cd16a nk 92 cells  (ATCC)

Journal: The Journal of Infectious Diseases

Article Title: Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy

doi: 10.1093/infdis/jiad421

Figure Lengend Snippet: Increasing maternal and cord blood neutralizing and Fc effector functional magnitude and breadth were observed after infection, vaccination, and vaccination and infection in pregnancy. The bars depict the median of matched maternal (white, left) and cord (blue, right) blood levels of ( A ) neutralization (FRNT 50 ) against SARS-CoV-2 WA1 (infection n = 14, vaccine n = 13, vaccine + infection n = 19), Delta, and Omicron viruses (infection n = 12, vaccine n = 8, vaccine + infection n = 14); ( B ) ADNKA by CD107a, IFN-γ, and TNF-α; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. B – F , sample sizes are infection n = 20, vaccine n = 18, vaccine + infection n = 27. P values are adjusted for maternal age and body mass index using linear regression. ^ Marks significance after adjustment for multiple comparisons by Benjamini-Hochberg. The proportion of detectable functions was used to categorize individuals as a high, medium, or low responder . Polyfunctional breadth is depicted as the percentages of each type of responder within each group in ( G ) maternal and matched ( H ) cord samples. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT 50 , 50% reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.

Article Snippet: CD16a.NK-92 cells (PTA-6967, American Type Culture Collection) with brefeldin A (Biolegend), Golgi Stop (BD Biosciences), and anti-CD107a (clone H4A3, BD Biosciences) were then added (37°C, 5 hours).

Techniques: Functional Assay, Infection, Neutralization, Binding Assay, Activation Assay, Fluorescence

Journal: The Journal of Infectious Diseases

Article Title: Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy

doi: 10.1093/infdis/jiad421

Figure Lengend Snippet: Fc effector functions are preferentially transferred across the placenta compared to neutralizing activities. The bars depict the median of matched maternal (grey) and cord (blue) blood levels of ( A ) neutralization against live SARS-CoV-2 WA1, Delta, and Omicron; ( B ) ADNKA; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. Statistical significance was calculated by Wilcoxon-matched pairs test. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT, reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.

Article Snippet: CD16a.NK-92 cells (PTA-6967, American Type Culture Collection) with brefeldin A (Biolegend), Golgi Stop (BD Biosciences), and anti-CD107a (clone H4A3, BD Biosciences) were then added (37°C, 5 hours).

Techniques: Neutralization, Binding Assay, Activation Assay, Fluorescence

Journal: Antibodies

Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

doi: 10.3390/antib12030044

Figure Lengend Snippet: Flow cytometric gating to detect CD107a externalization. ( A – E ) sequential gating for CD107a-positive NK-92-CD16A cells incubated with antibody and Raji cells. The assay conditions were an NK to Raji E:T of 1:2, with or without 10 ng/mL GA101-GE antibody, and 40 min of incubation at 37 °C. ( A ) Starting NK and Raji cells; ( B ) gating for single cells; ( C ) gating for GFP-bright NK-92-CD16A cells; ( D ) gating for CD56 bright GFP positive double-positive cells; ( E ) gating for the % CD107a-positive cells. ( F ) NK cells with Rajis without mAb. The data are from experiment JCA044, illustrated in .

Article Snippet: Another laboratory, with NK-92-CD16A cells (obtained from Conkwest/NantKwest: San Diego, CA, USA), breast cancer target cells, and trastuzumab anti-HER2, found that blood NK cells were more effective for ADCC than their NK-92-CD16A cells [ ].

Techniques: Incubation

Journal: Antibodies

Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

doi: 10.3390/antib12030044

Figure Lengend Snippet: CD107a externalization after six assay conditions. The results are from JCA044 in which cells were cultured for 40 min, 1:2 NK to Raji target cells, without or with 0.04 or 10 ng/mL of GA-101 GE antibody or with phorbol myristic acid (PMA) and ionophore. ( A ) Unlabeled NK cells used to set gating for CD107a-positive cells. ( B – F ) Labeled cells. ( B ) NK-92-CD16A cells without Raji targets (repeat of F). ( C ) cells with PMA & calcium ionophore as a positive control. ( D ) NK activity towards Raji cells in the absence of anti-CD20 antibody. ( E ) NK plus Rajis with 0.04 ng/mL GA101 GE antibody. ( F ) NK plus Rajis with 10 ng/mL GA101-GE (repeat of E).

Article Snippet: Another laboratory, with NK-92-CD16A cells (obtained from Conkwest/NantKwest: San Diego, CA, USA), breast cancer target cells, and trastuzumab anti-HER2, found that blood NK cells were more effective for ADCC than their NK-92-CD16A cells [ ].

Techniques: Cell Culture, Labeling, Positive Control, Activity Assay

Journal: Antibodies

Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

doi: 10.3390/antib12030044

Figure Lengend Snippet: Assay by NK-92-CD16A cell externalization of CD107a for antibodies that can support ADCC. The Raji cells were pre-incubated with GA101 anti-CD20 antibodies, then NK-92-CD16A cells were added at a 1:2 effector NK to Raji target ratio (E:T), and the cells incubated for 40 min at 37 °C. NK and Raji cells were also incubated without antibodies to measure CD107a externalization associated with NK activity. ( A ) Antibody detection by NK CD107a vs. by fluorescent secondary anti-IgG to Raji-bound antibodies. For the anti-CD20 antibody bound to Raji cells, the Rajis were stained with AF647-labeled donkey anti-human IgG. Both PE-anti-CD107a and AF647 anti-human IgG were detected by flow cytometry. ( A1 ) Detection of target-cell bound antibody by NK CD107a or by fluorescent anti-human IgG. The EC 50 s for each method are indicated with arrows. EC 50 values are the effective concentrations of anti-CD20 that elicited 50% of maximum NK-92 antibody-specific (ADCC minus NK) CD107a externalization. The values for NK activity (without antibodies) are indicated by square symbols. ( A2 ) The median fluorescent intensities (MFIs) of the CD107a-positive NK cells or AF647-anti-human IgG labeled Raji cells. The NK MFIs are for only the CD107a-positive cells. ( B ) CD107a externalization in response to antibodies with different Fc-fucosylation. The antibodies are from one mAb clone, GA101. The WT antibody is ~10% afucosylated; the GE antibody 50% afucosylated. ( B1 ) EC 50 s for antibodies that differed in fucosylation. The EC 50 s associated with afucosylation were 20-fold apart in this experiment ( p < 0.05); similar differences were observed for three other experiments. ( B2 ) The MFIs of the CD107a positive cells. The MFIs are for the CD107a positive in ( B1 ). The CD107a per cell increased with afucosylation ( p < 0.001). ( C ) Antibody concentrations for CD107a externalization vs. death by ADCC. The CD107a values are the data of ( B1 ). ( C1 , C2 ) Antibody EC 50 s for CD107a vs. for cell death, with GA101-WT or GA101-GE antibody. NK CD107a was determined at an E:T of 1:2. ADCC-mediated death was determined with 51 Cr-Raji cells at an E:T of 20:1. Both assays were stopped at 40 min. The 95% confidence limits for each EC 50 are color-coded and indicated at the top of the graphs. ( D ) Antibody detection by NK-92CD16A cell CD107a or by ADCC by peripheral blood NK cells. The donors’ genotypes encoding CD16A AA158, either lower affinity for Fc-IgG phenylalanine (F) or higher affinity valine (V), are indicated. The CD16A-positive blood NK cell to target (E:T) ratios were 4:1, 3:1, and 1:1 for donors 030, 035, and 038, respectively.

Article Snippet: Another laboratory, with NK-92-CD16A cells (obtained from Conkwest/NantKwest: San Diego, CA, USA), breast cancer target cells, and trastuzumab anti-HER2, found that blood NK cells were more effective for ADCC than their NK-92-CD16A cells [ ].

Techniques: Incubation, Activity Assay, Staining, Labeling, Flow Cytometry

Journal: Antibodies

Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

doi: 10.3390/antib12030044

Figure Lengend Snippet: Conditions that affect CD107a externalization. ( A ) Time courses. The E:T was 1:2, and the antibody concentration was 1 µg/mL GA101 WT. ( A1 ) Increases in the percentage of CD107a-positive NK-92-CD16A cells. The NK activity without antibodies is included to show its increase after antibody-dependent activity was complete. The inset illustrates that the antibody-dependent fraction (Total % − NK % positive) was unchanged after 40 min. ( A2 ) Increases in CD107a expression. The MFIs are for the CD107a-positive cells from ( A1 ). ( A3 ) Side-by-side comparison of the % CD107a positive cells vs. CD107a. MFIs. The antibody-dependent data are from ( A1 , A2 ). ( B ) Effects of E:T ratios on CD107a externalization and death of Raji cells. The E:Ts varied from excess effectors to excess targets, as illustrated for two separate assays, one for NK CD107a externalization and another for ADCC by 51 Cr release. Both assays were for 40 min with 1 µg/mL GA101 WT antibody. ( B1 ) % CD107a-pos cells vs. death by ADCC. The percentage of cells with external CD107a paradoxically decreased with increased E:T ratios. Note: each datum for the % CD107a positive cells represents the % of a varying number of effector cells that increased two-fold for each E:T. The color-matched asterisks indicate p < 0.01compared to the E:T 1:8 values. ( B2 ) Frequencies of CD107a-positive NK cell numbers vs. numbers of target cells killed by ADCC. The numbers (instead of percentages) of CD107a-positive cells at each E:T of ( B1 ) were calculated and then re-expressed as percentages of the initial Raji cells, indicated in orange. The % Raji cell death is also from ( B1 ). The CD107a-positive NK cells far exceeded the dead Raji cells (as indicated by the two ordinate scales). At the E:T of 8:1, the ratio of CD107a-pos NK cells to dead Raji cells was 13:1.

Article Snippet: Another laboratory, with NK-92-CD16A cells (obtained from Conkwest/NantKwest: San Diego, CA, USA), breast cancer target cells, and trastuzumab anti-HER2, found that blood NK cells were more effective for ADCC than their NK-92-CD16A cells [ ].

Techniques: Concentration Assay, Activity Assay, Expressing

Journal: Antibodies

Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

doi: 10.3390/antib12030044

Figure Lengend Snippet: Variables that affect biosafety in future applications. Antibodies were heated to simulate inactivation of viruses. Formaldehyde treatment is routinely used to inactivate viruses. These assays were at an E:T of 1:2 for 40 min. ( A ) Heated antisera. GA101 WT antibody was heated with serum and then diluted with control heated serum to 1 μg/mL antibody in 10% heated human serum. The variables are indicated below each bar. ( B ) Formaldehyde fixation. Treatment was either (1) after addition of antibodies to Raji cells or (2) after the NK-92-CD16A-cells reacted with cell-bound antibodies. ( B ) Reactivity of the NK-92-CD16A cells to formaldehyde-treated GA101 IgG. Raji cells were treated with 1 or 0.01 µg/mL GA101 WT antibody, then 1% formaldehyde, washed, and used to stimulate NK-92-CD16A cells. The E:T was 1:2, and the assay was for 40 min. ( C ) Reactivity of PE-mAb anti-CD107a with formaldehyde-treated CD107a. Cells were treated with formaldehyde and washed prior to labeling.

Article Snippet: Another laboratory, with NK-92-CD16A cells (obtained from Conkwest/NantKwest: San Diego, CA, USA), breast cancer target cells, and trastuzumab anti-HER2, found that blood NK cells were more effective for ADCC than their NK-92-CD16A cells [ ].

Techniques: Labeling