Analysis of Th1/Th2 T-cell subsets.

Specific immune responses are mediated by activated CD4+ T-helper (Th) cells. Two major subsets, denoted Th1 and Th2, have been identified that are characterized by their distinctive cytokine secretion pattern and associated effector functions. The signature cytokines of Th1 and Th2 cells are interferon-gamma and interleukin-4, respectively. Because of the dominant role of Th cells in directing specific immunity, the analysis of Th subsets by means of determining their signature cytokines has contributed greatly to the progress that has been made in recent years in the understanding of protective as well as pathogenic immune responses. Several methods, such as reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, ELISpot, and intracellular flow cytometric analysis are used for the analysis of T-cell cytokines and, thus, of Th subsets. Here, we briefly discuss the advantages and disadvantages of these methods and describe in detail a standard protocol for the analysis of human Th subsets by means of detection of cytoplasmic cytokines by flow cytometry.