Triantafyllidou, Vasiliki

Introduction: V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA), a co-inhibitory immune checkpoint protein, is known for regulating adaptive immune responses. Here, we investigated the role of VISTA in atherosclerosis. Hypothesis: VISTA is constitutively expressed on hematopoietic cells, particularly T cells. VISTA functions as both a ligand and receptor to maintain T cell quiescence. We hypothesize that VISTA expression is protective against plaque formation by inhibiting CD4 + T cell activation. Methods: Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) was performed on aortic CD45 + cells of HCD fed ApoE-/- mice. Spatial transcriptomics (VISIUM) was performed on carotid plaques from patients with either asymptomatic or symptomatic cerebrovascular disease. Plaque burden, characterization, and flow cytometry of blood and lymphoid organs were performed on VISTA -/- ApoE -/- (VISTA KO) and VISTA +/+ ApoE -/- (WT) mice that were fed a HCD for 10 weeks. ApoE -/- mice treated with VISTA agonist 8G8 or IgG control (200ug, intra-peritoneal, 3 times per week for the last 6/10 weeks of HCD feeding). In vitro proliferation, migration, and functional T cell assays were performed. Results: We found that VISTA is expressed on plaque T cell populations, particularly regulatory T cells, gamma-delta T cells, and naïve T cells. In humans, individuals with symptomatic CVD have significantly reduced VISTA gene expression in carotid plaques. VISTA KO mice have increased total plaque area and necrotic core area compared to littermate WT controls. VISTA KO mice have increased peripheral CD4 + and CD8 + effector memory T (T EM ) cells with a corresponding decrease in CD4 + and CD8 + naïve T (T N ) cells and CD4 + regulatory T (T reg ) cells. Further, VISTA KO mice have a higher frequency of CD4 + T H 1 (T-bet + IFNy + ) cells and lower frequencies of IL-10 + CD4 + T cells and CD4 + T H 2 (GATA-3 + IL-13 + ) cells. In vitro assays showed that VISTA suppresses CD4 + T cell proliferation, IFNγ production, and migration. Finally, VISTA agonist treatment significantly decreased plaque and necrotic core areas in atherosclerotic mice. Conclusion: Our data reveals a novel role for VISTA in reducing plaque development and in suppressing T cell activation, proliferation, and migration. Further characterization of T-cell specific VISTA-deficient mouse models and mechanistic insights into how VISTA modulates T cells are currently being investigated.

BACKGROUND: The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme EZH2 (enhancer of zeste homolog 2) mediates the H3K27me3 (trimethylation of histone H3 lysine 27) and is pivotal in controlling T cell responses. METHODS: To detail the role of T cell EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe (apolipoprotein E)–deficient mice with CD (cluster of differentiation) 4 + or CD8 + T cell–specific Ezh2 deletion (Ezh2 cd4 -knockout [KO], Ezh2 cd8 -KO) were analyzed to unravel the role of T cell Ezh2 in atherosclerosis and T cell–associated immune status. RESULTS: EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2 cd4 -KO, but not Ezh2 cd8 -KO, mice showed reduced atherosclerosis with fewer advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4 + T cells drives atherosclerosis. In-depth analysis of CD4 + T cells of Ezh2 cd4 -KO mice revealed that absence of Ezh2 results in a type 2 immune response with increased Il-4 (interleukin 4) gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2 -deficient T cells polarized macrophages toward an anti-inflammatory phenotype. Single-cell RNA-sequencing of splenic T cells revealed that Ezh2 deficiency reduced naive, Ccl5 + (C-C motif chemokine ligand 5) and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift toward Th2 (type 2 T helper) effector CD4 + T cells in Ezh2 cd4 -KO mice and confirmed a profound increase in splenic iNKT cells with increased expression of Plzf (promyelocytic leukemia zinc finger), which is the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 ([zinc finger and BTB domain containing 16], the Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2 cd4 -KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3–chromatin immunoprecipitation followed by quantitative polymerase chain reaction showed that T cell–Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. CONCLUSIONS: Our study uncovers the importance of T cell EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4 + T cells drives type 2 immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis.

Abstract Background Transforming growth factor beta (TGFβ) and its receptors (TGFβRs) are widely expressed by most human and murine cells and are implicated in several physiological and pathological conditions. In the context of atherosclerosis, TGFβ has been identified as a pleiotropic molecule involved in both pro- and anti-atherogenic processes, with its effects varying depending on the cell type it signals through. Purpose Given that myeloid cells play a major role in atherogenesis, we sought out to unravel the involvement of myeloid TGFβ signaling in atherosclerosis. Methods Aortic leukocytes from ApoE-/- mice fed a Western diet (WD) for 20 weeks were subjected to CITE-Seq analysis to assess the expression profile of Tgfβ and Tgfβrs. To investigate the effect of myeloid Tgfβ signaling on atherosclerosis, LysMCre+Tgfβr1flox/floxTgfβr2flox/floxmT/mGflox/floxApoE-/- (M-Tgfβr1/2dkoApoE-/-) mice and their LysMCre- littermates (ApoE-/-) were fed a WD for 6 weeks. Leukocyte phenotyping was assessed by flow cytometry (FC) and qPCR. Atherosclerotic plaque size in the aortic root was assessed by immunohistochemistry. Results CITE-Seq revealed that aortic ApoE-/- leukocytes highly expressed Tgfb1, whereas Tgfb2 and Tgfb3 were very lowly expressed. Tgfbr1 was mainly expressed in myeloid cells with macrophages and neutrophils showing the highest expression. Tgfbr2 was primarily expressed in T and B cells, while still showing a high level of expression in macrophages. Importantly, Tgfbr3 was not expressed in myeloid cells. Thus, to study the effect of myeloid Tgfβ signaling on atherosclerosis and to mitigate the potential for aberrant signaling that might arise from deleting only one receptor, we generated ApoE-/- mice with myeloid lineage tracing mT/mG alleles and combined deletion of Tgfbr1 and Tgfbr2 in myeloid cells using the LysMCre model. FC analysis of eGFP+ cells, i.e., cells where LysMCre is active, revealed a ~60-80% Tgfbr1/2 deletion efficiency in bone marrow-derived macrophages (BMDM) and in blood, splenic and BM CD11b+ cells, which was accompanied by decreased Smad2/3 signaling. M-Tgfβr1/2dkoApoE-/- mice showed a ~35% increase in blood and splenic CD11b+ cells and a ~25% increase in splenic macrophages compared to ApoE-/- controls, while BM CD11b+ cells were not affected. Tgfbr1/2-deficient BMDMs had increased Il-6, Il-1b and CD38 expression compared to controls, suggesting that Tgfbr1/2-deficient macrophages have acquired a pro-inflammatory phenotype. Interestingly, M-Tgfβr1/2dkoApoE-/- mice had ~30% fewer blood and splenic T cells compared to controls, which was explained by decreased naïve CD4+ and CD8+ T cells. M-Tgfβr1/2dkoApoE-/- mice had a ~50% increase in atherosclerotic plaque burden compared to ApoE-/- controls. Conclusion Our data suggest that impaired myeloid Tgfβ signaling leads to a pro-atherogenic environment in ApoE-/- mice. These effects may be mediated by an altered crosstalk between T cells and Tgfβr1/2-deficient macrophages.

Abstract Background Atherosclerosis is a chronic inflammatory disease and the primary underlying cause of cardiovascular disease (CVD). Recently, intracellular metabolic pathways have been identified as master switches of immune cell function and thus seem promising targets for therapeutic interventions aimed at lowering inflammation and thereby atherogenesis. Decades of research have demonstrated the importance of the amino acid (AA) L-arginine (Arg) in CVD. Although most research has focused on endothelium-dependent effects of Arg by the nitric oxide synthase (NOS), Arg metabolism - mainly through its metabolizing enzyme arginase 1 (Arg1) - has also been shown to regulate immune cell responses independent of its endothelial functions. Despite these findings, the cell-specific contribution of Arg/Arg1 metabolism on atherogenesis is still largely unknown. Purpose Profiling the Arg metabolism in endothelial and immune cells to identify cell-specific changes during athero-progression. Methods To investigate Arg metabolism in atherogenesis, we fed apolipoprotein E deficient (Apoe-­/­-) mice a Western diet (WD) for 6 and 12 weeks. Chow diet-fed aged-matched Apoe-­/-­ mice were used as littermate controls and C57BL/6 mice as steady-state controls. Systemic and local Arg concentrations were measured by mass-spectrometry. Arg metabolism in organs was studied by qPCR. Arg1 expression in atherosclerotic lesions was examined by immunohistochemistry (IHC). To assess cell-specific expression of Arg-related enzymes, aortic leukocytes from WD-fed Apoe-­/-­ mice were subjected to CITE-seq analysis. The role of T cell Arg1 was determined by nor-NOHA inhibition in Jurkat cells. Results AA measurements showed that systemic Arg was reduced in early and advanced atherogenesis (p<0.02), and splenic Arg was reduced in advanced atherogenesis (p=0.006) compared with steady state. In advanced atherosclerosis, Arg1 was decreased in the liver but increased in the spleen and aorta, whereas Nos2 was increased in the liver and aorta compared with steady state. To identify the cell types driving these local changes, we performed IHC on atherosclerotic lesions. Arg1+ lesional macrophages accumulated in advanced compared to early lesions (p=0.03), whereas Arg1+ endothelial cells tended to decrease (p=0.2). Surprisingly, we also found an accumulation of Arg1+ CD4+ T cells in lesions and a further upward trend with athero-progression. CITE-seq confirmed a constitutive expression of Arg1 in lymphoid clusters. In vitro experiments with Jurkat cells showed that arginase inhibition suppressed T cell proliferation and promoted apoptosis and migration (all p<0.0001). Conclusion Our data indicate that Arg metabolism is strongly regulated during athero-progression and suggest a so far unknown role of T cell-intrinsic Arg/Arg1 metabolism in the disease. Murine atherosclerosis studies are now being performed to decipher the cell-specific contribution of Arg/Arg1 metabolism in atherosclerosis.