recombinant hpd l1  (BPS Bioscience)

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. B Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Luciferase, Activity Assay, Bioassay, Cell Culture, Expressing, Activation Assay, Positive Control, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-L1 MC38 cells following treatment with the indicated concentrations of TER for 72 h. B CD8 + T cells were isolated from tumors of hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. These tumor-infiltrating CD8 + T cells were co-cultured with hPD-L1 MC38 cells as target cells in the presence of TER for 72 h. Cell viability measured using the CCK assay is depicted. C PD-L1 expression in hPD-L1 MC38 cells, as assessed by western blot analysis using protein lysates from co-culture conditions. GAPDH was used as a loading control. D The levels of immune-related factors, including GrB, IL-2, and IFN-γ, measured in the co-culture supernatant by ELISA. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Isolation, Knock-In, Cell Culture, Expressing, Western Blot, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (10 or 30 mpk) for the indicated time. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured over time in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors treated with vehicle or TER (10 or 30 mpk). Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis of CD8 + T-cell populations in tumors from each treatment group. F PD-L1 expression in tumors from each group, as assessed by western blot analysis. GAPDH was used as a loading control. G IHC staining of tumor sections for immune-related markers, including CD8 + T cells and GrB. Representative images from each group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Knock-In, Flow Cytometry, Expressing, Western Blot, Control, Immunohistochemistry, Quantitation Assay, Marker

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (30 mpk) and received either an isotype control or a CD8 depletion antibody. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors over time following treatment with vehicle or TER (30 mpk) with or without CD8 depletion. Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis confirming CD8 + T-cell depletion in tumors from each treatment group. The proportion of CD8 + cells among total live cells was quantified. F IHC staining of tumor sections for CD8 + T cells and GrB. Representative images from each treatment group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant hPD-L1 (#71104, BPS Bioscience) was coated onto 96-well plates (Corning Inc., New York, NY, USA) at a concentration of 1 mg/mL in PBS and incubated overnight.

Techniques: Knock-In, Control, Flow Cytometry, Immunohistochemistry, Quantitation Assay, Marker

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: Flow cytometry analysis of ADP of Granta-519 B-cell lymphoma cells, treated with anti-CD20 RTX isotypes or human isotype control Abs (1.5 µg/ml), by MonoMac-6 cells at an E:T ratio of 1:1. a) Percentage phagocytosis of Granta-519 cells induced by single RTX-isotypes, or dual combinations of RTX-IgG2 with RTX-IgG1 or RTX-IgG3. Untreated cells (UT), and human Ab isotypes: hIgG1, hIgG2, hIgG3 were used as controls. For dual treatments, Granta-519 cells were pre-opsonized with 1.5 μg/mL of RTX-IgG2 for 30 min followed by 1.5 μg/mL of RTX-IgG1 or RTX-IgG3. Results are shown as mean ± standard error of the mean (SEM) of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test. b) Representative bivariate plots showing phagocytosis of RTX-treated CFSE-labelled Granta-519 target cells by CTFR-labelled MonoMac-6 effector cells. Phagocytosis was quantified as the percentage of double positive CFSE + CTFR + MonoMac-6 cells (square gate). Increased phagocytosis was observed when RTX-IgG2 was combined with RTX-IgG1 or RTX-IgG3.

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Flow Cytometry, Control

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: Granta-519 cells treated with STR (6 h) or RTX-IgG isotypes (30 min) were analyzed for apoptosis or necrosis compared to untreated cells (UT). Dimethyl sulfoxide (DMSO) was used as vehicle control of STR treatment. a) Percentage apoptosis in UT or treated Granta-519 cells. b) Representative bivariate plots of Granta-519 cells, showing apoptosis and necrosis in UT, and after treatment with RTX-isotypes or STR. Apoptotic cells were identified as Annexin V + DC-Violet ‒ cells, while double positive (Annexin V + DC-Violet + ) cells were identified as necrotic cells with compromised cell membrane. c) Percentage necrosis in UT or treated Granta-519 cells. Results in a) and c) show mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (ns = not significant).

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Control, Membrane

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: Flow cytometry analysis of phagocytosis of Granta-519 cells, untreated (UT) or incubated with STR for 6 h before addition of RTX isotypes or isotype controls (1.5 μg/mL), by MonoMac-6 cells (E:T ratio = 1:1). a) Percentage phagocytosis of UT Granta-519 cells, treated with STR, RTX-IgG1 or RTX-IgG3 or combinations thereof. Results shows mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test. b) Representative bivariate plots showing phagocytosis of CFSE-labelled Granta-519 target cells, treated with RTX isotypes alone or in combination with STR. Phagocytosis was quantified as the percentage of double positive CFSE + CTFR-MonoMac-6 cells (square gate). Increased phagocytosis was observed when RTX-IgG1 or RTX-IgG3 is combined with STR.

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: CD47 expression was evaluated in Granta-519 cells after incubation with STR (6 h) or treatment with RTX isotypes (1.5 μg/mL) (30 min). a) Representative histograms of CD47 expression in Granta-519 cells after treatment with RTX-IgG1, RTX-IgG2, RTX-IgG3, or STR. Grey dashed line indicates the level of CD47 on untreated cells (UT). b) Bar graph representation of fold change (left Y axis) and percentage difference (right Y axis) in CD47 expression on Granta-519 cells induced by STR or RTX isotypes, normalized to CD47 expression on UT. To obtain the fold change values, mean fluorescence intensity (MFI) of treated samples was first adjusted by subtracting MFI of isotype controls and then normalized to the MFI of UT samples. The percentage difference was calculated by the following formula: ((MFI treated sample – MFI UT )*100)/MFI UT ). Results show mean fold change ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test. c) Confocal microscopy images of CD47 expression in untreated, RTX-IgG1, RTX IgG2 and STR-treated Granta-519 cells. Cells were counterstained with Hoescht 33342 nucleus stain. Disruptions in CD47 cell surface pattern are indicated by white arrows. Scale bar: 10 μm.

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Expressing, Incubation, Fluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: Percentage phagocytosis of Granta-519 cells treated with RTX-IgG1 or RTX-IgG3, and in combination with blocking of CD47 by mouse anti-human CD47 Ab with a functional Fc domain (αCD47-fuFc, 1 μg/mL) or humanized anti-CD47 Ab with a silenced Fc domain (αCD47-siFc, 10 μg/mL), by MonoMac-6 cells (E:T ratio = 2:1). Data are presented as mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (ns = not significant).

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Blocking Assay, Functional Assay

Journal: bioRxiv

Article Title: Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

doi: 10.1101/2024.06.18.599534

Figure Lengend Snippet: Representative histograms of a) PD-L1 and b) CD59 expression on Granta-519 cells. c-d) Percentage phagocytosis of Granta-519 cells by MonoMac-6 cells (E:T ratio = 1:1), induced by c) human anti-PDL1 mAb (αPD-L1, 1.5 μg/mL) or d) mouse anti-human CD59 mAb (αCD59, 2.5 μg/mL) alone, or in combination with a pre-treatment with RTX-IgG2. Data are presented as mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (ns = not significant).

Article Snippet: For cell surface staining, the following mAbs were used: APC-conjugated mouse IgG2a anti-human CD20 (clone LT20, cat. no. H12155A, EuroBioScience, Friesoythe, Germany), PE-conjugated mouse IgG1 anti-human CD47 (clone CC2C6, cat. no. 323108, Biolegend), PE-conjugated mouse IgG2a anti-human CD59 (clone p282 (H19), cat. no. 304707, Biolegend), recombinant human IgG1 anti-human PD-L1 (cat. no. hpdl1-mab1, InvivoGen), APC-conjugated mouse anti-human IgG secondary Ab (cat. no. 562025, BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated mouse IgG2a anti-human SIRP-α Ab (clone 15-414, cat. no. 372109, Biolegend), FITC-conjugated mouse IgG2b anti-human CD32 Ab (clone IV.3, cat. no. 60012Fl.1, StemCell Technologies, Vancouver, BC, Canada).

Techniques: Expressing