A Rapid Fluorescence-Based Assay for Classification of iNKT Cell Activating Glycolipids

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 03/2011; 133(14):5198-201. DOI: 10.1021/ja200070u
Source: PubMed


Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.

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Available from: Pooja Arora
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    • "In addition, although we did not see any difference in cell viability during the ex vivo pulsing of DCs with and without CQ before transfer into recipient animals, we expect the stability of cells to be decreased rather than increased by CQ treatment since endosomal acidification is essential for normal cell survival. Furthermore, the total number of cell surface CD1d/glycolipid complexes is generally higher on APCs exposed to Th2-biasing glycolipids compared to those exposed to similar concentrations of Th1-biasing glycolipids, when measured by staining with the complex-specific L363 antibody (Im et al., 2009; Arora et al., 2011b). This indicates that NK cell transactivation depends on a qualitatively different initial response of iNKT cells, and not merely a difference in the quantitative strength of TCR signalling. "
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    ABSTRACT: Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure-activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these "stabilized" CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    Full-text · Article · Aug 2015 · Chemistry and Physics of Lipids
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    • "Recently, an α-GC analogue known as 7DW8-5 has been reported to be a potent activator of human NKT cells that performs well as an experimental vaccine adjuvant in nonhuman primates [31]. Similar to α-C-GC, the in vivo cytokine response to 7DW8-5 following systemic administration in mice reveals a bias toward strong IFN-γ production with relatively low, transient IL-4 secretion (Fig. S3) [32]. These properties have stimulated substantial interest in the development of 7DW8-5 as an adjuvant for human vaccination [33]. "
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    ABSTRACT: Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8 + T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8 + T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid a-galactosylceramide (a-GC) into rBCG-SIV gag significantly enhanced CD8 + T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of a-GC to enhance CD8 + T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an a-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8 + T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "All screening experiments were performed as previously described (Arora et al., 2011). Briefly, JAWS II cells were seeded in U bottom 96 well plates. "
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    ABSTRACT: Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, α-galactosyl ceramide (αGalCer), is a potential anticancer agent whose activity depends upon IFN-γ secretion. We report two analogs of αGalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-γ that correlates with detergent-resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen-presenting cells. Structural analysis on one of the analogs indicates that it is more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-γ-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility.
    Full-text · Article · Dec 2011 · Chemistry & biology
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