The glycosphingolipid sulfatide (SO3-3Galβ1Cer) is a demonstrated ligand for a subset of CD1d-restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet β-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic β-cells, is one of several natural ligands for type II CD1d-restricted NKT cells.

CD1d-restricted invariant natural killer T (iNKT) cells are known as potent early regulatory cells of immune responses. Besides the established roles in the regulation of inflammation and autoimmune disease, studies have shown that iNKT cells have important roles in tumor surveillance and the control of tumor metastasis. Here we found that absence of iNKT cells dramatically decreased the total number of intestinal polyps in APCMin/+ mice, a model for colorectal cancer. Polyp iNKT cells were enriched for IL-10 and IL-17 producing cells, showed a distinct phenotype being CD4+, NK1.1− CD44int and PD-1lo, and they were negative for the NKT cell transcription factor PLZF. Absence of iNKT cells was associated with a reduced frequency of Treg cells and lower expression levels of FoxP3 protein and transcript uniquely in the polyps, and a switch to an inflammatory macrophage phenotype. Moreover, in iNKT cell deficient APCMin/+ mice, expression of T helper (TH) 1-associated genes, such as IFN-γ and Nos2, was increased in polyps, concomitantly with elevated frequencies of conventional CD4+ and CD8+ T cells in this tissue. The results suggest that a population of regulatory iNKT cells locally promote intestinal polyp formation by enhancing Treg cells and immunosuppression of anti-tumor TH1-immunity.

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental autoimmune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of β-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated β-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingolipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells. antigen presenting molecule CD1d, expressed predominantly by hematopoietic cells. Upon activation, NKT cells display innatelike features such as rapid production of large amounts of both inflammatory and anti-inflammatory cytokines, including IFN-γ and IL-4, and they enhance DC maturation. Through these features, they are important in the initiation of innate immunity and also in the direction and regulation of the adaptive immune response resulting in the modulation C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2852 Sara Rhost et al. Eur. J. Immunol. 2012. 42: 2851-2860 of tumor immunity, autoimmunity, and microbial infections [1][2][3]. NKT cells can be activated both by microbial and endogenous lipids. The latter may be upregulated in antigen presenting cells (APCs) upon their activation, or be derived from other cells of the body and presented by APCs [4][5][6]. Sulfatide was among the first self-glycosphingolipids demonstrated to stimulate T lymphocytes [7]. Sulfatide is found in large amounts in the central nervous system, mainly located in the myelin, a target of the autoimmune process during multiple sclerosis. Indeed, the frequencies of sulfatide-reactive T cells were increased in multiple sclerosis patients [7], and sulfatide-reactive CD1d-restricted NKT cells accumulated in the CNS of mice during experimental autoimmune encephalomyelitis [8], a model for the human disease, suggesting that the cells were engaged in the disease process. This notion was supported by studies showing that sulfatide injection protected CD1d-sufficient mice from the disease, consistent with the hypothesis that that sulfatide-reactive NKT cells could suppress the autoimmune process ...

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic β cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4+ 24αβ type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4+ BDC2.5 NOD T cells in adoptive transfer experiments. CD4+ 24αβ NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αβ NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

Natural killer T (NKT) lymphocytes are implicated in the early response to microbial infection. Further, sulfatide, a myelin selfglycosphingolipid, activates a type II NKT cell subset and can modulate disease in murine models. We examined the role of NKT cells and the effect of sulfatide treatment in a murine model of Staphylococcus aureus sepsis. The lack of CD1d-restricted NKT cells did not alter survival after a lethal inoculum of S. aureus. In contrast, sulfatide treatment significantly improved the survival rate of mice with S. aureus sepsis, accompanied by decreased levels of tumor necrosis factor alpha and interleukin-6 in the blood. The protective effect of sulfatide treatment depended on CD1d but not on type I NKT cells, suggesting that activation of type II NKT cells by sulfatide has beneficial effects on the outcome of S. aureus sepsis in this model.

The glycosphingolipid α-galactosylceramide (α-GalCer) is a well-described immune activator with strong anti-tumor properties in animal models. It is presented on CD1d and acts by stimulating the invariant, type I, natural killer T (iNKT) lymphocytes to rapidly secrete TH1 and TH2 associated cytokines. This in turn promotes activation of a diversity of immune cells including natural killer (NK) cells with anti-tumor functions. Prior to tumor development, iNKT cells can also perform tumor surveillance and naturally protect from emergence of cancer. In contrast, we have recently demonstrated that iNKT cells naturally promote polyps in the spontaneous murine adenomatous polyposis coli (Apc) Apc Min /+ model for colon cancer, associated with suppressed TH1 immunity and enhanced immunoregulation. Here we investigated whether iNKT cell directed immunotherapy could subvert the polyp promoting function of iNKT cells and reduce polyp growth in this model. We treated Apc Min /+ mice with α-GalCer, or synthetic derivatives of this ligand (C-glycoside and C20:2) that have enhanced immunoregulatory properties. Treatment with iNKT cell ligands led to increased iNKT cell division, but reduced iNKT cell frequencies, lower NK1.1 expression and elevation of PD-1. Apc Min /+ mice that had been treated either long-term (5–15 weeks of age), or short-term (12–15 weeks of age) with α-GalCer demonstrated a significant decrease in polyp burden. Surprisingly, long-term treatment with the TH1 biasing ligand C-glycoside did not have significant effects on polyps, while long-term treatment with the TH2 biasing ligand C20:2 enhanced polyp growth. In stark contrast, short-term treatment with C20:2 led to reduction in polyp numbers and size. Reduced polyp burden after long-term treatment was associated with increased expression of genes indicating a pro-inflammatory polyp microenvironment. Polyp-reducing short-term treatment led to CD8 T cell activation specifically in polyps, and decreased tumor infiltrating and splenic macrophages, and a switch toward a pro-inflammatory phenotype. Thus, iNKT cell directed therapy could subvert the natural polyp enhancing function of iNKT cells, overcome immunosuppression, and reduce polyps. However, different iNKT cell activating ligands had opposite effects, and the timing of treatment had a major influence on outcomes.

Natural killer T (NKT) cells are αβ T cell receptor (TCR) expressing innate‐like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I‐like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi‐invariant Vα14‐Jα18 (mouse, Vα24‐Jα18 in humans) TCR reactive to the prototypic ligand α‐galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d‐presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18−/−MHCII−/− mice that harbour type II but not type I NKT cells, and CD1d−/−MHCII−/− mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4−CD8− (double negative, DN) TCRβ+ cells expressing NKG2D or NKG2A/C/E in Jα18−/−MHCII−/− mice compared with CD1d−/−MHCII−/− mice, suggesting that 30%‐50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+, while the DN cells were CD49b+ and CD62L+. Both subsets expressed the NKT cell‐associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN‐γ, but rarely IL‐4 and IL‐17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1‐like cytokine production.

The endogenous glycosphingolipid sulfatide is a ligand for CD1d-restricted type II natural killer T (NKT) lymphocytes. Through the action of these cells, sulfatide treatment has been shown to modulate the immune response in mouse models for autoimmune diseases, infections and tumour immunity. Sulfatide exists naturally in different organs including the pancreas, where sulfatide colocalizes with insulin within the Langerhans islet b-cells, targets for the immune destruction in type 1 diabetes (T1D). Human T1D patients, but not patients with type 2 diabetes nor healthy individuals, have autoantibodies against sulfatide in serum, suggesting that sulfatide induces an immune response in the natural course of T1D in humans. Here, we investigate sulfatide as an autoantigen and a modulator of autoimmune disease in the murine model for T1D, the non-obese diabetic (NOD) mice. We demonstrate that aged NOD mice displayed serum autoantibody reactivity to sulfatide; however, this reactivity did not correlate with onset of T1D. Repeated administration of sulfatide did not result in an increase in serum reactivity to sulfatide. Moreover, a multidose sulfatide treatment of female NOD mice initiated at an early (5 weeks of age), intermediate (8 weeks of age) or late (12 weeks of age) phase of T1D progression did not influence the incidence of disease. Thus, we demonstrate that a fraction of NOD mice develop autoantibody reactivity to sulfatide; however, we fail to demonstrate that sulfatide treatment reduces the incidence of T1D in this mouse strain.