In this review, we discuss recent data from our laboratory that address two aspects of major histocompatibility complex (MHC) class I-restricted antigen processing. First, we consider the nature of the peptide-loading complex, which is the assembly of proteins in the endoplasmic reticulum (ER) into which newly synthesized MHC class I-beta(2) microglobulin (beta(2)m) heterodimers are incorporated, and the mechanisms involved in MHC class I assembly and peptide loading that are facilitated by the peptide-loading complex. Second, we discuss mechanisms of cross-presentation, the phenomenon whereby extracellular and luminal protein antigens can be processed by antigen-presenting cells, particularly dendritic cells, and presented by MHC class I molecules to CD8(+) T cells. The focus of the discussion is mainly on the human MHC class I system.

Crosspresentation of exogenous antigens (Ags) to CD8(+) T cells by dendritic cells generally requires their entry into the cytosol. Here we show that both soluble and phagocytosed extracellular Ags accessed the cytosol via molecular components required for endoplasmic reticulum (ER)-associated degradation (ERAD). Exogenous Pseudomonas aeruginosa Exotoxin A, which inhibits protein translocation from the ER to the cytosol, abrogated crosspresentation. Exotoxin A also prevented the transporter associated with antigen processing (TAP) inhibitor, ICP47, from entering the cytosol and blocking TAP-mediated peptide transport. In an in vitro model of retrotranslocation, the AAA ATPase p97, an enzyme critical for ERAD, was the only cytosolic cofactor required for protein export from isolated phagosomes. Functional p97 was also required for crosspresentation but not conventional presentation. Thus, crosspresentation appears to result from an adaptation of the retrotranslocation mechanisms involved in the degradation of misfolded ER proteins.

Summary CD8+ T‐cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8+ T cells against these cell‐associated antigens, exogenous antigens must be cross‐presented by professional antigen‐presenting cells (APCs). While cross‐presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein‐based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(d,l‐lactide‐co‐glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow‐derived dendritic cells (BMDCs), the MHC class I presentation of PLGA‐encapsulated ovalbumin (OVA) stimulated T cell interleukin‐2 secretion at 1000‐fold lower concentration than soluble antigen and 10‐fold lower than antigen‐coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead‐associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle‐mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Chikungunya virus induces autophagy by triggering ER and oxidative stress, and this autophagy restricts apoptosis and viral propagation.

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Dendritic cells efficiently internalize exogenous protein antigens by fluid-phase uptake and receptor-mediated endocytosis. Such antigens contribute to cross-presentation by being translocated into the cytosol for proteasomal degradation, which liberates immunogenic peptides that can bind to major histocompatibility complex (MHC) class I molecules after being transported into the endoplasmic reticulum (ER). MHC class I–peptide complexes are then expressed on the cell surface and presented to CD8+ T cells. Here we show that internalized proteins can have an alternative fate. After internalization, proteins are first unfolded to allow translocation into the cytosol using a pathway related to ER-associated degradation (ERAD). Subsequently the unfolded proteins can undergo cytosolic refolding assisted by the chaperone Hsp90. These observations not only clarify the cellular processes regulating cytosolic access following endocytosis, but also demonstrate that functional proteins can potentially regain their activity in the cytosol of dendritic cells.

In cross-presentation by dendritic cells (DCs), internalized proteins are retrotranslocated into the cytosol, degraded by the proteasome, and the generated antigenic peptides bind to MHC class I molecules for presentation on the cell surface. Endoplasmic reticulum (ER) contribution to phagosomal membranes is thought to provide antigen access to the ER-associated degradation (ERAD) machinery, allowing cytosolic dislocation. Because the ERAD pathway is present in all cell types and exogenous antigens encounter an ER-containing compartment during phagocytosis, we postulated that forcing phagocytosis in cell types other than DCs would render them competent for cross-presentation. Indeed, FcR␥IIA expression endowed 293T cells with the capacity for both phagocytosis and ERAD-mediated crosspresentation of an antigen provided as an immune complex. The acquisition of this ability by nonprofessional antigen-presenting cells suggests that a function potentially available in all cell types has been adapted by DCs for presentation of exogenous antigens by MHC class I molecules. C ross-presentation refers to the binding of peptides derived from exogenous proteins by MHC class I molecules and their subsequent recognition by CD8 ϩ T cells (1). The process generally requires access of the exogenous antigen to the cytosol, and, like conventional MHC class I presentation, proteasomal activity (2). Resulting peptides are then translocated from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) for subsequent binding to MHC class I molecules (3).Dendritic cells (DCs) have been considered specialized antigenpresenting cells (APCs) for cross-presentation because they efficiently capture exogenous antigens and tightly regulate the pH and proteolytic activity of the endocytic pathway to minimize protein degradation (4-6). It has also been proposed that contribution of ER membrane to DC phagosomes allows exogenous antigens to access the ER-associated degradation (ERAD) machinery, normally used to dispose of misfolded proteins from the ER (7). Indeed, we showed that cross-presentation by DCs requires ERADmediated cytosolic translocation (8). Because all cell types are capable of ER quality control and use the ERAD pathway, we hypothesized that facilitating phagocytosis in nonprofessional APCs might promote ER recruitment to phagosomal membranes, rendering such cells competent for cross-presentation. Here, we show that expression of the Fc receptor Fc␥RIIA in the human 293T kidney cell line resulted in uptake of antibody-coated exogenous particles, ER contribution to phagosomes, and ERADmediated cross-presentation. Results 293T Cells Expressing Fc␥RIIA Efficiently Internalize Antibody-CoatedParticles. We generated 293T cells stably expressing an EGFPtagged version of human Fc␥RIIA (293T FcR-EGFP) (Fig. 1A) (9). Efficient internalization of antibody-coated particles by Fc␥R-expressing cells that are normally nonphagocytic has been described (10, 11), and confocal microscopy clearly sh...

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