Potent immune-modulating and anticancer effects of NKT cell stimulatory glycolipids

Alpha-galactosylceramide (alpha-GalCer), a glycolipid that stimulates natural killer T (NKT) cells to produce both T helper (Th)1 and Th2 cytokines, has shown antitumor effects in mice but failed in clinical trials. We evaluated 16 analogs of alpha-GalCer for their CD1-mediated T cell receptor (TCR) activation of na?ve human NKT cells and their anticancer efficacy. In vitro, glycolipids containing an aromatic ring in their acyl tail or sphingosine tail were more effective than alpha-GalCer in inducing Th1 cytokines/chemokines, TCR activation, and human NKT cell expansion. None of these glycolipids could directly stimulate human dendritic cell maturation, except for a glycolipid with an aromatic ring on the sphingosine tail. Here, we show that glycolipids activated the TCR of NKT cells with phosphorylation of CD3epsilon, ERK1/2, or CREB, which correlated with their induction of Th1 cytokines. Notably, the extent of NKT cell activation when glycolipid was presented by antigen-presenting cells was greater than when glycolipid was presented by non-antigen-presenting cells. In vivo, in mice bearing breast or lung cancers, the glycolipids that induced more Th1-biased cytokines and CD8/CD4 T cells displayed significantly greater anticancer potency than alpha-GalCer. These findings indicate that alpha-GalCer analogs can be designed to favor Th1-biased immunity, with greater anticancer efficacy and other immune-enhancing activities than alpha-GalCer itself.     

Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide

The antimetastatic effect of the CD1d-binding glycolipid, alpha-galactosylceramide (alpha-GalCer), is mediated by NK1.1(+)T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-gamma (IFN-gamma) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-gamma, and NK cells has been shown in the antimetastatic activity of alpha-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of alpha-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-gamma induction and control of lung metastases by alpha-GalCer. IL-18 was unnecessary for alpha-GalCer-related suppression of liver metastases. Most importantly, after adoptive transfer of alpha-GalCer-reactive NKT cells or NK cells into NKT cell-deficient, IFN-gamma-deficient, or RAG-1-deficient mice, it was demonstrated that the sequential production of IFN-gamma by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of alpha-GalCer.     

Glycolipid alpha-C-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice

alpha-Galactosylceramide (alpha-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with alpha-GalCer. We find that a recently described analogue, alpha-C-galactosylceramide (alpha-C-GalCer), more potently induces these innate and adaptive immune responses in mice. alpha-C-GalCer acts as a more effective trigger for IL-12 and IFN-gamma production, although it minimally elicits IL-4 and TNF-alpha release into the serum. Also, alpha-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that alpha-C-GalCer binds more stably to dendritic cells than alpha-GalCer and that dendritic cells loaded with alpha-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, alpha-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, alpha-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.     

The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition

Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.     

Enhancement of ligand-dependent activation of human natural killer T cells by lenalidomide: therapeutic implications

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand alpha-galactosylceramide (alpha-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-gamma. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.     

IFN-gamma-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand,alpha-galactosylceramide

Alpha-galactosylceramide (alpha-GalCer), which is a specific ligand for CD1d-restricted variable-alpha14 chain (V(alpha)14) natural killer T (NKT) cells, exerts a potent antitumor effect. We recently demonstrated that interferon-gamma (IFN-gamma) secreted by both NKT cells and NK cells plays a critical role in mediating the antimetastatic effect of alpha-GalCer; however, the IFN-gamma-dependent antitumor mechanisms remain poorly defined. In the present study, we demonstrate IFN-gamma-dependent inhibition of tumor angiogenesis by alpha-GalCer. In alpha-GalCer-treated mice, subcutaneous tumor growth and tumor-induced angiogenesis were inhibited in an IFN-gamma-dependent manner. The alpha-GalCer-activated splenic or hepatic mononuclear cells inhibited murine endothelial cell proliferation in vitro, and this inhibitory effect was mediated mostly by IFN-gamma produced by NKT cells and NK cells. NK cell depletion resulted in significant but partial inhibition of tumor growth and angiogenesis in vivo. These results suggest that the IFN-gamma-mediated inhibition of tumor angiogenesis is critically involved in the effector mechanisms of antitumor effects evoked by alpha-GalCer.     

Electric foot shock stress-induced exacerbation of alpha-galactosylceramide-triggered apoptosis in mouse liver

Recently, liver natural killer T (NKT) cells, which are specifically stimulated by alpha-galactosylceramide (alpha-GalCer), were found to play a critical role in intrahepatic immunity to several infections and certain hepatic disorders. However, the role of psychophysical stress on NKT cell-dependent liver injury induced by alpha-GalCer still remains to be elucidated. In this study, we employed inescapable electric foot shock as the mode of psychophysical stress and evaluated its effect on alpha-GalCer-induced hepatitis. Pre-exposure of 12 hours of foot shock stress before alpha-GalCer administration significantly enhanced alpha-GalCer-triggered increase in serum alanine aminotransferase levels, followed by increases in both liver caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive hepatocytes, thus indicating that the liver NKT cell-dependent apoptotic response was exacerbated by stress. Foot shock stress also significantly increased both the number of liver NKT cells and Fas expression levels on hepatocytes. Pretreatment with RU-486, a glucocorticoid (GC) receptor antagonist, completely reversed such stress-induced enhancement of the alpha-GalCer-triggered serum alanine aminotransferase and hepatocyte Fas antigen responses. In contrast, such a reversal effect was not found in the mice pretreated with naloxone, a micro-opioid receptor antagonist, which thus suggests that an elevation of endogenous GCs, but not beta-endorphin, as responsible for such stress-induced aggravation in mouse hepatitis models. In conclusion, foot shock stress-induced elevation of endogenous GCs exacerbates alpha-GalCer-initiated hepatic apoptosis through the expansion of liver NKT cells and the up-regulation of hepatocyte Fas antigen.     

Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d

Natural killer T (NKT) cells provide an innate-type immune response upon T cell receptor interaction with CD1d-presented antigens. We demonstrate through equilibrium tetramer binding and antigen presentation assays with Valpha14i-positive NKT cell hybridomas that the Sphingomonas glycolipid alpha-galacturonosyl ceramide (GalA-GSL) is a NKT cell agonist that is significantly weaker than alpha-galactosylceramide (alpha-GalCer), the most potent known NKT agonist. For GalA-GSL, a shorter fatty acyl chain, an absence of the 4-OH on the sphingosine tail and a 6'-COOH group on the galactose moiety account for its observed antigenic potency. We further determined the crystal structure of mCD1d in complex with GalA-GSL at 1.8-A resolution. The overall binding mode of GalA-GSL to mCD1d is similar to that of the short-chain alpha-GalCer ligand PBS-25, but its sphinganine chain is more deeply inserted into the F' pocket due to alternate hydrogen-bonding interactions between the sphinganine 3-OH with Asp-80. Subsequently, a slight lateral shift (>1 A) of the galacturonosyl head group is noted at the CD1 surface compared with the galactose of alpha-GalCer. Because the relatively short C(14) fatty acid of GalA-GSL does not fully occupy the A' pocket, a spacer lipid is found that stabilizes this pocket. The lipid spacer was identified by GC/MS as a mixture of saturated and monounsaturated palmitic acid (C(16)). Comparison of available crystal structures of alpha-anomeric glycosphingolipids now sheds light on the structural basis of their differential antigenic potency and has led to the design and synthesis of NKT cell agonists with enhanced cell-based stimulatory activities compared with alpha-GalCer.     

The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramatic in vivo expansion and surface phenotype alterations after NKT cell activation with alpha-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oral Salmonella infection. Our results indicate that in vivo activation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.     

Valpha24+ natural killer T-cell responses against T-acute lymphoblastic leukaemia cells: implications for immunotherapy

Human Valpha24+ natural killer T (NKT) cells correspond to mouse Valpha14+ NKT cells, both cell types use an invariant T-cell receptor-alpha chain and are activated by glycolipids in a CD1d-dependent manner. Mouse Valpha14+ NKT cells have been reported to have an antitumour effect in vivo. Human Valpha24+ NKT cells can kill a proportion of tumour cells in a CD1d-dependent manner in vitro. We report here that many human leukaemic T-cell lines express CD1d and can be directly killed by Valpha24+ NKT cells. This killing activity was enhanced in the presence of alpha-galactosylceramide (alpha-GalCer), a ligand of Valpha24+ NKT cells. Moreover, primary leukaemic T cells from five of eight T-cell acute lymphoblastic leukaemia (T-ALL) patients expressed CD1d and were good targets of Valpha24+ NKT cells. This cytotoxicity was increased in the presence of alpha-GalCer. Our results suggest that T-ALL is a good candidate for Valpha24+ NKT-cell-based immuno-cell therapy.     

Blockade of NKG2D on NKT cells prevents hepatitis and the acute immune response to hepatitis B virus

Hepatitis B virus (HBV) is a hepadnavirus that is a major cause of acute and chronic hepatitis in humans. Hepatitis B viral infection itself is noncytopathic, and it is the immune response to the viral antigens that is thought to be responsible for hepatic pathology. Previously, we developed a transgenic mouse model of primary HBV infection and demonstrated that the acute liver injury is mediated by nonclassical natural killer (NK)T cells, which are CD1d-restricted, but nonreactive to alpha-GalCer. We now demonstrate a role for NKG2D and its ligands in this nonclassical NKT cell-mediated immune response to hepatitis B virus and in the subsequent acute hepatitis that ensues. Surface expression of NKG2D and one of its ligands (retinoic acid early inducible-1 or RAE-1) are modulated in an HBV-dependent manner. Furthermore, blockade of an NKG2D-ligand interaction completely prevents the HBV- and CD1d-dependent, nonclassical NKT cell-mediated acute hepatitis and liver injury. This study has major implications for understanding activation of NKT cells and identifies a potential therapeutic target in treating hepatitis B viral infection.     

Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface

Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites.     

Activation and function of hepatic NK cells in hepatitis B infection: an underinvestigated innate immune response

Natural killer (NK) cells are abundant in the normal liver, accounting for around one-third of intrahepatic lymphocytes and are important in the defence against hepatitis B virus (HBV) infection as innate immune responses. In this review, we discuss the mechanisms of hepatic NK cell activity against HBV. Whether directly activated by HBV infection or indirectly activated by other lymphocytes such as NKT cells or antigen-presenting cells (APCs), hepatic NK cells exert their anti-viral functions by natural cytotoxicity and production of high levels of cytokines. However, activated NK cells play an important role in regulating adaptive immune responses by interaction with other lymphocytes such as T, B and APCs. In addition, NK cells may contribute to the lymphocyte-mediated liver injury during HBV infection that was previously considered to be mediated only by CD8+ T cells or/and NKT cells.     

Alpha-galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis

Recent studies have revealed significant efficacy of the marine sponge glycolipid, alpha-galactosylceramide (alpha-GalCer), in treatment of experimental metastatic cancers, infections, and autoimmune diseases. However, the capacity of alpha-GalCer to prevent tumor development had never, to our knowledge, been evaluated in mouse models of chemical- and oncogene-dependent carcinogenesis. In this study, we demonstrate that long-term administration of soluble alpha-GalCer, spanning the time of tumor initiation, inhibits primary tumor formation in three different models: methylcholanthrene-induced sarcomas, mammary carcinomas in Her-2/neu transgenic mice, and spontaneous sarcomas in p53-/- mice. Weekly treatment of mice with alpha-GalCer maintained lymphoid tissue natural killer cell and T cell activation and elevated serum IFN-gamma and IL-4 concentrations. Consistent with the antimetastatic activity of alpha-GalCer, prevention of methylcholanthrene-induced sarcoma was IFN-gammaand tumor necrosis factor-related apoptosis-inducing ligand dependent, but not perforin-dependent. Taken together, our results demonstrate that NK1.1+alphabetaTCR+ cell-based immune therapy can inhibit primary tumorigenesis.     

Gamma delta T cells contribute to immunity against the liver stages of malaria in alpha beta T-cell-deficient mice.

The functional role of gamma delta T cells (expressing the gamma delta heterodimeric T-cell receptor for antigen) in infectious diseases remains largely unknown. We have therefore attempted to define the possible role of these T cells in the immune response against the various developmental stages of malaria parasites. For this purpose, we monitored the immune response and the development of liver and blood stages of Plasmodium yoelii, a rodent malaria parasite, in immunized and nonimmunized alpha beta T-cell-deficient and gamma delta T-cell-deficient mice. Immunization of alpha beta T-cell-deficient mice with irradiated sporozoites induced an immune response that significantly inhibited the development of the parasite's liver stages. This inhibitory immune response was abolished by an antibody-mediated transient in vivo depletion of gamma delta T cells. Two gamma delta T-cell clones were derived from malaria-immunized alpha beta T-cell-deficient mice. The adoptive transfer of one of these gamma delta T-cell clones to normal mice inhibited the development of liver stages, following sporozoite inoculation. These results provide evidence for gamma delta T-cell-mediated protective immunity against parasites, in the absence of alpha beta T cells. As for the blood phase of the infection, both normal mice and gamma delta T-cell-deficient mice cleared the blood stages of the nonlethal strain of P. yoelii, while alpha beta T-cell-deficient mice failed to control the parasitemia.     

Fighting malaria with engineered symbiotic bacteria from vector mosquitoes

The most vulnerable stages of Plasmodium development occur in the lumen of the mosquito midgut, a compartment shared with symbiotic bacteria. Here, we describe a strategy that uses symbiotic bacteria to deliver antimalaria effector molecules to the midgut lumen, thus rendering host mosquitoes refractory to malaria infection. The Escherichia coli hemolysin A secretion system was used to promote the secretion of a variety of anti-Plasmodium effector proteins by Pantoea agglomerans, a common mosquito symbiotic bacterium. These engineered P. agglomerans strains inhibited development of the human malaria parasite Plasmodium falciparum and rodent malaria parasite Plasmodium berghei by up to 98%. Significantly, the proportion of mosquitoes carrying parasites (prevalence) decreased by up to 84% for two of the effector molecules, scorpine, a potent antiplasmodial peptide and (EPIP)(4), four copies of Plasmodium enolase-plasminogen interaction peptide that prevents plasminogen binding to the ookinete surface. We demonstrate the use of an engineered symbiotic bacterium to interfere with the development of P. falciparum in the mosquito. These findings provide the foundation for the use of genetically modified symbiotic bacteria as a powerful tool to combat malaria.     

CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses

A variety of tumor-derived antigens have been defined by IgG antibodies in tumor bearers' sera with serological identification of antigens by recombinant expression cloning (SEREX), a serological expression cloning method. The majority of these antigens show no structural abnormality and seem to be wild-type autoantigens. Coimmunization with DNA encoding these autoantigens and tumor-specific cytotoxic T lymphocytes epitopes heightened CD8+ T cell responses and increased resistance to tumor challenge in a CD4+ T cell-dependent manner. In contrast, immunization with these SEREX-defined autoantigens alone leads to heightened susceptibility to tumor challenge. This suppressive effect of immunization is mediated by CD4+ CD25+ T cells. In mice immunized with one of the SEREX-defined autoantigens, Dna J-like 2, the number of alpha-GalCer/CD1d tetramer+ CD3+ T cells [representing natural killer T (NKT) cells] was reduced in the pulmonary compartment, whereas no evident change in the number of other T cell subsets was observed. Experiments with Jalpha281-/- mice lacking most NKT cells indicate that NKT cells are primarily responsible for metastasis suppression and that their activity is inhibited by immunization with Dna J-like 2. We propose that SEREX identifies a pool of autoantigens that maintains and regulates immunological homeostasis via CD4+ CD25+ regulatory T cells.     

Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist alpha-galactosyl ceramide (alpha-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, alpha-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to alpha-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-gamma secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with alpha-GalCer. Because alpha-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.     

Application of Natural Killer T-Cells to Posttransplantation Immunotherapy

Graft-versus-host disease (GVHD) and graft-versus leukemia (GVL) effects are closely related to each other after allogeneic stem cell transplantation. This association exists because of the extensive and complicated interaction between cellular donor components and recipient components concomitant with cytokine storms. It has been demonstrated that part of this interaction may be related to the induction of a variety of regulatory cells, such as regulatory T-cells and natural killer T (NKT) cells. A lower number of NKT cells may be found in patients with autoimmune diseases, cancer, viral infection, and severe GVHD. When activated, NKT cells rapidly release suppressive cytokines, such as interleukin 4 (IL-4), IL-10, and IL-13, as well as inflammatory cytokines, such as interferon gamma and tumor necrosis factor alpha. NKT cells therefore act as a double-edged sword in their progressive or suppressive effects on diseases. Such contradictory phenomena may be related to the function or types of antigen-presenting cells (APCs) in response to their ligand. A single-dose injection of a ligand for NKT cells, alpha-galactosylceramide (alpha-GalCer), can induce immunity through fully mature dendritic cells in an antigen-specific manner. By contrast, multiple injections of alpha-GalCer would induce tolerance, which may be caused by immature APCs. This response suggests that the function of NKT cells can be determined by alpha-GalCer for controlling the immune response. Furthermore, activation of NKT cells followed by activation of APCs and IL-12 production may lead to activation of NK cells and suppress GVHD in mismatched major histocompatibility complex combinations or may induce GVL effects. Control and modification of NKT cell function may play an important role in regulating GVHD/GVL effects.