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A Human Whole‐Blood Assay for Analysis of T‐Cell Function by Quantification of Cytokine mRNA
Author(s) -
Härtel,
Bein,
Kirchner,
Klüter
Publication year - 1999
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.1999.00549.x
Subject(s) - whole blood , monoclonal antibody , taqman , microbiology and biotechnology , cytokine , cd28 , biology , antibody , t cell , immune system , monoclonal , stimulation , immunology , real time polymerase chain reaction , biochemistry , endocrinology , gene
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 β‐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 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 Therefore, whole blood was taken from healthy individuals ( n = 10) and aliquots for mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation.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 μ g/ml). Interferon‐γ and tumour necrosis factor‐α 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 .