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.

Toll-like receptor ligand activation. Healthy donor PBMC were activated as above for retroviral transduction with 50 ng/ml OKT-3 and 600 U/ml IL-2. In addition to the standard condition, TLR agonists 10 mg/ml polyI:C (TLR-3), 5 mg/ml Imiquimod (TLR-7), 1 mg/ml ssRNA40 (TLR-8), and 5 uM CpG (CpG2216; TLR-9) (Invivogen) were added during the first 48 h of PBMC activation. Following the 48 h incubation, the media was removed for transduction and stored at 280 C for luminex analysis. TLR ligands were not maintained in the maintenance medium and T cells were transduced with the HBV core 18-27-specific TCR and cultured in Aim-V 1 2% human AB serum 1 100 U/ml IL-2. TCR expression was monitored using HLA-A2-HBc18-27 pentamers and the functionality was monitored intracellular cytokine staining for IFN-c. Supernatants taken from 48 h activated cultures were analyzed for cytokine production using the MILLIPLEX Cytokine/Chemokine 42-Premixed Panel (Milipore). Luminex analysis was performed by the Immune Monitoring Core at the Singapore Immunology network.