Cd3 buv395 clone ucht1

Target HepG2 cells were pulsed with HBV surface envelope peptide S183-91 (FLLTRILTI, JPT Peptide Technologies) and irrelevant control HBV core peptide C18-27 (FLPSDFFPSV, JPT Peptide Technologies) at gradient concentrations for 1 h at 37°C. Cryopreserved effector T cells (eTCR+/rTCR−, eTCR+/rTCR±, and eTCR-/rTCR+) were thawed and cultured at an E:T ratio of 1:1, and 0.1 μg/mL Brefeldin A (Sigma) was added before overnight co-culture. A BD LSR Fortessa X20 flow cytometer (BD Biosciences) was used for cell acquisition, with FlowJo v10 (TreeStar) used to analyze phenotype and function of effector T cell groups. Phenotyping included intracellular staining with TNF-α FITC (clone MAb11, BD Biosciences, catalog no. 502906), MIP-1b PE (clone D21-1351, BD Biosciences, catalog no. 550078), IL-2 PerCP-eFluor 710 (clone MQ1-17H12, eBioscience, catalog no. 46-7029-42), GranzymeB AF700 (clone GB11, BD Biosciences, catalog no. 560213), IFNγ V450 (clone B27, BD Biosciences, catalog no. 560371), and surface staining with CD3 BUV395 (clone UCHT1, BD Biosciences, catalog no. 563546) and mouse TCR β constant-APC (clone H57-597, BioLegend, catalog no. 109211).

PBMC were thawed as described before, counted after centrifugation and stained with 1 μL/sample of CellTrace Violet (Thermofisher, USA, MA). After staining, cells were washed extensively, resuspended in R10 medium and aliquoted into a 24-well plate at 2x10⁶ cells/well (1mL final volume). Cells were then stimulated with either DMSO as negative control, SEB (0,1 mg/mL Sigma-Aldrich, USA, Mo) as positive control, S1a+b pool or S2a+b pool (0,3 mg/mL) or CEFX (1 μg/ml) for 5 days at 37°C 5% CO₂. After stimulation, cells were harvested and transferred into new FACS tubes to be stained with CD3 BUV395 clone UCHT1, CD4 BUV737 clone SK3, CD8 BV605 clone SK1 (all from BD) and CD19 APC-Vio770 clone LT19 (Miltenyi) in the presence of Brilliant Stain Buffer (BD) for 30′ at 4°C. Sample analysis was performed with FlowJo software (version 10.8.1, BD) following gating strategy depicted in Figure S10. For each donor, the percentage of cells proliferating to Spike peptides-pools were subtracted of their negative control and summed to provide total Spike proliferative response.