Journal: Blood Advances

Article Title: Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP ∗

doi: 10.1182/bloodadvances.2023012155

Figure Lengend Snippet: Schematic representation for obtaining scFvs that bind and block FcγRIIIA. BALB/c mice were immunized with the recombinant human FcγRIIIA, total splenic RNA was isolated, and genes encoding the V H and V L chains were amplified. A second polymerase chain reaction stitched V H and V L with a linker, and the products were cloned into a phagemid vector via Gibson assembly. E coli was then transformed with the constructs and a scFv-phage display library obtained. Five rounds of selection (R1, R2A, R2B, R3A, and R3B) were performed to select phages bound to FcγRIIIA with minimal cross-reactivity with FcγRIIA ( supplemental Figure 1 ). Selection of scFv was based on binding to NK cells by flow cytometry and inhibition of hIgG-FcγRIIIA interaction by homogeneous time-resolved fluorescence. Purified scFv were analyzed for binding to FcγRIIIA by ELISA and Octet; minimal cross-reactivity with the other human receptors and inhibition of hIgG-FcγRIIIA interaction (ie, FcγRIIIA blockers) were part of the selection process. The final antibody fragment, 17C02-scFv, was selected from 10 candidates based on these assessments as well as sequencing analysis to screen for glycosylation, oxidation, aggregation, deamidation/isomerization, and proteolytic sites to exclude scFv molecules with low biochemical stability.

Article Snippet: FcγRI (CD64, catalog number 10256-H08H), FcγRIIIA (CD16A, catalog number 10389-H27H), and FcγRIIIB (CD16B, catalog number 11046-H08C), as well as biotinylated-FcγRIIA (catalog number 10374-H27H-B) and biotinylated-FcγRIIIA (catalog number 10389-H27H1-B) were also purchased from Sino Biological.

Techniques: Blocking Assay, Recombinant, Isolation, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transformation Assay, Construct, Selection, Binding Assay, Flow Cytometry, Inhibition, Fluorescence, Purification, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Blood Advances

Article Title: Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP ∗

doi: 10.1182/bloodadvances.2023012155

Figure Lengend Snippet: Blocking capacity of 17C02-based molecules and FcγR utilization by THP-1-CD16A cells in the phagocytosis of IgG-opsonized human platelets. (A) Images of platelets sensitized with ITP serum and later incubated with THP-1-CD16A macrophages. Images were taken at the center of each well with Z-stacking. Phagocytosis was quantified using Imaris v9.6.0. The white arrows denote examples of phagocytosis of platelets; scale bar, 10 μm. (B) PI from 4 independent experiment are shown. Sensitization: “+” indicates platelets were incubated with normal human serum vs serum from patients with ITP. The PI was calculated as the number of platelets engulfed per 100 macrophages. The contribution of FcγRI, II, and III to phagocytosis was evaluated using Fc region deglycosylated blocking antibodies (final concentration of 10 μg/mL; 0.07 μM each): anti-FcγRI (clone 10.1), anti-FcγRIIA/B/C (clone AT10), or anti-FcγRIIIA (clone 3G8). The deglycosylated mouse IgG1 (clone MOPC-21), the deglycosylated mouse IgG2a (clone N/A-CP150), and human albumin were used as controls (final concentration of 0.07 μM). The blocking capacity of 17C02-based molecules was evaluated (17C02-albumin, 17C02-IgG1 OA , and deglycosylated 17C02-IgG2a) using the same comparative final molar concentration. Data are presented as the mean ± the standard deviation (n = 4-5). The statistical analysis was performed using Kruskal-Wallis and Dunn multiple comparison test (∗ P < .05).

Article Snippet: FcγRI (CD64, catalog number 10256-H08H), FcγRIIIA (CD16A, catalog number 10389-H27H), and FcγRIIIB (CD16B, catalog number 11046-H08C), as well as biotinylated-FcγRIIA (catalog number 10374-H27H-B) and biotinylated-FcγRIIIA (catalog number 10389-H27H1-B) were also purchased from Sino Biological.

Techniques: Blocking Assay, Incubation, Concentration Assay, Standard Deviation, Comparison

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Flow cytometry antibodies used.

Article Snippet: CD32-PEVio770 , Miltenyi , 130-097-506 , 2E1 , .

Techniques: Flow Cytometry, In Vivo, In Vitro

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Murlentamab opsonization of SKOV3-R2 + orients naïve macrophages and reprograms TAMs towards an M1-like profile. SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). ( A ) The proportion of macrophages expressing M1/M2 membrane markers (CD32, CD64, CD80, TLR2, CD163, CD36 and CD206) was determined by flow cytometry after three days of co-culture with SKOV3-R2 + cells. ( B ) The release of cytokines (IL1β, IL12, TNFα, IL6, IFNγ, IL10) and chemokines (CCL2, CCL4, CCL5, CXCL9 and CXCL10) in the culture medium was determined by AlphaLISA after three days of co-culture with SKOV3-R2 + cells. Data shown (boxplots) are the results from three different experiments (performed with three different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.

Article Snippet: CD32-PEVio770 , Miltenyi , 130-097-506 , 2E1 , .

Techniques: Labeling, Cell Culture, Derivative Assay, Expressing, Membrane, Flow Cytometry, Co-Culture Assay

Journal: Acta Neuropathologica Communications

Article Title: Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation

doi: 10.1186/s40478-020-00948-z

Figure Lengend Snippet: Human adult primary microglia express specific microglial markers. a Representative fluorescent photomicrographs of human primary microglia culture demonstrating cell morphology and purity of culture; Iba-1 (green), DAPI nuclear staining (blue). Scale bar represents 50 μm. b Flow cytometry analysis for CD11b, CD16, CD32 and CD64 confirmed purity of human microglia cultures. For each marker representative histograms are shown from one human microglia culture and bar graphs with percentage of positive cells from four independent microglia cultures (mean +/− SEM). Histograms and bar graphs show binding and cell positivity for anti-CD11b, −CD16, −CD32 and -CD64, respectively, compared to binding and cell positivity for nonspecific IgG

Article Snippet: Anti-human antibodies CD11b-PE-Vio770, CD64-Vio615, CD32-PE, CD16-VioBright and controls: REA control-Vio770, REA control-Vio615, REA control –PE, REA control-VioBright (MACS Miltenyi Biotec), anti-Iba1 (WAKO) were used for human microglia.

Techniques: Staining, Flow Cytometry, Marker, Binding Assay

Journal: Immunology

Article Title: Disease activity in systemic lupus erythematosus is associated with an altered expression of low-affinity Fc? receptors and costimulatory molecules on dendritic cells

doi: 10.1111/j.1365-2567.2009.03138.x

Figure Lengend Snippet: Fcγ receptors (FcγRs) on dendritic cells (DCs) from patients with systemic lupus erythematosus (SLE) present an expression pattern skewed towards an overactivated DC phenotype. Expression of the activating receptor CD32a (a) and of the inhibitory receptor CD32b (b) were analyzed on immature DCs (iDCs) or on DCs matured with 5 μg/ml of lipopolysaccharide (LPS) (mDCs) obtained from SLE patients or from healthy donors. Graphs represent the mean fluorescence intensity for each antibody staining (c) The ratio of expression between CD32a and CD32b was plotted for iDCs and mDCs, for SLE patients and healthy donors. White bars represent healthy donors (Control) and black bars represent SLE patients (SLE). The results show the mean ± standard error of the mean (SEM). *P < 0·05; **P < 0·01.

Article Snippet: AlexaFluor488-conjugated mAb anti-CD32a (clone IV.3) and anti-CD32b (clone 2B6) were obtained from MacroGenics, Inc. (Rockville, MD).

Techniques: Expressing, Fluorescence, Staining

Journal: Immunology

Article Title: Disease activity in systemic lupus erythematosus is associated with an altered expression of low-affinity Fc? receptors and costimulatory molecules on dendritic cells

doi: 10.1111/j.1365-2567.2009.03138.x

Figure Lengend Snippet: Alterations in the ratio of activating/inhibitory Fcγ receptors (FcγRs) correlate with the severity of systemic lupus erythematosus (SLE). The SLE Disease Activity Index (SLEDAI) was plotted for each of 20 patients against each respective CD32a/CD32b ratio for immature dendritic cells (iDCs) and for mature dendritic cells (mDCs). Statistical analysis shows a Spearman r value of 0·44 for iDCs, and a Spearman r value of 0·71 for iDCs (P values of 0·06 and 0·0006, respectively).

Article Snippet: AlexaFluor488-conjugated mAb anti-CD32a (clone IV.3) and anti-CD32b (clone 2B6) were obtained from MacroGenics, Inc. (Rockville, MD).

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Expansion of Immature Neutrophils During SIV Infection Is Associated With Their Capacity to Modulate T-Cell Function

doi: 10.3389/fimmu.2022.781356

Figure Lengend Snippet: Neutrophil priming during the course of SIV infection and cART. (A) Follow-up of blood CD62L-low neutrophil frequency and plasma viral loads in both groups. Values shown in the graphs indicate the mean with the SD of the CD62L-low frequency and the mean, with the range, of plasma viral load. The black curve represents the data points of the 12 animals from both groups before cART initiation. *p < 0.05, **p < 0.01 by the paired Friedman test with Dunn’s multiple comparison of the CD62L-low frequency compared to baseline values for the 6 animals studied in each group. The blue (upper graph) and orange (lower graph) curves represent the data in the early cART and late cART treated animals. The red curve shows the plasma viral load for the 12 animals till 28 dpi and for the 6 animals included in each group for the latter time point. (B) Representative flow cytometry data plots displaying CD62L and CD32a expression in CD66+ Lin- CD14- CDw125- neutrophils from one macaque before cART. (C, D) Frequency of CD62L-low PMNs in blood (C) and bone marrow (D) during different stages of infection and cART. The values in the graphs indicate the mean with the SD of each neutrophil population. *p < 0.05, **p < 0.01 by the paired Friedman test with Dunn’s multiple comparison and Mann Whitney tests for unpaired comparisons. (E, F) Frequency of CD64-high and HLADR+ PMNs in the blood for both groups. Before cART initiation for Group 1, both groups are represented together in the black line and then separated. The values in the graphs indicate the mean with the SD of the CD64-high or HLADR + PMN frequency. *p < 0.05, **p < 0.01 by the paired Friedman test with Dunn’s multiple comparison.

Article Snippet: Then, antibody staining was performed with the following antibodies for 15 min at room temperature: Core panel: CD11b (ICRF144, BD Bioscience), CD45 (D058-1283, BD Bioscience) CD3 (SP34.2, BD Bioscience), CD8a (RPAT8, BD Bioscience), HLADR (L234, Biolegend), and CD66abce (TET2, Myltenyi Biotec), CD20 (2H7, BD Bioscience); CD32a (FLI8.26, BD Bioscience), CD14 (M5E2, BD Bioscience), CD16 (3G8, BD Bioscience), CDw125 (A14, BD Bioscience); Priming panel: CD64 (10.1, BD Bioscience), CD62L (SK11, BD Bioscience), CD89 (A59, Biolegend); Maturation panel: CD49d (9F10, Biolegend), CD10 (HI10a, Biolegend), CD101 (BB27, Biolegend).

Techniques: Infection, Flow Cytometry, Expressing, MANN-WHITNEY