Anti cd45ra percp cy5

Approximately 2.0 × 10⁷ T cells were transduced with lentivirus, and trypan blue staining was used to count live cells every 24 h to draw cellular growth curves. BM38, 38BM and BCMA CAR expression was detected using biotinylated human BCMA (BC7-H82FO; ACRO Biosystems, USA), while CD38 CAR expression was detected using protein L (A033-1901; GeneScript, China), and all the samples were then stained with streptavidin-PE (405203; BioLegend, USA). Anti-CD3-APC (561811; BD, USA), anti-CD4-BB515(564419; BD), anti-CD8-PerCP (344708; BioLegend), anti-CD62L-FITC (555543; BD), anti-CD45RA-PerCP-Cy5.5(563,429; BD), anti-CD3-FITC (561806; BD) and anti-CD38-APC (555335; BD) were used for phenotype identification of the final CAR-T cell products. Data were collected using CytoFLEX S flow cytometer (Beckman, USA) and analyzed using FlowJo software (TreeStar, USA).

Human primary cell isolation and culture were performed as described previously [57 (link),58 (link)]. Briefly, PBMCs were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) from buffy coats of healthy male blood donors at the Shanghai Blood Center. nTregs were separated with a FACSAria II cell sorter (BD Biosciences, USA) using the monoclonal antibodies anti-CD4-FITC, anti-CD25-PE, and anti-CD127- PE-Cy7. Naïve T cells were gated from a population of CD4+ CD25- effector T cells and separated with anti-CD45RA-Percp-CY5.5 (all from BD Biosciences, USA). Induced Tregs (iTregs) were induced from naïve T cells with recombinant TGF-β (5 ng/ml) and IL-2 (100 U/ml). The purified human nTregs and iTregs were expanded with anti-CD3/CD28 beads (Invitrogen, USA) and 500 U/ml IL-2 (R&D, USA). Both cell types were cultured with X-VIVO 15 medium (Lonza, Cologne, Germany) supplemented with 10% heat-inactivated human AB serum (Irvine Scientific, USA), 1% GlutaMax, and 1% NaPyr (Both from GIBCO, Life Technologies, USA). Cell fraction purity was determined using intracellular FOXP3 staining with FOXP3-PE-A (eBioscience, USA), following the manufacturer’s instructions. FACS data were then analyzed using FlowJo software (Tree Star, USA).

PE-conjugated HLA-A2 tetramers refolded around the CMV pp65_495-503 epitope NLVPMVATV (NLV) were generated as described previously^{48 (link),49 (link)} and used at a final concentration of 10 μg/mL. Directly conjugated mAbs were obtained from commercial suppliers: (i) anti-CD3-APC-H7, anti-CD8-BV786, anti-CD14-BV510, anti-CD19-BV510, anti-CD45-RA-PerCP-Cy5.5, anti-CD57-APC, anti-CD127-BV421, anti-CD160-PerCP-Cy5.5, anti-CCR7-PE-Cy7, anti-CTLA-4-PE-Cy7, anti-BTLA-APC, anti-2B4-FITC, anti-2B4-PE, and anti-Tim-3-AF700 (BD Biosciences); (ii) anti-CD27-BV605 and anti-PD-1-PE/Dazzle 594 (BioLegend); and (iii) a panel of anti-TCR-Vβ-FITC and anti-TCR-Vβ-PE mAbs (Beckman Coulter). The acute effects of dasatinib administration were monitored using an IOTest Beta Mark TCR Repertoire Kit (BD Biosciences). Non-viable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Life Technologies). Data were acquired using a Fortessa X-20 flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.0.7 (Tree Star). Viable CD3⁺ CD8⁺ TCR-Vβ⁺ cells were sorted using an Influx^TM Cell Sorter (BD Biosciences).