Cd3 pe

Human CD3⁺T cells were transfected using Nucleofector™ technology (Amaxa, Cologne, Germany). Briefly, cells (1 × 10⁷) were resuspended in 0.1 mL supplemented Nucleofector solution at room temperature from the human T cell Nucleofector ™ kit. Each plasmid (5 μg; including the TCR Vα13-IRES-Vβ21 and TCR Vα18-IRES-Vβ21 recombinant plasmids and the negative control TCR empty vector) was mixed with 0.1 mL cell suspension, transferred to a 2.0 mm electroporation cuvette, and nucleofected using an Amaxa Nucleofector II apparatus according to the manufacturer's guidelines. Storage of the cell suspension in human T cell Nucleofector solution for longer than 20 min was avoided as this reduces cell viability and gene transfer efficiency. The cells were transfected using the U-014 program. The transfected T cells were immediately transferred to pre-warmed T cell medium OpTmizer^TM SFM and cultured in 12-well plates in a humidified incubator at 37°C and 5% CO₂. The culture medium was changed at 4–6 and 18 hours after transfection to T cell medium containing 200 IU/mL IL-2 and 2% FCS. The monoclonal antibodies 1 μg/mL OKT3 and 2 μg/mL CD28 were later added, and the cells were cultured for 48 hours. T cells were stained with CD3-PE and Vβ21.3-PE (Beckman Coulter, California, USA) to monitor TCR expression. Flow cytometry was performed using a FACS Canto flow cytometer (BD Biosciences).

PBMC exposed or not to RT33 or RT33 DR were processed for flow cytometry to assess cell death. Cells were labelled with a mix of anti-TCR-Vβ-FITC, -CD3-PE, and -CD4-PE-Cy7 mAbs (Beckman Coulter). Detection of apoptotic cells was performed using 7 AAD (BD Biosciences). Cells were analyzed on a CytoFlex cytometer (Beckman Coulter) and data treated with FlowJo software.