Quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-gamma production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c(+) DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 microm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-gamma production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.     

Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS*

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.     

Dendritic cells in the skin – potential use for melanoma treatment

Melanoma is an aggressive malignancy with poor prognosis. Eradication of tumor cells requires an effective interaction between melanoma cells and different players of the immune system. As the most potent professional antigen‐presenting cells, dendritic cells (DCs) play a pivotal role in mounting a specific immune response where their intratumoral and peritumoral density as well as their functional status are correlated with clinical staging of the disease and with patients’ survival. Under steady‐state conditions, internalization of apoptotic cells by immature DCs designates a state of tolerance to self‐antigens. Nevertheless, pathogens and necrotic cells interacting with pattern recognition receptors trigger downstream signaling pathways that evoke maturation of DCs, leading to the production of pro‐inflammatory cytokines. These mature DCs are essential for T‐cell priming and subsequent development of a specific immune response. Altered functions of DCs have an impact on the development of various disorders including autoimmune diseases and cancers. Herein, we focus on the checkpoints created throughout DCs antigen capturing and presentation to T cells, with subsequent development of either tolerance or immune response, with an emphasis on the role played by DCs in melanoma tumorigenesis and their therapeutic potential.     

Dendritic cells: arbiters of immunity and immunological tolerance

Dendritic cells (DCs) link innate immune sensing of the environment to the initiation of adaptive immune responses. Given their supreme capacity to interact with and present antigen to T cells, DCs have been proposed as key mediators of immunological tolerance in the steady state. However, recent evidence suggests that the role of DCs in central and peripheral T-cell tolerance is neither obligate nor dominant. Instead, DCs appear to regulate multiple aspects of T-cell physiology including tonic antigen receptor signaling, priming of effector T-cell response, and the maintenance of regulatory T cells. These diverse contributions of DCs may reflect the significant heterogeneity and "division of labor" observed between and within distinct DC subsets. The emerging complex role of different DC subsets should form the conceptual basis of DC-based therapeutic approaches toward induction of tolerance or immunization.     

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.     

T cell priming by tissue-derived dendritic cells: new insights from recent murine studies

Dendritic cells (DCs) act as sentinels in peripheral tissues, continuously scavenging for antigens in their immediate surroundings. Their involvement in T cell responses is generally thought to consist of a linear progression of events, starting with capture of antigen in peripheral tissues such as the skin followed by migration to draining lymphoid organs and MHC-restricted presentation of antigen-derived peptide to induce T cell priming. The role of tissue-derived DCs in the direct priming of immune responses has lately been challenged. It now appears that, at least in some instances, a non-migratory subtype of DCs in the secondary lymphoid tissue presents tissue-derived antigen to T cells. Here, we review recent developments in research on DC function in the priming of immune responses.     

Dendritic Cells from Spleen, Mesenteric Lymph Node and Peyer's Patch Can Induce the Production of Both IL-4 and IFN-γ from Primary Cultures of Naive CD4+ T Cells in a Dose-Dependent Manner

Dendritic cells (DCs) as antigen presenting cells can stimulate naive CD4⁺ T cells and initiate the primary immune response which controls Th1/Th2 development. It has been suggested that DCs derived from different tissues have distinct properties. We investigated whether DCs from mesenteric lymph nodes (MLN), Peyer's patches (PP) and spleen (SPL) could induce different responses of naive CD4⁺ T cells to varying doses of antigen by using a co-culture system of DCs and T cells. DCs from each tissue induced IL-4 secretion from naive CD4⁺T cells in the presence of low dose antigenic peptide, and induced IFN-γ production at high doses of antigen. When purified CD11c⁺/B220^? DCs were used, MLN-derived DCs induced a higher amount of IFN-γ secretion from naive CD4⁺ T cells, compared with SPL-derived DCs. We could not detect large differences in the expressions of costimulatory molecules on the surface of these two populations of DCs. On the other hand, we found that large amounts of IL-12 were secreted from MLN DCs in an antigen dose-dependent fashion. In conclusion, DCs from SPL, MLN and PP can induce the production of both IL-4 and IFN-γ from naive CD4⁺ T cells, depending on antigen dose. MLN-derived CD11c⁺/B220^? DCs induce higher IFN-γ production from naive CD4⁺ T cells than SPL-derived DCs, through efficient IL-12 secretion.     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets

Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.     

Spatial modelling of brief and long interactions between T cells and dendritic cells

In the early phases of an immune response, T cells of appropriate antigen specificity become activated by antigen-presenting cells in secondary lymphoid organs. Two-photon microscopy imaging experiments have shown that this stimulation occurs in distinct stages during which T cells exhibit different motilities and interactions with dendritic cells (DCs). In this paper, we utilize the Cellular Potts Model, a model formalism that takes cell shapes and cellular interactions explicitly into account, to simulate the dynamics of, and interactions between, T cells and DCs in the lymph node paracortex. Our three-dimensional simulations suggest that the initial decrease in T-cell motility after antigen appearance is due to "stop signals" transmitted by activated DCs to T cells. The long-lived interactions that occur at a later stage can only be explained by the presence of both stop signals and a high adhesion between specific T cells and antigen-bearing DCs. Furthermore, our results indicate that long-lasting contacts with T cells are promoted when DCs retract dendrites that detect a specific contact at lower velocities than other dendrites. Finally, by performing long simulations (after prior fitting to short time scale data) we are able to provide an estimate of the average contact duration between T cells and DCs.     

Gamma delta T cells and dendritic cells: close partners and biological adjuvants for new therapies

The knowledge of several signals influencing Dendritic Cell (DC) functions is crucial to manipulate the immune system for new vaccination therapies. Our recent findings provide a new model of intervention on DC system suggesting novel therapeutic implications. T, NK, and gammadelta T cell stimuli may enhance DC maturation, Th polarization and trigger the adaptive immune response. Regulatory effects of gammadelta T cells on inflammation and immune responses may be mediated by their interaction with DCs and they are analyzed in the last years in humans and mice. In humans, Vgamma9Vdelta2 T cells represent the most part of circulating gammadelta T cells and are activated by non-peptidic molecules derived from different microorganisms or abnormal metabolic routes. They share both NK-like and effector/memory T cell features, and among these the possibility to interact with DCs. Co-culture of immature DCs with activated Vgamma9Vdelta2 T cells allows DCs to acquire features of mature DCs complementing the migratory activity, up-regulating the chemokine receptors, and antigen presentation. Similarly to the NK-derived signals, DC activation is mostly mediated by soluble factors secreted by gammadelta T cells. Many non-peptidic molecules including nitrogen-containing bisphosphonates and pyrophosphomonoester drugs stimulate the activity of Vgamma9Vdelta2 T cells in vitro and in vivo. The relatively low in vivo toxicity of many of these drugs makes possible novel vaccine and immune-based strategies, through DCs, for infectious and neoplastic diseases.     

Dendritic cell-tumor fusion vaccine prevents tumor growth in vivo

Dendritic cells (DCs) are potent antigen presenting cells that are uniquely effective in generating primary immune responses. DCs that are manipulated to present tumor antigens induce antitumor immunity in animal models and preclinical human studies. A myriad of strategies have been developed to load tumor antigen effectively onto DCs. DC-tumor fusion presents a spectrum of tumor-associated antigens to helper T- and cytotoxic T-cell populations in the context of DC-mediated costimulatory signals. In this study, fusion cells (FCs) were generated with MCA-102 fibrosarcoma cells and murine bone marrow-derived myeloid DCs. The FCs coexpressed the DC-derived MHC class II and costimulatory molecules. The FCs also retained the functional properties of DCs and stimulated syngeneic T cell proliferation and interferon-gamma (IFN-gamma) production. Significantly, the results show that syngeneic T cells are primed by FCs to induce MHC class I-dependent lysis of MCA-102 fibrosarcoma. These findings indicate that fusions of tumor cells and DCs activate T-cell responses against syngeneic tumors.     

Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/-) DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Effects of d-allose on the endocytic activity of dendritic cells and the subsequent stimulation of T cells

We examined the effects of a rare sugar, d-allose, which is 6-carbon monosaccharide, on endocytosis and T cell stimulation by dendritic cells (DCs). The endocytosis of BCG-anti-BCG immune complexes by DCs markedly decreased in d-allose-containing medium. Co-culture with T cells (mixed leukocyte reaction, MLR) of DCs, which had been exposed to BCG in d-allose-supplemented medium, induced apoptosis of CD4⁺ T cells in a manner dependent on d-allose concentration. After the MLR, DCs cultured in the medium with d-allose expressed less CD40 and more Fas ligands than those cultured without d-allose. It was suggested that the functions of DCs, internalization, processing and the subsequent antigen presentation to T cells, are down-regulated via the action of d-allose.     

Immortalized dendritic cell line with efficient cross-priming ability established from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene

The uptake of an antigen and its presentation to specific T cells by dendritic cells (DCs) is a primary event in initiation of humoral and cellular immune responses as well as the induction of cytotoxic T cells (CTLs). DCs are induced by culturing bone marrow cells in the presence of GM-CSF. However, the resulting DCs are short-lived and the culture usually contains CD11c-negative non-DC cells, which adversely affects reproducibility and makes interpretation of the experimental results difficult. Therefore, it would be useful if DCs could be readily immortalized with their functions being retained. In this study we established a novel, immortalized murine DC line with antigen-presenting capacity in vitro as well as an augmenting effect on humoral and cellular immune responses in vivo, utilizing bone marrow cells from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene. In the presence of GM-CSF, the resulting DC line, termed SVDC, could be continuously subcultured for more than 12 months. When pulsed with OVA alone or OVA-IgG immune complexes via Fcgamma receptors, SVDC augmented OVA-specific T cell proliferation efficiently in vitro, and elicited OVA-specific IgG production in vivo on the adoptive transfer of pulsed SVDC into naive mice. Interestingly, SVDC exhibited significantly high cross-priming ability compared to DCs in a short-term culture, thus leading to their extremely high effectiveness in inducing anti-tumor immunity in vivo. Thus, SVDC is useful for the detailed characterization of antigen presentation, and for research on the various therapeutic benefits of DC vaccination to elicit specific immune responses in immunodeficiencies, infectious diseases and cancer.     

Ex vivo enhancement of antigen-presenting firnction of dendritic cells and its application for DC-based immunotherapy

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.     

Microglia and Microglia-Like Cell Differentiated from DC Inhibit CD4 T Cell Proliferation

The central nervous system (CNS) is generally regarded as a site of immune privilege, whether the antigen presenting cells (APCs) are involved in the immune homeostasis of the CNS is largely unknown. Microglia and DCs are major APCs in physiological and pathological conditions, respectively. In this work, primary microglia and microglia-like cells obtained by co-culturing mature dendritic cells with CNS endothelial cells in vitro were functional evaluated. We found that microglia not only cannot prime CD4 T cells but also inhibit mature DCs (maDCs) initiated CD4 T cells proliferation. More importantly, endothelia from the CNS can differentiate maDCs into microglia-like cells (MLCs), which possess similar phenotype and immune inhibitory function as microglia. Soluble factors including NO lie behind the suppression of CD4 T cell proliferation induced by both microglia and MLCs. All the data indicate that under physiological conditions, microglia play important roles in maintaining immune homeostasis of the CNS, whereas in a pathological situation, the infiltrated DCs can be educated by the local microenvironment and differentiate into MLCs with inhibitory function.     

Noncanonical autophagy in dendritic cells triggers CNS autoimmunity

Reactivation and expansion of myelin-reactive CD4⁺ T cells within the central nervous system (CNS) are considered to play a key role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). We demonstrated that accumulation of myelin-specific CD4⁺ T cells within the CNS and subsequent clinical disease development require autophagy related (ATG) protein-dependent phagocytosis in dendritic cells (DCs). Genetic ablation of this pathway impairs presentation of myelin-associated antigen following phagocytosis of injured, phosphatidylserine-exposing oligodendroglial cells. Thus, DCs use ATG-dependent phagocytosis for enhanced presentation of myelin antigen, thereby linking oligodendrocyte injury with antigen processing and T cell-pathogenicity during autoimmune CNS inflammation.     

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.