hpd 1 l25 t168  (R&D Systems)

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