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.
Uzel
G
G
Immunology Service, Warren Grant Magnuson Clinical Center, Bethesda, Maryland 20892, USA.
Frucht
D M
DM
Fleisher
T A
TA
Holland
S M
SM
D000818:Animals; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D016898:Interferon-alpha / Q000494:pharmacology; D018664:Interleukin-12 / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D050797:STAT4 Transcription Factor; D015534:Trans-Activators / Q000032:analysis* / Q000378:metabolism
D004268:DNA-Binding Proteins; D016898:Interferon-alpha; D050797:STAT4 Transcription Factor; C494091:Stat4 protein, mouse; D015534:Trans-Activators; D018664:Interleukin-12