title: Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T cells.
abstract: Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus C, Denmark.
authors.forename: C C
authors.initials: CC
authors.lastname: Fleischer
authors.forename: C
authors.initials: C
authors.lastname: Solling
authors.forename: M S
authors.initials: MS
authors.lastname: Petersen
authors.forename: R
authors.initials: R
authors.lastname: Agger
authors.forename: M E
authors.initials: ME
authors.lastname: Hokland
mesh_terms: D000328:Adult; D000818:Animals; D001774:Blood Chemical Analysis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D003587:Cytomegalovirus / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000097:blood / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology* / Q000821:virology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D007424:Intracellular Fluid / Q000737:chemistry; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D008875:Middle Aged; D013048:Specimen Handling
chemical_list: D007074:Immunoglobulin G; D007371:Interferon-gamma