CCR7 expression is negatively regulated by p66Shc through its pro-oxidant activity. (A) qRT-PCR analysis of CCR7 mRNA in purified peripheral B cells from either healthy donors (ctr; n = 8) or CLL patients with mutated (M; n = 29) or unmutated (U; n = 28) IGHV. The relative abundance of gene transcripts was determined on triplicate samples using the ddCt method and is expressed as the normalized fold expression (mean ± SD). ***P < .001 and **P < .01 by Mann-Whitney rank-sum test. (B) qRT-PCR analysis of CCR7 mRNA in MEC B cells stably transfected with empty vector (MEC ctr) or an expression construct encoding either wild-type p66Shc (MEC p66) or the S36A (MEC p66SA) or EE132/133QQ (MEC p66QQ) mutants. **P < .01. (C) qRT-PCR analysis of CCR7 mRNA in purified splenic B cells from wild-type (+/+) or p66Shc^−/− mice. The relative abundance of gene transcripts was determined on triplicate samples from at least 5 wild-type or p66Shc^−/− mice. **P < .01 by Mann-Whitney rank-sum test. (D) qRT-PCR analysis of CCR7 expression in PB B cells purified from CLL patients with either mutated (M; n = 7) or unmutated (U; n = 6) IGHV nucleofected with either empty vector (vector ctr) or an expression construct encoding p66Shc (p66). The analysis was carried out 48 hours after transfection. All samples were checked for reconstitution of p66Shc expression by qRT-PCR (data not shown). The relative abundance of gene transcripts was determined on triplicate samples from each patient using the ddCt method and is expressed as the normalized fold expression (mean ± SD; empty vector controls taken as 1 for all CLL samples). ***P < .001 and *P < .05.