Cd19 hib19

Co-cultures were set up with CAR-T cells and K562-HLA-G1 cells at an E:T ratio of 10:1 with no IL-2. Twenty-four hours after stimulation, 50 µL of medium were taken, stored for cytokine secretion analysis and replaced by complete RPMI. Seventy-two hours after stimulation, half of the medium was changed with complete RPMI supplemented with 50 U/mL of IL-2. Cells were maintained at 10⁶/mL. Twelve days after stimulation, some cells were used for flow cytometry analysis, whereas the rest was used for a new round of stimulation. A total of three consecutive stimulations were performed. For flow cytometry analysis, cells were labeled with antibodies directed against CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), CD62L (clone DREG-56, BD Bioscience) and CD45RA (clone HI-100, Invitrogen). In these experiments, the phenotype of CAR-T cells and activated NT autologous controls was also established 24 hours prior to the assay. At the end of the experiment, all recovered medium was analyzed for IFNγ, TNFα and IL-2 secretion using a cytometric bead array (CBA) kit (BD Biosciences).

CAR-T cells 15E7^CH2-CH3 (2×10⁵) and LFTT1^CH2-CH3 were seeded with 2×10⁵ K562-HLA-G1 in a U-bottom 96-well plate and centrifuged at 100 g during 1 min. Cells were incubated 1 hour at 37°C in a 5% CO₂ incubator then brefeldin A (5 µg/mL, BD Biosciences) was added. Cell were incubated 18 hours longer at 37°C in a 5% CO₂ incubator. For FACS analysis, cells were labeled with anti-CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), interferon (IFN)γ (clone 4S.B3, Biolegend), tumor necrosis factor (TNF)⍺ (clone MAb11, Biolegend), IL-2 (MQ1-17H12, Biolegend) and a viability dye.

Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

CAR detection was performed with an anti-FMC63 idiotype antibody (Miltenyi). CD3 (OKT3), CD4 (OKT4), CD8 (SK1), CCR7 (G043H7), CD95 (DX2), CD45RA (HI100), CD45RO (UCHL1), Tim3 (F38-2E2), LAG3 (7H2C65), CD25 (BC96), CD69 (FN50), CD14 (M5E2), CD11b, CD19 (HIB19), and the respective isotype control antibodies were purchased from BioLegend (San Diego, CA, USA). Measurement was performed on a Beckman Coulter (Brea, CA, USA) CytoFLEX flow cytometer. Viability was assessed via Zombie dye staining (BioLegend).