The gene expression signature induced in CLL cells by autologous activated T cells is very similar to that induced by CD40L stimulation. (A-C) Gene expression analysis (U133 plus 2.0 array, Affymetrix) of sorted CD20⁺ CD5⁺ CLL cells derived from PB or cultured with CD40L-expressing cells (CD40L) or Tact for 16 hours. Samples are numbers 2, 5B, 19, 24, and 27 as detailed in supplemental Table 1. (A) Principal component analysis is a statistical method for exploring large datasets by reducing the measurements (dimensions) to the few PCs that explain the main patterns. The coordinates obtained for the 3 PCs (PC1, PC2, and PC3) are depicted in 2-dimensional graphs. (B) Heatmap of 153 differentially expressed genes (ANOVA P < .01; minimum present calls >1; Bonferroni correction for multiple testing), depicting results for paired samples from the 5 patients mentioned above. (C) Venn diagram of the genes differentially expressed between Tact vs PB and CD40L vs PB. (D) The expression of different apoptotic mediators was analyzed by MLPA in the same samples as in panels A-C. Results are shown for relevant genes as relative expression to HK genes. (E) Purified CLL B cells were stained with DDAO and cultured for 48 hours either with CD40L-expressing fibroblasts (CD40L) or a control cell line (control) (left panel) or with autologous T cells, in the absence (resting T cells, Trest) or presence of agonistic antibodies against CD3 and CD28 (Tact) (right panel). CD95 expression was then assessed by flow cytometry on DDAO⁺ cells. Representative histograms are shown for each condition for sample 21.