title: Generation of trispecific cytotoxic T cells recognizing cytomegalovirus, adenovirus, and Epstein-Barr virus: an approach for adoptive immunotherapy of multiple pathogens.
abstract: Cytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Adoptive immunotherapy with donor-derived cytotoxic T cells (CTLs) directed against EBV or CMV prevents the clinical manifestations of these viruses. We have designed a protocol for the simultaneous generation of polyclonal CTL specific for CMV, Ad, and EBV, which could be used to restore immunity to multiple viruses after SCT. EBV-transformed lymphoblastoid cell lines (LCLs), transduced with an adenoviral vector carrying a transgene for the immunodominant CMV antigen pp65 (Ad5f35-pp65GFP), were used to stimulate peripheral blood mononuclear cells in 6 normal donors. We detected the simultaneous presence of CD8 CTL recognizing peptide epitopes from all 3 viruses by pentamer staining. Enzyme-linked immunospot assays demonstrated a median 29-fold (8 to 248), 47-fold (2 to 137), or 18-fold (5 to 29) increase in cells secreting interferon-gamma in response to CMV, adenoviral, or EBV antigens, respectively, compared with unmanipulated peripheral blood mononuclear cell, with concomitant loss of alloreactivity. The CTL lines showed cytotoxicity against autologous LCL alone and increased cytotoxicity to autologous LCLs pulsed with CMV pp65 peptides or infected with Ad. In summary, we have developed a protocol for the generation of CTL with trivirus specificity, enabling adoptive transfer of CTL recognizing multiple viruses to restore cellular immunity after SCT.
authors.affiliation: Molecular Immunology Unit, Institute of Child Health, Great Ormond Street Children's Hospital, London, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Karlsson
authors.forename: Jennifer
authors.initials: J
authors.lastname: Brewin
authors.forename: Christine
authors.initials: C
authors.lastname: Kinnon
authors.forename: Paul
authors.initials: P
authors.lastname: Veys
authors.forename: Persis J
authors.initials: PJ
authors.lastname: Amrolia
mesh_terms: D000256:Adenoviridae / Q000276:immunology*; D000938:Antigen-Presenting Cells / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology*; D003601:Cytotoxicity Tests, Immunologic; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D007111:Immunity, Cellular; D016219:Immunotherapy, Adoptive; D007371:Interferon-gamma / Q000096:biosynthesis; D010750:Phosphoproteins / Q000235:genetics; D033581:Stem Cell Transplantation; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D019076:Transgenes; D014763:Viral Matrix Proteins / Q000235:genetics
chemical_list: D000956:Antigens, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma