Expansion of human cytomegalovirus-specific T lymphocytes from unfractionated peripheral blood mononuclear cells with artificial antigen-presenting cells

BACKGROUND: The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (CMV)-specific cytotoxic T cells from unfractionated peripheral blood mononuclear cells (PBMNCs). STUDY DESIGN AND METHODS: Unfractionated PBMNCs from three HLA-A*0201-CMV-seropositive donors were stimulated with CMVpp65_495-503 peptide-loaded HLA-A*0201-immunoglobulin fusion protein (HLA-A2-Ig) based artificial antigen-presenting cells (aAPCs) on Day 1. Once a week the CMV-specific T cells were harvested and restimulated with fresh aAPCs. T-cell cultures were maintained for 28 days and then analyzed. RESULTS: With aAPCs and starting with 1 × 10⁷ freshly isolated PBMNCs that were less than 0.1 percent CMV-specific, more than 1 × 10⁷ T cells with a CMV-specific frequency greater than 93 percent in all donors tested were generated. Expanded CD8+ cytotoxic T lymphocytes were functionally active and showed antigen-specific secretion of interferon-γ and cytotoxic activity. No alloreactivity against unpulsed HLA-A*0201-positive cells was detected. CONCLUSION: Herein is reported the successful in vitro expansion of CMV-specific cytotoxic CD8+ T cells from unfractionated PBMNCs of healthy CMV-seropositive blood donors by the use of HLA-A2-Ig-based aAPCs. This study demonstrates that more than 1 × 10⁷ CMV-specific T cells can be generated from approximately 1 × 10⁷ unfractionated PBMNCs within 1 month under highly reproducible conditions.