A human whole-blood assay for analysis of T-cell function by quantification of cytokine mRNA

A whole blood assay was developed for T-lymphocyte analysis which allows th e quantification of induced cytokine mRNA expression. We applied a novel ki netic reverse transcription polymerase chain reaction method which directly measures product accumulation using Taqman technology. Quantitative result s were obtained by using beta-actin and cytokine standard curves generated from synthetic external standards. Since quantification relies on threshold cycles for fluorescence detection (C-t), this technique proved to be accur ate over a dynamic range of at least five orders of magnitude. To evaluate the method a study was undertaken to find optimal conditions fur whole-bloo d stimulation with soluble anti-CD3 monoclonal antibodies in the presence o f a costimulatory signal mediated by anti-CD28 monoclonal antibody. Therefo re, whole blood was taken from healthy individuals (n = 10) and aliquots fo r mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation. Op timal assay conditions were reached with 1 : 10 diluted heparinized whole b lood and after stimulation with equimolar amounts of anti-CD3 and anti-CD28 monoclonal antibodies (1 mu g/ml). Interferon-gamma and tumour necrosis fa ctor-alpha proved to be early response cytokines with peak expression at 4h . In contrast, interleukin (IL)-2, IL-4 and IL-10 required 8 h of stimulati on This novel whole-blood assay is potentially useful for monitoring T-cell -specific immune functions in a variety of clinical settings. Using whole b lood obviates the need for T-cell purification and may therefore closely ap proximate the state of responsiveness of circulating T cells in vivo.