Sulfatide-reactive type II NKT cells have been shown to regulate autoimmunity and anti-tumor immunity. Although, two major isoforms of sulfatide, C16:0 and C24:0, are enriched in the pancreas, their relative role in autoimmune diabetes is not known. Here, we report that sulfatide/CD1d-tetramer+ cells accumulate in the draining pancreatic lymph nodes, and that treatment of NOD mice with sulfatide or C24:0 was more efficient than C16:0 in stimulating the NKT cell-mediated transfer of a delay in onset from T1D into NOD.Scid recipients. Using NOD.CD1d−/− mice, we show that this delay of T1D is CD1d-dependent. Interestingly, the latter delay or protection from T1D is associated with the enhanced secretion of IL-10 rather than IFN-g by C24:0-treated CD4+ T cells and the deviation of the islet-reactive diabetogenic T cell response. Both C16:0 and C24:0 sulfatide isoforms are unable to activate and expand type I iNKT cells. Collectively, these data suggest that C24:0 stimulated type II NKT cells may regulate protection from T1D by activating DCs to secrete IL-10 and suppress the activation and expansion of type I iNKT cells and diabetogenic T cells. Our results raise the possibility that C24:0 may be used therapeutically to delay the onset and protect from T1D in humans.

SummaryProtection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with a-galactosylceramide (a-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of a-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of a-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened antiinflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c high CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (a-GalCer C26:0) that induces Th1-and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

SummaryWe have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist a-galactosylceramide C26:0 (a-GalCer) or its T helper type 2 (Th2)-biasing derivative a-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to a-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of a-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with a-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by a-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than a-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.