Graça Raposo

Proteomic Analysis of Dendritic Cell-Derived Exosomes: A Secreted Subcellular Compartment Distinct from Apoptotic Vesicles

Dendritic cells constitutively secrete a population of small (50–90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1α) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

Malignant effusions and immunogenic tumour-derived exosomes.

BACKGROUNDnExosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells.nnnMETHODSnWe isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes.nnnFINDINGSnMalignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules.nnnINTERPRETATIONnExosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.

Exosomes are released by cultured cortical neurones.

Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.

Shiga toxin induces tubular membrane invaginations for its uptake into cells

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

Exosomes as Potent Cell-Free Peptide-Based Vaccine. I. Dendritic Cell-Derived Exosomes Transfer Functional MHC Class I/Peptide Complexes to Dendritic Cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8+ T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1) DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8+ T cell priming in vitro; 2) exosomes require nature’s adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-γ (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.

BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes.

Recycling endosomes consist of a tubular network that emerges from vacuolar sorting endosomes and diverts cargoes toward the cell surface, the Golgi, or lysosome-related organelles. How recycling tubules are formed remains unknown. We show that recycling endosome biogenesis requires the protein complex BLOC-1. Mutations in BLOC-1 subunits underlie an inherited disorder characterized by albinism, the Hermansky-Pudlak Syndrome, and are associated with schizophrenia risk. We show here that BLOC-1 coordinates the kinesin KIF13A-dependent pulling of endosomal tubules along microtubules to the Annexin A2/actin-dependent stabilization and detachment of recycling tubules. These components cooperate to extend, stabilize and form tubular endosomal carriers that function in cargo recycling and in the biogenesis of pigment granules in melanocytic cells. By shaping recycling endosomal tubules, our data reveal that dysfunctionxa0of the BLOC-1-KIF13A-Annexin A2 molecular network underlies the pathophysiology of neurological and pigmentary disorders.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal exocytic vesicle. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11+ recycling and Rab27+ late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte–target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

Amutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype “chocolate” (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38G19V is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.

MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways.

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide‐loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen‐loaded DCs, which is triggered by cognately interacting antigen‐specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent‐resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.

Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes

Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky–Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky–Pudlak syndrome.