In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is downregulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly downregulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection. . They are localized to both peripheral and lymphatic tissues and sample their surroundings, internalizing, processing, and presenting captured antigens to T cells on MHC class I molecules (MHCI) and MHC class II molecules (MHCII). They distinguish between self-and foreign antigens using receptors of the innate immune system [e.g., Toll-like receptors (TLRs)], inducing immunity when antigen is captured in the presence of microbial products or inflammatory stimuli but tolerance in the absence of these signals (3). DCs exhibit dramatic functional and morphological changes, termed maturation, that maximize antigen presentation to T cells in response to such stimuli (1, 2). These changes involve acidification of the lysosomal compartment to optimize antigen processing, up-regulation of costimulatory molecules, and reorganization of MHCII from the late endocytic compartments to the plasma membrane for recognition by T cells (4, 5).Modulation of endocytosis also occurs during maturation. Immature DCs endocytose avidly through a variety of mechanisms, including "nonspecific" uptake by constitutive macropinocytosis and "specific" uptake via receptor-mediated endocytosis and phagocytosis (2). Macropinocytosis is transiently up-regulated immediately on the receipt of an inflammatory signal (6), but this is followed by its dramatic down-regulation, partly mediated by a reduction of the active form of the Rho GTPase, Cdc42 (7). Most studies of endocytosis in DCs have involved exposing cells to a high concentration of antigens or endocytic tracers. Although such assays mainly measure macropinocytosis, it is generally presumed that all forms of endocytosis are down-regulated in mature DCs. The idea seems to fit...

Dendritic cells (DCs) and B cells present antigen-derived peptides bound to MHC class II (MHC II) molecules for recognition by CD4-positive T lymphocytes. DCs control the intracellular traffic of peptide-MHC II complexes by regulating the ubiquitination of MHC II. In resting or "immature" DCs, ubiquitinated MHC II molecules are targeted to lysosomes, but upon pathogen-induced "maturation," ubiquitination is down-regulated and MHC II can accumulate on the plasma membrane of mature DCs. Although B cells constitutively ubiquitinate their MHC II, it unexpectedly remains at the surface. We find that DCs and B cells differ in MHC II-conjugated ubiquitin (Ub) chain length: four to six Ub in immature DCs vs. two to three in B cells. In both cell types, experimentally increasing Ub chain length led to efficient lysosomal transport of MHC II, whereas MHC II with fewer than two Ubs did not reach lysosomes. Thus, Ub chain length plays a crucial role in regulating the intracellular fate and function of MHC II in DCs and B cells.D endritic cells (DCs) and B lymphocytes are professional antigen-presenting cells (APCs) capable of stimulating efficient T-cell responses (1, 2). However, their approaches to antigen presentation differ in important respects. Whereas DCs are highly endocytic and internalize a wide variety of antigens, B cells take up and process only the single antigen recognized by their B-cell receptor. DCs are also distinguished by their ability to regulate antigen processing and presentation by "maturation" (3, 4). Immature DCs, found in peripheral tissues, are adept at endocytic uptake of antigen but do not efficiently generate peptide-MHC class II (MHC II) complexes or express them stably on the cell surface. In part, this is because MHC II in immature DCs is ubiquitinated on a single conserved lysine in the cytoplasmic domain of the β-chain (5, 6) by E3 ligases of the membrane-associated RING-CH (MARCH) family (7,8). Like other ubiquitinated membrane proteins (9), ubiquitinated MHC II molecules are targeted to and sequestered in multivesicular late endosomes and lysosomes. Upon receiving a maturation stimulus (e.g., Toll-like receptor agonist), however, ubiquitination ceases (5, 6) and peptide-MHC II complexes are translocated to and accumulate at the plasma membrane (10-13). In B cells, MHC II surface expression is always high despite also being ubiquitinated by MARCH ligases in naïve B cells (8).Internalization and down-regulation of receptor tyrosine kinases by ubiquitination is well known. Ligand binding activates the kinase, resulting in autophosophorylation and subsequent recruitment of soluble E3 ligases (e.g., Cbl) that ubiquitinate one or more acceptor lysines. The ubiquitin (Ub) moieties are recognized by Ub-interaction motif (UIM)-containing adapter molecules (e.g., epsins, eps15) that associate with clathrin-coated pits, leading to receptor internalization (14-18). Upon delivery to early endosomes, Ub is recognized by members of the endosomal sorting complex required for transport (ESCRT) complexe...