Journal: bioRxiv

Article Title: Lupus IgA1 autoantibodies synergize with IgG to enhance plasmacytoid dendritic cell responses to RNA-containing immune complexes

doi: 10.1101/2023.09.07.556743

Figure Lengend Snippet: Surface staining and flow cytometry analysis performed on PBMCs from healthy control (HC) donors. (A) Representative histograms of FcαR ( red ) and FcγRIIa ( blue ) expression on pDCs compared to mIgG1 isotype control ( grey ), with detection by the same secondary antibody. (B) Paired analysis between monocyte and pDC FcαR (left) and FcγRIIa (right) surface staining (n=12 HC donors). (C) Binding of IgA and IgG to pDCs was assessed using biotinylated human heat-aggregated IgA or heat-aggregated IgG and fluorescently labeled streptavidin for visualization by flow cytometry, Streptavidin (SA) alone ( grey ), heat-aggregated IgA ( red ) and heat-aggregated IgG ( blue ). (D) Direct ex vivo binding of IgA and IgG to pDCs was assessed by staining with anti-IgA ( red ) or anti-IgG antibody ( blue ) directly ex vivo. Control ( grey ) has no anti-IgA or anti-IgG detection antibody. ( A-D ) Staining was performed on thawed PBMCs and pDCs gated as shown in Supplementary Fig. 2B. (B) Ratio paired t-test (**** p<0.0001).

Article Snippet: To generate aggregated IgA or IgG, biotinylated IgG (Novus NBP1-96855) or IgA (Novus NBP1-97181) was heated at a concentration of 2 mg/mL for 35-45 min at 65 C. After live dead staining, heat aggregated antibody was diluted in staining buffer to a final concentration of 0.5 mg/mL, incubated with PBMCs for 30 min at 37C.

Techniques: Staining, Flow Cytometry, Control, Expressing, Binding Assay, Labeling, Ex Vivo

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet: (A) DMF-differentiated HL60 cells expressing FcγRIIA (HL60–2A) treated with β-glucan exhibited decreased affinity for hIgG-coated RBCs in the presence of LacCer C24 compared with LacCer C16 or vehicle. Non-specific binding as measured with RBCs without hIgG was negligible. (B) Lyn knockdown HL60–2A cells (Lyn-1) showed increased affinity for hIgG-coated RBCs compared to scRNA control in the presence of β-glucan but not in dextran. (C) SHP-1 knockdown HL60–2A cells showed similar results as Lyn-1 knockdown cells, with the addition of β-glucan leading to increased 2D affinity of knockdown cells for IgG. Conversely, there was a significant drop in affinity upon SHP-1 knockdown in the presence of dextran control. All measurements in (B) and (C) were in the presence of LacCer C24. Non-specific binding controls without hIgG are shown. A c K a values represent ligand-receptor density normalized adhesion frequencies. Data are average ± SD, and individual values are plotted. *p < 0.05, **p < 0.01, ***p < 0.001 using an unpaired Student’s t test.

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Expressing, Binding Assay, Knockdown, Control

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet: (A) DMF-differentiated HL60 cells expressing FcγRIIA (HL60–2A) were treated with vehicle or C24, dextran or β-glucan, and isotype or functional blocking FcγRIIA antibody (IV.3) as indicated. Cells were seeded on immobilized BSA-anti-BSA under static conditions and stained with phalloidin. The area of spread cells and representative images are shown. (B) DMF-differentiated HL60, HL60–2A with scRNA, Lyn shRNA-1 (Lyn-1), or Lyn shRNA-2 (Lyn-2) were evaluated as in (A). (C–E) Human neutrophils (C), HL60–2A cells (D) pretreated with C24 and β-glucan, or HL60–2A cells with indicated shRNAs (E) were treated with IV.3 followed with luminol and anti-mouse F(ab’) to induce cross-linking (XL), and ROS generation (relative light units [RLUs]) was monitored over time. Representative plots (left) and the peak level of ROS (right) for each condition that was normalized to the average of the F(ab’) untreated control are shown. (F) HL60 cells expressing wild-type FcγRIIA (WT) or FcγRIIA ΔITAM mutant were treated with LacCer C24 and β-glucan, dextran, IV.3, or isotype control as indicated and incubated with IgG-opsonized fluorescein isothiocyanate (FITC)-latex beads. Phagocytic index (percentage of FITC-positive cells over total cells) analyzed by FACS is shown. Bar graphs represent fold change compared to vehicle + dextran for (A), native for (B), and WT + dextran for (F). Dotted line represents the value of untreated HL60 for (A), (B), and (F) and no F(ab’) control for (C), (D), and (E). Data are average ± SEM, and individual values are plotted. *p < 0.05 using the Kruskal-Wallis test.

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Expressing, Functional Assay, Blocking Assay, Staining, shRNA, Control, Mutagenesis, Incubation

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet:

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Purification, Control, Recombinant, Phagocytosis Assay, Immunoprecipitation, Knock-Out, Software