Obinutuzumab

A FC500 and a Navios flow cytometer (both Beckman Coulter, Brea, USA) were used for multicolor immunofluorescence and functional tests.
Cells were stained in appropriate combinations according to use with following antibodies: anti-T-cell receptor (TCR) γδ-FITC [clone IMMU510], anti-CD3-r-Phycoerythrin-Texas Red (ECD) [clone UCHT1], anti-CD56-phycoerythrin-cyanine 5 (PC5) [clone N901], anti-CD27-phycoerythrin-cyanine 5 (PC5) [clone 1A4CD27], anti-CD56-phycoerythrin-cyanine 7 (PC7) [clone N901] (all Beckman Coulter, Brea, USA); anti-T-cell receptor (TCR) γδ-FITC [clone 11F2], anti-CD107a-PE [clone H4A3], anti-IFNγ−PE [clone 45-15], anti-CD45RA-PE [clone REA 562] (all Miltenyi Biotec, Bergisch Gladbach, Germany); and anti-perforin-PE [clone B-D48] (Biolegend, San Diego, USA).
As negative control, anti-IgG-PE [clone IS5-32F5] (Miltenyi Biotec, Bergisch Gladbach, Germany) was used.
γδ cells were defined as CD3⁺ TCR γδ⁺ MNC, NK cells as CD3^- CD56⁺ MNC and αβ T cells as CD3⁺ TCR γδ^- MNC.
For functional assay, the therapy grade monoclonal antibodies rituximab and obinutuzumab (both Roche, Grenzach-Wyhlen, Germany) as well as the IgG1-kappa control antibody (Sigma-Aldrich, St. Louis, USA) were used.

Freshly isolated PBMC from healthy donors were seeded in round-bottomed 96-well plates (50,000 cells/well) and cultured for 10 days in RPMI 1640 medium supplemented with 10% FCS, 1% l-glutamine, 1% penicillin/streptomycin, and 100 U/ml of human recombinant IL-2 (cat #: AF-200-02; Peprotech); after 2 days, irradiated Raji cells (3000 rad), incubated or not with an excess of rituximab, obinutuzumab, or wt-obinutuzumab (GA101-WT) (all from Roche) for 20 min at RT, were washed and added to the cultures (25,000 cells/well). Recombinant hIL-2-containing medium (100 IU/ml) was half-replaced every 2 days. At day 10, cells were harvested and used for phenotypic and functional analyses. For functional assays, 10 ng/ml of IL-15 (cat #: AF-200-15; Peprotech) was added at day 7.

Cells were incubated with different concentrations of dexamethasone (Sigma-Aldrich), hydrogen peroxide (Industrias Noriega, Asturias, Spain), obinutuzumab (a gift from Roche), 6-Aminonicotinamide (6AN) (Vitro, Madrid, Spain) or under conditions of decreasing concentrations of glutamine (Thermo Fisher Scientific) for different time points. Cell death was determined by staining with PE-conjugated Annexin V and 7-amino actinomycin D (7AAD) (Immunostep, Salamanca, Spain) and analyzed by flow cytometry. The percentage of cell death, including early apoptosis, late apoptosis and necrosis (Annexin V PE positive/7ADD negative, Annexin V PE positive/7ADD positive and Annexin V PE negative/7ADD positive cells) was calculated after subtracting the percentage of spontaneous death of cells incubated with the vehicle from the total cell death using the following formula: % specific lysis = [(% lysis of target cell - % spontaneous cell death)/(100% – % spontaneous cell death)] × 100.

Idelalisib was synthesized at Gilead Sciences, dissolved in DMSO at 10 mM, and stored at −20°C. Rituximab and obinutuzumab were provided by Hoffmann–La Roche (Basel, Switzerland). Palivizumab was used as a negative control and was produced at Gilead Sciences.

Cells were incubated with a concentration range of CD20-mAbs Rituximab or Obinutuzumab (Roche) in the presence of 20% human serum (purchased from University Hospital Brno) in 96-well plates in triplicates for three days. To assess cell viability, CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed as recommended by the manufacturer and samples were measured with Tecan Spark 10M system. Acquired values were normalized against PBS-treated wells and plotted as relative viability.