Journal: Frontiers in Immunology
Article Title: Dual checkpoint blockade of CD47 and LILRB1 enhances CD20 antibody-dependent phagocytosis of lymphoma cells by macrophages
doi: 10.3389/fimmu.2022.929339
Figure Lengend Snippet: Efficacy of dual checkpoint blockade of CD47 and LILRB1 with differentially polarized macrophages. (A) Left panel: Monocytes were differentiated with M-CSF (M0 macrophages), GM-CSF, LPS and IFN-γ (M1 macrophages) or M-CSF and IL-10 (M2c macrophages) and stained with PE-conjugated antibodies against CD163, CD80 (blue shaded peaks) or an isotype control antibody (black outlined peaks). Cell surface expression was analyzed by flow cytometry. One representative experiment is shown ( n = 3). Right panel: Cell surface expression of LILRB1, LILRB2 and SIRPα by M0, M1 and M2c macrophages was analyzed by calibrated flow cytometry. Data points indicate specific antibody binding capacity (SABC) for macrophage preparations from individual donors. Horizontal lines represent mean values ± SD ( n = 7). *P ≤ 0.05; ns, not significant; two way ANOVA and Tukey`s post hoc test. (B) Macrophages were differentiated in parallel from monocytes from seven donors towards M0, M1 or M2c phenotypes and analyzed in fluorescence microscopy-based ADCP assays using CFSE-labeled Carnaval cells (E:T cell ratio: 1:2). Efficacy was determined for rituximab (RTX) vs. phagocytosis in the absence of an antibody (w/o, first graph ), RTX + CD47-IgGσ (CD47) vs. RTX ( second graph ), and RTX + CD47-IgGσ + LILRB1-IgGσ (LILRB1) vs. RTX + CD47-IgGσ ( third graph ) and RTX + LILRB1-IgGσ vs. RTX ( fourth graph ). Antibodies were applied at a concentration of 10 µg/ml. Statistically significant differences are indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant; two-way ANOVA with Šidàk´s multiple comparisons test). (C) Live-cell imaging analysis of ADCP by M0, M1 and M2c macrophages in the presence of the indicated antibodies (each at a concentration of 10 µg/ml). HER2-IgGσ (IgGσ) was used in control reactions. Target cells were pHrodo ® labeled DG-75 cells (E:T cell ratio: 1:2). Data points represent means ± SD of the red object count per image of independent experiments using macrophages from six different donors that were polarized in parallel towards M0, M1 or M2c phenotypes (w/o, without antibody; #, statistically significant differences between RTX + CD47-IgGσ vs. RTX; *, statistically significant differences between RTX + CD47-IgGσ + LILRB1-IgGσ vs. RTX + CD47-IgGσ, two-way ANOVA and Fisher´s LSD test; P ≤ 0.05). (D) Macrophages were differentiated from peripheral monocytes in the presence of either M-CSF ( left graph ) or GM-CSF ( right graph ) for six days. Cells were left untreated or stimulated with IFN-γ and LPS for additional 48 h and analyzed for cell surface expression of LILRB1, LILRB2 and SIRPα by calibrated flow cytometry. Data points represent the specific antibody binding capacities (SABC) that were determined for individual macrophage preparations. Statistically significant differences between groups treated with IFN-γ and LPS and the control groups are indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; two-way ANOVA with Fisher´s LSD test). (E) Macrophages were differentiated with M-CSF or GM-CSF and analyzed without further stimulation (M0 macrophages; left graph ; n = 5) or after polarization with LPS and IFN-γ (M1 macrophages; right graph ; n = 4) in 2 h ADCP assays using CFSE-labeled Carnaval cells and the different antibodies as indicated. ADCP was analyzed by fluorescence microscopy. Data points indicate phagocytic index values for macrophages from individual donors. Horizontal lines represent mean values ± SD (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant; two-way ANOVA and Fisher´s LSD test).
Article Snippet: Twenty thousand macrophages per well were plated in M0, M1 or M2c macrophage differentiation media on 8 well µ-slides (Ibidi GmbH, Graefelfing, Germany) and incubated overnight.
Techniques: Staining, Expressing, Flow Cytometry, Binding Assay, Fluorescence, Microscopy, Labeling, Concentration Assay, Live Cell Imaging
