HBV-specific T cell proliferation assay

Details of the thymidine proliferation assay have been previously described^{44 (link),53 (link)}. Briefly, PBMCs from HBV donors were isolated and were cultured with RPMI containing 2% human AB serum with pen/strep for 14 days in the presence of medium, HBV overlapping peptides of S or Core, influenza overlapping peptides, as well as small molecules (0.5 μM), or antibodies (5 μg/μl), including atezolizumab and nivolumab^{44 (link)}. HBV genotype-specific overlapping 15-mer peptides (genotypes A–D) were synthesized by GenScript (NJ, USA). A total of 1 μCi of ³H thymidine (NET355001MC, PerkinElmer) was added for 18 h for its incorporation into newly synthesized chromosomal DNA before harvesting cells. ³H thymidine incorporation was counted by MicroBeta2 microplate scintillation counter (PerkinElmer, MA). A total of 50 μl of supernatants per well before adding ³H thymidine was used for measuring IFNγ production by ELISA (430107, BioLegend).