Optimization of culture conditions for HBV-specific T cell expansion in vitro from chronically infected patients

Hepatitis B virus (HBV) clearance depends on an effective adaptive immune response, especially HBV-specific T cell-mediated cellular immunity; however, it is difficult to produce enough HBV-specific T cells effectively.

In this work, we investigated the proportions of stimulated cells, serum, and culture media as the three primary factors to determine the most effective procedure and applied it to HLA-A2 (+) people. In parallel, we also examined the correlation between clinical parameters and HBV-specific immunity. Concerning amplification efficiency, 4 × 10⁵ cells stimulation was superior to 2 × 10⁶ cells stimulation, AIM-V medium outperformed 1640 medium, and fetal bovine serum (FBS) exceeded human AB serum under comparable conditions. As expected, this procedure is also suitable for developing HBV-specific CD8 + T cells in HLA-A2(+) individuals. Expanded HBV-specific T cell responses decreased with treatment time and were negatively correlated with HBV DNA and HBsAg. Furthermore, the number of HBV-specific IFN-γ + SFCs was strongly correlated with the ALT level and negatively correlated with the absolute lymphocyte count and the ALB concentration.

We confirm that stimulating 4 × 10⁵ PBMCs in AIM-V medium supplemented with 10% FBS is the best approach and that HBeAg, HBsAg, and ALB are independent predictors of HBV-specific T-cell responses.