The sequence of events in antigen presentation on MHC-I molecules is illustrated in Fig. 1, and the individual steps are described next.
Fig1. The major histocompatibility complex (MHC) class I pathway of antigen presentation. The steps in the processing of cytosolic proteins are described in the text. The proteins may be produced in the cytosol of an infected cell (or a tumor cell, not shown). Other proteins that are ingested into vesicles may be translocated into the cytosol and processed like cytosolic proteins, known as cross-presentation. β2m, β2 Microglobulin; ERAP, endoplasmic reticulum–associated peptidase; ER, endoplasmic reticulum; TAP, trans porter associated with antigen processing.
Sources of Protein Antigens Degraded in Proteasomes
Microbial proteins present in the cytosol that undergo proteasomal degradation are derived from microbes that either produce antigens in the cytosol of cells or whose antigens are ingested and then transferred to the cytosol. The same principles apply to tumor antigens. These cytosolic antigens come from a number of sources.
• All viruses replicate and survive in infected cells and thus synthesize proteins in the cytoplasm of the infected cell. These are among the most common type of microbial proteins that are processed by proteasomes and presented on MHC-I molecules. The MHC-peptide complexes are then recognized by differentiated, functional CTLs and the infected cells are killed.
• Some bacteria are internalized into phagosomes but are able to damage phagosome membranes and create pores through which the microbes and their antigens enter the cytosol. For instance, pathogenic strains of Listeria monocytogenes produce a protein called listeriolysin that enables the bacteria to escape from vesicles into the cytosol. (This escape is a mechanism that the bacteria may have developed to resist killing in phagolysosomes.) Once the antigens of the phagocytosed microbes are in the cytosol, they are processed in proteasomes like other cytosolic antigens.
• Some extracellular bacteria have type III secretion systems that inject bacterial proteins into the cytosol. Numerous pathogens, including Yersinia pestis, Salmonella typhi, Shigella dysenteriae, Vibrio cholerae, and Chlamydia species, inject signaling proteins into the host cytosol to manipulate host function and immunity to the pathogen’s advantage. This is a major mechanism of bacterial virulence.
• The products of mutated genes in tumors produce antigens in the cytosol of the tumor cells. As in virus-infected cells, display of these tumor antigens on MHC-I molecules enables CTLs to kill the tumor cells.
• Initiating immune responses against viruses and tumors requires antigen capture by DCs and transport of the antigen bearing DCs to secondary lymphoid organs, where the anti gens can be presented to naive CD8+ T cells. But most viruses infect cells other than DCs, and tumor antigens are produced in the tumor cells, not in DCs. DCs capture antigens and dis play them on MHC-I molecules by a process called cross presentation (or cross-priming).
In addition to these microbial antigens, proteins produced in the ER that either do not fold properly or fail to assemble correctly in this compartment are translocated out of the ER and are degraded in proteasomes. Some nuclear proteins are also degraded in proteasomes. These types of proteins are often found in damaged cells and tumors and may be involved in T-cell recognition and elimination of these cells.
Digestion of Proteins in Proteasomes
Cytosolic proteins are degraded in proteasomes to generate peptides that are able to bind to MHC-I molecules. Proteasomes are large multiprotein enzyme complexes with a broad range of proteolytic activity that are found in the cytoplasm and nuclei of most cells. A proteasome is a cylinder composed of a stacked array of two inner β rings and two outer α rings, each ring being composed of seven subunits, with a cap-like structure at each end of the cylinder. The proteins in the outer α rings are structural and lack proteolytic activity; in the inner β rings, three of the seven subunits (β1, β2, and β5) are the catalytic sites for proteolysis.
Proteasomes perform a basic housekeeping function in cells by degrading damaged or improperly folded proteins. Protein synthesis normally occurs at a rapid rate, with about six to eight amino acid residues being incorporated into elongating poly peptide chains every second. This process is error prone, and it is estimated that approximately 20% of newly synthesized proteins are misfolded. These newly translated but defective polypeptides, as well as proteins that are damaged by cellular stresses, are targeted for proteasomal degradation by covalent linkage of several copies of a small polypeptide called ubiquitin. Proteins with chains of four or more ubiquitins are recognized by the proteasomal cap and are then unfolded, the ubiquitin is removed, and the proteins are threaded through proteasomes, where they are degraded into peptides. The proteasome has broad substrate specificity and can generate a wide variety of peptides from cytosolic proteins (but usually does not degrade them completely into single amino acids).
The composition of proteasomes influences the peptides that are produced. The basic cellular function of proteasomes has been adapted for a role in antigen presentation. There are two types of proteasomes with specialized functions in the immune system. Immunoproteasomes are present in immune cells, such as DCs and other APCs. They contain three unique catalytic sub units in the β ring known as β1i, β2i, and β5i. The expression of these three subunits is increased in response to IFNs produced in innate and adaptive immune responses. The production of these subunits results in a change in the substrate specificity of the proteasome such that the peptides produced usually contain carboxy-terminal hydrophobic amino acids, such as leucine, valine, isoleucine, and methionine, or basic amino acids, such as lysine or arginine. These kinds of C termini enable peptides to bind with high affinity to MHC-I molecules. Thus, immunoproteasomes play an important role in generating peptides from foreign proteins that stimulate CD8+ T cells. The second type of proteasome is called the thymoproteasome because it is present in thymic epithelial cells. It contains a unique subunit called β5t, which confers upon it the ability to produce peptides that bind weakly to MHC-I molecules. in the thymus these peptides are derived from self proteins, and their low-affinity recognition is important for the process of positive selection, which preserves maturing T cells that recognize foreign antigens. In the absence of the β5t unit (e.g., in mice in which the gene is deleted), CD8+ T cells fail to mature because the peptides that positively select these cells are absent. Predictably, CD4+ T cells are not affected, because, as we discuss later, the peptides that are recognized by CD4+ cells are not generated in the proteasome.
Transport of Peptides From the Cytosol to the Endoplasmic Reticulum
Peptides generated by proteasomes are in the cytosol and are translocated by a specialized transporter into the ER, where newly synthesized MHC-I molecules are available to bind the peptides. This delivery is mediated by a dimeric protein located in the ER membrane called transporter associated with antigen processing (TAP), which is a member of the ABC transporter family of proteins, many of which mediate ATP-dependent transport of low-molecular-weight compounds across cellular membranes. Although the TAP heterodimer has a broad range of specificities, it optimally transports peptides ranging from 8 to 16 amino acids in length and containing carboxyl termini that are basic or hydrophobic. As mentioned earlier, these are the characteristics of the peptides that are generated in the proteasome and are able to bind to MHC-I molecules.
Assembly of Peptide–MHC Class I Complexes in the Endoplasmic Reticulum
Peptides translocated into the ER bind to newly synthesized MHC-I molecules that are associated with the TAP dimer through tapasin. On the luminal side of the ER membrane, the TAP protein associates with a protein called tapasin, which also has an affinity for newly synthesized empty MHC-I molecules. Tapasin is one part of a peptide-loading complex, which also contains a thiol oxidoreductase called ERp57 that can break and remake disulfide bonds in proteins and an ER luminal chaperone called calreticulin. Within this complex, tapasin forms a stable disulfide-bonded heterodimer with ERp57 and brings the TAP transporter adjacent to the MHC-I molecules that are awaiting the arrival of peptides.
MHC-I α chains and β2-microglobulin are synthesized in the ER. Appropriate folding of the nascent α chains is assisted by chaperone proteins, such as the membrane chaperone calnexin. Within the ER, the newly formed empty MHC-I dimers are attached to the peptide-loading complex. Peptides that enter the ER through TAP and peptides produced in the ER, such as signal peptides from membrane or secreted proteins, are often trimmed to the appropriate size for MHC binding by the ER-associated aminopeptidase (ERAP). The peptide is then able to bind to the cleft of the adjacent MHC-I molecule. The peptide-loading complex not only delivers peptides to newly synthesized MHC-I molecules but also selects pep tides that bind with high affinity to MHC-I molecules preferentially over low-affinity-binding peptides. This is a quality control mechanism in antigen processing. Once MHC-I molecules are loaded with peptide, they no longer have an affinity for tapasin, so the peptide-loaded MHC-I molecules are released and are able to exit the ER and be transported to the cell surface. In the absence of bound peptide, many of the newly formed α chain–β2-microglobulin dimers are unstable and cannot be transported efficiently from the ER to the Golgi complex. These misfolded empty MHC-I molecules are transported into the cytosol and eliminated by proteasomal degradation.
Peptides transported into the ER preferentially bind to MHC-I but not MHC-II molecules, for two reasons. First, newly synthesized MHC-I molecules are attached to the peptide loading complex in the ER, and they capture peptides rapidly as the peptides are transported into the ER by TAP. Second, as discussed later, the peptide-binding clefts of newly synthesized MHC-II molecules in the ER are blocked by a protein called the invariant chain.
Surface Expression of Peptide–MHC Class I Complexes
MHC-I molecules with bound peptides are structurally stable and are expressed on the cell surface. Stable peptide–MHC-I complexes produced in the ER are guided by chaperones to move through the Golgi complex and are transported to the cell surface in exocytic vesicles. Once expressed on the cell surface, the peptide–class I complexes may be recognized by peptide antigen–specific CD8+ T cells, with the CD8 coreceptor playing an essential role by binding to nonpolymorphic regions of the MHC-I molecule and initiating activating signals in the T cells. Several viruses and tumors have evolved mechanisms that interfere with class I assembly and peptide loading, emphasizing the importance of this pathway for antiviral and antitumor immunity.
Cross-Presentation of Internalized Antigens to CD8+ T
Cells Some dendritic cells can present ingested antigens on MHC-I molecules to CD8+ T lymphocytes. The initial response of naive CD8+ T cells, similar to CD4+ cells, requires that the antigens be presented by mature DCs in lymph nodes through which the naive T cells circulate. However, many viruses may infect only particular cell types in various tissues and not DCs, and these infected tissue cells may not be capable of traveling to lymph nodes or producing all the signals needed to initiate T-cell activation. Similarly, tumors arise from many different types of cells, and have to be presented to naive CD8+ T cells in lymph nodes by DCs.
Many conventional DCs (and other cell types) have the ability to ingest infected host cells, dead tumor cells, microbes, and microbial and tumor antigens and transport the ingested antigens from phagocytic vesicles into the cytosol, where they are processed by the proteasome. The antigenic peptides that are generated then enter the ER and bind to class I molecules, which display the antigens for recognition by CD8+ T lymphocytes (see Fig. 6.14). This process, mentioned earlier, is called cross-presentation (or cross-priming) to indicate that one type of cell, dendritic cells, can present the antigens of other infected or derived from dying cells or cell fragments and prime (or activate) naive CD8+ T lymphocytes specific for these antigens. The mechanisms by which antigens ingested into vesicles are trans ported into the cytosol are not fully defined. Once the CD8+ T cells have differentiated into CTLs, they kill infected host cells or tumor cells without the need for DCs or signals other than recognition of antigens. The same path way of cross-presentation is involved in initiating CD8+ T-cell responses to antigens of tumors and organ transplants.
