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.