Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids.
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ABSTRACT:
SUBMITTER: Deng S
PROVIDER: S-EPMC3308336 | biostudies-literature | 2011 Oct
REPOSITORIES: biostudies-literature
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Project description:NKT cells are innate-like T cells with powerful regulatory functions that are a promising target for immunotherapy. The efficacy of glycolipids, such as the prototypic NKT cell antagonist α-galactosylceramide (αGalCer), is currently being evaluated in clinical trials, but little is known about factors that target lipid antigens for CD1d presentation and NKT cell activation in vivo. Lipid uptake via the LDL receptor (LDLR) has been shown for digalactosylceramide; however, whether this pathway contributes to CD1d presentation of other important NKT cell agonists remains unclear. We therefore investigated receptor-mediated uptake pathways for CD1d presentation using a panel of structurally diverse lipid antigens. We found that uptake via scavenger receptors was essential for the CD1d presentation of αGalCer and Sphingomonas glycolipids. Moreover, in vivo NKT cell responses, i.e., cytokine production, proliferation, and NKT cell help for adaptive CD4+ and CD8+ T cells, required the uptake of αGalCer via scavenger receptor A. Importantly, our data indicate that structural characteristics of glycolipids determine their receptor binding and direct individual lipids toward different uptake pathways. These results reveal an important contribution of scavenger receptors in the selection of lipids for CD1d presentation and identify structural motifs that may prove useful for therapeutic NKT cell vaccination.
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Project description:The most potent foreign antigens for natural killer T cells (NKT cells) are ?-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ?-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated ?-linked agonists ?-galactosylceramide (?-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the ?-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the ?-linked self ligands to resemble the conformation of foreign ?-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ?-linked antigens.
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Project description:Glycolipids are potent activator of natural killer T (NKT) cells. The relationship between NKT cells and intestinal bacterial glycolipids in liver disorders remained unclear. We found that, in sharp contrast to specific pathogen-free (SPF) mice, germ-free (GF) mice are resistant to Concanavalin A (ConA)-induced liver injury. ConA treatment failed to trigger the activation of hepatic NKT cells in GF mice. These defects correlated with the sharply reduced levels of CD1d-presented glycolipid antigens in ConA-treated GF mice compared with SPF counterparts. Nevertheless, CD1d expression was similar between these two kinds of mice. The absence of intestinal bacteria did not affect the incidence of αGalCer-induced liver injury in GF mice. Importantly, we found the intestinal bacteria contain glycolipids which can be presented by CD1d and recognized by NKT cells. Furthermore, supplement of killed intestinal bacteria was able to restore ConA-mediated NKT cell activation and liver injury in GF mice. Our results suggest that glycolipid antigens derived from intestinal commensal bacteria are important hepatic NKT cell agonist and these antigens are required for the activation of NKT cells during ConA-induced liver injury. These finding provide a mechanistic explanation for the capacity of intestinal microflora to control liver inflammation.
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Project description:3-Acetamido-3,6-dideoxy-d-galactose (Fuc3NAc) and 3-acetamido-3,6-dideoxy-d-glucose (Qui3NAc) are unusual sugars found on the lipopolysaccharides of Gram-negative bacteria and on the S-layers of Gram-positive bacteria. The 3,4-ketoisomerases, referred to as FdtA and QdtA, catalyze the third steps in the respective biosynthetic pathways for these sugars. Whereas both enzymes utilize the same substrate, the stereochemistries of their products are different. Specifically, the hydroxyl groups at the hexose C-4' positions assume the "galactose" and "glucose" configurations in the FdtA and QdtA products, respectively. In 2007 we reported the structure of the apoform of FdtA from Aneurinibacillus thermoaerophilus, which was followed in 2014 by the X-ray analysis of QdtA from Thermoanaerobacterium thermosaccharolyticum as a binary complex. Both of these enzymes belong to the cupin superfamily. Here we report a combined structural and enzymological study to explore the manner in which these enzymes control the stereochemistry of their products. Various site-directed mutant proteins of each enzyme were constructed, and their dTDP-sugar products were analyzed by NMR spectroscopy. In addition, the kinetic parameters for these protein variants were measured, and the structure of one, namely, the QdtA Y17R/R97H double mutant form, was determined to 2.3-Å resolution. Finally, in an attempt to obtain a model of FdtA with a bound dTDP-linked sugar, the 3,4-ketoisomerase domain of a bifunctional enzyme from Shewanella denitrificans was cloned, purified, and crystallized in the presence of a dTDP-linked sugar analogue. Taken together, the results from this investigation demonstrate that it is possible to convert a "galacto" enzyme into a "gluco" enzyme and vice versa.
| S-EPMC5095699 | biostudies-literature
Project description:The use of impedance-based label free cell analysis is increasingly popular and has many different applications. Here, we report that a real-time cell analyzer (RTCA) can be used to study the stimulation of Natural Killer (NK) cells. Engagement of NK cells via plate-bound antibodies directed against different activating surface receptors could be measured in real time using the label-free detection of impedance. The change in impedance was dependent on early signal transduction events in the NK cells as it was blocked by inhibitors of Src-family kinases and by inhibiting actin polymerization. While CD16 was the only receptor that could induce a strong change in impedance in primary NK cells, several activating receptors induced changes in impedance in expanded NK cells. Using PBMCs we could detect T cell receptor-mediated T cell activation and CD16-mediated NK cell activation in the same sample. Performing a dose-response analysis for the Src-family kinases inhibitor PP1 we show that T cells are more sensitive to inhibition compared to NK cells. Our data demonstrate that the RTCA can be used to detect physiological activation events in NK cells in a label-free and real-time fashion.
| S-EPMC5862859 | biostudies-other
Project description:Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, ?-galactosyl ceramide (?GalCer), is a potential anticancer agent whose activity depends upon IFN-? secretion. We report two analogs of ?GalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-? 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 more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which 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-?-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility.
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Project description:Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.
| S-EPMC2519143 | biostudies-literature
Project description:Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.
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Project description:Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α-dependent structural changes in the F' roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α-galactosyl diacylglycerol-CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes.
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Project description:Some synthetic and bacterial glycolipids presented by CD1d specifically activate invariant NKT (iNKT) cells bearing an invariant V?14-J?18 (mouse) or V?24-J?18 (human) TCR. The antigenic glycolipids identified to date consist of two hydrophobic chains and an ?-glycoside in which the 2'-OH group is in the cis orientation toward the anomeric group, namely, either an ?-galactoside or an ?-glucoside. Several microbial ?-mannosyl glycolipids, in which the 2'-OH group is in the trans orientation, were herein examined to establish whether they have potential to activate iNKT cells. We found that ?-mannnosyl1-3 (6'-O-acyl ?-mannosyl)-1-1 monoacylglycerol and cholesteryl 6'-O-acyl ?-mannoside, found in Saccharopolyspora and Candida albicans, respectively, induced the activation of iNKT cells, dependent on CD1d. In contrast, ?-mannosyldiacylglycerol found in Streptococcus suis or ?-mannosylceramide demonstrated markedly less antigenicity for iNKT cells. The potentially antigenic ?-mannosyl glycolipids contributed to the protection of mice against infection with S. pneumoniae in which iNKT cells have previously been found to participate. Furthermore, these glycolipids induced the production of proinflammatory cytokines by macrophages, thereby suggesting their recognition by specific pattern recognition receptors (PRRs). Collectively, these results suggest that these microbial ?-mannosyl glycolipids are capable of being recognized by both the invariant TCR and PRRs and inducing immune responses.
| S-EPMC5574887 | biostudies-other