Glycolipids for natural killer T cells

Natural killer T cells (NKT cells) play a central role in regulating immune responses influencing conditions ranging from autoimmune to infectious diseases. NKT cell responses are induced by recognition of glycolipid antigens presented by CD1d, an antigen presentation protein. In the last 10 years great strides have been made in understanding the types of glycolipids recognized by NKT cells. These advances have included determination of the lipid and carbohydrate recognition requirements for stimulation and identification of "natural" antigens for these cells.     

Synthetic alpha-mannosyl ceramide as a potent stimulant for an NKT cell repertoire bearing the invariant Valpha19-Jalpha26 TCR alpha chain

A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.     

Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation: Implications for Natural Killer T-Cell Biology

α-1,3-Terminated galactose residues on glycoproteins and glycosphingolipids are recognized by natural anti-α-1,3-galactose antibodies in human serum and cause hyperacute rejection in pig-to-human xenotransplantation. Genetic depletion of α-1,3-galactosyl- transferase-1 in pigs abolishes the hyperacute rejection reaction. However, the isoglo- botriosylceramide (iGb3) synthase in pigs may produce additional α-1,3-terminated galactose residues on glycosphingolipids. In both α-1,3-galactosyltranserase-1 knockout mice and pigs, cytotoxic anti-α-1,3-galactose antibodies could be induced; thus, a paradox exists that anti-α-1,3-galactose antibodies are present in animals with functional iGb3 synthases. Furthermore, iGb3 has been found to be an endogenous antigen for natural killer T (NKT) cells, an innate type of lymphocyte that may initiate the adaptive immune responses. It has been reasoned that iGb3 may trigger the activation of NKT cells and cause the rejection of α-1,3-galactosyltransferase-1-deficient organs through the potent stimulatory effects of NKT cells on adaptive immune cells (see ref.^[20]). In this study, we examined the expression of iGb3 and the isoglobo-series glycosphingolipids in pig organs, including the heart, liver, pancreas, and kidney, by ion-trap mass spectrometry, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 μg/mL of the total iGb3/Gb3 mixture is present (see ref.^[35]). We did not detect iGb3 or other isoglobo-series glycosphingolipids in any of these organs, although they were readily detected in mouse and human thymus and dendritic cells. The lack of iGb3 and isoglobo-series glycosphingolipids in pig organs indicates that iGb3 is unlikely to be a relevant immune epitope in xenotransplantation.     

Effects of lipid chain lengths in alpha-galactosylceramides on cytokine release by natural killer T cells

Glycolipid presentation by CD1 proteins has emerged as an important aspect of antigen recognition, and presentation of alpha-glycosylceramides by CD1d to natural killer T cells has become a central focus in understanding how glycolipid presentation can influence immune responses. An alpha-galactosylceramide containing relatively long lipid chains has been the subject of intense study because, when presented by CD1d to natural killer T cells, it stimulates the release of both proinflammatory and immunomodulatory cytokines. Using an efficient synthesis of alpha-galactosylceramides, we have prepared a series of glycolipids in which the lipid chain lengths have been incrementally varied. The responses of natural killer T cells to these glycolipids have been determined, and we have found that truncation of the phytosphingosine lipid chain increases the relative amounts of immunomodulatory cytokines released. In similar fashion, the length of the acyl chain in alpha-galactosylceramides influences cytokine release profiles.     

NKT cell-dependent glycolipid–peptide vaccines with potent anti-tumour activity

It is known that T cells can eliminate tumour cells through recognition of unique or aberrantly expressed antigens presented as peptide epitopes by major histocompatibility complex (MHC) molecules on the tumour cell surface. With recent advances in defining tumour-associated antigens, it should now be possible to devise therapeutic vaccines that expand specific populations of anti-tumour T cells. However there remains a need to develop simpler efficacious synthetic vaccines that possess clinical utility. We present here the synthesis and analysis of vaccines based on conjugation of MHC-binding peptide epitopes to α-galactosylceramide, a glycolipid presented by the nonpolymorphic antigen-presenting molecule CD1d to provoke the stimulatory activity of type I natural killer T (NKT) cells. The chemical design incorporates an enzymatically cleavable linker that effects controlled release of the active components in vivo. Chemical and biological analysis of different linkages with different enzymatic targets enabled selection of a synthetic vaccine construct with potent therapeutic anti-tumour activity in mice, and marked in vitro activity in human blood.     

The stimulating adventure of KRN 7000

Associated with the CD1d protein, KRN 7000, a potent synthetic α-galactosylceramide, is known to activate the invariant NKT immune cells. This stimulation then leads to the production of different cytokines modulating a T(H)1/T(H)2 immune response balance involved in protection against several pathologies such as autoimmune diseases and cancers. Various efforts have been made toward the synthesis of simple and more functionalized analogues in order to selectively induce T(H)1 or T(H)2-type cytokine production. Since the discovery of KRN 7000, structure-activity relationships, crystallographic and modelling studies have pointed to the potential of several GalCer analogues in term of selective bioactivity, and have highlighted interesting elements in order to better understand the recognition and activation mechanisms of immune iNKT cells. By presenting an up-to-date library of analogues, collecting recent breakthroughs done in crystallography and molecular modelling, and relating them to the available biological results, we hope that this review will highlight and help the scientific community in their KRN research.     

Nootkatone,a Dietary Fragrant Bioactive Compound,Attenuates Dyslipidemia and Intramyocardial Lipid Accumulation and Favorably Alters Lipid Metabolism in a Rat Model of Myocardial Injury: An In Vivo and In Vitro Study

In the present study, we assessed whether nootkatone (NKT), a sesquiterpene in edible plants, can provide protection against dyslipidemia, intramyocardial lipid accumulation, and altered lipid metabolism in a rat model of myocardial infarction (MI) induced by subcutaneous injections of isoproterenol (ISO, 85 mg/kg) on days 9 and 10. The rats were pre- and co-treated with NKT (10 mg/kg, p.o.) administered daily for 11 days. A significant reduction in the activities of myocardial creatine kinase and lactate dehydrogenase, as well as non-enzymatic antioxidants, and alterations in lipids and lipoproteins, along with a rise in plasma lipid peroxidation and intramyocardial lipid accumulation, were observed in ISO-treated rats. ISO administration induced alterations in the activities of enzymes/expressions that played a significant role in altering lipid metabolism. However, NKT treatment favorably modulated all biochemical and molecular parameters altered by ISO and showed protective effects against oxidative stress, dyslipidemia, and altered lipid metabolism, attributed to its free-radical-scavenging and antihyperlipidemic activities in rats with ISO-induced MI. Additionally, NKT decreased the accumulation of lipids in the myocardium as evidenced from Oil red O staining. Furthermore, the in vitro observations demonstrate the potent antioxidant property of NKT. The present study findings are suggestive of the protective effects of NKT on dyslipidemia and the underlying mechanisms. Based on our findings, it can be suggested that NKT or plants rich in NKT can be promising for use as a phytopharmaceutical or nutraceutical in protecting the heart and correcting lipid abnormalities and dyslipidemia, which are risk factors for ischemic heart diseases.     

Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most prevalent. Multiple studies have demonstrated that galactosylceramides are more potent stimulators of NKT cells than their glucose isomers. To determine if this stereoselectivity is retained in the context of the C6-oxidized sugars found in bacterial glycolipids, we prepared two sets of gluco and galacto-glycolipids oxidized at their C6 positions and compared their NKT stimulatory properties. In the context of carboxylic acid groups at C6, gluco stereochemistry gave the more potent responses. We also prepared bacterial glycolipids containing more complex ceramide groups to determine if these chains impact NKT cell responses.     

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8⁺ iNKT cells in mice raised the question of whether CD8⁺ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8⁺ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Vα14⁺ transgenic mice, where the Vα14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8⁺ iNKT cells which respond to the NKT cell-specific glycolipid ligand α-galactosylceramide. Unlike conventional iNKT cells, CD8⁺ iNKT cells produce predominantly IFN-γ but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8⁺ iNKT cells in wild type mice. Our results suggest that CD8⁺ NKT cells do exist in mice, although their population size is quite small. Their T_h1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.     

Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.     

Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production

The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates T(H)1 and T(H)2 chemokine release. In this study, a variety of glycolipid ligands were attached to a microarray surface and their binding with dimeric CD1d was investigated. An alpha-galactosyl ceramide (alpha-GalCer) bearing a carbamate group at the 6'-OH position was tethered to the surface, and the dissociation constant on surface with CD1d was determined to reflect the multivalent interaction. Competition assays were then used to determine the dissociation constants (Ki) of new and intact glycolipids in solution. The 4-fluorophenyloctanoyl-modified alpha-GalCer (18) was found to bind most strongly with CD1d (Ki 0.21 microM), 2 orders of magnitude stronger than alpha-GalCer and more than three times more selective than alpha-GalCer for IFN-gamma release from NKT cells. Various alpha-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-gamma production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the T(H)1 pathway.     

Synthesis of Multivalent Galactosides as Targeting Ligand for Gene Delivery

A series of novel bifunctional glycolipid ligands designed to bind with high affinity and specificity to the asialoglycoprotein receptor (ASGP-R) has been synthesized and assayed in vitro on human hepatoma cells, HepG2, derived from parenchymal liver cells. The compounds bear five β-linked Gal moieties linked to the core scaffold, hexa-antennary alcohol, for interaction with the binding site of the ASGP-R. The liposome/DNA complexes containing the glycolipid ligands are efficiently recognized by ASGP-R and exhibited high affinity and transfection activity.     

The requirement of natural killer T-cells in tolerogenic APCs-mediated suppression of collagen-induced arthritis

TGF-β-induced tolerogenic-antigen presenting cells (Tol-APCs) could induce suppression of autoimmune diseases such as collagen-induced arthritis (CIA) and allergic asthma. In contrast, many studies have shown that NKT cells are involved in the pathogenesis of Th1-mediated autoimmune joint inflammation and Th2-mediated allergic pulmonary inflammation. In this study, we investigated the effect of CD1d-restricted NKT cells in the Tol-APCs-mediated suppression of autoimmune disease using a murine CIA model. When CIA-induced mice were treated with Tol-APCs obtained from CD1d^+/- or CD1d^-/- mice, unlike CD1d^+/- APCs, CD1d^-/- Tol-APCs failed to suppress CIA. More specifically, CD1d^-/- Tol-APCs failed to suppress the production of inflammatory cytokines and the induction of Th2 responses by antigen-specific CD4 T cells both in vitro and in vivo. Our results demonstrate that the presence of CD1d-restricted NKT cells is critical for the induction of Tol-APCs-mediated suppression of CIA.     

Sirtuins are crucial regulators of T cell metabolism and functions

It is well known that metabolism underlies T cell differentiation and functions. The pathways regulating T cell metabolism and function are interconnected, and changes in T cell metabolic activity directly impact the effector functions and fate of T cells. Thus, understanding how metabolic pathways influence immune responses and ultimately affect disease progression is paramount. Epigenetic and posttranslational modification mechanisms have been found to control immune responses and metabolic reprogramming. Sirtuins are NAD⁺-dependent histone deacetylases that play key roles during cellular responses to a variety of stresses and have recently been reported to have potential roles in immune responses. Therefore, sirtuins are of significant interest as therapeutic targets to treat immune-related diseases and enhance antitumor immunity. This review aims to illustrate the potential roles of sirtuins in different subtypes of T cells during the adaptive immune response.Subject terms: Acetylation, T cells     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Rapid identification of immunostimulatory alpha-galactosylceramides using synthetic combinatorial libraries

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.     

Chemical and immunological studies of cell surfaces from normal and transformed cells.

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-125I or [3H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.     

Modification in the side chain of solomonsterol A: discovery of cholestan disulfate as a potent pregnane-X-receptor agonist

Seven synthetic analogues of the PXR (pregnane-X-receptor) potent natural agonist solomonsterol A were prepared by total synthesis. Their activity toward PXR was assessed by transactivation and RT-PCR assays. The study discloses cholestan disulfate (8) as a new, simplified agonist of PXR. By in vitro studies on hepatic cells we have demonstrated that this compound is a potent PXR agonist and functional characterization in human macrophages and hepatic stellate cells provided evidence that cholestan disulfate (8) has the ability to modulate the immune response triggered by bacterial endotoxin as well as to counter-activate hepatic stellate cell activation induced by thrombin. Because inhibition of immune-driven circuits might have relevance in the treatment of inflammation and liver fibrosis, the present data support the development of cholestan disulfate (8) in preclinical models of inflammatory diseases.     

Recent Advancements on Immunomodulatory Mechanisms of Resveratrol in Tumor Microenvironment

Immunomodulation of the tumor microenvironment is emerging as an important area of research for the treatment of cancer patients. Several synthetic and natural agents are being investigated for their ability to enhance the immunogenic responses of immune cells present in the tumor microenvironment to impede tumor cell growth and dissemination. Among them, resveratrol, a stilbenoid found in red grapes and many other natural sources, has been studied extensively. Importantly, resveratrol has been shown to possess activity against various human diseases, including cancer. Mechanistically, resveratrol has been shown to regulate an array of signaling pathways and processes involving oxidative stress, inflammation, apoptosis, and several anticancer effects. Furthermore, recent research suggests that resveratrol can regulate various cellular signaling events including immune cell regulation, cytokines/chemokines secretion, and the expression of several other immune-related genes. In this review, we have summarized recent findings on resveratrol’s effects on immune regulatory cells and associated signaling in various cancer types. Numerous immunomodulatory effects of resveratrol suggest it may be useful in combination with other cancer therapies including immunotherapy for effective cancer management.     

Near-infrared exposure changes cellular responses to ionizing radiation

Near infrared (NIR) and X-rays are radiations from different sides of the wavelength spectrum but both are used during medical treatments, as they have severe impacts on cellular processes, including metabolism, gene expression, proliferation and survival. However, both radiations differ strictly in their consequences for exposed patients: NIR effects are generally supposed to be positive, mostly ascribed to a stimulation of metabolism, whereas X-ray leads to genetic instability, an increase of reactive oxygen species (ROS) and DNA damages and finally to cellular death by apoptosis in tumor cells. Since genomic stability after X-irradiation depends on the mitochondrial metabolism, which is well known to be regulated by NIR, we analyzed the impact of NIR on cellular responses of fibroblasts, retinal progenitor cells and keratinocytes to X-radiation. Our data show that previous exposure to naturally occurring doses of nonthermal NIR combined with clinically relevant X-ray doses leads to (1) increased genomic instability, indicated by elevated ratios of mitotic catastrophes, (2) increased ROS, (3) higher amounts of X-irradiated cells entering S-phase and (4) impaired DNA double-strand break repair. Taken together, our data show tremendous effects of NIR on cellular responses to X-rays, probably affecting the results of radiotherapy after NIR exposure during cancer treatment.