MDSC with or without Gal-8 or APOE treatment. CD11b + , CD33 + , HLA-DR low/− , and live monocytes were defined as M-MDSCs. Statistical results were obtained from 3 biological replicates and represented as mean ± SEM. (G) T cell proliferation assay showing that monocytes exposed to Gal-8 inhibited T cell function in a concentration-dependent manner. The T cell suppression rate represents the percentage of decreased proliferation rate compared to the control group (whose suppression rate was zero). Data were obtained from biological replicates and represented as mean ± SEM. See also Figure S2 and Tables S2 , , and .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: Flow Cytometry, Sequencing, RNA Sequencing Assay, Proliferation Assay, Cell Function Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Gal-8-LILRB4 interaction activates STAT3 and inhibits NF-κB pathway (A) Immune blotting of 3 potential protein tyrosine phosphatases (PTPs) downstream of LILRB4. Among the 3 PTPs, the phosphorylation level of SHP1 was significantly affected by Gal-8. The statistical plot shows the pSHP1/SHP ratio. (B and C) Immune blotting demonstrates the phosphorylation level of NF-κB and STAT3 with or without Gal-8 treatment in THP-1 (B) and MV411(C) cells. (D) Immune blotting of nuclear and extranuclear proteins of Vector and LILRB4 KD THP-1 cells. (E) Immune blotting of human CD14 + cells treated with or without Gal-8 for 48 or 72 h. The results were constant with what was observed in THP-1 and MV411 cell lines. (F) Immune blotting of S100A8/9 and SOCS3 in human CD14 + cells treated with or without Gal-8. (G and H) Immune blotting of TRAF6 ubiquitination in THP-1 cells with or without LILRB4 KD (G) and with or without Gal-8 treatment (H). The immune blotting was detected with an anti-K63 Ubi antibody. (I) NF-κB reporter gene signal intensity in THP-1 cells cocultured with Gal-8-overexpressing HEK293 cells or control HEK293 cells for 3 days before reporter signals were detected. (J) Immune blotting of ADAM17 expression alteration in THP-1 cells treated with different concentrations of Gal-8 and in Vector and LILRB4-KD THP-1 cells. Of all the statistical analysis of immune blotting results, data were obtained from 3 biological replicates and represented as mean ± SEM. See also Figure S3 .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: Plasmid Preparation, Expressing

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Gal-8 and LILRB4 interaction alters the microenvironment and promotes tumor growth in vivo (A) ELISA results detecting binding capacity of mouse LILRB4 and human Gal-8 proteins. Human Gal-8 was coated on ELISA plates and incubated with murine LILRB4-Fc protein. (B) Strategic diagram of tumor transplant mice model (n = 8). (C) B16 tumor volume. (D) Photograph of B16 tumor in vivo and ex vivo . (E) Survival curves of tumor-bearing mice. (F) Tumor-infiltrating M-MDSC level detected by flow cytometry assay. The proportion of M-MDSC to CD45 + CD11b + cells was statistically compared. (G and H) Ratio of M-MDSCs in the peripheral blood (G) and spleens (H) of mice bearing B16 tumors. (I‒L) Tumor infiltrating FOXP3 + Tregs and CD8 + T cells in tumor IHC assay. (I) Under a 40× objective lens, 5 fields of view were randomly captured on each tumor sample slide, and the number of FOXP3 + cells in these fields of view was counted and averaged, which was recorded as the FOXP3 + cell level of that sample. (J) Five 20× fields of view were randomly captured on each slide, and the area of positive staining was calculated using ImageJ and recorded as the CD8 + area level for that sample. The FOXP3 + and CD8 + level was statistically compared for each group of 8 samples. All of the statistical data mentioned above was represented as mean ± SEM. (K) FOXP3 + cells stained in B16 tumor (scale bar, 100 μm). (L) CD8 + cells stained in B16 tumor (scale bar, 250 μm). (M) Mechanistic diagram demonstrating downstream signaling of Gal-8-LILRB4. See also Figure S4 .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Ex Vivo, Flow Cytometry, Staining

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Anti-LILRB4 monoclonal antibodies that bind specific epitopes blocked Gal-8-LILRB4 interaction and tumor growth (A) The process of producing mouse anti-LILRB4 monoclonal antibodies from hybridoma. (B) Results of epitope binning and schematic diagram. Antibodies were categorized into 4 bins according to their binding epitope. (C) Clones 4–25 competed with other bin 4 clones but not clones from other bins to bind LILRB4 antigen in the BLI system. The shift of BLI did not increase when antibodies competed for the same epitope, whereas antibodies binding to a different epitope continued to bind to the antigen, further increasing the shift. (D) The blocking capacity of bin 4 antibodies represented by ELISA IC 50 . Statistical results were obtained from 3 replicate wells and represented as mean ± SEM. (E) The affinity curve of clones 3–11 and 4–15 antibodies detected and analyzed with the BLI system. (F) ELISA results demonstrating the linear epitope of the clones 3–11 antibody. The clones 3–11 antibody was shown to bind peptide P10 predominantly. (G) Epitope mapping of clones 3–11 antibody. Mutated amino acid sites with more significant interference on the ELISA binding signal were labeled darker in the figure. These amino acid sites and their binding signals were marked based on the molecular structure of the extracellular domain of LILRB4. (H) Flow cytometry assay revealed binding to THP-1 cells of the antibodies from different bins. (I) Flow cytometry assay of M-MDSC. The gating strategy was as described above. Compared with the mouse immunoglobulin G (msIgG), the clones 3–11 and 4–25 antibodies reduced the expansion of MDSC induced by Gal-8. (J) The Fab segment of clones 4–25 antibody reversed Gal-8-induced STAT3 activation and NF-κB inhibition in CD14 + monocytes. (K) Clones 4–25 antibody inhibited the growth of Gal-8 overexpressed tumors in vivo compared with isotype control. (L) Mechanism diagram of the blocking effect of anti-LILRB4 antibodies. See also Figure S5 and Tables S5 and .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: Binding Assay, Clone Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Activation Assay, Inhibition, In Vivo

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet: Anti-Gal-8 monoclonal antibody that blocked Gal-8-LILRB4 interaction had a similar effect on tumor growth with anti-LILRB4 antibody (A) Binding signals of Gal-8 antibody clones to human and cynomolgus antigens. The antibodies were developed by immunizing mice and identified by phage display technology. (B) Results of epitope binning by the BLI system. The result was analyzed and visualized by Cytoscape 3.9. (C) The blocking capacity of antibodies of different epitopes. The clone names for antibodies numbered 36 and 34 are A237 and A269, respectively. (D) Competitive binding of LILRB4 and A269 to human Gal-8. In the BLI system, the probe was coated with A269 antibody following association with Gal-8 (step 1). Afterward, the association of LILRB4 was blocked by A269 but not A237, another Gal-8 antibody (step 2), indicating that clone A269 blocked the binding of Gal-8 and LILRB4. (E and F) Binding kinetics of anti-Gal-8 antibody, clone A269 (E), and clone A237 (F) to human Gal-8 protein. A global fit of data was obtained from the association and dissociation phase with a 2-fold concentration series. (G‒J) Tumor growth curves and ex vivo tumor image for PBMC humanized A375 (G and H) and Hct116 (I and J) cell-line-derived tumor xenograft models. The drugs were given intraperitoneally once every 3 days as described. (K and L) Tissue microarray analysis of Gal-8 expression in melanoma clinical samples. IHC staining of Gal-8 on 46 melanoma samples was scored and categorized. (M and N) Treatment with the A269 antibody (anti-Gal-8) alone or in combination with atezolizumab (anti-PD-L1) in the MC38 in vivo transplant tumor model. MC38 cells overexpressing human Gal-8 were used to establish a subcutaneous graft tumor model. Drugs were given intraperitoneally once every 3 days. All of the statistical data in this figure were represented as mean ± SEM. See also Figure S6 .

Article Snippet: The high-affinity 96-well ELISA plate (42592, Costar) were coated with recombinant Gal-8 (10301-HNAE; Sino Biological) protein and incubated at 4°C overnight.

Techniques: Binding Assay, Clone Assay, Blocking Assay, Concentration Assay, Ex Vivo, Derivative Assay, Microarray, Expressing, Immunohistochemistry, In Vivo

Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) Data analysis with TIDE shows that LGALS8 plays an important role in T cell dysfunction in tumor microenvironment. (B) Spearman correlations between Galectin-8 expression and TILs. (C) TIMER was used to calculated MDSC fraction and correlation with LGALS8expression in the indicated types of tumors from the TCGA dataset. (D) Schematic procedure of phagocytosis assay. (E) Phagocytosis assay (FC) shows that Galectin-8 inhibits the ability of phagocytosis. F. Transcriptome analysis of CD14+ monocytes exposed with Galectin-8. (F) Heatmap of transcriptome sequencing data. Samples of 2 groups including control and gal-8 were analyzed. (G) Volcano plot of transcriptome sequencing data. Significantly regulated genes were labeled.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Expressing, Phagocytosis Assay, Sequencing, Labeling

Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) ELISA screening of potential Galectin-8 interactors revealed LILRB4. (B) 100X microscopic views of Immunofluorescence showing Galectin-8 binds to cell surface LILRB4. (C) CoIP of Galectin-8 and LILRB4. (D) Analysis of EC50 from ELISA test. Galectin-8 was coated by concentration scale, LILRB4-Fc was added to 1 μg/ml. (E) BLI assay showing the association-disassociation curve between Galectin-8 and LILRB4 with the kinetics constants. (F) Flow cytometry showing the fractions of MDSCs in PBMCs incubated with Gal-8, Gal-8 plus LILRB4 ectodomain, and APOE. (G) Flow cytometry showing the binding of Galectin8 to LILRB4 expressed on cell surface. (H) ELISA showing the binding between Galectin-8 and LILRB4 in presence of different concentrations of APOE.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Concentration Assay, Flow Cytometry, Incubation, Binding Assay

Journal: bioRxiv

Article Title: Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment

doi: 10.1101/2022.07.27.501694

Figure Lengend Snippet: (A) Phosphorylation of SHP1 was upregulated by the interaction of Galectin8 and LILRB4 in THP-1 cells. (B) THP-1 NFκB reporter cells cocultured with Galectin-8-overexpressed 293 cells rather than control 293 cells showed lower activation of NFκB signal. (C) Immune blotting demonstrates Gal-8 inhibited NFκB while activated STAT3 in THP-1 cells. (D) Nuclear protein extraction of NC/shLILRB4 THP-1 cells and immune blotting revealing effect of LILRB4 on NFκB and STAT3. (E) Immune blotting of human CD14+ cells. (F) Immune blotting of SHP-1 inhibitor, TPI, inhibited STAT3 phosphorylation in human CD14+ cells.

Article Snippet: Reagents involving recombinant protein Galectin-8 (10301-HNAE; SinoBiological), APOE (APE-H5246; Acrobiosystems), CD3ε (10977-H02H; SinoBiological), CTLA-4 (CT4-H5255; Acrobiosystems), CD28 (CD8-H525a; Acrobiosystems), CD96 (TAE-H5252; Acrobiosystems), LAG-3 (LA3-H5255; Acrobiosystems), TIM-3 (TM3-H5258; Acrobiosystems), CD40 (CD0-5253; Acrobiosystems), ICOS (ICS-H5258; Acrobiosystems), OX40 (OX0-H5255; Acrobiosystems), TIGHT (TIT-H5254; Acrobiosystems), LY86 (10242-H02H; SinoBiological), LILRB4 (16742-H02H; SinoBiological), CD27 (CD7-H5254; Acrobiosystems), PD-1 (10377-H02H; SinoBiological), CD8b (11031-HCCH; SinoBiological) were also purchased from the indicated suppliers.

Techniques: Activation Assay, Protein Extraction