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.

Patient samples and isolation. Blood was obtained from healthy donors under informed consent or purchased as anonymous buffy coats from the Blood Donation Center at the National University Hospital, Singapore. This study was approved by the Institutional Review Board at the National University Hospital, Singapore. Blood from resolved HBV patients was obtained at the Azienda Ospedaliero-Universitaria di Parma, Italy, under informed consent and approved by the local Ethics Committee. Blood from resolved SARS patients was collected at the Singapore General Hospital under informed consent approved by the Centralized Institutional Review Board of the Singapore Health Services Pte, Ltd. All experiments were performed in accordance with the guidelines of the ethics committees. Peripheral blood mononuclear cells (PBMCs) from donors were isolated by Ficoll-Hypaque density gradient centrifugation (GE healthcare) and HLA-typed to four digit resoltuion. Peptide-specific T cell expansion. PBMC with HLA matching the restriction of the synthetic peptides were expanded with 1 mg/ml of each peptide for 10 d in AIM-V medium (Invitrogen) with 2% human AB serum (Invitrogen) supplemented with 20 U/ml II-2 (R&D Systems). Cultures were tested for positive responses using intracellular cytokine staining for IFN-c. Briefly, T cells were stimulated for 5 h with 1 mg/ml of the peptide in the presence of 10 mg/ml of Brefeldin A. The cells were stained with anti-CD8 PE-Cy7 (BD Pharmingen) for 15 min on ice, wahsed and fixed using Cytofix/Cytoperm (BD Biosciences) for 20 min on ice. Following fixation/ permeabilization, cells were stained with anti-IFNc PE (BD Biosciences) for 30 min on ice. Cells were washed and then resuspended in 13 PBS for acquisition on the Facs Canto (BD Biosciences) and analyzed using FACs Diva software.