Clinical-scale expansion of human cytomegalovirus-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells requiring single-peptide stimulation and feeder cells but not additional antigen-presenting cells

BACKGROUND: The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (HCMV)-specific cytotoxic T cells from peripheral blood mononuclear cells (PBMNCs) without the aid of exogenous antigen-presenting cells (APCs) such as cultured dendritic cells. STUDY DESIGN AND METHODS: PBMNCs from three HLA-A*2402-seropositive donors were stimulated with HCMV pp65(341-350) peptide on Day 1 and then cultured with interleukin-2 and allogeneic feeder cells for 3 to 4 weeks. HCMV peptide-specific T cells were purified with HLA-A*2402/pp65(341-350) tetramer on Days 12 to 13 and harvested on Days 23 to 27. RESULTS: The initial numbers of PBMNCs were 2 x 10(7), 1.5 x 10(7), and 2.5 x 10(7) and the increases in HCMV peptide-specific T cells were 3.5 x 10(4)-, 2.0 x 10(3)-, and 1.1 x 10(3)-fold, respectively. The estimated final numbers of tetramer-positive cells were 9.1 x 10(7), 9.0 x 10(6), and 5.3 x 10(6), respectively. The purities of the tetramer-positive cell population in culture were 72.6, 75.0, and 80.9 percent, respectively. The cells killed peptide-pulsed B-lympoblastoid cell lines and secreted interferon-gamma in a HLA-restricted manner. They did not have natural killer cell activity or lymphokine activated killer cell activity. Most of them had an effector-memory phenotype. They did not express killer inhibitory receptors. CONCLUSION: This method makes it possible to obtain more than 1 x 10(7) HCMV-specific T cells from approximately 2 x 10(7) to 5 x 10(7) PBMNCs without exogenous APCs such as cultured dendritic cells.