Immune monitoring in whole blood using real-time PCR

Abstract

There is a need for simple and sensitive assays to assess innate and adaptive immune responses to microbial agents and vaccines. Herein, we describe a whole blood method allowing to measure the induction of cytokine synthesis at the mRNA level. The originality of this method consists in the combination of PAXgene™ tubes containing an mRNA stabilizer for blood collection, the MagNA Pure™ instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real-time PCR methodology on the Lightcycler™ for accurate and reproducible quantification of transcript levels. We first demonstrated that this method is adequate to measure the induction of interleukin (IL)-1β and IL-1 receptor antagonist (IL-1 RA) mRNA upon the addition of bacterial lipopolysaccharide (LPS) to whole blood. We then showed that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen. Finally, we demonstrated that this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1β and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.