Comprehensive Profiling of Age- and Immune Cell–Specific Signaling Activation Using Multiplex Phosphoflow

Immune phenotyping represents a pillar in diagnostics, characterization of new genetic defects, and understanding mechanisms of diseases. Cell population distribution often does not cover the intrinsic function changes that may contribute to disease. Outcome of signaling activation can be used as proxy for cell function. To overcome the limitation of sample availability and standardization of signaling assays, we developed a multiplex full spectrum cytometry phosphoflow assay allowing the study of 6 phospho-proteins representing BCR/TCR, MAPK, PI3K/Akt/mTOR and canonical NF-κB signaling pathways in 18 immune cell subpopulations. Maximal stimulation and temporal dynamics were studied in response to pan-stimuli, activating cells regardless of receptor, and targeted stimuli for T, B, and innate immune cells. We studied healthy individuals between 1-69 years and discovered subpopulations-specific responses. Furthermore, pediatric donors showed broad differences in B cell and T cell function compared to adults. Hence, we established a tool to assess multiple signaling pathways at once and provide age- and subpopulation-specific references for signaling outcome.