Info

aU P2-microglobulin

m

Types of APCs

All nucleated cells

Dendritic cells, mononuclear phagocytes, B lymphocytes; endothelial cells, thymic epithelium

Responsive T cells

CD8+ T cells

CD4+ T cells

Source of protein antigens

Cytosolic proteins (mostly synthesized in the cell; may enter cytosol from phagosomes)

Endosomal and lysosomal proteins (mostly internalized from extracellular environment)

Enzymes responsible for peptide loading of MHC

Cytosolic proteasome

Endosomal and lysosomal proteases (e.g., cathepsins)

Site of peptide loading of MHC

Endoplasmic reticulum

Specialized vesicular compartment

Molecules involved in transport of peptides and loading of MHC molecules

Chaperones, TAP in ER

Chaperones in ER; invariant chain in ER, Golgi and MIIC/CIIV; DM

APC, antigen-presenting cell; CIIV, class II vesicle; ER, endoplasmic reticulum; MHC, major histocompatibility complex; MIIC, MHC class II compartment; TAP, transporter associated with antigen processing.

characteristics required for associating with MHC molecules and to place these peptides in the same cellular location as the appropriate MHC molecules with available peptide-binding clefts. Peptide binding to MHC molecules occurs before cell surface expression and is an integral component of the biosynthesis and assembly of MHC molecules. In fact, as mentioned earlier, peptide association is required for the stable assembly and surface expression of class I and class II MHC molecules.

Protein antigens that are present in the cytosol (usually synthesized in the cell) generate class I-associated peptides that are recognized by CD8+ T cells, whereas antigens internalized from the extracellular environment into the vesicles of APCs generate peptides that are displayed by class II MHC molecules and recognized by CD4+ T cells. The different fates of cytosolic and vesicular antigens are due to the segregated pathways of biosynthesis and assembly of class I and class II MHC molecules (see Fig. 6-14 and Table 6-5). This fundamental difference between cytosolic and vesicular antigens has been demonstrated experimentally by analyzing the presentation of the same antigen introduced into APCs in different ways (Fig. 6-15). If a protein antigen is produced in the cytoplasm of APCs as the product of a transfected gene (modified so its protein product cannot enter the secretory pathway) or introduced directly into the cytoplasm of the APCs by osmotic shock, it is presented in the form of class I-associated peptides that are recognized by CD8+ T cells. In contrast, if the same protein is added in soluble form to APCs and endocytosed into the vesicles of the APCs, it is subsequently presented as class II-associated peptides and is recognized by antigen-specific CD4+ T cells.

We first describe these two pathways of antigen processing and then their functional significance.

The Class I MHC Pathway for Processing and Presentation of Cytosolic Proteins

Class I MHC-associated peptides are produced by the proteolytic degradation of cytosolic proteins, the transport of the generated peptides into the endoplasmic retic-ulum (ER), and their binding to newly synthesized class I molecules. This sequence of events is illustrated in Figure 6-16, and the individual steps are described next.

Sources of Cytosolic Protein Antigens

Most cytosolic protein antigens are synthesized within cells, and some are phagocytosed and transported into the cytosol. Foreign antigens in the cytosol may be the products of viruses or other intracellular microbes that infect such cells. In tumor cells, various mutated or overex-pressed genes may produce protein antigens that are recognized by class I-restricted CTLs (see Chapter 17). Peptides that are presented in association with class I molecules may also be derived from microbes and other particulate antigens that are internalized into phago-somes but escape into the cytosol. Some microbes 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 bacteria to escape from vesicles into the cytosol. (This escape is a mechanism that the bacteria may have evolved to resist killing by the microbicidal mechanisms of phagocytes, most of which are concentrated in pha-golysosomes.) Once the antigens of the phagocytosed microbes are in the cytosol, they are processed like other cytosolic antigens. In dendritic cells, some antigens that are ingested into vesicles enter the cytosolic class I

Antigen uptake

Antigen processing

Endogenous synthesis of foreign protein antigen

Transfection

Ovalbumin gene

Ovalbumin gene

Processed peptide bound to „ class I MHC

Artificial introduction of foreign protein antigen into cytoplasm

Osmotic shock

Osmotic shock

Processed peptide bound to class I MHC

Antigen uptake and release into Ovalbumin cytosol

Endocytosis of extracellular foreign protein antigen

Processed peptide bound to class I MHC

Class I MHC

Processed peptide bound to

Ovalbumin

Class II MHC

Ovalbumin

Antigen presentation to:

Class I- Class II-

restricted restricted

CD8+ cytolytic CD4+ helper

T cells T cells

Class I MHC

Processed peptide bound to

Class II MHC

FIGURE 6-15 Experimental demonstration of presentation of cytosolic and extracellular antigens. When a model protein antigen, ovalbumin, is synthesized intracellularly as a result of transfection of its gene modified to lack the N-terminal signal sequences (A) or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock (B), ovalbumin-derived peptides are presented in association with class I MHC molecules. When ovalbumin is added as an extracellular antigen to an APC that expresses both class I and class II MHC molecules, ovalbumin-derived peptides are presented only in association with class II molecules (C). The measured response of class I-restricted CTLs is killing of the APCs, and the measured response of class II-restricted helper T cells is cytokine secretion.

FIGURE 6-15 Experimental demonstration of presentation of cytosolic and extracellular antigens. When a model protein antigen, ovalbumin, is synthesized intracellularly as a result of transfection of its gene modified to lack the N-terminal signal sequences (A) or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock (B), ovalbumin-derived peptides are presented in association with class I MHC molecules. When ovalbumin is added as an extracellular antigen to an APC that expresses both class I and class II MHC molecules, ovalbumin-derived peptides are presented only in association with class II molecules (C). The measured response of class I-restricted CTLs is killing of the APCs, and the measured response of class II-restricted helper T cells is cytokine secretion.

pathway, in the process called cross-presentation that is described later. Other important sources of peptides in the cytosol are misfolded proteins in the ER that are translocated into the cytosol and degraded like other cytosolic proteins; this process is called ER-associated degradation.

Proteolytic Digestion of Cytosolic Proteins

The major mechanism for the generation of peptides from cytosolic protein antigens is proteolysis by the proteasome.

Proteasomes are large multiprotein enzyme complexes with a broad range of proteolytic activity that are found in the cytoplasm and nuclei of most cells. The proteasome appears as a cylinder composed of a stacked array of two inner P rings and two outer a rings, each ring being composed of seven subunits, with a cap-like structure at either end of the cylinder. The proteins in the outer a rings are structural and lack proteolytic activity; in the inner P rings, three of the seven subunits (P1, P2, and P5) are the catalytic sites for proteolysis.

The proteasome performs a basic housekeeping function in cells by degrading many damaged or improperly folded proteins. Protein synthesis normally occurs at a rapid rate, about six to eight amino acid residues being incorporated into elongating chains every second. The process is error prone, and it is estimated that approximately 20% of newly synthesized proteins are misfolded. These defective ribosomal products as well as older effete proteins are targeted for proteasomal degradation by covalent linkage of several copies of a small polypeptide called ubiquitin. Ubiquitinated proteins, with chains of four or more ubiquitins, are recognized by the protea-somal cap and are then unfolded, the ubiquitin is removed, and the proteins are "threaded" through pro-teasomes, 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 proteins completely into single amino acids). Interestingly, in cells treated with the cytokine IFN-y, there is increased transcription and synthesis of three novel catalytic subunits of the proteasome known as P 1i, P2i, and P5i, which replace the three catalytic subunits of the P ring of the proteasome. This results in a change in the substrate specificity of the proteasome

Production of proteins in the cytosol

Proteolytic Transport of Assembly of degradation peptides from peptide-class I of proteins cytosol to ER complexes in ER

Surface expression of peptide-class I complexes

Virus in cytoplasm

Synthesized viral protein

Synthesized viral protein

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