The MHC Class II Pathway for Presentation of Proteins Degraded in Acidic Vesicles The generation of MHC-II–associated peptides from endocytosed antigens involves the proteolytic degradation of internaized proteins in late endosomes and lysosomes and the binding of peptides to MHC-II molecules in these acidic vesicles. This sequence of events is illustrated in Fig. 1, and the individual steps are described next.
Fig1. The major histocompatibility complex (MHC) class II pathway of antigen presentation. The stages in the processing of extracellular antigens are described in the text. APC, Antigen-presenting cell; CLIP, class II–associated invariant chain peptide; ER, endoplasmic reticulum; HLA, human leukocyte antigen; Ii , invariant chain.
Ingestion of Protein Antigens Into Vesicles
Most MHC-II-associated peptides are derived from protein antigens that are ingested into and digested in endosomes and lysosomes in APCs. Proteins that are ingested into vesicles are most commonly extracellular proteins captured by endocytosis, pinocytosis, or phagocytosis.
Different APCs can bind native protein antigens in several ways and with varying efficiencies and specificities.
• DCs and macrophages express a variety of surface receptors, such as lectins, that recognize structures shared by many microbes. These APCs use the receptors to bind and internalize microbes efficiently.
• Macrophages also express receptors for the Fc portions of antibodies and receptors for the complement protein C3b, which bind antigens that are opsonized by antibodies or complement proteins.
• Another example of specific receptors on APCs is the surface Ig on B cells, the B-cell antigen receptor, which, because of its high affinity for antigens, can effectively mediate the internalization of these antigens even if they are present at very low concentrations.
After the bound protein antigens are internalized, they become localized in intracellular membrane-bound vesicles called endosomes. The endosomal pathway of intracellular protein traffic communicates with lysosomes, which are mem brane-bound enzyme-containing vesicles. Particulate microbes are internalized into vesicles called phagosomes, which may fuse with lysosomes, producing vesicles called phagolysosomes. Some microbes, such as mycobacteria and Leishmania, may survive and even replicate within phagosomes or endosomes, providing a persistent source of antigens in vesicular compartments.
Proteins other than those ingested from the extracellular milieu can also enter the MHC-II pathway.
• Some protein molecules destined for secretion may end up in the same vesicles as MHC-II molecules and may be processed instead of being secreted.
• Cytosolic proteins, membrane proteins, and proteins within membrane-bound compartments in the cell (including mitochondria) may all be processed and displayed by MHC-II molecules. In some cases, this may result from the enzymatic digestion of cytoplasmic contents in the process known as autophagy. In this pathway, cytosolic proteins as well as proteins within organelles in the cytoplasm are trapped within membrane-bound vesicles called autophagosomes, which fuse with lysosomes, and the cytoplasmic proteins are proteolytically degraded. The peptides generated by this route may be delivered to the same vesicular compartment as are peptides derived from ingested antigens. Autophagy is primarily a mechanism for degrading cellular proteins and recycling their products as sources of nutrients during times of stress. It also participates in the destruction of intracellular microbes, which are enclosed in vesicles and delivered to lysosomes.
Proteolytic Digestion of Antigens in Acidic Vesicles
Internalized proteins are degraded enzymatically in late endosomes and lysosomes to generate peptides that are able to bind to the peptide-binding clefts of MHC-II molecules. The degradation of protein antigens in vesicles is mediated by proteases that have acidic pH optima. The most abundant proteases of late endosomes are cathepsins, which are thiol and aspartyl proteases with broad substrate specificities. Several cathepsins contribute to the generation of peptides for the class II pathway. Partially degraded or cleaved proteins bind to the open-ended clefts of MHC-II molecules and are then trimmed enzymatically to their final size.
Biosynthesis and Transport of MHC Class II Molecules to Endosomes
MHC-II molecules are synthesized in the ER and transported to endosomes with an associated protein, the invariant chain (Ii ), which occupies the peptide-binding clefts of the newly synthesized MHC-II molecules (Fig.2). The α and β chains of MHC-II molecules are coordinately synthesized and associate with each other in the ER. The folding and assembly of MHC-II molecules are aided by ER-resident chaperones, such as calnexin. The Ii associates with MHC-II dimers in the ER and directs newly formed MHC-II molecules from the trans-Golgi to late endosomes and lysosomes, where internalized proteins have been proteolytically degraded into peptides. The Ii is a trimer composed of three 30-kD subunits, each of which binds one newly synthesized MHC-II αβ heterodimer in a way that blocks the peptide-binding cleft and prevents it from accepting peptides. As a result, MHC-II molecules cannot bind and present peptides they encounter in the ER, leaving such peptides to associate with MHC-I molecules. The MHC-II molecules are transported in vesicles from the ER to the Golgi. Vesicles budding from the trans-Golgi that contain the MHC-II–Ii complex are transported to lysosomes. Thus, MHC-II molecules encounter antigenic pep tides that have been generated by proteolysis of endocytosed proteins in lysosomes, and the peptide-MHC association occurs in these vesicles.
Fig2. The functions of major histocompatibility complex (MHC) class II–associated invariant chain (I i ) and human leukocyte antigen (HLA) DM. MHC-II with bound I i , or class II–associated I i peptide (CLIP), are transported into late endosomes and lysosomes, where the I i is degraded and the remaining CLIP is removed by the action of DM. Antigenic peptides generated in the vesicles are then able to bind to the MHC-II molecules. Another class II–like protein, called DO, may regulate the DM-catalyzed removal of CLIP (not shown). ER, endoplasmic reticulum.
Association of Processed Peptides With MHC Class II Molecules in Vesicles
Within the endosomal/lysosomal vesicles, the Ii dissociates from MHC-II molecules by the combined action of proteolytic enzymes and the HLA-DM molecule, and peptides derived from protein antigens are then able to bind to the available peptide-binding clefts of the MHC-II molecules (see Fig. 2). The same proteolytic enzymes that generate peptides from internalized proteins, such as cathepsins, also degrade the Ii , leaving only a 24 amino acid remnant called class II–associated invariant chain peptide (CLIP), which sits in the pep tide-binding cleft of the MHC-II molecule, which is relatively resistant to proteolysis. Enzymatic degradation of the trans membrane portion and cytosolic tail of Ii prevents tethering of MHC-II molecules to the lysosomal membrane, and this allows MHC-II–peptide complexes to bud out of the vesicles and go to the cell surface.
The HLA-DM molecule edits the repertoire of peptides being presented, favoring the display of peptides that bind with high affinity to MHC-II molecules. The displacement of CLIP and its replacement by a higher affinity antigenic pep tide in lysosomes are accomplished by the action of a molecule called HLA-DM (also called DM, or H-2M in the mouse), which is encoded within the MHC, has a structure similar to that of MHC-II molecules, and colocalizes with MHC-II molecules in late endosomes and lysosomes. Unlike MHC-II proteins, DM molecules are not polymorphic, do not have accessible peptide-binding clefts, and are not expressed on the cell surface. DM binds to the β chain of MHC-II molecules in the region where this chain forms the peptide-binding cleft and dislodges loosely bound peptides from the cleft. It thus acts as a peptide exchanger, facilitating the removal of CLIP and the addition of higher affinity peptides derived from protein antigens to MHC-II molecules. Peptides that bind to the MHC molecules with high affinity cannot be displaced by DM. Thus, the presence of DM is important for selecting peptides that bind strongly to MHC molecules in each individual and displaying these peptides to T cells.
Another dimeric MHC-II-like molecule, called HLA-DO, binds to HLA-DM in lysosomes and negatively regulates the function of DM. DM can only mediate peptide exchange after being released from DO. In response to cytokines and other stimuli produced during infections, DM levels rise while DO levels do not, resulting in more efficient peptide exchange and antigen presentation.
Expression of Peptide–MHC Class II Complexes on the Cell Surface
MHC-II molecules are stabilized by the bound peptides, and the stable peptide–class II complexes are delivered to the surface of the APC, where they are displayed for recognition by CD4+ T cells. The transport of MHC-II–peptide complexes to the cell surface is thought to occur by fusion of vesiculotubular extensions from the lysosome to the plasma membrane, through which the peptide-loaded MHC-II molecules are delivered to the cell surface. Once expressed on the APC surface, the peptide–class II complexes are recognized by peptide antigen–specific CD4+ T cells, with the CD4 coreceptor playing an essential role by binding to nonpolymorphic regions of the MHC-II molecule.
The amount of surface MHC-II–peptide complexes is regulated by modulation of MHC-II degradation. MHC-II molecules are normally recycled and degraded by the ubiquitin-proteasome system. A ubiquitin E3 ligase called MARCH-1 recognizes the tail of MHC-II molecules and targets them for degradation. In response to microbes and cytokines produced during infections, APCs shut off the expression of MARCH-1 and thus increase the amount of the relevant class II–peptide complexes on the cell surface.
