Journal: PLoS pathogens

Article Title: TGF-β1 down-regulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence.

doi: 10.1371/journal.ppat.1002594

Figure Lengend Snippet: Figure 2. Deficient NK cell function in IT patients. (A) Deficient NK cell cytotoxicity from IT patients. NK cell cytotoxicity was assessed in primary NK cells from IT, IA, and IN patients and healthy controls using a 51Cr-release assay with K562 target cells. (B) Results show the mean 6 SEM values in 19 patients and 5 controls. Values for IT patients were less than controls (P= 0.0384). (C) Positive correlation between NK cell cytotoxicity and the percentage of NK cells expressing NKG2D and 2B4. (D) Fresh PBMCs were stimulated with/without IL-12 as described in the Materials and Methods. After 16 h, IFN-c production was determined using flow cytometry by gating on CD32CD56+ NK cells. A representative dot plot displaying intracellular IFNc staining in NK cells with the subsets indicated is shown. (E) Cumulative data are shown. (F, G) Fresh PBMCs from patients before treatment (black) or after treatment (white) were stimulated with IL-12 for 16 h. IFNc (F) and CD107a (G) production was determined using flow cytometry by gating on CD32CD56+ NK cells. (H) Induction of ex vivo NK cell cytotoxic activity after in vivo administration of antiviral treatment with nucleoside analogues until ALT reached normal levels. NK cytotoxicity was determined ex vivo by measuring the lysis of 51Cr-labelled target cells before treatment (red) or after treatment (green). Cumulative data are shown. doi:10.1371/journal.ppat.1002594.g002

Article Snippet: Flow cytometric analysis Fresh human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll-Isopaque (Solarbio, China) gradient centrifugation.

Techniques: Cell Function Assay, Release Assay, Expressing, Flow Cytometry, Staining, Ex Vivo, Activity Assay, In Vivo, Analogues, Lysis

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: Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy adult donors using a Ficoll-Plaque density gradient centrifugation Biocoll (Biochrom).

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: Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy adult donors using a Ficoll-Plaque density gradient centrifugation Biocoll (Biochrom).

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: Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy adult donors using a Ficoll-Plaque density gradient centrifugation Biocoll (Biochrom).

Techniques: Activation Assay, Control, Derivative Assay