Jacquelyn Gervay-Hague

Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells

The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, α‐galactosylceramide (α‐GalCer) in a CD1d‐dependent manner. Because of the limited availability of α‐GalCer, there is a constant search for CD1d‐presented ligands that activate NKT cells. The α‐anomericity of the carbohydrate is considered to be an important requisite for the CD1d‐specific activation of NKT cells. The gram‐negative, lipopolysaccharide‐free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with α‐anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d‐specific manner. In addition, soluble CD1d–Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d‐specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states.

Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma.

Natural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) alpha chain rearrangement (Valpha14 Jalpha18 in mice; Valpha24 Jalpha18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the hosts innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b(+)Gr1(+)) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT-deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b(+)Gr1(+) cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.

A Nanoengineering Approach for Investigation and Regulation of Protein Immobilization

It is known that protein attachment to surfaces depends sensitively upon the local structure and environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both atomic force microscopy (AFM)-based lithography techniques, protein binding sites with well-defined local environments are designed and engineered with nanometer precision. Three proteins, goat antibiotin immunoglobulin G (IgG), lysozyme, and rabbit immunoglobulin G, are immobilized onto these engineered surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in antibody recognition, covalent attachment via primary amine residues and surface-bound aldehyde groups. This investigation indicates that AFM-based nanolithography enables the production of protein nanostructures, and more importantly, protein-surface interactions at a molecular level can be regulated by changing the binding domains and their local environment at nanometer scale.

Virus-induced inhibition of CD1d1-mediated antigen presentation: Reciprocal regulation by p38 and ERK

A critical component of the host’s innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.

Solution- and solid-phase oligosaccharide synthesis using glucosyl iodides: a comparative study

Glycosyl iodide donors have been used in both solid- and solution-phase syntheses yielding alpha-(1 --> 6)-linked glucosyl oligomers in highly efficient protocols. While the solid-phase strategy offers advantages in terms of ease of purification, it requires a total of 7.5 equiv of donor and approximately 12 h to complete the incorporation of one monosaccharide unit. In contrast, solution-phase methods require only 2.5 equiv of donor and 2-3 h reaction time per glycosylation. Moreover, since the reactions are virtually quantitative (> 90%) column chromatography of the material is facile. The overall advantages of solution-phase oligosaccharide synthesis were further illustrated in the convergent synthesis of a hexamer (methoxycarbonylmethyl 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6)-tetrakis-(2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6))-2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside) that was constructed from dimer donor iodides in a two-plus-two and a two-plus-four fashion.

Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo

We have investigated the role of the hosts CD1d‐dependent innate antitumor immune response in a murine T‐cell lymphoma model in vivo. We found that C57BL/6 wildtype (WT) mice inoculated with RMA/S cells transfected with murine CD1d1 died at the same rate as mice inoculated with vector‐transfected cells. In contrast, natural killer T (NKT) cell‐deficient CD1d or Jα18 knockout mice inoculated with CD1d‐transfected RMA/S cells survived significantly longer than mice inoculated with vector‐transfected RMA/S cells, implicating the involvement of invariant NKT (iNKT) cells in inhibiting antitumor activity in vivo. In contrast to the mutant mice, which produced more of the proinflammatory cytokines IFN‐γ and GM‐CSF, WT mice produced significantly elevated amounts of IL‐13. Antitumor activity in the knockout mice was not due to the development of CD1d‐specific cytotoxic T lymphocytes or circulating antibodies. However, iNKT cell numbers were elevated in tumor‐bearing mice. Thus, iNKT cells may be playing a negative role in the hosts antitumor immune response against T‐cell lymphomas in a CD1d‐dependent manner.

Inhibition of CD1d1-mediated antigen presentation by the vaccinia virus B1R and H5R molecules

Vaccinia virus (VV) has been most commonly used as the vaccine to protect individuals against the causative agent of smallpox (variola virus), but it also uses a number of strategies meant to evade or blunt the hosts antiviral immune response. Natural killer T (NKT) cells are a subset of immunoregulatory CD1d‐restricted T lymphocytes believed to bridge the innate and adaptive immune responses. It is shown here that the VV‐encoded molecules, B1R and H5R, play a role in the ability of VV to inhibit CD1d‐mediated antigen presentation to NKT cells. These are the first poxvirus‐encoded molecules identified that can play such a role in the evasion of an important component of the innate immune response.

Current Developments in HIV Chemotherapy

HIV infection is the leading cause of death worldwide and despite major advances in treatment, more new cases were diagnosed in 2004 than any previous year. Current treatment regimens are based on the use of two or more drugs from two or more classes of inhibitors termed highly active antiretroviral therapy (HAART). Although HAART is capable of suppressing viral loads to undetectable levels, problems of toxicity, patient adherence, and particularly the emergence of drug‐resistant viruses continues to spur the development of new chemotherapeutics to combat HIV. Clinical candidates from the four existing classes of inhibitors are presented in this review along with lead compounds against new viral targets, with special emphasis on HIV integrase.

Construction of di-scFv through a trivalent alkyne–azide 1,3-dipolar cycloaddition

Heterofunctional azide and alkyne PEG-linkers have been synthesized and site specifically conjugated to scFv via a reactive thiol functionality; two scFv were coupled by copper catalyzed 1,3-dipolar cycloaddition to make divalent scFv (di-scFv) with an inter-scFv distance defined to provide divalent binding; antigen binding was maintained for the di-scFv construct and increased several times compared to that of the parent scFv; the cycloaddition reaction reported herein represents an important ligation strategy to covalently link macromolecular proteins and retain sensitive structural conformations.

One-pot syntheses of immunostimulatory glycolipids.

Glycolipids containing alpha-linked galactosyl and glucosyl moieties have been shown to possess unique immunostimulatory activity creating a need for access to diverse and anomerically pure sources of these compounds for immunological studies. To meet this demand, glycosyl iodides were enlisted in the synthesis of these biologically relevant glycoconjugates. In the first-generation protocol, per-O-benzyl galactosyl iodide was efficiently coupled with activated sphingosine acceptors, but fully functionalized ceramides were found to be unreactive. To overcome this obstacle, per-O-trimethylsilyl glycosyl iodides were investigated and shown to undergo highly efficient coupling with ceramide and glycerol ester acceptors. Contrary to what has been observed with other donors, we detected little difference between the reactivity of glucosyl and galactosyl iodides. The trimethylsilyl protecting groups play a dual role in activating the donor toward nucleophilic attack while at the same time providing transient protection: the silyl groups are readily removed upon methanolysis. All reactions proceeded with complete acceptor regioselectivity, eliminating the need for additional protecting group manipulations, and the desired alpha-anomers were formed exclusively. This three-step, one-pot synthetic platform provides rapid access to an important class of immunostimulatory molecules including the first reported synthesis of the glucosyl analogue of the bacterial antigen BbGL-II.