Impaired S1P1 and S1P2 expression in CLL B cells from patients with unmutated IGHV. (A,C) qRT-PCR analysis of S1P1 (A) or S1P2 (C) mRNA in purified peripheral B cells from either healthy donors (ctr; n = 13) or CLL patients with mutated (M; n = 28 for S1P1; n = 25 for S1P2) or unmutated (U; n = 42 for S1P1; n = 36 for S1P2) IGHV. The relative abundance of gene transcripts was determined on triplicate samples using the ddCt method and is expressed as normalized fold expression (mean ± SD). (B,D) Flow cytometric analysis of surface S1P1 (B) or S1P2 (D) on purified peripheral B cells from either healthy donors (ctr; n = 10 for S1P1; n = 11 for S1P2) or CLL patients with mutated (M; n = 18 for S1P1; n = 16 for S1P2) or unmutated (U; n = 18 for S1P1; n = 16 for S1P2) IGHV. The data in the histograms are expressed as the mean fluorescence intensity (MFI) ± SD. Representative FACS profiles of S1P1 (B) or S1P2 (D) are shown. Specificity controls for each donor/patient included a sample incubated with secondary Ab alone (α-mouse) and, for S1P1 stainings, a sample preincubated with FTY720 to induce receptor down-regulation. ***P < .001; **P < .01; and *P < .05 by Mann-Whitney rank-sum test.