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

Journal: Frontiers in Immunology

Article Title: Sex-dependent differences in type I IFN-induced natural killer cell activation

doi: 10.3389/fimmu.2023.1277967

Figure Lengend Snippet: Type I IFN subtypes modulate natural killer cells in-vitro . Changes (presented as deltaΔ) in (A) activation measured by CD69+ expression on NK cells, and in (B) degranulation measured by CD107a+ expression on NK cells. Representative dot plots are shown on right side for CD69+ (A) and CD107a+ (B) expression on NK cells. PBMCs were stimulated with respective Type I IFN subtypes for 4h followed by a degranulation assay for 5h with K562 cell line. Plotted percentage delta values were calculated by subtracting the corresponding unstimulated condition with K562 cell co-cultures from each experiment. (C) Spearman correlation coefficient (r) between delta changes in activation (CD69) and delta changes in degranulation (CD107a) of NK cells after IFN-stimulation. Paired t test; non parametric Wilcoxon test; n=10. Spearman correlation; 100 XY pairs. *p<0.05; **p<0.005.

Article Snippet: NK cells were isolated from PBMCs using negative selection kit EasySep human NK cell enrichment kit II (Catalog no. 19055, StemCell Technologies).

Techniques: In Vitro, Activation Assay, Expressing, Degranulation Assay

Journal: Frontiers in Immunology

Article Title: Sex-dependent differences in type I IFN-induced natural killer cell activation

doi: 10.3389/fimmu.2023.1277967

Figure Lengend Snippet: Sex differences in NK cell activation after TLR7/8 stimulation. (A, B) Poly-IFNα ELISA quantification of supernatants after overnight stimulation with 1ug/mL TLR7/8 agonist (CL097) of male and female-derived PBMCs (A) and pDCs (B) . Average of duplicates are represented in box and whisker plots, n=13 (A) and n=12 (B) . Mann-Whitney test. (C) Representation of transwell co-culture system. Overnight culture of NK cells, plated in upper chamber, with stimulated or unstimulated PBMCs or pDCs, plated in lower chamber. Image created with BioRender.com . (D, E) Relative activation levels measured by CD69+ NK cells normalized to their corresponding control condition (untreated). NK cells were cultured with control stimulation, as well as co-cultured with unstimulated or stimulated (CL097) PBMCs (D) or pDCs (E) from male and female donors in transwell system. Total of 16 independent donors in (D) or 12 independent donors in (E) , consisting of equal numbers of male and female donors. Absolute expression levels relative to untreated condition. Paired sets of a male- and female-donors were matched for each experiment including controls. Wilcoxon test paired (solid line); 2way ANOVA test (dotted line); *p<0.05**; p<0.005; ***p<0.0005; ****p<0.00005.

Article Snippet: NK cells were isolated from PBMCs using negative selection kit EasySep human NK cell enrichment kit II (Catalog no. 19055, StemCell Technologies).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Whisker Assay, MANN-WHITNEY, Co-Culture Assay, Control, Cell Culture, Expressing

Journal: Frontiers in Immunology

Article Title: Sex-dependent differences in type I IFN-induced natural killer cell activation

doi: 10.3389/fimmu.2023.1277967

Figure Lengend Snippet: NK cell-intrinsic sex differences in response to TLR7/8 stimulation. (A, B) Relative activation of CD69+ NK cells, separated by sex, after overnight cultures with control stimulations, as well as co-cultures with unstimulated and CL097-stimulated PBMCs (A) or pDCs (B) in transwell system. Activation levels were relativized to their own control condition (untreated). (C, D) Relative activation levels measured by CD69+ NK cells by further separating the sex of NK cells- and PBMCs-derived donors (C) or pDCs-derived donors (D) . Activation levels were relativized to their own control condition (untreated). Total of 16 independent donors in (D) or 12 independent donors in (E) , consisting of equal number of male and female donors. Paired sets of a male- and female-donors were matched for each experiment, including controls. Male NK cells (yellow); female NK cells (turquoise). Wilcoxon test paired (solid line); 2way ANOVA test (dotted line); ns, non-significant; *p<0.05; **p<0.005; ***p<0.0005; ****p<0.00005.

Article Snippet: NK cells were isolated from PBMCs using negative selection kit EasySep human NK cell enrichment kit II (Catalog no. 19055, StemCell Technologies).

Techniques: Activation Assay, Control, Derivative Assay