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.

To confirm cytotoxic function, redirected cells specific for the HBV core18-27 epitope were sorted based on TCR V beta and CD8 expression. Sorted Vbeta1CD81 T cells were co-cultured overnight in 96 well flat-bottom plates with 20,000 HepG2 cells expressing luciferase plus either the HBV core antigen or an irrelevant antigen, HBV surface antigen, at 155 and 1510 effector5target ratios. Following the incubation, media was removed and cells were washed once with PBS. Steady-Glo reagent (Promega) was added to each well and incubated for 5 min at RT in dark to allow cell lysis. Steady-Glo reagent was transferred into a black 96-well plate and luminescence was measured using a Biotek Synergy 4 microplate reader. Results were expressed as % lysis 5 100% 2 (luminescence remaining after lysis (HepG2 1 T cells)/maximum luminescence (HepG2 alone))% and calculated as mean of triplicate measurements 1/2 standard deviation.