Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Vα14+ NK T cells, and a panel of Vα3/Vα8+ CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d+ antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-γ and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases.

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Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Invariant NKT (iNKT) cells recognize glycolipid Ags, such as the marine sponge-derived glycosphingolipid ␣-galactosylceramide (␣GalCer) presented by the CD1d protein. In vivo activation of iNKT cells with ␣GalCer results in robust cytokine production, followed by the acquisition of an anergic phenotype. Here we have investigated mechanisms responsible for the establishment of ␣GalCer-induced iNKT cell anergy. We found that ␣GalCer-activated iNKT cells rapidly up-regulated expression of the inhibitory costimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of ␣GalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of ␣GalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1/PD-L pathway failed to prevent bacterial-or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1/PD-L interactions enhanced the antimetastatic activities of ␣GalCer. Collectively, our findings reveal a critical role for the PD-1/PD-L costimulatory pathway in the ␣GalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.

TCR-mediated recognition of β-linked self-glycolipids bound to CD1d is poorly understood. Here, we have characterized the TCR repertoire of a CD1d-restricted type II NKT cell subset reactive to sulfatide involved in the regulation of autoimmunity and antitumor immunity. The sulfatide/CD1d-tetramer + cells isolated from naïve mice show an oligoclonal TCR repertoire with predominant usage of the Vα3/Vα1-Jα7/Jα9 and Vβ8.1/Vβ3.1-Jβ2.7 gene segments. The CDR3 regions of both the α- and β-chains are encoded by either germline or nongermline gene segments of limited lengths containing several conserved residues. Presence of dominant clonotypes with limited TCR gene usage for both TCR α- and β-chains in type II NKT cells reflects specific antigen recognition not found in the type I NKT cells but similar to the MHC-restricted T cells. Although potential CD1d-binding tyrosine residues in the CDR2β region are conserved between most type I and type II NKT TCRs, CDR 1α and 3α regions differ significantly between the two subsets. Collectively, the TCR repertoire of sulfatide-reactive type II NKT cells exhibits features of both antigen-specific conventional T cells and innate-like cells, and these findings provide important clues to the recognition of β-linked glycolipids by CD1d-restricted T cells in general.