Lipid and carbohydrate modifications of α-galactosylcer-amide differently influence mouse and human type I natural killer T cell activation

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from alpha-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type INKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators

Immune and Structural Studies of Synthetic Invariant Natural Killer T cell Glycosphingolipid Activators

Invariant Natural Killer T (iNKT) cells comprise a small fraction of immune cells capable of responding within hours when stimulated by glycosphingolipid (GSL) antigens (Ags) and can impact the immune system months later. These iNKT cells respond to GSLs that are presented by the antigen presenting molecule CD1d. The iNKT cell driven immune cytokine response can alter depending on the GSL that is presented by CD1d. The GSL can lead to the production of a T helper type 1 (Th1) or a T helper type 2 (Th2) response characterized by IFN-gamma and IL-4 cytokines. Many synthetic GSL analogs of the most common iNKT cell antigen, alpha-Galactosylceramide (aGalCer) were used in this study to determine immunological and biochemical properties of Ags capable of activating iNKT cells. Studies consisted of in vitro Ag binding models, crystallographic structural models and in vivo immunological studies. Ags that are categorized as Th1 iNKT cell cytokine skewers have properties correlated with this response. The Th1 Ags studied are presented by CD1d on dendritic antigen presenting cells (APCs) and the CD1d and GSL seem to form more contacts according to structural studies and the CD1d-GSL complexes are more biologically stable in vivo. The work from these studies adds the to growing information in the field regarding the nature of iNKT cell immune system skewin

Antigen specificity of invariant natural killer T-cells.

Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer

Antigen specificity of invariant natural killer T-cells

Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer

Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation

Activation of invariant (i) NKT cells with the model Ag alpha-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with a-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness

Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation

Activation of invariant natural killer T (iNKT) cells with the model antigen α-galactosylceramide (αGalCer) induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with αGalCer, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hypo-responsive state, however, remain poorly defined. Here, we show that Th1-biasing iNKT cell antigens could induce iNKT cell hypo-responsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing antigen OCH did not induce a hypo-responsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, while DCs and B cells have been reported to be essential for iNKT cell stimulation, neither DCs nor B cells were required to induce iNKT cell hypo-responsiveness. Therefore, our data indicate that while some bone marrow-derived cells could induce iNKT cell hypo-responsiveness, selective conditions, dependent on the structure and potency of the antigen, were required to induce hypo-responsiveness

A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production

Here we characterize a novel Ag for invariant natural killer T-cells (iNKT cells) capable of producing an especially robust Th1 response. This glycosphingolipid (GSL), DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), the only change being in a single atom, the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared to αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by DCs in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB061 compared to αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Our data are therefore consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result in part from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10 producing iNKT cells, which could counteract the benefits of increased, early IFN-γ production