Gavo-cel detection by flow cytometry in peripheral blood
HT
PBMCs were isolated from whole blood by density gradient centrifugation and cryopreserved. Thawed samples were stained with LIVE/DEAD Aqua (Thermo Fisher Scientific) and fluorochrome-conjugated antibodies specific for CD3-BUV395 (BD Biosciences, 564001, clone SK7, 1:50 dilution), CD4-BUV496 (BD Biosciences, 612936, clone SK3, 1:25 dilution), CD8-APC-Cy7 (BD Biosciences, 348793, clone SK1, 1:100 dilution), CD366/TIM3-PE (BD Biosciences, 565570, clone 7D3, 1:400 dilution), CD27-BV605 (BioLegend, 302829, clone O323, 1:12.5 dilution), CD45RA-BV711 (BioLegend, 304137, clone HI100, 1:400 dilution), CD45RO-Alexa-700 (BioLegend, 304217, clone UCHL1, 1:25 dilution), CCR7-PE-Cy7 (BioLegend, 353225, clone G043H7, 1:25 dilution), CD95-BV785 (BioLegend, 305645, clone DX2, 1:100 dilution), CD279/PD-1-BV421 (BioLegend, 329919, clone EH12.2H7, 1:25 dilution), CD223/LAG-3 BV650 (BioLegend, 369315, clone 11C3C65, 1:50 dilution) and an anti-VHH-AF488 for TRuC detection (GenScript, A01862, clone 96A3F5, 1:400 dilution). Flow cytometry was performed on an LSR flow cytometer (BD Biosciences). BD FACSDiva software version 9.0 was used to collect data, and the data were analyzed using FlowJo software version 10.8.1 (BD Biosciences). Viable (PBMC) cells were counted before acquisition by means of the automated Guava counting process using the Viacount Reagent (GuavaEasyCyte HT and GuavaSoft version 3.3). The gating strategy used for identifying TRuC T cells in post-infusion peripheral blood is shown in Supplementary Fig. 