Natural killer T (NKT) cells recognize glycosphingolipids presented by CD1d molecules and have been linked to defense against microbial infections. Previously defined foreign glycosphingolipids recognized by NKT cells are uniquely found in nonpathogenic sphingomonas bacteria. Here we show that mouse and human NKT cells also recognized glycolipids, specifically a diacylglycerol, from Borrelia burgdorferi, which causes Lyme disease. The B. burgdorferi-derived, glycolipid-induced NKT cell proliferation and cytokine production and the antigenic potency of this glycolipid was dependent on acyl chain length and saturation. These data indicate that NKT cells recognize categories of glycolipids beyond those in sphingomonas and suggest that NKT cell responses driven by T cell receptor-mediated glycolipid recognition may provide protection against diverse pathogens.

Natural killer T (NKT) cells recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell receptor (TCR), but the forces driving TCR conservation have remained uncertain. Here we show that NKT cells recognize diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells are required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is found at a low level in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR, and most important, they extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

The requirement for processing glycolipid antigens in T cell recognition was examined with mouse CD1d-mediated responses to glycosphingolipids (GSLs). Although some disaccharide GSL antigens can be recognized without processing, the responses to three other antigens, including the disaccharide GSL Gal(alpha1-->2)GalCer (Gal, galactose; GalCer, galactosylceramide), required removal of the terminal sugars to permit interaction with the T cell receptor. A lysosomal enzyme, alpha-galactosidase A, was responsible for the processing of Gal(alpha1-->2)GalCer to generate the antigenic monosaccharide epitope. These data demonstrate a carbohydrate antigen processing system analogous to that used for peptides and an ability of T cells to recognize processed fragments of complex glycolipids.

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription–polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of α-galactosylceramide (α-galcer) and endogenous antigens in mouse splenic and bone marrow–derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of α-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.

House dust contains antigens capable of activating mouse and human iNKT cells, contributing to allergen-induced airway inflammation.

Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3–deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3–deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II–reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.