Short-term culture of CD8 cells and intracellular cytokine staining.

CD8 T cells play an important role in the regulation of allergic disease. Human and murine CD8 T cells have been shown to be capable of differentiating into distinct subsets defined by cytokine profiles analogous to the Th1 and Th2 subsets and termed T cytotoxic 1 (Tc1, IFN-gamma producing) and 2 (Tc2, IL-4 producing). Effector cell phenotype can be analyzed in vitro on a single cell basis using intracellular cytokine staining and flow cytometry or analysis of other phenotypic markers. Human PBMC usually contain only very low percentages of effector cells which produce relatively high levels of cytokines required for this kind of analysis. It is therefore necessary to activate the T cells to induce rapid accumulation of cytoplasmic cytokines before analysis. This makes it difficult to analyze the antigen specificity of responding T cells but will indicate the type 1/type 2 bias of the population, reflecting previous exposures to antigen. In this chapter, we provide protocols for the generation of polarized populations of CD8 T effector cells using polyclonal stimulation and for their subsequent analysis by intracellular cytokine staining.