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Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKT FH ) cells are specialized to help B cells. However, the mechanisms of NKT FH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKT FH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1 + cells helped iNKT cell proliferation and NKT FH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKT FH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1 + cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKT FH cells. Interestingly, Gr-1 + cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies., Competing Interests: Competing interests statement:The authors declare no competing interest.

NKT cells recognize glycolipids presented by CD1d-expressing antigen-presenting cells (APCs) and include type I NKT cells with antitumor function and type II NKT cells, which have been reported to suppress the antitumor response. Some type II NKT cells recognize sulfatide, a glycosphingolipid with a sulfate modification of the sugar. Type I NKT cells recognize different glycosphingolipids. In this issue of the JCI, Nishio and colleagues showed that APCs could process sulfatide antigens, analogous to protein processing for peptide-reactive T cells. Antigen processing in lysosomes removed sulfate to generate a glycosphingolipid that stimulated type I NKT cells and thereby turned an antigen with no antitumor activity into one that not only stimulated type I NKT cells but also stimulated antitumor responses. These findings may extend to the development of glycolipid antigens that could stimulate anticancer responses via antigen processing by APCs.

Innate-like T cells, including invariant natural killer T cells, mucosal-associated invariant T cells, and γδ T cells, are present in various barrier tissues, including the lung, where they carry out protective responses during infections. Here, we investigate their roles during pulmonary pneumococcal infection. Following infection, innate-like T cells rapidly increase in lung tissue, in part through recruitment, but T cell antigen receptor activation and cytokine production occur mostly in interleukin-17-producing NKT17 and γδ T cells. NKT17 cells are preferentially located within lung tissue prior to infection, as are CD103 + dendritic cells, which are important both for antigen presentation to NKT17 cells and γδ T cell activation. Whereas interleukin-17-producing γδ T cells are numerous, granulocyte-macrophage colony-stimulating factor is exclusive to NKT17 cells and is required for optimal protection. These studies demonstrate how particular cellular interactions and responses of functional subsets of innate-like T cells contribute to protection from pathogenic lung infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4 + T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca 2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca 2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca 2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca 2+ buffering in NKT2 cells. In addition, the low Ca 2+ extrusion rate may also contribute to the higher Ca 2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca 2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells., (© 2021 The Authors.)

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.

Ample evidence exists for activation of invariant natural killer T (iNKT) cells in a sterile manner by endogenous ligands or microbial antigens from the commensal flora, indicating that iNKT cells are not truly self-tolerant. Their controlled autoreactivity state is disturbed in many types of sterile inflammatory disease, resulting in their central role in modulating autoimmune responses. This review focuses on sterile iNKT-cell responses that are initiated by metabolic triggers, such as obesity-associated inflammation and fatty liver disease, as a manifestation of metabolic disease and dyslipidemia, as well as ischemia reperfusion injuries and sickle cell disease, characterized by acute lack of oxygen and oxidative stress response on reperfusion. In the intestine, inflammation and iNKT-cell response type are shaped by the microbiome as an extended "self". Disease- and organ-specific differences in iNKT-cell response type are summarized and help to define common pathways that shape iNKT-cell responses in the absence of exogenous antigen.

Invariant natural killer T (iNKT) cells have been tested for their potential use in cancer immune therapy, but their low frequency has limited their use. In this issue, Zhu and colleagues propose to overcome this by engineering hematopoietic stem cells (HSCs) to provide a continual source of iNKT cells. (Zhu et al., 2019)., (Copyright © 2019. Published by Elsevier Inc.)

Invariant NKT (iNKT) cells are innate lipid-reactive T cells that develop and differentiate in the thymus into iNKT1/2/17 subsets, akin to T H 1/2/17 conventional CD4 T cell subsets. The factors driving the central priming of iNKT cells remain obscure, although strong/prolonged TCR signals appear to favor iNKT2 cell development. The Src homology 2 domain-containing phosphatase 1 (Shp1) is a protein tyrosine phosphatase that has been identified as a negative regulator of TCR signaling. In this study, we found that mice with a T cell-specific deletion of Shp1 had normal iNKT cell numbers and peripheral distribution. However, iNKT cell differentiation was biased toward the iNKT2/17 subsets in the thymus but not in peripheral tissues. Shp1-deficient iNKT cells were also functionally biased toward the production of T H 2 cytokines, such as IL-4 and IL-13. Surprisingly, we found no evidence that Shp1 regulates the TCR and Slamf6 signaling cascades, which have been suggested to promote iNKT2 differentiation. Rather, Shp1 dampened iNKT cell proliferation in response to IL-2, IL-7, and IL-15 but not following TCR engagement. Our findings suggest that Shp1 controls iNKT cell effector differentiation independently of positive selection through the modulation of cytokine responsiveness., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.

Invariant natural killer T cells (iNKT cells) are an innate-like T cell subset that expresses an invariant T cell receptor (TCR) α-chain and recognizes lipids presented on CD1d. They secrete diverse cytokines and can influence many types of immune responses. Despite having highly similar TCR specificities, iNKT cells differentiate in the thymus into distinct subsets that are analogous to T helper 1 (T H 1), T H 2 and T H 17 cell subsets. Additional iNKT cell subsets that may require peripheral activation have also been described, including one that produces IL-10. In general, iNKT cells are non-circulating, tissue-resident lymphocytes, but the prevalence of different iNKT cell subsets differs markedly between tissues. Here, we summarize the functions of iNKT cells in four tissues in which they are prevalent, namely, the liver, the lungs, adipose tissue and the intestine. Importantly, we explain how local iNKT cell responses at each site contribute to tissue homeostasis and protection from infection but can also contribute to tissue inflammation and damage.

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-γ-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-γ expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-γ production, but also the protective function of iNKT cells in arthritis.

Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs), probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro .

Invariant Natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after in vivo activation, allowing for the direct detection of a number of cytokines directly ex vivo. However, for some cytokines this approach is suboptimal. Here, we report technical variations that allow the improved detection of IL-4, IL-10, IL-13 and IL-17A ex vivo. Furthermore, we describe an alternative approach for stimulation of iNKT cells in vitro that allows a significantly improved detection of cytokines produced by iNKT cells. Together, these protocols allow the detection of iNKT cell cytokines ex vivo and in vitro with increased sensitivity.

ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7 -/- mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function.

Invariant natural killer T (iNKT) cells constitute a unique subset of innate-like T cells that have been shown to have crucial roles in a variety of immune responses. iNKT cells are characterized by their expression of both NK cell markers and an invariant T cell receptor (TCR) α chain, which recognizes glycolipids presented by the MHC class I-like molecule CD1d. Despite having a limited antigen repertoire, the iNKT cell response can be very complex, and participate in both protective and harmful immune responses. The protective role of these cells against a variety of pathogens has been particularly well documented. Through the use of these pathogen models, our knowledge of the breadth of the iNKT cell response has been expanded. Specific iNKT cell antigens have been isolated from several different bacteria, from which iNKT cells are critical for protection in mouse models. These responses can be generated by direct, CD1d-mediated activation, or indirect, cytokine-mediated activation, or a combination of the two. This can lead to secretion of a variety of different Th1, Th2, or Th17 cytokines, which differentially impact the downstream immune response against these pathogens. This critical role is emphasized by the conservation of these cells between mice and humans, warranting further investigation into how iNKT cells participate in protective immune responses, with the ultimate goal of harnessing their potential for treatment.

Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.

Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer., (Copyright © 2016 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness., (Copyright © 2015 by The American Association of Immunologists, Inc.)

In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycosphingolipids are a subgroup of glycolipids that contain an amino alcohol sphingoid base linked to sugars. They are found in the membranes of cells ranging from bacteria to vertebrates. This group of lipids is known to stimulate the immune system through activation of a type of white blood cell known as natural killer T cell (NKT cell). Here we summarize the extensive research that has been done to identify the structures of natural glycolipids that stimulate NKT cells and to determine how these antigens are recognized. We also review studies designed to understand how glycolipid variants, both natural and synthetic, can alter the responses of NKT cells, leading to dramatic changes in the global immune response., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Over the last two decades, it has been established that peptides are not the only antigens recognized by T lymphocytes. Here, we review information on two T lymphocyte populations that recognize nonpeptide antigens: invariant natural killer T cells (iNKT cells), which respond to glycolipids, and mucosal associated invariant T cells (MAIT cells), which recognize microbial metabolites. These two populations have a number of striking properties that distinguish them from the majority of T cells. First, their cognate antigens are presented by nonclassical class I antigen-presenting molecules; CD1d for iNKT cells and MR1 for MAIT cells. Second, these T lymphocyte populations have a highly restricted diversity of their T cell antigen receptor α chains. Third, these cells respond rapidly to antigen or cytokine stimulation by producing copious amounts of cytokines, such as IFNγ, which normally are only made by highly differentiated effector T lymphocytes. Because of their response characteristics, iNKT and MAIT cells act at the interface of innate and adaptive immunity, participating in both types of responses. In this review, we will compare these two subsets of innate-like T cells, with an emphasis on the various ways that lead to their activation and their participation in antimicrobial responses., (© 2015 Elsevier Inc. All rights reserved.)

Glycosylceramides in mammalian species are thought to be present in the form of β-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both β-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Invariant natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after activation, thereby impacting a wide variety of different immune reactions. However, strong activation of iNKT cells with α-galactosylceramide (αGalCer) reportedly induces a hyporeactive state that resembles anergy. In contrast, we determined here that iNKT cells from mice pretreated with αGalCer retain cytotoxic activity and maintain the ability to respond to TCR-dependent as well as TCR-independent cytokine-mediated stimulation. Additionally, αGalCer-pretreated iNKT cells acquired characteristics of regulatory cells, including production and secretion of the immunomodulatory cytokine IL-10. Through the production of IL-10, αGalCer-pretreated iNKT cells impaired antitumor responses and reduced disease in experimental autoimmune encephalomyelitis, a mouse model of autoimmune disease. Furthermore, a subset of iNKT cells with a similar inhibitory phenotype and function were present in mice not exposed to αGalCer and were enriched in mouse adipose tissue and detectable in human PBMCs. These data demonstrate that IL-10-producing iNKT cells with regulatory potential (NKT10 cells) represent a distinct iNKT cell subset.

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: Altered immune function in patients with renal failure results in both susceptibility to infection and increased inflammatory response. Invariant natural killer T (iNKT) cells are a conserved, immunoregulatory T lymphocyte subset that responds to lipid antigens with near-immediate cytokine production and cytotoxicity. iNKT cells are required for the antibacterial host response. Whether renal failure and renal replacement therapy alter iNKT cell abundance or phenotype has not been investigated., Methods: iNKT cells were studied by flow cytometry in the peripheral blood of patients with acute renal failure, chronic haemo- and peritoneal dialysis (PD), chronic kidney disease and after renal transplantation., Results: A very marked reduction in iNKT lymphocytes was found in acute renal failure before the first haemodialysis (HD) session. iNKT cells were depleted in end-stage renal disease patients receiving either HD or PD. iNKT cell depletion was accentuated after an HD session. Lesser degrees were observed in patients with non-dialysis-dependent chronic kidney disease. CD56 and CD161 NK cell marker expression was decreased in renal impairment. CD56(+) and CD161(+) iNKT cells produced more interferon-γ than negative cells of the same donor. Within the first year after kidney transplantation, the decrease in iNKT cells and their NK cell markers was reverted., Conclusions: We describe for the first time that iNKT lymphocytes are reduced in end-stage renal disease and further depleted by HD. iNKT cells are important for early host response including activation of other immune cells and their depletion may contribute to immune dysfunction in renal disease.

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells, NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However, whether similar mechanisms govern activation of these two cell types, as well as the significance of NKT cells for host resistance, remain unknown. In this article, we show that, although both NKT and NK cells are activated via cytokines, their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient, whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results, GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection, consistent with their virtual lack of IL-12 production, whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo, NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV, whereas NK cells were still activated. In turn, splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally, mice lacking NKT cells showed reduced control of MCMV, and depleting NK cells further enhanced viral replication. Taken together, our results show that NKT and NK cells have differing requirements for cytokine-mediated activation, and both can contribute nonredundantly to MCMV defense, revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses.

The majority of T lymphocytes, sometimes referred to as as mainstream or conventional T cells, are characterized by a diverse T cell antigen receptor (TCR) repertoire. They require antigen priming in order to become memory cells capable of mounting a rapid effector response. It has become established, however, that there are several distinct T cell lineages that exhibit a memory phenotype in the absence of antigen priming, even as they differentiate in the thymus. These lymphocytes typically express a markedly restricted TCR repertoire and their rapid response kinetics has led to their being described as innate-like T cells. In addition, several of these subsets typically express surface markers commonly found on natural killer cells, which has led to the moniker natural killer T cells (NKT cells). This review will describe our current understanding of the unique ways whereby transcription factors control the development and function of an abundant and widely studied lineage of NKT cells that recognizes glycolipid antigens.

Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18(-/-)) or wild-type mice, bacterial recovery significantly increased in Jα18(-/-) compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18(-/-) compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.

Invariant natural killer T cells (iNKT cells) are unique lymphocytes with characteristic features, such as expression of an invariant T-cell antigen receptor (TCR) α-chain, recognition of glycolipid antigens presented by CD1d molecules, and ability to rapidly produce large amounts of cytokines, including interferon-γ (IFN-γ) and interleukin 4 (IL-4) upon TCR stimulation. Many studies have demonstrated that iNKT cells participate in immune response against diverse microbes, including bacteria, fungi, protozoan parasites, and viruses. Generally, these cells play protective roles in host defense against infections. However, in some contexts they play pathogenic roles, by inducing or augmenting inflammation. Recent reports show that iNKT cells recognize glycolipid antigens from pathogenic bacteria including Streptococcus pneumoniae, and they contribute to host defense against infection. iNKT cell responses to these microbial glycolipid antigens are highly conserved between rodents and humans, suggesting that iNKT cells are evolutionally conserved because their invariant TCR is useful in detecting certain pathogens. Furthermore, glycolipid-mediated iNKT cell activation during immunization has adjuvant activity, enhancing humoral and cell-mediated responses. Therefore, iNKT cell activation is an attractive target for developing new vaccines for infectious diseases.

Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169(+) macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169(+) macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169(-/-) mice and is macrophage-dependent, demonstrating that targeting CD169(+) macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.

Regulation and induction of anergy in NKT cells of the liver can inhibit autoimmune and antitumor responses by mechanisms that are poorly understood. We investigated the effects of PGE2, delivered by intestinal, mucus-derived, exosome-like nanoparticles (IDENs), on NKT cells in mice. In this study, we demonstrate that IDENs migrate to the liver where they induce NKT cell anergy. These effects were mediated by an IDENs' PGE2. Blocking PGE2 synthesis attenuated IDENs inhibition of induction of IFN-γ and IL-4 by α-galactosylceramide (α-GalCer)-stimulated liver NKT cells in a PGE2 E-type prostanoid 2/E-type prostanoid 4 receptor-mediated manner. Proinflammatory conditions enhanced the migration of IDENs to the liver where α-GalCer and PGE2 induced NKT anergy in response to subsequent α-GalCer stimulation. These findings demonstrate that IDENs carrying PGE2 can be transferred from the intestine to the liver, where they act as immune modulators, inducing an anergic-like state of NKT cells. These reagents might be developed as therapeutics for autoimmune liver diseases.

ATP-binding cassette transporter G1 (ABCG1) plays a role in the intracellular transport of cholesterol. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid Ags. In this study, we demonstrate that ABCG1 regulates iNKT cell development and functions in a cell-intrinsic manner. Abcg1(-/-) mice displayed reduced frequencies of iNKT cells in thymus and periphery. Thymic iNKT cells deficient in ABCG1 had reduced membrane lipid raft content, and showed impaired proliferation and defective maturation during the early stages of development. Moreover, we found that Abcg1(-/-) mice possess a higher frequency of Vβ7(+) iNKT cells, suggesting alterations in iNKT cell thymic selection. Furthermore, in response to CD3ε/CD28 stimulation, Abcg1(-/-) thymic iNKT cells showed reduced production of IL-4 but increased production of IFN-γ. Our results demonstrate that changes in intracellular cholesterol homeostasis by ABCG1 profoundly impact iNKT cell development and function.

The majority of mouse Vα14 invariant natural killer T (Vα14i NKT) cells produce several cytokines, including IFNγ and IL-4, very rapidly after activation. A subset of these cells, known as NKT17 cells, however, differentiates in the thymus to preferentially produce IL-17. Here, we show that the transcription factor-known as T helper, Poxviruses, and Zinc-finger and Krüppel family, (Th-POK)-represses the formation of NKT17 cells. Vα14i NKT cells from Th-POK-mutant helper deficient (hd/hd) mice have increased transcripts of genes normally expressed by Th17 and NKT17 cells, and even heterozygosity for this mutation leads to dramatically increased numbers of Vα14i NKT cells that are poised to express IL-17, especially in the thymus and lymph nodes. In addition, using gene reporter mice, we demonstrate that NKT17 cells from wild-type mice express lower amounts of Th-POK than the majority population of Vα14i NKT cells. We also show that retroviral transduction of Th-POK represses the expression of the Th17 master regulator RORγT in Vα14i NKT-cell lines. Our data suggest that NKT17-cell differentiation is intrinsically regulated by Th-POK activity, with only low levels of Th-POK permissive for the differentiation of NKT17 cells.

Background & Aims: Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells., Methods: We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments., Results: Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation., Conclusions: Intestinal microbes can affect iNKT cell phenotypes and functions in mice., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Glycolipid reactive natural killer T cells with an invariant TCR α-chain (iNKT cells) are a conserved population of T lymphocytes with a distinct anatomical distribution and functional properties. The differentiation pathway of iNKT cells branches off from mainstream thymocyte differentiation at the double positive stage, and recent work has revealed how signaling events early in the iNKT cell pathway imprint a memory-like behavior on these cells. Additionally, unique molecular interactions governing iNKT cell development and tissue distribution have been uncovered recently, building up our knowledge of the complex network of interactions that form this population. Novel autologous antigens for these cells have been identified, although it has not yet been resolved if there is single endogenous antigen responsible for both positive selection and/or peripheral activation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Invariant NKT (iNKT) cells are a conserved αβTCR(+) T cell population that can swiftly produce large amounts of cytokines, thereby activating other leukocytes, including neutrophilic granulocytes (neutrophils). In this study, we investigated the reverse relationship, showing that high neutrophil concentrations suppress the iNKT cell response in mice and humans. Peripheral Vα14 iNKT cells from spontaneously neutrophilic mice produced reduced cytokines in response to the model iNKT cell Ag α-galactosyl ceramide and expressed lower amounts of the T-box transcription factor 21 and GATA3 transcription factor than did wild-type controls. This influence was extrinsic, as iNKT cell transcription factor expression in mixed chimeric mice depended on neutrophil count, not iNKT cell genotype. Transcription factor expression was also decreased in primary iNKT cells from the neutrophil-rich bone marrow compared with spleen in wild-type mice. In vitro, the function of both mouse and human iNKT cells was inhibited by coincubation with neutrophils. This required cell-cell contact with live neutrophils. Neutrophilic inflammation in experimental peritonitis in mice decreased iNKT cell T-box transcription factor 21 and GATA3 expression and α-galactosyl ceramide-induced cytokine production in vivo. This was reverted by blockade of neutrophil mobilization. Similarly, iNKT cells from the human peritoneal cavity expressed lower transcription factor levels during neutrophilic peritonitis. Our data reveal a novel regulatory axis whereby neutrophils reduce iNKT cell responses, which may be important in shaping the extent of inflammation.

Natural killer T (NKT) cells are a T cell subpopulation that were named originally based on coexpression of receptors found on natural killer (NK) cells, cells of the innate immune system, and by T lymphocytes. The maturation and activation of NKT cells requires presentation of glycolipid antigens by CD1d, a cell surface protein distantly related to the major histocompatibility complex (MHC)-encoded antigen presenting molecules. This specificity distinguishes NKT cells from most CD4(+) and CD8(+) T cells that recognize peptides presented by MHC class I and class II molecules. The rapid secretion of a large amount of both Th1 and Th2 cytokines by activated NKT cells endows them with the ability to play a vital role in the host immune defense against various microbial infections. In this review, we summarize progress on identifying the sources of microbe-derived glycolipid antigens recognized by NKT cells and the biochemical basis for their recognition., (© 2012 New York Academy of Sciences.)

Background: Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection., Methodology and Principal Findings: Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5-/- mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5-/- mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d-/- mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922)., Conclusions/significance: These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.

Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, α-galactosyl ceramide (αGalCer), is a potential anticancer agent whose activity depends upon IFN-γ secretion. We report two analogs of αGalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-γ that correlates with detergent-resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen-presenting cells. Structural analysis on one of the analogs indicates that it is more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-γ-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens., Competing Interests: The authors have declared that no competing interests exist.

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.

Invariant NKT cells (iNKT cells) are a unique subset of T lymphocytes that rapidly carry out effector functions. In this study, we report that a majority of sterile house dust extracts (HDEs) tested contained antigens capable of activating mouse and human iNKT cells. HDEs had adjuvant-like properties in an ovalbumin (OVA)-induced asthma model, which were dependent on Vα14i NKT cells, as vaccinated animals deficient for iNKT cells displayed significantly attenuated immune responses and airway inflammation. Furthermore, the administration of HDEs together with OVA mutually augmented the synthesis of cytokines by Vα14i NKT cells and by conventional CD4(+) T cells in the lung, demonstrating a profound immune response synergy for both Th2 cytokines and IL-17A. These data demonstrate that iNKT cell antigens are far more widely dispersed in the environment than previously anticipated. Furthermore, as the antigenic activity in different houses varied greatly, they further suggest that iNKT cell responses to ambient antigens, particular to certain environments, might promote sensitization to conventional respiratory allergens.

NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α-dependent structural changes in the F' roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α-galactosyl diacylglycerol-CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.