Cd19 car detection reagent

Three to four days after transduction, CAR T cells were enriched with magnetic microbeads and separation columns from Miltenyi Biotec (Bergisch Gladbach, Germany) according to the manufacturer’s instructions. In brief, cells were labeled with either CD271 microbeads (from now on referred to as Standard microbeads) or LNGFR MACSelect microbeads (from now on referred to as Select microbeads) and separated on MS (maximal capacity: 1 × 10⁷ cells), LS (maximal capacity: 1 × 10⁸ cells), and LD (maximal capacity: 5 × 10⁸ cells) columns. The three fractions (preMACS, flowthrough, and postMACS) were flow cytometrically analyzed on a MACSQuant Analyzer X for EGFP, CAR expression, and ΔNGFR expression via staining with CD271-PE (clone ME20.4, Miltenyi Biotec).
The expression levels of CD19 CAR constructs were determined by flow cytometry using the biotin-coupled CD19 CAR detection reagent followed by staining with anti-biotin-PE or anti-bioti-APC monoclonal antibodies (all reagents from Miltenyi Biotec).

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).

CAR T cells (2×10⁵ cells per test) were labeled with antibodies at 4°C for 15 min. Flow cytometry was performed using a MACSQuant Analyser 10 Flow Cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany), and data were analyzed by FlowJo V.10.7.1 software (FlowJo). Cells were subjected to SSc-FSc and gated as the CD3⁺ T-cell population. For transfection efficiency: CD19 CAR detection reagent, anti-biotin PE, and CD3-APC (Miltenyi Biotec, Bergisch Gladbach, Germany); T-cell phenotype: CD3-PerCP (UCHT1), CD8-PE (SK1), (BD Bioscience, New Jersey, USA), CD4-APC (BioLegend, San Diego, California, USA); memory phenotype: CD3-PerCP (UCHT1), CD45RO-VioGreen (UCHL1), and CD62L-VioBlue (DREG-56) (BD Bioscience, New Jersey, USA); exhaustion: CD3-PerCP, PD1(CD279)-APC, TIM-3-PE, LAG3-PE, and TIGIT-APC (BioLegend, San Diego, California, USA); activation: CD3-PerCP, CD25-PE, and CD69-APC (BioLegend, San Diego, California, USA); and for cytolytic activity: CD3-PE (OKT3), CD19-APC (HIB19) (BioLegend, San Diego, California, USA), and 7AAD-PerCP (BD Bioscience, New Jersey, USA).

CAR T cells were longitudinally monitored in peripheral blood (PB) through multiparameter flow cytometry (MFC) using CD19 CAR detection reagent, as previously described,¹¹ (link) or CD19 CAR FMC63 Idiotype (REA1297) (both from Miltenyi Biotec). CAR T-cell differentiation in infusion product leftovers was assessed by MFC as previously described.¹¹ (link) Antibodies and gating strategies are detailed in supplemental Material. Data were acquired on BD FACSCanto II (BD Biosciences), MACSQuant Analyzer MQ10 or MQ16 (Miltenyi Biotec) and analyzed using FlowJo-v10 and MACSQuantify softwares.