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Mouse CD1-specific NK1 T cells: Development, Specificity, and Function

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Abstract

NK1 T cells are a specialized population of alpha/beta T cells that coexpress receptors of the NK lineage and have the unique potential to very rapidly secrete large amounts of cytokines, providing early help for effector cells and regulating the Th1 or Th2 differentiation of some immune responses. NK1 T cells express a restricted TCR repertoire made of an invariant TCR alpha chain, V alpha 14-J alpha 281, associated with polyclonal V beta 8, V beta 7, and V beta 2 TCR beta chains. NK1 T cells recognize the products of the conserved family of MHC class I-like CD1 genes, apparently in the absence of foreign antigens. Thus, this novel regulatory pathway, which straddles the innate and the adaptive immune systems, is unique in that its activation may not require associative recognition of antigen. Here, we review the specificity and function of mouse NK1 T cells, and we discuss the relationship of this lineage to mainstream T cells and NK cells.

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... Upon activation, NKT cells can rapidly produce a broad range of cytokines, making them contributors to various pathologies and instrumental in guiding immune responses. 1 Several subsets of NKT cells have been described and were shown to play various roles in normal and disease conditions because they produce different cytokines, and are found in multiple organs. 2 Mouse NKT cells are born from CD4 + CD8 + [doublepositive (DP)] cortical thymocyte progenitors that express a semi-invariant T-cell receptor (TCR) composed of an invariant Va14-Ja18 chain paired with one of three Vb segments (Vb8, 2 or 7). 3,4 These cells are positively selected by glycolipid-presenting nonclassical class I CD1d molecules expressed on neighboring DP cortical thymocytes. 1 After positive selection, NKT cells enter a developmental program distinct from mainstream T cells. ...
... 3,4 These cells are positively selected by glycolipid-presenting nonclassical class I CD1d molecules expressed on neighboring DP cortical thymocytes. 1 After positive selection, NKT cells enter a developmental program distinct from mainstream T cells. This program is driven by the transcription factor promyelocytic leukemia zinc finger (PLZF), which plays a significant role in the acquisition of their specific phenotypic and functional properties. ...
... 6,8,9 The prototypical mouse NKT cell initially described expresses markers typical of the NK lineage such as NK1.1. 1 These cells were shown to produce mainly interferon-c and to play a role in tumor rejection. CD1d tetramers were used to track NKT cells based on their specificity and to show that following selection mature HSA low NKT cells progress through three developmental stages marked by the expression of the surface markers NK1.1 and CD44. ...
Article
Almost 20 years ago, CD1d tetramers were developed to track invariant natural killer T (NKT) cells based on their specificity, and to define developmental steps during which differentiation markers and functional features are progressively acquired from early NKT cell precursor to fully mature NKT cell subsets. Based on these findings, a linear developmental model was proposed and subsequently used by all studies investigating the specific role of factors that control NKT cell development. More recently, based on intracellular staining patterns of lineage-specific transcription factors such as T-bet, GATA-3, promyelocytic leukemia zinc finger and RORγt, a lineage differentiation model was proposed for NKT cell development. Currently, studies on NKT cells development present lineage differentiation model data in addition to the linear maturation model. In the perspective presented here, we discuss current knowledge relating to NKT cell developmental models and particularly focus on the approaches and strategies, some of which appear nebulous, used to define NKT cell developmental stages and subsets.
... Conventional mature HSA high T cells express either CD4 or CD8, whereas mature HSA high NKT cells in mice express only CD4 or neither of these receptors (DN). These cells represent around 0.5% of the cells in the thymus; conventional CD4 and CD8 T cells represent around 15% of thymic cells [2]. NKT cells express a restricted TCR repertoire which contrasts with the polyclonal repertoire of alpha beta (αβ) TCRs expressed on conventional T cells. ...
... The NKT receptor is composed of an invariant Vα14 alpha chain paired with Jα18, Vα14-Jα18, and a variable beta chain essentially consisting of Vβ8, Vβ7, or Vβ2 [3]. Functionally, there are also differences between NKT and conventional T cells; the latter recognize peptides presented by MHCI and MHCII molecules, whereas NKT cells recognize glycolipids presented by the nonclassical MHCI molecule CD1d [2]. The study of NKT cells benefited greatly from the generation of CD1d tetramers and the identification of the NKT cell ligand α-galactosylceramide [4]. ...
... 1. Mature NKT cells express phenotypic and functional features of NK cells, e.g., they express NK1.1 and produce IFN-γ [2]. However, not all mouse strains express the NK1.1 marker (e.g., BALB/c mice). ...
Chapter
Mouse invariant natural killer T (NKT) cells are a subset of T lymphocytes which have been shown to play a significant role in innate and adaptive immune responses. Features of innate responses are attributed to these cells because they can be stimulated simultaneously with the same ligand to produce quickly and in large amount cytokines without prior immunization. Because these characteristics could be exploited for clinical applications, NKT cells have attracted considerable interest. Many studies have investigated the molecular mechanisms through which they are selected and differentiate. These studies are based on developmental models that serve as a scaffold to understand the specific roles played by various factors and to identify checkpoints during cellular development. Analysis of NKT cell precursors at the HSAhigh stage, stage 0, can reveal potential selection defects, whereas analysis of NKT cells at the HSAlow stage can shed light on defects in the maturation/differentiation of the different NKT cell subsets (NKT1, 2, and 17). Unlike HSAlow NKT cell subsets, HSAhigh NKT cell precursors are not accurately identified by flow cytometry because of their extreme rarity. Here, we describe an NKT cell enrichment strategy to identify unambiguously NKT cell precursors at the HSAhigh stage that can be used to assess their distribution and characteristics by multicolor flow cytometry.
... Antigenic peptides are presented by major histocompatibility complex (MHC) molecules widely expressed on professional antigen presenting cells (APCs) including dendritic cell (DCs), macrophages, and B cells (1,2). In contrast to conventional T cell activation, invariant natural killer T (iNKT) cells can recognize lipid or glycolipid antigens presented on CD1d, an MHC class I (MHC-I)-like molecule, and show prolonged cytokine production upon activation (3). CD1d is a cell surface glycoprotein comprising a heavy chain in non-covalent association with a 2-microglobulin light chain. ...
... iNKT cells can be distinguished based on their TCR expression. They share some markers such as CD161 (NK1.1 in mice) and NKR-P1 that are characteristics of natural killer cells (3). iNKT cells can produce a wide variety of cytokines including proinflammatory and anti-inflammatory cytokines with multiple effects on the outcome of immune reactions (5). ...
... WT C57BL/6 mice were purchased from Orient Bio (Seongnamsi, Gyeonggi-do, Korea). C57BL/6 CD1d −/− mice used in this study were provided by Albert Bendelac's lab (3). All animal experiment protocols adapted in this study were approved by the Institutional Animal Care and Use Committee of Korea University (KUIACUC-2018-25). ...
Article
Invariant natural killer T (iNKT) cells are involved in various autoimmune diseases. Although iNKT cells are arthritogenic, transforming growth factor beta (TGFβ)-treated tolerogenic peritoneal macrophages (Tol-pMφ) from wild-type (WT) mice are more tolerogenic than those from CD1d knock-out iNKT cell-deficient mice in a collagen-induced arthritis (CIA) model. The underlying mechanism by which pMφ can act as tolerogenic antigen presenting cells (APCs) is currently unclear. To determine cellular mechanisms underlying CD1d-dependent tolerogenicity of pMφ, in vitro and in vivo characteristics of pMφ were investigated. Unlike dendritic cells or splenic Mφ, pMφ from CD1d+/- mice showed lower expression levels of costimulatory molecule CD86 and produced lower amounts of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared to pMφ from CD1d-deficient mice. In a CIA model of CD1d-deficient mice, adoptively transferred pMφ from WT mice reduced the severity of arthritis. However, pMφ from CD1d-deficient mice were unable to reduce the severity of arthritis. Hence, the tolerogenicity of pMφ is a cell-intrinsic property that is probably conferred by iNKT cells during pMφ development rather than by interactions of pMφ with iNKT cells during antigen presentation to cognate T cells.
... Studies in the literature suggest that Tγδ and NK cells can regulate the differentiation of the Th1 and Th2 responses (62) and thus modulate the development of allergic lung diseases. Among the populations of CD4+ T cells, the Th2 phenotype represents a fundamental population because elimination of this subpopulation by antibodies reduces bronchial hyperreactivity and pulmonary eosinophilic infiltration (53,63). Several studies have shown a crucial role of T lymphocytes and the production of Th2 cytokines in the development of asthma (14,29,57,63). ...
... Among the populations of CD4+ T cells, the Th2 phenotype represents a fundamental population because elimination of this subpopulation by antibodies reduces bronchial hyperreactivity and pulmonary eosinophilic infiltration (53,63). Several studies have shown a crucial role of T lymphocytes and the production of Th2 cytokines in the development of asthma (14,29,57,63). However, the role of B cells in allergic asthma remains undefined, with the exception of the well-known ability of B lymphocytes to produce IgE-specific immunoglobulin after Th2 cell signaling (10,11). ...
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Introduction: Reports have shown that the onset of diabetes mellitus (DM) in patients previously diagnosed with asthma decreases asthmatic symptoms, whereas insulin aggravates asthma. The present study evaluated the modulatory effect of insulin on the development of allergic airway inflammation in diabetic mice. Materials and Methods: To evaluate the effects of relative insulin deficiency, an experimental model of diabetes was induced by a single dose of alloxan (50 mg/kg, i.v.). After 10 days, the mice were sensitized with ovalbumin [OVA, 20 μg and 2 mg of Al(OH)3, i.p.]. A booster immunization was performed 6 days after the first sensitization [20 μg of OVA and 2 mg of Al(OH)3, i.p.]. The OVA challenge (1 mg/mL) was performed by daily nebulization for 7 days. Diabetic animals were treated with multiple doses of neutral protamine Hagedorn (NPH) before each challenge with OVA. The following parameters were measured 24 h after the last challenge: (a) the levels of p38 MAP kinase, ERK 1/2 MAP kinases, JNK, STAT 3, and STAT 6 in lung homogenates; (b) the serum profiles of immunoglobulins IgE and IgG1; (c) the concentrations of cytokines (IL-4, IL-5, IL-10, IL-13, TNF-α, VEGF, TGF-β, and IFN-γ) in lung homogenates; (d) cells recovered from the bronchoalveolar lavage fluid (BALF); (e) the profiles of immune cells in the bone marrow, lung, thymus, and spleen; and (f) pulmonary mechanics using invasive (FlexiVent) and non-invasive (BUXCO) methods. Results: Compared to non-diabetic OVA-challenged mice, OVA-challenged diabetic animals showed decreases in ERK 1 (2-fold), ERK 2 (7-fold), JNK (phosphor-54) (3-fold), JNK/SAPK (9-fold), STAT3 (4-fold), the levels of immunoglobulins, including IgE (1-fold) and IgG1 (3-fold), cytokines, including Th2 profile cytokines such as IL-4 (2-fold), IL-5 (2-fold), IL-13 (4-fold), TNF-α (2-fold), VEGF (2-fold), and TGF-β (2-fold), inflammatory infiltrates (14-fold), T cells, NK cells, B cells and eosinophils in the bone marrow, lung, thymus and spleen, and airway hyperreactivity. STAT6 was absent, and no eosinophilia was observed in BALF. Insulin treatment restored all parameters. Conclusion: The data suggested that insulin modulates immune cell phenotypes and bronchial hyperresponsiveness in the development of allergic airway inflammation in diabetic mice.
... The precise function of NKT cells is currently unknown, but it is clear that they are able to secrete large amounts of cytokines, including IL-4 and IFNy, very rapidly after exposure to antigen (Yoshimoto et al. 1995). NKT cells express a relatively invariant T cell receptor repertoire, implying that what they recognise is highly conserved (Bendelac et al. 1997, Godfrey et al. 2000. ...
... In mice NKT cells recognise CD Id, which is highly homologous to human C D ld (Bendelac et al. 1995, Park andBendelac 2000). Although it has been suggested that mouse NKT cells recognise CDl in the absence of foreign antigens (Bendelac et al. 1997), there is good evidence that human CD lb and C D lc can present lipid and glycolipid antigens from M. ...
Thesis
Following ligand binding by a wide variety of cytokines and growth factors, a glycosylphosphatidyl inositol phospholipase D (GPI-PLD) cleaves free membrane glycosylphosphatidyl inositol (GPI) to liberate water-soluble inositol phosphoglycan (IPG) second messengers. IPG are released outside cells and mediate many of the immediate metabolic effects of insulin. Many other cytokines and growth factors use IPG to signal, including IL-2, NGF, IGF-1 and ACTH. The composite structure of IPG molecules is known and they consist of hexose, hexosamine, inositol, phosphate and divalent cations. The development of clinical tuberculosis in a susceptible host appears to involve a highly complex interaction between the infecting organism and the host immune system, with much or indeed all the tissue damage characteristic of the disease being immune mediated. Many Mycobacterium tuberculosis colonised individuals do not develop active disease, and there are demonstrable immunological differences between the asymptomatic immune carrier state and the state of active disease in both humans and animal models. Mycobacterium tuberculosis contains PLD activity and contains phosphatidyl inositol linked glycans that could theoretically give rise to IPG like structures. Such glycolipids include lipoarabinomannan (LAM), lipomannan (LM) and phosphoinositol mannoside (PIM). Previously it has been shown in our laboratory and that of our collaborators that two strains of the pathogen M. tuberculosis and a non-pathogen, M. vaccae, contain IPG- like biological activity. This mycobacteria derived material has been provisionally named phospho-oligosaccharide (POS). The aim of this study was to examine the hypothesis that mycobacteria may contain a homologous system to the mammalian GPI/IPG signalling mechanism. Using an established protocol for the purification of mammalian GPI a glycophospholipid (GPL) was isolated from M. tuberculosis and M. vaccae that showed similar characteristics to mammalian GPI. This GPL was a substrate for (glycosyl) phosphatidylinositol phospholipase C (GPI-PLC), contained phosphate, sugar residues and did not contain amino groups. GPL was biologically active in a cell free pyruvate dehydrogenase (PDH) phosphatase activation assay and induced inflammatory mediator release from monocyte/macrophage cells. GPL is distinct from LAM, LM and PIM because it was sensitive to GPI-PLC and incorporated radioactive galactose. POS fractions were isolated from mycobacteria that were biologically active in PDH phosphatase, lipogenesis, cell proliferation and nitric oxide production assays, contained carbohydrate, phosphate (except for one fraction) and amino groups. On the basis of amino group analysis, which revealed that POS contained amino groups and GPL did not, POS is not a cleavage product of mycobacterial GPL, as is the case with GPI and IPG in mammalian cells. However, there were interesting similarities in biological activity between GPL and POS. Serological studies were undertaken to characterise mycobacterial POS both structurally and immunologically. Although POS exhibit biological activity in systems where mammalian IPG are also active, there are clearly structural differences between POS and IPG because some antibodies that bind IPG do not bind POS. POS are immunogenic: administration of complete Freund's adjuvant induced an anti-POS antibody response in rabbits (but not in mice) and sera from tuberculosis patients contained significantly higher levels of anti-POS antibody than healthy controls. POS is proliferogenic for human peripheral blood mononuclear cells in vitro, in particular, POS activates human B cells in vitro causing a greater increase in B cell CD25 expression than T cell CD25 expression. Thus the material described has some interesting immunological properties, although its role in the pathogenesis of tuberculosis remains to be determined. The growth factors IL-2 and insulin have both been shown to signal using IPG cleaved from GPI, it is therefore intriguing that mycobacteria appear to contain a homologue of an immunologically relevant mammalian second messenger.
... NK and NKT cells are two key lymphocyte subsets in innate immunity. Although they share many common phenotypes and functions during their antitumor and pathogen-infectious responses, they are different based on their distinct lineage development, antigen recognition, tissue distribution, and unique regulatory mechanisms in both physiological and pathological conditions [8][9][10]. NK cells are a subset of cytotoxic innate lymphoid cell regulated by activation and inhibition signals from membrane receptors [11]. ...
... In addition, levels of NKG2A in NKT cells in LNs were decreased from the tumor-bearing mice compared with tumor-free mice (Fig. 3A). The liver is a specific organ for NKT cells [9,10]. We found that NKT cells in livers from tumor-berating mice were upregulated NKG2A but no changes of NKG2D and NKp46 compared with those in the tumor-free mice. ...
Article
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Natural killer (NK) and natural killer T (NKT) cells are two important cell subsets of the innate immune system. NK and NKT cells share many phenotypes and functions for anti-tumor immunity; however, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here we report that NK and NKT cells have distinct properties, metabolic profiles, and functions during tumor development. Using the mouse E0771 breast cancer and B16 melanoma models, we found that both NK and NKT cells are dynamically involved in the immune responses to cancer but have distinct distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, both NK and NKT cells exhibit effector properties. In the later cancer stages, NK and NKT cells have impaired cytotoxic capacities and dysfunctional states. NK cells become senescent cells, while NKT cells, other than invariant NKT (iNKT) cells, are exhausted in the advanced cancers. In contrast, iNKT cells develop increases in activation and effector function within the breast tumor microenvironment. In addition, senescent NK cells have heightened glucose and lipid metabolism, but exhausted NKT cells display unbalanced metabolism in tumor microenvironments of both breast cancer and melanoma tumor models. These studies provide a better understanding of the dynamic and distinct functional roles of NK and NKT cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK and NKT cells for cancer treatment.
... NKT cell development occurs in the thymus, where they acquire effector functions as they mature [1][2][3][4]. In mice, NKT cells are quite enriched within the T lymphocyte compartments of liver and adipose tissues while lower percentages are found in spleen, blood and bone marrow. ...
... Mice deficient of PLZF are almost devoid of NKT cells while the CD4 T cells in T cell-specific PLZF transgenic mice show memory and effector program similar to NKT cells [5,6]. NKT cells are CD1d-restricted in that they recognize lipid antigens presented by CD1d, an MHC-I-like antigen-presenting molecule [2]. NKT cells are known to particularly recognize and react to a glycosphingolipid a-galactoceramide (aGalCer), presented by CD1d, as such, CD1d-aGalCer tetramers are used to stain and recognize NKT cells. ...
Article
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Type I or invariant natural killer T cells belong to a unique lineage of innate T cells, which express markers of both T lymphocytes and NK cells, namely T cell receptor (TCR) and NK1.1 (CD161C), respectively. Thus, apart from direct killing of target cells like NK cells, and they also produce a myriad of cytokines which modulate the adaptive immune responses. Unlike traditional T cells which carry a conventional αβ TCR, NKT cells express semi-invariant TCR – Vα14-Jα18, coupled with Vβ8, Vβ7 and Vβ2 in mice. In humans, the invariant TCR is composed of Vα24-Jα18, coupled with Vβ11.
... The Journal of Immunology, 2021, 207: 809823. I nvariant NKT (iNKT) cell subsets are a unique regulatory ab T cell population selected by self-lipid Ags presented by the nonclassical MHC molecule CD1d (1). Stimulation of NKT cells by a-galactosylceramide (a-GalCer), a synthetic CD1d ligand recognized by all NKT cell subsets, triggers the release of multiple cytokines; the specific mixture depends on the NKT cell subset (2). ...
Article
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The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.
... Previous studies have shown that splenic lymphocyte has an important role during the process of cell-mediated immunity based on the CD4 + , CD8 + , and invariant natural killer T (iNKT) cell [27][28][29]. The iNKT cells are a distinct population of innate T lymphocytes selected in the thymus [30,31]. They have an important function of rapidly secreting cytokines and an innate T cell population capable of activating and steering adaptive immune responses [28]. ...
Article
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The purpose of this study was to investigate whether the Dictyophora echinovolvata spore polysaccharides (DESP) affect the immunity in immunocompromised mice induced by cyclophosphamide (CTX). The healthy female Kunming mice were randomly divided into six groups, including a normal control (NC) group, a positive control group, a model control (MC) group, and three groups treated with low-, intermediate-, and high-dose polysaccharide, respectively. A series of immunoregulatory properties were determined, including humoral and cellular immunity, immune function, and immune factors of mononuclear macrophages. Compared with NC and MC groups, treatment with DESP significantly increased the spleen index and decreased the thymus index; increased the serum concentrations of immunoglobulin (Ig)A, IgG, IgM, hemolysin, IL-1β, and IL-2; delayed the allergic reaction; and improved the splenic lymphocyte transformation ability; and enhanced the phagocytosis of macrophages and the ability to secrete IL-6, TNF-α, caspase-1, and NO with DESP supplementation. These results indicated that DESP might have a good regulatory effect on CTX-induced immunodeficiency in mice, adjust the body’s immune imbalance, and improve the symptoms of low immunity.
... Invariant natural killer T cells (iNKT) cell subsets are a unique regulatory αβ T-cell population selected by self-lipid antigens presented by the nonclassical MHC molecule CD1d [1]. Stimulation of iNKT cells by α-galactosylceramide (αGC), a synthetic CD1dligand recognized unanimously by all iNKT cell subsets, results in the release of various cytokines depending on the iNKT cell subset [2]. ...
Article
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It is established that iNKT cells are a cell type that require strong T cell receptor (TCR) signal for their proper development and represent a model for thymic agonist selection. The nature of the signal perceived by iNKT cells promoting their specification is not well understood. To address this question we analyzed iNKT cell development in relevant TCR Vα14‐Jα18 alpha chain transgenic mice (Vα14Tg). In CD4‐Vα14Tg mice, where the transgene is driven by CD4 promoter, we identified a block in iNKT cell development at early developmental stages due to a reduced expression of key transcription factors accompanied with a reduced TCR expression levels. This indicates that TCR signal strength control iNKT cell differentiation. Importantly, we found in wild type mice that early precursors of iNKT cells express higher TCR levels compared to positively selected precursors of mainstream T cells showing that TCR levels could contribute to the strength of iNKT cell TCR signaling. Overall, our study highlights TCR signal strength associated with a higher TCR density as an important regulator of iNKT cell lineage specification. This article is protected by copyright. All rights reserved
... Although iNKTs develop from the same precursor, their development is different from classical T cells, owing to their inherent autoreactivity and hybrid expression of an ab TCR as well as the inhibitory MHC specific NK receptors (161). Thymocyte progression to DP status appears similar between classical and iNKT cells, and generation of the invariant iNKT TCR seems to be random, where no preferential joining of the Va14 to Ja18 occurs in mice (162). ...
Thesis
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Mycobacterium tuberculosis (Mtb) continues to cause a global health pandemic and the emergence of new drug-resistant strains requires new therapeutic interventions. Mtb is characterised by a lipid-rich cell wall, thus T cells that respond to lipid antigens are likely to play a critical role in host immunity. I investigate the hypothesis that lipid-responsive CD1b- and CD1drestricted T cells may play critical roles in host immunity to tuberculosis (TB). Invariant natural killer T cells (iNKTs) are a CD1d restricted T cell population found in humans and macaques and previous in vitro and in vivo studies have identified their key anti-Mtb roles. I focus on the macaque model of Mtb infection, due to their accurate recapitulation of human disease, to identify iNKT correlates of protection. Using validated methods to identify iNKTs, and systematic analyses across four genetically distinct groups, I first demonstrate that peripheral blood iNKT frequency associates with disease susceptibility. I also observe skewed iNKT CD4+ /CD8+ subsets in animals more susceptible to Mtb. Furthermore, I demonstrate that CD8+ iNKTs associate with better disease outcome in both the Chinese cynomolgus and Indian Rhesus macaque. My results warrant further investigation into the mechanisms behind the observed iNKT based correlates in macaque TB infection. CD1b, the second lipid presenting molecule that I have focused on, is expressed on the surface of professional antigen presenting cells (APCs) and I show for the first time its expression in the human lung granuloma. It presents mycolic acids (MA) from the Mtb cell wall to Germ-line-Encoded-Mycolyl lipid specific T cells (GEMs). I use multiple in vitro cellular models to study the structure-function relationships of a large panel (>20) of synthetic Mtb MAs, representative of the major families of MAs in the Mtb cell wall. My results demonstrate that structurally diverse MA lipid tails vary in their ability to stimulate GEM T cells in a mechanism which is independent of antigen processing. The mycolic acid antigenic potential is dictated by the type of meromycolate tail functional group and relative stereochemistry. In silico molecular modelling of MAs onto CD1b support my in vitro observations and suggest that stimulatory MAs form seemingly more stable CD1b lipid complexes. Finally, my observations are further corroborated in ex vivo T cell responses from TB patient clinical samples and increased cytokine responses after stimulation with different mycolic acids. Taken together, our findings delineate immunogenic Mtb lipids which paves the way for lipid based anti-Mtb vaccines and therapeutics targeting GEM T cells.
... Cette sous-population est caractérisée par l'utilisation d'un segment gamma V particulier (Haas et al., 1993). associé à une chaine Vβ8, Vβ7 ou Vβ2) (Bendelac et al., 1995(Bendelac et al., , 1997 Tilloy et al., 1999;. ...
Thesis
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Le surpoids et l’obésité touchent plus de 1,9 milliard d’adultes à travers le monde et pourraient atteindre 3,3 milliards de personnes dans une dizaine d’année. L’obésité est associée à une inflammation tissulaire et systémique chronique de bas grade, qui contribuent à l’apparition de la résistance à l’insuline. Récemment, notre laboratoire a mis en évidence des anomalies d’une nouvelle population de lymphocytes T innés, les cellules MAIT (Mucosal Associated Invariant T) chez des patients obèses et/ou ayant un diabète de type 2 (T2D). Les cellules MAIT sont des lymphocytes T non conventionnels, qui expriment un récepteur des cellules T (TCR) avec une chaîne alpha invariante. Leur TCR reconnait la molécule d’histocompatibilité de classe 1 non classique MR1, présentant des métabolites dérivés de la voie de biosynthèse des vitamines B, notamment les vitamines B2 et B9. Dans cette étude, nous utilisons les modèles murins pour analyser le rôle des cellules MAIT dans le developpement du T2D. Au cours de l’obésité induite par un régime riche en graisse, les cellules MAIT du tissu adipeux viscéral (TA) et de l’iléon sont activées de façon précoce et anormale et produisent plus de cytokines pro-inflammatoires (i.e. IL-17, TNFa et l’IFN?). De plus, l’augmentation de la fréquence tissulaire des MAIT chez des souris Va19 transgéniques conduit à l’apparition de la résistance à l’insuline et à une intolérance au glucose, au cours de l’obésité. A l’inverse, les souris obèses déficientes en MAIT, MR1-/-, sont protégées contre ces anomalies métaboliques. Une fréquence élevée de MAIT est associée au changement de macrophages M2 (anti-inflammatoires) en M1 (inflammatoires) et à une infiltration des cellules NK et des LTaß-CD8 au niveau du TA. Par ailleurs, les MAIT contrôlent la fréquence des Treg, ILC2 et ILC3 dans l’iléon et des Treg, ILC2 et éosinophiles dans le tissu adipeux. La modification de la fréquence des ILC2 et ILC3 est associée à la production intestinale d’IL-33 et d’IL-25. De plus, nous montrons que le rôle délétère des MAIT dans le développement du T2D est associé à une dysbiose intestinale. Finalement, des expériences de transfert de flores intestinales montrent que cette dysbiose intestinale est en partie responsable des anomalies immunitaires et métaboliques.
... NKT cells are subdivided into three clusters; iNKT cells (type I), diverse NKT cells (type II), and NKT-like cells . The TCR in iNKT cells has a semi-invariant TCRα chain and a restricted β-chain-Vα14Jα18 in the mouse and Vα24Jα18 in humans (Bendelac et al. 1997;Benlagha et al. 2000;Matsuda et al. 2000). Unlike conventional T cells, which are activated by peptides, iNKT cells recognize lipid antigens loaded on major histocompatibility complex class I (MHC I)-related protein CD1d through TCR (Beckman et al. 1994;Huh et al. 2018). ...
Article
In obesity, adipose tissue undergoes dynamic remodeling processes such as adipocyte hypertrophy, hypoxia, immune responses, and adipocyte death. However, whether and how invariant natural killer T (iNKT) cells contribute to adipose tissue remodeling are elusive. In this study, we demonstrate that iNKT cells remove unhealthy adipocytes and stimulate the differentiation of healthy adipocytes. In obese adipose tissue, iNKT cells were abundantly found nearby dead adipocytes. FasL-positive adipose iNKT cells exerted cytotoxic effects to eliminate hypertrophic and pro-inflammatory Fas-positive adipocytes. Furthermore, in vivo adipocyte-lineage tracing mice model showed that activation of iNKT cells by alpha-galactosylceramide promoted adipocyte turnover, eventually leading to potentiation of the insulin-dependent glucose uptake ability in adipose tissue. Collectively, our data propose a novel role of adipose iNKT cells in the regulation of adipocyte turnover in obesity.
... Accumulating several lines of evidences which obtained from the late 80's to 90's had been integrated into the establishment of novel immune cell lineage 'NKT cells' (Bendelac et al., 1997;Bendelac et al., 2007;Godfrey et al., 2004;Godfrey et al., 2010;Macdonald., 2007;Taniguchi et al., 2003;Terabe. & Berzofsky, 2008). ...
... We finally investigated the sources of endogenous FasL in our scenario of LPS-induced inflammation since various possible sources have been reported depending on the experimental model used (Tsutsui et al., 1999;Takeda et al., 2000). NKT cells are quite abundant in the liver constituting 20-30% of the liver T cells (Bendelac et al., 1997). They have previously been implicated in liver damage during hepatitis (Takeda et al., 2000). ...
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Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1β, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1β induces increased caspase-3 activity but keeps cells alive. We now report that IL-1β and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1β and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1β and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection.
... Natural killer T (NKT) cells share properties of both T and natural killer (NK) cells [6][7][8]. It is possible to distinguish two main NKT cell subsets both reacting to lipid-based antigens presented by the atypical MHC-I (-like) molecule CD1d on antigen presenting cells (APCs) [9,10]. ...
Article
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This review is a comprehensive analysis of the effects of SARS-CoV-2 infection on Unconventional T cells and innate lymphoid cells (ILCs). COVID-19 affected patients show dysregulation of their adaptive immune systems, but many questions remain unsolved on the behavior of Unconventional cells and ILCs during infection, considering their role in maintaining homeostasis in tissue. Therefore, we highlight the differences that exist among the studies in cohorts of patients who in general were categorized considering symptoms and hospitalization. Moreover, we make a critical analysis of the presence of particular clusters of cells that express activation and exhausted markers for each group in order to bring out potential diagnostic factors unconsidered before now. We also focus our attention on studies that take into consideration recovered patients. Indeed, it could be useful to determine Unconventional T cells’ and ILCs’ frequencies and functions in longitudinal studies because it could represent a way to monitor the immune status of SARS-CoV-2-infected subjects. Possible changes in cell frequencies or activation profiles could be potentially useful as prognostic biomarkers and for future therapy. Currently, there are no efficacious therapies for SARS-CoV-2 infection, but deep studies on involvement of Unconventional T cells and ILCs in the pathogenesis of COVID-19 could be promising for targeted therapies.
... NKT cells recognize glycolipid antigens, such as α-galactosylceramide (αGalCer), bridge innate and adaptive immunity and modulate immune responses in autoimmunity, malignancies and infection [2][3][4] . NKT cells can produce large amounts of both Th1 and Th2 cytokines as an immediate response to TCR ligation 5,6 . However, NKT cells have also been shown to display cytotoxic activity, in a mechanism similar to that of NK cells 7 . ...
Article
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Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4−CD8− (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25− (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44−CD25− (DN3) and CD44− CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3− and surface CD3+TCR-β− cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.
... Mouse NKT cells were first described as expressing a semi-invariant Vα14-Jα18/Vβ8,Vβ7,Vβ2 αβ T cell receptor (Vα14-Jα18/Vβ TCR) and NK-lineage markers, such as receptors belonging to the CD94/NKG2 or LY49 families (2). In a few mouse strains such as C57BL/6 where NKT cells have been initially extensively studied, they also express the NK1.1 receptor. ...
Article
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Following positive selection, NKT cell precursors enter an “NK-like” program and progress from an NK– to an NK+ maturational stage to give rise to NKT1 cells. Maturation takes place in the thymus or after emigration of NK– NKT cells to the periphery. In this study, we followed the fate of injected NKT cells at the NK– stage of their development in the thymus of a series of mice with differential CD1d expression. Our results indicate that CD1d-expressing cortical thymocytes, and not epithelial cells, macrophages, or dendritic cells, are necessary and sufficient to promote the maturation of thymic NKT1 cells. Migration out of the thymus of NK– NKT cells occurred in the absence of CD1d expression, however, CD1d expression is required for maturation in peripheral organs. We also found that the natural ligand Isoglobotriosylceramide (iGb3), and the cysteine protease Cathepsin L, both localizing with CD1d in the endosomal compartment and crucial for NKT cell positive selection, are also required for NK– to NK+ NKT cell transition. Overall, our study indicates that the maturational transition of NKT cells require continuous TCR/CD1d interactions and suggest that these interactions occur in the thymic cortex where DP cortical thymocytes are located. We thus concluded that key components necessary for positive selection of NKT cells are also required for subsequent maturation.
... IL-6 stimulates immunoglobulin secretion and it can be produced within a few hours of infection by macrophages after ingestion of bacteria (Fraser et al. 1998;. IFN-y is secreted by effector Thl cells after appropriate peptidergic stimuli in conjunction with MHC class II and leads via macrophage activation to extensive inflammatory processes that also enable the killing of intracellular pathogens (Bendelac et al. 1997;). Mice were administered subcutaneously with PLA nanoparticles prepared by prepared by w/o/w solvent evaporation method with or without alum co encapsulation and 2.5% m/v PVA or 0.75% chitosan as external phase stabilizing agent or free antigen plus alum or free antigen plus chitosan or free antigen. ...
Thesis
Soluble antigens and antigens synthesized by recombinant DNA techniques are weakly immunogenic and need protection against passage through the mucosal epithelium. Encapsulation of antigens into polymeric carriers is one of the techniques developed for delivering these agents. However, this technology needs further improvements to enhance the immune responses and stabilization of the antigen within the carriers. The incorporation of adjuvants to enhance immune responses is well known, and therefore incorporation of adjuvants with the polymeric carriers could also be beneficial. The focus of this study, therefore, was to co-encapsulate known and unknown adjuvants with polymeric particles. Polymeric particulate carriers were prepared using the solvent evaporation or spray drying technique. The biodegradable polymers selected were polylactic acid and polycaprolactone. Diphtheria toxoid (DT) and hepatitis B surface antigen (HBsAg) were used as model antigens. The adjuvants used for co-encapsulation with antigens were penetration enhancers and mineral compounds. These adjuvants were either mixed with the polymer in the organic phase or in the internal phase during the preparation of the particles. The penetration enhancers used in the study were tocopherol acetate (TA), tocopherol nicotinate (TN), D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), cholic acid, lithocholic acid and melittin. Mineral compounds such as alum, zinc sulphate and zinc oxide were also co-encapsulated to evaluate their effect on the physicochemical properties and adjuvancy of the carriers. As surface charge plays an important role in the particle uptake, chitosan was used as an external phase stabilizer in some formulations to impart a positive charge on the particles. The penetration enhancers used here have not been used to co-encapsulate antigens in polymeric particles before, and there is not much data available for co-encapsulation of mineral compounds. The formulations were characterized with respect to size, surface charge, encapsulation efficiency, uptake in the cell lines and toxicity. These formulations were also evaluated in mice for antibody responses and cytokine analysis. Incorporation of tocopherol derivatives into the particulate formulations significantly increased the encapsulation efficiency of the antigen. The in vitro toxicity studies showed that these formulations were non-toxic. In vivo, the HBsAg-loaded particles containing TPGS significantly improved the serum antibody levels when compared with free HBsAg. TA and TN also improved the serum antibody levels when administered intramuscularly and intranasally. However, the intramuscular responses were higher than the intranasally administered formulations. DT-loaded particles containing TPGS significantly improved the antibody levels. Co-encapsulation of zinc sulphate and alum in nanoparticles improved the encapsulation efficiency of HBsAg. Sizes were in the range of 300 to 500nm for these formulations. The serum immune responses of particles containing zinc sulphate were equivalent to particles containing alum. However, the intranasal route elicited much lower antibody responses when compared with the intramuscular groups. Particles coated with chitosan significantly improved the antibody levels. The co-encapsulation of bile salts (cholic acid or lithocholic acid) and melittin along with HBsAg did not have any effect on the encapsulation efficiency. Particles containing bile salts had a higher cellular uptake compared with particles without bile salts. Particles containing bile salts or melittin significantly improved the antibody levels when compared with free antigen and antigen plus cholera toxin B.
... NKT | glucose | OXPHOS | PLZF I nvariant natural killer T (NKT) cells are lymphocytes sharing characteristics of innate and adaptive immune cells and acting as a bridge between the two immune responses. The hallmark of NKT cells is their ability to produce a copious amount of proinflammatory and immune modulatory cytokines very rapidly after antigenic stimulation (1,2). NKT cells are CD1d-restricted T cells that respond to a variety of glycolipids (3). ...
Article
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Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells’ glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function.
... Invariant Natural Killer T (NKT) cells express highly restricted T cell receptor repertoire and share characteristics of both T cells and natural killer (NK) cells (1). NKT cells arise from a common precursor of CD4 + CD8 + double positive thymocytes which have undergone TCR gene rearrangement and expression. ...
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Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for antigen specific activation of T cells, but little is known about what role ROS play in NKT cells. In the current study, we investigated the role of ROS in NKT cell function. We found that ROS levels are similar among CD4, CD8 and NKT cell subsets in the thymus. However, NKT cells, but neither CD4 nor CD8 T cells, showed dramatically increased ROS in the spleen and liver but not in adipose tissues. ROS in the peripheral NKT cells were primarily produced by NADPH oxidases not mitochondria. Accordingly, ROS-high NKT cells were susceptible to oxidative stress and underwent apoptotic cell death. Furthermore, ROS play an important role in regulating the inflammatory function of NKT cells because antioxidant treatment of NKT cells showed reduced frequencies of IFN-γ ⁺ and IL-17 ⁺ cells. In line with this, freshly isolated ROS-high NKT cells had more NKT1 and NKT17 cells but less NKT2 than ROS-low cells. These characteristics are regulated by promyelocytic leukemia zinc finger (PLZF) as evidenced by low ROS in NKT cells from PLZF haplodeficient mice and also from adipose tissues that do not express PLZF. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Together, our study revealed for the first time that ROS regulate NKT cell functions through PLZF.
... We recently demonstrated that ChAcαG produced by H. pylori is presented by the non-classical MHC class Ib molecules CD1d following phagocytosis and processing of the microbes by APCs, and recognized by invariant TCR-bearing NKT cells [62,138]. Murine invariant Vα14-Jα18 TCR + and their human counterpart invariant Vα24-Jα18 TCR + NKT (iNKT) cells are a subset of T lymphocytes [139] that are selected by non-classical MHC class I molecule CD1d in the thymus. These lymphocytes were reported to respond to marine sponge α-galactosylceramide (GalCer) presented by CD1d on APCs [140]. ...
Article
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Steryl glycosides (SGs) are sterols glycosylated at their 3β-hydroxy group. They are widely distributed in plants, algae, and fungi, but are relatively rare in bacteria and animals. Glycosylation of sterols, resulting in important components of the cell membrane SGs, alters their biophysical properties and confers resistance against stress by freezing or heat shock to cells. Besides, many biological functions in animals have been suggested from the observations of SG administration. Recently, cholesteryl glucosides synthesized via the transglycosidation by glucocerebrosidases (GBAs) were found in the central nervous system of animals. Identification of patients with congenital mutations in GBA genes or availability of respective animal models will enable investigation of the function of such endogenously synthesized cholesteryl glycosides by genetic approaches. In addition, mechanisms of the host immune responses against pathogenic bacterial SGs have partially been resolved. This review is focused on the biological functions of SGs in mammals taking into consideration their therapeutic applications in the future.
... In the thymus, IL-15 expression contributes to the development and cytotoxic features of CD8 T cells (13,14), and it is also critical for iNKT cell development and differentiation (46)(47)(48). Based on studies of thymic iNKT cell development, it was assumed that medullary epithelial cells would be the source of intrathymic IL-15, since the medulla is the site of final maturation of iNKT cells which requires IL-15 (49,50). ...
Article
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IL-15 is a cytokine of the common γ-chain family that is critical for natural killer (NK), invariant natural killer T (iNKT), and CD8 memory T cell development and homeostasis. The role of IL-15 in regulating effector T cell subsets, however, remains incompletely understood. IL-15 is mostly expressed by stromal cells, myeloid cells, and dendritic cells (DCs). Whether T cells themselves can express IL-15, and if so, whether such T cell-derived IL-15 could play an autocrine role in T cells are interesting questions that were previously addressed but answered with mixed results. Recently, three independent studies described the generation of IL-15 reporter mice which facilitated the identification of IL-15-producing cells and helped to clarify the role of IL-15 both in vitro and in vivo. Here, we review the findings of these studies and place them in context of recent reports that examined T cell-intrinsic IL-15 expression during CD4 effector T cell differentiation.
... While Metelitsa et al. has reported that the presence of NKTs within the primary tumor site is associated with improved prognosis in patients with NBL [14][15][16]; its prognostic values for MYCN-non-amplified NBL versus MYCNamplified NBL remain to be elucidated. NKTs are CD1d-restricted in that they can be directly cytotoxic against CD1d + cells [17]. Although the majority of human tumors express low levels of CD1d to escape from NKT cytotoxicity, NKTs have been shown to overcome immune evasion by modulation of the TME via inhibition of tumor-associated macrophages (TAMs) [14]. ...
Article
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Immune tumor microenvironment (TME) in neuroblastoma (NBL) contributes to tumor behavior and treatment response. T cells and natural killer (NK) cells have been shown to play important roles in the neuroblastoma TME. However, few reports address the clinical relevance of natural killer T cells (NKTs) and interleukin-15 (IL-15), one of the crucial cytokines controlling the activation and expansion of NK/NKT cells, in NBL. In this study, we examined NKT immunoscores and IL-15 expression in both MYCN-amplified and MYCN-non-amplified NBL to correlate with clinical outcomes such as event-free survival (EFS) and overall survival (OS). From Gene Expression Omnibus (GEO) datasets GSE45480 (n = 643) and GSE49711 (n = 493), we found that NKT immunoscore and IL-15 expression were both significantly lower in MYCN-amplified NBL, and similar results were observed using our clinical NBL samples (n = 53). Moreover, NBL patients (GEO dataset GSE49711 and our clinical samples) with both lower NKT immunoscore and IL-15 expression exhibited decreased EFS and OS regardless of MYCN gene amplification status. Multivariate analysis further showed that the combination of low NKT immunoscore and low IL-15 expression level was an independent prognostic factor for poor EFS and OS in our NBL patients. These findings provide the rationale for the development of strategy to incorporate IL-15 and NKT cell therapy into the treatment regimen for neuroblastoma.
... NKT cells are also efficient regulators of early host responses. NKT cells are a specialized population of T cells that co-express restricted T cell receptors (TCR) and receptors of the NK lineage (Bendelac et al. 1997). It appears that NKT cells do not play a critical role in restricting early acutephase infection but may play a role in helping to activate immune responses that control viral replication at later stages (Van Dommelen et al. 2003). ...
Thesis
Bis heute sind die beiden Ratten-Cytomegalovirus (RCMV)-Isolate RCMV-England (RCMV-E) und RCMV-Berlin (RCMV-B) die einzig bekannten Viren, die ein Homolog von XCL1 kodieren, einem g-Chemokin, das vom Virus kopiert wurde um das Chemokin-Netzwerk des Wirts zu beeinträchtigen. Wie das Wirtshomolog lockt vXCL1 ausschließlich dendritische Zellen (DC) an, die den XC-Chemokinrezeptor 1 (XCR1) exprimieren. In dieser Arbeit wurde untersucht, inwieweit RCMV die XCL1-XCR1-Achse nutzt, um DC zu infizieren und sich im Wirt auszubreiten. In der Ratte konnten zwei DC-Hauptpopulationen identifiziert werden, XCR1+ CD4- und XCR1- CD4+ DC. Es konnte gezeigt werden, dass Überstände von RCMV-infizierten embryonalen Rattenfibroblasten ausschließlich die XCR1+ CD4- Population anlocken. Darüber hinaus konnte nachgewiesen werden, dass RCMV DC infiziert. Durch die Anreicherung wurden DC aktiviert. Während der Infektion inhibierte RCMV die Hochregulation von Reifungsmarkern, einschließlich CD40, CD86 und CCR7. Unabhängig von vXCL1 scheint RCMV die DC-Funktionalität durch das Herunterregulieren von Reifungsgenen zu lähmen. Um die Rolle von XCR1 und die Funktion von vXCL1 in vivo zu analysieren, wurden Xcr1+/+ und Xcr1-/- Ratten mit RCMV-B wt und RCMV-B D-vxcl1 infiziert. Während das XCR1- Expressionsniveau einen Einfluss auf die Geschwindigkeit der RCMV-Verbreitung in den Speicheldrüsen hatte, führte das Fehlen von vXCL1 zu einer starken Abnahme der Virusausbreitung. Die DC-Migration in die Speicheldrüsen war sowohl von vXCL1 als auch von XCR1 abhängig und war reduziert, wenn vXCL1 und XCR1 nicht vorhanden waren. Während der Infektion wurden CD8+ T-Zellen in die Speicheldrüsen rekrutiert. Diese Migration blieb jedoch aus, wenn Xcr1+/+ Ratten mit RCMV-B D-vxcl1 infiziert wurden. Zusammenfassend besitzt RCMV die Fähigkeit DC unabhängig von der vXCL1-Expression zu infizieren. RCMV verwendet vXCL1, um XCR1+ DC anzulocken, was entscheidend für die Virusausbreitung in die Speicheldrüsen zu sein scheint.
... Based on the surface expression, type I can be further subdivided into CD4+, CD8+, and double negative CD4−CD8−(DN) [58][59][60][61]. CD4+ NKT cells generally have a broader repertoire of responses when stimulated with the pro-inflammatory cytokines such as IL-12 or IL-2 [59][60][61]. ...
Article
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Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.
Article
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Lymphocytes are an integral component of the immune system. Classically, all lymphocytes were thought to perpetually recirculate between secondary lymphoid organs and only traffic to non-lymphoid tissues upon activation. In recent years, a diverse family of non-circulating lymphocytes have been identified. These include innate lymphocytes, innate-like T cells and a subset of conventional T cells. Spanning the innate-adaptive spectrum, these tissue-resident lymphocytes carry out specialized functions and cross-talk with other immune cell types to maintain tissue integrity and homeostasis both at the steady state and during pathological conditions. In this review, we provide an overview of the heterogeneous tissue-resident lymphocyte populations, discuss their development, and highlight their functions both in the context of microbial infection and cancer.
Thesis
Tay-Sachs disease (TSD) is an autosomal-recessive genetic disorder which results in the dysfunction of the metabolic enzyme hexosaminidase A (HexA). It leads to severe lysosomal storage of acidic glycosphingolipid, namely ganglioside GM2, and early fatalities for humans with the infantile on-set form. Despite fifty years of research, to date there is no effective treatment beyond palliative care. It was found that mouse models of HexA deficiency display only moderate GM2 accumulation, which was connected with a late onset neuronal phenotype. Therefore neuraminidases were investigated as possible bypass enzymes for the degradation of GM2 and offer a new opportunity for therapeutic approaches in humans. However, to assess the extent of side effects for such a therapeutic bypass, the substrate specificity and ganglioside (GG) turnover has to be defined in detail. This work presents the development of an HILIC based LC MS2 method as well as mass spectrometry imaging (MSI) using DESI (QqQ)MS2 and MALDI TOF to monitor GG pattern changes in mouse brains. The HILIC MS2 analysis of mouse brain tissue with neuraminidase 3 or 4 deficiency in the background of TSD as well as combined knockouts of GG synthesis enzymes revealed an overlapping but distinct substrate processing for the neuraminidases Neu3 and Neu4. MSI of the same tissue samples displayed similar patterns in spatial neural GM2 accumulation that suggest rather a broad distribution of these sialidases in mouse brain. Proposed neuroinflammation and demyelination in mouse brains of TSD led to a modulated HILIC MS2 method with which hexosylceramide isomer separation of GG precursor β-glucosylceramide (β-GlcCer) and prominent myelin sheath component β-galactosylceramide (β-GalCer) was achieved. Decreased levels of β-GalCer as a marker for demyelination in brains of TSD combined with neuraminidase deficiency could not be observed at the age of 6 month. Furthermore, proof of concept study and screening of various WT mouse tissues revealed the adaptability of this method. Even α-anomeric HexCers could be separated from mammalian β-anomers. In contrast to the mentioned β-HexCers, invariant natural killer T cells are activated most effectively when recognizing galactosylceramide with an α-glycosidic linkage appearing on the cell surface receptor CD1d of antigen presenting cells. One natural bacterial source of this compound in contact with our body is Bacteroides fragilis, a bacterial member of the human gut microbiome. This work highlights the detection and separation of α-GalCer(d17:0;h17:0) in B.fragilis and three other bacteria of the human gut microbiome β-HexCers. Very recent preliminary studies indicate the identification of an α-glycosidic GalCer in the mouse microbiome with the proposed structure of BdS α-GalCer(d18:0;h16:0).
Conference Paper
The developed systems for storage of blood and its components are necessary to ensure the availability of these components to the patients. Red blood cell (RBC) components, platelet (PL) components and fresh frozen plasma (FFP) are among the most commonly used in transfusion medicine. During storage of the RBC units in blood banks an accumulation of the storage lesion occurs which can result in the unwanted transfusion-related complications to the recipient. As the red blood cells are stored, they undergo a series of metabolic, oxidative and structural changes. The RBC transfusion are widely used in premature infants, patients in intensive care units and patients with hematological diseases who require multiple transfusions. The current state of knowledge concerning the comparison of the parameters of oxidative stress before and after RBC transfusion in these patients is negligible. Keywords: oxidative stress, red blood cell transfusion, reactive oxygen species, transfusion medicine Opracowane systemy przechowywania krwi i jej składników są niezbędne aby zapewnić dostępność tych składników pacjentom. Do najczęściej stosowanych w transfuzjologii składników krwi należą koncentraty krwinek czerwonych (KKCz), koncentraty krwinek płytkowych (KKP) oraz osocze świeżo mrożone (FFP). Podczas przechowywania KKCz w ośrodkach krwiodawstwa i krwiolecznictwa dochodzi do akumulacji zmian wynikających z czasu przechowywania, które mogą być przyczyną wystąpienia poważnych powikłań poprzetoczeniowych u biorcy. W przechowywanych krwinkach zachodzą zmiany metaboliczne, oksydacyjne i strukturalne. Metody leczenia z użyciem KKCz są powszechnie stosowane u wcześniaków, pacjentów na oddziałach intensywnej terapii oraz u pacjentów ze schorzeniami hematologicznymi, którzy wymagają wielokrotnych przetoczeń. Aktualny stan wiedzy dotyczący porównania parametrów stresu oksydacyjnego przed i po przetoczeniu KKCz w tych grupach pacjentów jest znikomy. Słowa kluczowe: stres oksydacyjny, przetoczenie koncentratu krwinek czerwonych, reaktywne formy tlenu, transfuzjologia
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In this review, we summarize the mechanisms by which sphingolipids modulate virus multiplication and the host innate immune response, using a number of host-virus systems as illustrative models. Sphingolipids exert diverse functions, both at the level of the viral life cycle and in the regulation of antiviral immune responses. Sphingolipids may influence viral replication in three ways: by serving as (co)receptors during viral entry, by modulating virus replication, and by shaping the antiviral immune response. Several studies have demonstrated that sphingosine kinases (SphK) and their product, sphingosine-1-phosphate (S1P), enhance the replication of influenza, measles, and hepatitis B virus (HBV). In contrast, ceramides, particularly S1P and SphK1, influence the expression of type I interferon (INF-I) by modulating upstream antiviral signaling and enhancing dendritic cell maturation, differentiation, and positioning in tissue. The synthetic molecule α-galactosylceramide has also been shown to stimulate natural killer cell activation and IFN-γ secretion. However, to date, clinical trials have failed to demonstrate any clinical benefit for sphingolipids in the treatment of cancer or HBV infection. Taken together, these findings show that sphingolipids play an important and underappreciated role in the control of virus replication and the innate immune response.
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The inability to elicit strong and durable cellular responses is a major obstacle in the development of successful vaccines, in particular those against malaria. In this regard, the generation of novel adjuvants that will potently boost cell-mediated immunity induced by candidate vaccines is helpful. We and others have found a glycolipid, called α-galactosylceramide (α-GalCer), which could be presented on CD1d expressed by antigen-presenting cells (APCs) and stimulate natural killer T (NKT) cells. This triggers the activation/maturation of APCs, particularly dendritic cells (DCs). By activating NKT cells and subsequently DCs, α-GalCer has been shown to enhance adaptive immune responses, particularly of CD8+ T cells, induced by the vaccines. More recently, we identified an analogue of α-GalCer, which can display a potent adjuvant activity in conjunction with malaria vaccines in mice and non-human primates. It is anticipated that CD1d-binding, NKT cell-stimulating glycolipids will be tested as adjuvants in humans in the near future.
Chapter
Coordination of the innate immune and adaptive system during the inflammation stage is essential to successful wound healing. The process of acute wound healing can be divided into three phases: inflammation, proliferation, and remodeling. It is during the first phase, inflammation, that cells of the innate immune system generate an antimicrobial state and mount host defenses, resulting in the removal of pathogens and wound debris. Adaptive immune cells, in particular T cells, generate cytokines, which regulate expression of antimicrobial peptides and proteins and cutaneous immune cells relevant to wound healing. At the end of the inflammation phase, proinflammatory cells give way to anti-inflammatory cells, which leads to the resolution of inflammation and the initiation of repair during the proliferative phase. If proinflammatory signals are overt and resolution of inflammation is dysfunctional, non-healing wounds can develop.
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Interleukin (IL)-4 plays a central role in determining the phenotype of naïve CD4+ T cells by promoting their differentiation into IL-4-producing T helper type 2 (Th2) cells, which are crucial for the induction of allergic inflammation. However, to date, the potential sources of “primary IL-4” in vivo, as distinguished from IL-4 produced by Th2 cells, remain unclear. Here, I describe the research I carried out in collaboration with Dr. William E. Paul to identify “primary IL-4”-producing cells and Th2 cell differentiation in vivo.
Article
Often referred to as the “Swiss Army knife” of the immune system, invariant natural killer T (iNKT) cells are a subpopulation of T lymphocytes stimulated by the synthetic glycolipid α‐galactosylceramide (αGalCer) when in complex with the CD1d receptor of antigen presenting cells. Through their ability to produce T H 1 and T H 2 cytokines and co‐stimulate several other lymphocytes, iNKT cells have emerged as central players in directing the immune response in a range of physiological processes, such as infections, autoimmune diseases, and cancer. Over the years, synthetic chemistry has advanced the field of iNKT cell stimulation with the development of more efficient approaches to prepare αGalCer, and, additionally, with the chemical synthesis of αGalCer analogs in the search of better T H 1/T H 2 cytokine skewing compounds for therapeutic applications. Here we review the strategies for the synthesis of αGalCer and its analogs, including synthetic probes, together with the most important advances in the understanding of the mechanism of action of these compounds, as a guide to the available tools for interrogating the iNKT cell–αGalCer–CD1d complex and inspiration for future research.
Chapter
Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes characterized by the expression of an invariant T-cell antigen receptor rearrangement, and the recognition of glycolipid antigens presented by CD1d, a nonpolymorphic major histocompatibility complex class I-like antigen-presenting molecule. After activation iNKT cells rapidly produce copious amounts of Th1, Th2, and Th17 cytokines. As a consequence of their vigorous and early response, iNKT cells have been implicated in a dazzling variety of different immune reactions, ranging from responses to pathogens and tumors to autoimmune diseases. In this review we will focus on the diverse roles of iNKT cells in autoimmune diseases and will consider the proposed mechanisms whereby they exert their influence.
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Skin is a fundamental component of our host defense system that provides a dynamic physical and chemical barrier against pathogen invasion and environmental insults. Cutaneous barrier function is mediated by complex interactions between structural cells such as keratinocytes and diverse lineages of immune cells. In contrast to the protective role of these intercellular interactions, uncontrolled immune activation can lead to keratinocyte dysfunction and psoriasis, a chronic inflammatory disease affecting 2% of the global population. Despite some differences between human and murine skin, animal models of psoriasiform inflammation have greatly informed clinical approaches to disease. These studies have helped to identify the interleukin (IL)-23-IL-17 axis as a central cytokine network that drives disease. In addition, they have led to the recent description of long-lived, skin-resident innate lymphocyte and lymphoid cells that accumulate in psoriatic lesions. Although not completely defined, these populations have both overlapping and unique functions compared to antigen-restricted αβ T lymphocytes, the latter of which are well-known to contribute to disease pathogenesis. In this review, we describe the diversity of innate lymphocytes and lymphoid cells found in mammalian skin with a special focus on αβ T cells, Natural Killer T cells and Innate Lymphoid cells. In addition, we discuss the effector functions of these unique leukocyte subsets and how each may contribute to different stages of psoriasis. A more complete understanding of these cell types that bridge the innate and adaptive immune system will hopefully lead to more targeted therapies that mitigate or prevent disease progression.
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CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
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A bstract The paired measurement of RNA and surface protein abundance in single cells with CITE-seq is a promising approach to connect transcriptional variation with cell phenotypes and functions. However, each data modality exhibits unique technical biases, making it challenging to conduct a joint analysis and combine these two views into a unified representation of cell state. Here we present Total Variational Inference (totalVI), a framework for the joint probabilistic analysis of paired RNA and protein data from single cells. totalVI probabilistically represents the data as a composite of biological and technical factors such as limited sensitivity of the RNA data, background in the protein data, and batch effects. To evaluate totalVI, we performed CITE-seq on immune cells from murine spleen and lymph nodes with biological replicates and with different antibody panels measuring over 100 surface proteins. With this dataset, we demonstrate that totalVI provides a cohesive solution for common analysis tasks like the integration of datasets with matched or unmatched protein panels, dimensionality reduction, clustering, evaluation of correlations between molecules, and differential expression testing. totalVI enables scalable, end-to-end analysis of paired RNA and protein data from single cells and is available as open-source software.
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It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4-CD8-TCRαβ+, double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5high DN thymocytes that preferentially yield TCRαβ+ CD8αα intestinal lymphocytes, we find that mature CD5low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4+CD8+ (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells.
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The immune system comprises two major arms: the innate immune system, which is activated following the detection of danger molecules by highly conserved receptors, and a second line of defense known as adaptive immunity, which provides an enormous anticipatory repertoire of antigen-specific effector cells and antibodies. The liver is an important immune organ, and liver-derived products can trigger the innate and adaptive immune system to initiate, mediate, regulate, and resolve systemic inflammation. The unique anatomy and microanatomy of the liver enables the generation of distinct immune responses. Generally, the immune responses elicited in the liver are skewed toward tolerance in response to the constant exposure of an enormous antigen load from the gut. An important mechanism leading to liver tolerance is antigen presentation by nonprofessional and/or immature hepatic antigen-presenting cells. Autoimmune diseases occur when a specific adaptive immune response is mounted against self-antigens. A combination of genetic and environmental factors plays a role in the pathogenesis of autoimmune disorders. The development of autoimmunity specifically in the liver, a tolerogenic organ, is a great paradox. The reasons for that are largely obscured currently and hopefully will be uncovered utilizing novel scientific methods in the near future.
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This book “Concise notes in Biochemistry for Physiotherapy and Allied Health Sciences graduates” is a comprehensive yet concise textbook of biochemistry concepts. It primarily targets students pursuing courses in physiotherapy and allied health sciences field having biochemistry in their course but not in-depth. It is suitable for the readers of undergraduate and post-graduate courses in biomedical, paramedical, and allied health sciences such as Nursing, Optometry, MLT, etc. This book is authored in a manner to develop interest among students to facilitate effortless understanding of the subject. Further, the key points of each topic are also projected having a pointwise summary. This book will also provide job seekers of various examinations and interviews with a quick revision of biochemistry at a glance.
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Pharmaceutical Biotechnology is the discipline that encompasses all proficiencies required for conducting research in inventing, developing and registering of biological drugs. Pharma firms apply bioengineering procedures in finding lead compounds and formulating drugs, including- recombinant technology, gene therapy, pharmacogenomics and genetic analysis. Along with that, drug designing is severely dependent on handling and altering organisms, generally at molecular level. All these procedures provide initial inputs in formulating drugs, studying the best administration routes, possible side-effects, release and metabolism of new remedial drugs, mediators for medicinal investigations and in gene therapy for rectifying the signs of heritable disorders. Besides drug development, the pharmaceutical companies also need to deal with many ethical issues in healthcare practices, following strict standards and procedures in conducting laboratory experiments as well as clinical trial on patients and launching of drugs. This chapter is an attempt to provide initial clarity about various procedures required for introducing new products in the market.
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Gastrointestinal (GI) cancers represent a complex array of cancers that affect the digestive system. This includes liver, pancreatic, colon, rectal, anal, gastric, esophageal, intestinal and gallbladder cancer. Patients diagnosed with certain GI cancers typically have low survival rates, so new therapeutic approaches are needed. A potential approach is to harness the potent immunoregulatory properties of natural killer T (NKT) cells which are true T cells, not natural killer (NK) cells, that recognize lipid instead of peptide antigens presented by the non-classical major histocompatibility (MHC) molecule CD1d. The NKT cell subpopulation is known to play a vital role in tumor immunity by bridging innate and adaptive immune responses. In GI cancers, NKT cells can contribute to either antitumor or protumor immunity depending on the cytokine profile expressed and type of cancer. This review discusses the complexities of the role of NKT cells in liver, colon, pancreatic and gastric cancers with an emphasis on type I NKT cells.
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Immune response (Ir) genes, originally proposed by Baruj Benacerraf to explain differential antigen-specific responses in animal models, have become synonymous with the major histocompatibility complex (MHC). We discovered a non-MHC-linked Ir gene in a T cell receptor (TCR) locus that was required for CD8(+) T cell responses to the Plasmodium berghei GAP5040-48 epitope in mice expressing the MHC class I allele H-2D(b). GAP5040-48-specific CD8(+) T cell responses emerged from a very large pool of naive Vβ8.1(+) precursors, which dictated susceptibility to cerebral malaria and conferred protection against recombinant Listeria monocytogenes infection. Structural analysis of a prototypical Vβ8.1(+) TCR-H-2D(b)-GAP5040-48 ternary complex revealed that germline-encoded complementarity-determining region 1β residues present exclusively in the Vβ8.1 segment mediated essential interactions with the GAP5040-48 peptide. Collectively, these findings demonstrated that Vβ8.1 functioned as an Ir gene that was indispensable for immune reactivity against the malaria GAP5040-48 epitope.
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Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long-lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen-specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is amplified in BALB/c as compared to DBA/2, C3H, or C57BL/6 mice whereas tested. These findings support the hypothesis that continuous release of low amounts of protein antigens from pathogenic microorganisms may polarize the immune response toward a Th2 phenotype in susceptible mouse strains.
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A minor subset of immature (CD4-,8-) thymocytes that lack expression of the B2A2 antigen was found to express low levels of surface TCR protein as detected by mAbs F23.1 and KJ16 (reacting with protein products of the V beta 8 gene family). Interestingly, F23.1/KJ16 determinants were expressed on a two- to three-fold higher proportion of B2A2- thymocytes than mature lymph node T cells in four independent haplotypes. When expanded in short-term culture with PMA and calcium ionophore, B2A2- thymocytes retained their overexpression of F23.1/KJ16 determinants and showed a fivefold elevated level (relative to lymph node) of V beta 8-specific mRNA. Taken together, these findings suggest that expression of TCR V beta genes, like Ig genes, is developmentally regulated.
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The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses.
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We demonstrate here the presence of a distinct mature CD4+8- T cell subset in mouse thymus. This subset, termed "Thy0," is delineated by the absence of 3G11 expression from about half of the 6C10-/HSAlow/- fraction of CD4+8- thymic cells. Thy0 is detectable from the neonatal period and largely contributes the Th0-type diverse cytokine production previously reported for the HSAlow/-CD4+ thymic population. Further, cells expressing the T cell receptor V beta 8 gene family are found at increasing frequency in Thy0 with age, comprising 40-60% of Thy0 in adult BALB/c mice. This alteration of V beta 8+ cell frequency is unique to Thy0, since no other CD4+ subset in thymus or spleen shows such V beta 8 overusage. All functional CD4+ T cell subsets, including Thy0, show appropriate V beta clonal deletion associated with endogenous superantigens. Thus, it appears that Thy0 is an intrathymically generated secondary cell subset produced after CD4+ T cell selection.
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In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.
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Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.
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During their differentiation in the mouse thymus, CD4+8- cells undergo several of the sequential changes observed upon normal activation of mature, peripheral CD4+ lymphocytes. Expression of CD69, an early activation marker, is first observed on a minority of cells at the T cell receptor (TCR)lo/med double-positive stage, is maximal (50-90%) on heat-stable antigen (HSA)hi TCRhi double-positive, HSAhi TCRmed CD4+8lo, and HSAhi TCRhi CD4+8- cells, and is downmodulated at the mature HSAlo CD4+8- stage. In contrast, CD44, a late activation marker, is selectively expressed at the HSAlo stage. The set of lymphokines that CD4+8- thymocytes can produce upon stimulation also characteristically expands from mainly interleukin 2 (IL-2) at the HSAhi stage, to IL-2 and very large amounts of IL-4, IL-5, IL-10, and interferon gamma (IFN-gamma) at the HSAlo stage. 1 in 30 HSAlo CD4+8- adult thymocytes secrete IL-4 upon stimulation through their TCR. This frequency is 25% of the frequency of IL-2 producers, about 100-fold above that of peripheral (mainly resting) CD4+ T cells. With time after their generation in organ culture, CD4+8- thymocytes lose their capacity to secrete IL-4, IL-5, and IFN-gamma, but not IL-2. Similarly, the frequency of IL-4, but not of IL-2, producers progressively decreases after emigration to the periphery as judged by direct comparison between thymic and splenic CD4+ cells in newborns, or by following the fate of intrathymically labeled CD4+8- cells in adults after their migration to the spleen. This sequence suggests that thymic selection results from an activation process rather than a simple rescue from death at the double-positive stage, and shows that the functional changes induced after intrathymic activation, although transient, are still evident after export to the periphery.
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In male mice that express a transgenic alpha/beta T cell receptor (TCR) specific for a male-specific peptide presented by class I Db major histocompatibility complex (MHC) molecules, we describe an unusual lineage of alpha/beta T cells that are thymus dependent but do not require selection by Db MHC molecules on thymic epithelium in the absence of the specific peptide (positive selection). These cells express the transgenic alpha/beta TCR and have the CD4-8- or CD4-8low phenotype. Cells with the latter phenotype are only detected when hemopoietic cells express both the male-specific peptide as well as Db MHC molecules. In fact, these cells are autoreactive, as they expand relatively slowly after transfer into male nude mice. Also in male but not female alpha/beta TCR transgenic mice, the CD8+ cells with the transgenic TCR bear the Pgp1 marker characteristic of mature T cells activated by antigen. CD4-8- as well as CD4-8low cells do not respond significantly when cultured with male stimulator cells but proliferate vigorously when stimulated by TCR antibodies. By this latter criterion, cells in the periphery of male alpha/beta TCR transgenic mice differ from mature male-specific T cells from female alpha/beta TCR transgenic, which become intrinsically anergic when transferred into male nude mice and cannot be stimulated significantly by TCR antibodies. Thus, intrathymic deletion does not eliminate all autoreactive T cells and it is possible that cells with an apparently "benign" autoreactivity may be involved in certain forms of autoimmunity.
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A distinctive variable region 14-positive (V14+) alpha chain (V alpha 14+) of the T-cell antigen receptor is predominantly expressed in multiple mouse subspecies. The V alpha 14 family has two members, V alpha 14.1 and V alpha 14.2, which differ by only three amino acids at positions 50-52. Based on the EcoRI restriction fragment length polymorphism of the gene encoding V alpha 14, mice can be divided into three groups: type I with an 11.2-kilobase (kb) fragment, type II with a 2.0-kb fragment, and type III with the 2.0-kb and 11.2-kb fragments. Usage of V alpha 14-J alpha 281, where J alpha 281 is an alpha-chain joining segment, with a one-base N region dominates at the level of 0.02-1.5% of alpha chains in all laboratory strains, Mus musculus castaneus, and Mus musculus domesticus but not in Mus musculus molossinus, Mus musculus musculus, and Mus spicilegus samples. The preferential V alpha 14-J alpha 281 expression seems to be due to positive selection because the V-J junctional region is always glycine, despite the ability of the V alpha 14 gene to associate with J alpha other than J alpha 281. As V alpha 14-J alpha 281 expression is independent of known major histocompatibility complex antigens, including H-2, TLA, Qa, and HMT, the selecting ligand must be a monomorphic molecule of the mouse, expressed in a subspecies-specific manner. Additional observations, such as the expression of homogeneous V alpha 14-J alpha 281 in athymic mice, suggest that the positive selection of V alpha 14+ T cells occurs extrathymically.
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Nucleotide sequences of VJ (variable-joining) junctional regions of V14+ alpha-chain T-cell receptor genes show that most V alpha 14+ T cells use one alpha chain (V alpha 14J alpha 281 with a one-nucleotide N region, which is frequently used in keyhole limpet hemocyanin-specific suppressor T-cell hybridomas) in unprimed mice. Moreover, the frequency of this alpha-chain expression was greater than 1.5% of the total alpha chains found in laboratory strains, including B10 congenic mice. This is about 10(4) times higher than was expected. The V14J281 alpha-chain expression was relatively low but was significant in CD4+/CD8+ immature thymocytes and became quite high in mature single-positive T cells, implying that this alpha chain is selected during T-cell maturation. V14J281 expression increased with time after birth and reached a maximum at around 5 weeks of age. The ligand seems to be a self molecule and to be present in laboratory strains but to be absent in a wild mouse, Mus musculus molossinus, because bone marrow chimeras clearly showed that bone marrow cells derived from Mus musculus molossinus negative for this alpha chain raised V14J281-positive T cells in a C57BL/6 environment. The above results suggest that there are some selection mechanisms for this cell type other than those for conventional alpha beta T cells and also that the homogenous VJ junction of the V14J281 alpha chain plays a pivotal role in the selection of the T cell and its ligand reactivity.
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Human CD1 antigens have a similar tissue distribution and overall structure to (mouse) TL. However recent data from human CD1 suggest that the mouse homologue is not TL. Since no human TL has been conclusively demonstrated, we have analysed the murine CD1 genes. Two closely linked genes are found in a tail to tail orientation and the limited polymorphism found shows that, as in humans, the CD1 genes are not linked to the MHC. Both genes are found to be equally transcribed in the thymus, but differentially in other cell types. The expression in liver, especially, does not parallel CD1 in humans. This demonstrates conclusively that CD1 and TL are distinct and can co-exist in the same thymus. It is paradoxical that despite the structural similarity between mouse and human CD1, the tissue distribution of human CD1 is closer to TL. The possibility of a functional convergence between MHC molecules and CD1 is discussed.
Article
MAJOR histocompatibility complex (MHC) class I and class II molecules bind immunogenic peptides and present them to lymphocytes bearing the alpha beta T-cell antigen receptor (TCR)(1-4). An analogous antigen-presenting function also has been proposed for the non-MHC-encoded CDI molecules: a family of non-polymorphic, beta(2)-microglobulin-associated glycoproteins(5-8) expressed on most professional antigen-presenting cells(9-11). In support of this hypothesis, CD1 molecules are recognized by selected CD4(-)CD8(-)alpha beta or gamma delta TCR(+) T-cell clones(12-14), and we have recently shown that CD1 molecules restrict the recognition of foreign microbial antigens by alpha beta TCR(+) T cells(10). But the substantial structural divergence of CD1 from MHC class I and class II molecules(7), raises the possibility that the antigens presented by the CD1 system may differ fundamentally from those presented by MHC-encoded molecules. Here we report that a purified CD1b-restricted antigen of Mycobacterium tuberculosis presented to alpha beta TCR(+) T cells is mycolic acid, a family of alpha-branched, beta-hydroxy, long-chain fatty acids found in mycobacteria(15-16). This example of non-protein microbial antigen recognition suggests that alpha beta TCR(+) T cells recognize a broader range of antigens than previously appreciated and that at least one member of the CD1 family has evolved the ability to present lipid antigens.
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Natural killer (NK) cells play an important role in immune response by producing interferon gamma (IFN-gamma) as well as exhibiting cytotoxic function. IFN-gamma produced by NK cells has been suggested to be involved in differentiation of T helper cells. On the other hand, the NKR-P1 molecule was recently identified as one of the important NK cell receptors, and it recognizes certain kinds of oligosaccharides on target cells and triggers NK cells for cytotoxicity. In the present study, we found that NK cells produce great amounts of IFN-gamma upon cross-linking of the NKR-P1 molecule. In contrast, stimulation of NK cells with IL-2 induced proliferation without producing IFN-gamma. Similar to NK cells, NK1.1+ T cells also produced IFN-gamma upon NKR-P1 cross-linking. NK1.1+ T cells produced IFN-gamma but not interleukin 4 (IL-4) upon NKR-P1 cross-linking, whereas they secreted both IFN-gamma and IL-4 upon T cell receptor cross-linking. These results indicate that NKR-P1 is a receptor molecule on NK and NK1.1+ T cells that induces not only cytotoxicity but also IFN-gamma production. Our findings provide a new pathway for IFN-gamma production by NK and NK1.1+ T cells through NKR-P1 molecules; it may be essential for immune regulation.
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We demonstrate here the presence of a distinct mature CD4+8- T cell subset in mouse thymus. This subset, termed "Thy0," is delineated by the absence of 3G11 expression from about half of the 6C10-/HSAlow/- fraction of CD4+8- thymic cells. Thy0 is detectable from the neonatal period and largely contributes the Th0-type diverse cytokine production previously reported for the HSAlow/-CD4+ thymic population. Further, cells expressing the T cell receptor V beta 8 gene family are found at increasing frequency in Thy0 with age, comprising 40-60% of Thy0 in adult BALB/c mice. This alteration of V beta 8+ cell frequency is unique to Thy0, since no other CD4+ subset in thymus or spleen shows such V beta 8 overusage. All functional CD4+ T cell subsets, including Thy0, show appropriate V beta clonal deletion associated with endogenous superantigens. Thus, it appears that Thy0 is an intrathymically generated secondary cell subset produced after CD4+ T cell selection.
Article
A comparison of the genes encoding the CD1 leucocyte differentiation antigens in man and mouse shows important differences which prompted us to analyze theCD1 genes of the rabbit. We have found that the rabbit genome contains multipleCD1 loci. Upon cloning and sequencing, one of these loci was found to encode the known rabbit CD1-like antigen (R-Ta) and to be closely related to the humanCD1b gene, which is absent in the mouse, while a second rabbit gene is closely related to both the humanR3 and the mouseCD1 genes. The data reinforce the notion of the existence of two classes ofCD1 genes, one of which is conserved in all species, while the other, albeit also evolutionarily old, has been deleted in mice as well as in other rodents.
Article
The present report describes developmental, phenotypic and functional features of unconventional CD4+ TCRαβ lymphocytes. In C57BL/6 mice, the majority of liver lymphocytes expressing intermediate intensity of TCRαβ (TCRαβint) are CD4+NK1.1+ and express a highly restricted TCR Vβ repertoire, dominated by Vβ8 with some contribution by Vβ7 and Vβ2. Although these cells express the CD4 co-receptor, they are present in H2-l Aβ (Aβ)+/− gene disruption mutants but are markedly reduced in β2-microglobulin (β2m)−/− mutant mice and hence are β2m dependent. Thymocytes expressing the CD4+NK1.1+ TCRα β phenotype are also (β2m) contingent, suggesting that these two T lymphocyte populations are related. The CD4+NK1.1+TCRαβ lymphocytes in liver and thymus share several markers such as LFA-1+, CD44+, CD5+, LECAM-1− and IL-2Rαβa∼. The CD4+NK1.1 + TCRαβint liver lymphocytes were not detected in athymic nuinu mice. We conclude that β2m expression is crucial for development of the CD4+NK1.1+ TCRαβint liver lymphocytes and that thymus plays a major role. CD4+ TCRαβint liver lymphocytes were also identified in NK1.1+ mouse strains, there lacking the NK1.1 marker. We assume that the NK1.1 molecule is a characteristic marker of the CD4+TCRα"int liver lymphocytes in NK1.1+ mouse strains, although its expression is not obligatory for their development. The liver lymphocytes from +2m+/−, but not from +2m−/−mice are potent IL-4 producers in response to CD3 or TCRαβ engagement and the IL-4 production by liver lymphocytes was markedly reduced by treatment with anti-NK1.1 mAb. We conclude that the CD4+NK1.1+ TCRαβint liver lymphocytes are capable of producing IL-4 in response to TCR stimulation.
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Small numbers of T cells have been isolated from the normal mouse liver and many of these are of the CD4⁻CD8⁻TCRαβ⁺ phenotype. Larger numbers of such cells are present in the livers of mice homozygous for the Ipr mutation and the liver has been proposed to be the site of an extrathymlc T cell development pathway that is expanded in Ipr/lpr mice. Using a modified separation procedure that increases the liver T cell yield, we have been able to characterize a subset of CD4⁻CD8⁻TCRαβintermediate T cells that express the B220 epltope of the CD45 molecule, and resemble in this and many other ways the accumulating T cells in Ipr lymph nodes. These cells are an actively dividing population and even in healthy, unmanipulated mice a large proportion of them are undergoing apoptosis. We propose the model that the normal liver is a major site for T cell destruction and that the Ipr defect results in failure of this process with leakage of B220⁺CD4⁻CD8⁻TCRαβ⁺ cells from the liver to peripheral lymphoid tissues, particularly lymph nodes.
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Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.
Article
MAJOR histocompatibility complex (MHC) class I and class II molecules bind immunogenic peptides and present them to lymphocytes bearing the αβ T-cell antigen receptor (TCR)1–4. An analogous antigen-presenting function also has been proposed for the non-MHC-encoded GDI molecules5, a family of non-polymorphic, β2-microglobulin-associated glycoproteins5–8 expressed on most professional antigen-presenting cells9–11. In support of this hypothesis, CD1 molecules are recognized by selected CD4−CD8− αβ or γδ8TCR+ T-cell clones12–14, and we have recently shown that GDI molecules restrict the recognition of foreign microbial antigens by αβTGR+ T cells10. But the substantial structural divergence of GDI from MHC class I and class II molecules7, raises the possibility that the antigens presented by the GDI system may differ fundamentally from those presented by MHC-encoded molecules. Here we report that a purified CDlb-restricted antigen of Mycobacterium tuberculosis presented to αβTCR+ T cells is mycolic acid, a family of α-branched, β-hydroxy, long-chain fatty acids found in mycobacteria15,16. This example of non-protein microbial antigen recognition suggests that αβTCR+ T cells recognize a broader range of antigens than previously appreciated and that at least one member of the GDI family has evolved the ability to present lipid antigens.
Article
MOLECULES encoded by the human GDI locus on chromosome 1 (ref. 33) are recognized by selected CD4−8− T-cell clones expressing either αβ or γδ T-cell antigen receptors1,2. The known structural resemblance of GDI molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHG) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that GDI may represent a family of antigen-presenting molecules separate from those encoded in the MHC1,5,6. Here we report that the proliferative and cytotoxic responses of human CD4−8− αβTCR+ T cells specific for Mycobacterium tuberculosis can be restricted by GDlb, one of the four identified protein products of the GDI locus. The responses of these T cells to M. tuberculosis seemed not to involve MHG encoded molecules, but were absolutely dependent on the expression of GDlb by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class Il-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of GDI molecules and suggest that the GDI family plays a role in cell-mediated immunity to microbial pathogens.
Article
The origin of autoreactive CD4−CD8− T cells is largely unknown. In TCR transgenic (Tg) mice expressing the cognate class I MHCantigen, CD4−CD8−T cells differed depending on characteristics of Tg-TCR/antigen interaction. Tg-TCRJCD3lo CD4−CD8− T cells expressing the NK1.1 marker were observed only for a Tg-TCR whose stimulation by antigen was independent of CD8. Unlike normal T cells, which have essentially TCR-associated ζ homodimers, these cells had a high proportion of TCR-associated ζ-FcϵRlγ heterodimers. They were also characterized by an unusually high content of FcϵRlγ mRNA and low content of mRNA encoding CD3ϵ, CD3γ, CD3δ, and ζ. Based on their phenotype and selection requirements, it is proposed that CD4−CD8− thymic precursor cells can be driven along the CD4−CD8−NK1.1+ pathway following coreceptor-independent TCR signaling at an intrathymic stage when FcϵRlγ and CD3 components are coexpressed.
Article
Immune responses dominated by interleukin-4 (IL-4)-producing T helper type 2 (TH2) cells or by interferon γ (IFN-γ)-producing T helper type 1 (TH1) cells express distinctive protection against infection with different pathogens. Interleukin-4 promotes the differentiation of naïve CD4+ T cells into IL-4 producers and suppresses their development into IFN-γ producers. CD1-specific splenic CD4+NK1.1+ T cells, a numerically minor population, produced IL-4 promptly on in vivo stimulation. This T cell population was essential for the induction of IL-4-producing cells and for switching to immunoglobulin E, an IL-4-dependent event, in response to injection of antibodies to immunoglobulin D.
Article
A monoclonal antibody against V14+ α-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/lgG3 protein containing murine TCR V α 14J α 281 in place of the V H D H J H of an lgG3-λ1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 α-chain. The anti-mouse Vα14 antibody precipitated TCRαβ molecules from Triton X-100-solubilized extracts of 125l-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3θe molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3ϵ, which is a novel family component of the CD3 complex and is associated closely with the CD3ζ-ζ homodimer as well as TCRαβ or TCRγθ.
Article
The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.
Article
Most, but not all, Vβ8.1+ T cells respond to Mls-1 and are clonally deleted in the thymus of Mls-1-expressing animals. To formally examine the role of the TCR α-chain in reactivity and tolerance to Mls-1, we have analyzed Mls-1 reactivity in a large panel of CD4+ hybridomas generated from TCR Vβ8.1 transgenic mice, that express an identical, potentially Mls-1- reactive Mls-1-reactive β-chain. The data show that the α-chain strongly influences the Mls-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, Vα11+ hybridomas were biased toward Mls-1 reactivity and Vα11+ T cells were correspondingly absent from the peripheral repertoire of Mls-1-expressing transgenic mice. Vα2+ hybridomas, on the other hand, were biased against Mls-1 reactivity, and Vα2+ T cells were correspondingly amplified in the Mls-1-expressing transgenic mice. Structural analysis of the α-chains revealed that the Mls-1 reactivity of the Vα11+ hybridomas segregated precisely with family member, such that Vα11.1+ hybridomas were Mls-1-reactive and Vα11.3+ hybridomas were not Mls-1-reactive. On the other hand, there was not a clear correlation between family member and Mls-1 reactivity in the Vα2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the Vα11+ hybridomas correlated precisely with family member and with Mls-1 reactivity. In contrast, there was not a clear correlation with Vα2+ α-chain structure and SEB reactivity. Also, there was no correlation between Mls-1 reactivity and SEB reactivity in individual Vα2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR α-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of Mls-1-reactive T cells in normal and transgenic mice.
Article
Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.
Article
In male mice expressing a transgenic αβ TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male ‘double transgenic’ mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2− population. This implies that, unlike the majority of αβ T cells, TCR+CD4−CD8−cells in TCR transgenic mice are not subject to deletion.
Article
What pushes a T-cell response towards a predominantly TH1 or TH2 phenotype? Several factors have been proposed, including the properties of antigens, dose of antigen, site of exposure and ongoing immune response in the host. Here, Sergio Romagnani presents new evidence to indicate a determining role for the 'natural' immune response, including NK cells and cells of the mast cell/basophil lineage, in the subsequent 'specific' T-cell response.
Article
During differentiation in the thymus, immature T cells progress through an ordered sequence of developmental stages that are best characterized by variable expression of the co-receptor molecules CD4 and CD8. Crosslinking of T cell receptor (TCR) molecules on precursor thymocytes was found to block their differentiation into CD4+CD8+ cells by eliminating messenger RNA's encoding two families of developmentally important molecules: the co-receptor molecules CD4 and CD8 and the recombination activating genes 1 and 2. TCR-induced post-transcriptional regulation in early thymocytes was specific for selective messenger RNA's, required protein synthesis, and was itself developmentally regulated. These data identify a post-transcriptional mechanism that is influenced by TCR signals and that regulates early thymocyte development.
Article
The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double-negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E-expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection.
Article
CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha-beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets.
Article
Although peripheral naive cells only secrete IL-2 upon primary stimulation, their presumptive immediate precursors, HSAlow CD4+8- thymocytes, can produce a large amount of the set of lymphokines usually associated with preactivated or memory CD4+ lymphocytes: IL-4, IL-5, IL-10 and gamma-IFN. This phenotype can be attributed to true virgin thymocytes and not only to recirculating lymphocytes, because it is found in newborn thymuses and in fetal thymic organ culture. This mature stage of CD4+8- thymocytes is itself preceded by an immature stage (HSAhigh) where only IL-2 and small amounts of gamma-IFN can be elicited by the combination of calcium ionophore and phorbol ester, but not by TCR cross-linking. CD8+4- thymocytes pass through a similar immature HSAhigh stage, where their pattern of lymphokine secretion is not yet differentiated from that of CD4+8- HSA high thymocytes. The subsets acquire their specific profiles at the HSAlow stage. We propose that recent thymic CD4+8- emigrant cells include a significant proportion of Th0 type cells, and that their role is critical to prime the immune system for IL-4 production, as well as to explain the longstanding observations of synergy between helper cell subpopulations in the periphery.
Article
CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.
Article
The CD1 antigens are a family of differentiation antigens found predominantly, but not exclusively, in the human thymus. Although three antigens (CD1a-c) are described by monoclonal antibodies, five genes (CD1A-E) are found in the human genome. The cloning of the mouse CD1 genes (Bradbury, A., Belt, K.T., Nery, T.M., Milstein, C. and Calabi, F., EMBO J. 1988. 7:3081) demonstrated the presence of homologues to human CD1D, but not to any of the other human CD1 genes. In this work we have examined the expression of mouse CD1D mRNA in the thymus and shown that it is predominantly cortical, as is the expression of the CD1 antigens in man. Somewhat surprisingly, we also find that most CD1D mRNA in the mouse thymus is unspliced. Despite this, we have also been able to show, using a polyclonal antiserum directed against a bacterial fusion protein, the existence of the expected protein product.
Article
The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.
Article
The cluster of differentiation-1 (CD1) antigens are major histocompatibility complex (MHC) class I-like glycoproteins belonging to the immunoglobulin supergene family. Initially described in humans, more recently putative CD1 encoding genes have been identified in several other species, including the mouse where it has been clearly demonstrated that CD1 mRNA is expressed. However, in the mouse both its unusually wide tissue distribution and the prevalence of incompletely spliced RNA have raised the possibility that the mRNA did not encode a functional protein. We have utilized a rabbit polyclonal antiserum raised against an Escherichia coli-expressed recombinant murine CD1 fusion protein to characterize the murine CD1 protein. Here we demonstrate that the antiserum binds specifically to a set of glycoproteins (49,000-55,000 MW) which contain a common core protein with both a size (36,000 MW) and tissue distribution in accordance with those predicted. During thymic ontogeny, this protein is highly expressed by Day 14 of embryonic development and persists into adulthood, while its pattern of expression in other organs changes significantly during development. Thus, the mouse provides an amenable model system for the study of CD1 function.
Article
A human intestinal intraepithelial lymphocyte (IEL) T cell line was established from jejunum to characterize the structure and function of the alpha beta T cell antigen receptors (TCRs) expressed by this population. Single-sided polymerase chain reaction (PCR) amplification cloning and quantitative PCR amplification of the TCR chains from the cell line and from fresh IELs demonstrated that IELs were oligoclonal. The IEL T cell line exhibited CD1-specific cytotoxicity and a dominant IEL T cell clone was CD1c-specific. Thus, human jejunal intraepithelial lymphocytes are oligoclonal and recognize members of the CD1 gene family.
Article
The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue.
Article
We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.