title: A human whole-blood assay for analysis of T-cell function by quantification of cytokine mRNA.
abstract: A whole blood assay was developed for T-lymphocyte analysis which allows the quantification of induced cytokine mRNA expression. We applied a novel kinetic reverse transcription polymerase chain reaction method which directly measures product accumulation using Taqman technology. Quantitative results were obtained by using beta-actin and cytokine standard curves generated from synthetic external standards. Since quantification relies on threshold cycles for fluorescence detection (Ct), this technique proved to be accurate over a dynamic range of at least five orders of magnitude. To evaluate the method a study was undertaken to find optimal conditions for whole-blood stimulation with soluble anti-CD3 monoclonal antibodies in the presence of a costimulatory signal mediated by anti-CD28 monoclonal antibody. Therefore, whole blood was taken from healthy individuals (n = 10) and aliquots for mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation. Optimal assay conditions were reached with 1 : 10 diluted heparinized whole blood and after stimulation with equimolar amounts of anti-CD3 and anti-CD28 monoclonal antibodies (1 microgram/ml). Interferon-gamma and tumour necrosis factor-alpha proved to be early response cytokines with peak expression at 4 h. In contrast, interleukin (IL)-2, IL-4 and IL-10 required 8 h of stimulation. This novel whole-blood assay is potentially useful for monitoring T-cell-specific immune functions in a variety of clinical settings. Using whole blood obviates the need for T-cell purification and may therefore closely approximate the state of responsiveness of circulating T cells in vivo.
authors.affiliation: Institute of Immunology and Transfusion Medicine, University of Lübeck Medical School, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Härtel
authors.forename: G
authors.initials: G
authors.lastname: Bein
authors.forename: H
authors.initials: H
authors.lastname: Kirchner
authors.forename: H
authors.initials: H
authors.lastname: Klüter
mesh_terms: D001681:Biological Assay / Q000379:methods*; D016207:Cytokines / Q000032:analysis* / Q000097:blood / Q000276:immunology; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D016207:Cytokines; D012333:RNA, Messenger