Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.

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.

Recognition of lyso-phospholipids by human natural killer T lymphocytes.

Natural killer T (NKT) cells are a subset of T lymphocytes with potent immunoregulatory properties. Recognition of self-antigens presented by CD1d molecules is an important route of NKT cell activation; however, the molecular identity of specific autoantigens that stimulate human NKT cells remains unclear. Here, we have analyzed human NKT cell recognition of CD1d cellular ligands. The most clearly antigenic species was lyso-phosphatidylcholine (LPC). Diacylated phosphatidylcholine and lyso-phosphoglycerols differing in the chemistry of the head group stimulated only weak responses from human NKT cells. However, lyso-sphingomyelin, which shares the phosphocholine head group of LPC, also activated NKT cells. Antigen-presenting cells pulsed with LPC were capable of stimulating increased cytokine responses by NKT cell clones and by freshly isolated peripheral blood lymphocytes. These results demonstrate that human NKT cells recognize cholinated lyso-phospholipids as antigens presented by CD1d. Since these lyso-phospholipids serve as lipid messengers in normal physiological processes and are present at elevated levels during inflammatory responses, these findings point to a novel link between NKT cells and cellular signaling pathways that are associated with human disease pathophysiology.

Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Natural killer T-cell autoreactivity leads to a specialized activation state.

Natural killer T (NKT) cells are innate-like T cells that recognize specific microbial antigens and also display autoreactivity to self-antigens. The nature of NKT-cell autoreactive activation remains poorly understood. We show here that the mitogen-activated protein kinase (MAPK) pathway is operative during human NKT-cell autoreactive activation, but calcium signaling is severely impaired. This results in a response that is biased toward granulocyte macrophage colony-stimulating factor (GM-CSF) secretion because this cytokine requires extracellular signal-regulated kinase (ERK) signaling but is not highly calcium dependent, whereas interferon-gamma (IFN-gamma), interleukin (IL)-4, and IL-2 production are minimal. Autoreactive activation was associated with reduced migration velocity but did not induce arrest; thus, NKT cells retained the ability to survey antigen presenting cells (APCs). IL-12 and IL-18 stimulated autoreactively activated NKT cells to secrete IFN-gamma, and this was mediated by Janus kinase-signal transducers and activators of transcription (JAK-STAT)-dependent signaling without induction of calcium flux. This pathway did not require concurrent contact with CD1d(+) APCs but was strictly dependent on preceding autoreactive stimulation that induced ERK activation. In contrast, NKT-cell responses to the glycolipid antigen alpha-galactosyl ceramide (alpha-GalCer) were dampened by prior autoreactive activation. These results show that NKT-cell autoreactivity induces restricted cytokine secretion and leads to altered basal activation that potentiates innate responsiveness to costimulatory cytokines while modulating sensitivity to foreign antigens.

Rapid identification of immunostimulatory alpha-galactosylceramides using synthetic combinatorial libraries.

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d.

The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.

Synthesis and evaluation of sphinganine analogues of KRN7000 and OCH.

[structures: see text] The phytosphingosine-containing alpha-galactosylceramides (alpha-GalCers), KRN7000 and OCH, have been shown to activate NKT cells via interaction with CD1d, a member of the CD1 family of antigen presenting proteins. Evidence from KRN7000 stimulation of NKT cells suggests that alpha-GalCers may have applications in the treatment or prevention of a range of viral, bacterial, and autoimmune conditions. Moreover, OCH, a truncated analogue of KRN7000, appears to induce a T(H)2 bias, which could have implications for the treatment of autoimmune and inflammatory conditions. We have prepared the direct sphinganine-containing analogues of KRN7000 and OCH, 1 and 2, and found them to be comparable in activity to the parent compounds in inducing the release of IL-2, IL-4, and IFNgamma. In addition, compound 2 leads to a cytokine bias similar to that seen with OCH. This is significant because sphinganines are more easily accessed than phytosphingosines, which should facilitate SAR studies.

The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition.

Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.

Adsorption and detection of some phenolic compounds by rice husk ash of Kenyan origin.

Rice husk ash (RHA) obtained from a rice mill in Kenya has been used as an inexpensive and effective adsorbent (and reagent) for the removal (and detection) of some phenolic compounds in water. The abundantly available rice mill waste was used in dual laboratory-scale batch experiments to evaluate its potential in: (i) the removal of phenol, 1,3-dihydroxybenzene (resorcinol) and 2-chlorophenol from water; and (ii) the detection of 1,2-dihydroxybenzene (pyrocatechol) and 1,2,3-trihydroxybenzene (pyrogallol) present in an aqueous medium. The studies were conducted using synthetic water with different initial concentrations of the phenolic compounds. The effects of different operating conditions (such as contact time, concentration of the phenolic compounds, adsorbent quantity, temperature, and pH) were assessed by evaluating the phenolic compound removal efficiency as well as the extent of their color formation reactions (where applicable). RHA exhibits reasonable adsorption capacity for the phenolic compounds and follows both Langmuir and Freundlich isotherm models. Adsorption capacities of 1.53 x 10(-4), 8.07 x 10(-5), and 1.63 x 10(-6) mol g(-1) were determined for phenol, resorcinol and 2-chlorophenol, respectively. Nearly 100% adsorption of the phenolic compounds was possible and this depended on the weight of RHA employed. For the detection experiments, pyrocatechol and pyrogallol present in water formed coloured complexes with RHA, with the rate of colour formation increasing with temperature, weight of RHA, concentration of the phenolic compounds and sonication. This study has proven that RHA is a useful agricultural waste product for the removal and detection of some phenolic compounds.