Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Analysis of the SARS-CoV-2-specific ADCC responses. Analysis of the kinetics of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response in plasma obtained from hospitalized patients with COVID-19 ( n = 19) collected at different time points: 3 ±1 ( n = 7), 6 ±1 ( n = 14), 9 ±1 ( n = 15), 12 ±1 ( n = 18), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptoms onset or nonhospitalized, mildly ill patients with COVID-19 ( n = 9) 28 to 31 (median: 30) days after symptom onset. Each plasma sample as well as each of 6 control samples from SARS-CoV-2 seronegative persons was individually tested against 26 different CD56 + CD16 + NK cell preparations from SARS-CoV-2 seronegative healthy blood donors and the percentage of CD107a (A), perforin (B), IFNγ (C), or TNFα (D) positive cells was assessed using flow cytometry. Percentage of all CD107a (A), IFNγ (C), or TNFα (D) positive cells, as well as only highly perforin-expressing cells (B) was assessed. All samples were normalized to the same SARS-CoV-2 seronegative control. From each sample, the median of independent experiments was calculated. Data are shown as mean values (±95% CI). ADCC positive plasma samples were identified using 6 SARS-CoV-2 seronegative controls. Fold change in positive cells at each time point were compared either between hospitalized patients with COVID-19 (indicated as a red bar) or mildly diseased nonhospitalized patients with COVID-19 (indicated as a blue bar) and seronegative controls (indicated as a black bar) using analysis of variance and Dunn’s post test (hospitalized patients with COVID-19) or Mann-Whitney test (nonhospitalized patients with COVID-19). P < .05 was considered significant. IFNγ, interferon gamma; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Control, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Impact of the FcγRIIIa-158-V/F variants on the ADCC responses. Analysis of the extent of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response with plasma obtained from hospitalized patients with COVID-19 ( n = 19) 6 ±1 ( n = 10), 9 ±1 ( n = 14), 12 ±1 ( n = 14), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptom onset (A-D) or nonhospitalized patients with COVID-19 ( n = 9) 28 to 31 (median: 30 days) days after symptom onset (E-H). Each plasma sample from hospitalized and nonhospitalized patients with COVID-19 was stimulated with CD56 + CD16 + NK cells from 26 healthy blood donors (FcγRIIIa-158-V/V: n = 5, FcγRIIIa-158-V/F: n = 12, FcγRIIIa-158-F/F: n = 9) and the MFI of CD107a (A,E), perforin (B,F), IFNγ (C,G), or TNFα (D,H) positive cells were assessed using flow cytometry. All samples were normalized to the same nonhospitalized SARS-CoV-2 seropositive control. MFI of all CD107a (A), IFNγ (C) or TNFα (D) positive cells, as well as of only high perforin-expressing cells (B) was assessed. Data are shown as mean values (±95% CI). Fold change MFI at each time point was compared between assays using FcγRIIIa-158-V/V, FcγRIIIa-158-V/F, and FcγRIIIa-158-F/F variant expressing NK cells, respectively using a ANOVA (A-D) or a paired t test (E-H). P < .05 was considered significant. ANOVA, analysis of variance; d.p.s.o, days post symptom onset; IFNγ, interferon gamma; MFI, mean fluorescence intensity; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Flow Cytometry, Control, Expressing, Variant Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma

doi: 10.3390/ijms23094717

Figure Lengend Snippet: Blocking the membranal-PCNA on MM cell lines enhances IFN-γ secretion and degranulation of NK cells. ( A ) Primary NK cells from a healthy donor were incubated with U266 cell lines and mAb 14-25-9 or mIgG1 for 18 h using different ratios of effector:target (1:1 and 1:3). IFN-γ was measured by ELISA. ( B ) Primary NK cells from a healthy donor were incubated either with RPMI 8226 or U266 cell lines and mAb 14-25-9 or mIgG1 for 5 h. Surface CD107a was measured by flow cytometry, to detect NK cells that had degranulated. Mouse IgG1 acts as a background control (red bars) in comparison to mAb 14-25-9 (blue bars). Dead cells were discriminated against using 7-AAD. Data is shown from three independent replicates and the experiment was repeated three times. Two-way ANOVA test showed p ** < 0.01 and p *** < 0.001. Statistical analysis and design were performed using Prism GraphPad v8 (California).

Article Snippet: Cells were isolated using a human negative selection-based NK cell isolation kit (EasySep-19615, STEMCELL).

Techniques: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma

doi: 10.3390/ijms23094717

Figure Lengend Snippet: Blocking the membranal-PCNA on primary BM mononuclear cells from MM patients enhanced the activation of NK cells. ( A ) NKp44 isoform 1-transduced NK92 cells (NK92-44-1) were incubated either with Pat#MM2 or Pat#MM7 primary cells for 18 h. ( B ) Primary NK cells from a healthy donor were incubated either with Pat#MM2 or Pat#MM7 primary cells for 18 h. ( C ) Primary NK cells from another unrelated healthy donor were incubated either with Pat#MM3.1 or Pat#MM4.1 primary cells for 18 h. Mouse IgG1 was used as a background control (red bars) for mAb 14-25-9 (blue bars). IFN-γ was measured by ELISA. Data is shown of three technical replicates. Two-way ANOVA test showed p * < 0.05, p ** < 0.01, and p *** < 0.001. Statistical analysis and design were performed using Prism GraphPad v8.

Article Snippet: Cells were isolated using a human negative selection-based NK cell isolation kit (EasySep-19615, STEMCELL).

Techniques: Blocking Assay, Activation Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay