CD44 promotes CLL cell survival in vitro. (A) The apoptosis-associated decrease (relative to day 0) of isotype-corrected CD44 surface expression by flow cytometry (mean ± SEM MFI at 2 days, 0.83 ± 0.07) in 5 human CLL samples is reversed by supplementation of prosurvival IL-4/CD40L (mean ± SEM MFI at 2 days, 1.17 ± 0.08; P < .05). (B) Coculture of 4 human CLL samples with the BMSC line NKtert leads to strong promotion of cell survival paralleled by increased CD44 surface expression (MFI), which is more marked in the direct feeder layer coculture (2.36 ± 0.45 at 2 days and 3.36 ± 1.20 at 7 days [mean ± SEM]; P < .05) than in cultures with 2-day-old NKtert-conditioned supernatant (1.63 ± 0.27 at 2 days and 2.71 ± 1.18 at 7 days [mean ± SEM]; P < .05). (C-D) The natural CD44 ligands HA and CS increase CLL cell viability and prevent apoptosis in vitro as analyzed in 19 human CLL samples. (C) A cytokine cocktail containing IL-4 and soluble CD40L consistently promotes ATP activation (mean ± SEM, 2.07 ± 0.27; P < .0001). Supplementation of media with additional HA or CS for the same 36 hours variably induces increased ATP activity and thus can synergize with the effects of IL-4/CD40L (mean ± SEM fold-changes to medium-only: 4.99 ± 3.91 for HA and 5.81 ± 3.77 for CS). (D) HA and CS partially prevent apoptosis (mean ± SEM fold-change to medium-only: 0.49 ± 0.09 for HA and 0.50 ± 0.10 for CS; P < .05) to a similar degree than does IL-4/CD40L (mean ± SEM, 0.51 ± 0.13; P < .05). (E) Double-target nucleofection experiments of scrambled/CD44 siRNAs in parallel to empty vector/complementary DNA for myr-AKT in 3 primary human CLL samples demonstrate the prosurvival relevance of CD44. CD44 knockdown decreases ATP activity (control: 2335 ± 308, CD44-siRNA: 1433 ± 328). Sole transfection of myr-AKT increases viability (4610 ± 1056), which is significantly reverted by CD44 knockdown (2134 ± 681). All values represent mean ± SEM, P < .0005, paired Student’s t test.