Development and application of 'phosphoflow' as a tool for immunomonitoring

Flow cytometry has revolutionized our ability to monitor immune responses by allowing us to simultaneously track a variety of cell surface and intracellular markers in discrete cell subsets in a highly sensitive and reproducible manner. This is especially critical in this new era of vaccinology trying to tackle the growing problems of chronic viral infections and cancer that not only evade host immune responses, but can negatively manipulate vaccine-induced immune responses. Thus, understanding how lymphocyte signaling is altered under normal and pathological conditions has become more critical. Over the last decade, a new flow cytometry technology called 'phosphoflow' (also sometimes called 'phosflow'), is rapidly developing for tracking multiple intracellular signaling molecules in the immune system at a single-cell level. Antibodies and reagents for tracking both tyrosine-phosphorylated and serine/threonine-phosphorylated signaling intermediaries in key immune signaling pathways have been developed, and phosphoflow is now starting to be applied to a wide variety of both preclinical and clinical studies on lymphocyte responses, as well as the functioning of cancer cells and virally infected cells. Here, we review the development of phosphoflow technology, its modern applications in the field of immunomonitoring and its current limitations. We then provide a perspective on the future of phosphoflow and a vision of how it can be applied to emerging critical questions in human vaccinology and public health.