eNAMPT is present in CLL plasma and can be produced by activated CLL cells. (A) Box plot showing eNAMPT concentrations measured with a quantitative ELISA assay performed on plasma samples from CLL patients (n = 130) or HDs of a comparable age (n = 20). (B) Regression line showing a positive correlation between NAMPT mRNA levels (x-axis) and plasmatic eNAMPT (y-axis) in 25 CLL patients. Pearson coefficient (r) and the corresponding P value are noted. (C) Regression line showing a positive correlation between lymphocytosis (x-axis) and plasmatic eNAMPT (y-axis) in 31 CLL patients. Pearson coefficient (r) and the corresponding P value are noted. (D) ELISA assay showing eNAMPT concentrations in supernatants (SN) from cultures of purified CLL cells cultured with the indicated stimuli (n = 8, 24 hours). (E) The presence of eNAMPT was confirmed by western blot performed on concentrated (×10) albumin-depleted CLL plasma samples (#1-#3) and culture supernatants (#4-#7) from different CLL patients with high (>20 ng/mL, #4-#6) or low (<5 ng/mL, #7) eNAMPT levels, as detected by ELISA assay. The condition (–) and (+) medium indicates concentrated (×10) albumin-depleted RPMI +10% FCS alone (–) or with 20 ng of rNAMPT (+). #3D, #4D, #6D indicate NAMPT-deprived fractions by immunoprecipitation from #3 CLL plasma, or from supernatants #4 and #6. rNAMPT was loaded as control. (F) Time course of eNAMPT activity determined in the plasma of a prototype CLL patient, as described in supplemental Methods.