Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs downregulate T-cell responses to induce/ maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosomeshuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. IntroductionCellular miRNAs are released membrane free 1 or packaged inside microvesicles (0.1-1 m) shed by the plasma membrane 2,3 or within nanovesicles (Ͻ 100nm) derived from the endocytic pathway known as exosomes. 4,5 Exosomes are generated as intraluminal vesicles by reverse budding of the membrane of multivesicular bodies (MVBs). Release of exosomes occurs when MVBs fuse their limiting membrane with the plasma membrane. [6][7][8][9] Dendritic cells (DCs) are APCs with the ability to regulate adaptive immunity. Whereas immature DCs down-regulate T-cell responses, mature DCs promote activation, proliferation, and differentiation of effector T cells. 10 Communication between DCs is essential to amplify their tolerogenic and immunogenic functions. 11,12 This DC-to-DC interaction is mediated through cell-tocell contact, soluble mediators, exchange of plasma membrane patches, 13,14 nanotubules, 15 and interaction with apoptotic cellderived vesicles 16 and exosomes. 17,18 Although the mechanisms have not been elucidated, it has been reported that DCs acquire proteins/peptides from other cells via exosomes. [17][18][19] Recently, it has been suggested that transfer of exosome-shuttle miRNAs might constitute a mechanism of cell-tocell communication that regulates mRNA translation 20 or, alternatively, a way to dispose of "unwanted" miRNAs. 21 An important unanswered question in the field is how exosome-shuttle miRNAs, transported inside the vesicles, are delivered into the cytosol of the acceptor cells, a problem we have investigated in this study with the use of DCs. Addressing this point has been challenging because (1) the composition of DC exosomes depends on the maturation of the DC of origin 22,23 ; (2) there is limited information on intercellular communication via "endogenous" (instead of exogenously added...

IntroductionDendritic cells (DCs) are antigen (Ag)-presenting cells (APCs) that function as biosensors of the cellular microenvironment by detecting the presence of signals that determine T-cell tolerance or immunity. 1,2 To accomplish this task, DCs acquire extracellular Ags by receptor-mediated endocytosis, macropinocytosis, or phagocytosis [3][4][5] ; by incorporation of microvesicles shed from the surface of neighboring cells, 6,7 and by their recently described interaction with nanovesicles (Յ 100 nm) termed "exosomes." [8][9][10][11][12] Exosomes are formed by reverse budding of the membrane of late endosomes [13][14][15] or multivesicular bodies (MVBs) and are released to the extracellular space by fusion of MVB with the plasma membrane. [13][14][15] Originally described in neoplastic cell lines, 16 exosomes also are produced by leukocytes and epithelial cells. [17][18][19][20][21][22] Although the function of exosomes still is poorly understood, exosomes are a source of Ag for APCs and participate in Ag presentation to T lymphocytes. 11,12 High concentrations of exosomes expressing major histocompatibility complex (MHC) and costimulatory molecules activate T-cell clones and T-cell lines weakly 10,13 and fail to stimulate naive T cells. 9,11 This impaired naive T-cell stimulatory ability of exosomes has been attributed to their low T-cell receptor-cross-linking capacity (inadequate for naive T-cell activation) and their small size and membrane composition. 10 However, in the presence of DCs, exosomes increase their ability to stimulate T cells. 10,11,23,24 The mechanism of interaction of extracellular exosomes with DCs is unknown. Although there is evidence that exosomes may transfer functional MHC-I/peptide complexes to DCs, 24 it is unclear whether exosomes cluster or fuse with DCs or if they are internalized and processed, as occurs with vesicles derived from apoptotic cells. [2][3][4][5] Herein we demonstrate that exosomes are internalized efficiently by DCs. Targeting of exosomes to DCs depends on ligands on the exosome and DC surface and is independent of complement factors. Once internalized by DCs, exosomes are sorted into recycling endosomes and then through late endosomes/lysosomes. By this mechanism, DCs process and present peptides derived from the internalized exosomes to T cells. In vivo, blood-borne exosomes are captured by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. In the steady state, uptake of circulating exosomes by splenic DCs does not induce DC maturation and does not prevent CD40-induced DC activation in vivo. Our results demonstrate that blood-borne allogeneic exosomes are efficiently targeted, internalized, and processed by splenic DCs in vivo, a phenomenon followed by presentation of exosome-derived allopeptides by CD8␣ ϩ DCs to CD4 ϩ T cells. Since allogeneic exosomes are a rich source of alloMHC and are targeted and processed in vivo by host DCs (without inducing their activation), intravenous administration of donor-derived exosomes may constitut...

Under steady-state conditions, internalization of self-antigens embodied in apoptotic cells by dendritic cells (DCs) resident in peripheral tissue followed by DC migration and presentation of self-peptides to T cells in secondary lymphoid organs are key steps for induction and maintenance of peripheral T-cell tolerance. We show here that, besides this traffic of apoptotic cells mediated by peripheral tissueresident DCs, splenic marginal zone DCs rapidly ingest circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8␣ during their mobilization to T-cell areas of splenic follicles. Because apoptotic cells activate complement and some complement factors are opsonins for phagocytosis and play roles in the maintenance of peripheral tolerance, we investigated the role of complement receptors (CRs) in relation to phagocytosis of apoptotic cells by DCs. Apoptotic cell uptake by marginal zone DCs was mediated in part via CR3 (CD11b/CD18) and, to a lesser extent, CR4 (CD11c/CD18) and was reduced significantly in vivo in hypocomplementemic animals. Following phagocytosis of apoptotic cells, DCs exhibited decreased levels of mRNA and secretion of the proinflammatory cytokines interleukin 1␣ (IL-1␣), IL-1␤, IL-6, IL-12p70, and tumor necrosis factor ␣ (TNF-␣), without effect on the anti-inflammatory mediator transforming growth factor ␤1 (TGF-␤1). This selective inhibitory effect was at least partially mediated through C3bi-CD11b/CD18 interaction.