Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections

Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M.; Bertoletti, Antonio; Gehring, Adam J.

Redirected T cell functionality was tested 7 d post transduction. Either HLA-A0201 T2 cells or HLA-matched immortalized EBV B cell lines were used as antigen presenting cells (APCs) to test functionality of the TCR redirected T cells. APCs were loaded with 1 mg/ml for each peptide for 1 h at room temperature. APCs were washed with HBSS and co-cultured with TCR redirected T cells overnight in Aim-V 2% Abs with 2 mg/ml brefeldin A (Sigma). Cells were stained with CD8-PECy7 and fixed with cytofix/cytoperm (BD Biosciences). Cells were then stained with anti-IFN-c-APC, TNF-a-Alexa488 (BD Biosciences) or IL-2-PE (R&D Systems) for 30 min on ice with staining buffer 1 0.1% saponin (Sigma). Cytokine producing cells were analyzed on a BD FACs Canto flow cytometer.