MHC class II αβ dimers associate with the invariant chain (Ii) in the endoplasmic reticulum (ER), traffic through the Golgi apparatus and are delivered to the plasma membrane. Ii–MHC class II complexes are internalized by clathrin-mediated endocytosis and traffic to multivesicular antigen-processing compartments. Some of these complexes are sorted into the intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), in which sequential Ii proteolysis leads to the generation of a fragment of Ii, termed class II-associated invariant chain peptide (CLIP), which remains in the peptide-binding groove of MHC class II. CLIP is removed from CLIP–MHC class II complexes by the enzyme HLA-DM, which is present in the MVB internal and limiting membranes, thereby facilitating peptide binding onto nascent MHC class II. The activity of HLA-DM is regulated by HLA-DO, but the mechanism of regulation remains unknown. ILV-associated peptide–MHC class II somehow associates with the MVB limiting membrane, and endosomal–lysosomal tubules directed towards the plasma membrane either directly fuse or give rise to transport vesicles that fuse with the plasma membrane. MVB membranes are rich in the lipids that constitute lipid raft membrane microdomains, and fusion of peptide–MHC class II from MVB-derived membranes with the plasma membrane leads to the deposition of lipid raft-associated peptide–MHC class II directly into the plasma membrane. In circumstances in which an entire MVB fuses with the plasma membrane, the ILVs of MVBs are released from the APC in the form of exosomes. Surface expressed peptide–MHC class II can be internalized through a clathrin-independent endocytosis pathway and can be targeted for lysosomal degradation or can recycle back to the plasma membrane.

Antigens can enter the endocytic pathway of antigen-presenting cells (APCs) by several distinct mechanisms. Receptor-mediated endocytosis via clathrin-coated vesicles requires antigen binding to one of several receptors on APCs — such as lectin receptors — which results in their internalization into early endosomes. Macropinocytosis, which is a form of cell ‘drinking’, is an actin-dependent process that leads to the uptake of soluble material into the cell in a macropinosome. In each of these processes, the internalized early endosomes eventually fuse with multivesicular late endosomal–lysosomal antigen-processing compartments. It is in these compartments that internalized antigen proteolysis and peptide–MHC class II complex formation takes place. Phagocytosis is an endocytic process in which opsonized particles bind to various receptors and, following actin-dependent membrane reorganization around the particle, enter cells in membrane-derived phagosomes. Phagosomes are not particularly rich in proteases or MHC class II and, after Toll-like receptor (TLR)-induced fusion with lysosomes or potentially with MHC class II-containing late endosomal–lysosomal compartments, the resulting phagolysosome generates peptide–MHC class II complexes. Autophagy is a process by which membranes (often derived from the endoplasmic reticulum (ER)) envelop cytosolic antigens to form an autophagosome. Upon autophagosome fusion with lysosomal compartments, the resulting autophagolysosome generates peptide–MHC class II complexes. It is important to remember that this is a schematic and that each APC does not contain only one early endosome and one antigen-processing compartment; there is a gradient of endosomes containing varying amounts of the essential components that are necessary to generate peptide–MHC class II complexes in every cell and it is for this reason that the nature of the antigen-processing compartment used by different antigens will differ.

a | In immature dendritic cells (DCs), up to 75% of all MHC class II molecules reside in lysosome-like antigen-processing compartments. It is in these compartments that peptide–MHC class II complexes are generated (albeit slowly in immature DCs). The predominant intracellular localization of MHC class II in immature DCs is due to robust synthesis of invariant chain (Ii)–MHC class II complexes and the movement of these complexes to antigen-processing compartments. Although some peptide–MHC class II complexes formed in these compartments do traffic to the plasma membrane, much of the MHC class II that is synthesized in immature DCs is degraded in lysosomes. Maturation of DCs by pathogen uptake and/or Toll-like receptor (TLR) stimulation leads a transient burst in Ii–MHC class II biosynthesis, efficient generation of peptide–MHC class II complexes that traffic to the plasma membrane in tubular endosomes and degradation of residual MHC class II present in lysosomes. Fully mature conventional DCs do not synthesize Ii–MHC class II and therefore express very little MHC class II in antigen-processing compartments. Arrow thickness indicates prominence of a particular pathway and dashed arrows indicate minor pathways. In mature DCs, any internalized MHC class II is simply recycled to the plasma membrane where peptide–MHC class II complexes can interact with naive antigen-specific CD4⁺ T cells. Note that after DC activation, structures such as antigen-processing compartments still exist in the mature DC, but peptide–MHC class II complexes do not accumulate in these compartments. b | In resting B cells, large amounts of MHC class II are present on the plasma membrane and in peripheral early endosomal vesicles. Binding of antigen to the B cell receptor (BCR) triggers BCR endocytosis and recruits the peripheral MHC class II-containing vesicles towards the antigen–BCR-containing lysosome-like antigen-processing compartments. Internalized antigen is processed into peptides in these compartments and the resulting peptide–MHC class II complexes traffic to the plasma membrane for recognition by primed antigen-specific CD4⁺ T cells. TCR, T cell receptor.

After arrival at the cell surface, peptide–MHC class II complexes are internalized into early endosomes by clathrin-independent endocytosis in both immature and mature dendritic cells (DCs) at the same rate (k_int); however, whether this is true in all antigen-presenting cell (APC) subsets remains to be determined. The E3 ubiquitin ligase MARCH1 is constitutively expressed by resting B cells and DCs, and its expression can be enhanced by exposure of monocytes or macrophages to interleukin-10 (IL-10; not shown). In immature DCs, ubiquitylation of internalized peptide–MHC class II complexes by MARCH1 at the plasma membrane and in early endosomes targets these complexes for lysosomal degradation. By contrast, inefficient ubiquitylation of peptide–MHC class II complexes by MARCH1 in B cells prevents their delivery to lysosomes and instead redirects them back to the plasma membrane (indicated by the dashed arrow). Activation of immature DCs rapidly terminates MARCH1 expression and peptide–MHC class II ubiquitylation. As internalized peptide–MHC class II complexes are not ubiquitylated in mature DCs, these complexes are spared from lysosomal degradation and enter a re-iterative recycling pathway back to the plasma membrane (indicated by the thicker arrow).