Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors.

Helicobacter pylori causes gastritis, which has been attributed to the development of H. pylori-specific T cells during infection. However, the mechanism underlying innate immune detection leading to the priming of T cells is not fully understood, as H. pylori evades TLR detection. Here, we report that H. pylori metabolites modified from host cholesterol exacerbate gastritis through the interaction with C-type lectin receptors. Cholesteryl acyl α-glucoside (αCAG) and cholesteryl phosphatidyl α-glucoside (αCPG) were identified as noncanonical ligands for Mincle (Clec4e) and DCAR (Clec4b1). During chronic infection, H. pylori-specific T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial burdens remained unchanged. Furthermore, a mutant H. pylori strain lacking αCAG and αCPG exhibited an impaired ability to cause gastritis. Thus H. pylori-specific modification of host cholesterol plays a pathophysiological role that exacerbates gastric inflammation by triggering C-type lectin receptors.

Cholesteryl 6-O-acyl-α-glucosides from diverse Helicobacter spp. signal through the C-type lectin receptor Mincle.

Helicobacter spp. are Gram-negative bacteria that cause a spectrum of disease in the gut, biliary tree and liver. Many Helicobacter spp. produce a range of cholesteryl α-glucosides that have the potential to act as pathogen associated molecular patterns. We report a highly stereoselective α-glucosylation of cholesterol using 3,4,6-tri-O-acetyl-2-O-benzyl-d-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate, which allowed the synthesis of cholesteryl α-glucoside (αCG) and representative Helicobacter spp. cholesteryl 6-O-acyl-α-glucosides (αCAGs; acyl = C12:0, 14:0, C16:0, C18:0, C18:1). All αCAGs, irrespective of the nature of their acyl chain composition, strongly agonised signalling through the C-type lectin receptor Mincle from human and mouse to similar degrees. By contrast, αCG only weakly signalled through human Mincle, and did not signal through mouse Mincle. These results provide a molecular basis for understanding of the immunobiology of non-pylori Helicobacter infections in humans and other animals.

Design of potent Mincle signalling agonists based on an alkyl ß-glucoside template.

The innate immune receptor Mincle senses lipid-based molecules derived from pathogens, commensals and altered self. Based on emerging structure-activity relationships we design simple alkyl 6-O-acyl-ß-d-glucosides that are effective agonists of Mincle and signal with potency on par with the prototypical ligand trehalose dimycolate.

α-Glucuronosyl and α-glucosyl diacylglycerides, natural killer T cell-activating lipids from bacteria and fungi.

Natural killer T cells express T cell receptors (TCRs) that recognize glycolipid antigens in association with the antigen-presenting molecule CD1d. Here, we report the concise chemical synthesis of a range of saturated and unsaturated α-glucosyl and α-glucuronosyl diacylglycerides of bacterial and fungal origins from allyl α-glucoside with Jacobsen kinetic resolution as a key step. These glycolipids are recognized by a classical type I NKT TCR that uses an invariant Vα14-Jα18 TCR α-chain, but also by an atypical NKT TCR that uses a different TCR α-chain (Vα10-Jα50). In both cases, recognition is sensitive to the lipid fine structure, and includes recognition of glycosyl diacylglycerides bearing branched (R- and S-tuberculostearic acid) and unsaturated (oleic and vaccenic) acids. The TCR footprints on CD1d loaded with a mycobacterial α-glucuronosyl diacylglyceride were assessed using mutant CD1d molecules and, while similar to that for α-GalCer recognition by a type I NKT TCR, were more sensitive to mutations when α-glucuronosyl diacylglyceride was the antigen. In summary, we provide an efficient approach for synthesis of a broad class of bacterial and fungal α-glycosyl diacylglyceride antigens and demonstrate that they can be recognised by TCRs derived from type I and atypical NKT cells.

Distinct CD1d docking strategies exhibited by diverse Type II NKT cell receptors.

Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F'-pocket-docking mode that contrasts sharply with the previously determined A'-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens.

A carbon tetrachloride-free synthesis of N-phenyltrifluoroacetimidoyl chloride.

N-Phenyltrifluoroacetimidoyl chloride (PTFAI-Cl) is a reagent widely used for the preparation of glycosyl N-phenyltrifluoroacetimidates. However, the most commonly applied method requires carbon tetrachloride, a hepatotoxic reagent that has been phased out under the Montreal Protocol. We report a new synthesis of N-phenyltrifluoroacetimidoyl chloride (PTFAI-Cl) using dichlorotriphenylphosphane and triethylamine.

Quantitation in the regioselectivity of acylation of glycosyl diglycerides: total synthesis of a Streptococcus pneumoniae α-glucosyl diglyceride.

The fidelity of acylation regioselectivity in the synthesis of mixed glycosyl diacylglycerols can be accurately measured by quantitative 13C NMR spectroscopy using a 1-13C-labelled fatty acid and a paramagnetic relaxation enhancement agent. Exquisite regioselectivity is achieved using a stepwise acylation/substitution of a glycosyl ß-bromohydrin, which is applied to the total synthesis of Streptococcus pneumoniae Glc-DAG-s2.

Immune sensing of microbial glycolipids and related conjugates by T cells and the pattern recognition receptors MCL and Mincle.

Microbes produce a wide range of small molecule glycoconjugates that constitute unique molecular signatures. These molecules are recognized by a range of detection systems, triggering immune responses to microbial pathogens and commensals. The antigen-presenting molecules of the CD1 class, CD1a-d, capture lipidic molecules and present them to diverse and innate-like T cell populations including natural killer T cells and germline-encoded mycolyl reactive T cells. The antigen-presenting molecule MR1 captures vitamin B metabolites and presents them to mucosal associated invariant T cells. In both cases, recognition of the small molecule-antigen presenting molecule complexes occurs through T cell receptors on the surface of T lymphocytes. The pattern recognition receptors macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle) receptors sense glycolipids and through signalling initiate cellular activation, shaping immune responses to peptide antigens, including the differentiation of naïve T cells into conventional effector T helper cells. In this review, we provide an overview of the diverse structures of immunogenic lipidic molecules and vitamin B metabolites and their recognition by select systems of the immune system. Future advances in our understanding of the roles of such molecules in innate and adaptive immune responses will require the coordinated efforts of synthetic and natural products chemists, immunologists and biologists.