Figure 1

CD157 modulates viability in primary AML cells. Mononuclear cells from AML patients were maintained ex vivo for 24 h under standard culture conditions (untreated) or treated with (A, B) SY11B5 anti-CD157 mAb or (B) control mIgG, then were subjected to AnnexinV/PI staining and flow cytometry analysis. Each dot represents a single patient. p < 0.0001 and p = 0.02, Wilcoxon’s signed-rank test. (C) Flow cytometry analysis of a bone marrow sample from a representative AML patient. Density plots show the distribution of each population composing the mononuclear cell fraction (X axis = FSC, Y axis = SSC) at time zero (T0), after 24 h in standard medium (untreated, top panels), with mIgG (bottom left panel) or SY11B5 anti-CD157 mAb (bottom right panel). Lymphocytes (FSC^low/SSC^low) and blasts (FSC^dim/SSC^dim) were gated according to morphological parameters. Percentage indicates the frequency of each population compared to total acquired events. (D) Pro-survival effect of increasing concentrations of SY11B5 anti-CD157 mAb or mIgG at 24 h in primary AML cells from a representative patient. Black bar represents the percentage of viable untreated cells. (E) Pro-survival effect of SY11B5 anti-CD157 mAb compared with mIgG. The percentage of live cells at each time point compared to time zero is shown. The graph is a representative PrestoBlue assay performed in quadruplicate. **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Sidak’s multiple comparison test. Western blot analysis of phosphorylation levels of the indicated proteins of (F) PI3K/AKT/mTOR pathway and (G) Bcl-2 apoptotic pathway, following CD157 stimulation for 24 h with SY11B5 anti-CD157 (10 µg/ml) mAb or mIgG (10 µg/ml), used as isotype-matched control. β-Actin was used as loading control. A representative Western blot is shown out of four patients analysed with similar results. Samples analysed in panel F and G derive from the same experiment and were processed in parallel. Uncropped blot image is provided in Supplementary Fig. S8. (H) AML samples were treated for 24 h with vehicle or AraC (10 µM) in the presence of anti-CD157 mAb or mIgG (both at 10 µg/ml). The percentage of apoptotic cells was measured by AnnexinV/PI staining and flow cytometry analysis. p values were determined by Wilcoxon’s signed-rank test.