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Cerebral malaria is a devastating complication of Plasmodium falciparum infection. Its pathogenesis is complex, involving both parasite- and immune-mediated events. CD8+ T cells play an effector role in murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection. We have identified a highly immunogenic CD8 epitope in glideosome-associated protein 50 that is conserved across rodent malaria species. Epitope-specific CD8+ T cells are induced during PbA infection, migrating to the brain just before neurological signs manifest. They are functional, cytotoxic and can damage the blood-brain barrier in vivo. Such CD8+ T cells are also found in the brain during infection with parasite strains/species that do not induce neuropathology. We demonstrate here that PbA infection causes brain microvessels to cross-present parasite antigen, while non-ECM-causing parasites do not. Further, treatment with fast-acting anti-malarial drugs before the onset of ECM reduces parasite load and thus antigen presentation in the brain, preventing ECM death. Thus our data suggest that combined therapies targeting both the parasite and host antigen-presenting cells may improve the outcome of CM patients. [ABSTRACT FROM AUTHOR]

CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood. Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells. [ABSTRACT FROM AUTHOR]

Effectiveness of immune surveillance of intracellular viruses and bacteria depends upon a functioning antigen presentation pathway that allows infected cells to reveal the presence of an intracellular pathogen. The antigen presentation pathway uses virtually all endogenous polypeptides as a source to produce antigenic peptides that are eventually chaperoned to the cell surface by MHC class I molecules. Intriguingly, MHC I molecules present peptides encoded not only in the primary open reading frames but also those encoded in alternate reading frames. Here, we review recent studies on the generation of cryptic pMHC I. We focus on the immunological significance of cryptic pMHC I, and the novel translational mechanisms that allow production of these antigenic peptides from unconventional sources. [ABSTRACT FROM AUTHOR]

Within cells of their host, many bacteria and parasites inhabit specialized compartments, such as a modified phagosome for Mycobacterium tuberculosis or a parasitophorous vacuole for Toxoplasma gondii. These locations could exclude microbial material from entry into the MHC class I surveillance pathway. Remarkably, however, under these circumstances, cells can still signal the presence of invading pathogens to circulating CD8+ T cells, which typically play a key role in protection against such intracellular organisms. Here, we review MHC I presentation pathways in various contexts, ranging from model antigens in non-infectious settings to pathogen-infected cells. We suggest that presentation of intracellular pathogens can be described as not just one, but several distinct pathways; perhaps because diverse pathogens have evolved different strategies to interact with host cells. [ABSTRACT FROM AUTHOR]

Many prokaryotic and eukaryotic parasites multiply in specialized subcellular niches in the host cell. The invading microbes hijack key cellular functions to establish the intracellular niches but, unlike viruses, do not need the protein synthesis machinery of host cells to replicate. Circulating CD8+ T cells provide protective immunity by recognizing pathogen-derived peptide major histocompatibility complex class I molecules (pMHC I) expressed by infected cells. Here, we review studies on the complex and varied pathways that produce the appropriate pMHC I as ligands for the CD8+ T cells. We also discuss possible explanations for the curious observations that CD8+ T cells are specific for fewer pMHC I ligands in parasite infections compared to the diversity of pMHC I ligands in viral infections. [ABSTRACT FROM AUTHOR]

MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance. [ABSTRACT FROM AUTHOR]

Discovery of immunologically relevant antigens in prostate cancer forms the basis for developing more potent active immunotherapy. We report here a strategy using the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which allows for the functional identification of immunogenic prostate tumor antigens with relevance for human immunotherapy. Using a combination of active tumor vaccination in the presence of CTL-associated antigen 4 (CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to expression clone the first T cell-defined TRAMP tumor antigen, called Spas-i (stimulator of prostatic adenocarcinoma specific I cells-i). Spas-i expression was increased in advanced primary TRAMP tumors. We show that the immunodominant SPAS-i epitope SNC9-H8 arose from a point mutation in one allele of the gene in TRAMP tumor cells, and that immunization with dendritic cells pulsed with SNC9-H8 peptide resulted in protection against TRAMP-C2 tumor challenge. In humans, the Spas-i ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. Additionally, we identified a nonmutated HLA-A2-binding epitope in the human ortholog SH3GLB2, which primed T cells from healthy HLA-A2+ individuals in vitro. Importantly, in vitro-primed T cells also recognized naturally processed and presented SH3GLB2. Our findings demonstrate that our in vivo CTLA-4 blockade-based T cell expression cloning can identify immunogenic cancer antigens with potential relevance for human immunotherapy. [ABSTRACT FROM AUTHOR]

The MHC class I molecules present thousands of peptides (pMHC I) on the cell surface for immune surveillance by CD8 T cells. The pMHC I repertoire normally contains peptides of perfect length and sequences suitable for binding each MHC I. The peptides are made by first fragmenting cytoplasmic proteins. The fragments are then transported into the endoplasmic reticulum (ER), where they are trimmed to appropriate length by the ER aminopeptidase associated with antigen processing (ERAAP) to generate the final pMHC I. Here, we review studies on the role of ERAAP in generating pMHC I from endogenous or viral proteins and their ability to elicit CD8 T cell responses. The absence of ERAAP profoundly disrupts the pMHC I repertoire which can have major consequences on the immune responses to endogenous and viral antigens. [Copyright &y& Elsevier]

Immunosurveillance by cytotoxic T cells requires that cells generate a diverse spectrum of peptides for presentation by major histocompatibility complex (MHC) class I molecules. Those peptides are generated by proteolysis, which begins in the cytoplasm and continues in the endoplasmic reticulum by the unique aminopeptidase ERAAP. The overall extent to which trimming by ERAAP modifies the peptide pool and the immunological consequences of ERAAP deficiency are unknown. Here we show that the peptide-MHC repertoire of ERAAP-deficient mice was missing many peptides. Furthermore, ERAAP-deficient cells presented many unstable and structurally unique peptide-MHC complexes, which elicited potent CD8+ T cell and B cell responses. Thus, ERAAP is a 'quintessential editor' of the peptide-MHC repertoire and, paradoxically, its absence enhances immunogenicity. [ABSTRACT FROM AUTHOR]

Summary: Intracellular proteins are degraded in the antigen processing pathway to generate peptide-loaded MHC I complexes (pMHC I) for immune surveillance. The characteristics of the final pMHC I are clear but those of their precursors and their potential binding partners remain poorly defined. By using a unique method to biochemically detect preprocessed ovalbumin-derived antigenic peptides, we find that cells generate large, C-terminally extended proteolytic intermediates that are associated with the α isotype of hsp90 chaperone. Knockdown of hsp90α expression by siRNA resulted in the loss of these intermediates and decreased presentation of the final pMHC I on the cell surface. Generation of pMHC I was also inhibited by knockdown of the cochaperone CHIP that interacts with heat shock proteins, ubiquitinates their clients, and delivers them to the proteasome. Thus, hsp90α can serve as a chaperone for precursors of pMHC I at an early stage in the antigen processing pathway. [Copyright &y& Elsevier]

Major histocompatibility complex (MHC) class I molecules present thousands of peptides to allow CD8+ T cells to detect abnormal intracellular proteins. The antigen-processing pathway for generating peptides begins in the cytoplasm, and the MHC molecules are loaded in the endoplasmic reticulum. However, the nature of peptide pool in the endoplasmic reticulum and the proteolytic events that occur in this compartment are unclear. We addressed these issues by generating mice lacking the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). We found that loss of ERAAP disrupted the generation of naturally processed peptides in the endoplasmic reticulum, decreased the stability of peptide–MHC class I complexes and diminished CD8+ T cell responses. Thus, trimming of antigenic peptides by ERAAP in the endoplasmic reticulum is essential for the generation of the normal repertoire of processed peptides. [ABSTRACT FROM AUTHOR]

Activating the immune system to trigger a specific response is a major challenge in vaccine development. In particular, activating sufficient cytotoxic T lymphocyte-mediated cellular immunity, which is crucial for the treatment of many diseases including cancer and AIDS, has proven to be especially challenging. In this study, antigens were encapsulated in acid-degradable polymeric particle carriers to cascade cytotoxic T lymphocyte activation. To target dendritic cells, the most potent antigen-presenting cells, the particle carriers, were further conjugated with monoclonal antibodies. A series of ex vivo and in vivo studies have shown increased receptor-mediated uptake of antibody-conjugated particles by dendritic cells as well as migration of particle-carrying dendritic cells to lymph nodes and stimulation of naïve T cells leading to enhanced cellular immune response as confirmed by specific cell lysis and IFN-γ secretion. [ABSTRACT FROM AUTHOR]

The end result of the antigen-processing pathway is the display of peptide-bound major histocompatibility complex I (pMHC I) molecules. The pMHC I molecules are expressed on the cell surface where they can be surveyed by CD8+ T cells for abnormal proteins. MHC I molecules present a large repertoire of peptides that fit perfectly in their binding grooves and represent the otherwise hidden intracellular contents. Many peptides originate as defective ribosomal products in the cytoplasm. In a stepwise manner, the antigen-processing pathway generates and protects the proteolytic intermediates until they yield the final peptides that can fit the MHC I in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site--like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5' to 3' specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2-independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes. [ABSTRACT FROM AUTHOR]

MHC class I molecules present precisely cleaved peptides of intracellular proteins on the cell surface. For most antigenic precursors, presentation requires transport of peptide fragments into the ER, but the nature of the cytoplasmic peptides and their chaperones is obscure. By tracking proteolytic intermediates in living cells, we show that intracellular proteolysis yields a mixture of antigenic peptides containing only N-terminal flanking residues for ER transport. Some of these peptides were bound to the group II chaperonin TRiC and were protected from degradation. Destabilization of TRiC by RNA interference inhibited the expression of peptide-loaded MHC I molecules on the cell surface. Thus, the TRiC chaperonin serves a function in protecting proteolytic intermediates in the MHC I antigen processing pathway. [Copyright &y& Elsevier]

The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigenpresenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200-500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The aciddegradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. [ABSTRACT FROM AUTHOR]