Guangwu Chen

Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity

Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4-CD8-, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.

A Single Early Activation of Invariant NK T Cells Confers Long-Term Protection against Collagen-Induced Arthritis in a Ligand-Specific Manner

The glycosphingolipid α-galactosylceramide (α-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of α-GalCer (α-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of α-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog α-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-γ in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-γ release induced by either α-GalCer or α-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-γ release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas α-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from α-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-γ-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.

Identification of C-glycoside analogues that display a potent biological activity against murine and human invariant natural killer T cells

We have recently shown that α‐C‐galactosylceramide (α‐C‐GalCer) stimulates invariant natural killer T (iNKT) cells and preferentially induces a T helper 1 (Th1)‐type response in mice. However, α‐C‐GalCer was found to be a rather weak ligand against human iNKT cells in vitro. Therefore, in this study, we sought to identify a compound that displays a strong stimulatory activity against human iNKT cells, by determining the biological activities of several C‐glycoside analogues. From the in vitro screening assays, we found that almost all C‐glycoside analogues, which have an E‐alkene linker between sugar and lipid moieties, are able to activate human iNKT cells and to induce the maturation and activation of human dendritic cells through iNKT‐cell activation. In summary, although α‐galactosylceramide (α‐GalCer) remains the strongest iNKT‐cell ligand, our study identified E‐alkene‐linked C‐glycoside analogues as potent human iNKT‐cell stimulants, and indicated that these analogues could be used as a therapeutic agent in the future for diseases resolved by Th1‐type responses.

E and Z α‐C‐Galactosylceramides by Julia–Lythgoe–Kocienski Chemistry: A Test of the Receptor‐Binding Model for Glycolipid Immunostimulants

potent immunostimulant activity of this family has been a subject of great interest. At the organism level, these molecules have been shown to have potent therapeutic effects against many diseases, ranging from cancer to malaria to diabetes. At the molecular level, glycolipid 1 has been shown to act as a connecting ligand presented by the CD1d molecule of antigen-presenting cells to the murine Va14 receptor and the human Va24 receptor of natural killer T (NKT) cells. Upon recognition of the galactosyl ceramide in the context of CD1d, the NKT cell then is stimulated to produce interferon-g (IFN-g), interleukin-4 (IL-4), and interleukin-2 (IL-2). Interleukin-12 (IL12) is secreted by antigen-presenting cells, such as dendritic cells, upon activation of NKT cells. Through secondary stimulation of other innate and adaptive immunocytes, galactosylcerACHTUNGTRENNUNGamide-induced NKT cell activation results in disease suppression. The balance between the secreted levels of IFN-g, a TH1 cytokine, and IL-4, a TH2 cytokine, through antagonistic effects, might have an important impact on the ultimate immunotherapeutic effect of NKT cell stimulation. Developments for Oglycoside analogues include i) in the sugar, inter alia, switching the 6-hydroxymethyl of the galactose to an uronic acid and the 3-hydroxyl to sulfate ii) in the lipid chains, varying chain lengths and degrees of unsaturation. Interestingly, these variations result in relatively small changes in the IFN-g and IL-4 ACHTUNGTRENNUNGbalance compared to the parent l, and only one analogue showed greater promise than the parent. The most dramatic effect in the O-glycoside series was seen when the sphingosine tail was shortened from 14 to 5 carbon atoms; this led to a ACHTUNGTRENNUNGsignificant increase in the IL-4/IFN-g ratio (due to a decrease in IFN-g secretion). In the mouse encephalomyelitis disease model, this change results in an improved therapeutic effect. Recently, two independent reports of X-ray crystallographic ACHTUNGTRENNUNGdeterminations of O-galactosylceramides bound to the CD1d molecule have appeared. Key features include hydrogen bonding between the protein and several ligand sites: the gal 2 and 3 OH’s, the side-chain 3 and 4 OH’s, and the glycosidic oxygen. Also featured are the lipid chains of the ligand, which are buried in channels composed of hydrophobic amino acids. Most pertinent to this research, the bound conformation of the linker region between the galactose and the ceramide reveals “anti” dihedral angles of ~1708. Our laboratory has focused on a deep-seated structural change, the preparation of C-glycosides. Analogue 2 caused an increase in the IFN-g/IL-4 ratio (due to decreased secretion of IL-4) at the cytokine level, in contrast to all other analogues reported; at the organism level, analogue 2 exhibited a dramatic increase in potency in suppressing malaria and melanoma in murine models. We have previously reported on the syntheses of 2 and related materials using the Ramberg–BHcklund method and an olefin cross-metathesis approach. Prior to the X-ray reports described above, we reasoned that C analogues with a cis or trans linker might supply evidence for a preferred ligand-binding geometry. Here we report on our studies using the “one-pot” version of the Julia olefin synthesis to obtain both E and Z isomers 3 and 4 (Scheme 1), which serve to test the ligand-binding model suggested by the X-ray data. Also of interest is the synthetic chemistry aim of determining the utility of Julia chemistry for C-glycoside synthesis. The one-pot version of the Julia reaction, or Julia–Lythgoe– Kocienski (JLK) reaction, has gained popularity for the total synthesis of natural products since its emergence about a decade ago. Its impressive stereoselectivity and multifunctional compatibility make it an essential complement to other methods for direct transformation of carbonyl groups into substituted olefins. We report here a convergent construction of a-C-galactosylceramide analogues that is a challenging test of the JLK method owing to issues of base sensitivity of both partners in our scheme. The a-orientated sugar aldehyde (5) is susceptible to anomeric epimerization and elimination to form endo glycal aldehyde (vide infra). The heteroaryl sulfones (6, 7) with a b-amido-substituted chiral center are also vulnerable to b-elimination. Preparations of sugar aldehyde 5 were initially realized by modifying a literature procedure (Scheme 2). Starting from methyl galactoside, perbenzylation followed by treatment with 3-(trimethylsilyl)prop-1-yne and trimethylsilyltriflate (TMSOTf) or BF3·OEt2/TMSOTf afforded the sugar allene intermediate 8. Subsequent conversion to the alcohol 9 was carried out by [a] Dr. G. Chen, Prof. Dr. R. W. Franck Department of Chemistry, Hunter College of CUNY 695 Park Avenue, New York, NY 10021 (USA) Fax: (+1)212-772-5332 E-mail : rfranck@hunter.cuny.edu [b] Prof. Dr. M. Tsuji Department of Medical Parasitology New York University School of Medicine 341 East 25th Street, New York, NY 10010 (USA) [c] M. Chien, Prof. Dr. M. Tsuji Aaron Diamond AIDS Research Center, The Rockefeller University 455 First Avenue, New York, NY 10016 (USA)

Invariant TCR Rather Than CD1d Shapes the Preferential Activities of C-Glycoside Analogues Against Human Versus Murine Invariant NKT Cells

C-glycoside analogues of α-galactosylceramide were shown to activate both human and mouse invariant NKT (iNKT) cells. Among these analogues, GCK152, which has an aromatic ring in the acyl chain, exhibited a stronger stimulatory activity against human iNKT cells and a much weaker activity against murine iNKT cells than GCK127 that has an almost identical fatty acyl chain as α-galactosylceramide. In this study, we have found that invariant TCR (invTCR) expressed by iNKT cells, but not CD1d expressed by APCs, command the species-specific preferential activity of C-glycosides, and that their preferential activity against human vs murine iNKT cells correlate with the binding affinity of glycolipid-CD1d complex to invTCR of respective iNKT cells rather than that of glycolipid to human or murine CD1d molecules. Overall, the structural difference of invTCR appears to supersede those of CD1d molecule in shaping the strength of the biological activity of C-glycoside analogues.