Optimization of whole blood antigen-specific cytokine assays for CD4(+) 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 eva luation involves three-color flow cytometric analysis of cytokine productio n in individual CD4(+) T cells. Methods: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic e xperiments 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 mu g/ml and for a period of at least 6 h, the first 2 h in the absenc e of 10 mu g/ml Brefeldin A. This period of incubation can be made more con venient by the use of a "timed cooling" device, whereby the samples are aut omatically cooled and held at 4 degrees C at the end of incubation. Such ti nted cooling does not affect backgrounds or the proportion of responding ce lls. For certain samples, a high background can be reduced by adding fourth -color reagents. They identify and allow for elimination of monocytes and a ctivated 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 les s than 10%; interassay variability is less than 25%). Cytometry 40:60-68, 2 000. (C) 2000 Wiley-Liss, Inc.