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Background:Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation‚ notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies.Methods:We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity.Results:In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression ofCCR7andL-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express theHLA-DRAsurface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4HLA-DRA, 2 clusters expressed a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expressed AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression.Conclusions:Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

BACKGROUND: Multifood Oral Immunotherapy (mOIT) with adjunctive anti-IgE (omalizumab, Xolair(®)) treatment affords safe, effective, and rapid desensitization to multiple foods, although the specific immune mechanisms mediating this desensitization remain to be fully elucidated. METHODS: Participants in our phase 2 mOIT trial (NCT02643862) received omalizumab from baseline to week 16 and mOIT from week 8 to week 36. We compared the immune profile of PBMCs and plasma taken at baseline, week 8 and week 36 using high-dimensional mass cytometry, component-resolved diagnostics, the indirect basophil activation test, and Luminex. RESULTS: We found (i) decreased frequency of IL4(+) peanut-reactive CD4(+) T cells and a marked downregulation of GPR15 expression and CXCR3 frequency among γδ and CD8(+) T cell subsets at week 8 during the initial, omalizumab-alone induction phase; (ii) significant upregulation of the skin-homing receptor CCR4 in peanut-reactive CD4(+) T and Th2 effector memory (EM) cells and of cutaneous lymphocyte-associated antigen (CLA) in peanut-reactive CD8(+) T and CD8(+) EM cells (iii) downregulation of CD86 expression among antigen-presenting cell subsets; and (iv) reduction in pro-inflammatory cytokines, notably IL-17, at week 36 post-OIT. We also observed significant attenuation of the Th2 phenotype post-OIT, defined by downregulation of IL-4 peanut-reactive T cells and OX40 in Th2EM cells, increased allergen component-specific IgG(4)/IgE ratio, and decreased allergen-driven activation of indirectly sensitized basophils. CONCLUSIONS: This exploratory study provides novel comprehensive insight into the immune underpinnings of desensitization through omalizumab-facilitated mOIT. Moreover, this study provides encouraging results to support the complex immune changes that can be induced by OIT.

Background: The atherosclerotic plaque is a complex niche composed of immune cells including T cells and macrophages as well as vascular smooth muscle cells. How T cells interact with the plaque microenvironment is not well understood. Objective: To map the crosstalk between T cells, macrophages, and smooth muscle cells. Methods/Results: We performed single cell RNAseq using the 10x transcriptomics platform in coronary plaque samples from 12 patients, who underwent heart transplantation. We found a high proportion of macrophages as well as T cells, many of which display a memory phenotype that was confirmed by immunohistochemistry. Ligand-receptor computational analysis demonstrated significant cross talk between T cells, macrophages, and smooth muscle cells. One cytokine of interest is amphiregulin (AREG), which has been shown to mediate tissue healing and fibrosis. Of note, AREG expression appears to increase as coronary plaques progress from early lipid-rich stages to more advanced stages. To elucidate the effects of AREG on smooth muscle cells, we treated human coronary artery smooth muscle cells (hCASMCs) in vitro with recombinant AREG and found increased cell proliferation compared to control (P Conclusion: Taken together, our findings suggest that cross talk between T cells and smooth muscle cells, partially through AREG production by T cells, may contribute to plaque progression.

Background: Vascular thrombosis has been described in patients actively infected with influenza and, more recently, with the coronavirus. The risk may persist during the convalescence period. The underlying mechanisms mediating these events remain unclear. Objective: To determine whether human coronary plaque T cells reactive to influenza virus are also activated by self-proteins. Methods/Results: Using single cell targetRNAseq, we mapped the T cell repertoire in 19 patients with a range of coronary artery disease severity. To determine the potential peptides activating coronary plaque T cell receptors (TCRs), we matched TCR alpha and beta identity with HLA restriction to a database of known specificities. We found that plaque TCRs were potentially activated by viral epitopes, including those from the influenza M1 protein. We used a computational algorithm to identify self-proteins in the endothelial cells and vascular smooth muscle cells that may have peptides with similar homologies to viral epitopes predicted to bind our plaque TCRs. We then introduced plaque TCRs into the Jurkat T cells, exposed these cells to T2 (HLA-2 antigen presenting cells), viral peptides (e.g., M1) and self-epitopes (e.g., TSPAN17; ZIP9), and demonstrated T cell cross reactivity in vitro ( Figure 1 ). TSPAN17 is a transmembrane protein that regulates VE-cadherin expression and promotes T cell transmigration. Zip9, in contrast, is a protein that regulates zinc homeostasis, serves as an androgen receptor on endothelial cells, and has been shown to promote endothelial proliferation. Conclusion: Taken together, our findings suggest that T cell cross reactivity may be a potential mediator of viral-induced arterial thrombosis.

Background: Atherosclerosis is considered a chronic inflammatory disorder characterized by the presence of T cells and macrophages in the plaque. Given that T cells are the master regulators of the immune system, the possible role of T cells in mediating disease warrants further investigation. Objective: To map the T cell transcriptome in coronary artery plaque. Methods/Results: Using single cell RNAseq, we mapped the transcriptome of immune cells within the coronary atherosclerotic plaque in 12 patients with various disease stages. In addition to macrophages, we find an abundance of plaque T cells that appear to be enriched in plaque compared to blood ( Figure 1A ). Two plaque-enriched CD8 effector memory clusters (CD8 CTL Tem1 and CD8 CTL Tem2) display the highest proportion of activated cells (e.g., HLA-DRA+). These clusters are characterized by high expression of the pro-inflammatory cytokines (e.g., CCL3, CCL4, CCL5, and IFNG) and cytolytic markers (e.g.,GZMA, GZMH, GZMM, NKG7). CD8 CTL Tem1, however, has higher expression of GZMK, a cytolytic marker recently associated with inflammaging. CD8 CTL Tem2, on the other hand, has a higher expression of perforin and granulysin, two pore-forming proteins that mediate cell killing by enabling granzyme entry. Notably, CD8 CTL Tem2 appears to track with plaque progression, increasing as plaques mature from lipid-rich to more complex lesions then declining as plaques became more stable and calcified post-rupture. Specifically, this cluster displayed two-fold enrichment in complex plaques compared to other plaque phenotypes (12.1% vs. 6.9%, p=0.0069) ( Figure 1B ). This pro-inflammatory and cytolytic signature is also more apparent in the activated compared to nonactivated cells in this cluster. Conclusion: Taken together, our findings suggest that a pro-inflammatory CD8 T cell cluster characterized by a trifecta of cytolytic enzymes distinguishes complex plaque and deserves further investigation.

SummaryγδT cells contribute to host immune defense uniquely; but how they function in different stages (e.g., acute versus chronic) of a specific infection remains unclear. As the role ofγδT cells in early, activeMycobacterium tuberculosis(Mtb) infection is well documented, we focused on elucidating theγδT cell response in persistent or controlled Mtb infection. Systems analysis of circulatingγδT cells from a South African adolescent cohort identified a distinct population of CD8+γδT cells that expanded in this state. These cells had features indicative of persistent antigenic exposure but were robust cytolytic effectors and cytokine/chemokine producers. While theseγδT cells displayed an attenuated response to TCR-mediated stimulation, they expressed Natural Killer (NK) cell receptors and had robust CD16 (FcγRIIIA)-mediated cytotoxic response, suggesting alternative ways forγδT cells to control this stage of the infection. Despite this NK- like functionality, the CD8+γδT cells consisted of highly expanded clones, which utilized TCRs with different Vγ/δpairs. Theses TCRs could respond to an Mtb-lysate, but not to phosphoantigens, which are components of Mtb-lysate that activateγδT cells in acute Mtb infection, indicating that the CD8+γδT cells were induced in a stage-specific, antigen-driven manner. Indeed, trajectory analysis showed that theseγδT cells arose from naive cells that had traversed distinct differentiation paths in this infection stage. Importantly, increased levels of CD8+γδT cells were also found in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may lead to similarγδT cell responses.

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen–MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis ( Mtb ) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8 + T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8 + T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8 + T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

Background: Despite advances in medical therapy, heart attack and stroke remain leading causes of morbidity and mortality. Biological, epidemiological, and clinical studies have shown that many patients (up to 33% in some trials) still have residual inflammatory risk that is associated with plaque rupture, even in the setting of low LDL. Methods and Results: In this study, we analyzed the T cell landscape of coronary atherosclerotic plaque to determine their role in the development of atherosclerosis and its adverse sequelae. We recruited 17 patients undergoing heart transplantation. From each explanted heart, we obtained plaques from the major coronary arteries, digested them to form a single cell suspension, sorted the arteries for CD4 and CD8 T cells into 96 well plates, and submitted them for single cell genotyping and phenotyping ( Figure 1 ). Although peripheral blood from these patients also contains T cell clones, plaque CD8 T cell clones appear to function differently than blood (e.g., producing more BCL6, TNF-, and interferon gamma) further supporting that this is not a bystander effect. We found CD4 and CD8 T cells with identical TCR sequence across the arteries of the same patient, suggesting CD8T cells are purposely recruited to the plaques. When we analyzed the percent clonality based upon location and disease severity, we find that clonality is not dependent on location and is present at all severity levels, especially, in early disease (e.g., AHA Type I-IV lesions. Conclusion: CD8 T cells are clonally expanded in atherosclerotic plaque and appear to function differently than blood, suggesting these cells are purposefully recruited.

Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population(1). Although fewer than one in ten individuals eventually progress to active disease(2), tuberculosis is a leading cause of death from infectious disease worldwide(3). Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments. Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes.

Significance T cells are a central component of our immune system. They express a T cell receptor (TCR) on their surface, which detects pathogens and stimulates the T cell to initiate an immune response. Upon exposure to a specific pathogen, some T cells are activated and clonally expand. These pathogen-specific clonally expanded T cells, however, are generally rare and difficult to isolate. We have developed a technology (SELECT-seq) to isolate this rare population and to analyze their transcriptome and TCR composition. Therefore, our work may be the key to achieving a better understanding of pathogen-specific T cell clonality and function., To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen–MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

The spelling of author Qianting Yang was corrected; the affiliation of author Stephanus T. Malherbe was corrected; and graphs in Fig. 4b and c were corrected owing to reanalysis of the data into the correct timed intervals.

This review describes the adjuvanticity of novel diterpenoids (synthetic phytol derivatives) compared to some commercially available adjuvants. The efficacy of the phytol-derived immunostimulants was evaluated in terms of their ability to activate innate immunity, amplify various antigen-specific immune responses, and engender immunological memory with no discernible adverse effects in both competent and immune-deficient mice. The profile that emerges out of these studies reveals that the phytol derivatives are excellent immunostimulants, superior to a number of commercial adjuvants in terms of long-term memory induction and activation of both innate and acquired immunity. Additionally, the phytol-derived compounds have no cumulative inflammatory or toxic effects even in immuno-compromised mice.