Liam R. Cox

Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18+ invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

Activation of iNKT cells by a distinct constituent of the endogenous glucosylceramide fraction

Significance Invariant natural killer T (iNKT) cells are a specialized subset of T cells that recognizes lipids, rather than peptides, as antigens. Recognition of both endogenous and exogenous lipids by iNKT cells contributes to immune responses during infection, cancer, autoimmune disease, and allergic disease. The endogenous lipids recognized by iNKT cells in most contexts, however, remain unclear. In this report, we characterize the lipid antigen activity found in mammalian milk and tissues. Our data suggest that activity is related to a minor component of the glucosylceramide fraction. Whether contributed from endogenous sources or from the diet, this rare, yet potent lipid activity may play an important role in driving immune responses. Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of β-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of β-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with β-GlcCer in mammals functions as a lipid self antigen for iNKT cells.

A Single Subset of Dendritic Cells Controls the Cytokine Bias of Natural Killer T Cell Responses to Diverse Glycolipid Antigens

Summary Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

1,3-Diethynylallenes: Carbon-rich modules for three-dimensional acetylenic scaffolding

0-catalyzed cross-coupling of substrates, which bear bispropargylic leaving groups with silyl-protected alkynes, has provided access to a variety of 1,3-diethynylallenes, a new family of modules for three-dimensional acetylenic scaffolding. In enantiomerically pure form, these C-rich building blocks could provide access ± by oxidative oligomerization ± to a fascinating new class of helical oligomers and polymers with all-carbon backbones (Fig. 2). In the first of two routes, a bispropargylic epoxide underwent ring opening during S2-type cross-coupling, and the resulting alkoxide was silyl-protected, providing 1,3-diethynylallenes ()-8, ()-12 (Scheme 3), and ()-15 (Scheme 5). A more general approach involved bispropargylic carbonates or esters as substrates (Scheme 6 ± 8), and this route was applied to the preparation of a series of 1,3diethynylallenes to investigate how their overall stability against undesirable [2 2] cycloaddition is affected by the nature of the substituents at the allene moiety. The investigation showed that the 1,3-diethynylallene chromophore is stable against [2 2] cycloaddition only when protected by steric bulk and when additional electron delocalization is avoided. The regioselectivity of the cross-coupling to the bispropargylic substrates is entirely controlled by steric factors: attack occurs at the alkyne moiety bearing the smaller substituent (Schemes 9 and 10). Oxidative Hay coupling of the terminally mono-deprotected 1,3-diethynylallene ()-49 afforded the first dimer 50, probably as a mixture of two diastereoisomers (Scheme 12). Attempts to prepare a silyl-protected tetraethynylallene by the new methodology failed (Scheme 13). Control experiments (Schemes 14 ± 16) showed that the Pd 0 -catalyzed cross-coupling to butadiyne moieties in the synthesis of this still-elusive chromophore requires forcing conditions under which rapid [2 2] cycloaddition of the initial product cannot be avoided.

Tricarbonyliron complexes: an approach to acyclic stereocontrol

π-Allyltricarbonyliron lactone complexes, η4-dienetricarbonyliron complexes and their relatives offer an interesting approach to the problem of acyclic stereocontrol. Functional groups appended to the organic ligand frequently adopt a preferred conformation. This, combined with the steric bulk of the Fe(CO)3 moiety provides a means for controlling the addition of reagents to such pendant functionality in a defined manner. Thus addition of nucleophiles to aldehydes and ketones affords a route to diastereoisomerically pure secondary and tertiary alcohols while olefinic functionality in the side-chain can be utilised in stereoselective dihydroxylations, Diels–Alder and Michael addition reactions. Just as the formation of arene Cr(CO)3 complexes modifies reactivity at the α-position of arene substituents, the Fe(CO)3 group of η4-diene and trimethylenemethane tricarbonyliron complexes can be used to stabilise an adjacent positive charge. Trapping of the carbocation resulting from ionisation of an α-carbinol occurs with high diastereoselectivity, providing an unusual and useful stereoselective SN1-type reaction. Such highly stereoselective reactions have been put to good use in the preparation of a number of biologically interesting natural products.

Synthesis of a Versatile Building Block for the Preparation of 6-N-Derivatized α-Galactosyl Ceramides: Rapid Access to Biologically Active Glycolipids

A concise route to the 6-azido-6-deoxy-α-galactosyl-phytosphingosine derivative 9 is reported. Orthogonal protection of the two amino groups allows elaboration of 9 into a range of 6-N-derivatized α-galactosyl ceramides by late-stage introduction of the acyl chain of the ceramide and the 6-N-group in the sugar headgroup. Biologically active glycolipids 6 and 8 have been synthesized to illustrate the applicability of the approach.

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

Significance Understanding how to optimize lipid-loading onto CD1d molecules is important to better harness invariant natural killer T (iNKT) cells’ central role at the interface between innate and adaptive immunity. We report that the lipid transfer proteins saposins play an essential role in modulating human iNKT cell autoreactivity to antigen-presenting cells activated by inflammatory stimuli. Lipid-loading occurs in an endo-lysosomal compartment, where saposins work as “lipid editors,” capable of fine-tuning loading and unloading of CD1d molecules and increasing the off-rate of CD1d-bound lipids. Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the off-rate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a “lipid editor,” capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity.

Synthesis and biological activity of α-galactosyl ceramide KRN7000 and galactosyl (α1→2) galactosyl ceramide

Graphical abstract The synthesis and biological activity of α-galactosyl ceramide (α-GalCer) and Gal(α1→2)GalCer is reported.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2−/− mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

Disruption of the serine/threonine protein kinase H affects phthiocerol dimycocerosates synthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components.