A Human Whole-Blood Assay for Analysis of T-Cell Function by Quantification of Cytokine mRNA

Authors


HaraldKlüter, MD Institute of Immunology and Transfusion Medicine, University of Lübeck Medical School, Ratzeburger Allee 160, 23552 Lübeck, Germany

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 β-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 Therefore, whole blood was taken from healthy individuals (n = 10) and aliquots for mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation.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 μg/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.

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