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

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 5 IL-18/poly I:C-primed NK cells kill hepatic stellate cells in a TRAIL- involved degranulation manner. IL-18 and poly I:C-primed NK cells were treated by anti-TRAIL or isotype antibody before coculture. HSC death was measured (A), and NK cell degranulation was evaluated by CD107a expression (C). Soluble TRAIL was added to cell coculture, the cell death of HSCs (B) and CD107a expression of NK cells (D) was shown. Results were shown as mean ± SEM of 6 independent experiments performed with 6 different donor NK cells. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test. The kinetics of the activated NK cell-mediated LX2 cell apoptosis were observed by using live-cell imaging (E).

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Expressing, Live Cell Imaging

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 6 IL-18/poly I:C-primed hepatic NK cells kill HSCs in a TRAIL-involved degranulation manner. Liver NK cells were purified from speci- men, then primed by IL-18 and/or poly I:C before cocultured with primary HSCs as above. CD107a expression of liver NK cells (A) and PI/AnnexinV level of primary HSCs (B) were detected. Anti-TRAIL or isotype antibody were used to treat IL-18/poly I:C-primed liver NK cells before cocultured with primary HSCs, then CD107a expression of liver NK cells (C) and the cell death of primary HSCs (D) was measured. Results were shown as mean ± SEM of 5 independent experiments performed with 5 different donor NK cells. *P < 0.05, **P < 0.01, paired t-test.

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Purification, Expressing