Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells.

In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune responses efficiently, have developed a unique membrane transport pathway linking the lumen of endocytic compartments and the cytosol. Endosome-to-cytosol transport is restricted to dendritic cells, specific to internalized antigens and selective for the size of the transported molecules. Thus, in dendritic cells, internalized antigens gain access to the cytosolic antigen-processing machinery and to the conventional MHC class I antigen-presentation pathway.

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.

Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo.

Dendritic cells (DC) can provide all the known costimulatory signals required for activation of unprimed T cells and are the most efficient and perhaps the critical antigen presenting cells in the induction of primary T cell-mediated immune responses. It is now shown that mouse cell lines with many of the features of DC can be generated using the MIB phi 2-N11 retroviral vector transducing a novel envAKR-mycMH2 fusion gene. The immortalized dendritic cell line (CB1) displays most of the morphologic, immunophenotypic, and functional attributes of DC, including constitutive expression of major histocompatibility complex (MHC) class II molecules, costimulatory molecules B7/BB1, heat stable antigen, intracellular adhesion molecule 1, and efficient antigen-presenting ability. Granulocyte/macrophage colony-stimulating factor (GM-CSF) proved to be effective in increasing MHC class II molecule expression and in enhancing presentation of native protein antigens. In comparison with macrophages, CB1 dendritic cells did not exhibit phagocytic and chemotactic activity in response to various stimuli and lipopolysaccharide activation was ineffective in inducing tumor necrosis factor alpha or interleukin 1 beta production. CB1 cells, pulsed with haptens in vitro and injected into naive mice were able to induce delayed-type hypersensitivity responses, further increased with pretreatment with GM-CSF, indicating that these cells may represent an immature, rather than a mature DC. The ability of CB1 to prime T cells in vivo could provide a tool to design novel immunization strategies.

T-cell lymphoma induction by radiation leukemia virus in athymic nude mice.

We report the development of extrathymic lymphoblastic lymphomas in RadLV-inoculated congenitally athymic nude mice. Thus, a leukemogenic virus which appears to require the presence of a thymus for its replication in normothymic mice can infect and transform target cells in the absence of this organ in the athymic host. The cells of one of these lymphomas have been established in vitro as a permanent cell line, BALB/Nu1. This cell line as well as a lymphoma induced in NIH/Swiss nude mice exhibit several T-cell markers, including terminal deoxynucleotidyl transferase activity, Thy-1.2, and Ly-2.2, but not Ly-1.2 nor TL. Ig determinants were not detected. The characteristics of the tumor cells support the view that cells with T-cell markers may normally exist in nude mice and undergo neoplastic transformation and clonal expansion after infection with a leukemogenic virus. The alternative possibility that virus-induced differentiation of prothymocytes may lead to the expression of Thy-1.2 and Ly-2.2 antigens is also considered. BALB/Nu1 cells release large numbers of type C viral particles. The virus, designated radiation leukemia virus (RadLV)/Nu1, has RTase activity and the protein profile characteristic of murine leukemia virus (MuLV). In radioimmunoassays, it cross-reacts completely with RadLV/VL3, a virus obtained from RadLV-induced C57BL/Ka thymic lymphoma cells in culture, and slightly with a xenotropic virus (BALB:virus-2) and with AKR MuLV. On inoculation into C57BL/Ka mice it has thymotropic and leukemogenic activity. In vitro it is B-tropic, poorly fibrotropic, and has limited xenotropic activity. Thus, RadLV/Nu1 appears to be biologically and serologically similar or identical to its parent virus, RadLV.

Ig-specific T cell receptor-transgenic T cells are not deleted in the thymus and are functional in vivo.

The mechanisms that induce T cell tolerance to circulating self-proteins are still controversial, and both the deletion and selection of autoreactive T cells have been observed in the thymus of transgenic mouse models. To address the question of the induction of tolerance to circulating self-constituents, a T cell receptor-transgenic mouse specific for the serum protein immunoglobulin (Ig) gamma and (IgG2ab) was generated. The choice of an allotype-specific T cell also allowed the generation of transgenic control mice not expressing the self-antigen. It was found that the transgenic T cells were not deleted in the thymus, did not become tolerant in the periphery, and regulated the function of gamma 2ab-positive B cells as shown by the lack of IgG2ab protein in the serum of the transgenic mice. In spite of this activity in vivo, the transgenic T cells did not proliferate in vitro in response to the allotype-specific peptide. Interestingly, antigen-specific T cell proliferation could be restored if the transgenic mice were previously challenged to induce IgG2ab responses. After this challenge, IgG2ab protein in the serum of the transgenic mice could be partially restored, although still remaining much lower than in control mice. In addition, there was a dramatic increase in serum IgE levels, suggesting that newly generated gamma 2ab-secreting B cells can be induced to switch to IgE in the presence of allotype-specific T cells. These results indicate that Ig-specific T cells may represent a late-acting form of T cell help for the regulation of the IgG2a-to-IgE class switch.

Dendritic cell survival and maturation are regulated by different signaling pathways.

Although dendritic cell (DC) activation is a critical event for the induction of immune responses, the signaling pathways involved in this process have not been characterized. In this report, we show that DC activation induced by lipopolysaccharide (LPS) can be separated into two distinct processes: first, maturation, leading to upregulation of MHC and costimulatory molecules, and second, rescue from immediate apoptosis after withdrawal of growth factors (survival). Using a DC culture system that allowed us to propagate immature growth factor-dependent DCs, we have investigated the signaling pathways activated by LPS. We found that LPS induced nuclear translocation of the nuclear factor (NF)-kappaB transcription factor. Inhibition of NF-kappaB activation blocked maturation of DCs in terms of upregulation of major histocompatibility complex and costimulatory molecules. In addition, we found that LPS activated the extracellular signal-regulated kinase (ERK), and that specific inhibition of MEK1, the kinase which activates ERK, abrogated the ability of LPS to prevent apoptosis but did not inhibit DC maturation or NF-kappaB nuclear translocation. These results indicate that ERK and NF-kappaB regulate different aspects of LPS-induced DC activation: ERK regulates DC survival whereas NF-kappaB is responsible for DC maturation.

Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo.

The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces. The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of debate. Here we show the interaction, and consequences, of different forms of C. albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C. albicans saprophytism and infections.

Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses.

Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34(+)-derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC-lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1beta and tumor necrosis factor alpha. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1beta. Importantly, it is possible to reduce the production of IL-1beta and interferon (IFN)-gamma during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-gamma secretion is independent of IL-12.

The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18).

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the alpha(M)beta(2) integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the alpha(M)beta(2) integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with alpha(M)beta(2) (CD11b/CD18) but not in cells transfected with the vector alone or with the alpha(X)beta(2) (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.

Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization.

Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.

Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; non-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli.

The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.

Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594-600). To unravel the molecular basis of exosome-induced immune stimulation, we now analyze the regulation of their production during DC maturation and characterize extensively their protein composition by peptide mass mapping. Exosomes contain several cytosolic proteins (including annexin II, heat shock cognate protein hsc73, and heteromeric G protein Gi2alpha), as well as different integral or peripherally associated membrane proteins (major histocompatibility complex class II, Mac-1 integrin, CD9, milk fat globule-EGF-factor VIII [MFG-E8]). MFG-E8, the major exosomal component, binds integrins expressed by DCs and macrophages, suggesting that it may be involved in exosome targeting to these professional antigen-presenting cells. Another exosome component is hsc73, a cytosolic heat shock protein (hsp) also present in DC endocytic compartments. hsc73 was shown to induce antitumor immune responses in vivo, and therefore could be involved in the exosome's potent antitumor effects. Finally, exosome production is downregulated upon DC maturation, indicating that in vivo, exosomes are produced by immature DCs in peripheral tissues. Thus, DC-derived exosomes accumulate a defined subset of cellular proteins reflecting their endosomal biogenesis and accounting for their biological function.

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells.

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.

Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominant Lpsd allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

Impaired calcineurin signaling in myeloid cells results in downregulation of pentraxin-3 and increased susceptibility to aspergillosis.

Treatment of post-transplant patients with immunosuppressive drugs targeting the calcineurin-nuclear factor of activated T cells (NFAT) pathway, including cyclosporine A or tacrolimus, is commonly associated with a higher incidence of opportunistic infections, such as Aspergillus fumigatus, which can lead to severe life-threatening conditions. A component of the A. fumigatus cell wall, ß-glucan, is recognized by dendritic cells (DCs) via the Dectin-1 receptor, triggering downstream signaling that leads to calcineurin-NFAT binding, NFAT translocation, and transcription of NFAT-regulated genes. Here, we address the question of whether calcineurin signaling in CD11c-expressing cells, such as DCs, has a specific role in the innate control of A. fumigatus. Impairment of calcineurin in CD11c-expressing cells (CD11ccrecnb1loxP) significantly increased susceptibility to systemic A. fumigatus infection and to intranasal infection in irradiated mice undergoing bone marrow transplant. Global expression profiling of bone marrow-derived DCs identified calcineurin-regulated processes in the immune response to infection, including expression of pentraxin-3, an important antifungal defense protein. These results suggest that calcineurin inhibition directly impairs important immunoprotective functions of myeloid cells, as shown by the higher susceptibility of CD11ccrecnbloxP mice in models of systemic and invasive pulmonary aspergillosis, including after allogeneic bone marrow transplantation. These findings are relevant to the clinical management of transplant patients with severe Aspergillus infections.

Dendritic cell interactions and cytokine production.

The dendritic cell lineage comprises cells at various stages of functional maturation that are able to induce and regulate the immune response against antigens and thus function as initiators of protective immunity. The signals that determine the given dendritic cell functions depend mostly on the local microenvironment and on the interaction between dendritic cells and microorganisms. These interactions are complex and very different from one pathogen to another; nevertheless, both shared and unique responses have been observed using global genomic analyses. In this review, we have focused on the study of host-pathogen interactions using a genome-wide transcriptional approach with a focus on cytokine family members.

Activation of the M-CSF gene in mouse macrophages immortalized by retroviruses carrying a v-myc oncogene.

We have recently immortalized murine brain macrophages (microglial cells) with a complex of retroviruses (3RV) transducing separately the myc and mil oncogenes. Surprisingly, the immortalized cells harboured an exogenous v-myc oncogene, but no v-mil sequences. The transformed macrophage cell lines grew in vitro without the addition of exogenous growth factors and were also able to grow in vivo in nude mice. In addition, they released oncogenic retroviruses able to immortalize mouse macrophages from primary splenic or thymic cultures. Molecular cloning of the provirus (VN-11) harboured in a microglial clone demonstrated that no cell-derived sequences apart from an avian v-myc gene were transduced by the recombinant retrovirus. When cells were tested for production of myeloid growth factors, they were found to transcribe and synthesize the Macrophage-Colony Stimulating Factor (M-CSF). The correlation between viral infection and activation of the M-CSF gene was tested using a M-CSF dependent cell line from which growth factor independent clones could be readily obtained after infection. The synthesis of M-CSF was detected only in cells expressing the avian v-myc protein. These data support the hypothesis that, in our conditions, macrophages can be immortalized by the expression of v-myc and the concomitant establishment of an autocrine loop triggered by viral infection.