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.