Optimization of whole blood antigen-specific cytokine assays for CD4⁺ T cells

Background: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4⁺ T cells. Methods: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors. Results: CMV is most effective as a stimulating antigen when used at a dose of 5 μg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 μg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a 'timed cooling' device, whereby the samples are automatically cooled and held at 4°C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining. Conclusions: These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra- assay variability is less than 10%; interassay variability is less than 25%). (C) 2000 Wiley-Liss, Inc.