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CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans. [ABSTRACT FROM AUTHOR]

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-&#x03B3 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. [ABSTRACT FROM AUTHOR]