title: Activation requirements of circulating antigen-specific human CD8(+) memory T cells probed with insect cell-based artificial antigen-presenting cells.
abstract: We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines.
authors.affiliation: Department of NBE Discovery, Boehringer Ingelheim Austria, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Guelly
authors.forename: Zaruhi
authors.initials: Z
authors.lastname: Küpcü
authors.forename: Doris
authors.initials: D
authors.lastname: Zalusky
authors.forename: Margarete
authors.initials: M
authors.lastname: Karner
authors.forename: Margit
authors.initials: M
authors.lastname: Zehetner
authors.forename: Tamás
authors.initials: T
authors.lastname: Schweighoffer
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D018122:B7-1 Antigen / Q000235:genetics / Q000276:immunology; D016367:Baculoviridae; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology*; D006304:Health Status; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007156:Immunologic Memory / Q000276:immunology*; D007313:Insecta; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D010455:Peptides / Q000276:immunology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D018411:Spodoptera / Q000166:cytology; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D018122:B7-1 Antigen; D015789:HLA-A2 Antigen; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; D018799:Intercellular Adhesion Molecule-1