Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function,... Show moreTissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis.To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (RAG1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks.Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content.TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model. Show less
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... Show moreViral 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.\nThe 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.\nVirus-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.\nThis 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. Show less
Smit, V.; Mol, J. de; Schaftenaar, F.H.; Depuydt, M.A.C.; Postel, R.J.; Smeets, D.: Verheijen, F.W.M.; ... ; Foks, A.C. 2023
Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact... Show moreAging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans.\nHere, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leukocytes of young versus aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood.\nCollectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease. Show less
Atherosclerosis is a lipid-driven chronic infammatory disease; however, whether it can be classifed as an autoimmune disease remains unclear. In this study, we applied single-cell T cell receptor... Show moreAtherosclerosis is a lipid-driven chronic infammatory disease; however, whether it can be classifed as an autoimmune disease remains unclear. In this study, we applied single-cell T cell receptor seqencing (scTCR-seq) on human carotid artery plaques and matched peripheral blood mononuclear cell samples to assess the extent of TCR clonality and antigen-specifc activation within the various T cell subsets. We observed the highest degree of plaque-specifc clonal expansion in efector CD4+ T cells, and these clonally expanded T cells expressed genes such as CD69, FOS and FOSB, indicative of recent TCR engagement, suggesting antigen-specifc stimulation. CellChat analysis suggested multiple potential interactions of these efector CD4+ T cells with foam cells. Finally, we integrated a published scTCR-seq dataset of the autoimmune disease psoriatic arthritis, and we report various commonalities between the two diseases. In conclusion, our data suggest that atherosclerosis has an autoimmune compondent driven by autoreactive CD4+ T cells. Show less
Cytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with... Show moreCytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established. Single-cell RNA sequencing was performed on human carotid atherosclerotic plaques of 18 patients to identify specific T cell populations (2). Next, human femoral and carotid atherosclerotic plaques (n=95) and matched blood samples (n=49) were analyzed by flow cytometry for the presence of CD4+GZMB+ T cells. Plaque morphology was assessed by Movat’s Pentachrome staining. A distinct cytotoxic GZMB+ PRF1+ CD28- CD4+ T cell cluster was identified using single-cell RNA sequencing. Furthermore, flow cytometry analysis showed that the percentage of GZMB+CD4+ T cells was significantly elevated in plaque compared to blood (Blood: 12.17±2.0 vs. Plaque: 17.40±1.0; P=0.0002). Moreover, a significant positive correlation was observed between the percentage of GZMB+CD4+ T cells in blood versus plaque (P=0.031). In line with the proinflammatory character of these cells, we found a positive association of GZMB+CD4+ T cells (P=0.036) with necrotic core size, whereas no correlation was found with this subtype in the circulation. In this study we have shown an enrichment of cytotoxic CD4+ T cells in atherosclerotic lesions, which positively correlate with necrotic core size. Future studies are aimed at elucidating the role of these cells in advanced atherosclerosis. Show less
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... Show moreThe 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. Show less
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... Show moreThe 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 Fc epsilon-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 Fc epsilon R, 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. Show less
Kritikou, E.; Heijden, T. van der; Swart, M.; Duijn, J. van; Slütter, B.; Wezel, A.; ... ; Bot, I. 2019
Mast cells (MCs) are potent innate immune cells that aggravate atherosclerosis through the release of proinflammatory mediators inside atherosclerotic plaques. Similarly, CD4+ T cells are... Show moreMast cells (MCs) are potent innate immune cells that aggravate atherosclerosis through the release of proinflammatory mediators inside atherosclerotic plaques. Similarly, CD4+ T cells are constituents of the adaptive immune response and accumulate within the plaques following lipid-specific activation by APCs. Recently it has been proposed that these two cell types can interact in a direct manner. However, no indication of such an interaction has been investigated in the context of atherosclerosis. In our study, we aimed to examine whether MCs can act as APCs in atherosclerosis, thereby modulating CD4+ T cell responses. We observed that MCs increased their MHC class II expression under hyperlipidemic conditions both in vivo and in vitro. Furthermore, we showed that MCs can present Ags in vivo via MHC class II molecules. Serum from high-fat diet–fed mice also enhanced the expression of the costimulatory molecule CD86 on cultured MCs, whereas OVA peptide–loaded MCs increased OT-II CD4+ T cell proliferation in vitro. The aortic CD4+ and TH1 cell content of atherosclerotic mice that lack MCs was reduced as compared with their wild-type counterparts. Importantly, we identified MCs that express HLA-DR in advanced human atheromata, indicating that these cells are capable of Ag presentation within human atherosclerotic plaques. Therefore, in this artice, we show that MCs may directly modulate adaptive immunity by acting as APCs in atherosclerosis. Show less
