PROJECT SUMMARY Atherosclerotic cardiovascular disease (CVD) is the main cause of morbidity and mortality worldwide. Thrombotic complications in atherosclerosis often lead to severe clinical events (myocardial infarction and stroke) and are mostly the consequence of active chronic vessel wall inflammation, characterized by presence of abundant plaque macrophages. Immunological studies have elucidated that macrophage dynamics in atherosclerosis is a complex systemic process which, after initial production of monocytes in the bone marrow, involves (i) monocyte egress (E) from the bone marrow and spleen, and subsequent plaque (ii) monocyte recruitment (R), resulting in increased (iii) macrophage accumulation (A). Although many different aspects of macrophage dynamics in ischemic heart disease have been elucidated, our current knowledge of the complex systemic interactions between immune organs and the vessel wall is exclusively based on snapshot immunological assays. In the absence of suitable in vivo readouts, an all-encompassing view on these different processes is difficult to acquire. The goal of our application is to develop an integrated multimodality imaging platform based on fluorine (19F) magnetic resonance imaging (MRI) and nanobody positron emission tomography (PET) that allows studying all aspects of macrophage dynamics in atherosclerosis, through the use of monocyte/macrophage-specific MRI and PET probes. We will employ these techniques to quantitatively map macrophage dynamics in atherosclerotic mice during disease progression and after novel nanoimmunotherapeutic intervention. 19F-MRI will be used to track monocyte egress (E) from the spleen, a process that happens gradually (during atherosclerosis progression) (Aim 1A). Monocyte recruitment (R) to and macrophage accumulation (A) in atherosclerotic plaques will be instead quantified using PET imaging of radiolabeled nanobodies (antibody fragments with ideal pharmacokinetics for vessel wall imaging) targeted against vascular cell adhesion molecule 1 (VCAM1) (R) and macrophage mannose receptor (MMR) (A) (Aim 1B). Combined 19F-MRI and nanobody PET will be used to map macrophage dynamics during atherosclerosis progression (Aim 1C), and treatment with a novel nanoimmunotherapy based on TRAF6 inhibition - which we show to specifically impair monocyte migration - to either slow down atherosclerosis progression (Aim 2A) or induce atherosclerosis regression (Aim 2B). We foresee that our findings will set the foundation for future studies of macrophage dynamics and targeted immunotherapies in the context of plaque thrombosis and cardiovascular events, for an improved stratification of cardiovascular risk.