Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: IgA mediates higher tumor cell lysis than IgG with human neutrophils. (A–D) Specific lysis of tumor cells by isolated human neutrophils (E:T = 40:1) using a 4 h 51 Cr release assay. Anti-CD20 Abs were used for Ramos and EL4-CD20, anti-HER2 Abs were used for SK-BR-3 and Ba/F3-HER2, anti-EGFR Abs were used for A431 and A1207 target cells. (A–C) Specific lysis of indicated target cells using a broad titration range of IgA and IgG antibodies. (D) Antibody concentrations were identical (10 μg/mL) for IgG and IgA antibodies against all target cells except for Ramos (13.3 μg/mL) and A1207 cells (1 μg/mL). Ctrl indicates condition without antibody (or 0.001 μg/mL IgG1-anti-CD20 for Ramos cells). One representative graph is shown for n = 4–6 independent experiments with different healthy donors. p < 0.001: *** , p < 0.0001: **** , unpaired Student's t test. (E) Stills from live cell microscopy of adhered A431-HER2 cells (calcein labeled) and neutrophils in the presence of 5 μg/mL anti-HER2 IgA2 or IgG (Trastuzumab) and TO-PRO-3 (red fluorescence), time in minutes, image acquisition every 30 s for 1.5 h.

Article Snippet: Labeling of the beads was checked with RPE-labeled anti-IgA (Southern Biotech, 2052-09), and PE-labeled anti-IgG (Southern Biotech, 2042-09).

Techniques: Lysis, Isolation, Release Assay, Titration, Microscopy, Labeling, Fluorescence

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Blocking FcγRIIIb only marginally improves tumor cell lysis. (A) Quantitative expression of FcγR and FcαRI on human neutrophils as analyzed by flow cytometry (Qifikit), n = 6–11 healthy donors. (B) 3 h 51 Cr release assays using EL4-CD20 with anti-CD20 IgA2 or IgG1 (rituximab), SK-BR-3 with anti-HER2 IgA1 or IgG1 and A431 with anti-EGFR IgA2 or IgG1, all at 10 ug/mL. 3G8 F(ab′) 2 fragments (1 ug/mL final concentration) were added 15 min prior to start of the ADCC assays. Statistics: one-way ANOVA with Tukey's multiple comparison test, p < 0.05: * , p < 0.01: ** , p < 0.0001: **** . Ctrl indicates no antibody added.

Article Snippet: Labeling of the beads was checked with RPE-labeled anti-IgA (Southern Biotech, 2052-09), and PE-labeled anti-IgG (Southern Biotech, 2042-09).

Techniques: Blocking Assay, Lysis, Expressing, Flow Cytometry, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Neutrophils display similar binding characteristics to IgA- or IgG-associated surfaces. (A) Example of relative binding of a donor of which calcein labeled neutrophils were allowed to associate to anti-CD20 IgA2 or IgG1 (clone UMAB001) coated 96 well plate. Remaining calcein fluorescence was measured after repeating wash steps. Calcein fluorescence before the first wash was set 100%. Ctrl indicates that no antibody was used during coating. (B–D) As in A , but data of 6–9 different donors at wash 10 are combined per target being anti-CD20 IgA2 or IgG1 (clone UMAB001) or anti-HER2 IgA2 or IgG1. Each color indicates data from the same donor. Statistics: two-way ANOVA with Tukey's multiple comparison test, p < 0.0001: **** . (E) Examples images of neutrophils binding to albumin, anti-CD20 IgA, or IgG coated Dynabeads (ratio beads:neutrophils = 5:1). Rosettes are defined as cells binding 5 or more beads. (F,G) Quantification of rosettes of anti-CD20- or anti-HER2-coated Dynabeads with neutrophils. One representative of n = 3 individual experiments is shown. Differences were not significant according to one-way ANOVA with Tukey's multiple comparison test. (H) Binding traces for calcein labeled neutrophils binding to anti-CD20 IgA- (red) or IgG- (black) opsonized Daudi cells. One representative of n = 5 experiments is shown. (I) Binding association and (J) average half-life of the Daudi:neutrophils complex pooled from 5 different donors. Statistics: paired Student's t -test.

Article Snippet: Labeling of the beads was checked with RPE-labeled anti-IgA (Southern Biotech, 2052-09), and PE-labeled anti-IgG (Southern Biotech, 2042-09).

Techniques: Binding Assay, Labeling, Fluorescence

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Strong p-ERK signal in neutrophils elicited by IgA. (A,B) Time-dependent increase and decrease of p-ERK signal in neutrophils after exposure to anti-CD20 or anti-HER2 IgA2- or IgG1-coated Dynabeads. Actin detection was performed on the same blot after destroying the p-ERK or total ERK signal. (C,D) Quantification of signaling defined as the ratio of normalized p-ERK/actin over normalized total ERK/actin. Data was normalized against the 0 min time point. (E) 4 h 51 Cr release assays using SK-BR-3 with anti-HER2 IgA2 or IgG1, both at 1 ug/mL. Where indicated, pharmacological inhibitors of signaling factors were included (wortmannin at 0.5 μM, Ly294002 and U0126 both at 20 μM). Statistics: two-way ANOVA with Tukey's multiple comparisons test p < 0.05: * , p < 0.01: ** , p < 0.001: *** , p < 0.0001: **** .

Article Snippet: Labeling of the beads was checked with RPE-labeled anti-IgA (Southern Biotech, 2052-09), and PE-labeled anti-IgG (Southern Biotech, 2042-09).

Techniques:

Journal: Frontiers in Immunology

Article Title: Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

doi: 10.3389/fimmu.2019.00704

Figure Lengend Snippet: Model for superior IgA-mediated tumor cell killing by neutrophils. Neutrophils express the low affinity Fc receptors FcγRIIIb, FcγRIIa, and FcαRI. Both FcγRIIa and FcαRI are activating Fc receptors, because upon ligand (antibody Fc domain) binding, clustering and recruitment of kinases (e.g., Lyn), they signal through their ITAM domains and elicit cellular effector functions. The relatively high FcγRIIIb expression on neutrophils could possibly interfere with the activating capacity of FcγRIIa by competing for Fc domain binding and preventing proper formation of signaling platforms and/or organization within the lipid bilayer. This results in a poor ability of FcγRIIa to initiate sufficient signaling in neutrophils. Despite the low FcαRI expression on neutrophils, they can still engage clustered IgA Fc domains to a similar degree as IgG Fc domains bind the FcγRs. IgA can, however, bind FcαRI bivalently resulting in more stable binding and recruitment of in total 4 ITAMs. This scenario would initiate a robust ITAM signaling necessary for activating effector functions, including trogoptosis to eliminate tumor cells. This is further illustrated by the signaling inhibitors wortmannin, Ly294002 and U0126 that prevent tumor cell lysis in vitro . The question mark refers to yet unclear processes involving signaling, intracellular Ca 2+ , actin-myosin contraction, and immune cell-tumor cell interactions that lead to ADDC by neutrophils (trogoptosis) .

Article Snippet: Labeling of the beads was checked with RPE-labeled anti-IgA (Southern Biotech, 2052-09), and PE-labeled anti-IgG (Southern Biotech, 2042-09).

Techniques: Binding Assay, Expressing, Lysis, In Vitro