It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.

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.

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.

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.

Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosisinducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.

A defect in RelB, a member of the Rel/nuclear factor (NF)-κB family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-κB–inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/− × aly/+ compound heterozygous mice. After stimulation with α-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/− or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of Vα14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the μ subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.