Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays
Figure 1
Comparison of intracellular and cell surface markers after treatment of cells with two different fixation/permeabilization buffers. Assessment of cytokine secretion and cytotoxic factor expression in CD8+ T-cells. Briefly, thawed PBMC from a healthy donor was cultured (1 hour at 37°C) in presence of anti-CD107a and Staphylococcus enterotoxin B (SEB; Sigma-Aldrich, Munich, Germany, used at 2 μg/mL) or PHA (HA16, Murex Biotech, Dartford, UK, used at 1.5 μg/mL) in presence of costimulatory antibodies (CD28 and CD49d). After the addition of brefeldin A (Golgi Plug) and monensin (Golgi stop) (Becton Dickinson, San Jose, CA, USA), cells were incubated for additional 5 hours. Following stimulation, final 2 mM EDTA was added to each well and incubated for 15 minutes. Cells were then incubated for 30 min at 4°C with surface antibodies (CD8), fixed, and permeabilized with the previously mentioned lysing/permeabilization buffers and stained with fluorescently labelled antibodies directed against IL-2 and TNF-α. Samples were then acquired on a FacsCanto flow cytometer instrument (BD Biosciences) and analyzed by FACSDiva and/or FlowJo software (Tree Star, Ashland). (a) Bar graph showing the percentages of total CD107a+, TNF-α +, and IL-2+ analyzed within CD8+ gated cells. (b) Bar graph showing the polyfunctionality of CD8+ T-cells upon SEB stimulation (Boolean analysis). As negative controls, we included untreated cell (only costimuli).
