Abstract
Intracellular phosphorylation regulates immune cell function and requires precise measurement. Mass cytometry (CyTOF) resolves the spectral overlap limitations of flow cytometry, enabling simultaneous, high-dimensional profiling of signaling networks. To eliminate processing artifacts that impede clinical translation, a whole blood “vein‑to‑tube” workflow was developed to capture early T cell activation while preserving the native microenvironment. A secondary analytical pipeline established a framework to assess JAK/STAT signaling fidelity in whole-blood and cryopreserved PBMCs. Comparisons between matrices confirmed biological fidelity, identifying cryopreservation-induced baseline quiescence that necessitates targeted normalization to detect pathological deviations.
Pipeline feasibility was validated by profiling known STAT1-GOF variants, confirming elevated basal pSTAT1 and diminished pSTAT3. Furthermore, this approach exposed constitutive NF‑κB activation, demonstrated by baseline IκBα degradation and a failure to activate following LPS stimulation. This inflammatory exhaustion signature provides a probable mechanistic link to chronic autoinflammation and secondary infection susceptibility.
Applying this validated framework to an uncharacterized IQGAP1 A754T variant unveiled signaling dissociation in T cells. Phospho-CyTOF revealed stimulation responses of membrane-proximal (pPLCγ2 and pAKT) and distal (pS6) proteins indicative of conformational dysregulation, providing a potential mechanistic blueprint for clinical granulopenia and compensatory lymphoid accumulation experienced by patients.
This infrastructure advances single‑cell analysis beyond descriptive immunophenotyping, enabling rare immune diagnostics and functional precision medicine.