Fig. 2

Effect of idelalisib on fresh primary ATL cells and non-malignant cells from ATL patients. a Flow-cytometry gating strategy. Uncultured PBMCs were screened using a panel of monoclonal antibodies (Beckman Coulter IOTest Beta mark) to identify the TCR-Vβ subunit expressed by the malignant clone, as previously described [13]. The viability of ATL cells was assayed after staining with annexin V (BioLegend) and Fixable Dead Cell Stain (Thermo Fisher). b Flow-cytometric assay in a patient with unfavorable chronic type. The gate with AnnexinV¯ and Live/Dead¯ stains, highlighted in red, indicates viable cells, and the gate with AnnexinV⁺, highlighted in blue, shows apoptotic cells. c The viability of ATL cells was assayed at day 0–10 by flow cytometry. PBMCs were freshly isolated from ATL patients (n = 7) and CD8 positive cells were depleted using Dynabeads (Invitrogen). The live and apoptotic cells were quantified in the population of TCR-VβX⁺CD7⁻ ATL cells. Y axis refers to the viability ratio between treated and untreated cells of time-matched samples from the same individuals. d CD4⁺ cells were selected from freshly isolated PBMCs and pre-incubated with or without idelalisib for 30 min. These cells were subsequently cultured with or without 50 μg/ml of CCL22 (R&D SYSTEMS) for 2 h and the whole cell lysates extracted for immunoblotting of AKT and AKT phosphorylated at Ser473. e Freshly isolated PBMCs from ATL patients (n = 6) were pre-incubated with or without idelalisib for 30 min, after depletion of CD8+ cells. These cells were subsequently cultured with or without 50 μg/ml of CCL22 for 0–10 days. All flow cytometric assays were performed in duplicate