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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: C2 Domains of Munc13-4 Are Crucial for Ca 2+ -Dependent Degranulation and Cytotoxicity in NK Cells.

doi: 10.4049/jimmunol.1800426

Figure Lengend Snippet: FIGURE 3. Rescue of defective degranulation of Munc13-4 KO NK cells with lentivirus expressing WT Munc13-4. Degranulation assays of Munc13-4 KO pri- mary NK cells that were transduced with lentivirus expressing EmGFP or WT Munc13-4–EmGFP. Degran- ulation was triggered with 2.5 mM ionomycin + 0.1 mM PMA for 6 h, and surface expression of CD107a was measured by FACS analysis. (A) Contour plots of FACS analysis illustrating induction of CD107a surface ex- pression in NK1.1+ primary NK cells that were trans- duced with EmGFP or WT Munc13-4 in unstimulated (top) and stimulated (bottom) condition. (B) Overlay of CD107a histograms in unstimulated (black) and stimu- lated (gray) NK cells. (C) A bar graph showing CD107a degranulation from EmGFP-rescued (white) and WT Munc13-4–rescued (black) KO NK cells. Error bars in- dicate SEM (n = 4).

Article Snippet: Isolation and culture of primary NK cells from mouse spleen MagCellect Mouse NK Cell Isolation Kit (catalog no. MAGM210) (R&D Systems, Minneapolis, MN) was used, following manufacturer’s instructions for isolation.

Techniques: Expressing, Transduction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: C2 Domains of Munc13-4 Are Crucial for Ca 2+ -Dependent Degranulation and Cytotoxicity in NK Cells.

doi: 10.4049/jimmunol.1800426

Figure Lengend Snippet: FIGURE 4. Double and quadruple muta- tions in the C2 domains of Munc13-4 alters Ca2+ sensitivity of degranulation from NK cells. Degranulation assays of Munc13-4 KO primary NK cells that were transduced with lentivirus expressing EmGFP alone, WT, C2 domain double, or quadruple mu- tant Munc13-4–EmGFP. Degranulation was triggered with 2.5 mM ionomycin + 0.1 mM PMA for 6 h and surface expression of CD107a was measured by FACS analysis. (A) Contour plots of FACS analysis illus- trating induction of CD107a surface ex- pression in NK1.1+ primary NK cells in 2.2 mM (left) and 10 mM (right) extracel- lular Ca2+. (B) Overlay of CD107a histo- grams in 2.2 mM (black) and 10 mM (gray) NK cells. (C) A bar graph showing CD107a degranulation from rescued NK cells in 2.2 mM (white) and 10 mM (black). Error bars indicate SEM (n 5 4). *p , 0.05.

Article Snippet: Isolation and culture of primary NK cells from mouse spleen MagCellect Mouse NK Cell Isolation Kit (catalog no. MAGM210) (R&D Systems, Minneapolis, MN) was used, following manufacturer’s instructions for isolation.

Techniques: Transduction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: C2 Domains of Munc13-4 Are Crucial for Ca 2+ -Dependent Degranulation and Cytotoxicity in NK Cells.

doi: 10.4049/jimmunol.1800426

Figure Lengend Snippet: FIGURE 5. Single-point mutations in the C2 domains of Munc13-4 alter Ca2+ sensi- tivity of degranulation from NK cells. De- granulation assays of Munc13-4 KO primary NK cells that were transduced with lentivirus expressing EmGFP alone, WT, or C2 domain single-mutant Munc13-4 EmGFP. Degranula- tion was triggered with 2.5 mM ionomycin + 0.1 mM PMA for 6 h and surface expression of CD107a was measured by FACS analysis. (A) Contour plots of FACS analysis illustrat- ing induction of CD107a surface expression in NK1.1+ primary NK cells in 2.2 mM (left) and 10 mM (right) extracellular Ca2+. (B) Overlay of CD107a histograms in 2.2 mM (black) and 10 mM (gray) NK cells. (C) A bar graph showing CD107a degranulation from rescued NK cells in 2.2 mM (white) and 10 mM (black). Error bars indicate SEM (n 5 4). *p , 0.05.

Article Snippet: Isolation and culture of primary NK cells from mouse spleen MagCellect Mouse NK Cell Isolation Kit (catalog no. MAGM210) (R&D Systems, Minneapolis, MN) was used, following manufacturer’s instructions for isolation.

Techniques: Transduction, Expressing, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: C2 Domains of Munc13-4 Are Crucial for Ca 2+ -Dependent Degranulation and Cytotoxicity in NK Cells.

doi: 10.4049/jimmunol.1800426

Figure Lengend Snippet: FIGURE 6. Mutations in C2 domains of Munc13-4 drastically reduces cytotoxicity of NK cells. (A) Control and Munc13-4 KO NK cells (effector cells) were incubated with Calcein Blue AM–loaded K562 cells (target cells) at indicated E:T ratios for 4 h, and the amount of Calcein Blue AM was measured. Percentage of lysis was calculated by dividing the amount of Calcein Blue AM by the total amounts (2% Triton X-100) 3 100%. Error bars indicate SEM (n = 12). (B) Cytotoxicity assay of Munc13-4 KO NK cells that are transduced with lentivirus expressing EmGFP, WT, or indicated C2 domain mutants. E:T ratios were 50:1 and 12.5:1. Error bars indicate SEM (n = 9).

Article Snippet: Isolation and culture of primary NK cells from mouse spleen MagCellect Mouse NK Cell Isolation Kit (catalog no. MAGM210) (R&D Systems, Minneapolis, MN) was used, following manufacturer’s instructions for isolation.

Techniques: Control, Incubation, Lysis, Cytotoxicity Assay, Transduction, Expressing