Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Primary human CD4 T cells from eight different donors ( n = 8) were separately infected with lab‐adapted and primary HIV‐1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry 4 days after infection. Uninfected CD4 T cells were determined as CD4‐positive and HIV‐1 p24‐negative, infected cells as CD4‐negative and p24‐positive. Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with NL4‐3, JR‐CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity ( x ‐axis). Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles), and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4‐3: n = 8; JR‐CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7–8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary AB‐only control ( y ‐axis). Data information: Wilcoxon signed‐rank test. Values for non‐infected and infected cells of the same donor are connected with lines. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Infection, Expressing, Flow Cytometry, Incubation, Fluorescence, Comparison, Control

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Degranulation of primary human NK cells after co‐culture with various target cells in the presence or absence of αTRAIL or αDR4/5. Comparison of CD107a expression after co‐culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry ( n = 10 different donors per condition). Each data point represents the mean of two technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation ( y ‐axis) after co‐culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 10 different donors per condition). Comparison of CD107a expression after co‐culture with autologous HIV‐I‐infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry ( n = 9 different donors per condition). Each data point represents the mean of two technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation ( y ‐axis) after co‐culture with autologous HIV‐I‐infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 9 different donors per condition). Representative histograms (overlay, left panel) and bar graphs ( n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of three technical replicates. Comparison of CD107a expression after co‐culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each) or 20 µg/ml isotype control (Iso) using flow cytometry ( n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a + NK cells ( y ‐axis). Box plots display inhibition of degranulation after co‐culture with 721.221 target cells in the presence of either isotype or αDR4/5 as relative reduction compared to no antibody ( n = 9 different donors per condition). Data information: Wilcoxon signed‐rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Co-Culture Assay, Comparison, Expressing, Control, Flow Cytometry, Fluorescence, Inhibition, Infection

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Degranulation of primary human NK cells after incubation with plate‐coated antibodies or whole proteins. Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry ( n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity ( y ‐axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated antibodies of different concentrations ( x ‐axis). Comparison of CD107a expression after incubation with plate‐coated DR4 protein, DR5 protein, or human IgG using flow cytometry ( n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity ( y ‐axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated proteins of different concentrations ( x ‐axis). Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Correlation analysis between relative frequency of CD107a + NK cells and granzyme B concentration ( n = 53, data points obtained from A, B, and C, 11 different donors). Comparison of CD107a expression after incubation with plate‐coated DcR1 protein, osteoprotegerin (OPG), or human IgG using flow cytometry ( n = 9 different donors). Box plots showing relative frequency of CD107a + NK cells ( y ‐axis) after incubation with plate‐coated proteins of different concentrations ( x ‐axis). Data information: Wilcoxon signed‐rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, C, E) Each data point represents the mean of two technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Incubation, Comparison, Expressing, Flow Cytometry, Fluorescence, Concentration Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: Lysis of different target cells in co‐culture with NK cells was quantified in various cytotoxicity assays. Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining ( y ‐axis) after co‐culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference in target cells remaining ( y ‐axis) between αTRAIL and isotype conditions displayed as p.p. ( n = 12 different donors). Each data point represents the mean of at least two technical replicates. Left panel: Representative contour plots showing the percentage of .221‐DR4/5KO (control) and .221‐Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221‐DR4/5KO and .221‐Cas9 cells ( y ‐axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221‐Cas9 cells displayed as percent ( n = 12 different donors). Each data point represents the mean of at least three technical replicates. Left panel: Representative contour plots showing the percentage of Raji‐pSIP (control) and Raji‐DR5 ++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji‐pSIP and Raji‐DR5 ++ ( y ‐axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji‐DR5 ++ cells displayed as % ( n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed‐rank test. Experiments were performed in four batches with three different donors each. “No NK” control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated “No NK” control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Source data are available online for this figure.

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Lysis, Co-Culture Assay, Control

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet: The expression of DR4 and DR5 was assessed by flow cytometry. 721.221 and Raji cells were labeled with LIVE/DEAD Fixable Near‐IR Stain, followed by incubation with biotin‐conjugated mouse anti‐human DR4 or DR5, and then labeled with Streptavidin‐BV421. Expression was quantified as fluorescence intensity. Representative histogram of DR4 (light orange) and DR5 (dark orange) expression in comparison to the Streptavidin‐only control (grey) or the FMO control (dashed line). Upper panel (from left to right): untransduced 721.221 cells, Cas9‐transduced .221s, and DR4/5 double knockout .221s. Lower panel (from left to right): untransduced Raji cells, Raji cells transduced with an empty vector (pSIP), and Raji cells overexpressing DR5.

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Expressing, Flow Cytometry, Labeling, Staining, Incubation, Fluorescence, Comparison, Control, Double Knockout, Transduction, Plasmid Preparation

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet:

Article Snippet: Mouse α‐human CD262 (DR5) Biotin (clone DJR2‐4) , Miltenyi Biotec , Cat#130‐097‐303; RRID:AB_2656745.

Techniques: Generated, Recombinant, Control, Sequencing, Staining, Software, Selection, Enzyme-linked Immunosorbent Assay, Marker

Journal: Blood

Article Title: TRAIL mediates and sustains constitutive NF-κB activation in LGL leukemia

doi: 10.1182/blood-2017-09-808816

Figure Lengend Snippet: TRAIL is overexpressed in LGL leukemia. (A) TRAIL gene expression levels in PBMC from T-LGL patients (triangles; n = 37), CD8+ cells from normal subjects (circles, n = 5), and Temra cells from normal subjects (squares, n = 3) in the GSE42664 Affymetrix data set.18 (B) Quantitative real-time PCR was performed to measure levels of TRAIL mRNA in CD8+ cells from T-LGL patients (n = 11) or purified CD8+ from normal donors (n = 3). Relative TRAIL mRNA expression was normalized to 18S. Data are presented as mean ± SEM. *P < .05 indicates significant difference between LGL leukemia patients and normal donors (Student t test). (C) Immunoblot analysis of TRAIL protein in purified CD8+ cells from patients with T-LGL leukemia (n = 6) vs CD8+ cells from normal (n) donors (n = 6) and purified NK cells from patients with NK-LGL leukemia (n = 4) vs purified NK cells isolated from normal donors (n = 4) and PBMCs from normal donors (n = 2). Loading of protein was confirmed by probing for GAPDH or β-actin. Vertical lines within the leukemic groups indicate regions where samples were removed from the image based on poor protein extract quality as indicated by loading controls. (D) CD8+ cells from a T-LGL patient or normal (NL) donor were stained with TRAIL antibodies and visualized using light microscopy (original magnification ×400). Rabbit IgG antibody was used as a negative control. Data are representative of 3 experiments conducted with cells from 3 independent patients. (E) Serum levels of TRAIL were determined using an ELISA assay. Sera were tested from T-LGL leukemia patients (squares, n = 24; ANOVA, *P < .0001 T-LGL vs normal donor), NK-LGL leukemia patients (triangles, n = 10; ANOVA, *P < .0001, NK-LGL vs normal donor), or normal donors (circles, n = 24). cDNA, complementary DNA.

Article Snippet: TRAIL determination by enzyme-linked immunosorbent assay (ELISA) TRAIL protein levels in serum samples from LGL leukemia patients and normal controls were determined using a Human TRAIL Quantikine ELISA kit (R&D Systems).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Purification, Expressing, Western Blot, Isolation, Staining, Light Microscopy, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Blood

Article Title: TRAIL mediates and sustains constitutive NF-κB activation in LGL leukemia

doi: 10.1182/blood-2017-09-808816

Figure Lengend Snippet: TRAIL induces NF-κB activation and nuclear translocation in LGL leukemia cells that are blocked by proteasome inhibitors. (A) EMSA demonstrating NF-κB activity in nuclear extracts from T-LGL patient PBMCs (n = 4) and NK-LGL patient PBMCs (n = 3) treated with either saline or rhTRAIL (10 ng/mL) for 2 hours. (B) NF-κB p50 or p65 protein ICC staining as visualized by light microscopy in CD8+ cells from an LGL leukemia patient compared with CD8+ cells from a normal donor (original magnification ×1000). Cells were treated with control (NTC), TNF-α (positive control, 10 ng/mL), or rhTRAIL (10 ng/mL). Brown staining represents NF-κB p50 or p65 protein. Data are representative of 3 experiments conducted with cells from 3 independent patients. (C) NF-κB p65 protein ICC staining as visualized by light microscopy in CD3+/CD8+/DcR2− and CD3+/CD8+/DcR2+ cells from an LGL leukemia patient (original magnification ×400). Cells were treated with either vehicle control (NTC) or rhTRAIL (10 ng/mL). Brown staining represents NF-κB p65 protein. Data are representative of experiments conducted with cells from 2 independent patients. (D) NF-κB p50 ELISA of PBMC nuclear protein extracts from T-LGL leukemia patients (n = 4) that were treated with sera from normal donor or sera from LGL patients. Patient cells treated with LGL sera were cotreated with vehicle, TRAIL neutralizing antibody (Neut Ab), or IgG control antibody. (E) EMSA demonstrating NF-κB activity in nuclear extracts from T-LGL patient PBMCs (n = 3) treated with vehicle (DMSO), rhTRAIL (10 ng/mL), or TRAIL (10 ng/mL) plus bortezomib (5 nM). (F) EMSA demonstrating NF-κB activity in nuclear extracts from patient cells from a T-LGL or NK-LGL patient treated with rhTRAIL (10 ng/mL) or rhTRAIL (10 ng/mL) plus increasing doses of ixazomib (0-100 nM). (G) NF-κB p50 or p65 protein ICC staining as visualized by light microscopy in CD8+ cells from normal control or LGL leukemia (original magnification ×400). Cells were pretreated with either bortezomib (5 nM) or DMSO for 2 hours followed with the treatment of rhTRAIL (10 ng/mL) or rhTNF-α (10 ng/mL positive control). Brown staining represents NF-κB p50 or p65 protein. Data are representative of 3 experiments conducted with cells from 3 independent patients.

Article Snippet: TRAIL determination by enzyme-linked immunosorbent assay (ELISA) TRAIL protein levels in serum samples from LGL leukemia patients and normal controls were determined using a Human TRAIL Quantikine ELISA kit (R&D Systems).

Techniques: Activation Assay, Translocation Assay, Activity Assay, Saline, Staining, Light Microscopy, Control, Positive Control, Enzyme-linked Immunosorbent Assay