Regulatory authorities have recognized the importance of stimulating T cell responses to influenza

and have encouraged the exploration of T cell assays for evaluating vaccine efficacy in general [27] and [28] and, in particular, influenza vaccines including those aimed to protect against avian influenza [29] and [30]. However, standardized and reproducible assays of influenza-specific T cell responses that are too needed to make significant progress in the development of improved influenza vaccines have yet to be validated [29]. Herein, we report the validation of standardized assays of T cell responses that are likely to correlate with protection against influenza [13], [14] and [31]. The assays are based on the detection of effector molecules produced by peripheral see more blood mononuclear cells (PBMC) after ex vivo stimulation with live influenza virus. By using multiplex technology, multiple cytokines including IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, GM-CSF, IFN-γ, and TNF-α, could efficiently be detected in one sample of PBMC culture supernatant. In addition, a detection assay for granzyme B activity, an essential Selleckchem RG7420 effector molecule in the cytotoxic response of CD8+ T cells against virus-infected target cells [32], was validated in lysates of these virus-stimulated PBMC. The validation process was preceded by rigid standardization

of the assays and on-site training of the laboratory technicians following standard operating procedures (SOP) [33]. This work comprised determination of specificity, accuracy,

linearity, range, detection limit, intermediate precision, and robustness by three European and one Canadian laboratory. The validation results showed that these assays of the T cell response to influenza were reproducible and could measure the levels of granzyme B and cytokines in an accurate and specific manner. Human PBMC were isolated from buffy coats of healthy individuals by Lymphoprep (Axis Shield, Oslo, Norway) density centrifugation at 950 × g for 20 min. The PBMC were washed several times with PBS until the supernatant was clear. Subsequently, the PBMC were frozen in multiple aliquots in 90% FCS (Hyclone, Logan, Utah)/10% DMSO (Sigma–Aldrich, St. Louis, USA) and stored at −135 °C. Buffy coats were retrieved in accordance with the human experimental guidelines of Sanquin Blood Bank North West Region (project number S03.0015-X). Influenza Phosphoprotein phosphatase H3N2 A/Wisconsin/67/2005 was produced by infecting MDCK cells. As negative control (mock) medium of uninfected MDCK cells was used. The participating laboratories in alphabetical order, not in order in results, were: 1. National Centre for Epidemiology (NCE), Budapest, Hungary Frozen PBMC were thawed in AIM V medium and rested by incubation for 4 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Pilot experiments showed that this resting period is essential to obtain responses similar to responses with fresh cells (data not shown). Subsequently, PBMC (1.