Background: Atherosclerosis is a chronic inflammatory disease which drives the formation of luminal plaques in arteries. It is now widely accepted that T cells play a pivotal role in the atherosclerotic inflammatory environment. Genes driving T cell activation are known to be regulated by the polycomb repressive complex 2 (PRC2), that mediates the methylation of Histone 3 Lysine 27 (H3K27) epigenetic mark. The major component of the PRC2 is represented by the Enhancer of Zeste Homolog 2 (EZH2), which catalyzes the addition of methyl groups to H3K27.

Aim: We hypothesize that EZH2 mediates T cell activation and modulates T-helper (Th) cell polarization and thus atherogenesis.

Methods: Human atherosclerotic plaque specimens were collected from patients undergoing carotid endarterectomy (samples were obtained from the Munich Vascular Biobank, Technical University Munich) and classified as stable or ruptured. EZH2 gene expression was measured via qPCR. The specimens were subjected to single cell RNA sequencing (scRNA-Seq) to correlate EZH2 expression to immune cell subtypes. To study the effect of EZH2 in atherosclerosis, we generated mice with Ezh2 flanked by loxP-sites sensitive to Cre-mediated inactivation in T cells. All mice were backcrossed to Apolipoprotein E (Apoe^-/-) mice and set on high fat diet to have hyperlipidemic Ezh2^fl/flCd4^CreApoe^-/-, Ezh2^fl/flCd8^CreApoe^-/-mice and respective controls. EZH2 deficiency in T cells was confirmed by mass spectrometry and western blot analysis. Atherosclerotic plaque progression and immune cell phenotypes were assessed by histology, flow cytometry (FC), gene expression analysis and (sc)RNA-Seq. 

Results: EZH2 expression in human carotid endarterectomy specimens was increased 10-fold in ruptured plaques compared to stable plaques (p<0.0001). ScRNA-Seq revealed that the increase in EZH2 was associated with T cell clusters. Female Ezh2^fl/flCd4^Cre mice developed less atherosclerotic
plaques in the aortic root compared to wildtype (WT) littermates (p=0.001). However, no differences were observed between Ezh2^fl/flCd8^Cre mice and controls. FC analysis of Ezh2^fl/flCd4^Cre spleen and lymph nodes revealed a shift from naive to effector CD4⁺ T cells, and the majority were Th2 cells (p<0.0001). Moreover, plasma levels of lL4 were increased (p=0.0079), accompanied by increased  aortic IL4 mRNA levels (p=0.05) in Ezh2^fl/flCd4^Cre mice. To unravel mechanisms driving the type 2 immune response, bulk RNA-Seq of CD4⁺ T cells and scRNA-Seq of splenic CD3⁺ T cells from Ezh2^fl/flCd4^Cre mice and controls revealed that CD4⁺ T cell EZH2 deficiency caused an upregulation of promyelocytic leukemia zinc finger (Plzf, known as ZBTB16), a transcription factor of NKT cells. Via FC we confirmed the increase in splenic NKT cells in Ezh2^fl/flCd4^Cre mice compared to WT littermates. Likewise, Plzf mRNA levels were also significantly increased in the aorta of Ezh2^fl/flCd4^Cre mice. These NKT cells showed “Th2-like” phenotype (NKT2), producing high IL4.

Conclusion: Our study demonstrates that CD4⁺ T cell specific EZH2 deficiency skews the immune response towards type 2 immune response, driven by Th2 or NKT2 cell subtypes resulting in athero-protection.

Clinical Outlook: Nowadays little is known on cell-specific therapies able to dampen inflammation during atherosclerosis. Thus, we aim at exploring whether the epigenetic mark H3K27me3 in T cells may be a promising therapeutic target for more tailored cardiovascular immunotherapies.