cd56 subsets  (Miltenyi Biotec)

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: PLoS ONE

Article Title: Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3 + CD56 + Subset through the Co-Culturing Dendritic Cells

doi: 10.1371/journal.pone.0078980

Figure Lengend Snippet: The CIK cells (A) at 1.0×10 5 cells/well from each condition were incubated with the attached HubCCA1 cells (5,000 cells/well) for 4 h before the PI assay. The CIK cell preparations comprised untreated condition, direct sunitinib treatment, macrophage co-culture, sunitinib-treated macrophage co-culture, iDC co-culture, and sunitinib-treated iDC co-culture, mDC co-culture, and sunitinib-treated mDC co-culture. The isolated CD3 + CD56 + cells (B) were studied in similar fashion. These included untreated CD3 + CD56 + cells, direct sunitinib treatment, mDC co-culture, and sunitinib-treated mDC co-culture. * and ** designate data with significant different from those of the untreated CIK cells at the same effector to target (E:T) ratio with p<0.05 and <0.01 respectively.

Article Snippet: An aliquot of CIK cells (1.0×10 8 cells) on day 14 was purified for CD3 + CD56 + subset using CD3 Microbeads kit and CD56 Microbeads kit (Miltenyi Biotec, Germany) according to the manufacturer instruction.

Techniques: Incubation, Co-Culture Assay, Isolation

Journal: PLoS ONE

Article Title: Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3 + CD56 + Subset through the Co-Culturing Dendritic Cells

doi: 10.1371/journal.pone.0078980

Figure Lengend Snippet: The studied subpopulations included CD3 + CD56 + (A), Th17 (RORC + IL-17 + , B) and Treg (CD4 + CD25 + Foxp3 + , C) subsets. The corresponding dot plot analysis demonstrated the gating of each subset. The CIK cells were either exposed to sunitinib directly, primed with mDCs or primed with sunitinib-pretreated DCs.

Article Snippet: An aliquot of CIK cells (1.0×10 8 cells) on day 14 was purified for CD3 + CD56 + subset using CD3 Microbeads kit and CD56 Microbeads kit (Miltenyi Biotec, Germany) according to the manufacturer instruction.

Techniques:

Journal: PLoS ONE

Article Title: Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3 + CD56 + Subset through the Co-Culturing Dendritic Cells

doi: 10.1371/journal.pone.0078980

Figure Lengend Snippet: The CD3 + CD56 + subset that had been directly exposed to sunitinib or co-cultured with sunitinib-pretreated mDCs were analyzed for the expression of IFN-γ, T-bet, IL-4, GATA-3, RORC, STAT3, and IDO.

Article Snippet: An aliquot of CIK cells (1.0×10 8 cells) on day 14 was purified for CD3 + CD56 + subset using CD3 Microbeads kit and CD56 Microbeads kit (Miltenyi Biotec, Germany) according to the manufacturer instruction.

Techniques: Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3 + CD56 + Subset through the Co-Culturing Dendritic Cells

doi: 10.1371/journal.pone.0078980

Figure Lengend Snippet: The CD3 + CD56 + cells from each condition were incubated with the attached HubCCA1 cells (5,000 cells/well) for 4 h before the PI assay. These conditions included the untreated CD3 + CD56 + cells, αIFN-γ treatment to CD3 + CD56 + cells, CD3 + CD56 + cells primed with mDC, αIFN-γ treatment to CD3 + CD56 + cells primed with mDC, CD3 + CD56 + cells primed with sunitinib-pretreated mDC, and αIFN-γ treatment to CD3 + CD56 + cells primed with sunitinib-pretreated mDC. * designates conditions that provided statistically difference after αIFN-γ treatment at the same E:T ratio with p<0.05.

Article Snippet: An aliquot of CIK cells (1.0×10 8 cells) on day 14 was purified for CD3 + CD56 + subset using CD3 Microbeads kit and CD56 Microbeads kit (Miltenyi Biotec, Germany) according to the manufacturer instruction.

Techniques: Incubation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Proliferation of NK cells in response to stimulation with OVCAR-3 cells expressing 4-1BBL and IL-12. a Flow cytometric confirmation of 4-1BBL expression by OVCAR-3 cells 48 h after infection with Ad-4-1BBL (open histogram), versus uninfected OVCAR-3 cells (shaded). b Level of IL-12 secreted in 1 ml medium between 48 and 72 h post-infection by 1 × 105 OVCAR-3 cells infected with Ad-IL-12 as described, Ad-GFP-infected or uninfected cells as controls, measured by ELISA. c Fresh PBMC from healthy donors or RCC patients, or TAL from patients with ovarian carcinoma, were stained with anti-CD3 and anti-CD56, and analysed by flow cytometry; typical results are shown. d Non-adherent PBMC from 15 healthy donors were co-cultured with OVCAR-3 cells pre-infected with adenovirus vectors expressing GFP, IL-12, 4-1BBL, or 4-1BBL + IL12, or mock-infected. After 7 days, viable lymphocytes were counted by haemocytometer and characterised by flow cytometry to identify T cells (CD3+CD56−), NKT cells (CD3+CD56+) or NK cells (CD3−CD56+), enabling calculation of their expansion relative to the starting populations. e Expansion of NK cells from PBMC of 13 RCC patients (RCC) or TAL from 3 ovarian cancer patients (OvCa) when stimulated and analysed as in d. Results for each cell type were analysed by one-way ANOVA and Bonferroni’s multiple comparison post-test; *P < 0.05 for comparison with GFP control; † P < 0.05 for comparison of 4-1BBL + IL-12 with IL-12 alone

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Cell Culture, Comparison, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Phenotype of expanded NK cells. PBMC from healthy donors, stimulated for 7 days as in Fig. 1, were stained for CD3 and CD56 to identify NK cells, and with additional antibodies as indicated. a Scatter and histogram plots illustrating gating of CD56dim and CD56bright NK cells, and the change from predominantly CD56dim cells at day 0, to CD56bright after 7 days stimulation with 4-1BBL + IL-12. b Scatter plots of CD3 versus CD56 for all stimulation conditions; c–j histogram plots gated on CD3−CD56+ NK cells, showing expression of CD56, CD16, CCR7, CD62L, CXCR3, NKG2D, CD158a (KIR2DL1/S1) and CD158e (KIR3DL1) as indicated (heavy line, open; thin line, shaded isotype controls)

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Staining, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Long-term proliferation and viability of NK cells in response to stimulation. Non-adherent PBMC from 6 healthy donors (a) and 6 RCC patients (b) were cultured for 7 days with OVCAR-3 cells expressing 4-1BBL + IL-12, 4-1BBL, IL-12, GFP or mock-infected, and expansion of the NK cell population was determined. Cultures were then redistributed and IL-2 added to a final concentration of 20U/ml. NK cell number was determined again at day 14 of culture, and cultures re-stimulated with OVCAR-3 pre-infected with the same adenoviral vectors, in the continued presence of IL-2. NK cell number was determined again at day 21 of culture. Symbols indicate mean ± SD. *P < 0.05, **P < 0.01, paired t test compared with previous time-point. c Analysis of CD3 and CD56 in a representative 4-1BBL + IL-12 stimulated culture, at each time-point. d Gating strategy to determine the viability of NK cells via Syto-16 and PI staining. Cells were first identified using side scatter and CD56 staining and subsequently gated on the CD3− population. NK cells staining Syto16+PI− were deemed live, double negative cells were considered to be apoptotic and Syto16−PI+ cells scored as necrotic. e Representative data for day 7 of culture. Results from 6 healthy donors at day 7 (f) and 14 (g) of culture (mean ± SD)

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Cell Culture, Expressing, Infection, Concentration Assay, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Proliferation, induction of 4-1BB expression and phenotypic conversion of isolated NK cells. a Negatively isolated NK cells were labelled with CFSE then co-cultured for 7 days with OVCAR-3 cells expressing GFP (as control), 4-1BBL, IL-12 or 4-1BBL + IL-12, prior to flow cytometric analysis (gated on CD56+ cells; results are typical of 3 donors). b Negatively isolated NK cells or adherent cell-depleted PBMC were stimulated with OVCAR-3 cells expressing 4-1BBL, IL-12 or GFP (as indicated) for 3 days, before 4-1BB expression on NK cells (CD3−CD56+) was determined by flow cytometry; 4-1BB expression on NK cells in freshly isolated PBMC (day 0) is shown for comparison. Gated on CD3−CD56+ cells; results are typical of 4 donors. c Negatively isolated NK cells were cultured for 3 days alone or in the presence or absence of recombinant human IL-12 (rhIL-12, 20 ng/ml) and OVCAR-3 cells pre-infected with adenovirus expressing GFP (GFPOVCAR) or IL-12 (IL-12OVCAR), or mock-infected (OVCAR). Expression of 4-1BB by the NK cells was then determined by flow cytometry (ANOVA, Dunnett’s multiple comparisons test vs. NK only control). d Negatively isolated CD56+ NK cells were magnetically selected for CD16+ cells; the resulting CD56dim CD16+ population was analysed immediately for CD3 and CD56 expression, or stained with CFSE prior to stimulation with OVCAR-3 cells pre-infected with Ad-4-1BBL + Ad-IL-12, Ad-IL-12 or Ad-GFP vectors as indicated for 7 days, and CFSE dilution in CD56+ cells analysed by flow cytometry; results typical of 2 donors. e CD56dim CD16+ NK cells were isolated by fluorescence activated cell sorting, then analysed immediately (Day 0) or co-cultured with OVCAR-3 cells expressing 4-1BBL + IL-12, IL-12 or GFP as control, for 7 days. Expression of CD56, CD16 and CD62L were determined by flow cytometry, gated on CD3− lymphocytes. Results are typical of 4 donors

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Expressing, Isolation, Cell Culture, Control, Flow Cytometry, Comparison, Recombinant, Infection, Staining, Fluorescence, FACS

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Functionality of NK cells in cultures stimulated with 4-1BBL, IL-12 or both. PBMC from healthy donors or RCC patients were stimulated for 7 days with OVCAR-3 cells pre-infected with adenovirus vectors expressing 4-1BBL, IL-12 or GFP as indicated or mock-infected. Cultured lymphocytes from healthy donors (a) or RCC patients (b) were then replated with 1 × 105 cells in 0.2 ml fresh medium and cultured overnight. Production of IFNγ was measured by ELISA (data points indicate different donors). a, b analysed by ANOVA, Dunnett’s multiple comparisons (with the GFP control). c Alternatively, after the 7 days stimulation, cells from healthy donors were cultured for 5 h in the presence of 10 μg/ml brefeldin A, and IFNγ assessed by intracellular staining and flow cytometry. Typical plots are shown, demonstrating IFNγ staining of the lymphocyte population gated as indicated to show T cells (CD3+CD56−), NKT cells (CD3+CD56+) and NK cells (CD3−CD56+). Cytotoxic function was assessed in a standard 5 h chromium release assay with cultured cells from healthy donors (d) and RCC patients (e) as effectors against K562 targets (means ± SD of triplicate wells); data are typical of at least 3 donors

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Infection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Staining, Flow Cytometry, Release Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Long-term proliferation of functional human NK cells, with conversion of CD56 dim NK cells to a CD56 bright phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12

doi: 10.1007/s00262-011-1122-3

Figure Lengend Snippet: Enhanced secondary response to targets of NK cells previously stimulated with 4-1BBL + IL-12. PBMC from healthy donors were co-cultured for 7 days with OVCAR-3 cells pre-infected with adenovirus vectors expressing 4-1BBL + IL-12. The lymphocyte populations were then passaged and maintained for a further 14 days without further stimulation. These pre-stimulated cultures containing an expanded population of NK cells or unstimulated PBMC from the same donors (which had been cryopreserved) were then stimulated with either (uninfected) OVCAR-3 or K562 cells or with no target cells as indicated, for 6 h. a Shows representative plots of intracellular IFNγ staining versus CD56 gated on CD3−CD56+ lymphocytes. Data from all 4 donors are summarised showing the proportion (b) and mean fluorescent intensity (c) of NK (CD3−CD56+) cells expressing IFNγ, following incubation with the indicated targets. As an indicator of cytotoxicity, degranulation of the NK cells was monitored via CD107a staining; d shows representative plots of CD107a versus CD56 (gated on CD3−CD56+ cells); e, f show data from all 5 donors, indicating CD107a+ cells as a per cent of total NK (CD3−CD56+) cells (e), and as a per cent of CD56bright NK cells (f). Data analysed by ANOVA, Bonferroni’s multiple comparison test

Article Snippet: Whole NK cell population and CD56 dim CD16 + NK cell subset isolation For some experiments, total purified NK cells were negatively isolated from PBMC using a Miltenyi Biotec CD56 + CD16 + NK Cell isolation kit and, when specified, further selected for CD16 + cells.

Techniques: Cell Culture, Infection, Expressing, Staining, Incubation, Comparison