Randal D. Goff

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.

Synthesis and NKT cell stimulating properties of fluorophore- and biotin-appended 6"-amino-6"-deoxy-galactosylceramides.

Alpha-galactosylceramides are potent stimulators of human T cells. Stimulation occurs through binding of the glycolipids by CD1d, presentation to T cells, and formation of a CD1d-glycolipid-T cell receptor complex. To facilitate the elucidation of the structural features of glycolipids necessary for T cell stimulation, alpha-galactosylceramides have been prepared with small molecules appended at the C6 position of the sugar. The appended molecules do not significantly influence the abilities of the glycolipids to stimulate T cells. [reaction: see text]

Synthesis and evaluation of stimulatory properties of Sphingomonadaceae glycolipids

Glycosphingolipids (GSLs) from the Sphingomonadaceae family of bacteria have been reported to be potent stimulators of natural killer T cells. These glycolipids include mono-, tri- and tetraglycosylceramides. Here we have prepared the GSL-1 to GSL-4 series of glycolipids and tested their abilities to stimulate natural killer T cells. Among these glycolipids, only GSL-1 (1) is a potent stimulator. Using a series of synthetic diglycosylceramides, we show that oligoglycosylceramides from Sphingomonadaceae are not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poor substrates for lysosomal acyltransfer enzymes.

Enhancing the Divergent Activities of Betulinic Acid via Neoglycosylation

Using neoglycosylation, the impact of differential glycosylation upon the divergent anticancer and anti-HIV properties of the triterpenoid betulinic acid (BA) was examined. Each member from a library of 37 differentially glycosylated BA variants was tested for anticancer and anti-HIV activities. Enhanced analogs for both desired activities were discovered with the corresponding antitumor or antiviral enhancements diverging, on the basis of the appended sugar, into two distinct compound subsets.

Enhancement of cyclopamine via conjugation with nonmetabolic sugars.

The Veratrum alkaloid cyclopamine, an inhibitor of cancer stem cell growth, was used as a representative scaffold to evaluate the inhibitory impact of glycosylation with a group of nonmetabolic saccharides, such as d-threose. In a five-step divergent process, a 32-member glycoside library was created and assayed to determine that glycosides of such sugars notably improved the GI50 value of cyclopamine while metabolic sugars, such as d-glucose, did not.

Alpha Anomers of iGb3 and Gb3 Stimulate Cytokine Production by Natural Killer T Cells

Natural killer T cells (NKT cells) respond to presentation of specific glycolipids with release of a variety of proinflammatory and immunomodulatory cytokines. The repertoire of glycolipid antigens for these cells includes alpha-glycosylceramides, alpha-glycosyldiacylglycerols, and the triglycosylceramide iGb3. Two features of iGb3 set it apart from these other antigens: (i) three sugars are required for stimulation and (ii) the glycosidic bond between ceramide and the proximal sugar is beta in iGb3, whereas it is alpha in other antigens. We have synthesized the alpha versions of iGb3 and Gb3 and demonstrate that they are effective antigens for NKT cells and that they do not require lysosomal processing to the monoglycosylceramides for stimulation. These triglycosylceramides constitute a new class of antigen that stimulates NKT cells comparably to monoglycosylceramides.