NKT cells are thought of as a bridge between innate and adaptive immunity. In this study, we demonstrate that mouse NKT cells are activated in response to Escherichia coli LPS, and produce IFN-γ, but not IL-4, although activation through their TCR typically induces both IL-4 and IFN-γ production. IFN-γ production by NKT cells is dependent on LPS-induced IL-12 and IL-18 from APC. LPS induced IFN-γ production by NKT cells does not require CD1d-mediated presentation of an endogenous Ag and exposure to a combination of IL-12 and IL-18 is sufficient to activate them. In mice that are deficient for NKT cells, innate immune cells are activated less efficiently in response to LPS, resulting in the reduced production of TNF and IFN-γ. We propose that in addition to acting as a bridge to adaptive immunity, NKT cells act as an early amplification step in the innate immune response and that the rapid and complete initiation of this innate response depends on the early production of IFN-γ by NKT cells.

Certain glycolipid Ags for Vα14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, α-galactosylceramide. Although the CD1d-exposed portions of OCH and α-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-γ, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.

Invariant NK T (iNKT) cells influence the response to viral infections, although the mechanisms are poorly defined. In this study we show that these innate-like lymphocytes secrete IFN-γ upon culture with CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but it does not require CD1d expression. iNKT cells also produce IFN-γ in response to mouse CMV infection. Their mechanism of mouse CMV detection is quite similar to that of CpG, requiring both TLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an Ag-independent manner, suggesting they may play a broad role in antipathogen defenses despite their limited TCR repertoire.

The ER aminopeptidase associated with antigen processing, ERAAP, is essential for trimming peptides presented by MHC I molecules. ERAAP inhibition by cytomegalovirus causes immune evasion, and ERAAP polymorphisms are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that ERAAP inhibition rapidly induced presentation of the FL9 peptide by the Qa-1b MHC Ib molecule. Antigen-experienced T cells specific for the Qa-1b-FL9 complex were frequent in naïve mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8+ T cell response specific for the Qa-1b-FL9- complex. MHC Ib-restricted cytolytic effectors specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, non-classical peptide-Qa-1b complexes direct cytotoxic T cells to targets with defective antigen processing in the ER.

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.

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of α-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-γ, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-γ production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, α-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.

The MHC class I (MHC-I) molecules ferry a cargo of peptides to the cell surface as potential ligands for CD8+ cytotoxic T cells. For nearly 20 years, the cargo has been described as a collection of short 8-9 mer peptides, whose length and sequences were believed to be primarily determined by the peptide-binding groove of MHC-I molecules. Yet the mechanisms for producing peptides of such optimal length and composition have remained unclear. In this study, using mass spectrometry, we determined the amino acid sequences of a large number of naturally processed peptides in mice lacking the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). We find that ERAAP-deficiency changed the oeuvre and caused a marked increase in the length of peptides normally presented by MHC-I. Furthermore, we observed similar changes in the length of viral peptides recognized by CD8+ T cells in mouse CMV-infected ERAAP-deficient mice. In these mice, a distinct CD8+ T cell population was elicited with specificity for an N-terminally extended epitope. Thus, the characteristic length, as well as the composition of MHC-I peptide cargo, is determined not only by the MHC-I peptide-binding groove but also by ERAAP proteolysis in the endoplasmic reticulum.

The peptide repertoire presented by classical as well as non-classical MHC I molecules is altered in the absence of the ER aminopeptidase associated with antigen processing (ERAAP). To characterize the extent of these changes, peptides from cells lacking ERAAP were eluted from the cell surface and analyzed by high-throughput mass spectrometry. We found that the majority of peptides found in WT cells were retained in the absence of ERAAP. In contrast, a subset of “ERAAP-edited” peptides was lost in WT cells, and ERAAP-deficient cells presented an unique “unedited” repertoire. A substantial fraction of MHC-associated peptides from ERAAP-deficient cells contained N-terminal extensions and had a different molecular composition than those from WT cells. We found that the number and immunogenicity of peptides associated with non-classical MHC I was increased in the absence of ERAAP. Conversely, only peptides presented by classical MHC I were immunogenic in ERAAP-sufficient cells. Finally, MHC I peptides were also derived from different intracellular sources in ERAAP-deficient cells.