Effect of fluvastatin on ADCC and TNFα release triggered with rituximab or trastuzumab.

<p>(A). Effect of fluvastatin on ADCC triggered with humanized antibodies. Cytolytic activity of NK cell clones was analyzed upon treatment with fluvastatin in ADCC assay with rituximab (rit) or trastuzumab (tra) using the C1R CD20⁺ or SKBR3 HER2⁺ tumor cell lines at 2∶1 NK/tumor cell ratio respectively. Nil: basal cytolysis in absence of humanized antibody. Results are expressed as %⁵¹Cr specific release and are the mean±SD of 30 NK cell clones tested from six donors. (B). TNFα released in the NK-tumor cell co-culture after 24 h incubation was assessed by ELISA. Nil: basal level of TNFα produced by unstimulated NK cell clones. Results are expressed as pg/ml and are the mean±SD of TNFα present in SN from 15 NK cell clones. (C). Cytotoxic effect of TNFα released by NK cells in co-cultures with CIR and rituximab or with SKBR3 and trastuzumab antibody. Cytotoxicity was assessed on WEHI164 cell line sensitive to TNFα-mediated killing. In some experiments to block the cytotoxic effect of TNFα a saturating amount of anti-TNFα polyclonal antibody (5 µg/ml) was added (medium plus anti-TNFαAb). *p<0.0001. (D). Expression of CD20 on the cell surface of C1R, Raji and CLL cells (one patients out of ten analyzed) or HER2 on SKBR3 and BT474 breast adenocarcinomas cell lines upon incubation for three days with solvent (upper panels) or 10 µM of fluvastatin (lower panels). Cells were labelled with either anti-CD20 mAb or anti-HER2 mAb followed by alexafluor647 conjugated isotype specific GAM (thick lines). Thin line: cells labelled with an unrelated mAb matched for isotype. Results are expressed as Log far red fluorescence intensity (a.u.) vs number of cells and are representative of three independent experiments for CIR, Raji, SKBR3 and BT474 cell lines or 10 CLL patients. (E). ADCC mediated by ex-vivo NK cells incubated for 36 h with solvent or 10 µM fluvastatin, as indicated, of CIR or SKBR3 cell lines untreated (white columns) or treated with fluvastatin (for three day at 10 µM, black columns). Results are expressed as %⁵¹Cr specific release and are the mean±SD of 6 donors of NK cells.