Recognize Lipid Antigens Research Articles

Chapter 8 The Role of NKT Cells in Tumor Immunity

NKT cells are a relatively newly recognized member of the immune community, with profound effects on the rest of the immune system despite their small numbers. They are true T cells with a T cell receptor (TCR), but unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a nonclassical MHC molecule. As members of both the innate and adaptive immune systems, they bridge the gap between these, and respond rapidly to set the tone for subsequent immune responses. They fill a unique niche in providing the immune system a cellular arm to recognize lipid antigens. They play both effector and regulatory roles in infectious and autoimmune diseases. Furthermore, subsets of NKT cells can play distinct and sometimes opposing roles. In cancer, type I NKT cells, defined by their invariant TCR using Valpha14Jalpha18 in mice and Valpha24Jalpha18 in humans, are mostly protective, by producing interferon-gamma to activate NK and CD8(+) T cells and by activating dendritic cells to make IL-12. In contrast, type II NKT cells, characterized by more diverse TCRs recognizing lipids presented by CD1d, primarily inhibit tumor immunity. Moreover, type I and type II NKT cells counter-regulate each other, forming a new immunoregulatory axis. Because NKT cells respond rapidly, the balance along this axis can greatly influence other immune responses that follow. Therefore, learning to manipulate the balance along the NKT regulatory axis may be critical to devising successful immunotherapies for cancer.

Adrénoleucodystrophie liée à l'X

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder. ALD is characterized by progressive demyelination within the central and peripheral nervous system, adrenal insufficiency (Addison's disease) and accumulation of very-long-chain fatty acids (VLCFA) in plasma, fibroblasts and tissues. The overall incidence of ALD is 1:17,000 including hemizygotes and heterozygotes who are frequently symptomatic. There are two main ALD phenotypes: 1) a cerebral demyelinating form which affects boys between 5–12 years, but also 35% of adult males; 2) a form that mainly involves the spinal cord (adrenomyeloneuropathy, AMN) in adult males between 20–50 years and 50% of heterozygous women after the age of 40 years. AMN presents with progressive spastic paraparesis. Addison's disease may be the first symptom of ALD in boys and adult males. These patients are at risk to develop cerebral ALD or AMN for life. ALD results from mutations in the ABCD1 gene without correlation between genotype and phenotype. The diagnosis of ALD relies upon the measurement of plasma VLCFA levels that allows the identification of 100% affected males and of 80–95% heterozygous women. Because of these false-negative, it is therefore mandatory to search for a mutation in the ABCD1 gene in all women at risk to be heterozygous for ALD. The ABCD1 gene encodes a peroxisomal transmembrane protein (ALD protein) with the structure of an half ATP-binding cassette transporter. It is possible that ALD protein imports VLCFA or VLCFA-CoA into peroxisomes in which they are degraded by a peroxisomal β-oxidation system. Elongation of VLCFAs is enhanced in fibroblasts from ALD patients and likely contributes to the load of VLCFA in tissues. The underlying mecanisms that lead to cerebral demyelination, axonal degeneration in spinal cord and adrenal insufficiency are unknown. The « toxic » role of VLCFA accumulation remains to be demonstrated. The mecanisms that lead to the inflammatory reaction in cerebral ALD might involve abnormal acylation of gangliosides and phospholipidis by VLCFA that would result in immune reaction of brain macrophages and astrocytes bearing CD1 molecules that recognize lipid antigens. De novo mutation of ABCD1 occurs in less than 8% of ALD patients. The genetic counseling aims to identify: 1) women who are at risk to be heterozygous; 2) neurologically asymptomatic boys. It is only at this stage that allogeneic bone marrow transplantation has clinical benefit; 3) ALD patients who have Addison's disease that can lead to sudden death. Prenatal diagnosis (chorionic villus samples, cultured amniotic fluid cells) relies upon DNA based mutation detection techniques, expression of ALD protein and measurement of VLCFA levels. Allogeneic bone marrow transplantation is the only treatment that provides a permanent cure when the procedure is performed at an early stage of cerebral demyelination, i.e when the patients are asymptomatic despite abnormal brain MRI. Treatment of Addison's disease is mandatory but does not modify the course of neurological symptoms. Dietary therapy failed to halt the neurologic progression in cerebral ALD and AMN. It might have a partial preventive effect in boys treated before 6 years of age.

Activation of Invariant NKT Cells by Toll-like Receptor 9-Stimulated Dendritic Cells Requires Type I Interferon and Charged Glycosphingolipids

Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-gamma, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged beta-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.

Role of NKT Cells in the Digestive System. III. Role of NKT cells in intestinal immunity

Natural killer T (NKT) cells are a small subset of unconventional T cells that recognize lipid antigens presented by the nonclassical major histocompatibility complex (MHC) class I molecule CD1d. NKT cells are involved in the host response to a variety of microbial pathogens and likely commensals. In the intestine, invariant and noninvariant NKT cells can be found among intraepithelial lymphocytes and in the lamina propria. Activation of intestinal NKT cells by CD1d-expressing intestinal epithelial cells and professional antigen-presenting cells may contribute to induction of oral tolerance and protection from mucosal infections. On the other hand, sustained and uncontrolled activation of NKT cells may play a pivotal role in the pathogenesis of inflammatory bowel disease. Here we review the current literature on intestinal NKT cells and their function in the intestine in health and disease.

MR1‐restricted Vα19i T cells – a second population recognizing lipid antigens?

There is increasing evidence that T cells recognizing lipid antigens contribute to the immunological regulation of different disease conditions including autoimmunity. The best-known subset is CD1d-restricted lipid-reactive T cells characterized by the expression of an invariant TCRalpha chain. Much less is known about the biology of another invariant T cell subset, which is restricted to the MHC class I-like molecule MR1. A beneficial role of MR1-restricted T cells has been suggested in a mouse EAE model. However, the nature of antigens that can be presented by MR1 to this invariant T cell subset remained largely unclear. An article in this issue of the European Journal of Immunology presents strong indications that derivatives of alpha-mannosyl ceramide (alpha-ManCer), i.e. glycolipids, can serve as ligands for MR1-restricted invariant T cells. In addition to that, the structure of the alpha-ManCer sphingosine chain influences the Th1-Th2 polarization of the cytokine response. These important new findings will foster further research on the identity of physiological ligands for MR1-restricted T cells and on their relation with immunoregulation. See accompanying article: (http://dx.doi.org/10.1002/eji.200636689).

Lower prevalence of circulating natural killer T cells in patients with angina: a potential novel marker for coronary artery disease

Atherosclerosis is an inflammatory disease. Natural killer T cells are a unique lymphocyte subset that can recognize lipid antigens presented by CD1d and secrete copious amounts of pro-atherogenic cytokines such as interferon-gamma. We have previously shown that natural killer T cells accelerate atherosclerosis in mice and macrophages incubated with oxidized low-density lipoproteins induce natural killer T cells to produce interferon-gamma. Thus, whether the prevalence of natural killer T cells in peripheral blood is altered in patients with angina pectoris and its correlation with coronary risk factors was determined. Cell profiling was performed using flow cytometry in patients with stable angina, unstable angina (Braunwald IIIB), and healthy controls. Natural killer T cells in peripheral blood were identified by the expression of natural killer T specific invariant T cell receptor alpha-chain (Valpha24) and T cell receptor beta-chain (Vbeta11). Prevalence of natural killer T (Valpha24-Vbeta11 double positive) cells was significantly decreased in patients with unstable angina and stable angina compared with that in controls. No significant differences were observed in the prevalence between unstable and stable angina. Reduction of natural killer T cells was independently associated with the presence of angina. Lower prevalence of circulating natural killer T cells is related to the presence of coronary artery disease. As T cell receptor down-regulation or apoptosis after natural killer T cell activation and subsequent interferon-gamma release may contribute to atherogenesis, natural killer T cells can become a novel therapeutic target for the prevention and treatment of atherosclerotic vascular diseases.

How T lymphocytes recognize lipid antigens

Recognition of lipid antigens by T lymphocytes is well established. Lipids are recognized by T cells when presented in association with CD1 antigen-presenting molecules. Both microbial and self lipids stimulate specific T lymphocytes, thus participating in immune reactions during infections and autoimmune diseases. The immune system uses a variety of strategies to solubilise lipid antigens, to facilitate their internalization, processing, and loading on CD1 molecules. Recent studies in the field of lipid antigen presentation have revealed new mechanisms which allow the immune system to sense lipids as stimulatory antigens.

Serum lipoproteins: Trojan horses of the immune response?

T cells recognizing lipid antigens presented by CD1 molecules have an important role in the immune response. Several lipid antigens for CD1-restricted T cells have been identified, as have some rules of CD1 loading and CD1-restricted presentation. Little is known, however, about the delivery of lipid antigens from either extracellular compartments or CD1-negative cells to CD1-expressing antigen-presenting cells (APCs). A recent study provides evidence for a role for apolipoprotein E in binding lipid antigens and delivering them to APCs.

CD1d-dependent activation of NKT cells aggravates atherosclerosis.

Adaptive and innate immunity have been implicated in the pathogenesis of atherosclerosis. Given their abundance in the lesion, lipids might be targets of the atherosclerosis-associated immune response. Natural killer T (NKT) cells can recognize lipid antigens presented by CD1 molecules. We have explored the role of CD1d-restricted NKT cells in atherosclerosis by using apolipoprotein E–deficient (apoE−/−) mice, a hypercholesterolemic mouse model that develops atherosclerosis. ApoE−/− mice crossed with CD1d−/− (CD1d−/−apoE−/−) mice exhibited a 25% decrease in lesion size compared with apoE−/− mice. Administration of α-galactosylceramide, a synthetic glycolipid that activates NKT cells via CD1d, induced a 50% increase in lesion size in apoE−/− mice, whereas it did not affect lesion size in apoE−/−CD1d−/− mice. Treatment was accompanied by an early burst of cytokines (IFNγ, MCP-1, TNFα, IL-2, IL-4, IL-5, and IL-6) followed by sustained increases in IFNγ and IL-4 transcripts in the spleen and aorta. Early activation of both T and B cells was followed by recruitment of T and NKT cells to the aorta and activation of inflammatory genes. These results show that activation of CD1d-restricted NKT cells exacerbates atherosclerosis.

Activation of a Nonclassical NKT Cell Subset in a Transgenic Mouse Model of Hepatitis B Virus Infection

NKT cells are specialized cells of the immune system that bear both T cell and NK cell markers. Classical NKT cells display TCRs of restricted heterogeneity (Vα14-Jα281) and recognize lipid antigens (e.g., α-galactosyl ceramide) presented by nonpolymorphic CD1 molecules. Recently, other nonclassical NKT subsets have been recognized, including NKT cells not reactive with CD1d-α-galactosyl ceramide complexes. The biological functions of these cells are unknown. Here, we show that nonclassical NKT cells that are CD1d restricted but nonreactive to α-GalCer are activated in response to hepatocytes expressing hepatitis B viral antigens in a transgenic mouse model of acute hepatitis B virus infection. Our results document the first in vivo function for such nonclassical NKT cells and suggest a role for these cells in the host response to HBV infection.

AUTOIMMUNE RESPONSES TO GLYCOLIPIDS IN PARANEOPLASTIC NEUROPATHY: REPORT OF TWO CASES

Glycolipids (GL) have been implicated as potential antigens in autoimmune neuropathies, but their role in paraneoplastic neuropathy has been poorly investigated. We describe two patients affected by lung neoplasia and subacute sensori‐motor axonal neuropathy with high titres of IgG auto‐antibodies against GM1, GD1b, GQ1b, GD1a, GD2, GM2, GM3, GD3, asialo‐GM1 and sulfatide. The first patient showed prevalent sensory axonal neuropathy, ophthalmoplegia and dysphagia. Small cell lung carcinoma was bioptically discovered and treated with chemotherapy and irradiation, which yielded clinical stabilization and decline of anti‐GL IgG titre in a two years follow‐up. The second patient complained of subacute, progressive distal hypostenia. Neurologic examination showed areflexic, hypotonic tetraparesis with marked wasting, fasciculations, mimicking lower motor neuron disease. In addition to anti‐GL IgG, we detected expansion of peripheral γ/δ T lymphocytes (3% of total T cells, n.v. <0.5%), a T cell subset known to recognize lipid antigens. Lung adenocarcinoma was discovered and surgically removed. After two months, neuropathy stabilized and both anti‐GL IgG and γ/δ T lymphocytes markedly declined. Immunohistochemistry on this patients' tumor showed expression of GM1, GD1a, GD2, GM2, GM3, GD3 on neoplastic cells, which were infiltrated by IgG and CD8 and γ/δ T lymphocytes. The association of neoplasia, peripheral neuropathy and autoimmune responses to GL observed in our patients proposes a previously underestimated role for GL as putative antigens in paraneoplastic neuropathy. GL should be considered as candidate antigens in paraneoplastic neuropathy and anti‐GL IgG should be evaluated in patients with subacute sensory and/or motor neuropathy.

Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis.

Natural killer (NK) T cells recognize lipid antigens in the context of the major histocompatibility complex (MHC) class 1-like molecule CD1 and rapidly secrete large amounts of the cytokines interferon (IFN)-gamma and interleukin (IL)-4 upon T cell receptor (TCR) engagement. We have asked whether NK T cell activation influences adaptive T cell responses to myelin antigens and their ability to cause experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. While simultaneous activation of NK T cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) and myelin-reactive T cells potentiates EAE in B10.PL mice, prior activation of NK T cells protects against disease. Exacerbation of EAE is mediated by an enhanced T helper type 1 (Th1) response to myelin basic protein and is lost in mice deficient in IFN-gamma. Protection is mediated by immune deviation of the anti-myelin basic protein (MBP) response and is dependent upon the secretion of IL-4. The modulatory effect of alpha-GalCer requires the CD1d antigen presentation pathway and is dependent upon the nature of the NK T cell response in B10.PL or C57BL/6 mice. Because CD1 molecules are nonpolymorphic and remarkably conserved among different species, modulation of NK T cell activation represents a target for intervention in T cell-mediated autoimmune diseases.

The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice.

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

CD1-restricted NK T cells protect nonobese diabetic mice from developing diabetes.

NK T cells are a unique subset of T cells that recognize lipid antigens presented by CD1d. After activation, NK T cells promptly produce large amounts of cytokines, which may modulate the upcoming immune responses. Previous studies have documented an association between decreased numbers of NK T cells and the progression of some autoimmune diseases, suggesting that NK T cells may control the development of autoimmune diseases. To investigate the role of NK T cells in autoimmune diabetes, we crossed CD1 knockout (CD1KO) mutation onto the nonobese diabetic (NOD) genetic background. We found that male CD1KO NOD mice exhibited significantly higher incidence and earlier onset of diabetes compared with the heterozygous controls. The diabetic frequencies in female mice showed a similar pattern; however, the differences were less profound between female CD1KO and control mice. Early treatment of NOD mice with alpha-galactosylceramide, a potent NK T cell activator, reduced the severity of autoimmune diabetes in a CD1-dependent manner. Our results not only suggest a protective role of CD1-restricted NK T cells in autoimmune diabetes but also reveal a causative link between the deficiency of NK T cells and the induction of insulin-dependent diabetes mellitus.

Repeat catheterization in ventricular septal defect and pulmonary hypertension.

NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer⁺) and type II (sulfatide/CD1d-tetramer⁺) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4⁺/CD8⁺T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.