Application of phospho-CyTOF to characterize immune activation in patients with sickle cell disease in an ex vivo model of thrombosis

Jeffrey Glassberg, Adeeb H. Rahman, Mohammad Zafar, Caroline Cromwell, Alexa Punzalan, Juan Jose Badimon, Louis Aledort

Sickle cell disease (SCD) is a genetic disease caused by mutations in the beta globin gene, and inflammation plays a key role in driving many aspects of disease pathology. Early immune activation is believed to be associated with hemodynamic stresses and thrombus formation as cells traffic through blood vessels. We applied an extracorporeal perfusion system to model these effects ex vivo, and combined this with a phospho-CyTOF workflow to comprehensively evaluate single-cell signatures of early activation across all major circulating immune subsets. These approaches showed immune activation following passage through the perfusion chamber, most notably in monocytes, which exhibited platelet aggregation and significantly elevated expression of multiple phospho-proteins. Overall, these studies outline a robust and broadly applicable workflow to leverage phospho-CyTOF to characterize immune activation in response to ex vivo or in vivo perturbations and may facilitate identification of novel therapeutic targets in SCD and other inflammatory diseases.