title: Development of an artificial-antigen-presenting-cell-based assay for the detection of low-frequency virus-specific CD8(+) T cells in whole blood, with application for measles virus.
abstract: Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8(+) T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8(+) T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-gamma) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8(+) T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8(+) T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8(+) T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-gamma mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8(+) T cells in vaccine trials. The technology should be transferable to analysis of CD8(+) T-cell responses to other antigens.
authors.affiliation: W Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Zaza M
authors.initials: ZM
authors.lastname: Ndhlovu
authors.forename: Monika
authors.initials: M
authors.lastname: Angenendt
authors.forename: Diana
authors.initials: D
authors.lastname: Heckel
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Schneck
authors.forename: Diane E
authors.initials: DE
authors.lastname: Griffin
authors.forename: Mathias
authors.initials: M
authors.lastname: Oelke
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology; D001769:Blood / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D015234:HLA-A Antigens / Q000276:immunology; D015789:HLA-A2 Antigen; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007371:Interferon-gamma / Q000096:biosynthesis; D008458:Measles Vaccine / Q000276:immunology*; D008459:Measles virus / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D000956:Antigens, Viral; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D008458:Measles Vaccine; D007371:Interferon-gamma