Supplementary Figure 2 from RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

PDF file - 264K, (A) The ability of MM cells to release sRANKL does not influence NK reactivity induced by RANK-Fc-ADCC. The increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells using RANKL surface-positive MM cells that do (+sRANKL) or do not (-sRANKL) release sRANKL as targets upon treatment with RANK-Fc-ADCC was calculated. To this end, specific lysis (E:T ratio 40:1) or cytokine release (E:T ratio 1:1) of NK cells in the presence of isotyp control-treated target cells was set to 1 in each individual data set. No statistically significant difference (p=0.39 and p=1, respectively; Mann-Whitney U-Test) was observed upon analysis of results from 8 (cytotoxicity) and 6 (cytokine release) independent experiments. (B) Surface levels of RANKL do not correlate with induction of NK reactivity by RANK-Fc-ADCC. Expression of RANKL on patient MM (circles) and CLL cells (triangles) was correlated with the increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells upon treatment with RANK-Fc-ADCC calculated by as described in A. Each symbol represents the result of one independent experiment. The number of independent experiments, correlation coefficients (Pearson correlation) and p values (T-Test) are indicated. (C) NK reactivity against RANKL surface-negative MM cells is not affected by the fusion proteins. RPMI 8226 MM cells (left) and primary MM cells (right) that lack RANKL surface expression were incubated with allogeneic NK cells in the presence or absence of 10mug/ml of the indicated RANK fusion proteins or isotype control. Cytotoxicity was determined by 2h Europium release assays (upper panels) and IFN-gamma levels in supernatants were analyzed after 24h by ELISA (lower panels). (D) RANKL transfectants were cultured with allogeneic NK cells in the presence or absence of RANK-Fc-ADCC or isotype control (10�g/ml each) and the indicated concentrations of rRANKL. Cytotoxicity was determined by 2h Europium release assays (left) and IFN-� levels in supernatants were analyzed after 24h by ELISA (right). One repesentative experiment each of a total of three with similar results is shown in C and D.