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.