Detection of intracellular phosphorylated STAT-4 by flow cytometry.

The convergence of innate and adaptive immunity is critical for host defense, allowing for early protection and the generation of specific responses. STAT-4 is at that point of convergence, unifying the IFNalpha and IL-12 pathways. Activation of STAT-4 is crucial to T cell polarization, B cell and NK cell activation, and the control of intracellular pathogens. However, techniques to detect phosphorylated STAT-4 are cumbersome and require many cells. We have developed a flow cytometric detection technique to investigate IL-12 signaling in human peripheral blood mononuclear cells. Using different polyclonal antibodies that recognize either total STAT-4 protein or tyrosine-phosphorylated STAT-4, we can easily detect IL-12 and IFNalpha signaling in PHA/IL-2 blasts derived from peripheral blood lymphocytes. This technique not only allows us to evaluate IL-12 signaling, but it is also less time consuming and labor intensive than alternative methods. Using this flow cytometry-based method, we should be able to detect patients with defects in IL-12 receptor signal transduction, who typically present with disseminated nontuberculous mycobacterial infections.