title: Rapid, large-scale generation of highly pure cytomegalovirus-specific cytotoxic T cells for adoptive immunotherapy.
abstract: Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific cytotoxic T cell (CTL) clones can restore immunity in allogeneic stem cell transplant recipients, providing protection against CMV disease. Current methods for selecting and expanding CMV-specific T cell clones are technically difficult, making adoptive T cell therapy impractical for routine clinical use. In this study, we describe a method for ex vivo generation and expansion of high-purity CMV-specific CTL using peptide-pulsed dendritic cells as antigen-presenting cells. Generation of CMV-specific CTL in numbers sufficient for clinical use in the time span of 4 weeks was accomplished in 6 of 8 CMV-seropositive donors. Examination of pp65 specificity by HLA/peptide tetramer staining demonstrated that a purity of greater than 95% peptide-specific cells could be obtained after two weekly stimulations and retained after further expansion for 3-4 weeks. Median expansion of total cell number was greater than 500-fold and expansion of peptide-specific CTL by tetramer staining was greater than 1.7 x 10(5)-fold. Four weeks after initiating CTL culture, we were able to generate greater than 10(9) total cells that specifically lysed target cells loaded with CMV peptide and cells infected with CMV. This simple and rapid method for generating high-purity CMV-specific CTL for adoptive immunotherapy is currently being examined for routine clinical use for allogeneic stem cell transplantation.
authors.affiliation: Westmead Institute for Cancer Research, Westmead Millennium Institute and Department of Chemical Engineering, University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Foster
authors.forename: David J
authors.initials: DJ
authors.lastname: Gottlieb
authors.forename: Marina
authors.initials: M
authors.lastname: Marangolo
authors.forename: Anna
authors.initials: A
authors.lastname: Bartlett
authors.forename: Yi-Chiao
authors.initials: YC
authors.lastname: Li
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Barton
authors.forename: Jose A
authors.initials: JA
authors.lastname: Romagnoli
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Bradstock
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology; D018929:Cell Culture Techniques; D003587:Cytomegalovirus / Q000378:metabolism*; D003712:Dendrites / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005805:Genes, MHC Class I / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008957:Models, Genetic; D009000:Monocytes / Q000276:immunology; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000276:immunology*; D033581:Stem Cell Transplantation / Q000379:methods; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000276:immunology*
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa