surface recombinant human gal 8 binding  (R&D Systems)

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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: circHMGCS1–016 regulates the miR-1236-3p/GAL-8 and CD73 axis in the ICC cells. A . The overlapped differentiated proteins were shown. For differentially expressed proteins (left panel), the GO and KEGG analysis were performed; B . Western blot for CD73 and GAL-8 proteins in modified RBE and QBC939 cells; C . Putative binding site of miR-1236-3p with respect to GAL-8 and CD73 via StarBase v3.0. D . The luciferase activity of pLG3-CD73 or GAL-8 in the 293 T cells co-transfected with miR-1236-3p. Data are representative of 3 independent tests (** p < 0.01, *** p < 0.001, n.s. p > 0.05); E . The levels of CD73 or GAL-8 proteins were determined by western blot in the ICC cells with different miR-1236-3p or circHMGCS1–016 expression; F . The level of GAL-8 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); G . The level of sCD73 in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent tests(*** p < 0.001); H . The level of adenosine concentration in the supernatant from ICC cells with different miR-1236-3p or circHMGCS1–016 expression was determined; Data are representative of 3 independent test; I and J. A co-culture showed the supernatant from ICC cells overexpressing circHMGCS1–016 inhibited the CD4 + and CD8 + T cell proliferation; Data are representative of 3 independent tests (*** p < 0.001, n.s. p > 0.05); K. Chemokine chips and ELISA were employed to determine the different chemokines in the supernatant between RBE-control, RBE-circHMGCS1–016 and RBE-shmiR-1236-3p groups

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Western Blot, Modification, Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: GAL-8 and CD73 interference restraints the function of circHMGCS1–016 in ICC cells. A . The relative level of CD73 and GAL-8 was measured by qRT-PCR in the ICC cells; Data are representative of 3 independent tests; B . The expression of CD73 and GAL-8 were modified by lentivirus-mediated knockdown in RBE-circHMGCS1–016 cells. Data are representative of 3 independent tests(*** p < 0.001); C . The efficacy of GAL-8/CD73 interference was analyzed by western blot; D . Invasion assay was performed to detect the invasion ability of RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference (Bar = 200 μm). Data are representative of 3 independent tests(*** p < 0.001); E . Colony formation assay was performed to detect the ability of colony formation in RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference. Data are representative of 3 independent tests(*** p < 0.001); F . The level of GAL-8 in the supernatant of RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; Data are representative of 3 independent tests(*** p < 0.001); G . The level of adenosine concentration in the supernatant of RBE-circHMGCS1_016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; Data are representative of 3 independent tests(** p < 0.01); H . A co-culture showed the supernatant from RBE-circHMGCS1–016 cells inhibited the CD4 + and CD8 + T cell proliferation compared to RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference (*** p < 0.001); I . Chemokine chips and ELISA were employed to determine the different chemokines in the supernatant between RBE-circHMGCS1–016 cells and RBE-circHMGCS1–016 cells with CD73 and GAL-8 interference; J . A model for circHMGCS1–016 driven ICC development and established the immune privilege microenvironment

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Quantitative RT-PCR, Expressing, Modification, Western Blot, Invasion Assay, Colony Assay, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: The relationship among circHMGCS1–016, CD73 and GAL-8 in ICC tissues. A . CD73 and GAL-8 levels in 40 pairs of ICC and matched adjacent non-tumor tissues. Data was shown as log 2 (T/N) ; B . Representative images for CD73 and GAL-8 staining of ICC and matched adjacent nontumor tissues (Bar = 100 μm); C . Representative images for circHMGCS1–016, CD73, GAL-8, CD4 and CD8 staining of ICC tissues (Bar = 200 μm); D . Correlation analysis showed the positive relationship between circHMGCS1–016 and CD73( R 2 = 0.7379, p = 3.45E-18), circHMGCS1–016 and GAL-8 ( R 2 = 0.6747, p = 5.87E-16). But circHMGCS1_016 expression was adversely related to the level of CD8 ( R 2 = 0.1299, p = 6.45E-17) and CD4 ( R 2 = 0.2157, p = 1.17E-14). The relationship between CD8 and CD73( R 2 = 0.3711, p = 1.81E-12), CD8 and GAL-8 ( R 2 = 0. 4244, p = 2.12E-23) was also analyzed (*** p < 0.001)

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

doi: 10.1186/s13046-021-02095-2

Figure Lengend Snippet: Higher levels of circHMGCS1–016 correlated with resistance to anti-PD1 therapy in mice and ICC patients. A . Representative images of the RBE orthotopic ICC tumors from humanized NSG mice ( n = 3 /group). B . Tumor growth volume of the RBE orthotopic planted humanized mice from each group (*** p < 0.001, ** p < 0.01); C . The level of GAL-8 in the serum of the RBE orthotopic ICC tumors from each group; Data are representative of 3 independent tests (*** p < 0.001, * p < 0.05, n.s. p > 0.05); D . The level of adenosine concentration in the serum of mice planted with the RBE orthotopic ICC tumors; Data are representative of 3 independent tests(** p < 0.01, * p < 0.05); E . The CD8 + and CD4 + T cells in the blood of mice planted with the RBE orthotopic ICC tumors (*** p < 0.001); F. At study endpoint, the CD8 + and CD4 + T cells in IgG and PD-1 treatment group were analyzed by immunohistochemistry (Bar = 200 μm); G . Representative ICC cases from 12 patients who received PD-1 antibody treatment were analyzed by IHC staining for CD8 and circHMGCS1–016 (Bar = 200 μm); H . Twelve patients were divided into two groups according to circHMGCS1–016 expression, and patients in CR, PR, SD, and PD were shown in each group. I . The efficacy of PD1 antibody immunotherapy was assessed by MRI based on RECIST1.1. J . The number of CD8 + cells was significantly different between two groups(** p < 0.01)

Article Snippet: Rabbit polyclonal to human CD73 antibody (1:1000, ab237757, Abcam, USA), Rabbit monoclonal [EPR3610] to human GAL-8 antibody (1:1000, ab92742, Abcam, USA) and CD4 (1:1000, ab203034, Abcam, USA) were used in immunohistochemistry (IHC).

Techniques: Concentration Assay, Immunohistochemistry, Expressing