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.

Building a virus-specific T cell receptor library. We used a panel of previously identified viral epitopes from HBV, EBV, CMV, FLU and SARS to expand T cells from healthy donors or patients with resolved HBV and SARS infections (Table 1) . Antigen-specific T cells were identified using matching HLA-pentamers/tetramers or the CD107a degranulation assay and clonal populations were derived by limiting dilution cloning or sorting T cells using antibodies specific for the variable region of TCR beta chains. Total RNA was extracted from sorted clones and the wild type TCR alpha and beta genes were cloned using rapid amplification of cDNA ends (RACE) PCR with TCR constant region gene specific primers. The TCRs were cloned into the MP-71 retroviral vector 18 and tested for expression in primary human T cells.