Anti cd3 bv421

Analysis was performed using a BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FCS Express 6 software. Antibodies used included anti-CD5 PerCP/Cy5.5, anti-CD3 BV421, anti-γδ T cell receptor (TCR) phycoerythrin (PE) and anti-CD69 APC-Cy7 (BD Biosciences, San Jose, CA). CD5-Fc fusion protein (G&P Biosciences, Santa Clara, CA) and CD19-Fc fusion protein (ACROBiosystems, Newark, DE) were used to detect anti-CD5 constructs and anti-CD19 constructs, respectively, with a secondary anti-IgG Fc antibody (Jackson Immunoresearch Laboratories, West Grove, PA), as previously described.⁶ (link) Violet Proliferation Dye 450 (VPD450) was used to label the target cells in the cytotoxicity and co-culture studies, and cell death was assessed using eFluor 780 (described below). Degranulation of γδ T cells was detected using anti-CD107a APC (BD Biosciences, San Jose, CA).

CD19-CAR- and CD19-NSCAR-modified γδ T cells were cultured with 697 cells in 12 × 75 mm FACS tubes at an E:T ratio of 5:1 in a total volume of 250 μL and incubated for 12 h at 37°C in 5% CO₂. 697 cells were labeled with VPD450 using the manufacturer’s protocol prior to co-culture. Following the incubation, cells were stained for flow cytometry to analyze cell surface expression of CD107a using antibodies including anti-CD3 BV421, anti-γδ TCR PE, anti-CD107a APC (BD Biosciences, San Jose, CA), and viability dye eFluor 780 (Thermo Fisher Scientific, Waltham, MA).

For MDSCs, isolated PBMCs were stained with anti‐CD3‐BV421 (UCHT1/562426), CD19‐BV421 (HIB19/562440), CD56‐BV421 (NCAM16.2/562751), CD20‐BV421 (2H7/562873), CD11b‐BB515 (ICRF44/564517), CD15‐PerCP‐Cy 5.5 (HI98/560828), CD14‐APC (M5E2/555399), and HLA‐DR‐PE (G46‐6/555812) antibodies (BD Biosciences, San Jose, CA, USA) for 45 min. For lectin‐type oxidized LDL receptor 1 (Lox‐1) expression on PMN‐MDSCs, lox‐1‐BV421 (15C4/358610) (Biolegend, San Diego, CA, USA) was used. For Treg cells, isolated PBMCs were stained with anti‐CD4‐FITC (RPA‐T4/555346), CD25‐APC (M‐A251/555434), CD45RA‐PerCP‐Cy 5.5 (HI100/563429), and Foxp3‐PE (259D/C7/560046) antibodies (BD Biosciences) for 45 min. For CD8⁺ T cells, isolated PBMCs were stained with anti‐CD8‐APC (RPA‐T8/555369) (BD Biosciences), CD39‐PE‐Cy7 (A1/328212) (Biolegend), and PD‐1‐PerCP‐Cy5.5 (EH12.1 /561273) (BD Biosciences) antibodies. Samples were washed twice and then read on a BD FACSVerse (BD Biosciences) flow cytometer. Dead cells were excluded using 7‐Amino‐Actinomycin D (7AAD) (Biolegend). Gating strategies for PMN‐MDSCs, M‐MDSCs, and CD8⁺ T cells are shown in Supporting information Fig. 1A‐C. All the process of T‐cell assays and flow cytometry analysis (linear axis) adhered to the guidelines of MIATA compliant and Cossarizza [30].

Peripheral blood mononuclear cells were isolated from blood samples of the convalescent COVID-19 patient samples through Ficoll density gradient centrifugation and B cells were extracted by magnetic-activated cell sorting using the B Cell Isolation Kit II human (Miltenyi Biotec) according to the manufacturer’s instructions. The enriched B cells were first incubated with the Viability 405/452 Fixable Dye (Miltenyi Biotec) for 15 min at room temperature, subsequently for 30 min on ice with in-house produced recombinant His- and Fc-tagged SARS-CoV-2 Spike RBD-SD1 protein and, and finally with anti-CD3-BV421 (BD Biosciences), anti-CD14-BV421 (BD Biosciences), anti-CD16-BV421 (BD Biosciences), anti-CD27-PE-Cy7 (BD Biosciences), anti-CD19-FITC (Miltenyi Biotec), anti-His-PE (Miltenyi Biotec) and anti-His-APC (Miltenyi Biotec) for 30 min on ice. AutoMACS Rinsing Solution (Miltenyi Biotec) supplemented with 0.5% BSA (Miltenyi Biotec) was used to prepare the RBD and antibody solutions and to wash the cells after the different staining steps. Single cells were sorted directly into lysis buffer that consisted of 0.5 × PBS, 10 mM DTT and 2 U/μl RNasin Plus (Promega) with a BD FACSAria™ Fusion (BD Biosciences), immediately frozen on dry ice and stored at −80 °C.