Adaptive Immune Function Research Articles

Immunophenotypic analysis of lymphocyte subsets in newborns with biotinidase deficiency.

Biotin is a vital micronutrient that plays a role in metabolic homeostasis and the regulation of innate and adaptive immune system functions. Biotinidase deficiency (BTD) leads to impairment in biotin-dependent immune functions. This study focused on immunophenotypic analysis of lymphocyte subsets in newborns with BTD. A total of 181 (95 female and 86 male; 114 had BTD and 67 were healthy) newborns underwent biotinidase enzyme activity, molecular and lymphocyte immunophenotyping analyses. BTD is classified into four biochemical phenotypes: profound, partial, heterozygous and normal. The following lymphocyte subsets were studied in all participants: total B lymphocyte (CD19), total T lymphocyte (CD3), helper T lymphocyte (CD3/CD4), cytotoxic T lymphocyte (CTL) (CD3/CD8), natural killer T lymphocyte (CD16/56) and a T-lymphocyte activation marker (HLA-DR). The percentages of lymphocyte subsets were similar in newborns with and without BTD. In all newborns with BTD, the mean CD3/CD4 levels were higher in females, while the CD3/CD8 levels were higher in males (P<.001 for each). In female and male newborns, the CD3/CD4 levels were 53.83±9.46 and 16.82±5.19, respectively, and the CD3/CD8 levels were 48.80±8.65 and 21.48±6.02, respectively. A moderate negative correlation was found between CD3/CD4 and CD3/CD8 in female and male newborns (rfemale =-0.488, rmale =-0.574, P<.001). This study showed that although there were no differences in the lymphocyte subsets in newborns with BTD, the CD3/CD4 levels were higher in females, and the CD3/CD8 levels were higher in males. In addition, there was a negative correlation between the CD3/CD4 and CD3/CD8 levels in both genders. Although these results indicate sexual dimorphism between CD3/CD4 and CD3/CD8 levels, whether this dissociation is unique to BTD in newborns is not fully clear.

Sensorimotor dysfunction in a mild mouse model of cortical contusion injury without significant neuronal loss is associated with increases in inflammatory proteins with innate but not adaptive immune functions.

Traumatic brain injury is a leading cause of mortality and morbidity in the United States. Acute trauma to the brain triggers chronic secondary injury mechanisms that contribute to long-term neurological impairment. We have developed a single, unilateral contusion injury model of sensorimotor dysfunction in adult mice. By targeting a topographically defined neurological circuit with a mild impact, we are able to track sustained behavioral deficits in sensorimotor function in the absence of tissue cavitation or neuronal loss in the contused cortex of these mice. Stereological histopathology and multiplex enzyme-linked immunosorbent assay proteomic screening confirm contusion resulted in chronic gliosis and the robust expression of innate immune cytokines and monocyte attractant chemokines IL-1β, IL-5, IL-6, TNFα, CXCL1, CXCL2, CXCL10, CCL2, and CCL3 in the contused cortex. In contrast, the expression of neuroinflammatory proteins with adaptive immune functions was not significantly modulated by injury. Our data support widespread activation of innate but not adaptive immune responses, confirming an association between sensorimotor dysfunction with innate immune activation in the absence of tissue or neuronal loss in our mice.

Comparative evaluation of memory T cells in COVID-19 patients and the predictive role of CD4+CD8+ double positive T lymphocytes as a new marker.

The COVID-19 pandemic has affected the entire world, posing a serious threat to human health. T cells play a critical role in the cellular immune response against viral infections. We aimed to reveal the relationship between T cell subsets and disease severity. 40 COVID-19 patients were randomly recruited in this cross-sectional study. All cases were confirmed by quantitative RT-PCR. Patients were divided into two equivalent groups, one severe and one nonsevere. Clinical, laboratory and flow cytometric data were obtained from both clinical groups and compared. Lymphocyte subsets, CD4+ and CD8+ T cells, memory CD4+ T cells, memory CD8+ T cells, naive CD4+ T cells, effector memory CD4+ T cells, central memory CD4+ T cells, and CD3+CD4+ CD25+ T cells were significantly lower in severe patients. The naive T cell/CD4 + EM T cell ratio, which is an indicator of the differentiation from naive T cells to memory cells, was relatively reduced in severe disease. Peripheral CD4+CD8+ double-positive T cells were notably lower in severe presentations of the disease (median DP T cells 11.12 µL vs 1.95 µL; p< 0.001). As disease severity increases in COVID-19 infection, the number of T cell subsets decreases significantly. Suppression of differentiation from naive T cells to effector memory T cells is the result of severe impairment in adaptive immune functions. Peripheral CD4+CD8+ double-positive T cells were significantly reduced in severe disease presentations and may be a useful marker to predict disease severity.

Analysis of Antimicrobial Activity of Monocytic Myeloid-Derived Suppressor Cells in Infection with Mycobacterium tuberculosis and Human Immunodeficiency Virus.

Myeloid-derived suppressor cells (MDSC) encompass a subset of myeloid cells, which suppress both innate and adaptive immune functions. Since Mycobacterium tuberculosis (M. tuberculosis) can infect these cells, interest has emerged to study the antimicrobial response of MDSC to mycobacteria causing tuberculosis. Reactive oxygen species (ROS) are critical mediators to control intracellular replication of M. tuberculosis and MDSC express high levels of these effector molecules. Here we describe the flow cytometric assessment of total cellular ROS produced by MDSC in response to infection with M. tuberculosis and compare it with the ROS activity of non-MDSC myeloid cells. To further understand the dynamics of host-pathogen interactions, we provide details on methods for measurement of the intracellular replication of M. tuberculosis within MDSC. Of note, these procedures were adopted for primary MDSC and non-MDSC subsets isolated from human immunodeficiency virus (HIV)-uninfected or HIV-infected individuals, in vitro infected with M. tuberculosis to mimic M. tuberculosis mono- or HIV-M. tuberculosis coinfection, respectively.

Adrenergic regulation of immune cell function and inflammation.

The sympathetic nervous system integrates the functions of multiple organ systems by regulating their autonomic physiological activities. The immune system is regulated both locally and systemically by the neurotransmitters epinephrine and norepinephrine secreted by the adrenal gland and local sympathetic neurons. Immune cells respond by activation of adrenergic receptors, primarily the β2-adrenergic receptor, which signal through heterotrimeric G-proteins. Depending upon the cell type, adrenergic signaling regulates a variety of functions in immune cells ranging from cellular migration to cytokine secretion. Furthermore, due to the diurnal oscillation of systemic norepinephrine levels, various immune functions follow a circadian rhythmic pattern. This review will highlight recent advances in our understanding of how the sympathetic nervous system regulates both innate and adaptive immune functions and how this regulation is linked to circadian rhythms.

Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment.

The prenatal period is a critical window for the development of autism spectrum disorder (ASD). The relationship between prenatal nutrients and gestational gene expression in mothers of children later diagnosed with ASD or non‐typical development (Non‐TD) is poorly understood. Maternal blood collected prospectively during pregnancy provides insights into the effects of nutrition, particularly one‐carbon metabolites, on gene pathways and neurodevelopment. Genome‐wide transcriptomes were measured with microarrays in 300 maternal blood samples in Markers of Autism Risk in Babies‐Learning Early Signs. Sixteen different one‐carbon metabolites, including folic acid, betaine, 5′‐methyltretrahydrofolate (5‐MeTHF), and dimethylglycine (DMG) were measured. Differential expression analysis and weighted gene correlation network analysis (WGCNA) were used to compare gene expression between children later diagnosed as typical development (TD), Non‐TD and ASD, and to one‐carbon metabolites. Using differential gene expression analysis, six transcripts (TGR‐AS1, SQSTM1, HLA‐C, and RFESD) were associated with child outcomes (ASD, Non‐TD, and TD) with genome‐wide significance. Genes nominally differentially expressed between ASD and TD significantly overlapped with seven high confidence ASD genes. WGCNA identified co‐expressed gene modules significantly correlated with 5‐MeTHF, folic acid, DMG, and betaine. A module enriched in DNA methylation functions showed a suggestive protective association with folic acid/5‐MeTHF concentrations and ASD risk. Maternal plasma betaine and DMG concentrations were associated with a block of co‐expressed genes enriched for adaptive immune, histone modification, and RNA processing functions. These results suggest that the prenatal maternal blood transcriptome is a sensitive indicator of gestational one‐carbon metabolite status and changes relevant to children's later neurodevelopmental outcomes.Lay SummaryPregnancy is a time when maternal nutrition could interact with genetic risk for autism spectrum disorder. Blood samples collected during pregnancy from mothers who had a prior child with autism were examined for gene expression and nutrient metabolites, then compared to the diagnosis of the child at age three. Expression differences in gene pathways related to the immune system and gene regulation were observed for pregnancies of children with autism and non‐typical neurodevelopment and were associated with maternal nutrients.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Injecting Immunosuppressive M2 Macrophages Alleviates the Symptoms of Periodontitis in Mice.

Periodontitis is the second most common oral disease affecting tooth-supporting structures. The tissue damage is mainly initiated by the excessive secretion of proinflammatory cytokines by immune cells. Macrophages are a type of antigen-presenting cells that influence the adaptive immunity function. We used a unique set of cytokines, i.e., a combination of IL-4, IL-13, and IL-10, to stimulate macrophages into a subset of M2 polarization cells that express much higher levels of ARG-1, CD206, and PDL-2 genes. The cells’ anti-inflammatory potential was tested with mixed-lymphocyte reaction assay, which showed that this subset of macrophages could increase IL-2 secretion and suppress IL-17, IL-6, and TNF-α secretion by splenocytes. The gram-negative bacterial species Porphyromonas gingivalis was used to initiate an inflammatory process in murine periodontal tissues. In the meantime, cell injection therapy was used to dampen the excessive immune reaction and suppress osteoclast differentiation during periodontitis. Maxilla was collected and analyzed for osteoclast formation. The results indicated that mice in the cell injection group exhibited less osteoclast activity within the periodontal ligament region than in the periodontitis group. Moreover, the injection of M2 macrophages sustained the regulatory population ratio. Therefore, the M2 macrophages induced under the stimulation of IL-4, IL-13, and IL-10 combined had tremendous immune modulation ability. Injecting these cells into local periodontal tissue could effectively alleviate the symptom of periodontitis.

From Classical to Unconventional: The Immune Receptors Facilitating Platelet Responses to Infection and Inflammation.

Simple SummaryPlatelets are small and numerous cells, second only to red blood cells in the bloodstream. They have been recognized mainly for their contributions to blood clotting, however, we now know that they also play important roles in our responses to infection and inflammation. In this extensive review, we cover the “classic” platelet receptors involved in blood clotting and “unconventional” immune receptors that platelets possess and how both groups contribute to our immune responses. Platelet receptors can interact with bacteria and affect the behaviors of white blood cells which fight infection. Additionally, these receptors can be involved in autoimmune conditions, when the body mounts an immune response against itself. Continued research on these interactions could lead to development of new treatments against numerous diseases.Platelets have long been recognized for their role in maintaining the balance between hemostasis and thrombosis. While their contributions to blood clotting have been well established, it has been increasingly evident that their roles extend to both innate and adaptive immune functions during infection and inflammation. In this comprehensive review, we describe the various ways in which platelets interact with different microbes and elicit immune responses either directly, or through modulation of leukocyte behaviors.

Failure of CD4 T Cell-Deficient Hosts To Control Chronic NontyphoidalSalmonellaInfection Leads to Exacerbated Inflammation, Chronic Anemia, and Altered Myelopoiesis

Immunocompromised patients are more susceptible to recurrent nontyphoidal Salmonella (NTS) bacteremia. A key manifestation of HIV infection is the loss of CD4 T cells, which are crucial for immunity to Salmonella infection. We characterized the consequences of CD4 T cell depletion in mice where virulent Salmonella establish chronic infection, similar to chronic NTS disease in humans. Salmonella-infected, CD4-depleted 129X1/SvJ mice remained chronically colonized for at least 5 weeks, displaying increased splenomegaly and more severe splenitis than infected mice with CD4 T cells. Mature erythrocytes, immature erythroid cells, and phagocytes accounted for the largest increase in splenic cellularity. Anemia, which is associated with increased mortality in Salmonella-infected humans, was exacerbated by CD4 depletion in infected mice and was accompanied by increased splenic sequestration of erythrocytes and fewer erythropoietic elements in the bone marrow, despite significantly elevated levels of circulating erythropoietin. Splenic sequestration of red blood cells, the appearance of circulating poikilocytes, and elevated proinflammatory cytokines suggest inflammation-induced damage to erythrocytes contributes to anemia and splenic retention of damaged cells in infected animals. Depleting CD4 T cells led to increased myeloid cells in peripheral blood, spleen, and bone marrow, as well as expansion of CD8 T cells, which has been observed in CD4-depleted humans. This work describes a mouse model of Salmonella infection that recapitulates several aspects of human disease and will allow us to investigate the interplay of innate and adaptive immune functions with chronic inflammation, anemia, and susceptibility to Salmonella infection.

A possible application of hinokitiol as a natural zinc ionophore and anti-infective agent for the prevention and treatment of COVID-19 and viral infections

Zinc and the combination with zinc ionophore have been reported in basic research and several clinical investigations as a potentially viable and economical preventive and therapeutic options for COVID-19 treatment. Zinc is a vital microelement that actively supports respiratory epithelium barrier integrity, innate and adaptive immune functions, and inflammatory regulations. Moreover, zinc may also prevent viral entry, suppress viral replication, and mitigate the damages due to oxidative stress and hyperinflammatory reaction in patients with respiratory infections. Hinokitiol (β-thujaplicin) is a natural monoterpenoid and is considered as a safe zinc ionophore to help zinc transport into cells. It has been widely used in skin and oral care, and therapeutic products for its potent antiviral, antimicrobial, antifungal, anti-inflammatory, and anticancer applications.The ongoing COVID-19 pandemic and the significant morbidity and mortality exist in the high-risk group of patients associated with other respiratory infections such as influenza, respiratory syncytial virus, and dengue fever. There is an urgent need for the development of inexpensive, safe, and effective therapeutics to prevent and treat these viral infections. Considering that hydroxychloroquine (HCQ), the most studied zinc ionophore drug for COVID-19, is linked to potentially serious side effects, we propose the implementation of hinokitiol as a zinc ionophore and anti-infective agent for the prevention and treatment of COVID-19 and other viral infections.

Galectin-9 regulates HTR8/SVneo function via JNK signaling.

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

The importance of bone marrow and the immune system in driving increases in blood pressure and sympathetic nerve activity in hypertension.

What is the topic of this review? This manuscript provides a review of the current understanding of the role of the sympathetic nervous system in regulation of bone marrow-derived immune cells and the effect that the infiltrating bone marrow cells may have on perpetuation of the sympathetic over-activation in hypertension. What advances does it highlight? We highlight the recent advances in understanding of the neuroimmune interactions both peripherally and centrally as they relate to blood pressure control. The sympathetic nervous system (SNS) plays a crucial role in maintaining physiological homeostasis, in part by regulating, integrating and orchestrating processes between many physiological systems, including the immune system. Sympathetic nerves innervate all primary and secondary immune organs, and all cells of the immune system express β-adrenoreceptors. In turn, immune cells can produce cytokines, chemokines and neurotransmitters capable of modulating neuronal activity and, ultimately, SNS activity. Thus, the essential role of the SNS in the regulation of innate and adaptive immune functions is mediated, in part, via β-adrenoreceptor-induced activation of bone marrow cells by noradrenaline. Interestingly, both central and systemic inflammation are well-established hallmarks of hypertension and its co-morbidities, including an inflammatory process involving the transmigration and infiltration of immune cells into tissues. We propose that physiological states that prolong β-adrenoreceptor activation in bone marrow can disrupt neuroimmune homeostasis and impair communication between the immune system and SNS, leading to immune dysregulation, which, in turn, is sustained via a central mechanism involving neuroinflammation.

Crosstalk Between Platelets and Microbial Pathogens.

Platelets, small anucleate cells circulating in the blood, are critical mediators in haemostasis and thrombosis. Interestingly, recent studies demonstrated that platelets contain both pro-inflammatory and anti-inflammatory molecules, equipping platelets with immunoregulatory function in both innate and adaptive immunity. In the context of infectious diseases, platelets are involved in early detection of invading microorganisms and are actively recruited to sites of infection. Platelets exert their effects on microbial pathogens either by direct binding to eliminate or restrict dissemination, or by shaping the subsequent host immune response. Reciprocally, many invading microbial pathogens can directly or indirectly target host platelets, altering platelet count or/and function. In addition, microbial pathogens can impact the host auto- and alloimmune responses to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. In this review, we discuss the mechanisms that contribute to the bidirectional interactions between platelets and various microbial pathogens, and how these interactions hold relevant implications in the pathogenesis of many infectious diseases. The knowledge obtained from “well-studied” microbes may also help us understand the pathogenesis of emerging microbes, such as SARS-CoV-2 coronavirus.

Viral Evasion of the Complement System and Its Importance for Vaccines and Therapeutics.

The complement system is a key component of innate immunity which readily responds to invading microorganisms. Activation of the complement system typically occurs via three main pathways and can induce various antimicrobial effects, including: neutralization of pathogens, regulation of inflammatory responses, promotion of chemotaxis, and enhancement of the adaptive immune response. These can be vital host responses to protect against acute, chronic, and recurrent viral infections. Consequently, many viruses (including dengue virus, West Nile virus and Nipah virus) have evolved mechanisms for evasion or dysregulation of the complement system to enhance viral infectivity and even exacerbate disease symptoms. The complement system has multifaceted roles in both innate and adaptive immunity, with both intracellular and extracellular functions, that can be relevant to all stages of viral infection. A better understanding of this virus-host interplay and its contribution to pathogenesis has previously led to: the identification of genetic factors which influence viral infection and disease outcome, the development of novel antivirals, and the production of safer, more effective vaccines. This review will discuss the antiviral effects of the complement system against numerous viruses, the mechanisms employed by these viruses to then evade or manipulate this system, and how these interactions have informed vaccine/therapeutic development. Where relevant, conflicting findings and current research gaps are highlighted to aid future developments in virology and immunology, with potential applications to the current COVID-19 pandemic.

Fc Gamma Receptors and Their Role in Antigen Uptake, Presentation, and T Cell Activation.

The cellular uptake, intracellular processing, and presentation of foreign antigen are crucial processes for eliciting an effective adaptive host response to the majority of pathogens. The effective recognition of antigen by T cells requires that it is first processed and then presented on MHC molecules that are expressed on other cells. A critical step leading to the presentation of antigen is delivering the foreign cargo to an intracellular compartment where the antigen can be processed and loaded onto MHC molecules. Fc-gamma receptors (FcγRs) recognize IgG-coated targets, such as opsonized pathogens or immune complexes (ICs). Cross-linking leads to internalization of the cargo with associated activation of down-stream signaling cascades. FcγRs vary in their affinity for IgG and intracellular trafficking, and therefore have an opportunity to regulate antigen presentation by controlling the shuttling and processing of their cargos. In this way, they critically influence physiological and pathophysiological adaptive immune cell functions. In this review, we will cover the contribution of FcγRs to antigen-presentation with a focus on the intracellular trafficking of IgG-ICs and the pathways that support this function. We will also discuss genetic evidence linking FcγR biology to immune cell activation and autoimmune processes as exemplified by systemic lupus erythematosus (SLE).

The NF-κB Signaling Pathway, the Microbiota, and Gastrointestinal Tumorigenesis: Recent Advances.

Gastrointestinal (GI) cancers, especially gastric cancer and colorectal cancer (CRC), represent a major global health burden. A large population of microorganisms residing in the GI tract regulate physiological processes, such as the immune response, metabolic balance, and homeostasis. Accumulating evidence has revealed the alteration of microbial communities in GI tumorigenesis. Experimental studies in cell lines and animal models showed the functional roles and molecular mechanisms of several bacteria in GI cancers, including Helicobacter pylori in gastric cancer as well as Fusobacterium nucleatum, Escherichia coli, Peptostreptococcus anaerobius, and Bacteroides fragilis in CRC. The transcriptional factor NF-κB plays a crucial role in the host response to microbial infection through orchestrating innate and adaptive immune functions. Moreover, NF-κB activity is linked to GI cancer initiation and development through its induction of chronic inflammation, cellular transformation and proliferation. Here, we provide an overview and discussion of modulation of the NF-κB signaling pathway by microbiota, especially infectious bacteria, in GI tumorigenesis, with a major focus on gastric cancer and CRC.

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39.

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two β (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγin combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγplus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.

Polymorphisms of HLA-B: influences on assembly and immunity.

The major histocompatibility class I (MHC-I) complex functions in innate and adaptive immunity, mediating surveillance of the subcellular environment. In humans, MHC-I heavy chains are encoded by three genes: the human leukocyte antigen (HLA)-A, HLA-B, and HLA-C. These genes are highly polymorphic, which results in the expression, typically, of six different HLA class I (HLA-I) proteins on the cell surface, and the presentation of diverse peptide antigens to CD8+ T cells for broad surveillance against many pathogenic conditions. Recent studies of HLA-B allotypes show that the polymorphisms, not surprisingly, also significantly impact protein folding and assembly pathways. The use of non-canonical assembly routes and the generation of non-canonical HLA-B conformers has consequences for immune receptor interactions and disease therapies.

1675-P: Adipocyte Adaptive Immune Cell Function and Exhaustion Contribute to the Drop in Adipose Tissue (AT) Regulatory T-Cells (Tregs) in Human Obesity

Background: AT Tregs are decreased in obese mice. We have previously shown that preventing this decline improves insulin sensitivity, despite persistent obesity, underscoring a key role for AT Tregs in systemic metabolism. Therefore, we sought to examine AT Treg changes in human subjects and elucidate potential mechanisms responsible for these changes. Methods: We obtained visceral (VAT) and subcutaneous adipose tissue (SAT) at elective surgery in 54 obese and 18 lean subjects, and SAT via liposuction in 15 lean subjects before and after 2 weeks high fat diet (HFD). Results: Obese individuals had 67% less Treg abundance in VAT (p&amp;lt;0.001) and 79% in SAT (p&amp;lt;0.001); Tregs as %CD4+ cells correlated with insulin sensitivity and inversely with BMI. Subjects ingesting a HFD experienced a 50% drop in SAT Tregs (p&amp;lt;0.001), which correlated with decreased insulin sensitivity by clamp. To understand this drop, we next performed gene expression analyses on isolated adipocytes, AT T cells, and VAT Tregs. Obese adipocytes had profound increases in major histocompatibility class II (MHCII) genes, but decreases in fatty acid oxidation and mitochondrial genes, suggesting a functional switch from a primary metabolic to an adaptive immune cell. These changes were associated with decreased AT Treg abundance, and increased insulin resistance and BMI. T cells revealed increased Tbet, a proinflammatory CD4+ Th1 cell marker, and decreased FOXP3, a Treg marker, consistent with our demonstration that human adipocytes can present antigen to activate CD4+ Th1 cells. Obese vs. lean VAT Tregs also had a 3-fold (p&amp;lt;0.05) increase in the T cell exhaustion marker, program cell death protein-1 (PD-1), and a 75% (p&amp;lt;0.05) decrease in CD127, which is attenuated in exhausted T cells. Discussion: Thus, human adipocyte immune cell activity, combined with Treg exhaustion, contributes to the drop in AT Tregs and strongly affects metabolism. Disclosure D. Bradley: None. A.M. Blaszczak: None. A. Smith: None. A.D. Jalilvand: None. V.P. Wright: None. K. Wyne: Advisory Panel; Self; Novo Nordisk Inc. Research Support; Self; Sanofi. S. Noria: None. J. Joseph: None. J.Z. Liu: None. W. Hsueh: None. Funding American Diabetes Association (1-16-ICTS-049 to W.H.); National Institutes of Health (KL2TR001068, R01HL135622)