7aad 7 aminoactinomycin

PBS57-loaded and unloaded human CD1d tetramers were obtained from the National Institutes of Health Tetramer Core Facility (Atlanta, USA). The following antibodies were purchased from BD Biosciences (Franklin Lakes, USA) or BioLegend (San Diego, USA): anti-CD19 (HIB19), anti-CD25 (BC96), anti-CD28 (CD28.2), anti-CD3 (HIT3a, OKT3), anti-CD4 (RPA-T4, OKT4), anti-CD44 (IM7), anti-CD45RA (HI100), anti-CD69 (FN50), anti-CD8 (HIT8a), anti-CTLA-4 (BNI3), anti-IgG1 (mouse, MOPC-21), anti-IgG2b (mouse, MPC-11), anti-IgG1 (mouse, MOPC-21), anti-IgG2b (mouse, MOPC-173), anti-KLRG1 (14C2A07), anti-LAG-3 (11C3C65), anti-PD-1 (EH12.2H7), anti-PD-L1 (MIH2), anti-PD-L2 (24F.10C12), anti-TIGIT (A15153G), anti-TIM-3 (F38-2E2). A goat F(ab’)2 anti-human IgG (H+L)-F(ab’)2-fragment (polyclonal, Jackson Immunoresearch, Ely, UK) and the CD19-CAR detection reagent (Miltenyi Biotech) were used to determine the percentage of CAR-expressing cells. Fluorescence minus one controls were used for proper gating. To stain dead cells, eBioscience Fixable Viability Dyes eFluor 506, 780 (ThermoFisher Scientific) and 7-AAD (7-aminoactinomycin, BD Biosciences) were used. Data were acquired on a BD LSRFortessa cell analyzer (BD Biosciences) and analyses were performed with FlowJo V.10.2 (Tree Star, Ashland, USA).