Acd3 pe cy7

Antibody staining was performed in FACS Buffer (PBS with 2% FCS, 2 mM EDTA and 0.01% NaN3) using the following antibodies: anti-(a) CD11c-BV650, aMHCII (I-A/I-E)-Pacific Blue, aCD3-Pe-Cy7, aGr-1-AF647, aCD69-PE and aVα2-APC were from Biolegend (San Diego, CA, USA); aCD62L-PE-Cy7 and aCD44-APC-eFluor780 were from eBioscience (San Diego, CA, USA); aCD40-PE, aCD86-FITC, aVβ5.1/5.2-FITC, aNK1.1-PE, aSiglecF-PE-CF594, aCD19-APC-H7 and aCD4-BV605 were from Becton Dickinson (Franklin Lakes, NJ, USA); while aCD8-FITC and aCD16/32 hybridoma supernatant were prepared in house. Data were acquired on a LSRII SORP (Becton Dickinson, San Jose, CA, USA) or LSR Fortessa SORP (Becton Dickinson), and analysed using FlowJo Software v 9.9 (FlowJo LLC, Ashland, OR, USA). 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (Molecular Probes, Eugene, OR, USA) was used to exclude dead cells.

Leucocyte cones of healthy donors were purchased from National Health Service Blood and Transplant (NHSBT, UK), with consent for non-clinical use. Work was performed under approval of the Human Tissue Authority (HTA license 12642). Whole blood was extracted from each cone and diluted to 50 mL with sterile PBS. PBMCs were isolated by Ficoll gradient centrifugation using SepMate 50 (85450, StemCell) and Ficoll® Paque Plus (GE17-1440-02, Merk) layering 25 mL of whole blood mixture to each SepMate 50. The cells were centrifuged at 1200 g for 20 min. The buffy coat was extracted and washed twice with sterile PBS. PBMCs were resuspended at 2 × 10⁷/mL in cell separation buffer (20144, StemCell) and incubated with 3 μg/2 × 10⁵ cells of biotinylated JOVI (ANC-101-030, Ancell) for 10 min. Samples were centrifuged at 400 g for 5 min and then washed with separation buffer before following EasySep™ Release Human Biotin Positive Selection Kit (17653, StemCell) protocol. The unbound (TRBC2⁺) fraction were harvested from the first incubation on the magnetic rack. The bound (TRBC1⁺) fraction was collected by following the protocol as stated. Isolation was confirmed via flow cytometry, staining with aCD3-PE/Cy7 (317334, Biolegend) and Streptavidin-APC (405243, Biolegend).

Isolated TRBC1⁺ or TRBC2⁺ T cells were resuspended at 1 × 10⁶ cells/mL in R10 and stimulated with TransAct (Miltenyi Biotec; 130-111-160), 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). 24 h after, cells were collected, plated at a density of 1 × 10⁶ cells per well (1 mL) on retronectin-coated (Takara, T100B) 6-well plates in the presence of retroviral supernatant at an MOI of 1. Total volume was adjusted to 3 mL using R10 supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). The plate were centrifuged at 1000 g for 40 min. 24 h post spinoculation, the T cells were harvested and re-plated in complete R10 media supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). Transduction efficiency was determined on day 5 after transduction, and further experiments were commenced on days 5–9 after transduction. CAR expression was assessed by staining with aCD3-PE/Cy7 (317334, Biolegend) and QBend10 APC (FAB7227A, R&D System).