Validation of gene expression observed by microarray. (A) Concordant with data seen on microarray, by quantitative PCR there was decreased expression of NFRKB in CD4 cells and VAMP2 in CD8 cells from CLL patients compared with healthy donors. The figure represents data from CD4 cells from 6 CLL patients and 5 healthy donors and CD8 cells from 4 CLL patients and 6 healthy donors. Statistical significance was assessed in a 2-tailed Student’s t test. (B) Decreased expression of NF-κBp65 in CD4 cells and Rho-GAP p190 proteins in CD8 cells from CLL patients compared with healthy donors. The left 2 lanes represent protein expression in CD4 or CD8 cells from 2 CLL patients (C1 and C2), and the right 2 lanes represent 2 healthy donors (H1 and H2). The expression of proteins was normalized by GAPDH expression level and is shown as protein bands and densitometric intensity of each band. The figure is representative of 3 additional experiments performed on 6 different donors, all showing a similar pattern (P < 0.05). (C) Intracytoplasmic expression of the GP1 gene product granzyme B, detected in CD8 cells from CLL patients and healthy donors by flow cytometry and fluorescent microscopy. To obtain at least 99% CD8 cell population, cells were purified using magnetically labeled negative cell-depletion antibodies. High expression of granzyme B in CD8 cells from healthy donors (CD8-FITC⁺ granzyme B–PE⁺, orange-brown) was observed compared with that in CD8 cells from CLL patients (CD8-FITC⁺ granzyme B–PE^–, green). The figure is representative of experiments performed with 4 different patients and healthy donors (P < 0.05).