CD1d presents lipid-based antigens (Ag) that are recognised by the semi-invariant T cell receptor (TCR) expressed on Natural Killer T (NKT) cells. While the TCR α-chain is typically invariant, the TCR β-chain expression is more diverse, particularly in mice where at least three different Vβ chains are commonly expressed. We report the structures of Vα14-Vβ8.2 and Vα14-Vβ7 NKT TCRs in complex with CD1d-α-galactosylceramide (α-GalCer), as well as a 2.5 Å structure of the human NKT TCR-CD1d-α-GalCer complex. Both Vβ8.2 and Vβ7 NKT TCRs, as well as the human NKT TCR, ligated CD1d-α-GalCer in a broadly similar manner, thereby highlighting the evolutionarily-conserved nature of this interaction. However, differences within the Vβ domains of the Vβ8.2 and Vβ7 NKT TCR-CD1d complexes not only resulted in altered TCR-β-CD1d-mediated contacts, but also surprisingly modulated recognition mediated by the invariant α-chain. Mutagenesis studies revealed the differing contributions of Vβ8.2 and Vβ7 residues within the CDR2β loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR Vβ usage in NKT cells.

NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4 ؊ NK1.1 ؊ NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2-3 h of activation. On intrathymic transfer these cells develop into mature CD4 ؊ NK1.1 ؉ but not into CD4 ؉ NK1.1 ؉ NKT cells, indicating that CD4 ؊ NK1.1 ؊ NKT cells include an IL-17-producing subpopulation, and also mark the elusive branch point for CD4 ؉ and CD4 ؊ NKT cell sublineages.cytokines ͉ CD1d ͉ thymus ͉ T cell

[IL]-4, IL-10, IL-13). 4,9-11 NKT cells have also been reported to exhibit direct cytotoxicity against tumor target cells, 8,[12][13][14] which has made it difficult to predict the consequences of their activation in vivo but nonetheless has caused much speculation that they play a central role in immunoregulation. Accumulated experimental evidence has supported their role in promoting innate antitumor immunity 8,14-17 while paradoxically suppressing acquired antitumor immunity 18 and autoimmunity 19 and maintaining some forms of tolerance. [20][21][22] These results have ensured continued debate and confirmed the central role these cells play in the immune system. 23,24 The ␣-galactosylceramide (␣-GalCer) is chemically and functionally analogous to natural glycolipids first purified from marine sponges on the basis of their antitumor properties against B16 melanoma. 25,26 ␣-GalCer is presented by CD1d, leading to specific stimulation of NKT cells. 4,10,11,27 The antitumor effect of ␣-GalCer is observed against various tumor cells of different origins, including melanomas, lung, colon, and renal cell carcinomas, erythroleukemias, and other hematopoietic malignancies. 25,26,[28][29][30] In vivo, the antitumor activities of ␣-GalCer and IL-12 are similar, whereas in vitro it has been demonstrated that the production of IFN-␥ by NKT cells in response to ␣-GalCer requires IL-12 produced by dendritic cells (DCs) 31 and direct contact between NKT cells and DCs through CD40-CD40 ligand interactions. Both ␣-GalCer and low doses of IL-12 are strong inducers of NKT cell activity and will not exert their antitumor activities in the absence of NKT cells. 8,[15][16][17] Despite these findings, little is known about the precise sequence of events and factors involved in ␣-GalCerinduced tumor suppression.We know that recognition of the ␣-GalCer-CD1d complex leads to NKT cell activation. 10,11,27 This results in rapid production (within hours) of Th1 and Th2 cytokines by NKT cells, 32 rapid elimination of NKT cells after they produce cytokines, [33][34][35][36] proliferation and activation of NK cells 4,10,32,37 and subsequent IFN-␥ production, 38 and bystander activation of immune responses mediated by conventional T cells and B cells. 32,33,[39][40][41][42][43] NK cell activation was at least partly dependent on IFN-␥. 32,33,39 Although ␣-GalCer For personal use only. on May 9, 2018. by guest www.bloodjournal.org From has been shown to induce perforin-dependent cytotoxicity by NKT and NK cells, the significance of this pathway in vivo is less clear. Our previous study 30 and more recently that of Hayakawa et al 38 have demonstrated that the antimetastatic effect of ␣-GalCer was impaired in NK cell-depleted or IFN-␥-deficient mice. Collectively, these results indicate an important role for NKT and NK cells and the cytokine IFN-␥ after ␣-GalCer administration. Despite these clues, none of the previous studies have defined the sequence of events after ␣-GalCer treatment, nor was it known whether IFN-␥ production by NKT cells, NK...