New insights into pathways for CD1-mediated antigen presentation

Recent studies of CD1 structure and intracellular trafficking have demonstrated significant differences among the CD1 isoforms (CD1a, CD1b, CD1c and CD1d). The molecular and structural basis for the differential trafficking of CD1 molecules has also been delineated. These observations broaden our understanding of why the immune system has evolved multiple CD1 isoforms to survey different cellular compartments for lipid antigen presentation, to provide host defense against the microbial world and to offer immunoregulation with relevance to tumor immunity and autoimmunity.     

Separate pathways for antigen presentation by CD1 molecules

The ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.     

CD1 and lipid antigens: intracellular pathways for antigen presentation

Recently, different members of the CD1 family of MHC-like molecules have been shown to sample different intracellular compartments to present lipid and glycolipid antigens to T cells. Emerging models suggest that CD1 may have evolved to monitor the integrity of membrane lipids and/or to present microbial lipid antigens to both alpha beta and gamma delta T cells.     

Apoptosis-induced inhibition of CD1d-mediated antigen presentation: different roles for caspases and signal transduction pathways

The stimulation of programmed cell death can either enhance or inhibit antigen presentation by classic major histocompatibility complex molecules. In the current study, we report that the induction of apoptosis by topoisomerase I inhibition or elevation of intracellular ceramide levels substantially impairs CD1d-mediated antigen presentation. In the former case, such a reduction occurred via the regulation of both the p38 mitogen-activated protein kinases and protein kinase C delta signal transduction pathways as well as the caspase cascade, whereas the latter was p38-(but not caspase)-dependent. Confocal microscopic analysis showed an altered intracellular distribution of CD1d following the inhibition topoisomerase I or by an increase in intracellular ceramide levels, that was prevented by p38 and caspase inhibitors. Thus, the induction of apoptosis in antigen presenting cells severely compromises CD1d-mediated antigen presentation by multiple mechanisms.     

Lipid antigen presentation in the immune system; lessons learned from CD 1 d knockout mice

Summary: CD I molecules represent a distinct lineage of antigen-presenting molecules chat are evolutionarily related to the classical major histocompatility complex (MHC) dass I and class II molecules, Unlike the classical MHC products that bind peptides, GDI molecules have evolved Co bind lipids and glycolipids, Murine and human CD Id molecules can present glycolipid antigens such as α-galactosylceramide (α-GalCer) to CD 1d-restricced natural killer (NK) T cells. Using CD 1d knockout mice we demonstrated chat CDI d expression is required for the development of NK T cells. These animals were also deficient in the rapid production of inter-leukin-4 and intcrferon-γ in response to stimulation by anti-CD3 antibodies. Despite these defects, CD Id knockout animals were able to generate strong T-helper type 1 (TH1) and TH2 responses. Spleen cells from these animals neither proliferated nor produced cytokines in response to stimulation by α-GalCer, Repeated injection of α-GalCer into wild-type but not CD 1 d mutant mice was able to clear metastatic tumors. We further showed that α-GalCer can inhibit disease in diabetes-prone non-obese diabetic mice. Collectively, these findings with CD ld knockout animals indicate a critical role for CD 1 d-dependent T cells in various disease conditions, and suggest that α-GalCer may be useful for therapeutic intervention in these diseases.     

The versatility of the CD1 lipid antigen presentation pathway

The family of non‐classical major histocompatibility complex (MHC) class‐I like CD1 molecules has an emerging role in human disease. Group 1 CD1 includes CD1a, CD1b and CD1c, which function to display lipids on the cell surface of antigen‐presenting cells for direct recognition by T‐cells. The recent advent of CD1 tetramers and the identification of novel lipid ligands has contributed towards the increasing number of CD1‐restricted T‐cell clones captured. These advances have helped to identify novel donor unrestricted and semi‐invariant T‐cell populations in humans and new mechanisms of T‐cell recognition. However, although there is an opportunity to design broadly acting lipids and harness the therapeutic potential of conserved T‐cells, knowledge of their role in health and disease is lacking. We briefly summarize the current evidence implicating group 1 CD1 molecules in infection, cancer and autoimmunity and show that although CD1 are not as diverse as MHC, recent discoveries highlight their versatility as they exhibit intricate mechanisms of antigen presentation.     

Saposin C is required for lipid presentation by human CD1b

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.     

Bacterial entry to the splenic white pulp initiates antigen presentation to CD8+ T cells

The spleen plays an important role in host-protective responses to bacteria. However, the cellular dynamics that lead to pathogen-specific immunity remain poorly understood. Here we examined Listeria monocytogenes (Lm) infection in the mouse spleen via in situ fluorescence microscopy. We found that the redistribution of Lm from the marginal zone (MZ) to the periarteriolar lymphoid sheath (PALS) was inhibited by pertussis toxin and required the presence of CD11c(+) cells. As early as 9 hr after infection, we detected infected dendritic cells in the peripheral regions of the PALS and clustering of Lm-specific T cells by two-photon microscopy. Pertussis toxin inhibited both Lm entry into the PALS and antigen presentation to CD8(+) T cells. Our study suggests that splenic dendritic cells rapidly deliver intracellular bacteria to the T cell areas of the white pulp to initiate CD8(+) T cell responses.     

Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing

Summary: During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen‐presenting cells (APCs) to modulate immune responses to foreign antigens and guide self‐tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a γ‐interferon‐inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome‐associated membrane protein‐2a and heat‐shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.     

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.     

Novel pathways of antigen presentation for the maintenance of memory

Follicular dendritic cells (FDC) store native antigen for long periods in lymphoid follicles and so provide a source of continued stimulation for specific B cells. The expression of MHC class II by FDC suggested they might act as antigen-presenting cells for MHC class II-restricted T cells. We show here, however, that the MHC class II molecules found on their surface are not synthesized by the FDC but are picked up from surrounding B cells in germinal centres. Although FDC by themselves cannot present native antigen to T cells, acquired MHC class II-peptide complexes can be recognized by T cells. The true physiological role of FDC seems to be as long-term antigen depots. We demonstrate that antigen localized onto FDC in vivo can be retrieved by antigen-specific B cells, which in turn process and present it to T cells. These presentation pathways are likely to be crucial in both the maintenance of long-term immune responses and the continued survival of memory cells.     

Requirement for invariant chain in macrophages for Mycobacterium tuberculosis replication and CD1d antigen presentation

Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis-encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis-infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii(-/-)) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii(-/-) macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis-infected Ii(-/-) macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis-containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.     

The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules

The endosomes and lysosomes of antigen-presenting cells host the processing and assembly reactions that result in the display of peptides on major histocompatibility complex (MHC) class II molecules and lipid-linked products on CD1 molecules. This environment is potentially hostile for T cell epitope and MHC class II survival, and the influence of regulators of protease activity and specialized chaperones that assist MHC class II assembly is crucial. At present, evidence indicates that individual proteases make both constructive and destructive contributions to antigen processing for MHC class II presentation to CD4 T cells. Some features of CD1 antigen capture within the endocytic pathway are also discussed.     

Targeting antigen to MHC Class I and Class II antigen presentation pathways for malaria DNA vaccines

An effective malaria vaccine which protects against all stages of Plasmodium infection may need to elicit robust CD8(+) and CD4(+) T cell and antibody responses. To achieve this, we have investigated strategies designed to improve the immunogenicity of DNA vaccines encoding the Plasmodium yoelii pre-erythrocytic stage antigens PyCSP and PyHEP17, by targeting the encoded proteins to the MHC Classes I and II processing and presentation pathways. For enhancement of CD8(+) T cell responses, we targeted the antigens for degradation by the ubiquitin (Ub)/proteosome pathway following the N-terminal rule. We constructed plasmids containing PyCSP or PyHEP17 genes fused to the Ub gene: plasmids where the N-terminal antigen residues were mutated from the stabilizing amino acid methionine to destabilizing arginine, plasmids where the C-terminal residues of Ub were mutated from glycine to alanine, and plasmids in which the potential hydrophobic leader sequences of the antigens were deleted. For enhancement of CD4(+) T cell and antibody responses, we targeted the antigens for degradation by the endosomal/lysosomal pathway by linking the antigen to the lysosome-associated membrane protein (LAMP). We found that immunization with DNA vaccine encoding PyHEP17 fused to Ub and bearing arginine induced higher IFN-gamma, cytotoxic and proliferative T cell responses than unmodified vaccines. However, no effect was seen for PyCSP using the same targeting strategies. Regarding Class II antigen targeting, fusion to LAMP did not enhance antibody responses to either PyHEP17 or PyCSP, and resulted in a marginal increase in lymphoproliferative CD4(+) T cell responses. Our data highlight the antigen dependence of immune enhancement strategies that target antigen to the MHC Class I and II pathways for vaccine development.