Drugs such as chloroquine that raise the pH of endosomes, making them less acidic, inhibit the presentation of intravesicular antigens, suggesting that acid proteases are responsible for processing internalized antigen. These proteases include the cysteine proteases—so called because they use a cysteine in their catalytic site—known as cathepsins B, D, S, and L, of which L is the most active. Antigen processing can be mimicked to some extent by the digestion of proteins with these enzymes in vitro at acid pH. Cathepsins S and L may be the predominant proteases in the processing of vesicular antigens; mice that lack cathepsin B or cathepsin D process antigens normally, whereas mice with no cathepsin S show some deficiencies, including in cross-presentation. Asparagine endopeptidase (AEP), a cysteine protease cleaving after asparagines, is important for processing some antigens, such as the tetanus toxin antigen for MHC class II presentation, but is not required in all cases where antigens contain asparagine residues near their relevant epitopes. It is likely that the overall repertoire of peptides produced within the vesicular pathway reflects the activities of the many proteases present in endosomes and lysosomes. Disulfide bonds, particularly intramolecular disulfide bonds, help in the denaturation process and facilitate proteolysis in endosomes. The enzyme IFN-γ-induced lysosomal thiol reductase (GILT) is present in endosomes and functions by breaking and re-forming disulfide bonds in the antigen-processing pathway. The various endosomal proteases act in a largely redundant and nonspecific manner to digest regions of the polypeptide that have become accessible to proteolysis by denaturation and previous steps of degradation. The peptides generated vary in sequence and abundance throughout the endocytic pathway, so that MHC class II molecules can bind and present many different peptides from these compartments.
A significant number of the self-peptides bound to MHC class II molecules arise from common proteins that are cytosolic in location, such as actin and ubiquitin. The most likely way in which cytosolic proteins are processed for MHC class II presentation is by the natural process of protein turnover known as autophagy, in which damaged organelles and cytosolic proteins are delivered to lysosomes for degradation. Here their peptides could encounter MHC class II molecules present in the lysosome membranes, and the resulting peptide:MHC class II complex could be transported to the cell surface via endolysosomal tubules (see Fig. 6.4). Autophagy is constitutive, but it is increased by cellular stresses such as starvation, when the cell catabolizes intracellular proteins to obtain energy. In microautophagy, cytosol is continuously internalized into the vesicular system by lysosomal invaginations, whereas in macroautophagy, which is induced by starvation, a double-membraned autophagosome engulfs cytosol and fuses with lysosomes. A third autophagic pathway uses the heat-shock cognate protein 70 (Hsc70) and the lysosome-associated membrane protein-2 (LAMP-2) to transport cytosolic proteins to lysosomes. Autophagy has been shown to be involved in the processing of the Epstein–Barr virus nuclear antigen 1 (EBNA-1) for presentation on MHC class II molecules. Such presentation enables cytotoxic CD4 T cells to recognize and kill B cells infected with Epstein–Barr virus.
6-7 The invariant chain directs newly synthesized MHC class II molecules to acidified intracellular vesicles.