However, several reports have shown better antigen-specific T-cell responses by IFN-γ ELISPOT using cryopreserved PBMCs when cells are rested (overnight) prior to performing the assay (Kierstead et al., 2007, Britten et al., 2008, Janetzki et al., 2008, Boaz

et al., 2009 and Kutscher et al., 2013). It has been suggested that this improvement was due to the fact that a fraction of cells undergoing death is eliminated with resting, thus allowing more accurate counts of viable cells used for the assay and eliminating interference by non-viable cells, resulting Dabrafenib in decreased background noise. On the other hand, a recent study argues that resting is not a universally applicable approach selleck inhibitor to improve the performance of cryopreserved PBMC for detection of low frequency T-cell responses (Kuerten et al., 2012), which underlines the importance of establishing whether resting could benefit immune monitoring in a specific read-out. In our case, overnight antigen stimulation (Tstim) compared

to the standard 6 h improved the magnitude of the RT-specific CD8+ T-cell response following ICS on PBMCs, without affecting the cytokine profile. A previous study has shown that a PBMC Tstim of 6 h gave the highest values for intracellular cytokine production (Horton et al., 2007). Another study using ICS on whole blood has found that the optimal time for antigen stimulation was 18 h (Hanekom et al., 2004). The ICS assay on whole blood was previously developed and used in several clinical trials (Agnandji et al., 2011, Day et al., 2013 and Harenberg Idoxuridine et al., 2013). An advantage of using whole blood instead of PBMCs is the smaller sample volumes needed when blood is used without preliminary PBMC purification, making this type of assay particularly attractive in children, in settings where testing at frequent time

intervals is planned or for trials conducted in areas with high HIV endemicity, where access to liquid nitrogen might be problematic (Agnandji et al., 2011, Mallone et al., 2011, Day et al., 2013 and Harenberg et al., 2013). Given the good correlations for CD8+ T-cell responses between ICS performed on whole blood compared to frozen PBMCs, we have demonstrated that the whole blood ICS assay is a valid tool to study HIV-1-infected individuals. The performance of PBMC vs whole blood T-cell assays has been previously assessed, with contradictory findings. Suni et al. showed that the whole blood method consistently yielded higher T-cell frequencies than the method using purified PBMCs (Suni et al., 1998). However, Hoffmeister et al. obtained higher T-cell frequencies with PBMCs than with whole blood, and the responses were sometimes detected only with PBMCs (Hoffmeister et al., 2003).