title: Quantitation of human influenza virus-specific cytotoxic T lymphocytes: correlation of cytotoxicity and increased numbers of IFN-gamma producing CD8+ T cells.
abstract: To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-gamma specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic 51Cr-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-gamma production by IFN-gamma-specific ELISPOT and ELISA, while IFN-gamma mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h 51Cr-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-gamma spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-gamma SFCs directly correlated with levels of secreted IFN-gamma and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-gamma mRNA in T cells isolated from 7 day cultures. Separate aliquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high 51Cr release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-gamma SFCs, and amount of IFN-gamma in culture supernatants. Studies with purified T cell subsets showed that elevated IFN-gamma SFCs, IFN-gamma synthesis, and cytotoxic activity were associated with CD4-CD8+ T cells but not with the CD4+CD8- T cell subset.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Immunobiology Vaccine Center, University of Alabama at Birmingham 35294.
authors.forename: S
authors.initials: S
authors.lastname: Di Fabio
authors.forename: I N
authors.initials: IN
authors.lastname: Mbawuike
authors.forename: H
authors.initials: H
authors.lastname: Kiyono
authors.forename: K
authors.initials: K
authors.lastname: Fujihashi
authors.forename: R B
authors.initials: RB
authors.lastname: Couch
authors.forename: J R
authors.initials: JR
authors.lastname: McGhee
mesh_terms: D000328:Adult; D016827:CD8 Antigens; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D008297:Male; D009975:Orthomyxoviridae / Q000276:immunology*; D016133:Polymerase Chain Reaction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D016827:CD8 Antigens; D007371:Interferon-gamma