A method to increase tetramer staining efficiency of CD8+ T cells with MHC-peptide complexes: therapeutic applications in monitoring cytotoxic T lymphocyte activity during hepatitis B and C treatment

Abstract

The development of peptide–MHC tetrameric complexes heralds a new era in the study of antigen-specific T cells and their role in viral infections. However, the frequencies of tetramer-staining CD8+ T cells in fresh peripheral blood mononuclear cells (PBMCs) are usually below 1% in patients with chronic hepatitis B and C viruses (HBV and HCV) as well as human immunodeficiency virus (HIV) infections, which makes difficult the comparison and sequential evaluation of different individuals. Thus, the development of a method to enumerate efficiently antigen–specific CD8+ T cells will be clinically beneficial in monitoring the antiviral cellular immunity during therapy. We report here a modified CRI-p culture method (cytotoxic T lymphocyte response index of the epitope–peptide method), using a panel of peptides to stimulate PBMCs in bulk culture. The modified CRI-p cultured cells were, in turn, subjected to fluorescence-activated cell sorter (FACS) analysis, tetramer staining or T-cell functional assays to quantify the antiviral immunity of HLA-A2 (+) HBV and HCV patients receiving antiviral therapies. The results obtained showed that patients with a sustained response had a significantly higher increase in the frequencies of tetramer staining of virus-specific CD8+ T cells than did nonresponders. This method permits semi-quantitative determination of the relative strength of CTL activity against a panel of peptides and provides a large number of cells for FACS analysis from a single blood sampling. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. The mechanisms involved in this method are discussed.

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