Surface receptors identify mouse NK1.1+ T cell subsets distinguished by function and T cell receptor type

Natural killer T (NKT) lymphocytes rapidly produce several cytokines, including IL-4 and IFN-gamma, upon activation, and act as regulatory cells at an early interphase of innate and adaptive immune responses. They have been implicated as important elements in diverse immune responses including the regulation of autoimmune disease, the immune response to infections, and the prevention of tumor metastasis. The broad spectrum of their activities suggested that functionally different subsets of NKT cells may exist. We demonstrate two functionally distinct splenic NKT populations identified by the expression of CD49b and CD69, respectively. Each NKT subset was represented by the amplified transgenic NKT cell population in a distinct transgenic mouse line expressing a CD1d-restricted TCR. CD49bhigh CD69- NKT cells, termed NKT1 cells by us, were high producers of IFN-gamma after stimulation, but essentially devoid of IL-4-synthesizing cells. Most NKT1 cells used diverse (non-Valpha14-canonical) TCR. The CD69+ CD49(-/low) NKT cell population, which we term NKT2, produced large quantities of IL-4 and substantial amounts of IFN-gamma upon activation and were dominated by cells using the canonical Valpha14-Jalpha18 T cell receptor. Knowledge of the unique roles of the different NKT cell subsets in specific situations will be essential for our understanding of NKT cell biology.     

Mechanism of CD1d-restricted natural killer T cell activation during microbial infection

CD1d-restricted natural killer T (NKT) cells are important for host defense against a variety of microbial pathogens. How and when these T cells become activated physiologically during infection remains unknown. Our data support a model in which NKT cells use a unique activation mechanism not requiring their recognition of microbial antigens. Instead, weak responses to CD1d-presented self antigens were amplified by interleukin 12 made by dendritic cells in response to microbial products, resulting in potent interferon-gamma secretion. NKT cells were among the first lymphocytes to respond during Salmonella typhimurium infection, and their activation in vivo also depended on interleukin 12 and CD1d recognition. We propose this mechanism of activation as a major pathway responsible for the rapid activation of NKT cells in different microbial infections.     

Regulation of murine cerebral malaria pathogenesis by CD1d-restricted NKT cells and the natural killer complex

NKT cells are specialized cells coexpressing NK and T cell receptors. Upon activation they rapidly produce high levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and are therefore postulated to influence T(H)1/T(H)2 immune responses. The precise role of the CD1/NKT cell pathway in immune response to infection remains unclear. We show here that CD1d-restricted NKT cells from distinct genetic backgrounds differentially influence T(H)1/T(H)2 polarization, proinflammatory cytokine levels, pathogenesis, and fatality in the P. berghei ANKA/rodent model of cerebral malaria. The functional properties of CD1d-restricted NKT cells vary according to expression of loci of the natural killer complex (NKC) located on mouse chromosome 6, which is shown here to be a significant genetic determinant of murine malarial fatalities.     

The role of CD1d-restricted NK T lymphocytes in the immune response to oral infection with Salmonella typhimurium

CD1d-restricted natural killer T (NKT) cells belong to the innate-like lymphocytes which respond rapidly to stress and infectious challenge. We have studied murine CD1d-restricted NKT cells in the early immune response to virulent Salmonella enterica serovar Typhimurium after oral infection. In the liver and spleen, neutrophil and macrophage numbers had increased several-fold by day 5 post-infection, while the frequency of B and T lymphocytes decreased. These cellular changes occurred independently of CD1d-restricted NKT cells, and further, CD1d-restricted T cells did not influence the bacterial load. However, in CD1d(+) mice NK1.1(+) T cells and invariant CD1d-restricted T cells were activated by the infection, as demonstrated by an increase in size, up-regulation of CD69 and production of IFN-gamma. The NK1.1 antigen was down-modulated on these cells during the course of infection, while TCR levels were unaffected. While dendritic cells (DC) up-regulated CD1d-levels upon 24 h of in vitro exposure to the bacteria, increased CD1d expression was not evident on DC in vivo during infection. Furthermore, in vitro re-stimulation of CD1d-restricted T cells isolated from infected mice demonstrated a significant skewing of the cytokine profile, with suppressed IL-4 and increased IFN-gamma production.     

A molecular basis for NKT cell recognition of CD1d-self-antigen

The antigen receptor for natural killer T?cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3???resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.     

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.     

Molecular profiling reveals distinct functional attributes of CD1d-restricted natural killer (NK) T cell subsets

CD1d-restricted natural killer T (NKT) cells can have multiple effects on an immune response, including the activation, regulation and attraction of innate immune cells, and modulation of adaptive immunity. Recent studies reveal that there are distinct subsets of NKT cells which selectively perform some of the functions attributed to CD1d-restricted cells, but the mechanisms underlying these functional differences have not been resolved. Our aim in this study was to identify novel NKT cell associated traits that would provide important insight into NKT cell activation and function. To this end, we have performed gene expression profiling of two separate subsets of NKT cells, analyzing genes differentially expressed in these cells compared to conventional CD4(+)NK1.1(-) T cells. We identify different sets of genes over expressed in each of the two NKT cell types, as well as genes that are common to the two CD1d-restricted NKT cell populations analyzed. A large number of these genes are highly relevant for NKT cell development, activation and function. Each NKT subtype displayed a unique set of chemokine receptors, integrins and molecules related to effector function, supporting the notion that distinct NKT cells can be selectively engaged and have diverse functions in different types of immune reactions.     

Invariant and noninvariant natural killer T cells exert opposite regulatory functions on the immune response during murine schistosomiasis

CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) alpha chain (Valpha14Jalpha18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jalpha18-/- (exclusively deficient in iNKT cells) and CD1d-/- (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response.     

Primary infection of C57BL/6 mice with Plasmodium yoelii induces a heterogeneous response of NKT cells

NKT cells are a population of innate-like lymphocytes that display effector functions and immunoregulatory properties. We characterized the NKT cell response induced in C57BL/6 mice during a primary infection with Plasmodium yoelii sporozoites. We observed a heterogeneous NKT cell response that differed between liver and spleen. Hepatic NKT cells found in infected livers consisted mainly of CD1d-dependent CD4+ and double-negative (DN) NKT cells, whereas CD1d-independent NKT cells exhibiting a TCR(high) CD4(high) phenotype were prominent among splenic NKT cells during the infection. Hepatic and splenic NKT cells isolated from infected mice were activated and secreted mainly gamma interferon and tumor necrosis factor alpha in response to stimulation. Finally, P. yoelii-activated hepatic DN NKT cells inhibited the parasite's liver stage in a CD1d-dependent manner in vitro. However, experiments using B6.CD1d-deficient mice showed that CD1d and CD1d-restricted NKT cells are not necessary to control the parasite's development in vivo during neither the preerythrocytic stage nor the erythrocytic stage. Thus, our results show that a primary P. yoelii infection induces a heterogeneous and organ-specific response of NKT cells and that CD1d-dependent NKT cells play a minor role in the control of the development of Plasmodium in vivo in our model.     

Diverse CD1d-restricted T cells: diverse phenotypes, and diverse functions

Invariant CD1d-restricted T cells express NK cell markers and use a limited TCR repertoire. Here, we describe a second CD1d-restricted T cell subset that uses a diverse TCR repertoire. These T cells can also express NK cell markers and function similarly to invariant T cells. The antigens recognized by the diverse subset are likely to be different from those recognized by invariant TCRs. The variable NK1.1 antigen expression on these T cell populations limits its usefulness in identifying CD1d-restricted T cells. Lastly, the discovery of antigens recognized by diverse CD1d-restricted T cells will provide insight into their role in normal and pathological immune responses.     

NKG2D performs two functions in invariant NKT cells: direct TCR-independent activation of NK-like cytolysis and co-stimulation of activation by CD1d

Invariant NKT cells are important in the activation and regulation of immune responses. They can also function as CD1d-restricted killer cells. However, the role of activating innate NK-cell receptors expressed on NKT cells in triggering cytolytic function is poorly characterized. Here, we initially confirmed that the cellular stress-ligand receptor NKG2D is expressed on CD4- NKT cells, whereas most CD4+ NKT cells lack this receptor. Interestingly, NKG2D+ NKT cells frequently expressed perforin, and both NKG2D and perforin localized at the site of contact with NKG2D ligand-expressing target cells. CD4- NKT cells degranulated in response to NKG2D engagement in a redirected activation assay independent of stimulation via their invariant TCR. NKT cells killed P815 cells coated with anti-NKG2D mAb and CD1d-negative K562 tumor target cells in an NKG2D-dependent manner. Furthermore, NKG2D engagement co-stimulated TCR-mediated NKT-cell activation in response to endogenous CD1d-presented ligands or suboptimal levels of anti-CD3 triggering. These data indicate that the CD4- subset of human NKT cells can mediate direct lysis of target cells via NKG2D engagement independent of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells.     

Different subsets of natural killer T cells may vary in their roles in health and disease

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class‐I‐like molecule. Most type I NKT cells express a semi‐invariant T‐cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR‐α dominates CD1d‐lipid recognition by type I NKT cells, TCR‐α and TCR‐β contribute equally to CD1d‐lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine‐dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen‐presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes.     

The CD1d-binding glycolipid alpha-galactosylceramide enhances humoral immunity to T-dependent and T-independent antigen in a CD1d-dependent manner

Specific interaction of class II/peptide with the T-cell receptor (TCR) expressed by class II-restricted CD4+ T helper (Th) cells is essential for in vivo production of antibodies reactive with T-dependent antigen. In response to stimulation with CD1d-binding glycolipid, Valpha14+ TCR-expressing, CD1d-restricted natural killer T (NKT) cells may provide additional help for antibody production. We tested the hypothesis that the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances production of antibodies reactive with T-dependent antigen in vivo. alpha-GC enhanced antibody production in vivo in a CD1d-dependent manner in the presence of class II-restricted Th cells and induced a limited antibody response in Th-deficient mice. alpha-GC also led to alterations in isotype switch, selectively increasing production of immunoglobulin G2b. Further analysis revealed that alpha-GC led to priming of class II-restricted Th cells in vivo. Additionally, we observed that alpha-GC enhanced production of antibodies reactive with T-independent antigen, showing the effects of NKT cells on B cells independently of Th cells. Our data show that NKT cells have multiple effects on the induction of a humoral immune response. We propose that NKT cells could be exploited for the development of novel vaccines where protective antibody is required.     

A role for CD1d-restricted NKT cells in injury-associated T cell suppression

Natural killer T (NKT) cells are known to modulate T cell responses during autoimmunity, tolerance, and antitumor immunity; however, their potential role in regulating the immune response to injury has not been reported. Using a murine model of burn injury, we investigated whether CD1d-restricted NKT cells played a role in the T cell suppression that occurs early after injury. A functional role for CD1d stimulation of NKT cells in the injury-related immune suppression was demonstrated by experiments in which the suppression of antigen (Ag)-specific delayed-type hypersensitivity and in vitro T cell-proliferative responses were prevented if mice were given anti-CD1d monoclonal antibody (mAb) systemically just before injury. The CD1d-NKT cell-dependent suppression of the T cell response after injury occurred in the absence of quantitative changes in NKT cells themselves or CD1d(+) Ag-presenting cells. We observed that elevated production of the immunosuppressive cytokine interleukin (IL)-4 correlated with burn-induced immune dysfunction, and we found that NKT cells but not conventional T cells were the source of IL-4 early after injury. Lastly, we observed that the injury-induced production of NKT cell-derived IL-4 could be blocked by systemic treatment of burn-injured mice with anti-CD1d mAb. Together, our results reveal a novel mechanism involving CD1d stimulation of NKT cells in the onset of T cell suppression that occurs subsequent to injury.     

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.     

Vα14 NKT cell-mediated anti-tumor responses and their clinical application

A unique lymphocyte population, Vα14 NKT cells, has recently been revealed to be a key player in the immune responses against tumors. Activation of Vα14 NKT cells affects various cell types, particularly dendritic cells (DCs), NK cells, CD4 Th1 cells, and CD8 cytotoxic T cells in the innate and acquired immune systems, eventually resulting in the enhanced activation of NKT cell-mediated cellular cascade in the anti-tumor responses. The specific ligand, α-galactosylceramide (α-GalCer), effectively stimulates mouse and human NKT cells, making NKT cells an ideal target for the development of cancer immunotherapy. Clinical trials using α-GalCer have actually started in several centers in the world. In this review, we summarize the Vα14 NKT cell-mediated cellular cascade in the anti-tumor response in mice and discuss potential clinical applications of α-GalCer-pulsed DC therapy.     

The natural killer T lymphocyte: a player in the complex regulation of autoimmune diabetes in non-obese diabetic mice

Manipulation of the immune response to specifically prevent autoaggression requires an understanding of the complex interactions that occur during the pathogenesis of autoimmunity. Much attention has been paid to conventional T lymphocytes recognizing peptide antigens presented by classical major histocompatibility complex (MHC) class I and II molecules, as key players in the destructive autoreactive process. A pivotal role for different types of regulatory T lymphocytes in modulating the development of disease is also well established. Lately, CD1d-restricted natural killer T (NKT) lymphocytes have been the subject of intense investigation because of their ability to regulate a diversity of immune responses. The non-classical antigen presenting molecule CD1d presents lipids and glycolipids to this highly specialized subset of T lymphocytes found in both humans and mice. From experimental models of autoimmunity, evidence is accumulating that NKT cells can protect from disease. One of the best studied is the murine type 1 diabetes model, the non-obese diabetic (NOD) mouse. While the NKT cell population was first recognized to be deficient in NOD mice, augmenting NKT cell activity has been shown to suppress the development of autoimmune disease in this strain. The mechanism by which CD1d-restricted T cells exert this function is still described incompletely, but investigations in NOD mice are starting to unravel specific effects of NKT cell regulation. This review focuses on the role of CD1d-restricted NKT cells in the control of autoimmune diabetes.     

Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers

The liver regulates lipid homeostasis and is enriched with natural killer T (NKT) cells that respond to lipid antigens. Optimal maturation and activation of NKT cells requires their interaction with lipid antigens that are presented by cluster of differentiation-1 (CD-1) molecules on antigen-presenting cells. Hepatocytes express CD1d and present lipid antigens to NKT cells. Depletion and dysregulation of hepatic NKT cells occurs in mice with fatty livers. Herein, we assess whether reduced CD1d content on steatotic hepatocytes contributes to fatty liver-associated NKT cell abnormalities. We show that despite expressing normal levels of CD1d mRNA, fatty hepatocytes from ob/ob mice have significantly less CD1d on their plasma membranes than normal hepatocytes. This has functional significance because ob/ob hepatocytes are less able to activate CD1d-restricted T-cell responses in vitro, and CD1d-reactive NKT cells are reduced in ob/ob livers. Events in the endoplasmic reticulum (ER) normally regulate CD1d trafficking to plasma membranes. Hepatic steatosis has been associated with ER stress. To determine if ER stress reduces CD-1 accumulation on hepatocytes, we evaluated hepatic ER stress in ob/ob mice and treated cultured hepatocytes and lean mice with tunicamycin to induce ER stress. Lipid accumulation and ER stress occurred in the livers of both ob/ob and tunicamycin-treated mice. Tunicamycin caused dose-dependent decreases in hepatocyte CD1d, inhibited hepatocyte activation of CD1d-restricted T-cell responses, depleted liver populations of CD1d-reactive NKT cells and promoted Th-1 polarization of hepatic cytokine production. In conclusion, ER stress-related decreases in hepatocyte CD1d contribute to NKT cell dysregulation in fatty livers.     

Inhibition of CD1d1-mediated antigen presentation by the vaccinia virus B1R and H5R molecules

Vaccinia virus (VV) has been most commonly used as the vaccine to protect individuals against the causative agent of smallpox (variola virus), but it also uses a number of strategies meant to evade or blunt the host's antiviral immune response. Natural killer T (NKT) cells are a subset of immunoregulatory CD1d-restricted T lymphocytes believed to bridge the innate and adaptive immune responses. It is shown here that the VV-encoded molecules, B1R and H5R, play a role in the ability of VV to inhibit CD1d-mediated antigen presentation to NKT cells. These are the first poxvirus-encoded molecules identified that can play such a role in the evasion of an important component of the innate immune response.     

CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system

Natural Killer T cells are a distinct lymphocyte lineage that regulates a broad range of immune responses. NKT cells recognize glycolipids presented by the non-classical MHC molecule CD1d. Structural insight into the TCR/glycolipid/CD1d tri-complex has revealed an unusual and unexpected mode of recognition. Recent studies have also identified some of the signaling events during NKT cell development that give NKT cells their innate phenotype. Pathogen-derived glycolipid antigens continue to be found, and new mechanisms of NKT cell activation have been described. Finally, NKT cells have been shown to be remarkably versatile in function during various immune responses. Whether these extensive functional capacities can be attributed to a single population sensitive to environmental cues or if functionally distinct NKT cell subpopulations exist remains unresolved.