Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: A simple culture system using tetrameric HLA-peptide complexes

Background. Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. Methods. CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65_495-503 tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4⁺ T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. Result. Starting from 50 mL of blood, we generated > 10⁷/m² tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was > 75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-γ. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. Discussion. This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.