p66Shc deficiency enhances IL-9 production in CLL cells. qRT-PCR analysis of IL-9 mRNA (A) and quantification by ELISA of IL-9 released in the culture supernatants (B) from B cells purified from healthy donors (HD; n = 17) and CLL patients (n = 52), grouped as M-CLL (n = 33) and U-CLL (n = 19), according to the mutational IGHV status (M, mutated; U, unmutated). (C) Correlation between mRNA levels of p66Shc and mRNA levels of IL-9 in B cells purified from CLL patients (n = 52). qRT-PCR analysis of IL-9 mRNA (left panels) and quantification by ELISA (right panels) of IL-9 released in the culture supernatants of B cells purified from CLL patients and transfected with empty vector (vect; n = 6) or with a vector encoding WT p66Shc (p66; n = 6) (D) or mutated p66Shc (p66QQ; n = 5) (E). (F) qRT-PCR analysis of CXCL9, CXCL10, CXCL11, and CCL2 mRNA in HS-5 human stromal cells cultured for 48 hours with conditioned supernatants from CLL B-cell transfectants (vect SN, n = 6 and p66 SN, n = 6). (G) Migration of leukemic cells from CLL patients (n = 4) measured after a 3 hour-treatment with supernatants of HS-5 stromal cells conditioned for 48 hours with culture supernatants of CLL B cell transfectants (vect, n = 5; p66, n = 5), washed, and cultured in fresh medium for additional 48 hours. The data were obtained from duplicate samples from each CLL patient (± standard deviation [SD]). (H) qRT-PCR analysis of CXCL9, CXCL10, CXCL11, and CCL2 mRNA in HS-5 stromal cells cultured for 48 hours with conditioned supernatants of CLL B cell transfectants (vect, n = 3; p66, n = 3), in the presence of 0.1 ng/mL isotype-control antibody (ctr Ab) or 0.1 ng/mL neutralizing anti–IL-9 monoclonal antibody (IL-9 Ab). The relative gene transcript abundance was determined from triplicate samples using the dCt method. Data are mean ± SD. ****P ≤ .0001, ***P ≤ .001, **P ≤ .01, *P ≤ .05. Mann-Whitney rank-sum test (A-B,F), paired t test (D-E), 2-way ANOVA with Tukey’s post hoc correction, multiple comparisons (G-H). ns, not significant.