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Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multiomics approaches. Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases. Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data (AlliGateR). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations. Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are the master regulators of immunity and they have been implicated in different disease states such as infection, autoimmunity and cancer. Since their discovery, many studies have focused on understanding Treg development, differentiation, and function. While there are many players in the generation and function of truly suppressive Tregs, the role of checkpoint pathways in these processes have been studied extensively. In this paper, we systematically review the role of different checkpoint pathways in Treg homeostasis and function. We describe how co-stimulatory and coinhibitory pathways modulate Treg homeostasis and function and highlight data from mouse and human studies. Multiple checkpoint pathways are being targeted in cancer and autoimmunity; therefore, we share insights from the clinic and discuss the effect of experimental and approved therapeutics on Treg biology. [ABSTRACT FROM AUTHOR]

Food allergy is a serious public health problem because of its high incidence and risk. Probiotics can induce immune regulation in patients with allergic diseases, but its mechanism is not fully clear. In this paper, b-lactoglobulin (bLG)-sensitized mice were used as models to explore the mechanism of Bifidobacterium animalis KV9 (KV9) and Lactobacillus vaginalis FN3 (FN3) on reducing allergic reactions and regulating immune cell function. The results showed that oral administration of KV9 and FN3 significantly reduced the scores of allergic symptoms, hypothermia symptoms, and serum levels of bLG-specific immunoglobulins E (b-LG-sIgE), histamine, and mast cell protease in allergic mice. Flow cytometry analysis of intestinal dendritic cells (DCs) showed that the proportion of CD11c+major histocompatibility complex (MHC)-II+DCs, CD11c+CD80+DCs, and CD11c+ CD86+DCs increased after KV9 and FN3 intervention, indicating that the strains induced immature DCs and decreased the antigen-presenting capacity of DCs. Meanwhile, the toll-like receptor 4 (TLR4)-NF-kB signaling pathway was activated in DCs. The secretion of interleukin-12 (IL-12) was significantly increased, while interleukin-4 (IL-4) was decreased by DCs after KV9 and FN3 intervention, indicating that DCs have the potential to promote T-cell differentiation into T helper type 1 (Th1) cells. Furthermore, the proportion of CD3+CD8−IFN-g+ T cells in the spleen increased, while CD3+CD8−IL-4+T cells decreased after oral administration of KV9 and FN3, correcting the T helper type 2 (Th2)-skewed immune responses. These results indicate that KV9 and FN3 reduce b-LGinduced allergic symptoms in mice, and suggest that the two potential probiotics might be used as an alternative therapeutic agent for mitigating food allergy. [ABSTRACT FROM AUTHOR]

For the skin immune system, γδ T cells are important components, which help in defensing against damage and infection of skin. Compared to the conventional αβ T cells, γδ T cells have their own differentiation, development and activation characteristics. In adult mice, dendritic epidermal T cells (DETCs), Vγ4 and Vγ6 γδ T cells are the main subsets of skin, the coordination and interaction among them play a crucial role in wound repair. To get a clear overview of γδ T cells, this review synopsizes their derivation, development, colonization and activation, and focuses their function in acute and chronic wound healing, as well as the underlining mechanism. The aim of this paper is to provide cues for the study of human epidermal γδ T cells and the potential treatment for skin rehabilitation. [ABSTRACT FROM AUTHOR]

Stroke is the second leading cause of global death and is characterized by high rates of mortality and disability. Oxidative stress is accompanied by other pathological processes that together lead to secondary brain damage in stroke. As the major component of the brain, glial cells play an important role in normal brain development and pathological injury processes. Multiple connections exist in the pathophysiological changes of reactive oxygen species (ROS) metabolism and glia cell activation. Astrocytes and microglia are rapidly activated after stroke, generating large amounts of ROS via mitochondrial and NADPH oxidase pathways, causing oxidative damage to the glial cells themselves and neurons. Meanwhile, ROS cause alterations in glial cell morphology and function, and mediate their role in pathological processes, such as neuroinflammation, excitotoxicity, and blood-brain barrier damage. In contrast, glial cells protect the Central Nervous System (CNS) from oxidative damage by synthesizing antioxidants and regulating the Nuclear factor E2-related factor 2 (Nrf2) pathway, among others. Although numerous previous studies have focused on the immune function of glial cells, little attention has been paid to the role of glial cells in oxidative stress. In this paper, we discuss the adverse consequences of ROS production and oxidative-antioxidant imbalance after stroke. In addition, we further describe the biological role of glial cells in oxidative stress after stroke, and we describe potential therapeutic tools based on glia cells. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is an organelle equipped with mechanisms for proper protein folding, trafficking, and degradation to maintain protein homeostasis in the secretory pathway. As a defense mechanism, perturbation of ER proteostasis by ER stress agents activates a cascade of signaling pathways from the ER to the nucleus known as unfolded protein response (UPR). The primary goal of UPR is to induce transcriptional and translational programs to restore ER homeostasis for cell survival. As such, defects in UPR signaling have been implicated as a key contributor to multiple diseases including metabolic diseases, degenerative diseases, inflammatory disorders, and cancer. Growing evidence support the critical role of ER stress in regulating the fate as well as the magnitude of the immune response. Moreover, the availability of multiple UPR pharmacological inhibitors raises the hope that targeting UPR can be a new strategy for immune modulation and immunotherapy of diseases. This paper reviews the principal mechanisms by which ER stress affects immune cell biology and function, with a focus of discussion on UPR-associated immunopathology and the development of potential ER stress-targeted therapeutics. [ABSTRACT FROM AUTHOR]

Cellular metabolism is a crucial determinant of immune cell fate and function. Extensive studies have demonstrated that metabolic decisions influence immune cell activation, differentiation, and cellular capacity, in the process impacting an organism’s ability to stave off infection or recover from injury. Conversely, metabolic dysregulation can contribute to the severity of multiple disease conditions including autoimmunity, alloimmunity, and cancer. Emerging data also demonstrate that metabolic cues and profiles can influence the success or failure of adoptive cellular therapies. Importantly, immunometabolism is not one size fits all; and different immune cell types, and even subdivisions within distinct cell populations utilize different metabolic pathways to optimize function. Metabolic preference can also change depending on the microenvironment in which cells are activated. For this reason, understanding the metabolic requirements of different subsets of immune cells is critical to therapeutically modulating different disease states or maximizing cellular function for downstream applications. Fatty acid oxidation (FAO), in particular, plays multiple roles in immune cells, providing both pro- and anti-inflammatory effects. Herein, we review the major metabolic pathways available to immune cells, then focus more closely on the role of FAO in different immune cell subsets. Understanding how and why FAO is utilized by different immune cells will allow for the design of optimal therapeutic interventions targeting this pathway. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a complex three-dimensional structure composed of proteins, glycans, and proteoglycans, constituting a critical component of the tumor microenvironment. Complex interactions among immune cells, extracellular matrix, and tumor cells promote tumor development and metastasis, consequently influencing therapeutic efficacy. Hence, elucidating these interaction mechanisms is pivotal for precision cancer therapy. T lymphocytes are an important component of the immune system, exerting direct anti-tumor effects by attacking tumor cells or releasing lymphokines to enhance immune effects. The ECM significantly influences T cells function and infiltration within the tumor microenvironment, thereby impacting the behavior and biological characteristics of tumor cells. T cells are involved in regulating the synthesis, degradation, and remodeling of the extracellular matrix through the secretion of cytokines and enzymes. As a result, it affects the proliferation and invasive ability of tumor cells as well as the efficacy of immunotherapy. This review discusses the mechanisms underlying T lymphocyte-ECM interactions in the tumor immune microenvironment and their potential application in immunotherapy. It provides novel insights for the development of innovative tumor therapeutic strategies and drug. [ABSTRACT FROM AUTHOR]

Tumors pose a significant global public health challenge, resulting in numerous fatalities annually. CD8+ T cells play a crucial role in combating tumors; however, their effectiveness is compromised by the tumor itself and the tumor microenvironment (TME), resulting in reduced efficacy of immunotherapy. In this dynamic interplay, extracellular vesicles (EVs) have emerged as pivotal mediators, facilitating direct and indirect communication between tumors and CD8+ T cells. In this article, we provide an overview of how tumor-derived EVs directly regulate CD8+ T cell function by carrying bioactive molecules they carry internally and on their surface. Simultaneously, these EVs modulate the TME, indirectly influencing the efficiency of CD8+ T cell responses. Furthermore, EVs derived from CD8+ T cells exhibit a dual role: they promote tumor immune evasion while also enhancing antitumor activity. Finally, we briefly discuss current prevailing approaches that utilize functionalized EVs based on tumor-targeted therapy and tumor immunotherapy. These approaches aim to present novel perspectives for EV-based tumor treatment strategies, demonstrating potential for advancements in the field. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are a unique subset of T cells that recognizes metabolites derived from the vitamin B2 biosynthetic pathway. Since the identification of cognate antigens for MAIT cells, knowledge of the functions of MAIT cells in cancer, autoimmunity, and infectious diseases has been rapidly expanding. Recently, MAIT cells have been found to contribute to visual protection against autoimmunity in the eye. The protective functions of MAIT cells are induced by T-cell receptor (TCR)-mediated activation. However, the underlying mechanisms remain unclear. Thus, this mini-review aims to discuss our findings and the complexity of MAIT cell-mediated immune regulation in the eye. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy has made impressive advances in improving the outcome of patients affected by malignant diseases. Nonetheless, some limitations still need to be tackled to more efficiently and safely treat patients, in particular for those affected by solid tumors. One of the limitations is related to the immunosuppressive tumor microenvironment (TME), which impairs antitumor immunity. Efforts to identify targets able to turn the TME into a milieu more auspicious to current immuno-oncotherapy is a real challenge due to the high redundancy of the mechanisms involved. However, the insulin-like growth factor 1 receptor (IGF1R), an attractive drug target for cancer therapy, is emerging as an important immunomodulator and regulator of key immune cell functions. Here, after briefly summarizing the IGF1R signaling pathway in cancer, we review its role in regulating immune cells function and activity, and discuss IGF1R as a promising target to improve anti-cancer immunotherapy. [ABSTRACT FROM AUTHOR]

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8-, and TCR1lowCD8- subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8- subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted g and d chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the d chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent g-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the g and d chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function. [ABSTRACT FROM AUTHOR]

Intrauterine fetal demise (IUFD) – fetal loss after 20 weeks – affects 6 pregnancies per 1,000 live births in the United States, and the majority are of unknown etiology. Maternal systemic regulatory T cell (Treg) deficits have been implicated in fetal loss, but whether mucosal immune cells at the maternal-fetal interface contribute to fetal loss is under-explored. We hypothesized that the immune cell composition and function of the uterine mucosa would contribute to the pathogenesis of IUFD. To investigate local immune mechanisms of IUFD, we used the CBA mouse strain, which naturally has mid-late gestation fetal loss. We performed a Treg adoptive transfer and interrogated both pregnancy outcomes and the impact of systemic maternal Tregs on mucosal immune populations at the maternal-fetal interface. Treg transfer prevented fetal loss and increased an MHC-IIlow population of uterine macrophages. Single-cell RNAsequencing was utilized to precisely evaluate the impact of systemic Tregs on uterine myeloid populations. A population of C1q+, Trem2+, MHC-IIlow uterine macrophages were increased in Treg-recipient mice. The transcriptional signature of this novel uterine macrophage subtype is enriched in multiple studies of human healthy decidual macrophages, suggesting a conserved role for these macrophages in preventing fetal loss. [ABSTRACT FROM AUTHOR]

In recent years, the central role of cell bioenergetics in regulating immune cell function and fate has been recognized, giving rise to the interest in immunometabolism, an area of research focused on the interaction between metabolic regulation and immune function. Thus, early metabolic changes associated with the polarization of macrophages into pro-inflammatory or proresolving cells under different stimuli have been characterized. Tumor-associated macrophages are among the most abundant cells in the tumormicroenvironment; however, it exists an unmet need to study the effect of chemotherapeutics on macrophage immunometabolism. Here, we use a systems biology approach that integrates transcriptomics and metabolomics to unveil the immunometabolic effects of trabectedin (TRB) and lurbinectedin (LUR), two DNA-binding agents with proven antitumor activity. Our results show that TRB and LUR activate human macrophages toward a pro-inflammatory phenotype by inducing a specific metabolic rewiring program that includes ROS production, changes in the mitochondrial inner membrane potential, increased pentose phosphate pathway, lactate release, tricarboxylic acids (TCA) cycle, serine and methylglyoxal pathways in human macrophages. Glutamine, aspartate, histidine, and proline intracellular levels are also decreased, whereas oxygen consumption is reduced. The observed immunometabolic changes explain additional antitumor activities of these compounds and open new avenues to design therapeutic interventions that specifically target the immunometabolic landscape in the treatment of cancer. [ABSTRACT FROM AUTHOR]

Costimulatory receptors on immune cells represent attractive targets for immunotherapy given that these molecules can increase the frequency of individual protective immune cell populations and their longevity, as well as enhance various effector functions. 4-1BB, a member of the TNF receptor superfamily, also known as CD137 and TNFRSF9, is one such molecule that is inducible on several cell types, including T cells and NK cells. Preclinical studies in animal models have validated the notion that stimulating 4-1BB with agonist reagents or its natural ligand could be useful to augment conventional T cell and NK cell immunity to protect against tumor growth and against viral infection. Additionally, stimulating 4-1BB can enhance regulatory T cell function and might be useful in the right context for suppressing autoimmunity. Two human agonist antibodies to 4-1BB have been produced and tested in clinical trials for cancer, with variable results, leading to the production of a wealth of second-generation antibody constructs, including bi- and multi-specifics, with the hope of optimizing activity and selectivity. Here, we review the progress to date in agonism of 4-1BB, discuss the complications in targeting the immune system appropriately to elicit the desired activity, together with challenges in engineering agonists, and highlight the untapped potential of manipulating this molecule in infectious disease and autoimmunity. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy worldwide and is associated with a poor prognosis. Sophisticated molecular mechanisms and biological characteristics need to be explored to gain a better understanding of HCC. The role of metabolites in cancer immunometabolism has been widely recognized as a hallmark of cancer in the tumor microenvironment (TME). Recent studies have focused on metabolites that are derived from carbohydrate, lipid, and protein metabolism, because alterations in these may contribute to HCC progression, ischemia-reperfusion (IR) injury during liver transplantation (LT), and post-LT rejection. Immune cells play a central role in the HCC microenvironment and the duration of IR or rejection. They shape immune responses through metabolite modifications and by engaging in complex crosstalk with tumor cells. A growing number of publications suggest that immune cell functions in the TME are closely linked to metabolic changes. In this review, we summarize recent findings on the primary metabolites in the TME and post-LT metabolism and relate these studies to HCC development, IR injury, and post-LT rejection. Our understanding of aberrant metabolism and metabolite targeting based on regulatory metabolic pathways may provide a novel strategy to enhance immunometabolism manipulation by reprogramming cell metabolism. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising treatment option for several hematologic cancers. However, efforts to achieve the same level of therapeutic success in solid tumors have largely failed mainly due to CAR-T cell exhaustion and poor persistence at the tumor site. Although immunosuppression mediated by augmented programmed cell death protein-1 (PD-1) expression has been proposed to cause CAR-T cell hypofunction and limited clinical efficacy, little is known about the underlying mechanisms and immunological consequences of PD-1 expression on CAR-T cells. With flow cytometry analyses and in vitro and in vivo anti-cancer T cell function assays, we found that both manufactured murine and human CAR-T cell products displayed phenotypic signs of T cell exhaustion and heterogeneous expression levels of PD- 1. Unexpectedly, PD-1high CAR-T cells outperformed PD-1low CAR-T cells in multiple T cell functions both in vitro and in vivo. Despite the achievement of superior persistence at the tumor site in vivo, adoptive transfer of PD-1high CAR-T cells alone failed to control tumor growth. Instead, a PD-1 blockade combination therapy significantly delayed tumor progression in mice infused with PD-1high CAR-T cells. Therefore, our data demonstrate that robust T cell activation during the ex vivo CAR-T cell manufacturing process generates a PD-1high CAR-T cell subset with improved persistence and enhanced anti-cancer functions. However, these cells may be vulnerable to the immunosuppressive microenvironment and require combination with PD-1 inhibition to maximize therapeutic functions in solid tumors. [ABSTRACT FROM AUTHOR]

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M (LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M (0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophagelike cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-β mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells. [ABSTRACT FROM AUTHOR]

Feed accounts for the largest portion (65-70%) of poultry production costs. The feed formulation is generally improved to efficientlymeet the nutritional needs of chickens by reducing the proportion of crude protein (CP) and metabolizable energy (ME) levels in the diet. Although many studies have investigated the production performance during dietary restriction, there is a lack of research on the mechanisms by which immune cell function is altered. This study examined the effects of ME and CP restriction in the chicken diet on serumimmunoglobulins and expression of immune function genes in spleen. Changes in serum immunoglobulins and immune-related gene expression were analyzed in 216 YS-909 broilers fed with 9 different dietary treatments, including experimental treatment diets containing low, standard, and high levels of ME or CP in the diet. At 42 days of age, serum immunoglobulins and expression of spleen immune genes in 6 female chickens selected randomly from each dietary treatment (3×3 factorial arrangement) group were measured by enzyme-linked immunosorbent assay (ELISA) and transcriptomic analysis using RNA sequencing, respectively. The results showed that the IgM level in the low ME group chickens was significantly (p < 0.05) lower than that in other groups. In addition, immune-related genes, such as MX1, USP18, TLR4, IFNG and IL18 were significantly upregulated when the dietary nutrient density was reduced, which may put the body in an inflammatory state. This study provided general information on the molecular mechanism of the spleen immune response to variable nutrient density. [ABSTRACT FROM AUTHOR]

The lymph node is a highly structured organ that mediates the body’s adaptive immune response to antigens and other foreign particles. Central to its function is the distinct spatial assortment of lymphocytes and stromal cells, as well as chemokines that drive the signaling cascades which underpin immune responses. Investigations of lymph node biology were historically explored in vivo in animal models, using technologies that were breakthroughs in their time such as immunofluorescence with monoclonal antibodies, genetic reporters, in vivo two-photon imaging, and, more recently spatial biology techniques. However, new approaches are needed to enable tests of cell behavior and spatiotemporal dynamics under well controlled experimental perturbation, particularly for human immunity. This review presents a suite of technologies, comprising in vitro, ex vivo and in silico models, developed to study the lymph node or its components. We discuss the use of these tools to model cell behaviors in increasing order of complexity, from cell motility, to cell-cell interactions, to organ-level functions such as vaccination. Next, we identify current challenges regarding cell sourcing and culture, real time measurements of lymph node behavior in vivo and tool development for analysis and control of engineered cultures. Finally, we propose new research directions and offer our perspective on the future of this rapidly growing field. We anticipate that this review will be especially beneficial to immunologists looking to expand their toolkit for probing lymph node structure and function. [ABSTRACT FROM AUTHOR]

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages. [ABSTRACT FROM AUTHOR]

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration. [ABSTRACT FROM AUTHOR]

The small nucleolar RNA host genes (SNHGs) are a group of genes that can be transcript into long non-coding RNA SNHG (lncSNHG) and further processed into small nucleolar RNAs (snoRNAs). Although lncSNHGs and snoRNAs are well established to play pivotal roles in tumorigenesis, how lncSNHGs and snoRNAs regulate the immune cell behavior and function to mediate anti-tumor immunity remains further illustrated. Certain immune cell types carry out distinct roles to participate in each step of tumorigenesis. It is particularly important to understand how lncSNHGs and snoRNAs regulate the immune cell function to manipulate anti-tumor immunity. Here, we discuss the expression, mechanism of action, and potential clinical relevance of lncSNHGs and snoRNAs in regulating different types of immune cells that are closely related to anti-tumor immunity. By uncovering the changes and roles of lncSNHGs and snoRNAs in different immune cells, we aim to provide a better understanding of how the transcripts of SNHGs participate in tumorigenesis from an immune perspective. [ABSTRACT FROM AUTHOR]

Exosomes are membranous vesicles actively secreted by almost all cells and they deliver certain intracellular molecules, including nucleic acids, proteins, and lipids, to target cells. They are also considered to be good carriers for drug delivery due to their biocompatibility, high permeability, low immunogenicity, and low toxicity. Exosomes from immune cells were also reported to have immunomodulatory activities. Herein we evaluated the application of exosomes derived from expanded natural killer cells (eNK-EXO) for the treatment of ovarian cancer (OC). We demonstrate that eNK-EXO express typical protein markers of natural killer (NK) cells, can be preferentially uptaken by SKOV3 cells, and display cytotoxicity against OC cells. Furthermore, eNK-EXO loaded with cisplatin could sensitize drugresistant OC cells to the anti-proliferation effect of cisplatin. In addition, we show that eNK-EXO could activate NK cells from immunosuppressive tumor microenvironment, the mechanism of which is explored by transcriptional analysis. In summary, eNK-EXO exhibit anti-tumor activity against OC on its own, could be used to deliver cisplatin and enhance its cytotoxic effect against drug-resistant OC cells and also reverse the immunosuppression of NK cells, which may lead to great prospect of using eNK-EXO in the treatment of OC in the clinic. Our work also builds a strong foundation for further evaluation of eNK-EXO in other solid tumor therapies. [ABSTRACT FROM AUTHOR]

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Rechallenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-ofconcept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken. [ABSTRACT FROM AUTHOR]

Background: Psoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood. Methods: In this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development. Results: By analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice. Conclusions: Overall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells. [ABSTRACT FROM AUTHOR]

T cells rapidly transition from a quiescent state into active proliferation and effector function upon exposure to cognate antigen. These processes are tightly controlled by signal transduction pathways that influence changes in chromatin remodeling, gene transcription, and metabolism, all of which collectively drive specific T cell memory or effector cell development. Dysregulation of any of these events can mediate disease and the past several years has shown unprecedented novel approaches to understand these events, down to the single-cell level. The massive explosion of sequencing approaches to assess the genome and transcriptome at the single cell level has transformed our understanding of T cell activation, developmental potential, and effector function under normal and various disease states. Despite these advances, there remains a significant dearth of information regarding how these events are translated to the protein level. For example, resolution of protein isoforms and/or specific post-translational modifications mediating T cell function remains obscure. The application of proteomics can change that, enabling significant insights into molecular mechanisms that regulate T cell function. However, unlike genomic approaches that have enabled exquisite visualization of T cell dynamics at the mRNA and chromatin level, proteomic approaches, including those at the single-cell level, has significantly lagged. In this review, we describe recent studies that have enabled a better understanding of how protein synthesis and degradation change during T cell activation and acquisition of effector function. We also highlight technical advances and how these could be applied to T cell biology. Finally, we discuss future needs to expand upon our current knowledge of T cell proteomes and disease. [ABSTRACT FROM AUTHOR]

Introduction: In sarcoidosis, peripheral lymphopenia and anergy have been associated with increased inflammation and maladaptive immune activity, likely promoting development of chronic and progressive disease. However, the molecular mechanisms that lead to reduced lymphocyte proportions, particularly CD4+ T-cells, have not been fully elucidated. We posit that paradoxical peripheral lymphopenia is characterized by a dysregulated transcriptomic network associated with cell function and fate that results from altered transcription factor targeting activity. Methods: Messenger RNA-sequencing (mRNA-seq) was performed on peripheral blood mononuclear cells (PBMCs) from ACCESS study subjects with sarcoidosis and matched controls and findings validated on a sarcoidosis case-control cohort and a sarcoidosis case series. Preserved PBMC transcriptomic networks between case-control cohorts were assessed to establish cellular associations with gene modules and define regulatory targeting involved in sarcoidosis immune dysregulation utilizing weighted gene co-expression network analysis and differential transcription factor involvement analysis. Network centrality measures identified master transcriptional regulators of subnetworks related to cell proliferation and death. Predictive models of differential PBMC proportions constructed from ACCESS target gene expression corroborated the relationship between aberrant transcription factor regulatory activity and imputed and clinical PBMC populations in the validation cohorts. Results: We identified two unique and preserved gene modules significantly associated with sarcoidosis immune dysregulation. Strikingly, increased expression of a monocyte-driven, and not a lymphocyte-driven, gene module related to innate immunity and cell death was the best predictor of peripheral CD4+ T-cell proportions. Within the gene network of this monocyte-driven module, TLE3 and CBX8 were determined to be master regulators of the cell death subnetwork. A core gene signature of differentially over-expressed target genes of TLE3 and CBX8 involved in cellular communication and immune response regulation accurately predicted imputed and clinical monocyte expansion and CD4+ T-cell depletion. Conclusions: Altered transcriptional regulation associated with aberrant gene expression of a monocyte-driven transcriptional network likely influences lymphocyte function and survival. Although further investigation is warranted, this indicates that crosstalk between hyperactive monocytes and lymphocytes may instigate peripheral lymphopenia and underlie sarcoidosis immune dysregulation and pathogenesis. Future therapies selectively targeting master regulators, or their targets, may mitigate dysregulated immune processes in sarcoidosis and disease progression. [ABSTRACT FROM AUTHOR]

Upon antigen stimulation and co-stimulation, CD4+ T lymphocytes produce soluble factors that promote the activity of other immune cells against pathogens or modified tissues; this task must be performed in presence of a variety of environmental cytokines, nutrient, and oxygen conditions, which necessarily impact T cell function. The complexity of the early intracellular processes taking place upon lymphocyte stimulation is addressed by means of a mathematical model based on a network that integrates variable microenvironmental conditions with intracellular activating, regulatory, and metabolic signals. Besides the phenotype subsets considered in previous works (Th1, Th2, Th17, and Treg) the model includes the main early events in differentiation to the TFH phenotype. The model describes how cytokines, nutrients and oxygen availability regulate the differentiation of naïve CD4+ T cells into distinct subsets. Particularly, it shows that elevated amounts of an alltype mixture of effector cytokines under optimal nutrient and oxygen availability conduces the system towards a highly-polarized Th1 or Th2 state, while reduced cytokine levels allow the expression of the Th17, Treg or TFH subsets, or even hybrid phenotypes. On the other hand, optimal levels of an alltype cytokine mixture in combination with glutamine or tryptophan restriction implies a shift from Th1 to Th2 expression, while decreased levels of the Th2-inducing cytokine IL-4 leads to the rupture of the Th1-Th2 axis, allowing the manifestation of different (or hybrid) subsets. Modeling proposes that, even under reduced levels of pro-inflammatory cytokines, the sole action of hypoxia boost Th17 expression. [ABSTRACT FROM AUTHOR]

Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver's overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNg responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNg secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas AMSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation. [ABSTRACT FROM AUTHOR]

Cytotoxic CD8 T cells are crucial for the host antigen-specific immune response to viral pathogens. Here we report the identification of an essential role for the serine/arginine-rich splicing factor (SRSF) 1 in CD8 T cell homeostasis and function. Specifically, SRSF1 is necessary for the maintenance of normal CD8 T lymphocyte numbers in the lymphoid compartment, and for the proliferative capacity and cytotoxic function of CD8 T cells. Furthermore, SRSF1 is required for antigen-specific IFN-g cytokine responses in a viral infection challenge in mice. Transcriptomics analyses of Srsf1-deficient T cells reveal that SRSF1 controls proliferation, MAP kinase signaling and IFN signaling pathways. Mechanistically, SRSF1 controls the expression and activity of the Mnk2/p38-MAPK axis at the molecular level. Our findings reveal previously unrecognized roles for SRSF1 in the physiology and function of cytotoxic CD8 T lymphocytes and a potential molecular mechanism in viral immunopathogenesis. [ABSTRACT FROM AUTHOR]

Recently, accumulating evidence has elucidated the important role of T cells with stem-like characteristics in long-term maintenance of T cell responses and better patient outcomes after immunotherapy. The fate of TSL cells has been correlated with many physiological and pathological human processes. In this review, we described present advances demonstrating that stem-like T (TSL) cells are central players in human health and disease. We interpreted the evolutionary characteristics, mechanism and functions of TSL cells. Moreover, we discuss the import role of distinct niches and how they affect the stemness of TSL cells. Furthermore, we also outlined currently available strategies to generate TSL cells and associated affecting factors. Moreover, we summarized implication of TSL cells in therapies in two areas: stemness enhancement for vaccines, ICB, and adoptive T cell therapies, and stemness disruption for autoimmune disorders. [ABSTRACT FROM AUTHOR]

Foxa2, a member of the Forkhead box (Fox) family of transcription factors, plays an Important role in the regulation of lung function and lung tissue homeostasis. FOXA2 expression is reduced in the lung and airways epithelium of asthmatic patients and in mice absence of Foxa2 from the lung epithelium contributes to airway inflammation and goblet cell hyperplasia. Here we demonstrate a novel role for Foxa2 in the regulation of T helper differentiation and investigate its impact on lung inflammation. Conditional deletion of Foxa2 from T-cells led to increased Th2 cytokine secretion and differentiation, but decreased Th1 differentiation and IFN-γ expression in vitro. Induction of mouse allergic airway inflammation resulted in more severe disease in the conditional Foxa2 knockout than in control mice, with increased cellular infiltration to the lung, characterized by the recruitment of eosinophils and basophils, increased mucus production and increased production of Th2 cytokines and serum IgE. Thus, these experiments suggest that Foxa2 expression in T-cells is required to protect against the Th2 inflammatory response in allergic airway inflammation and that Foxa2 is important in T-cells to maintain the balance of effector cell differentiation and function in the lung. [ABSTRACT FROM AUTHOR]

IgM memory B cells, are a peculiar subset of memory B cells, which probably originates in the spleen and outside germinal centers and provide a rapid line of defence against mucosal infections. Their role in counteracting COVID-19 is still elusive but, recent evidence, mainly boosted by studies on spleen function/involvement in COVID-19, seems to support the notion that this subset of memory B cells could exert a protective role against this virus, along with other coronaviruses, particularly in the acute setting of the infection, as outlined by worst clinical outcomes observed in unvaccinated patients with impaired IgM B memory response and spleen function. Herein we critically summarise the current landscape of studies on IgM memory B cells, focusing on the clinical impact of their depletion, by comparing the COVID-19-related splenic dysfunction with other hypo- and asplenic conditions and by adding recent data on follow-up studies and postulate a mechanistic explanation for their reduced numbers. The early detection of an impaired IgM memory B cell response in patients with COVID-19 may contribute to their improved care through different strategies, such as through tailored vaccine strategies, prompt hospital admission and/or administration of anti-infective treatments, thus resulting in an better prognosis, although at present management algorithms are still unavailable. Moreover, further studies with longer follow-up are needed to assess the evolution of COVID-19-associated/exacerbated immune deficit. [ABSTRACT FROM AUTHOR]

The traditional view of the nuclear envelope (NE) was that it represented a relatively inert physical barrier within the cell, whose main purpose was to separate the nucleoplasm from the cytoplasm. However, recent research suggests that this is far from the case, with new and important cellular functions being attributed to this organelle. In this review we describe research suggesting an important contribution of the NE and its constituents in regulating the functions of cells of the innate and adaptive immune system. One of the standout properties of immune cells is their ability to migrate around the body, allowing them to carry out their physiological/pathophysiology cellular role at the appropriate location. This together with the physiological role of the tissue, changes in tissue matrix composition due to disease and aging, and the activation status of the immune cell, all result in immune cells being subjected to different mechanical forces. We report research which suggests that the NE may be an important sensor/transducer of these mechanical signals and propose that the NE is an integrator of both mechanical and chemical signals, allowing the cells of the innate immune system to precisely regulate gene transcription and functionality. By presenting this overview we hope to stimulate the interests of researchers into this often-overlooked organelle and propose it should join the ranks of mitochondria and phagosome, which are important organelles contributing to immune cell function. [ABSTRACT FROM AUTHOR]

Macrophages are a population of immune cells functioning in antigen presentation and inflammatory response. Research has demonstrated that macrophages belong to a cell lineage with strong plasticity and heterogeneity and can be polarized into different phenotypes under different microenvironments or stimuli. Many macrophages can be recruited by various cytokines secreted by adipose tissue. The recruited macrophages further secrete various inflammatory factors to act on adipocytes, and the interaction between the two leads to chronic inflammation. Previous studies have indicated that adipose tissue macrophages (ATMs) are closely related to metabolic diseases like obesity and diabetes. Here, we will not only conclude the current progress of factors affecting the polarization of adipose tissue macrophages but also elucidate the relationship between ATMs and human diseases. Furthermore, we will highlight its potential in preventing and treating metabolic diseases as immunotherapy targets. [ABSTRACT FROM AUTHOR]

Iguratimod (IGU) is a novel disease modified anti-rheumatic drug, which has been found to act directly on B cells for inhibiting the production of antibodies in rheumatoid arthritis (RA) patients. Follicular helper T (Tfh) cells, a key T cell subsets in supporting B cell differentiation and antibody production, have been shown to play critical roles in RA. However, whether IGU can inhibit RA Tfh cells which further restrains B cell function remains unclear. Here, we aimed to explore the roles of IGU in regulating RA circulating Tfh (cTfh) cell function and investigate the potential mechanism associated with cell glucose metabolism. In our study, we found that IGU could act on RA-CD4+ T cells to reduce T cell-dependent antibody production. IGU decreased the percentage of RA cTfh cells and the expression of Tfh cell-related molecules and cytokines which were involved in B cell functions. Importantly, our data showed that IGU significantly restrained the cTfh cell function by inhibiting glucose metabolism, which relied on Hif1α-HK2 axis. In summary, we clarified a new target and mechanism of IGU by restraining RA cTfh cell function via inhibiting Hif1α-HK2-glucose metabolism axis. Our study demonstrates the potential application of IGU in the treatment of diseases related to abnormal metabolism and function of Tfh cells. [ABSTRACT FROM AUTHOR]

The nutritional status of dairy cows and the metabolism of specific nutrients are critical regulators of immune cell function. Around the time of parturition, mobilization of body lipid and muscle helps compensate for the decrease in nutrient intake and the increased requirements of the mammary gland for lactation. An end-result of these processes is the marked increase in circulating concentrations of fatty acids (FA), which are a major risk factor for immune dysfunction. In food animal species such as dairy cows, any disturbance in nutritional or immunological homeostasis leads to deleterious feedback loops that can further risk health, efficiency of nutrient use, and compromise availability of safe and nutritious dairy foods for humans. Despite substantial progress with respect to regulation of innate immunity, such knowledge for adaptive immunity is scarce. To help bridge this gap in knowledge, we sought to study the role of calcium release-activated calcium modulator ORAI1 activation in T cells systemic immune function in vivo. CD4+ T cells were isolated from peripheral blood of dairy cows diagnosed as healthy or with ketosis, a common metabolic disorder of FA metabolism. Results revealed that levels of intracellular Ca2+ and reactive oxygen species (ROS) along with the abundance of store-operated Ca2+ entry (SOCE) moiety increased during ketosis. Further, plasma concentrations of inflammatory cytokines were elevated, the balance of Th17/Treg cells was disrupted, mitochondrial function impaired, and the abundance of mitophagy-related proteins in CD4+ T cells altered during ketosis. Molecular characterization of the direct effects of FA was evaluated in CD4+ T cells isolated from the spleen of 1-day-old calves. Enhanced supply of FA increased intracellular Ca2+ and ROS concentrations, upregulated the abundance of proteins associated with mitochondrial dynamics and ORAI1. Intermediates of mitophagy accumulated and the balance of Th17/Treg cells also was affected by the supply of FA. These negative effects were attenuated by silencing or inhibition of ORAI1 in CD4+ T cells. Together, data indicated that physiological states that lead to increases in systemic concentrations of FA could impact adaptive immunity negatively through ORAI1 regulated intracellular Ca2+, ROS balance, and increased effector functions of Th17 cells. [ABSTRACT FROM AUTHOR]

In early lactation, an energy deficit leading to a negative energy balance (NEB) is associated with increased susceptibility to disease and has been shown to be an important factor during transition in dairy cows. L-carnitine as a key factor in the mitochondrial transport of fatty acids and subsequently for β-oxidation and energy release is known to modulate mitochondrial biogenesis and thus influence metabolism and immune system. In the current study, we characterized hematological changes around parturition and investigated the potential effects of dietary L-carnitine supplementation on immune cell functions. For this approach, dairy cows were assigned either to a control (CON, n = 30) or an L-carnitine group [CAR, n = 29, 25 g rumen-protected L-carnitine per cow and day (d)]. Blood samples were taken from d 42 ante partum (ap) until d 110 post-partum (pp), with special focus and frequent sampling from 0.5 to72 h post-calving to clarify the impact of L-carnitine supplementation on leukocyte count, formation of reactive oxygen species (ROS) in polymorphonuclear cells (PMN) and peripheral mononuclear cells (PBMC) and their phagocytosis activity. Blood cortisol concentration and the capacity of PBMC proliferation was also investigated. All populations of leukocytes were changed during the peripartal period, especially granulocytes showed a characteristic increase up to 4 h pp. L-carnitine supplementation resulted in increased levels of eosinophils which was particularly pronounced one day before to 4 h pp , indicating a possible enhanced support for tissue repair and recovery. Non-supplemented cows showed a higher phagocytic activity in PBMC as well as a higher phagocytic capacity of PMN during the most demanding period around parturition, which may relate to a decrease in plasma levels of non-esterified fatty acids reported previously. L-carnitine, on the other hand, led to an increased efficiency to form ROS in stimulated PMN. Finally, a short period around calving proved to be a sensitive period in which L-carnitine administration was effective. [ABSTRACT FROM AUTHOR]

The NF-κB transcription factor family controls the transcription of many genes and regulates a number of pivotal biological processes. Its activity is regulated by the IκB family of proteins. Bcl-3 is an atypical member of the IκB protein family that regulates the activity of nuclear factor NF-κB. It can promote or inhibit the expression of NF-κB target genes according to the received cell type and stimulation, impacting various cell functions, such as proliferation and differentiation, induction of apoptosis and immune response. Bcl-3 is also regarded as an environment-dependent cell response regulator that has dual roles in the development of B cells and the differentiation, survival and proliferation of Th cells. Moreover, it also showed a contradictory role in inflammation. At present, in addition to the work aimed at studying the molecular mechanism of Bcl-3, an increasing number of studies have focused on the effects of Bcl-3 on inflammation, immunity and malignant tumors in vivo. In this review, we focus on the latest progress of Bcl-3 in the regulation of the NF-κB pathway and its extensive physiological role in inflammation and immune cells, which may help to provide new ideas and targets for the early diagnosis or targeted treatment of various inflammatory diseases, immunodeficiency diseases and malignant tumors. [ABSTRACT FROM AUTHOR]

Regulatory B cells (Bregs) have been highlighted in very different pathology settings including autoimmune diseases, allergy, graft rejection, and cancer. Improving tools for the characterization of Bregs has become the main objective especially in humans. Transitional, mature B cells and plasma cells can differentiate into IL-10 producing Bregs in both mice and humans, suggesting that Bregs are not derived from unique precursors but may arise from different competent progenitors at unrestricted development stages. Moreover, in addition to IL-10 production, regulatory B cells used a broad range of suppressing mechanisms to modulate the immune response. Although Bregs have been consistently described in the literature, only a few reports described the molecular aspects that control the acquisition of the regulatory function. In this manuscript, we detailed the latest reports describing the control of IL-10, TGFβ, and GZMB production in different Breg subsets at the molecular level. We focused on the understanding of the role of the transcription factors STAT3 and c-MAF in controlling IL-10 production in murine and human B cells and how these factors may represent an important crossroad of several key drivers of the Breg response. Finally, we provided original data supporting the evidence that MAF is expressed in human IL-10- producing plasmablast and could be induced in vitro following different stimulation cocktails. At steady state, we reported that MAF is expressed in specific human B-cell tonsillar subsets including the IgD+ CD27+ unswitched population, germinal center cells and plasmablast. [ABSTRACT FROM AUTHOR]

Post-transcriptional gene regulation by RNA-binding proteins (RBPs) is important in the prevention of inflammatory and autoimmune diseases. With respect to T cell activation and differentiation, the RBPs Roquin-1/2 and Regnase-1 play pivotal roles by inducing degradation and/or translational silencing of target mRNAs. These targets encode important proinflammatory mediators and thus Roquin and Regnase-1 functions dampen cellular programs that can lead to inflammation and autoimmune disease. Recent findings demonstrate direct physical interaction of both RBPs. Here, we propose that cooperativity of trans -acting factors may be more generally used to reinforce the regulatory impact on selected targets and promote specific cell fate decisions. We develop this concept for Roquin and Regnase-1 function in resting and activated T cells and discuss the involvement in autoimmunity as well as how the therapeutic potential can be used in anti-tumor therapies. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID19) pandemic has left researchers scrambling to identify the humoral immune correlates of protection from COVID-19. To date, the antibody mediated correlates of virus neutralization have been extensively studied. However, the extent that non-neutralizing functions contribute to anti-viral responses are ill defined. In this study, we profiled the anti-spike antibody subtype/subclass responses, along with neutralization and antibody-dependent natural killer cell functions in 83 blood samples collected between 4 and 201 days post-symptoms onset from a cohort of COVID-19 outpatients. We observed heterogeneous humoral responses against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Overall, anti-spike profiles were characterized by a rapid rise of IgA and sustained IgG titers. In addition, strong antibody-mediated natural killer effector responses correlated with milder disease and being female. While higher neutralization profiles were observed in males along with increased severity. These results give an insight into the underlying function of antibodies beyond neutralization and suggest that antibody-mediated natural killer cell activity is a key function of the humoral response against the SARS-CoV-2 spike protein. [ABSTRACT FROM AUTHOR]

The Pellino family is a novel and well-conserved E3 ubiquitin ligase family and consists of Pellino1, Pellino2, and Pellino3. Each family member exhibits a highly conserved structure providing ubiquitin ligase activity without abrogating cell and structure-specific function. In this review, we mainly summarized the crucial roles of the Pellino family in pattern recognition receptor-related signaling pathways: IL-1R signaling, Toll-like signaling, NOD-like signaling, T-cell and B-cell signaling, and cell death-related TNFR signaling. We also summarized the current information of the Pellino family in tumorigenesis, microRNAs, and other phenotypes. Finally, we discussed the outstanding questions of the Pellino family in immunity. [ABSTRACT FROM AUTHOR]

A wealth of innate and adaptive immune cells and hormones are involved in mounting tolerance towards the fetus, a key aspect of successful reproduction. We could recently show that the specific cross talk between the pregnancy hormone progesterone and dendritic cells (DCs) is significantly engaged in the generation of CD4+ FoxP3+ regulatory T (Treg) cells while a disruption led to placental alterations and intra-uterine growth restriction. Apart from progesterone, also glucocorticoids affect immune cell functions. However, their functional relevance in the context of pregnancy still needs clarification. We developed a mouse line with a selective knockout of the glucocorticoid receptor (GR) on DCs, utilizing the cre/flox system. Reproductive outcome and maternal immune and endocrine adaptation of Balb/c-mated C57Bl/6 GRflox/floxCD11ccre/wt (mutant) females was assessed on gestation days (gd) 13.5 and 18.5. Balb/c-mated C57Bl/6 GRwt/wtCD11ccre/wt (wt) females served as controls. The number of implantation and fetal loss rate did not differ between groups. However, we identified a significant increase in fetal weight in fetuses from mutant dams. While the frequencies of CD11c+ cells remained largely similar, a decreased expression of co-stimulatory molecules was observed on DCs of mutant females on gd 13.5, along with higher frequencies of CD4+ and CD8+ Treg cells. Histomorphological and gene expression analysis revealed an increased placental volume and an improved functional placental capacity in mice lacking the GR on CD11c+ DCs. In summary, we here demonstrate that the disrupted communication between GCs and DCs favors a tolerant immune microenvironment and improves placental function and fetal development. [ABSTRACT FROM AUTHOR]

FOXP3 is the master transcription factor in both murine and human FOXP3+ regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein in scurfy mice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenic FOXP3 mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4+ T cells (FOXP3KOGFP CD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KOGFP CD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells. [ABSTRACT FROM AUTHOR]

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The adipokine apelin (APLN) plays critical roles in several cellular functions, including angiogenesis. We report that APLN treatment of RA synovial fibroblasts (RASFs) increased angiopoietin-1 (Ang1) expression. Ang1 antibody abolished endothelial progenitor cell (EPC) tube formation and migration in conditioned medium from APLN-treated RASFs. We also found significantly higher levels of APLN and Ang1 expression in synovial fluid from RA patients compared with those with osteoarthritis. APLN facilitated Ang1-dependent EPC angiogenesis by inhibiting miR-525-5p synthesis via phospholipase C gamma (PLCγ) and protein kinase C alpha (PKCα) signaling. Importantly, infection with APLN shRNA mitigated EPC angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with collagen-induced arthritis. APLN is therefore a novel therapeutic target for RA. [ABSTRACT FROM AUTHOR]

Lipid-derived signaling molecules known as eicosanoids have integral roles in mediating immune and inflammatory processes across metazoans. This includes the function of prostaglandins and their cognate G protein-coupled receptors (GPCRs) to employ their immunological actions. In insects, prostaglandins have been implicated in the regulation of both cellular and humoral immune responses, yet in arthropods of medical importance, studies have been limited. Here, we describe a prostaglandin E2 receptor (Ag PGE2R) in the mosquito Anopheles gambiae and demonstrate that its expression is most abundant in oenocytoid immune cell populations. Through the administration of prostaglandin E2 (PGE2) and AgPGE2R- silencing, we demonstrate that prostaglandin E2 signaling regulates a subset of prophenoloxidases (PPOs) and antimicrobial peptides (AMPs) that are strongly expressed in populations of oenocytoids. We demonstrate that PGE2 signaling via the Ag PGE2R significantly limits both bacterial replication and Plasmodium oocyst survival. Additional experiments establish that PGE2 treatment increases phenoloxidase (PO) activity through the increased expression of PPO1 and PPO3 , genes essential to anti- Plasmodium immune responses that promote oocyst killing. We also provide evidence that the mechanisms of PGE2 signaling are concentration-dependent, where high concentrations of PGE2 promote oenocytoid lysis, negating the protective effects of lower concentrations of PGE2 on anti- Plasmodium immunity. Taken together, our results provide new insights into the role of PGE2 signaling on immune cell function and its contributions to mosquito innate immunity that promote pathogen killing. [ABSTRACT FROM AUTHOR]

There is increasing evidence that the metabolic status of T cells and macrophages is associated with severe phenotypes of chronic inflammation, including allergic inflammation. Metabolic changes in immune cells have a crucial role in their inflammatory or regulatory responses. This notion is reinforced by metabolic diseases influencing global energy metabolism, such as diabetes or obesity, which are known risk factors of severity in inflammatory conditions, due to the metabolic-associated inflammation present in these patients. Since several metabolic pathways are closely tied to T cell and macrophage differentiation, a better understanding of metabolic alterations in immune disorders could help to restore and modulate immune cell functions. This link between energy metabolism and inflammation can be studied employing animal, human or cellular models. Analytical approaches rank from classic immunological studies to integrated analysis of metabolomics, transcriptomics, and proteomics. This review summarizes the main metabolic pathways of the cells involved in the allergic reaction with a focus on T cells and macrophages and describes different models and platforms of analysis used to study the immune system and its relationship with metabolism. [ABSTRACT FROM AUTHOR]

Introduction: CCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren's syndrome (pSS) and indicated to play a role in pSS immunopathology. Here we delineate the CCR9+ Th cell-specific transcriptome to study the molecular dysregulation of these cells in pSS patients. Methods: CCR9+, CXCR5+ and CCR9-CXCR5- Th cells from blood of 7 healthy controls (HC) and 7 pSS patients were FACS sorted and RNA sequencing was performed. Computational analysis was used to identify differentially expressed genes (DEGs), coherent gene expression networks and differentially regulated pathways. Target genes were replicated in additional cohorts. Results: 5131 genes were differentially expressed between CCR9+ and CXCR5+ Th cells; 6493 and 4783 between CCR9+ and CCR9-CXCR5- and between CXCR5+ and CCR9-CXCR5-, respectively. In the CCR9+ Th cell subset 2777 DEGs were identified between HC and pSS patients, 1416 and 1077 in the CXCR5+ and CCR9-CXCR5- subsets, respectively. One gene network was selected based on eigengene expression differences between the Th cell subsets and pathways enriched for genes involved in migration and adhesion, cytokine and chemokine production. Selected DEGs of interest (HOPX, SOX4, ITGAE, ITGA1, NCR3, ABCB1, C3AR1, NT5E, CCR5 and CCL5) from this module were validated and found upregulated in blood CCR9+ Th cells, but were similarly expressed in HC and pSS patients. Increased frequencies of CCR9+ Th cells were shown to express higher levels of CCL5 than CXCR5+ and CCR9-CXCR5- Th cells, with the highest expression confined to effector CCR9+ Th cells. Antigenic triggering and stimulation with IL-7 of the Th cell subsets co-cultured with monocytes strongly induced CCL5 secretion in CCR9+ Th cell cocultures. Additionally, effector CCR9+ Th cells rapidly released CCL5 and secreted the highest CCL5 levels upon stimulation. Conclusion: Transcriptomic analysis of circulating CCR9+ Th cells reveals CCR9-specific pathways involved in effector T cell function equally expressed in pSS patients and HC. Given the increased numbers of CCR9+ Th cells in the blood and inflamed glands of pSS patients and presence of inflammatory stimuli to activate these cells this suggests that CCR9-specific functions, such as cell recruitment upon CCL5 secretion, could significantly contribute to immunopathology in pSS. [ABSTRACT FROM AUTHOR]