Alternative Splicing of FOXP3 Controls Regulatory T Cell Effector Functions and Is Associated with Human Atherosclerotic Plaque Stability

Rationale: Regulatory T (Treg) cells suppress immune responses and have been shown to attenuate atherosclerosis. The Treg cell lineage specification factor FOXP3 is essential for Treg cells9 ability to uphold immunological tolerance. In humans, FOXP3 exists in several different isoforms, however, their specific role is poorly understood. Objective: To define the regulation and functions of the two major FOXP3 isoforms, FOXP3fl and FOXP3Δ2, as well as to establish whether their expression is associated with ischemic atherosclerotic disease. Methods and Results: Human primary T-cells were transduced with lentiviruses encoding distinct FOXP3 isoforms. The phenotype and function of these cells were analyzed by flow cytometry, in vitro suppression assays and RNA-sequencing. We also assessed the effect of activation on Treg cells isolated from healthy volunteers. Treg cell activation resulted in increased FOXP3 expression that predominantly was made up of FOXP3Δ2. FOXP3Δ2 induced specific transcription of GARP, which functions by tethering the immunosuppressive cytokine TGF-β to the cell membrane of activated Treg cells. RT-PCR was used to determine the impact of alternative splicing of FOXP3 in relation with atherosclerotic plaque stability in a cohort of over 150 patients that underwent carotid endarterectomy. Plaque instability was associated with a lower FOXP3Δ2 transcript usage, when comparing plaques from patients without symptoms and patients with occurrence of recent ( FOXP3 mRNA between these two groups. Conclusions: These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.