Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.

The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, a-galactosylceramide (a-GalCer) in a CD1d-dependent manner. Because of the limited availability of a-GalCer, there is a constant search for CD1d-presented ligands that activate NKT cells. The a-anomericity of the carbohydrate is considered to be an important requisite for the CD1d-specific activation of NKT cells. The gram-negative, lipopolysaccharide-free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with a-anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d-specific manner. In addition, soluble CD1d-Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d-specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states. IntroductionLipid antigens, including phospholipids and glycosphingolipids (GSL), are presented by CD1d -a subset of the CD1 family of MHC class I-like molecules -to specialized immune effector cells called NKT cells [1]. Located on a different chromosome than the MHC, five different CD1 genes encode CD1 molecules -CD1a, CD1b, CD1c, CD1d and CD1e -in humans, whereas only two homologues of CD1d (CD1d1 and CD1d2) are present in mice [2]. On the basis of the crystal structure of mouse CD1d1 [3], it is predicted that the fatty-acyl chains are buried in the hydrophobic pocket of the molecule with the hydrophilic head-group of the lipid antigen available outside the molecule for its interaction with the NKT cell receptor. Even though the lipidbinding groove of CD1d is widely accommodative of many lipid groups, it is believed that only the sugar structures in a-anomeric orientation are able to stimulate NKT cells [4]. Because of the lack of physiological glycolipids with terminal a-anomeric sugars, it is hypothesized that both altered selfglycolipids during a pathological process and exogenous antigenic glycolipids could be potential ligands presented by CD1d [4]. The presence of GSL in the cell wall of some bacterial strains indicates the possibility of these bacterial GSL being presented by CD1d. Although human CD1a, b and c molecules are known to present mycobacterial antigens to human NKT cells, there is a lack of substantial evidence to show that CD1d has the ability to present these microbial lipids [5]. In a recent study, Fischer et al. [6] were able to show that mycobacterial phosphoinositolmannosides (PIM) could bind to soluble CD1d and activate human and mouse NKT cells. However, only a fraction of NKT cells detected by agalactosylceramide (a-GalCer) could be stained by a CD1d tetramer loaded with mycobacterial PIM [6]. A recent study [7] was able to show the binding of synthetic a-GalNAc containing GSL to cell surface CD1d, but no functional studies were done t...

Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.