SUMMARY Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, α-galactosyl ceramide (αGalCer), is a potential anti-cancer agent whose activity depends upon IFN-γ secretion. Here we report two analogs of αGalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-γ that correlates with detergent resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen presenting cells. Structural analysis on one of the analogs indicates that it is bound more deeply inside the CD1d groove, suggesting tighter lipid-CD1d interactions. This is the first example where structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-γ inducing compounds, and since our compounds activate human NKT cells, they could have therapeutic utility.

Activation of invariant natural killer T (iNKT) cells with the model antigen α-galactosylceramide (αGalCer) induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with αGalCer, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hypo-responsive state, however, remain poorly defined. Here, we show that Th1-biasing iNKT cell antigens could induce iNKT cell hypo-responsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing antigen OCH did not induce a hypo-responsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, while DCs and B cells have been reported to be essential for iNKT cell stimulation, neither DCs nor B cells were required to induce iNKT cell hypo-responsiveness. Therefore, our data indicate that while some bone marrow-derived cells could induce iNKT cell hypo-responsiveness, selective conditions, dependent on the structure and potency of the antigen, were required to induce hypo-responsiveness.

Background:Several synthetic glycosphingolipids have been tested to determine their capacity to activate type I NKT cells. Results: Although the TCR binds with high affinity to all CD1d-presented glycolipids, only a few activate type I NKT cells in vivo. Conclusion: TCR binding affinity does not necessarily predict antigenicity in vivo.Significance: The prediction of the therapeutic efficacy of type I NKT cell antigens requires complementary assays.

The first rearrangement of 2-methyleneoxetanes to α,β-unsaturated methylketones is reported. It is proposed that when these substrates are heated, the corresponding oxetenes are formed and subsequently undergo electrocyclic ring-opening to methyl vinylketones. In particular, α-silyl-α,β-unsaturated methylketones were isolated in moderate to high yields and with high stereoselectivities. Based on the proposed mechanism, density functional theory explains the differential kinetics and stereoselectivities among substrates.

An unanticipated cleavage of 2-azido-2-(hydroxymethyl)oxetanes is reported. In attempts to oxidize the title oxetanyl alcohols to the corresponding carboxylic acids with RuO4, cleaved nitriles were formed as the sole isolable products, while a closely related tetrahydrofuran gave solely the expected carboxylic acid. Quantum chemical calculations suggest that the divergent outcomes are governed by conformational differences in the azidoalcohols.