title: Strategic use of an adenoviral vector for rapid and efficient ex vivo-generation of cytomegalovirus pp65-reactive cytolytic and helper T cells.
abstract: Cytomegalovirus (CMV) reactivation can cause severe complications for transplant patients. Such patients can be protected against CMV-associated diseases through reconstitution of donor-derived CMV-reactive cytolytic and helper T cells. We have developed a strategic protocol for efficient simultaneous generation of CMV-reactive CD8+ and CD4+ T cells ex vivo. The protocol uses peripheral blood lymphocytes (PBLs), antigen-modified mature dendritic cells (DCs) generated in only 3 d and an adenoviral vector encoding the CMV pp65 antigen (Adpp65) both as an endogenous and exogenous source of antigen. PBLs stimulated once with Adpp65-transduced DCs (endogenously expressed pp65) resulted in preferential activation and expansion of pp65-specific CD8+ T cells while PBLs stimulated with DCs pulsed with cell lysate from Adpp65-transduced autologous monocytes (exogenously expressed pp65) yielded pp65-specific CD4+ T cells. Stimulation with double-modified DCs efficiently activated and expanded cytolytic and helper T cells simultaneously. The frequency of T cells producing interferon-gamma in response to pp65 increased after one stimulation on average 9.6-fold to 4.3% for CD8+ T cells and 25.8-fold to 6.5% for CD4+ T cells. This implies that sufficient number of pp65-specific cytolytic and helper T cells for adoptive transfer may be obtained in only 2 weeks.
authors.affiliation: Clinical Immunology Division, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Ole
authors.initials: O
authors.lastname: Forsberg
authors.forename: Björn
authors.initials: B
authors.lastname: Carlsson
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Tötterman
authors.forename: Magnus
authors.initials: M
authors.lastname: Essand
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000956:Antigens, Viral / Q000276:immunology; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D014161:Transduction, Genetic; D014763:Viral Matrix Proteins / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa