Activation of Dendritic Cells Alters the Mechanism of MHC Class II Antigen Presentation to CD4 T Cells.
ABSTRACT: Both immature and mature dendritic cells (DCs) can process and present foreign Ags to CD4 T cells; however, the mechanism by which MHC class II (MHC-II) in mature DCs acquires antigenic peptides remains unknown. To address this, we have studied Ag processing and presentation of two distinct CD4 T cell epitopes of the influenza virus hemagglutinin coat protein by both immature and mature mouse DCs. We find that immature DCs almost exclusively use newly synthesized MHC-II targeted to DM+ late endosomes for presentation to influenza virus-specific CD4 T cells. By contrast, mature DCs exclusively use recycling MHC-II that traffics to both early and late endosomes for antigenic peptide binding. Rab11a knockdown partially inhibits recycling of MHC-II in mature DCs and selectively inhibits presentation of an influenza virus hemagglutinin CD4 T cell epitope generated in early endosomes. These studies highlight a "division of labor" in MHC-II peptide binding, in which immature DCs preferentially present Ags acquired in Rab11a- DM+ late endosomes, whereas mature DCs use recycling MHC-II to present antigenic peptides acquired in both Rab11a+ early endosomes and Rab11a- endosomes for CD4 T cell activation.
Project description:Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.
Project description:Antigen presentation by dendritic cells (DCs) stimulates naive CD4+ T cells, triggering T cell activation and the adaptive arm of the immune response. Newly synthesized major histocompatibility complex class II (MHC-II) molecules accumulate at MHC-II-enriched endosomal compartments and are transported to the plasma membrane of DCs after binding to antigenic peptides to enable antigen presentation. In DCs, MHC-II molecules are included in tetraspanin-enriched microdomains (TEMs). However, the role of tetraspanin CD9 in these processes remains largely undefined. Here, we show that CD9 regulates the T cell-stimulatory capacity of granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent bone marrow-derived DCs (BMDCs), without affecting antigen presentation by fms-like tyrosine kinase 3 ligand (Flt3L)-dependent BMDCs. CD9 knockout (KO) GM-CSF-dependent BMDCs, which resemble monocyte-derived DCs (MoDCs), induce lower levels of T cell activation than wild-type DCs, and this effect is related to a reduction in MHC-II surface expression in CD9-deficient MoDCs. Importantly, MHC-II targeting to the plasma membrane is largely impaired in immature CD9 KO MoDCs, in which MHC-II remains arrested in acidic intracellular compartments enriched in LAMP-1 (lysosome-associated membrane protein 1), and MHC-II internalization is also blocked. Moreover, CD9 participates in MHC-II trafficking in mature MoDCs, regulating its endocytosis and recycling. Our results demonstrate that the tetraspanin CD9 specifically regulates antigenic presentation in MoDCs through the regulation of MHC-II intracellular trafficking.
Project description:Cell division cycle 42 (Cdc42) is a member of the Rho GTPase family and has pivotal functions in actin organization, cell migration and proliferation. Cdc42 has been shown to regulate antigen (Ag)-uptake in immature dendritic cells (DC) and controls their migration from tissues to lymph nodes. Previous reports demonstrated that Cdc42 is inactivated upon DC-maturation to avoid continued Ag-acquisition. To further study the molecular mechanisms of DC-control by Cdc42, we used bone marrow-derived DCs from Cdc42-deficient mice. We show that Cdc42-deficient DCs are phenotypically mature without additional maturation stimuli, as they upregulate CD86 from intracellular storages to the cell surface. They also accumulate invariant chain (Ii)-MHC class II complexes at the cell surface, which cannot efficiently present peptide Ag for priming of Ag-specific CD4 T cells. Lack of Cdc42 in immature DCs does not allow MHC class II maturation, as lysosomal Cathepsins are lost into the supernatant and Ii-MHC class II complexes cannot mature. Therefore Cdc42-deficient DCs are "pseudomature" and lose most functional hallmarks of antigen-presenting cells. Our results propose that Cdc42 keeps DCs in an immature state, while downregulation of Cdc42-activity during maturation facilitates generation of CD86+MHCII+ mature DCs.
Project description:The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface expression, intracellular distribution, and survival of pMHC-II in DCs. DCs isolated from March-I-KO mice express very high levels of pMHC-II on the plasma membrane even before DC activation. Although ubiquitination does not affect the kinetics of pMHC-II endocytosis from the surface of DCs, the survival of pMHC-II is enhanced in DCs obtained from March-I-deficient and MHC-II ubiquitination-mutant mice. Using pMHC-II-specific mAb, we show that immature DCs generate large amounts of pMHC-II that are remarkably stable under conditions in which pMHC-II ubiquitination is blocked. Thus, the cellular distribution and stability of surface pMHC-II in DCs is regulated by ubiquitin-dependent degradation of internalized pMHC-II.
Project description:The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.
Project description: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 down-regulation 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 down-regulate 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.
Project description:Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain-major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag's) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II-processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface.
Project description:Antigen processing for presentation by major histocompatibility complex class II (MHCII) molecules requires the latter to travel through the endocytic pathway together with its chaperons: the invariant chain (Ii) and DM. Nevertheless, the nature of the compartments where MHCII molecules travel to acquire peptides lacks definition regarding molecules involved in intracellular vesicular trafficking, such as Rab small GTPases. We aimed to define which Rab proteins are present during the intracellular transport of MHCII, DM, and Ii through the endocytic pathway on their route to the cell surface during dendritic cell (DC) maturation. We examined, by means of three-color confocal microscopy, the association of MHCII, DM, and Ii with Rab5, Rab7, Rab9, and Rab11 during the maturation of bone marrow-derived or spleen DC in response to LPS as an inflammatory stimulus. Prior to the stage of immature DC, MHCII migrated from diffuse small cytoplasmic vesicles, predominantly Rab5+Rab7- and Rab5+Rab7+ into a pericentriolar Rab5+Rab7+Rab9+ cluster, with Rab11+ areas. As DC reached the mature phenotype, MHCII left the pericentriolar endocytic compartments toward the cell surface in Rab11+ and Rab9+Rab11+ vesicles. The invariant chain and MHCII transport pathways were not identical. DM and MHCII appeared to arrive to pericentriolar endocytic compartments of immature DC through partially different routes. The association of MHCII molecules with distinct Rab GTPases during DC maturation suggests that after leaving the biosynthetic pathway, MHCII sequentially traffic from typical early endosomes to multivesicular late endosomes to finally arrive at the cell surface in Rab11+ recycling-type endosomes. In immature DCs, DM encounters transiently MHCII in the Rab5+Rab7+Rab9+ compartments, to remain there in mature DC.
Project description:The Rab GTPase family is a major regulator of membrane traffic in eukaryotic cells. The Rab11 subfamily plays important roles in specific trafficking events such as exocytosis, endosomal recycling, and cytokinesis. SH3BP5 and SH3BP5-like (SH3BP5L) proteins have recently been found to serve as guanine nucleotide exchange factors (GEF) for Rab11. Here, we report the crystal structures of the SH3BP5 GEF domain alone and its complex with Rab11a. SH3BP5 exhibits a V-shaped structure comprising two coiled coils. The coiled coil composed of ?1, and ?4 is solely responsible for the Rab11a binding and GEF activity. SH3BP5 pulls out and deforms switch I of Rab11a so as to facilitate the GDP release from Rab11a. SH3BP5 interacts with the N-terminal region, switch I, interswitch, and switch II of Rab11a. SH3BP5 and SH3BP5L localize to Rab11-positive recycling endosomes and show GEF activity for all of the Rab11 family but not for Rab14. Fluorescence-based GEF assays combined with site-directed mutagenesis reveal the essential interactions between SH3BP5 and Rab11 family proteins for the GEF reaction on recycling endosomes.
Project description:As sentinels of the immune system, dendritic cells (DCs) continuously generate and turnover antigenic peptide-MHC class II complexes (pMHC-II). pMHC-II generation is a complex process that involves many well-characterized MHC-II biosynthetic intermediates; however, the mechanisms leading to MHC-II turnover/degradation are poorly understood. We now show that pMHC-II complexes undergoing clathrin-independent endocytosis from the DC surface are efficiently ubiquitinated by the E3 ubiquitin ligase March-I in early endosomes, whereas biosynthetically immature MHC-II-Invariant chain (Ii) complexes are not. The inability of MHC-II-Ii to serve as a March-I substrate is a consequence of Ii sorting motifs that divert the MHC-II-Ii complex away from March-I(+) early endosomes. When these sorting motifs are mutated, or when clathrin-mediated endocytosis is inhibited, MHC-II-Ii complexes internalize by using a clathrin-independent endocytosis pathway and are now ubiquitinated as efficiently as pMHC-II complexes. These data show that the selective ubiquitination of internalizing surface pMHC-II in March-I(+) early endosomes promotes degradation of "old" pMHC-II and spares forms of MHC-II that have not yet loaded antigenic peptides or have not yet reached the DC surface.