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

✍ Scribed by Laurel E. Nomura; Joshua M. Walker; Holden T. Maecker

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 296 KB
• Volume: 40
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(20000501)40:1<60::aid-cyto8>3.0.co;2-j

✦ Synopsis

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 ϩ T cells. Methods: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFN␥), tumor necrosis factor alpha (TNF␣), 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 g/ml and for a period of at least 6 h, the first 2 h in the absence of 10 g/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%).