Abstract. Invariant natural killer T (iNKT) cells play a protective role in the development of certain autoimmune diseases. However, their precise role in the pathogenesis of autoimmune arthritis remains unclear. In this study, we examined the possible contribution of iNKT cells in collageninduced arthritis (CIA) by using iNKT cell-deficient mice (Jα281 -/-mice). CIA in these mice was markedly suppressed and interleukin (IL)-17 production was reduced in a native type II collagen (CII)-specific T cell response. Draining lymph nodes of CII-immunized Jα281 -/-mice contained a significantly low number of IL-17-producing T helper cells. To determine whether iNKT cells produce IL-17, we measured IL-17 by enzyme-linked immunosorbent assay in iNKT cells stimulated with the ligand, α-galactosylceramide (α-GalCer). Notably, splenocytes from Jα281 -/-mice stimulated in this way were negative for IL-17, whereas those from C57BL/6 mice produced IL-17. Immunostaining for IL-17 in iNKT cells confirmed intracellular staining of the protein.RT-PCR analysis showed that iNKT cells expressed retinoidrelated orphan receptor γT and IL-23 receptor. Moreover, cell sorting demonstrated that NK1.1 -iNKT cells were the main producers of IL-17 compared with NK1.1 + iNKT cells. IL-17 production by iNKT cells was induced by IL-23-dependent and -independent pathways, since iNKT produced IL-17 when stimulated with either IL-23 or α-GalCer alone. Our findings indicate that iNKT cells are producers and activators of IL-17 via IL-23-dependent and -independent pathways, suggesting that they are key cells in the pathogenesis of CIA through IL-17.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

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SummaryAlpha-carba-GalCer (RCAI-56), a novel synthetic analogue of agalactosylceramide (a-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-g. IFN-g exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of a-carba-GalCer on collageninduced arthritis (CIA) in mice. First, we confirmed that a-carba-GalCer selectively induced IFN-g in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and a-carbaGalCer. The incidence and clinical score of CIA were significantly lower in a-carba-GalCer-treated mice. Anti-IFN-g antibodies abolished the beneficial effects of a-carba-GalCer, suggesting that a-carba-GalCer ameliorated CIA in an IFN-g-dependent manner. Treatment with a-carba-GalCer reduced anti- Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with a-carba-GalCer. In addition, we observed higher IFN-g production by NK T cells in a-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that a-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that a-carbaGalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response. CII antibody production [immunoglobulin (Ig)G and IgG2a] and CIIreactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-g-producing T cells to IL-17-producing T cells (Th1/

ObjectiveInvariant natural killer T (iNKT) cells regulate collagen-induced arthritis (CIA) when activated by their potent glycolipid ligand, alpha-galactosylceramide (α-GalCer). Glucose-6-phosphate isomerase (GPI)-induced arthritis is a closer model of human rheumatoid arthritis based on its association with CD4+ T cells and cytokines such as TNF-α and IL-6 than CIA. Dominant T cell epitope peptide of GPI (GPI325-339) can induce arthritis similar to GPI-induced arthritis. In this study, we investigated the roles of activation of iNKT cells by α-GalCer in GPI peptide-induced arthritis.MethodsArthritis was induced in susceptible DBA1 mice with GPI peptide and its severity was assessed clinically. The arthritic mice were treated with either the vehicle (DMSO) or α-GalCer. iNKT cells were detected in draining lymph nodes (dLNs) by flow cytometry, while serum anti-GPI antibody levels were measured by enzyme-linked immunosorbent assay. To evaluate GPI peptide-specific cytokine production from CD4+ T cells, immunized mice were euthanized and dLN CD4+ cells were re-stimulated by GPI-peptide in the presence of antigen-presenting cells.Resultsα-GalCer induced iNKT cell expansion in dLNs and significantly decreased the severity of GPI peptide-induced arthritis. In α-GalCer-treated mice, anti-GPI antibody production (total IgG, IgG1, IgG2b) and IL-17, IFN-γ, IL-2, and TNF-α produced by GPI peptide-specific T cells were significantly suppressed at day 10. Moreover, GPI-reactive T cells from mice immunized with GPI and α-GalCer did not generate any cytokines even when these cells were co-cultured with APC from mice immunized with GPI alone. In vitro depletion of iNKT cells did not alter the suppressive effect of α-GalCer on CD4+ T cells.Conclusionα-GalCer significantly suppressed GPI peptide-induced arthritis through the suppression of GPI-specific CD4+ T cells.