title: Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines.
abstract: Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.
authors.affiliation: Department of Gene Vectors, GSF - National Research Center for Environment and Health, Munich, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Wiesner
authors.forename: Caroline
authors.initials: C
authors.lastname: Zentz
authors.forename: Markus H
authors.initials: MH
authors.lastname: Hammer
authors.forename: Mark
authors.initials: M
authors.lastname: Cobbold
authors.forename: Florian
authors.initials: F
authors.lastname: Kern
authors.forename: Hans-Jochem
authors.initials: HJ
authors.lastname: Kolb
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hammerschmidt
authors.forename: Reinhard
authors.initials: R
authors.lastname: Zeidler
authors.forename: Andreas
authors.initials: A
authors.lastname: Moosmann
mesh_terms: D000328:Adult; D017951:Antigen Presentation / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D002461:Cell Line, Transformed; D002999:Clone Cells; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D018984:Epitopes, T-Lymphocyte; D006680:HLA Antigens; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa