Title of Invention	COMPOUND FOR ACTIVATING NKT CELLS
Abstract	ABSTRACT Disclosed are methods for activating an NKT cell, methods of stimulating an immune response in a subject, methods of improving vaccine efficacy, and methods of treating an infection. Also disclosed are methods of promoting tumor rejection, treating cancer, modulating autoimmunity and inhibiting allergen-induced hypersensitivity in subjects. The methods include contacting an NKT cell with a bacterial glycolipid complexed with a CD1 molecule to activate the NKT cell. The bacterial glycolipid may be derived from a member of the Class Alphaproteobacteria.

Title of Invention

COMPOUND FOR ACTIVATING NKT CELLS

Abstract

ABSTRACT Disclosed are methods for activating an NKT cell, methods of stimulating an immune response in a subject, methods of improving vaccine efficacy, and methods of treating an infection. Also disclosed are methods of promoting tumor rejection, treating cancer, modulating autoimmunity and inhibiting allergen-induced hypersensitivity in subjects. The methods include contacting an NKT cell with a bacterial glycolipid complexed with a CD1 molecule to activate the NKT cell. The bacterial glycolipid may be derived from a member of the Class Alphaproteobacteria.

Full Text	COMPOUND FOR ACTIVATING NKT CELLS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application 60/648,153 filed on January 28, 2005. The provisional application is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with United States government support awarded by the National Institutes of Health, National Institute of Allergy and Infectious Disease (Grant No. AI053725). The United States government has certain rights in this invention. INTRODUCTION The CD1d molecule is a member of the CD1 family of b2 microglobulin- associated molecules. In contrast to class I and II major histocompatibility complex (MHC) molecules that present protein antigens to CD8+ and CD4+ T cells, respectively, CD1 molecules have evolved to capture and process both foreign and self lipid antigens for display to T cells. CD1a, -b, and -c molecules have been shown to present foreign microbial antigens to human TCRab T cells. In contrast, CDId-restricted T cells, or NKT cells, are a population of innate-like memory/effector cells expressing both NK receptors and a conserved, semi- invariant TCR (Va14-Ja18/Vb8 in mice and Va24-Ja18/Vb11 in humans). Like NK cells, NKT cells constitutively express mRNA but not protein for IFN-g, evidencing their poised effector stage. NKT cells have been implicated in suppression of autoimmunity and graft rejection, promotion of resistance to pathogens, and promotion of tumor immunity. While NKT cells are known to respond to a-GalactosylCeramide (aGal-Cer), a surrogate ligand derived from a marine sponge, lack of knowledge of their natural antigens has previously precluded understanding of the mechanisms of their peripheral activation and recruitment, as well as their thymic development. The inventors have previously identified a natural endogenous antigen, isoglobotrihexosylceramide (iGb3), which is presented to NKT cells by LPS- activated dendritic cells. This work suggests that iGb3 is a primary ligand for NKT cells. However, the partial diversity of the b-chain of the TCR suggests that multiple natural antigen specificity may be possible. 2 SUMMARY Described herein is the inventors' surprising discovery that glycolipids derived from members of the Class Alphaproteobacteria also act as natural ligands of CD1d molecules to activate NKT cells. In one aspect, the invention provides a method of activating an NKT cell comprising contacting the NKT cell with a bacterial glycolipid complexed with a CD1d molecule. In some embodiments, the bacterial glycolipid may be derived from a member of the class Alphaproteobacteria. In another aspect, the invention provides a method of inducing cytokine expression by an NKT cell comprising contacting a T-cell receptor of the NKT cell with a bacterial glycolipid complexed with a CD1d molecule. In yet another aspect, the invention provides a method of stimulating an immune response in a subject comprising administering to the subject an effective amount of NKT cells activated by contacting a T-cell receptor of the NKT cells with a bacterial glycolipid complexed with a CD1d molecule. In further aspects, the invention provides methods of improving vaccine efficacy, promoting tumor rejection, modulating autoimmunity, inhibiting allergen- induced hypersensitivity, and treating an infection in a subject by administration of an effective amount of a bacterial glycolipid derived from a member of the Class Alphaproteobacteria. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A depicts CD1d-dependent IFH-g secretion by mouse and human NKT cells stimulated with heat-killed bacteria or aGal-Cer. Mean and standard deviation of 3 experiments. FIG. 1B depicts NKT cell proliferation in a spleen cell culture stimulated with heat-killed bacteria or aGal-Cer. Data points show means and standard deviations from 3 separate experiments. FIG. 1C depicts NKT cell proliferation in response to bacterial stimuli or aGal-Cer. Upper row, CD1d-aGal-Cer/B220 staining of spleen cells with NKT cell gate and percentage as indicated. Lower row, CFSE dilution profile of 5x103 gated NKT cells. FIG. 2A depicts IFN-g released by whole spleen cells cultured with heat killed Salmonella typhimurium, Sphingomonas capsulata, and Ehrlichia muris for 48 hours. Left panel, data shown as percentage of wild type control. Right panel, data shown as mean and standard deviation of two to three separate experiments. 3 FIG. 2B depicts the blockade of human NKT cell responses to DC plus antigen by lectin IB4. Similar data obtained in two experiments. FIG. 2C depicts stimulation of mouse NKT cell responses to bacterial antigen presented by Hexb+/- or Hexb-/- DC. Similar data obtained in two experiments. FIG. 3A depicts structures of synthetic Sphingomonas cell wall antigens. PBS 50 is a control b-glucuronosylceramide. FIG. 3B depicts the IFN-g response of a human Va24-Ja18 NKT line and fresh purified mouse NKT cells stimulated by synthetic lipid antigens and DC. Data shown are the mean and standard deviation of two separate experiments. FIG. 3C depicts CD1d tetramer staining of human NKT (upper row) and mouse spleen cells (lower row) with synthetic glycolipids. NKT cell gate and percentages are as indicated. FIG. 4A depicts in vivo activation of NKT cells 24 hours after intravenous infection with Sphingomonas (1x107), Ehrlichia (1x108) and Salmonella (1x106). Similar results were obtained in 2 experiments. FIG. 4B depicts IFN-g production by NKT cells in response to Salmonella. The difference between Hexb+/+ and Hexb-1- was significant for Salmonella (p=0.001). Three mice per group were analyzed and similar results obtained in 2 independent experiments. FIG. 4C depicts bacterial burden in the lungs of CD1d+/- and CD1d-/- mice after infection with the indicated CFU of Sphingomonas (each bar represents 4 to 5 mice). Fold increase and p values are indicated. Two representative experiments are shown. FIG. 4D depicts acute lethality in mice after inoculation of a high dose of 5x108 Sphingomonas capsulata. Separate experiments comparing CD1d+/- and CDId-/- (n=24 each, p shown. FIG. 4E depicts acute serum release of IFN-g and IL-12 p40 in heterozygous and homozygous CD1d and Ja18 mutant mice and littermate controls after infection with 1x107 Sphingomonas capsulata. Similar results were obtained in 2 independent experiments. FIG. 4F depicts Ehrlichia PCR counts in lungs, livers and spleens of CD1d+/- and CD1d-/- mice recovered at day 2 and day 7 post-infection (each bar represents 3 mice). Fold increase and p values are indicated. One representative experiment is shown. 4 FIG. 5 depicts several synthetic glycolipids derived from bacteria of the class Alphaproteobacteria. FIG. 6 depicts an exemplary synthetic scheme for glycolipid PBS 61. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS CD1- restricted T cells carry out both effector and helper functions and interact with a variety of cell types, including macrophages, dendritic cells, NK cells, T cells and B cells, thereby contributing to both innate and adaptive immune responses. A subset of these T cells, NKT cells, also known as CD Id-restricted T cells or CD1d tetramer+ T cells, are characterized by invariant TCRa chains, self lipid reactivity and rapid effector responses. These cells play an important role in a number of immune functions, including antimicrobial responses, antitumor immunity and in regulating the balance between tolerance and autoimmunity. In the absence of foreign antigens, NKT cells are stimulated by exposure to CD1+ antigen presenting cells, such as monocytes, dendritic cells (DC) and macrophages. Classes of self-antigens that can be presented to and recognized by NKT cells include phospholipids, such as phosphatidylinositol, phosphatidylethanolamine and phophatidylglycerol, as well as sphingolipids. However, not all classes elicit a response in NKT cells in terms of cytokine release. NKT cells also are known to recognize a-galactosylceramide (aGal-Cer), a glycosphingolipid found in marine sponges. This molecule has no known immunological or other physiological function in mammals, but is widely used by investigators to study NKT activation. Prior to the present invention, activation of NKT by direct presentation of microbial glycolipids was not known. NKT cells are rapidly activated upon stimulation by CD1d presented polar lipid antigens. "Activation," as the term is used herein and in the art, refers to secretion by NKT cells of IFN-g, IL-4, IL-2, IL-10, IL-13, GM-CSF or TNF-a, or combinations of these cytokines, upon contact with CD1d presented stimulatory antigens. Alternatively, "activation" may refer to upregulated expression of cell- surface markers for activated T-cells, for example, CD69. Activation of NKT cells in accordance with the invention comprises contacting an NKT cell, or more specifically, a T cell receptor (TCR) of the NKT cell, with a CD1d-complexed bacterial polar lipid. Glycolipids are suitable species of polar lipids. Thus, in some embodiments, activation of NKT cells comprises contacting an NKT cell with a bacterial glycolipid derived from a member of the Class Alphaproteobacteria. "A T cell receptor of an NKT cell," as the term is used herein, refers to the conserved, semi-invariant TCR of NKT cells comprising, e.g., 5 Va14-Ja18/vb8 in mice and VaT24-Ja18/Vb11 in humans. "Contacting," as used herein, refers to the in vitro addition of bacterial glycolipid in solution to immobilized, soluble, or insoluble CD1d molecules, or to the in vivo administration of bacterial glycolipid to a subject having antigen presenting cells which express cell surface CD1d molecules. Activation of NKT cells may be measured in vitro or ex vivo by any suitable method. An example of an in vitro test permitting evaluation of NKT cell activation is co-culturing NKT cells with antigen presenting cells (APC), such as dendritic cells (DC), in the presence of a bacterial glycolipid activator or putative activator, and subsequently assaying for IFN-g or other secreted cytokines in the supernatant. Alternatively, activation of NKT cells can be measured ex vivo by administering a bacterial glycolipid antigen to a subject or by administering CD1d+ antigen presenting cells after ex vivo contact with bacterial glycolipids to a subject. The NKT cells from these subjects can be isolated by, e.g., CD1d-tetramer staining and gating via flow cytometry, and subsequently assayed for surface CD69 (early T-cell activation antigen) and/or intracellular IFN-g by suitable methods. Alphaproteobacteria is a class in the phylum Proteobacteria comprised mostly of bacteria having two major phenotypes: purple non-sulfur bacteria and aerobic bacteriochlorophyll-containing bacteria. Bacterial members of the class of Alphaproteobacteria are primarily isolated from soil, lakes or ponds. Several members are known human pathogens. The class Alphaproteobacteria includes six orders: Rhodospirillales, Rickettsiales, Rhodobacterales, Sphingomonadales, Caulobacterales and Rhizobiales (Garrity, GM et al., Taxonomic Outline of the Procaryotic Genera, BERGEY'S MANUAL of Systematic Bacteriology, 2nd Ed, April 2001, incorporated herein by reference). Bacterial glycolipids which may be useful in activating NKT cells may be derived from members of any of these orders. However, members of orders Rickettsiales, Sphingomonadales and Rhizobiales are contemplated to be particularly suitable. The order Rickettsiales includes three families: Rickettsiaceae, Ehrlichiaceae and Holosporaceae. Polar lipids derived from members of Ehrlichiaceae in the genus Ehrlichia are contemplated to be suitably used in methods of the invention. For example, E. muris- derived glycolipids may be suitable. The order Sphingomonadales includes the family Sphingomonadaceae. Glyclolipids derived from members of this family in the genus Sphingomonas, for example, from S. capsulata, are contemplated to be suitable. 6 The order Rhizobiales includes ten families: Rhizobiaceae, Bartonellaceae, Brucellaceae, Phyllobacteriaceae, Methylocystaceae, Beijerinckiaceae, Bradyrhizobiaceae, Hyphomicrobiaceae, Methylobacteriaceae and Rhodobiaceae. Glycolipids derived from members of Brucellaceae in the genus Brucella are contemplated to be suitably used in methods of the invention. Sphingomonas capsulata is a pathogen of the Alphaproteobacteria class which is a gram-negative, lipopolysaccharide (LPS)-negative bacteria whose cell wall lipids have been extensively characterized. Glycolipids derived from the cell walls of these bacteria may be used to activate NKT cells in accordance with the invention. Similarly, members of the genus Ehrlichia are gram-negative, LPS-negative bacteria whose cell wall lipids may be used to activate NKT cells. Although the cell membrane lipids of Ehrlichia are not as well-characterized as those of Sphingomonas capsulata, it is contemplated that members of this genus will function to activate NKT cells in suitable activation assays, as well as in vivo. Brucella is another genus in this class known to be pathogenic. The four species of this genus that can infect humans include B. abortus, B. suis, B. melitensis and B. canis. Brucellosis disease in humans is characterized as either an acute febrile disease or a persistent disease with a wide variety of symptoms. It is a true zoonosis in that virtually all human infections are acquired from animals. Subclinical infection is common. In contrast to Erlichia and Sphingomonas spp., the outer cell membrane comprises a dominant LPS component and three main groups of proteins. It is contemplated that particular fractions or components of these bacterial cell membranes may be used to directly activate NKT cells in accordance with the invention. As noted, bacterial glycolipids are suitably derived from bacteria of the class Alphaproteobacteria. "Derived from," refers to isolation and/or purification from bacterial sources, and also refers to de novo synthesis of bacterial compounds, or compounds rationally designed based on bacterial compounds, using suitable synthetic processes known in the art. As will be appreciated by one of ordinary skill in the art, "bacterial glycolipids" may also include heat killed or attenuated bacteria in the context of the methods of the invention. For example, contacting a NKT cell with a bacterial glycolipid suitably includes contacting a NKT cell with a heat killed or attenuated bacteria, as well as isolated or synthetic bacterial glycolipids. The term "glycolipid" designates any compound containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety such as an acylglycerol, a sphingoid, a ceramide (N-acylsphingoid) or a prenyl 7 phosphate. In particular, one or more saccharides bound to a ceramide moiety may be particularly useful in activating NKT cells. Bacterial glycolipids suitable for use in methods of activating NKT cells may be generally of the structural formula (I): wherein — indicates either a single bond wherein X is H or lower alkyl, or an ionic bond wherein X is a counter ion; R1 and R2 are independently selected from the group consisting of-H, -OH, a monosaccharide and an oligosaccharide; R3 is -H or - OH; R4 is -H or -OH or, together with R7, forms a double bond; R5 and R6 are independently C1-C30 alkyl, wherein the C1-C30 alkyl is saturated or unsaturated or comprises one or more cyclopropyl groups; and R7 is -H or, together with R4, forms a double bond. As used herein, the term "lower alkyl" is meant to refer to a straight or branched, saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms. Specific examples of such hydrocarbon radicals are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl, isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl or cyclopropyl. Also as used herein, a "counter ion" is any positively charged species that can associate via an ionic bond with a negatively charged carboxylate on the glycolipid. Some representative examples of suitable bacterial glycolipids for complexing with CD1d molecules and activating NKT cells are depicted in FIG. 5. PBS 30, PBS 45 and PBS 59 were synthesized based on known Sphingomonas cell membrane molecules and were found to activate NKT cells in vitro. Conversely, PBS 50 and PBS 60 do not activate NKT cells. The remaining compounds depicted in FIG. 5 were rationally designed based on the following features determined to be common among glycolipids capable of activating NKT cells: 1) an alpha-type 8 glycosidic linkage and 2) oxidation at the 6-position on the carbohydrate moiety of the glycolipid. In some embodiments, activation of NKT cells by administration of a bacterial glycolipid in accordance with the invention may provide a means by which an immune response may be stimulated in a subject. An "immune response" as used herein refers to any elevated level of humoral or cellular response that is measurable in a subject in comparison to the subject's baseline, or unstimulated, state. Methods of measuring both humoral and cellular immune responses are well- known in the art. As will be appreciated, the in vivo response of NKT cells is influenced, in part, by the cellular environment during activation. TH1 immune responses are characterized predominantly by release of, e.g., IL-2, 1FN-g, IL-12 and TNF-a. In contrast, TH2 cytokines predominantly include IL-4, IL -5, IL -6, IL - 10, and IL-13. The in vivo response of NKT cells may also be influenced by antigen concentration or prior, or repeated, antigen exposure. Activation may be further mediated by interactions with co-stimulatory molecules on NKT cells and APCs, e.g., CD40/CD40L interactions. In addition to cytokine secretion, activated NKT cells are potently cytolytic via release of perforin and granzymes, as well as granulysin, and can contribute directly to bacterial cell and/or tumor cell killing via secretion of these molecules. Thus, activating NKT cells in a subject by administration of an effective amount of a bacterial glycolipid to a subject may generate an anti-microbial immune response and thereby provide a means of treating an infection in the subject. The infection may be viral, bacterial or parasitic and the anti-microbial immune response may be sufficient to inhibit the growth of, or kill a microbe, including e.g., viruses, bacteria or parasites. Administration may be carried out by any method employed in the art, including intraperitoneal, intravenous, intramuscular, subcutaneous, transcutaneous, oral, nasopharyngeal or mucosal absorption, among others. As mentioned, methods of the invention may also be employed in the treatment of cancer, or in promoting tumor rejection, by inducing an antihyperproliferative immune response in a mammai. "Treating" or "treatment" of cancer in a mammal includes one or more of: (1) inhibiting growth of the cancer, i.e., arresting its development, (2) preventing spread of the cancer, i.e. preventing metastases, (3) relieving the cancer, i.e., causing regression of the cancer, (4) preventing recurrence of the cancer, (5) palliating symptoms of the cancer, and (6) promoting rejection of one or more solid tumors. In a particular embodiment, bacterial glycolipids in accordance with the invention can be administered as an adjuvant to improve vaccine efficacy when co- 9 administered with a vaccine. As used herein the term "co-administration" or "co- administering" refers to administration of at least two components concurrently, i.e., simultaneously in time, or sequentially, i.e., administration of one component, followed by administration of the other component. Adoptive transfer methods are based on administering cells that have been contacted with bacterial glycolipids ex vivo to stimulate an immune response in a subject. In some embodiments, the cells may be NKT cells that are activated ex vivo and injected into a subject to provide or enhance an immune response to, e.g., cancerous cells or microbes. In some embodiments, administration of activated NKT cells may induce an antihyperproliferative immune response to promote solid tumor rejection. In other embodiments, the cells may be antigen presenting cells that have been contacted with bacterial glycolipids ex vivo to allow complexing of the bacterial glycolipids with the CD1d molecules expressed by the antigen presenting cell, e.g., a dendritic cell. Antigen presenting cells can then be administered, e.g., by injection into the subject, to provide a suitable immune response. This method of administration allows for stimulation of the immune response with minimal exposure of the subject or the subject's cells to the bacterial glycolipids. Activation of NKT cells may also be employed in methods of modulating autoimmunity or inhibiting allergen-induced hypersensitivity. Both direct administration of bacterial glycolipids, as well as adoptive transfer methods are contemplated for these particular treatments. The following examples are provided to assist in a further understanding of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope thereof. Example 1. In vitro stimulation of NKT cells with heat-killed bacteria. Bacterial strains Sphingomonas capsulata (ATCC 14666) and Salmonella typhimurium R71 were grown in Mueller-Hinton Agar. Ehrlichia muris were prepared as described by Ismail N et al., J. Immunol. 172, 1786-1800 (2004), incorporated herein by reference. Bacteria were heat killed by 2-hour exposure to 74°C and 2.5 - 5x106 cfu equivalent/well were used for in vitro stimulation. Stimulation assays were performed with whole spleen cells (5x105 per 200 ml well) or with purified T cells and antigen presenting cells. T cell populations used in the assays comprised sorted CD1d-aGal-Cer+ mouse spleen cells (5x104 per 200ml well), human peripheral blood lymphocytes (PBL) (5x105 per 200ml well) (obtained 10 after Ficoll centrifugation of heparinized blood) or human NKT cell lines (2.5x105 per 200ml well). Human Va24 NKT cells were derived from PBL stimulated with aGal- Cer and were maintained by repeated rounds of stimulation with PHA and IL-2 in the presence of irradiated PBMC and EBV transformed B cells in vitro. Antigen presenting cells were dendritic cells that were derived from bone marrow, stimulated with GMCSF/IL-4 (2 ng/mL and 5 ng/mL, Biosource) and cultured at 2.5x105 per 200 ml well for mouse assays, and irradiated allogeneic human PBMC fresh or cultured for 5 days with recombinant human GMCSF/IL-4, (100 mg/mL of each cytokine, R&D Systems) (2x105 per 200 ml well) for human assays. Cells were washed twice and starved for 6 hours in medium alone before addition to the stimulation experiments. NKT cells were stimulated with heat-killed bacteria as indicated above for 48 hrs in 96 well round bottom plates in RPMI 1640 (Biofluids) supplemented with glutamine, antibiotics, 5x10-5 M 2-ME and 10% FCS (mouse studies) or 5% AB serum (human studies). Concentrations of mouse and human IFN-g. in the supernatant were measured at 48 hours using the respective ELISA kits (BD Bioscience, lower detection limit of 12.5 pg/ml). Whole spleen cells were stimulated for 6 days with 5x108 heat killed bacteria or 100 ng/mL aGal-Cer, and the frequency of CD1d-aGal-Cer+ NKT cells were measured at stimulation and 2,4 and 6 days post-stimulation. At 6 days post stimulation, CD1d-aGal-Cer, CFSE and aB220 (BD Pharmingen) labeling and staining procedures were performed and cells were analyzed by FACS. To generate CD1d-aGal-Cer tetramers, a mixture of 5 ml of aGal-Cer (from 1mg/ml stock solution in DMSO), 10 ml of PBS 0.5% Tween 20, 10 ml of biotinylated CD1d (1 mg/ml), and 75 ml of PBS was incubated at 37°C for 1 hr, and lipid-loaded CD1d was purified by centrifugation dialysis and complexed with streptavidin-APC. (Benlagha K. et al., J. Exp. Med. 191, 1895-1903 (2000).) Cells were analyzed on a FACSCalibur (BD Biosciences) with CellQuest software. Results are reported in FIGS. 1A-C. Mouse CD1d tetramer-sorted NKT cells co-cultured with fresh bone marrow derived CD1+/- or CD1-/- DC secreted IFN-g in a CD1d-dependent manner when stimulated with heat killed Sphingomonas and Erlichia, as well as control Salmonella and aGal-Cer. (FIG. 1A, left.) Similarly, human NKT cells co-cultured with PBMC-derived DC secreted IFN-g in a CD1d- dependent manner upon stimulation, where CD1d dependence was illustrated using blocking with 1 mg/mL anti-CD1d antibodies or control IgGl (FIG. 1A, right.) Whole spleen cell suspensions cultured in the presence of heat-killed bacteria for 6 days showed a marked expansion and proliferation of NKT cells, only slightly less than that induced by pure aGal-Cer. (FIG. 1B-C.) 11 Example 2. Differential requirements for the IFN-Y response to Sphingomonas and Ehrlichia versus Salmonella. Whole spleen cells co-cultured with DC of genotype MyD88-1-, Triflps2/lps2 and MyD88-1-Trif lps2/lps2 (lacking one or the two adaptors MyD88 and TRIF for TLR signaling) or CD1-l- were stimulated for 48 hours with 5x106 heat killed Salmonella, Sphingomonas or Ehrlichia. Concentrations of mouse and human IFN-y. in the supernatant were measured at 48 hours using the respective ELISA kits (BD Bioscience, lower detection limit of 12.5 pg/ml). DC were pulsed with heat-killed bacteria, prepared as described in Example 1 and added to human NKT cell preparations in the presence of IB4 (Griffonia Simplicifolia isolectin B4) (Vector Laboratories) which binds the terminal disaccharide of iGb3, but does not bind to aGal-Cer. IFN-y production was measured at 48 hours. Hexb-l- DC, which fail to generate iGb3 in the lysosome because they lack the b-hexosaminidase needed to remove the terminal GalNAc of iGb4, the precursor of iGb3, were pulsed with heat-killed bacteria as described above and added to NKT cell cultures. IFN-Y production was measured at 48 hours. Results are reported in FIGS. 2A-C. In the whole spleen cell culture assay, Salmonella-induced IFN-Y was drastically reduced to 2-15% of control, on average, in the absence of either one or the two TLR adaptors (FIG. 2A). In sharp contrast, the splenic IFN-Y response to LPS-negative Ehrlichia and Sphingomonas was largely independent of MyD88 and TRIF. CD1d-l- spleen cells lacking NKT cells failed to respond to Sphingomonas and Ehrlichia, whereas the response to Salmonella was only marginally reduced (FIG. 2A, left). Likewise, wild type NKT cells co-cultured with MyD88-deficient DC responded to Sphingomonas and Ehrlichia but not Salmonella (FIG. 2A, right). Altogether, these results suggested that in total spleens exposed to heat-killed Salmonella, IFN-K production was initiated after TLR signaling of antigen presenting cells and subsequent recruitment of NKT cells as well as other cell-types such as NK cells.. In contrast, IFN-Y stimulation by Ehrlichia and Sphingomonas was primarily dependent on NKT cells and CD1d with minimal contribution of TLR. Similarly, lectin IB4 binding did not impair the stimulation of NKT cells by DC pulsed with heat-killed Ehrlichia or Sphingomonas, consistent with direct recognition of a distinct microbial antigen. However, the lectins readily blocked stimulation by Salmonella (FIG. 2B), suggesting that for the Salmonella NKT response, endogenous iGb3 is the likely ligand. 12 Hexb-l- DC pulsed with heat-killed Ehrlichia or Sphingomonas stimulated NKT cells as well as wild-type DC. (FIG. 2C) In contrast, Salmonella-pulsed Hexb-l- DC did not stimulate NKT cells. Together, the results identify the endogenous ligand iGb3, rather than a microbial antigen, as the target of NKT cells in their response to Salmonella infection. Example 3. NKT cell stimulatory response to synthetic glycolipid antigens. a-glucuronosylceramide (PBS 30) and a-galacturonosylceramide (PBS 59), derived from known Sphingomonadaceae cell membrane antigens, were synthesized as described in Example 5. PBS 50, a b-glucuronosylceramide, served as a control compound. The structures of these compounds are shown in FIG. 3A. The immunological properties of the above compounds in NKT cells were measured. Human Va24-Ja18 NKT cells and fresh purified mouse NKT cells were co-cultured with DC pulsed with aGal-Cer or synthetic glycolipid at concentrations ranging from 0.001 to 1000 ng/mL. IFN-Y production was measured at 48 hours as described above. CD1d tetramers were prepared as described in Example 1 using synthetic glycolipids PBS 30, PBS 59 and PBS 50 and oGal-Cer, and were used to stain human NKT cells and mouse spleen cells. Results are shown in FIGS. 3 B-C. Both a-glucuronosylceramide (PBS 30) and to a lesser degree, a-galacturonosylceramide (PBS 59) strongly activated mouse and human NKT cell proliferation as well as IFN-y secretion, whereas control b-glucuronosylceramide (PBS 50) did not (FIG. 3B). Tetramers of CD1d-a- glucuronosylceramide (PBS 30) stained all human NKT cells and ~25% of mouse NKT cells (FIG. 3C). Thus, these findings revealed that the lipids replacing LPS in the cell wall of some species of Gram-negative bacteria may be directly recognized by the conserved TCR of innate-like NKT cells. Example 4. In vivo role of NKT cells during microbial infection. CD1d-l- mice were generated at the University of Chicago, Ja18-l- mice were obtained from Dr. Taniguchi, Chiba University (Japan) and Hexb-l- mice were obtained from R. Proia, National Institutes of Health. All mice were in the C57/BL6 background. In all cases, littermates obtained from heterozygous matings were genotyped by PCR and used for comparative analysis. All mice were raised in a pathogen-free environment at University of Chicago according to the Institutional Animal Care and Use Committee guidelines. 13 Six- to seven-week old C57/BL6 mice were intravenously inoculated with 100 ml Sphingomonas (1x107), Ehrlichia (1x108) or Salmonella (1x106) suspended in PBS. Twenty-four hours post-infection, isolated NKT cells gated as tetramer+/B220- were analyzed by FACS for surface CD69 (early T-cell activation antigen) and intracellular IFN-Y. Results, shown in FIG. 4A, confirm that NKT cells are activated and secrete IFN-Y within 24 hours after infection in vivo. To determine whether hexb is required for antigen processing in response to Salmonella and Sphingomonas infection in vivo, Hexb+/-and Hexb+/- littermates were challenged intraperitoneally with 5x106 Sphingomonas or Salmonella. Two hours post-challenge, 5x106 CFSE-labelled Va14 transgenic thymocytes were intrasplenically injected in a volume of 50ml (Bendelac A. et al., J. Exp. Med. 184, 1285-12293 (1996), incorporated herein by reference). At 24 hours post-challenge, intracellular staining for IFN-Y was performed. Results are shown in FIG. 4B. The difference between Hexb+/- and Hexb+/- was statistically significant only for Salmonella challenged mice, demonstrating that IFN-Y production by NKT cells in response to Salmonella infection requires lysosomal iGb3, whereas the response of NKT cells to Sphingomonas does not require lysosomal iGb3. To characterize the role of NKT cells in controlling infection in vivo, Ja18-/- and CD1-/- mice and their littermate controls were injected intravenously with either 5x106 or 1x106 Sphingomonas. Bacterial burden in the lungs was assessed at intervals indicated in FIG. 4C. Bacterial counts were performed after tissue homogenization in 0.5% Triton X-100 and cultured for colony formation. The results demonstrate that both Ja18-/- and CD1-/- mice had delayed bacterial clearance compared to heterozygous littermate controls, with up to 12-14 times higher bacterial load in the lung at early time points. For survival experiments, Ja18-/- and CD1-/- mice and their littermate controls were injected intravenously with a high dose of 5x108 Sphingomonas. Dead or moribund (euthanized) mice were recorded every 2-4 hours post-infection. The results, shown in FIG. 4D, demonstrate that infection with a high dose of Sphingomonas was rapidly lethal in wild-type mice, whereas a majority of NKT deficient mice survived. To test whether lethality was associated with cytokine release, Sphingomonas (1x107) was intravenously injected in Ja18-/- and CD1-/- mice and their littermate controls. At intervals specified in FIG. 4E, serum levels of IFN-Y and IL-12 p40 were measured. The results indicate that the lethal outcome in wild-type mice was associated with the explosive release of IFN-Y and IL-12 in the serum, whereas NKT deficient mice produced significantly less cytokines. 14 For Ehrlichia infection experiments, mice were infected intraperitoneally with 500ml of a 10-1 dilution of Ehrlichia muris stock. The Ehrlichia load in the lungs, livers and spleens of CD1d-/- and control Itttermates was determined by real-time PCR of the Ehrlichia dsb gene (Ismail, N. et al., J. Immunol. 172, 1786-1800 (2004)) at 2 and 7 days post-infection. Results, reported in FIG. 4F, show that NKT deficient mice demonstrate an inability to clear Ehrlichia. Example 5. Synthesis of bacterial glycolipid PBS 61. FIG. 6 depicts a suitable route of synthesis for PBS 61. To a vigorously stirred solution of compound "1" (Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y. Angew. Chem. Int. Ed. 2001, 40, 4725-4728.) (453 mg, 0.976 mmol) in CH2CI2 (3 mL) and water (1.5 mL) was added TEMPO (60.8 mg, 0.390 mmol) and bis(acetoxy)iodobenzene (BAIB) (345 mg, 1.07 mmol) to produce intermediate compound "2" in FIG. 6. Additional BAIB (345 mg, 1.07 mmol) was added after 1 hour. The reaction was stirred until TLC indicated complete conversion of the starting material (~1.5 hour). The reaction mixture was extracted with CH2CI2 twice and the combined organic layers were dried over MgSO4 and concentrated. A short flash column (SiO2, CH3OH/CH2CI21:10) afforded crude glucoronic acid. A solution of crude glucoronic acid in CH2CI2 (3 mL) was treated with a freshly prepared ethereal solution of diazomethane until the evolution of gas ceased. The reaction mixture was then treated with AcOH (2 mL) and concentrated in vacuo. Flash column chromatography (SiO2, EtOAc/hexanes 1:4-1:3) afforded corresponding methyl glucuronate (186 mg, 0.378 mmol, yield 39% of two steps). 1H NMR (CDCI3) 57.31-7.27 (m, 4 H), 6.87-6.86 (m, 4 H), 4.83-4.67(m, 4 H), 4.49 (d, J = 9.8 Hz, 1 H), 3.87-3.80 (m, 2 H), 3.78 (s, 3 H), 3.51 (t, J = 7.8 Hz, 1 H), 3.39 (t, J = 8.8 Hz, 1 H), 2.80-2.70 (m, 2 H), 1.32 (t, J = 7.3 Hz, 3 H). 13C NMR (CDCI3) d169.65, 159.47, 159.40, 130.65, 130.10, 129.71, 114.02, 113.89, 86.01, 84.83, 80.36, 75.28, 75.26, 71.91, 55.34, 52.79, 25.29, 15.13. High resolution fast atom bombardment mass spectrometry (thioglycerol + Na + matrix) m/e ([M + Na]+) 515.1716 (100.0%); calculated 515.1714. The methyl glucuronate (186 mg, 0.378 mmol) was dissolved in CH2CI2 (10 mL) and Et3N (0.5 mL) followed by the introduction of a catalytic amount of DMAP (20 mg) and Ac2O (0.2 mL). The solvent was removed in vacuo after 12 hours and the residue was chromatographed (SiO2, EtOAc/hexane 1:4) to afford the product (172 mg, 0.329 mmol, 87% yield) as clear oil. 1H NMR (CDCI3) d57.31-7.18 (m, 4 H), 6.88-6.85 (m, 4 H), 5.12 (t, J = 9.8 Hz, 1 H), 4.83-4.61(m, 4 H), 4.47 (d, J = 9.8 Hz, 1 H), 3.86 (d, J = 10.3 Hz, 1 H), 3.80 (s, 3 H), 3.71 (s, 3 H), 3.65 (t, J = 8.8 Hz, 1 H), 3.49 (t, J = 8.8 Hz, 1 H), 2.82-2.68 (m, 2 H), 1.95 (s, 3 H), 1.32 15 (t, J = 7.3 Hz, 3 H). 13C NMR (CDCI3) d169.72, 167.94, 159.63, 159.48, 130.38, 130.29, 130.02, 129.66, 113.99, 85.56, 82.80, 80.61, 76.64, 75.49, 75.22, 71.33, 55.47, 52.92, 25.17, 20.89, 15.15. High resolution fast atom bombardment mass spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 557.1827 (100.0%); calculated 557.1822. To prepare intermediate compound "6," a mixture of compound "2" (172 mg, 0.329 mmol), compound "3" (150 mg, 0.328 mmol), and 2,6-di-tert-butyl-4- methylpyridine (67.6 mg, 0.329 mmol) in toluene (3 mL) was stirred with 4 Å molecular sieves (300 mg) for 1 h at room temperature. Next, dimethyl(methylthio)sulfonium triflate (66.8 mg, 0.329 mmol) was added, and stirring was continued for 8 hours. The mixture was concentrated and passed through a SiO2 plug using 1:1 EtOAc/hexanes. The solvent was removed in vacuo and the residue was chromatographed (SiO2, EtOAc/hexane 1:5-1:4) to afford the product "4" (61.1 mg, 0.0657 mmol, mixture of a-b-anomers, 20% yield). A solution of compound "4" (61.1 mg, 0.0657 mmol, mixture of a-b-anomers) in pyridine (10 mL) and water (2 mL) was treated with a stream of hydrogen sulfide for 15 minutes. The solution was stirred for 12 hours, and then hydrogen sulfide was bubbled again for 15 minutes. The reaction mixture was stirred for another 12 hours. The solvent was evaporated under vacuum and the residue was co-evaporated with toluene. The residue was dissolved in CH2CI2 (10 mL) followed by the introduction of 5 (43.1 mg, 0.131 mmol). A solution of dicyclohexylcarbodiimide (DCC) (27.0 mg, 0.131 mmol) and dimethylaminopyridine (DMAP) (6.3 mg, 0.052 mmol) in CH2CI2 was added, and stirring was continued for 6 hours. The mixture was concentrated and passed through a SiO2 plug using 1:1 EtOAc/hexanes. The solvent was removed in vacuo and the residue was chromatographed (SiO2, EtOAc/hexane 1:5-1:4) to afford the product "6" (22.3 mg, 0.0184 mmol, 28% yield of a-anomer). 1H NMR (CDCI3) d8.07- 8.04 (m, 2 H), 7.61-7.58 (m, 1 H), 7.47-7.44 (m, 2 H), 7.27-7.16 (m, 4 H), 6.87-6.81 (m, 4 H), 6.67 (d, J = 7.8 Hz, 1 H), 5.36-5.29 (m, 3 H), 5.18 (t, J = 5.9 Hz, 1 H), 5.00 (t, J = 9.3 Hz, 1 H), 4.79 (d, J = 3.4 Hz, 1 H), 4.74-4.54(m, 4 H), 4.52-4.49 (m, 1 H), 4.14 (d, J = 9.8 Hz, 1 H), 3.85-3.74 (m, 8 H), 3.69-3.62 (m, 4 H), 3.55 (dd, J = 9.3, 3.4 Hz, 1 H), 2.01-1.96 (m, 7 H), 1.88-1.78 (m, 2 H), 1.36-1.09 (m, 55 H), 0.90-0.87 (m, 6 H). 13C NMR (CDCI3) d177.19, 170.04, 169.84, 168.71, 166.45, 159.60, 159.40, 133.53, 130.76, 130.01, 129.46, 128.73, 114.03, 113.94, 98.56, 78.49, 78.19, 74.49, 73.97, 73.18, 71.18, 69.18, 67.96, 55.48, 52.92, 50.91, 49.38, 38.99, 34.16, 32.14, 31.99, 29.99, 29.86, 29.80, 29.65, 29.57, 29.19, 27.42, 27.30, 25.81, 25.57, 25.15, 24.84, 22.90, 20.91, 14.34. High resolution fast atom bombardment 16 mass spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 1236.7557 (100.0%); calculated 1236.7539. Preparation of PBS-61: Compound "6" (22.3 mg, 0.0184 mmol) was dissolved in tetrahydrofuran (THF) (1 mL) and water (0.5 mL) followed by the introduction of trifluoroacetic acid (TFA) (2 mL). The reaction was stirred until TLC indicated complete conversion of the starting material to a lower spot (~1.0 h). The reaction mixture was diluted by toluene and then concentrated in vacuo. The dialcohol was obtained as a clear glass (10.0 mg, 0.0103 mmol, yield 56%) after column chromatography (SiO2, MeOH/CH2CI2 1:40-1:24) 1H NMR (CDCI3) d8.02- 8.00 (m, 2 H), 7.64-7.61 (m, 1 H), 7.50-7.46 (m, 2 H), 6.67 (d, J = 7.8 Hz, 1 H), 5.35- 5.33 (m, 2 H), 5.22-5.18 (m, 1 H), 5.08 (t, J = 6.4 Hz, 1 H), 4.98 (t, J = 9.8 Hz, 1 H), 4.82 (d, J = 3.9 Hz, 1 H), 4.51-4.50 (m, 1 H), 4.22 (d, J = 9.8 Hz, 1 H), 3.99 (dd, J = 10.2, 3.4 Hz, 1 H), 3.93 (t, J = 9.8 Hz, 1 H), 3.72 (s, 3 H), 3.58 (dd, J = 9.3, 3.4 Hz, 1 H), 3.40 (dd, J = 10.3, 7.4 Hz, 1 H), 2.11 (s, 3 H), 2.05-1.98 (m, 4 H), 1.88-1.78 (m, 2 H), 1.36-1.09 (m, 64 H), 0.89-0.86 (m, 6 H). 13C NMR (CDCI3) d177.99, 170.94, 170.44, 168.67, 167.14, 134.07, 130.15, 130.01, 129.19, 128.92, 99.47, 74.98, 74.60, 72.23, 71.76, 71.51, 69.23, 68.35, 53.06, 51.61, 39.11, 32.28, 32.13, 31.99, 31.78, 29.86, 29.73, 29.63, 29.57, 29.40, 29.19, 27.42, 27.22, 25.68, 25.07, 22.91, 22.87, 20.97, 14.35. High resolution fast atom bombardment mass spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 996.6404 (100.0%); calculated 996.6388. The dialcohol (10.0 mg, 0.0103 mmol) was dissolved in MeOH (1 mL) and THF (1 mL) followed by addition of NaOMe (0.2 mL of 1 M NaOMe solution in MeOH) and 3 drops of water. The mixture was stirred for 12 hours and then water (2 mL) was added. The reaction mixture was concentrated in vacuo and the residue was chromatographed (SiO2, CHCI3/MeOH/H2O 60:30:4) to afford PBS-61 (5.0 mg, 0.069 mmol, 67% yield). 1H NMR (DMSO-d6 0.7ml with 1 drop of DCI and 3 drops of D2O, 55 °C) d5.36-5.34 (m, 2 H), 4.79 (d, J = 3.4 Hz, 1 H), 3.94 (t, J = 5.9 Hz, 1 H), 3.88 (d, J = 9.7 Hz, 1 H), 3.82-3.79 (m, 1 H), 3.71-3.63 (m, 2 H), 3.58-3.56 (m, 1 H), 3.50 (t, J = 9.3, 1 H), 3.38 (t, J = 9.3 Hz, 1 H), 3.30 (dd, J = 9.3, 3.4 Hz, 1 H), 2.01-1.99 (m, 4 H), 1.60-1.55 (m, 2 H), 1.36-1.09 (m, 64 H), 0.90-0.87 (m, 6 H). 13C NMR (DMSO-d6 0.7ml with 1 drop of DCI and 3 drops of D2O, 55 °C) d174.21, 171.39, 130.29, 100.46, 100.38, 73.35, 72.37, 71.54, 69.98, 68.02, 53.22, 41.09, 34.93, 34.22, 31.92, 31.75, 31.56, 29.93,29.71, 29.32, 28.89, 27.26, 25.70, 24.97, 22.68, 14.54. High resolution fast atom bombardment mass spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 752.5289 (100.0%); calculated 752.5284. 17 All publications, patents and patent applications referenced in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications, patents and patent applications are herein expressly incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference. In case of conflict between the present disclosure and the incorporated patents, publications and references, the present disclosure should control. We claim: 1. A glycolipid of formula (I): wherein the glycolipid is selected from the following compounds: - PBS-49, wherein X is methyl; R1 is-H; R2 is-OH; R3 is-OH; is-H; R 5 is C11 alkyl; R6 is Cn alkyl; and R1 is -H; - PBS-45, wherein X isH; R 1 is-H; R2 is -OH; R3 is -OH; is-H; R5 is Cll alkyl; R6 is C13 alkyl; and R1 is-H; - PBS-30, wherein X isH; R1 is-H; R2 is -OH; R3 is -OH; is-H; Rs is C1 6 alkyl; R6 is C1s alkyl; and R1 is-H; - PBS-29, wherein X is H; R1 is -H; R2 is -OH; R3 is-H;is -OH; Rs is C23 alkyl; R6 is C13 alkyl; and R7 is-H; - PBS-62, wherein X isH; Rt is-H; R2 is -OH; R3 is -OH; is-H; Rs is C23 alkyl; R6 is C1s alkyl comprising one double bond; and R7 is-H; and - PBS-65, wherein X isH; R1 is-H; R2 is -OH; R3 is -OH; is-H; Rs is C 11 alkyl; R6 is C 13 alkyl comprising one cyclopropyl group; and R7 is -H. 2. A vaccine adjuvant comprising the glycolipid of formula (I) as claimed in claim 1. ABSTRACT Disclosed are methods for activating an NKT cell, methods of stimulating an immune response in a subject, methods of improving vaccine efficacy, and methods of treating an infection. Also disclosed are methods of promoting tumor rejection, treating cancer, modulating autoimmunity and inhibiting allergen-induced hypersensitivity in subjects. The methods include contacting an NKT cell with a bacterial glycolipid complexed with a CD1 molecule to activate the NKT cell. The bacterial glycolipid may be derived from a member of the Class Alphaproteobacteria.

Full Text

COMPOUND FOR ACTIVATING NKT CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application 60/648,153
filed on January 28, 2005. The provisional application is incorporated herein by
reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with United States government support awarded by
the National Institutes of Health, National Institute of Allergy and Infectious Disease
(Grant No. AI053725). The United States government has certain rights in this
invention.
INTRODUCTION
The CD1d molecule is a member of the CD1 family of b2 microglobulin-
associated molecules. In contrast to class I and II major histocompatibility complex
(MHC) molecules that present protein antigens to CD8+ and CD4+ T cells,
respectively, CD1 molecules have evolved to capture and process both foreign and
self lipid antigens for display to T cells. CD1a, -b, and -c molecules have been
shown to present foreign microbial antigens to human TCRab T cells. In contrast,
CDId-restricted T cells, or NKT cells, are a population of innate-like
memory/effector cells expressing both NK receptors and a conserved, semi-
invariant TCR (Va14-Ja18/Vb8 in mice and Va24-Ja18/Vb11 in humans). Like NK
cells, NKT cells constitutively express mRNA but not protein for IFN-g, evidencing
their poised effector stage. NKT cells have been implicated in suppression of
autoimmunity and graft rejection, promotion of resistance to pathogens, and
promotion of tumor immunity.
While NKT cells are known to respond to a-GalactosylCeramide (aGal-Cer),
a surrogate ligand derived from a marine sponge, lack of knowledge of their natural
antigens has previously precluded understanding of the mechanisms of their
peripheral activation and recruitment, as well as their thymic development.
The inventors have previously identified a natural endogenous antigen,
isoglobotrihexosylceramide (iGb3), which is presented to NKT cells by LPS-
activated dendritic cells. This work suggests that iGb3 is a primary ligand for NKT
cells. However, the partial diversity of the b-chain of the TCR suggests that multiple
natural antigen specificity may be possible.
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SUMMARY
Described herein is the inventors' surprising discovery that glycolipids
derived from members of the Class Alphaproteobacteria also act as natural ligands
of CD1d molecules to activate NKT cells.
In one aspect, the invention provides a method of activating an NKT cell
comprising contacting the NKT cell with a bacterial glycolipid complexed with a
CD1d molecule. In some embodiments, the bacterial glycolipid may be derived from
a member of the class Alphaproteobacteria.
In another aspect, the invention provides a method of inducing cytokine
expression by an NKT cell comprising contacting a T-cell receptor of the NKT cell
with a bacterial glycolipid complexed with a CD1d molecule.
In yet another aspect, the invention provides a method of stimulating an
immune response in a subject comprising administering to the subject an effective
amount of NKT cells activated by contacting a T-cell receptor of the NKT cells with a
bacterial glycolipid complexed with a CD1d molecule.
In further aspects, the invention provides methods of improving vaccine
efficacy, promoting tumor rejection, modulating autoimmunity, inhibiting allergen-
induced hypersensitivity, and treating an infection in a subject by administration of
an effective amount of a bacterial glycolipid derived from a member of the Class
Alphaproteobacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A depicts CD1d-dependent IFH-g secretion by mouse and human NKT
cells stimulated with heat-killed bacteria or aGal-Cer. Mean and standard deviation
of 3 experiments.
FIG. 1B depicts NKT cell proliferation in a spleen cell culture stimulated with
heat-killed bacteria or aGal-Cer. Data points show means and standard deviations
from 3 separate experiments.
FIG. 1C depicts NKT cell proliferation in response to bacterial stimuli or
aGal-Cer. Upper row, CD1d-aGal-Cer/B220 staining of spleen cells with NKT cell
gate and percentage as indicated. Lower row, CFSE dilution profile of 5x103 gated
NKT cells.
FIG. 2A depicts IFN-g released by whole spleen cells cultured with heat
killed Salmonella typhimurium, Sphingomonas capsulata, and Ehrlichia muris for 48
hours. Left panel, data shown as percentage of wild type control. Right panel, data
shown as mean and standard deviation of two to three separate experiments.
3

FIG. 2B depicts the blockade of human NKT cell responses to DC plus
antigen by lectin IB4. Similar data obtained in two experiments.
FIG. 2C depicts stimulation of mouse NKT cell responses to bacterial
antigen presented by Hexb+/- or Hexb-/- DC. Similar data obtained in two
experiments.
FIG. 3A depicts structures of synthetic Sphingomonas cell wall antigens.
PBS 50 is a control b-glucuronosylceramide.
FIG. 3B depicts the IFN-g response of a human Va24-Ja18 NKT line and
fresh purified mouse NKT cells stimulated by synthetic lipid antigens and DC. Data
shown are the mean and standard deviation of two separate experiments.
FIG. 3C depicts CD1d tetramer staining of human NKT (upper row) and
mouse spleen cells (lower row) with synthetic glycolipids. NKT cell gate and
percentages are as indicated.
FIG. 4A depicts in vivo activation of NKT cells 24 hours after intravenous
infection with Sphingomonas (1x107), Ehrlichia (1x108) and Salmonella (1x106).
Similar results were obtained in 2 experiments.
FIG. 4B depicts IFN-g production by NKT cells in response to Salmonella.
The difference between Hexb+/+ and Hexb-1- was significant for Salmonella
(p=0.001). Three mice per group were analyzed and similar results obtained in 2
independent experiments.
FIG. 4C depicts bacterial burden in the lungs of CD1d+/- and CD1d-/- mice
after infection with the indicated CFU of Sphingomonas (each bar represents 4 to 5
mice). Fold increase and p values are indicated. Two representative experiments
are shown.
FIG. 4D depicts acute lethality in mice after inoculation of a high dose of
5x108 Sphingomonas capsulata. Separate experiments comparing CD1d+/- and
CDId-/- (n=24 each, p shown.
FIG. 4E depicts acute serum release of IFN-g and IL-12 p40 in heterozygous
and homozygous CD1d and Ja18 mutant mice and littermate controls after infection
with 1x107 Sphingomonas capsulata. Similar results were obtained in 2 independent
experiments.
FIG. 4F depicts Ehrlichia PCR counts in lungs, livers and spleens of CD1d+/-
and CD1d-/- mice recovered at day 2 and day 7 post-infection (each bar represents 3
mice). Fold increase and p values are indicated. One representative experiment is
shown.
4

FIG. 5 depicts several synthetic glycolipids derived from bacteria of the class
Alphaproteobacteria.
FIG. 6 depicts an exemplary synthetic scheme for glycolipid PBS 61.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
CD1- restricted T cells carry out both effector and helper functions and
interact with a variety of cell types, including macrophages, dendritic cells, NK cells,
T cells and B cells, thereby contributing to both innate and adaptive immune
responses. A subset of these T cells, NKT cells, also known as CD Id-restricted T
cells or CD1d tetramer+ T cells, are characterized by invariant TCRa chains, self
lipid reactivity and rapid effector responses. These cells play an important role in a
number of immune functions, including antimicrobial responses, antitumor immunity
and in regulating the balance between tolerance and autoimmunity.
In the absence of foreign antigens, NKT cells are stimulated by exposure to
CD1+ antigen presenting cells, such as monocytes, dendritic cells (DC) and
macrophages. Classes of self-antigens that can be presented to and recognized by
NKT cells include phospholipids, such as phosphatidylinositol,
phosphatidylethanolamine and phophatidylglycerol, as well as sphingolipids.
However, not all classes elicit a response in NKT cells in terms of cytokine release.
NKT cells also are known to recognize a-galactosylceramide (aGal-Cer), a
glycosphingolipid found in marine sponges. This molecule has no known
immunological or other physiological function in mammals, but is widely used by
investigators to study NKT activation. Prior to the present invention, activation of
NKT by direct presentation of microbial glycolipids was not known.
NKT cells are rapidly activated upon stimulation by CD1d presented polar
lipid antigens. "Activation," as the term is used herein and in the art, refers to
secretion by NKT cells of IFN-g, IL-4, IL-2, IL-10, IL-13, GM-CSF or TNF-a, or
combinations of these cytokines, upon contact with CD1d presented stimulatory
antigens. Alternatively, "activation" may refer to upregulated expression of cell-
surface markers for activated T-cells, for example, CD69.
Activation of NKT cells in accordance with the invention comprises
contacting an NKT cell, or more specifically, a T cell receptor (TCR) of the NKT cell,
with a CD1d-complexed bacterial polar lipid. Glycolipids are suitable species of
polar lipids. Thus, in some embodiments, activation of NKT cells comprises
contacting an NKT cell with a bacterial glycolipid derived from a member of the
Class Alphaproteobacteria. "A T cell receptor of an NKT cell," as the term is used
herein, refers to the conserved, semi-invariant TCR of NKT cells comprising, e.g.,
5

Va14-Ja18/vb8 in mice and VaT24-Ja18/Vb11 in humans. "Contacting," as used
herein, refers to the in vitro addition of bacterial glycolipid in solution to immobilized,
soluble, or insoluble CD1d molecules, or to the in vivo administration of bacterial
glycolipid to a subject having antigen presenting cells which express cell surface
CD1d molecules.
Activation of NKT cells may be measured in vitro or ex vivo by any suitable
method. An example of an in vitro test permitting evaluation of NKT cell activation is
co-culturing NKT cells with antigen presenting cells (APC), such as dendritic cells
(DC), in the presence of a bacterial glycolipid activator or putative activator, and
subsequently assaying for IFN-g or other secreted cytokines in the supernatant.
Alternatively, activation of NKT cells can be measured ex vivo by administering a
bacterial glycolipid antigen to a subject or by administering CD1d+ antigen
presenting cells after ex vivo contact with bacterial glycolipids to a subject. The
NKT cells from these subjects can be isolated by, e.g., CD1d-tetramer staining and
gating via flow cytometry, and subsequently assayed for surface CD69 (early T-cell
activation antigen) and/or intracellular IFN-g by suitable methods.
Alphaproteobacteria is a class in the phylum Proteobacteria comprised
mostly of bacteria having two major phenotypes: purple non-sulfur bacteria and
aerobic bacteriochlorophyll-containing bacteria. Bacterial members of the class of
Alphaproteobacteria are primarily isolated from soil, lakes or ponds. Several
members are known human pathogens.
The class Alphaproteobacteria includes six orders: Rhodospirillales,
Rickettsiales, Rhodobacterales, Sphingomonadales, Caulobacterales and
Rhizobiales (Garrity, GM et al., Taxonomic Outline of the Procaryotic Genera,
BERGEY'S MANUAL of Systematic Bacteriology, 2nd Ed, April 2001, incorporated
herein by reference). Bacterial glycolipids which may be useful in activating NKT
cells may be derived from members of any of these orders. However, members of
orders Rickettsiales, Sphingomonadales and Rhizobiales are contemplated to be
particularly suitable.
The order Rickettsiales includes three families: Rickettsiaceae,
Ehrlichiaceae and Holosporaceae. Polar lipids derived from members of
Ehrlichiaceae in the genus Ehrlichia are contemplated to be suitably used in
methods of the invention. For example, E. muris- derived glycolipids may be
suitable.
The order Sphingomonadales includes the family Sphingomonadaceae.
Glyclolipids derived from members of this family in the genus Sphingomonas, for
example, from S. capsulata, are contemplated to be suitable.
6

The order Rhizobiales includes ten families: Rhizobiaceae, Bartonellaceae,
Brucellaceae, Phyllobacteriaceae, Methylocystaceae, Beijerinckiaceae,
Bradyrhizobiaceae, Hyphomicrobiaceae, Methylobacteriaceae and Rhodobiaceae.
Glycolipids derived from members of Brucellaceae in the genus Brucella are
contemplated to be suitably used in methods of the invention.
Sphingomonas capsulata is a pathogen of the Alphaproteobacteria class
which is a gram-negative, lipopolysaccharide (LPS)-negative bacteria whose cell
wall lipids have been extensively characterized. Glycolipids derived from the cell
walls of these bacteria may be used to activate NKT cells in accordance with the
invention.
Similarly, members of the genus Ehrlichia are gram-negative, LPS-negative
bacteria whose cell wall lipids may be used to activate NKT cells. Although the cell
membrane lipids of Ehrlichia are not as well-characterized as those of
Sphingomonas capsulata, it is contemplated that members of this genus will
function to activate NKT cells in suitable activation assays, as well as in vivo.
Brucella is another genus in this class known to be pathogenic. The four
species of this genus that can infect humans include B. abortus, B. suis, B.
melitensis and B. canis. Brucellosis disease in humans is characterized as either an
acute febrile disease or a persistent disease with a wide variety of symptoms. It is a
true zoonosis in that virtually all human infections are acquired from animals.
Subclinical infection is common. In contrast to Erlichia and Sphingomonas spp., the
outer cell membrane comprises a dominant LPS component and three main groups
of proteins. It is contemplated that particular fractions or components of these
bacterial cell membranes may be used to directly activate NKT cells in accordance
with the invention.
As noted, bacterial glycolipids are suitably derived from bacteria of the class
Alphaproteobacteria. "Derived from," refers to isolation and/or purification from
bacterial sources, and also refers to de novo synthesis of bacterial compounds, or
compounds rationally designed based on bacterial compounds, using suitable
synthetic processes known in the art. As will be appreciated by one of ordinary skill
in the art, "bacterial glycolipids" may also include heat killed or attenuated bacteria
in the context of the methods of the invention. For example, contacting a NKT cell
with a bacterial glycolipid suitably includes contacting a NKT cell with a heat killed or
attenuated bacteria, as well as isolated or synthetic bacterial glycolipids.
The term "glycolipid" designates any compound containing one or more
monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety
such as an acylglycerol, a sphingoid, a ceramide (N-acylsphingoid) or a prenyl
7

phosphate. In particular, one or more saccharides bound to a ceramide moiety may
be particularly useful in activating NKT cells.
Bacterial glycolipids suitable for use in methods of activating NKT cells may
be generally of the structural formula (I):

wherein — indicates either a single bond wherein X is H or lower alkyl, or an ionic
bond wherein X is a counter ion; R1 and R2 are independently selected from the
group consisting of-H, -OH, a monosaccharide and an oligosaccharide; R3 is -H or -
OH; R4 is -H or -OH or, together with R7, forms a double bond; R5 and R6 are
independently C1-C30 alkyl, wherein the C1-C30 alkyl is saturated or unsaturated or
comprises one or more cyclopropyl groups; and R7 is -H or, together with R4, forms
a double bond. As used herein, the term "lower alkyl" is meant to refer to a straight
or branched, saturated or unsaturated hydrocarbon radical having 1 to 4 carbon
atoms. Specific examples of such hydrocarbon radicals are methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl, isobutenyl, isopropenyl,
formyl, acetyl, propionyl, butyryl or cyclopropyl. Also as used herein, a "counter ion"
is any positively charged species that can associate via an ionic bond with a
negatively charged carboxylate on the glycolipid.
Some representative examples of suitable bacterial glycolipids for
complexing with CD1d molecules and activating NKT cells are depicted in FIG. 5.
PBS 30, PBS 45 and PBS 59 were synthesized based on known Sphingomonas cell
membrane molecules and were found to activate NKT cells in vitro. Conversely,
PBS 50 and PBS 60 do not activate NKT cells. The remaining compounds depicted
in FIG. 5 were rationally designed based on the following features determined to be
common among glycolipids capable of activating NKT cells: 1) an alpha-type
8

glycosidic linkage and 2) oxidation at the 6-position on the carbohydrate moiety of
the glycolipid.
In some embodiments, activation of NKT cells by administration of a
bacterial glycolipid in accordance with the invention may provide a means by which
an immune response may be stimulated in a subject. An "immune response" as
used herein refers to any elevated level of humoral or cellular response that is
measurable in a subject in comparison to the subject's baseline, or unstimulated,
state. Methods of measuring both humoral and cellular immune responses are well-
known in the art. As will be appreciated, the in vivo response of NKT cells is
influenced, in part, by the cellular environment during activation. TH1 immune
responses are characterized predominantly by release of, e.g., IL-2, 1FN-g, IL-12
and TNF-a. In contrast, TH2 cytokines predominantly include IL-4, IL -5, IL -6, IL -
10, and IL-13. The in vivo response of NKT cells may also be influenced by antigen
concentration or prior, or repeated, antigen exposure. Activation may be further
mediated by interactions with co-stimulatory molecules on NKT cells and APCs,
e.g., CD40/CD40L interactions.
In addition to cytokine secretion, activated NKT cells are potently cytolytic via
release of perforin and granzymes, as well as granulysin, and can contribute directly
to bacterial cell and/or tumor cell killing via secretion of these molecules.
Thus, activating NKT cells in a subject by administration of an effective
amount of a bacterial glycolipid to a subject may generate an anti-microbial immune
response and thereby provide a means of treating an infection in the subject. The
infection may be viral, bacterial or parasitic and the anti-microbial immune response
may be sufficient to inhibit the growth of, or kill a microbe, including e.g., viruses,
bacteria or parasites. Administration may be carried out by any method employed in
the art, including intraperitoneal, intravenous, intramuscular, subcutaneous,
transcutaneous, oral, nasopharyngeal or mucosal absorption, among others.
As mentioned, methods of the invention may also be employed in the
treatment of cancer, or in promoting tumor rejection, by inducing an
antihyperproliferative immune response in a mammai. "Treating" or "treatment" of
cancer in a mammal includes one or more of: (1) inhibiting growth of the cancer, i.e.,
arresting its development, (2) preventing spread of the cancer, i.e. preventing
metastases, (3) relieving the cancer, i.e., causing regression of the cancer, (4)
preventing recurrence of the cancer, (5) palliating symptoms of the cancer, and (6)
promoting rejection of one or more solid tumors.
In a particular embodiment, bacterial glycolipids in accordance with the
invention can be administered as an adjuvant to improve vaccine efficacy when co-
9

administered with a vaccine. As used herein the term "co-administration" or "co-
administering" refers to administration of at least two components concurrently, i.e.,
simultaneously in time, or sequentially, i.e., administration of one component,
followed by administration of the other component.
Adoptive transfer methods are based on administering cells that have been
contacted with bacterial glycolipids ex vivo to stimulate an immune response in a
subject. In some embodiments, the cells may be NKT cells that are activated ex
vivo and injected into a subject to provide or enhance an immune response to, e.g.,
cancerous cells or microbes. In some embodiments, administration of activated
NKT cells may induce an antihyperproliferative immune response to promote solid
tumor rejection. In other embodiments, the cells may be antigen presenting cells
that have been contacted with bacterial glycolipids ex vivo to allow complexing of
the bacterial glycolipids with the CD1d molecules expressed by the antigen
presenting cell, e.g., a dendritic cell. Antigen presenting cells can then be
administered, e.g., by injection into the subject, to provide a suitable immune
response. This method of administration allows for stimulation of the immune
response with minimal exposure of the subject or the subject's cells to the bacterial
glycolipids.
Activation of NKT cells may also be employed in methods of modulating
autoimmunity or inhibiting allergen-induced hypersensitivity. Both direct
administration of bacterial glycolipids, as well as adoptive transfer methods are
contemplated for these particular treatments.
The following examples are provided to assist in a further understanding of
the invention. The particular materials and conditions employed are intended to be
further illustrative of the invention and are not limiting upon the reasonable scope
thereof.
Example 1. In vitro stimulation of NKT cells with heat-killed bacteria.
Bacterial strains Sphingomonas capsulata (ATCC 14666) and Salmonella
typhimurium R71 were grown in Mueller-Hinton Agar. Ehrlichia muris were prepared
as described by Ismail N et al., J. Immunol. 172, 1786-1800 (2004), incorporated
herein by reference. Bacteria were heat killed by 2-hour exposure to 74°C and 2.5 -
5x106 cfu equivalent/well were used for in vitro stimulation.
Stimulation assays were performed with whole spleen cells (5x105 per 200 ml
well) or with purified T cells and antigen presenting cells. T cell populations used in
the assays comprised sorted CD1d-aGal-Cer+ mouse spleen cells (5x104 per 200ml
well), human peripheral blood lymphocytes (PBL) (5x105 per 200ml well) (obtained
10

after Ficoll centrifugation of heparinized blood) or human NKT cell lines (2.5x105 per
200ml well). Human Va24 NKT cells were derived from PBL stimulated with aGal-
Cer and were maintained by repeated rounds of stimulation with PHA and IL-2 in the
presence of irradiated PBMC and EBV transformed B cells in vitro. Antigen
presenting cells were dendritic cells that were derived from bone marrow, stimulated
with GMCSF/IL-4 (2 ng/mL and 5 ng/mL, Biosource) and cultured at 2.5x105 per 200
ml well for mouse assays, and irradiated allogeneic human PBMC fresh or cultured
for 5 days with recombinant human GMCSF/IL-4, (100 mg/mL of each cytokine, R&D
Systems) (2x105 per 200 ml well) for human assays. Cells were washed twice and
starved for 6 hours in medium alone before addition to the stimulation experiments.
NKT cells were stimulated with heat-killed bacteria as indicated above for 48
hrs in 96 well round bottom plates in RPMI 1640 (Biofluids) supplemented with
glutamine, antibiotics, 5x10-5 M 2-ME and 10% FCS (mouse studies) or 5% AB
serum (human studies). Concentrations of mouse and human IFN-g. in the
supernatant were measured at 48 hours using the respective ELISA kits (BD
Bioscience, lower detection limit of 12.5 pg/ml).
Whole spleen cells were stimulated for 6 days with 5x108 heat killed bacteria
or 100 ng/mL aGal-Cer, and the frequency of CD1d-aGal-Cer+ NKT cells were
measured at stimulation and 2,4 and 6 days post-stimulation.
At 6 days post stimulation, CD1d-aGal-Cer, CFSE and aB220 (BD
Pharmingen) labeling and staining procedures were performed and cells were
analyzed by FACS. To generate CD1d-aGal-Cer tetramers, a mixture of 5 ml of
aGal-Cer (from 1mg/ml stock solution in DMSO), 10 ml of PBS 0.5% Tween 20, 10
ml of biotinylated CD1d (1 mg/ml), and 75 ml of PBS was incubated at 37°C for 1 hr,
and lipid-loaded CD1d was purified by centrifugation dialysis and complexed with
streptavidin-APC. (Benlagha K. et al., J. Exp. Med. 191, 1895-1903 (2000).) Cells
were analyzed on a FACSCalibur (BD Biosciences) with CellQuest software.
Results are reported in FIGS. 1A-C. Mouse CD1d tetramer-sorted NKT cells
co-cultured with fresh bone marrow derived CD1+/- or CD1-/- DC secreted IFN-g in a
CD1d-dependent manner when stimulated with heat killed Sphingomonas and
Erlichia, as well as control Salmonella and aGal-Cer. (FIG. 1A, left.) Similarly,
human NKT cells co-cultured with PBMC-derived DC secreted IFN-g in a CD1d-
dependent manner upon stimulation, where CD1d dependence was illustrated using
blocking with 1 mg/mL anti-CD1d antibodies or control IgGl (FIG. 1A, right.) Whole
spleen cell suspensions cultured in the presence of heat-killed bacteria for 6 days
showed a marked expansion and proliferation of NKT cells, only slightly less than
that induced by pure aGal-Cer. (FIG. 1B-C.)
11

Example 2. Differential requirements for the IFN-Y response to Sphingomonas
and Ehrlichia versus Salmonella.
Whole spleen cells co-cultured with DC of genotype MyD88-1-, Triflps2/lps2 and
MyD88-1-Trif lps2/lps2 (lacking one or the two adaptors MyD88 and TRIF for TLR
signaling) or CD1-l- were stimulated for 48 hours with 5x106 heat killed Salmonella,
Sphingomonas or Ehrlichia. Concentrations of mouse and human IFN-y. in the
supernatant were measured at 48 hours using the respective ELISA kits (BD
Bioscience, lower detection limit of 12.5 pg/ml).
DC were pulsed with heat-killed bacteria, prepared as described in Example
1 and added to human NKT cell preparations in the presence of IB4 (Griffonia
Simplicifolia isolectin B4) (Vector Laboratories) which binds the terminal
disaccharide of iGb3, but does not bind to aGal-Cer. IFN-y production was
measured at 48 hours.
Hexb-l- DC, which fail to generate iGb3 in the lysosome because they lack
the b-hexosaminidase needed to remove the terminal GalNAc of iGb4, the precursor
of iGb3, were pulsed with heat-killed bacteria as described above and added to NKT
cell cultures. IFN-Y production was measured at 48 hours.
Results are reported in FIGS. 2A-C. In the whole spleen cell culture assay,
Salmonella-induced IFN-Y was drastically reduced to 2-15% of control, on average,
in the absence of either one or the two TLR adaptors (FIG. 2A). In sharp contrast,
the splenic IFN-Y response to LPS-negative Ehrlichia and Sphingomonas was
largely independent of MyD88 and TRIF. CD1d-l- spleen cells lacking NKT cells
failed to respond to Sphingomonas and Ehrlichia, whereas the response to
Salmonella was only marginally reduced (FIG. 2A, left). Likewise, wild type NKT
cells co-cultured with MyD88-deficient DC responded to Sphingomonas and
Ehrlichia but not Salmonella (FIG. 2A, right). Altogether, these results suggested
that in total spleens exposed to heat-killed Salmonella, IFN-K production was
initiated after TLR signaling of antigen presenting cells and subsequent recruitment
of NKT cells as well as other cell-types such as NK cells.. In contrast, IFN-Y
stimulation by Ehrlichia and Sphingomonas was primarily dependent on NKT cells
and CD1d with minimal contribution of TLR.
Similarly, lectin IB4 binding did not impair the stimulation of NKT cells by DC
pulsed with heat-killed Ehrlichia or Sphingomonas, consistent with direct recognition
of a distinct microbial antigen. However, the lectins readily blocked stimulation by
Salmonella (FIG. 2B), suggesting that for the Salmonella NKT response,
endogenous iGb3 is the likely ligand.
12

Hexb-l- DC pulsed with heat-killed Ehrlichia or Sphingomonas stimulated
NKT cells as well as wild-type DC. (FIG. 2C) In contrast, Salmonella-pulsed Hexb-l-
DC did not stimulate NKT cells.
Together, the results identify the endogenous ligand iGb3, rather than a
microbial antigen, as the target of NKT cells in their response to Salmonella
infection.
Example 3. NKT cell stimulatory response to synthetic glycolipid antigens.
a-glucuronosylceramide (PBS 30) and a-galacturonosylceramide (PBS 59),
derived from known Sphingomonadaceae cell membrane antigens, were
synthesized as described in Example 5. PBS 50, a b-glucuronosylceramide, served
as a control compound. The structures of these compounds are shown in FIG. 3A.
The immunological properties of the above compounds in NKT cells were
measured. Human Va24-Ja18 NKT cells and fresh purified mouse NKT cells were
co-cultured with DC pulsed with aGal-Cer or synthetic glycolipid at concentrations
ranging from 0.001 to 1000 ng/mL. IFN-Y production was measured at 48 hours as
described above.
CD1d tetramers were prepared as described in Example 1 using synthetic
glycolipids PBS 30, PBS 59 and PBS 50 and oGal-Cer, and were used to stain
human NKT cells and mouse spleen cells.
Results are shown in FIGS. 3 B-C. Both a-glucuronosylceramide (PBS 30)
and to a lesser degree, a-galacturonosylceramide (PBS 59) strongly activated
mouse and human NKT cell proliferation as well as IFN-y secretion, whereas control
b-glucuronosylceramide (PBS 50) did not (FIG. 3B). Tetramers of CD1d-a-
glucuronosylceramide (PBS 30) stained all human NKT cells and ~25% of mouse
NKT cells (FIG. 3C). Thus, these findings revealed that the lipids replacing LPS in
the cell wall of some species of Gram-negative bacteria may be directly recognized
by the conserved TCR of innate-like NKT cells.
Example 4. In vivo role of NKT cells during microbial infection.
CD1d-l- mice were generated at the University of Chicago, Ja18-l- mice were
obtained from Dr. Taniguchi, Chiba University (Japan) and Hexb-l- mice were
obtained from R. Proia, National Institutes of Health. All mice were in the C57/BL6
background. In all cases, littermates obtained from heterozygous matings were
genotyped by PCR and used for comparative analysis. All mice were raised in a
pathogen-free environment at University of Chicago according to the Institutional
Animal Care and Use Committee guidelines.
13

Six- to seven-week old C57/BL6 mice were intravenously inoculated with
100 ml Sphingomonas (1x107), Ehrlichia (1x108) or Salmonella (1x106) suspended in
PBS. Twenty-four hours post-infection, isolated NKT cells gated as tetramer+/B220-
were analyzed by FACS for surface CD69 (early T-cell activation antigen) and
intracellular IFN-Y. Results, shown in FIG. 4A, confirm that NKT cells are activated
and secrete IFN-Y within 24 hours after infection in vivo.
To determine whether hexb is required for antigen processing in response to
Salmonella and Sphingomonas infection in vivo, Hexb+/-and Hexb+/- littermates were
challenged intraperitoneally with 5x106 Sphingomonas or Salmonella. Two hours
post-challenge, 5x106 CFSE-labelled Va14 transgenic thymocytes were
intrasplenically injected in a volume of 50ml (Bendelac A. et al., J. Exp. Med. 184,
1285-12293 (1996), incorporated herein by reference). At 24 hours post-challenge,
intracellular staining for IFN-Y was performed. Results are shown in FIG. 4B. The
difference between Hexb+/- and Hexb+/- was statistically significant only for
Salmonella challenged mice, demonstrating that IFN-Y production by NKT cells in
response to Salmonella infection requires lysosomal iGb3, whereas the response of
NKT cells to Sphingomonas does not require lysosomal iGb3.
To characterize the role of NKT cells in controlling infection in vivo, Ja18-/-
and CD1-/- mice and their littermate controls were injected intravenously with either
5x106 or 1x106 Sphingomonas. Bacterial burden in the lungs was assessed at
intervals indicated in FIG. 4C. Bacterial counts were performed after tissue
homogenization in 0.5% Triton X-100 and cultured for colony formation. The results
demonstrate that both Ja18-/- and CD1-/- mice had delayed bacterial clearance
compared to heterozygous littermate controls, with up to 12-14 times higher
bacterial load in the lung at early time points.
For survival experiments, Ja18-/- and CD1-/- mice and their littermate controls
were injected intravenously with a high dose of 5x108 Sphingomonas. Dead or
moribund (euthanized) mice were recorded every 2-4 hours post-infection. The
results, shown in FIG. 4D, demonstrate that infection with a high dose of
Sphingomonas was rapidly lethal in wild-type mice, whereas a majority of NKT
deficient mice survived.
To test whether lethality was associated with cytokine release,
Sphingomonas (1x107) was intravenously injected in Ja18-/- and CD1-/- mice and
their littermate controls. At intervals specified in FIG. 4E, serum levels of IFN-Y and
IL-12 p40 were measured. The results indicate that the lethal outcome in wild-type
mice was associated with the explosive release of IFN-Y and IL-12 in the serum,
whereas NKT deficient mice produced significantly less cytokines.
14

For Ehrlichia infection experiments, mice were infected intraperitoneally with
500ml of a 10-1 dilution of Ehrlichia muris stock. The Ehrlichia load in the lungs,
livers and spleens of CD1d-/- and control Itttermates was determined by real-time
PCR of the Ehrlichia dsb gene (Ismail, N. et al., J. Immunol. 172, 1786-1800 (2004))
at 2 and 7 days post-infection. Results, reported in FIG. 4F, show that NKT
deficient mice demonstrate an inability to clear Ehrlichia.
Example 5. Synthesis of bacterial glycolipid PBS 61.
FIG. 6 depicts a suitable route of synthesis for PBS 61. To a vigorously
stirred solution of compound "1" (Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y.
Angew. Chem. Int. Ed. 2001, 40, 4725-4728.) (453 mg, 0.976 mmol) in CH2CI2 (3
mL) and water (1.5 mL) was added TEMPO (60.8 mg, 0.390 mmol) and
bis(acetoxy)iodobenzene (BAIB) (345 mg, 1.07 mmol) to produce intermediate
compound "2" in FIG. 6. Additional BAIB (345 mg, 1.07 mmol) was added after 1
hour. The reaction was stirred until TLC indicated complete conversion of the
starting material (~1.5 hour). The reaction mixture was extracted with CH2CI2 twice
and the combined organic layers were dried over MgSO4 and concentrated. A short
flash column (SiO2, CH3OH/CH2CI21:10) afforded crude glucoronic acid. A solution
of crude glucoronic acid in CH2CI2 (3 mL) was treated with a freshly prepared
ethereal solution of diazomethane until the evolution of gas ceased. The reaction
mixture was then treated with AcOH (2 mL) and concentrated in vacuo. Flash
column chromatography (SiO2, EtOAc/hexanes 1:4-1:3) afforded corresponding
methyl glucuronate (186 mg, 0.378 mmol, yield 39% of two steps). 1H NMR (CDCI3)
57.31-7.27 (m, 4 H), 6.87-6.86 (m, 4 H), 4.83-4.67(m, 4 H), 4.49 (d, J = 9.8 Hz, 1 H),
3.87-3.80 (m, 2 H), 3.78 (s, 3 H), 3.51 (t, J = 7.8 Hz, 1 H), 3.39 (t, J = 8.8 Hz, 1 H),
2.80-2.70 (m, 2 H), 1.32 (t, J = 7.3 Hz, 3 H). 13C NMR (CDCI3) d169.65, 159.47,
159.40, 130.65, 130.10, 129.71, 114.02, 113.89, 86.01, 84.83, 80.36, 75.28, 75.26,
71.91, 55.34, 52.79, 25.29, 15.13. High resolution fast atom bombardment mass
spectrometry (thioglycerol + Na + matrix) m/e ([M + Na]+) 515.1716 (100.0%);
calculated 515.1714. The methyl glucuronate (186 mg, 0.378 mmol) was dissolved
in CH2CI2 (10 mL) and Et3N (0.5 mL) followed by the introduction of a catalytic
amount of DMAP (20 mg) and Ac2O (0.2 mL). The solvent was removed in vacuo
after 12 hours and the residue was chromatographed (SiO2, EtOAc/hexane 1:4) to
afford the product (172 mg, 0.329 mmol, 87% yield) as clear oil. 1H NMR (CDCI3)
d57.31-7.18 (m, 4 H), 6.88-6.85 (m, 4 H), 5.12 (t, J = 9.8 Hz, 1 H), 4.83-4.61(m, 4 H),
4.47 (d, J = 9.8 Hz, 1 H), 3.86 (d, J = 10.3 Hz, 1 H), 3.80 (s, 3 H), 3.71 (s, 3 H), 3.65
(t, J = 8.8 Hz, 1 H), 3.49 (t, J = 8.8 Hz, 1 H), 2.82-2.68 (m, 2 H), 1.95 (s, 3 H), 1.32
15

(t, J = 7.3 Hz, 3 H). 13C NMR (CDCI3) d169.72, 167.94, 159.63, 159.48, 130.38,
130.29, 130.02, 129.66, 113.99, 85.56, 82.80, 80.61, 76.64, 75.49, 75.22, 71.33,
55.47, 52.92, 25.17, 20.89, 15.15. High resolution fast atom bombardment mass
spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 557.1827 (100.0%);
calculated 557.1822.
To prepare intermediate compound "6," a mixture of compound "2" (172 mg,
0.329 mmol), compound "3" (150 mg, 0.328 mmol), and 2,6-di-tert-butyl-4-
methylpyridine (67.6 mg, 0.329 mmol) in toluene (3 mL) was stirred with 4 Å
molecular sieves (300 mg) for 1 h at room temperature. Next,
dimethyl(methylthio)sulfonium triflate (66.8 mg, 0.329 mmol) was added, and stirring
was continued for 8 hours. The mixture was concentrated and passed through a
SiO2 plug using 1:1 EtOAc/hexanes. The solvent was removed in vacuo and the
residue was chromatographed (SiO2, EtOAc/hexane 1:5-1:4) to afford the product
"4" (61.1 mg, 0.0657 mmol, mixture of a-b-anomers, 20% yield). A solution of
compound "4" (61.1 mg, 0.0657 mmol, mixture of a-b-anomers) in pyridine (10 mL)
and water (2 mL) was treated with a stream of hydrogen sulfide for 15 minutes. The
solution was stirred for 12 hours, and then hydrogen sulfide was bubbled again for
15 minutes. The reaction mixture was stirred for another 12 hours. The solvent was
evaporated under vacuum and the residue was co-evaporated with toluene. The
residue was dissolved in CH2CI2 (10 mL) followed by the introduction of 5 (43.1 mg,
0.131 mmol). A solution of dicyclohexylcarbodiimide (DCC) (27.0 mg, 0.131 mmol)
and dimethylaminopyridine (DMAP) (6.3 mg, 0.052 mmol) in CH2CI2 was added, and
stirring was continued for 6 hours. The mixture was concentrated and passed
through a SiO2 plug using 1:1 EtOAc/hexanes. The solvent was removed in vacuo
and the residue was chromatographed (SiO2, EtOAc/hexane 1:5-1:4) to afford the
product "6" (22.3 mg, 0.0184 mmol, 28% yield of a-anomer). 1H NMR (CDCI3) d8.07-
8.04 (m, 2 H), 7.61-7.58 (m, 1 H), 7.47-7.44 (m, 2 H), 7.27-7.16 (m, 4 H), 6.87-6.81
(m, 4 H), 6.67 (d, J = 7.8 Hz, 1 H), 5.36-5.29 (m, 3 H), 5.18 (t, J = 5.9 Hz, 1 H), 5.00
(t, J = 9.3 Hz, 1 H), 4.79 (d, J = 3.4 Hz, 1 H), 4.74-4.54(m, 4 H), 4.52-4.49 (m, 1 H),
4.14 (d, J = 9.8 Hz, 1 H), 3.85-3.74 (m, 8 H), 3.69-3.62 (m, 4 H), 3.55 (dd, J = 9.3,
3.4 Hz, 1 H), 2.01-1.96 (m, 7 H), 1.88-1.78 (m, 2 H), 1.36-1.09 (m, 55 H), 0.90-0.87
(m, 6 H). 13C NMR (CDCI3) d177.19, 170.04, 169.84, 168.71, 166.45, 159.60,
159.40, 133.53, 130.76, 130.01, 129.46, 128.73, 114.03, 113.94, 98.56, 78.49,
78.19, 74.49, 73.97, 73.18, 71.18, 69.18, 67.96, 55.48, 52.92, 50.91, 49.38, 38.99,
34.16, 32.14, 31.99, 29.99, 29.86, 29.80, 29.65, 29.57, 29.19, 27.42, 27.30, 25.81,
25.57, 25.15, 24.84, 22.90, 20.91, 14.34. High resolution fast atom bombardment
16

mass spectrometry (thioglycerol + Na+ matrix) m/e ([M + Na]+) 1236.7557
(100.0%); calculated 1236.7539.
Preparation of PBS-61: Compound "6" (22.3 mg, 0.0184 mmol) was
dissolved in tetrahydrofuran (THF) (1 mL) and water (0.5 mL) followed by the
introduction of trifluoroacetic acid (TFA) (2 mL). The reaction was stirred until TLC
indicated complete conversion of the starting material to a lower spot (~1.0 h). The
reaction mixture was diluted by toluene and then concentrated in vacuo. The
dialcohol was obtained as a clear glass (10.0 mg, 0.0103 mmol, yield 56%) after
column chromatography (SiO2, MeOH/CH2CI2 1:40-1:24) 1H NMR (CDCI3) d8.02-
8.00 (m, 2 H), 7.64-7.61 (m, 1 H), 7.50-7.46 (m, 2 H), 6.67 (d, J = 7.8 Hz, 1 H), 5.35-
5.33 (m, 2 H), 5.22-5.18 (m, 1 H), 5.08 (t, J = 6.4 Hz, 1 H), 4.98 (t, J = 9.8 Hz, 1 H),
4.82 (d, J = 3.9 Hz, 1 H), 4.51-4.50 (m, 1 H), 4.22 (d, J = 9.8 Hz, 1 H), 3.99 (dd, J =
10.2, 3.4 Hz, 1 H), 3.93 (t, J = 9.8 Hz, 1 H), 3.72 (s, 3 H), 3.58 (dd, J = 9.3, 3.4 Hz, 1
H), 3.40 (dd, J = 10.3, 7.4 Hz, 1 H), 2.11 (s, 3 H), 2.05-1.98 (m, 4 H), 1.88-1.78 (m,
2 H), 1.36-1.09 (m, 64 H), 0.89-0.86 (m, 6 H). 13C NMR (CDCI3) d177.99, 170.94,
170.44, 168.67, 167.14, 134.07, 130.15, 130.01, 129.19, 128.92, 99.47, 74.98,
74.60, 72.23, 71.76, 71.51, 69.23, 68.35, 53.06, 51.61, 39.11, 32.28, 32.13, 31.99,
31.78, 29.86, 29.73, 29.63, 29.57, 29.40, 29.19, 27.42, 27.22, 25.68, 25.07, 22.91,
22.87, 20.97, 14.35. High resolution fast atom bombardment mass spectrometry
(thioglycerol + Na+ matrix) m/e ([M + Na]+) 996.6404 (100.0%); calculated
996.6388. The dialcohol (10.0 mg, 0.0103 mmol) was dissolved in MeOH (1 mL)
and THF (1 mL) followed by addition of NaOMe (0.2 mL of 1 M NaOMe solution in
MeOH) and 3 drops of water. The mixture was stirred for 12 hours and then water
(2 mL) was added. The reaction mixture was concentrated in vacuo and the residue
was chromatographed (SiO2, CHCI3/MeOH/H2O 60:30:4) to afford PBS-61 (5.0 mg,
0.069 mmol, 67% yield). 1H NMR (DMSO-d6 0.7ml with 1 drop of DCI and 3 drops
of D2O, 55 °C) d5.36-5.34 (m, 2 H), 4.79 (d, J = 3.4 Hz, 1 H), 3.94 (t, J = 5.9 Hz, 1
H), 3.88 (d, J = 9.7 Hz, 1 H), 3.82-3.79 (m, 1 H), 3.71-3.63 (m, 2 H), 3.58-3.56 (m, 1
H), 3.50 (t, J = 9.3, 1 H), 3.38 (t, J = 9.3 Hz, 1 H), 3.30 (dd, J = 9.3, 3.4 Hz, 1 H),
2.01-1.99 (m, 4 H), 1.60-1.55 (m, 2 H), 1.36-1.09 (m, 64 H), 0.90-0.87 (m, 6 H). 13C
NMR (DMSO-d6 0.7ml with 1 drop of DCI and 3 drops of D2O, 55 °C) d174.21,
171.39, 130.29, 100.46, 100.38, 73.35, 72.37, 71.54, 69.98, 68.02, 53.22, 41.09,
34.93, 34.22, 31.92, 31.75, 31.56, 29.93,29.71, 29.32, 28.89, 27.26, 25.70, 24.97,
22.68, 14.54. High resolution fast atom bombardment mass spectrometry
(thioglycerol + Na+ matrix) m/e ([M + Na]+) 752.5289 (100.0%); calculated
752.5284.
17

All publications, patents and patent applications referenced in this
specification are indicative of the level of ordinary skill in the art to which this
invention pertains. All publications, patents and patent applications are herein
expressly incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually indicated by
reference. In case of conflict between the present disclosure and the incorporated
patents, publications and references, the present disclosure should control.
We claim:

1. A glycolipid of formula (I):

wherein the glycolipid is selected from the following compounds:

- PBS-49, wherein X is methyl; R1 is-H; R2 is-OH; R3 is-OH; is-H; R 5

is C11 alkyl; R6 is Cn alkyl; and R1 is -H;

- PBS-45, wherein X isH; R 1 is-H; R2 is -OH; R3 is -OH; is-H; R5 is

Cll alkyl; R6 is C13 alkyl; and R1 is-H;

- PBS-30, wherein X isH; R1 is-H; R2 is -OH; R3 is -OH; is-H; Rs is

C1 6 alkyl; R6 is C1s alkyl; and R1 is-H;

- PBS-29, wherein X is H; R1 is -H; R2 is -OH; R3 is-H;is -OH; Rs is

C23 alkyl; R6 is C13 alkyl; and R7 is-H;

- PBS-62, wherein X isH; Rt is-H; R2 is -OH; R3 is -OH; is-H; Rs is

C23 alkyl; R6 is C1s alkyl comprising one double bond; and R7 is-H; and

- PBS-65, wherein X isH; R1 is-H; R2 is -OH; R3 is -OH; is-H; Rs is

C 11 alkyl; R6 is C 13 alkyl comprising one cyclopropyl group; and R7 is -H.

2. A vaccine adjuvant comprising the glycolipid of formula (I) as

claimed in claim 1.

ABSTRACT

Disclosed are methods for activating an NKT cell, methods of stimulating an immune
response in a subject, methods of improving vaccine efficacy, and methods of treating an
infection. Also disclosed are methods of promoting tumor rejection, treating cancer,
modulating autoimmunity and inhibiting allergen-induced hypersensitivity in subjects. The
methods include contacting an NKT cell with a bacterial glycolipid complexed with a CD1
molecule to activate the NKT cell. The bacterial glycolipid may be derived from a member of
the Class Alphaproteobacteria.

Documents:

02840-kolnp-2007-abstract.pdf

02840-kolnp-2007-claims .pdf

02840-kolnp-2007-correspondence others.pdf

02840-kolnp-2007-description complete.pdf

02840-kolnp-2007-drawings.pdf

02840-kolnp-2007-form 1.pdf

02840-kolnp-2007-form 2.pdf

02840-kolnp-2007-form 3.pdf

02840-kolnp-2007-form 5.pdf

02840-kolnp-2007-international publication.pdf

02840-kolnp-2007-international search report.pdf

02840-kolnp-2007-others pct form.pdf

02840-kolnp-2007-priority document.pdf

2840-KOLNP-2007-(03-12-2012)-AMANDED PAGES OF SPECIFICATION.pdf

2840-KOLNP-2007-(03-12-2012)-CLAIMS.pdf

2840-KOLNP-2007-(03-12-2012)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(10-10-2012)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(17-10-2012)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(19-12-2011)-ABSTRACT.pdf

2840-KOLNP-2007-(19-12-2011)-AMANDED CLAIMS.pdf

2840-KOLNP-2007-(19-12-2011)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(19-12-2011)-DESCRIPTION (COMPLETE).pdf

2840-KOLNP-2007-(19-12-2011)-DRAWINGS.pdf

2840-KOLNP-2007-(19-12-2011)-FORM-1.pdf

2840-KOLNP-2007-(19-12-2011)-FORM-2.pdf

2840-KOLNP-2007-(19-12-2011)-FORM-3.pdf

2840-KOLNP-2007-(19-12-2011)-OTHER PATENT DOCUMENT.pdf

2840-KOLNP-2007-(19-12-2011)-OTHERS.pdf

2840-KOLNP-2007-(21-03-2014)-CLAIMS.pdf

2840-KOLNP-2007-(21-03-2014)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(23-09-2013)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(25-06-2012)-AMANDED CLAIMS.pdf

2840-KOLNP-2007-(25-06-2012)-AMANDED PAGES OF SPECIFICATION.pdf

2840-KOLNP-2007-(25-06-2012)-CORRESPONDENCE.pdf

2840-KOLNP-2007-(25-06-2012)-DESCRIPTION (COMPLETE).pdf

2840-KOLNP-2007-(25-06-2012)-FORM-1.pdf

2840-KOLNP-2007-(25-06-2012)-FORM-2.pdf

2840-KOLNP-2007-(25-06-2012)-OTHERS.pdf

2840-KOLNP-2007-ASSIGNMENT.pdf

2840-KOLNP-2007-CORRESPONDENCE 1.1.pdf

2840-KOLNP-2007-CORRESPONDENCE OTHERS-1.1.pdf

2840-kolnp-2007-form 18.pdf

2840-KOLNP-2007-FORM 3 1.1.pdf

2840-KOLNP-2007-FORM 3-1.1.pdf

2840-KOLNP-2007-PA.pdf

2840-KOLNP-2007-PETITION UNDER RULE 137.pdf

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Patent Number

259862

Indian Patent Application Number

2840/KOLNP/2007

PG Journal Number

14/2014

Publication Date

04-Apr-2014

Grant Date

28-Mar-2014

Date of Filing

03-Aug-2007

Name of Patentee

THE SCRIPPS RESEARCH INSTITUTE

Applicant Address

10550 NORTH TORREY PINES ROAD LA JOLLA CALIFORNIA

Inventors:

#	Inventor's Name	Inventor's Address
1	TEYTON, LUC	14226 PINEWOOD DRIVE DEL MAR CALIFORNIA 92014
2	SAVAGE, PAUL	1075 EAST MAPLE STREET MAPLETON, UTAH 84664
3	BENDELAC, ALBERT	1121 EAST 53RD STREET CHICAGO, ILLINOIS 60615

PCT International Classification Number

C12Q 1/00,C12N 13/00

PCT International Application Number

PCT/US2006/002781

PCT International Filing date

2006-01-26

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/648,153	2005-01-28	U.S.A.