A whole blood assay as a simple, broad assessment of cytokines and chemokines to evaluate human immune responses to Mycobacterium tuberculosis antigens

Whole-blood assays (WBA) are well-suited low-cost proxy-measures while keeping the physiological environment during search for 11 out of 16 serum biomarkers (scheme) after in vitro stimulation with specific tuberculosis antigens. •Broader assessment of biosignature profiles can be accurately, fast and feasibly achieved using WBA.•For in vitro IL-1β, IL-4, IL-12p70, IL-17 and GM-CSF levels, PBMC showed to be a better choice related to WBA.•WBA provide a more physiological environment, which mimic the human blood condition.•WBA is a reliable in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis antigens. In vitro stimulation of whole blood or isolated peripheral blood cells with specific antigens is used for several purposes. We sought to identify a reliable, reproducible, fast and feasible in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis. In contrast to peripheral blood mononuclear cell (PBMC) culture, a whole blood assay (WBA) provides a more physiological environment, which may provide a broader assessment of serum biomarker, biosignature profiles. Twenty-three asymptomatic individuals with M. tuberculosis infection were recruited. Total cells from the WBA (diluted 1:3 in completed RPMI) and PBMC (2×105cells/ml) plus M. tuberculosis Ag85A, Ag85B, ESAT-6 and Mycobacterium bovis 65kDa were characterized by flow cytometry, then added in 96-well plates and on day 5 plasma and supernatants were harvested for detection of 17 cytokines by a Luminex array system. There was agreement between PBMC and WBA for IL-2, IL-5, IL-6, IL-7, IL-13, IFN-γ, TNF-α, MCP-1 and MIP-1β. There was evidence toward higher IL-10 (p≤0.049) and G-CSF (p≤0.012) plasma production, and higher IL-1β (p≤0.048), IL-4 (p≤0.044), IL-12p70 (p≤0.006), IL-17 (p≤0.002) and GM-CSF (p≤0.049) production for PBMC vs. WBA. Both methods provided virtually no reaction to the internal, negative control. Due to technical issues linked to data out of range, IL-8 data were not considered. These results suggest that, depending on the method employed, PBMC and/or WBA techniques provide fine conditions for the model proposed and thus whole blood cultures are well-suited low-cost proxy-measures during search for serum biomarkers.