Abstract 612: High Dimensional Single-Cell Intracellular Signaling Identifies CCL5 as a Potential Key Driver of Myeloid Cell Reprogramming in Atherosclerotic Patients

Atherosclerosis is a disease characterized by chronic inflammation. However, identifying new molecularly targeted interventions in human atherosclerosis has proven difficult. In this study, we used phosphoCyTOF to comprehensively characterize functional responses of peripheral immune populations in human atherosclerosis at the single-cell level. Healthy PBMCs were exposed to plasma of patients with atherosclerosis (n=20) or healthy donors (n=10) and the activation of major signaling pathways across all major immune subsets using 18 surface markers and 10 intracellular phospho-proteins. Using viSNE we visualized and defined 10 major immune cell populations: B cells, Basophils, CD1c DCs, CD4 T cells, CD8 T cells, CD14 and CD16 monocytes, NK cells, NKT cells, and pDCs. Next, we evaluated the relative expression of 10 phospho-proteins (IkBa, pCREB, pERK1/2, pMAPKAP2, pp38, pPLCg2, pS6, pSTAT1, pSTAT3, and pSTAT5) across this immunological map and visualized functional pathways that were differentially induced in response to plasma of atherosclerotic patients vs. healthy donors. Monocytes exhibited the greatest immune activation, showing increased phosphorylation of p38, MAPKAP2, ERK1/2, CREB and S6 in response to atherosclerotic plasma. To identify plasma cytokines responsible for this effect, we measured a panel of 41 cytokines and identified CCL5, CXCL10 (IP-10), CXCL1 (GRO), PDGF-AA and PDGF-BB as the most differentially expressed in atherosclerotic vs. healthy plasma. PhosphoCyTOF analysis showed that only CCL5 reproduced the phosphorylation pattern induced by atherosclerotic plasma in monocytes, an effect that was completely inhibited by a CCL5 blocking antibody (Fig. 1). These results suggest that CCL5 in plasma of patients with atherosclerotic disease drives specific innate immune cell signaling functions that mark the inflammatory response in atherosclerotic disease.