Effect of anti–IL-7 mAb in cocultures and IL-7 expression and production by HMCLs and MM patients.

(A) Activated CD3⁺ cells were cocultured with HMCL RPMI-8226 in the presence or absence of anti–IL-7 polyclonal antibody (0.03 μg/mL) for 24 hours (i); a polyclonal anti-IgG was used as control. RANKL expression was analyzed by Western blot analysis. The figure is representative of 3 independent experiments; C indicates control (activated CD3⁺lymphocytes). Band intensity was quantified by densitometry and graphically presented (ii) as OD ratio of a representative experiment (optical density of RANKL normalized to the OD of actin). (B) Analysis of IL-7 mRNA expression in HMCLs (RPMI-8226, OPM-2, U266, XG-6) and fresh purified MM cells by RT-PCR. Bone marrow stromal cells (BMSCs) were used as positive control and PB MNCs from healthy subjects as negative control. (C) RT-PCR analysis of IL-7 expression in HMCL RPMI-8266 stimulated with IL-6 (20 ng/mL) for 24 hours. The intensity of the bands was quantified by densitometry; graph (ii) represents the mean OD of IL-7 normalized to the OD of β₂-microglobulin (OD ratio) of a representative experiment. Aliquots of conditioned media of normal bone marrow B cells or B leukemia cell line REH and HMCLs (RPMI-8226, U266, XG-6) incubated in presence or absence of IL-6 (20 ng/mL) were assessed for IL-7 levels by ELISA (iii). Graph at bottom represents the mean levels ± SE of 6 repeated experiments. *P < .001. (D) PB serum and BM plasma were obtained from MM patients and healthy subjects. IL-7 levels were detected by ELISA (plots represent individual value and bars the median levels).