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Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction: Viral infections have been associated with the progression of atherosclerosis and CD8 + T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8 + T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8 + T-cells in the atherosclerotic lesion., Methods: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8 + T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-β sequencing and single-cell T-cell receptor (α and β) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8 + T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined., Results: Virus-associated CD8 + T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P =0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8 + T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8 + T-cells were phenotypically highly similar to other CD8 + T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8 + T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion., Conclusions: This study suggests that virus-specific CD8 + T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms., Competing Interests: Disclosures None.

Background: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects., Methods: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr -/- and APOE*3-Leiden.CETP mice., Results: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose., Conclusions: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment., Competing Interests: Disclosures None.

Aims: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis., Methods and Results: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade., Conclusion: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production., Competing Interests: Conflict of interest: E.J.G., C.G., S.R., and D.L. are employed by and hold stocks or options in Cantargia AB. C.G., S.R., and D.L. are signed as co-inventors on a patent related to anti-IL1RAP monoclonal antibodies. D.E. received reagents related to the current study from Cantargia AB., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol, a final intermediate in cholesterol biosynthesis, has been identified as a selective LXR ligand that suppresses inflammation without inducing lipogenesis. Δ24-Dehydrocholesterol reductase (DHCR24) converts desmosterol into cholesterol, and we previously showed that the DHCR24 inhibitor SH42 increases desmosterol to activate LXR and attenuate experimental peritonitis and metabolic dysfunction-associated steatotic liver disease. Here, we aimed to evaluate the effect of SH42 on atherosclerosis development in APOE∗3-Leiden.CETP mice and low-density lipoproteins (LDL) receptor knockout mice, models for lipid- and inflammation-driven atherosclerosis, respectively. In both models, SH42 increased desmosterol without affecting plasma lipids. While reducing liver lipids in APOE∗3-Leiden.CETP mice, and regulating populations of circulating monocytes in LDL receptor knockout mice, SH42 did not attenuate atherosclerosis in either model., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Lipid nanoparticles (LNPs) have unlocked the potential of ribonucleic acid (RNA) therapeutics and vaccines. Production and large-scale manufacturing methods for RNA-LNPs have been established and rapidly accelerate. Despite this, basic research on LNPs is still required, due to their high assembly complexity and fairly new development, including research on lipid organization, transfection optimization, and in vivo behavior. Understanding fundamental aspects of LNPs that is, how lipid composition and physicochemical properties affect their biodistribution, cell recognition, and transfection, could propel their clinical development and facilitate overcoming current challenges. Herein, we review recent developments in the field of LNP technology and summarize the main findings focusing on nano-bio interactions., Competing Interests: Declaration of Competing Interest I hereby confirm that there is no conflict of interest regarding the paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically-approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: Nanomedicine provides a promising platform for manipulating dendritic cells (DCs) and the ensuing adaptive immune response. For the induction of regulatory responses, DCs can be targeted in vivo with nanoparticles incorporating tolerogenic adjuvants and auto-antigens or allergens., Methods: Here, we investigated the tolerogenic effect of different liposome formulations loaded with vitamin D3 (VD3). We extensively phenotyped monocyte-derived DCs (moDCs) and skin DCs and assessed DC-induced regulatory CD4+ T cells in coculture., Results: Liposomal VD3 primed-moDCs induced the development of regulatory CD4+ T cells (Tregs) that inhibited bystander memory T cell proliferation. Induced Tregs were of the FoxP3+ CD127low phenotype, also expressing TIGIT. Additionally, liposome-VD3 primed moDCs inhibited the development of T helper 1 (Th1) and T helper 17 (Th17) cells. Skin injection of VD3 liposomes selectively stimulated the migration of CD14+ skin DCs., Discussion: These results suggest that nanoparticulate VD3 is a tolerogenic tool for DC-mediated induction of regulatory T cell responses., Competing Interests: Authors BS and EdJ: Payment to institute by Health Holland and Samenwerkende Gezondheidsorganisaties SGF Grantnr: LSHM18065-SGF. Author RvR: Payment to institute by Health Holland and Samenwerkende Gezondheidsorganisaties SGF Grantnr: LSHM18065-SGF, Payment to institute by Health Holland − TKI-LSH PPP Allowance − Grant nr. LSHM19073, European Commission, NWO-TKI, AB Enzymes, Angany Inc., payment to self consulting fees received from HAL Allergy, Citeq BV, Angany Inc, Reacta Healthcare, and Mission MightyMe, payment to self for lectures, speakers bureaus, or educational events by HAL Allergy BV, ALK and ThermoFisher Scientific. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nagy, Lozano Vigario, Sparrius, van Capel, van Ree, Tas, de Vries, Geijtenbeek, Slütter, de Jong and for the DC4Balance consortium.)

Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG 1 /IgG 2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT., Competing Interests: HW and HS are employees of HAL Allergy BV. RR is a consultant for HAL Allergy BV, Citeq BV, Reacta Healthcare Ltd., Angany Inc., Mission MightyMe and AB Enzymes GmbH. RR reports speaker fees from HAL Allergy BV, ThermoFisher Scientific and ALK and has stock options of Angany Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Warmenhoven, Leboux, Bethanis, van Strien, Logiantara, van Schijndel, Aglas, van Rijt, Slütter, Kros, Jiskoot and van Ree.)

Gold nanoparticles (GNPs) can be manufactured in various shapes, and their size is programmable, which permits the study of the effects imposed by these parameters on biological processes. However, there is currently no clear evidence that a certain shape or size is beneficial. To address this issue, we have utilised GNPs and gold nanorods (GNRs) functionalised with model epitopes derived from chicken ovalbumin (OVA 257-264 and OVA 323-339 ). By using two distinct epitopes, it was possible to draw conclusions regarding the impact of nanoparticle shape and size on different aspects of the immune response. Our findings indicate that the peptide amphiphile-coated GNPs and GNRs are a safe and versatile epitope-presenting system. Smaller GNPs (∼15 nm in diameter) induce significantly less intense T-cell responses. Furthermore, effective antigen presentation via MHC-I was observed for larger spherical particles (∼40 nm in diameter), and to a lesser extent for rod-like particles (40 by 15 nm). At the same time, antigen presentation via MHC-II strongly correlated with the cellular uptake, with smaller GNPs being the least efficient. We believe these findings will have implications for vaccine development, and lead to a better understanding of cellular uptake and antigen egress from lysosomes into the cytosol., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Aims: CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis., Methods and Results: Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient mice revealed a pronounced increase in RORγt+CD8+ T cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/- low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice., Conclusion: These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T (Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cell migration by affecting the expression of specific membrane proteins, thereby decreasing Treg cell migration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has been shown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemia contributes to altered migration of Treg cells, in part, by affecting cellular metabolism., Methods and Results: Dyslipidemia was induced by feeding Ldlr-/- mice a western-type diet for 16-20 weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined. Dyslipidemia was associated with altered mTORC2 signalling in Treg cells, decreased expression of membrane proteins involved in migration, including CD62L, CCR7, and S1Pr1, and decreased Treg cell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemia inhibited mTORC1 signalling, induced PPARδ activation and increased fatty acid (FA) oxidation in Treg cells. Moreover, mass-spectrometry analysis of serum from Ldlr-/- mice with normolipidemia or dyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with a synthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FA oxidation-dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cell migration into the inflamed peritoneum and into atherosclerotic lesions in vitro., Conclusion: Altogether, our findings implicate that dyslipidemia does not contribute to atherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-like migratory phenotype in Treg cells., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed., Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis., Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 + and CD8 + T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells., Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

Atherosclerosis is the main pathology behind most cardiovascular diseases. It is a chronic inflammatory disease characterized by the formation of lipid-rich plaques in arteries. Atherosclerotic plaques are initiated by the deposition of cholesterol-rich LDL particles in the arterial walls leading to the activation of innate and adaptive immune responses. Current treatments focus on the reduction of LDL blood levels using statins, however the critical components of inflammation and autoimmunity have been mostly ignored as therapeutic targets. The restoration of immune tolerance towards atherosclerosis-relevant antigens can arrest lesion development as shown in pre-clinical models. In this review, we evaluate the clinical development of similar strategies for the treatment of inflammatory and autoimmune diseases like rheumatoid arthritis, type 1 diabetes or multiple sclerosis and analyse the potential of tolerogenic vaccines for atherosclerosis and the challenges that need to be overcome to bring this therapy to patients., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: The immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B- and T-lymphocyte attenuator (BTLA) is a novel co-receptor that negatively regulates the activation of B and T cells; however, there have been no reports of BTLA and its function in atherosclerosis or cardiovascular disease (CVD). We aimed to assess the dominant BTLA expressing leucocyte in CVD patients and to investigate whether BTLA has a functional role in experimental atherosclerosis., Methods and Results: We show that BTLA is primarily expressed on B cells in CVD patients and follicular B2 cells in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We treated Ldlr-/- mice that were fed a western-type diet (WTD) with phosphate-buffered saline, an isotype antibody, or an agonistic BTLA antibody (3C10) for 6 weeks. We report here that the agonistic BTLA antibody significantly attenuated atherosclerosis. This was associated with a strong reduction in follicular B2 cells, while regulatory B and T cells were increased. The BTLA antibody showed similar immunomodulating effects in a progression study in which Ldlr-/- mice were fed a WTD for 10 weeks before receiving antibody treatment. Most importantly, BTLA stimulation enhanced collagen content, a feature of stable lesions, in pre-existing lesions., Conclusion: Stimulation of the BTLA pathway in Ldlr-/- mice reduces initial lesion development and increases collagen content of established lesions, presumably by shifting the balance between atherogenic follicular B cells and atheroprotective B cells and directing CD4+ T cells towards regulatory T cells. We provide the first evidence that BTLA is a very promising target for the treatment of atherosclerosis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to their characteristic Fcε-receptor, causes the release of their cytoplasmic granules. These granules are filled with neutral proteases such as tryptase, but also with histamine and pro-inflammatory mediators. Mast cells accumulate in high numbers within human atherosclerotic tissue, particularly in the shoulder region of the plaque. These findings are largely based on immunohistochemistry, which does not allow for the extensive characterization of these mast cells and of the local mast cell activation mechanisms. In this study, we thus aimed to develop a new flow-cytometry based methodology in order to analyze mast cells in human atherosclerosis. We enzymatically digested 22 human plaque samples, collected after femoral and carotid endarterectomy surgery, after which we prepared a single cell suspension for flow cytometry. We were able to identify a specific mast cell population expressing both CD117 and the FcεR, and observed that most of the intraplaque mast cells were activated based on their CD63 protein expression. Furthermore, most of the activated mast cells had IgE fragments bound on their surface, while another fraction showed IgE-independent activation. In conclusion, we are able to distinguish a clear mast cell population in human atherosclerotic plaques, and this study establishes a strong relationship between the presence of IgE and the activation of mast cells in advanced atherosclerosis. Our data pave the way for potential therapeutic intervention through targeting IgE-mediated actions in human atherosclerosis., Competing Interests: The authors declare no conflict of interest.

Aims: T lymphocytes play an important role in atherosclerosis development, but the role of the CD8+ T-cell remains debated, especially in the clinically relevant advanced stages of atherosclerosis development. Here, we set out to determine the role of CD8+ T-cells in advanced atherosclerosis., Methods and Results: Human endarterectomy samples analysed by flow cytometry showed a negative correlation between the percentage of CD8+ T-cells and macrophages, suggesting a possible protective role for these cells in lesion development. To further test this hypothesis, LDLr-/- mice were fed a western-type diet (WTD) for 10 weeks to induce atherosclerosis, after which they received CD8α-depleting or isotype control antibody for 6 weeks. Depletion of CD8+ T-cells in advanced atherosclerosis resulted in less stable lesions, with significantly reduced collagen content in the trivalve area, increased macrophage content and increased necrotic core area compared with controls. Mechanistically, we observed that CD8 depletion specifically increased the fraction of Th1 CD4+ T-cells in the lesions. Treatment of WTD-fed LDLr-/- mice with a FasL-neutralizing antibody resulted in similar changes in macrophages and CD4+ T-cell skewing as CD8+ T-cell depletion., Conclusion: These findings demonstrate for the first time a local, protective role for CD8+ T-cells in advanced atherosclerosis, through limiting accumulation of Th1 cells and macrophages, identifying a novel regulatory mechanism for these cells in atherosclerosis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis., Methods and Results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells., Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity., Objectives: This study evaluates the potential of TRAF-STOP treatment in atherosclerosis., Methods: The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe -/- ) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages., Results: TRAF-STOP treatment of young Apoe -/- mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe -/- mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair "classical" immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe -/- mice., Conclusions: TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis., (Published by Elsevier Inc.)

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies. IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States., (Copyright © 2017 Post et al.)

Objective: Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1β. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE -/- mice., Approach and Results: First, we determined the efficacy of MCC950 in vitro. Bone marrow-derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1β release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide-induced interleukin-1β secretion, without affecting the tumor necrosis factor-α response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE -/- mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin-stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice., Conclusions: These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development., (© 2017 American Heart Association, Inc.)

A small number of tissue-resident memory T cells (Trm) provide potent protection against infections. Three recent studies by Ariotti et al. (2014), Schenkel et al. (2014a), and Iijima and Iwasaki (2014) report that Trm rapidly produce cytokines after infection and initiate a tissue-wide anti-viral state by instructing innate immune cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inducing memory CD8(+) T cells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8(+) T cells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8(+) T cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8(+) T cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8(+) T cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8(+) T cells, and resulted in superior protection against IAV., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Development of vaccines in autoimmune diseases has received wide attention over the last decade. However, many vaccines showed limited clinical efficacy. To enhance vaccine efficacy in infectious diseases, biocompatible and biodegradable polymeric nanoparticles have gained interest as antigen delivery systems. We investigated in mice whether antigen-encapsulated PLGA (poly-lactic-co-glycolic acid), PLGA-TMC (N-trimethyl chitosan) or TMC-TPP (tri-polyphosphate) nanoparticles can also be used to modulate the immunological outcome after nasal vaccination. These three nanoparticles enhanced the antigen presentation by dendritic cells, as shown by increased in vitro and in vivo CD4(+) T-cell proliferation. However, only nasal PLGA nanoparticles were found to induce an immunoregulatory response as shown by enhanced Foxp3 expression in the nasopharynx associated lymphoid tissue and cervical lymph nodes. Nasal administration of OVA-containing PLGA particle resulted in functional suppression of an OVA-specific Th-1 mediated delayed-type hypersensitivity reaction, while TMC-TPP nanoparticles induced humoral immunity, which coincided with the enhanced generation of OVA-specific B-cells in the cervical lymph nodes. Intranasal treatment with Hsp70-mB29a peptide-loaded PLGA nanoparticles suppressed proteoglycan-induced arthritis, leading to a significant reduction of disease. We have uncovered a role for PLGA nanoparticles to enhance CD4(+) T-cell mediated immunomodulation after nasal application. The exploitation of this differential regulation of nanoparticles to modulate nasal immune responses can lead to innovative vaccine development for prophylactic or therapeutic vaccination in infectious or autoimmune diseases.

Tolerance to experimental autoimmune encephalomyelitis (EAE) in SJL mice can be induced by immunization with a mannosylated form of the proteolipid protein (M-PLP139-151), despite the presence of CFA. The state of tolerance is characterized by poor delayed-type hypersensitivity responses and the absence of clinical EAE symptoms. In vivo monitoring of CFSE-labeled PLP139-151-specific TCR-transgenic (5B6) T cells revealed that immunization with M-PLP139-151 increases the clonal expansion of 5B6 T cells that do not develop full effector functions. Moreover, nonfunctional T cells obtained from M-PLP139-151-immunized mice showed poor blastogenesis and were unable to transfer EAE to naïve recipients. Nevertheless, the in vitro production of cytokines and chemokines associated with EAE was unaffected. Importantly, tolerance induced by M-PLP139-151 was abrogated by the administration of pertussis toxin, resulting in EAE development. Our results suggest that M-PLP139-151 inhibits EAE development by affecting the differentiation of T cells into encephalitogenic effector cells.