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During inflammation, recruited monocytes can differentiate either into macrophages or dendritic cells (DCs); however, little is known about the environmental factors that determine this cell fate decision. Low extracellular pH is a hallmark of a variety of inflammatory processes and solid tumors. Here, we report that low pH dramatically promotes the differentiation of monocytes into DCs (monocyte-derived DCs [mo-DCs]). This process is associated with a reduction in glucose consumption and lactate production, the upregulation of mitochondrial respiratory chain genes, and the inhibition of mTORC1 activity. Interestingly, we also find that both serum starvation and pharmacological inhibition of mTORC1 markedly promote the differentiation of mo-DCs. Our study contributes to better understanding the mechanisms that govern the differentiation of monocytes into DCs and reveals the role of both extracellular pH and mTORC1 as master regulators of monocyte cell fate. Fil: Erra Díaz, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Ochoa, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Merlotti Ippólito, Antonela. PSL Research University; Francia. Inserm; Francia Fil: Dantas, Ezequiel Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Mazzitelli, Ignacio Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Gonzalez Polo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Sabatte, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Amigorena, Sebastián. Psl Research University; Francia. Inserm; Francia Fil: Segura, Elodie. Psl Research University; Francia. Inserm; Francia Fil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina

Encounters between naive T lymphocytes and dendritic cells (DCs) bearing adequate co-stimulatory signals are rare. In this issue of Immunity, Brewitz et al. (2017) show that chemokines secreted by CD8+ T cells recruit myeloid and plasmacytoid DCs that in turn boost CD8+ T cell activation.

The induction of most CD8+ T cell responses by dendritic cells (DCs) requires the presentation of peptides from internalized antigen by class I MHC molecules. Increasing number of reports have shown that cross presentation is involved in transplant rejection, in immune responses to viral infections, in certain autoimmune diseases and cancer. The precise role of cross presentation in the initiation of immune responses in vivo, however, remains a matter of debate. This ongoing controversy is, at least in part, due to a lack of understanding of the molecular machinery that determine cross presentation pathways in terms of cell biology. The present review aims to summarize recent insights and advances that help enlighten the intracellular steps of antigen cross presentation in DCs.

SummaryIntracellular Toll-like receptor 3 (TLR3), TLR7, and TLR9 localize in endosomes and recognize single-stranded RNA and nucleotides from viruses and bacteria. This interaction induces their conformational changes resulting in the production of proinflammatory cytokines and upregulation of cell surface molecules. TLR9 requires a proteolytic cleavage for its signaling. Here, we report that myeloid and plasmacytoid dendritic cells (DCs) deficient for the asparagine endopeptidase (AEP), a cysteine lysosomal protease, showed a decrease in the secretion of proinflammatory cytokines in response to TLR9 stimulation in vitro and in vivo. Upon stimulation, full-length TLR9 was cleaved into a 72 kDa fragment and this processing was strongly reduced in DCs lacking AEP. Processed TLR9 coeluted with the adaptor molecule MyD88 and AEP after size exclusion chromatography. When expressed in AEP-deficient DCs, the 72 kDa proteolytic fragment restored TLR9 signaling. Thus, our results identify an endocytic protease playing a critical role in TLR processing and signaling in DCs.

Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.

Summary The initiation of cytotoxic immune responses requires the direct interaction between naive CD8 + T lymphocytes and dendritic cells (DCs). Multiphoton imaging in intact lymph nodes (LNs) showed that during priming, naive T cells and DCs establish sequentially brief (i.e., minutes) and long (hours) antigen-specific contacts. We show here that the expression of the Intercellular Adhesion Molecule-1 (ICAM-1) by mature DCs is critical for long-lasting contacts with CD8 + T cells but dispensable for short-lived antigen-specific interactions. Serial brief DC-T cell contacts induced early CD8 + T cell activation, proliferation, and differentiation into effector cytotoxic T lymphocytes in the first few days after immunization. ICAM-1-deficient mature DCs, however, failed to induce fully effective priming, because CD8 + T cells produced reduced amounts of interferon γ and were clonally depleted after 2 weeks. In addition, Icam1 −/− mice failed to respond to rechallenge. We conclude that ICAM-1-dependent long-lasting interactions between mature DCs and naive CD8 + T cells determine the survival of activated CD8 + T cells and the establishment of effective memory.

Exosomes are MHC-bearing vesicles secreted by a wide array of cells. We have previously shown that donor-haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC-mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short-term LF 15-0195 (LF) treatment, which blocks the maturation of DCs, so that donor-MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF-treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor-specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor-specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor-MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor-specific allograft tolerance.

The specificity of the immunological responses is achieved through the cooperation of three classes of cells: B and T lymphocytes, and dendritic cells (DCs). A critical, intensely studied interaction is that between DCs and T cells, during which the DC presents MHC-bound antigenic fragments to the T cell receptor (TCR). There has been recent excitement about the possibility of increasing the signal-to-noise ratio in the detection of cognate antigen-TCR couples, by the use of kinetic proofreading mechanisms. We examine here the signal-to-noise problem in a broader perspective, and in particular, address the question of possible "antigen purification" mechanisms, prior to their presentation to the T cells. Ways in which the DCs might concentrate, purify and preserve their load of captured antigens are considered: (i) If antigens can be transferred from one DC to another, in such a way that the richer a DC in antigen, the more it captures antigens from other DCs, the antigens may end up concentrated in a small subset of DCs, (ii) antigen purification may be achieved through recycling interactions between DCs and B cells. A DC would transmit to a B cell antigen mixtures, and the DC would recapture only the antigens which can bind to the B cell's antibodies and (iii) dendrites, when they are present, may play an essential role in recapturing the antigens that were used in interactions of DCs with T cells, B cells, or other DCs, thereby reducing antigen losses. More generally, we provide a personal interpretation of cell-to-cell antigen transfers, in terms of a strategy in which there is a progressive emergence, through multiple interactions, of subsets of cells of each type better and better prepared for the subsequent rounds of interactions.

A recent workshop on "Dendritic Cells: Biology and Therapeutic Applications," sponsored by the Juan March Foundation, brought together basic and clinical research scientists to discuss the mechanisms underlying the control of immune responses and tolerance by dendritic cells (DCs), as well as recent research in cancer immunotherapy based on DC vaccination. Particular emphasis was placed on antigen processing and presentation by DCs, C-type lectin antigen receptors, DC maturation and polarization of T cell responses, the control of immunity versus tolerance by DCs, the developmental origin of DCs, and the use of DCs in cancer immunotherapy.

Tumor cells release membrane vesicles, named exosomes, capable of specific cytotoxic T-lympho- cyte activation by transferring tumor antigens to dendritic cells. By contrast, the nonclassical human leucocyte anti- gen (HLA)-G class I molecule displays immunotolerant properties and can be ectopically expressed by tumor cells, thereby allowing their escape from immunosurveillance. We describe here that a melanoma cell line, named Fon, established from an HLA-G-positive melanoma biopsy, spontaneously expressed high levels of the HLA-G1 mem- brane-bound isoform. Exosomes released by Fon cells were purified and analyzed both for their density on sucrose gradient and their protein composition by Western blot- ting and flow cytometry. Besides the expression of well- described proteins such as Lamp-2, notably, these mela- noma-derived exosomes bore HLA-G1. In addition, exosomes harboring HLA-G1 were secreted by the HLA- G-negative M8 melanoma cells transfected with the HLA-G1 cDNA. Thus, the presence of tolerogenic HLA-G molecules on melanoma-derived exosomes may provide a novel way for tumors to modulate host's im- mune response. Human Immunology 64, 1064 -1072 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc.

Summary Background Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supematants. 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. Methods We 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. Findings Malignant 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. Interpretation Exosomes 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.

Dendritic cells are the most efficient antigen-presenting cells. They take up antigens and pathogens, generate MHC-peptide complexes, migrate from the sites of antigen acquisition to secondary lymphoid organs and, finally, they physically interact with and stimulate T lymphocytes. Indeed, dendritic cells are the only antigen-presenting cells that induce the activation of resting T cells, both in vitro and in vivo. Thus, dendritic cells initiate adaptive immune responses and determine tolerance. To do so, dendritic cells have developed unique membrane transport pathways. The molecular mechanisms responsible for the control of antigen uptake and processing, for the generation of MHC-peptide complexes and for their transport to the cell surface have been partially unraveled in the past two years.

The role of specific receptors in antigen internalization and presentation to helper T lymphocytes has been known for more than ten years. However, recent work indicates that internalization may not always be sufficient for antigen presentation. Indeed, antigen receptors such as B-cell receptors and Fc receptors may also be involved in the post-endocytic transport events that determine selectively the delivery of antigens to different endocytic compartments and thereby the presentation of different T-cell epitopes.

Resume Veritables professionnelles de la presentation des molecules etrangeres ou anormales au systeme immunitaire, les cellules dendritiques pourraient etre judicieusement employees en immunotherapie pour stimuler le rejet des tumeurs cancereusus. Les essais cliniques ont debute.

The association between antigenic peptides and MHC class II molecules represents a critical event in the initiation of the immune response to extracellular antigens. Understanding the molecular basis of antigen processing requires the characterization of the intracellular compartments, or ‘single bars’, in which immunogenic peptides are generated and loaded onto class II molecules. In the past year, something of a breakthrough occurred with the identification of specialized compartments that host antigen processing and/or peptide loading, designated ‘MHC class II compartment’ and ‘class II vesicles’. It is becoming increasingly clear, however, that these compartments are themselves heterogeneous and not always distinct from conventional endosomes and lysosomes.

SummaryIt is well understood that antigen-presenting cells (APCs) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent on IRF8, Zbtb46, and Batf3 transcription factors and are generated by GM-CSF and FLT3L cytokines. Regressing tumors have higher proportions of these cells, T-cell-dependent immune clearance relies on them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types.

Mouse immunoglobulin G-binding factors (IgG-BF) produced either by activated T cells (ATC) or by hybridoma T cells (T2D4) directly inhibit the in vitro IgG secretion by hybridoma B cells. This inhibition affects IgGI, IgG2a and IgG2b and can be detected as early as after 2 h incubation of the cells with IgG-BF eluted from non-equilibrium pH gradient electrophoresis gels. Moreover, IgG-BF also exert a strong growth-inhibitory effect on hybridoma B cells without any detectable immediate cytotoxicity. These results provide an experimental basis to analyze the molecular and biological effects induced by IgG-BF on B cells.

A microtiter plate assay to detect the effect of immunoglobulin binding factor (IBF) on Ig secretion by hybridoma B cells is described. This technique permits the analysis of Ig secretion by only 200-400 hybridoma B cells using a PFC assay and an ELISA, and therefore increases the IBF/cell number ratio. This increase allows the detection of a strong IBF-mediated inhibitory effect on Ig secretion by hybridoma B cells, which is otherwise difficult to obtain reproducibly. The technique is simple, does not require any transferring step and is convenient, since it permits large numbers of samples to be tested. It can be extended to test IBF for all isotypes or other lymphokines that affect Ig secretion.

Software has been developed which permits the quantitation of monoclonal antibodies secreted by B cell hybridomas. This program does not require the user to enter a large number of complex parameters and can be easily used without any previous computer experience. It fits all the experimental and standard curves by determining overlapping linear domains using the linear least-squares method. The program is based on logarithmic interpolations for determining Ig concentrations comparing experimental samples to Ig concentrations in standards. It provides a complete print-out of the data with editing options and is written in BASIC EDEX 4.0 Commodore computer language. It permits the accurate quantification of minute amounts of monoclonal antibodies and can be used to detect the inhibitory or enhancing effects of lymphokines or cytokines on Ig secretion by hybridoma B cells.