Interaction of dendritic cells with mycobacteria: where the action starts

Dendritic cells (DC) are the major antigen-presenting cells in the induction of cellular responses to intracellular pathogens, such as mycobacteria. Recent studies have shown that they also play a critical role in the regulation of immune responses. The interaction of DC with microbial antigens may be the controlling factor in the development of a Th1-orientated protective immunity. Analysis of the innate response of DC to mycobacteria and the involvement of the DC receptors in antigen recognition have highlighted the pivotal role of these cells in T-cell activation. Mycobacteria-infected DC have an enhanced capacity to release pro-inflammatory cytokines and chemokines and are potent inducers of interferon-gamma-producing cells in vivo. Therefore, DC manipulation for maximal antigen presentation and Th1 cytokine production may form the basis of a new generation of vaccines, with improved efficacy against mycobacterial infections.     

Toll样受体与树突状细胞介导的天然免疫和获得性免疫

树突状细胞(dendritic cells,DCs)作为迄今所发现的抗原提呈功能最强的一类抗原提呈细胞,是联结天然免疫和获得性免疫的桥梁。Toll样受体(Toll-like receptors,TLRs)是一类进化保守的胚系编码的模式识别受体,在DCs的抗原识别、递呈及激活T细胞等方面具有重要作用,是机体受外来抗原入侵后作出适当免疫反应的调控点。现就TLRs在不同DCs亚群中的分布、与DCs介导的天然免疫和获得性免疫的关系及DCs功能可塑性的分子基础作一综述。     

Killer dendritic cells and their potential for cancer immunotherapy

Known for years as the principal messengers of the immune system, dendritic cells (DC) represent a heterogeneous population of antigen presenting cells critically located at the nexus between innate and adaptive immunity. DC play a central role in the initiation of tumor-specific immune responses as they are endowed with the unique ability to take up, process and present tumor antigens to naïve CD4⁺ or CD8⁺ effector T lymphocytes. By virtue of the cytokines they produce, DC also regulate the type, strength and duration of T cell immune responses. In addition, they can participate in anti-tumoral NK and NKT cell activation and in the orchestration of humoral immunity. More recent studies have documented that besides their primary role in the induction and regulation of adaptive anti-tumoral immune responses, DC are also endowed with the capacity to directly kill cancer cells. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. First, the direct killing of malignant cells by DC may foster the release and thereby the immediate availability of specific tumor antigens for presentation to cytotoxic or helper T lymphocytes. Second, DC may participate in the effector phase of the immune response, potentially augmenting the diversity of the killing mechanisms leading to tumor elimination. This review focuses on this non-conventional cytotoxic function of DC as it relates to the promotion of cancer immunity and discusses the potential application of killer DC (KDC) in tumor immunotherapy.     

Role of dendritic cells in the immune response to T-independent antigens of type 2

Dendritic cells (DC) belong to the most effective antigen-presenting cells. Their role in the presentation of thymus-dependent antigens and initiation of primary immune response is well known. At the same time, participation of DC in the immune response to T-independent antigens of type 2 (TI-2 antigens) is poorly explored. In this work, the ability of DC to initiate the immune response to a TI-2 antigen α(1→3) dextran (Dex) is investigated. Mouse bone-marrow-derived DC were generated by culturing the precursors with GM-CSF and then DC were pulsed by TI-2 antigens. The pulse induced DC activation, as was verified by an increase in the number of CD80 and CD86 positive cells. Uptake of FITC-labeled Dex was examined by flow cytometry. At a concentration of FITC-Dex of 100 μg/10⁶ cells, the number of DC binding the antigen (Ag) reached “plateau”. DC charged by TI-2 antigens were mixed with normal mouse splenocytes and cultivated in RPMI-1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells were determined by ELISPOT method. The mixtures of splenocytes and naïve DC not charged by the Ag were used as control. It was shown that the increase in the numbers of AFC and IFC under the influence of naïve DC did not exceed 20%. On the contrary, the addition of DC pulsed by the Ag increased specific immune response more than twofold. The data obtained point to the direct interactions of DC with TI-2 antigens. Pulsed DC present TI-2 antigens to mouse splenocytes and induce specific and polyclonal B-cell activation, i.e., possess immunostimulating activity.     

Memory and effector T cells modulate subsequently primed immune responses to unrelated antigens

Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated antigen, depending on whether the second antigen was administered in the same or opposite type of adjuvant. The regulatory effects of the first wave of T cell immunity on the subsequent T cell responses to an unrelated antigen attenuated over time. Notably, following challenge with the second antigen, there was a mutual cross-regulation between the first and second wave of humoral responses to unrelated antigens. Thus, immunization with one antigen not only primes immune responses to that antigen, but also influences subsequently primed immune responses to unrelated antigens.     

Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4(+) T cells responding to HCV core in patients with chronic HCV infection. However, CD4(+) response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4(+) response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.     

Involvement of PPAR-γ and p53 in DHA-induced apoptosis in Reh cells

Dendritic cells (DCs) are professional antigen-presenting cells and have come to be appreciated as critical controllers of the immune response, especially T cell responses. Apart from presenting antigens to T cells, DCs carry out many other functions in regulating immunity. DC-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin (DC-SIGN) is a novel receptor that plays an important role in DC migration and adhesion, the inflammatory response, T cell activation, initiating the immune response, and immune escape of pathogens and tumors. DC-SIGN mediates DC binding to ICAM-3 on the T cell surface and ICAM-2 on the endothelial cell (EC) surface, and takes part in the initial interaction between DC and T cells or vascular ECs. The procedure of systematic evolution of ligands by exponential enrichment (SELEX) is a method in which single-stranded oligonucleotides are selected from a wide variety of sequences, based on their interaction with a target molecule. In this study, we selected DNA aptamers against DC-SIGN protein by SELEX, and measured their binding affinity for DC-SIGN. Finally, an appropriate aptamer with high affinity for DC-SIGN was obtained, and it blocked DC adhesion to ECs as effectively as anti-DC-SIGN monoclonal antibody.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.     

Dendritic cell dysfunction in cancer: a mechanism for immunosuppression

Several reports have demonstrated that tumours are not intrinsically resistant to the immune response. However, neoplasias commonly fail to initiate and maintain adequate immunity. A number of factors have been implicated in causing the failure, including aberrant antigen processing by tumour cells, anergy or deletion of T cells, and recruitment of inhibitory/regulatory cell types. It has been suggested that dysfunction of dendritic cells (DC) induced by the tumour is one of the critical mechanisms to escape immune surveillance. As a minor subset of leucocytes, DC are the key APC for initiating immune responses. DC are poised at the boundaries of the periphery and the inner tissues, sampling antigens of diverse origin. Following their encounter with antigen or danger signals, DC migrate to lymph nodes, where they activate effector cells essential for tumour clearance. Although the DC system is highly heterogeneous, the differentiation and function of DC populations is largely regulated by exogenous factors. Malignancies appear to exploit this by producing a plethora of immunosuppressive factors capable of affecting DC, thus exerting systemic effects on immune function. This review examines recent findings on the effects of tumour-derived factors inducing DC dysfunction and in particular examines the findings on alteration of DC differentiation, maturation and longevity as a potent mechanism for immune suppression in cancer.     

Emerging roles for scavenger receptor SREC-I in immunity

SREC-I is a class F scavenger receptor with key role in the immune response, particularly in antigen presenting cell (APC) such as macrophages and dendritic cells (DC). This receptor is able to mediate engulfment of dead cells as well as endocytosis of heat shock protein (HSP)–antigen complexes. SREC-I could thus potentially mediate the tolerizing influence of apoptotic cells or the immunostimulatory effects of HSP–peptide complexes, depending on context. This receptor was able to mediate presentation of external antigens, bound to HSPs through both the class II pathway as well as cross presentation via MHC class I complexes. In addition to its recently established role in adaptive immunity, emerging studies are indicating a broad role in innate immunity and regulation of cell signaling through Toll Like Receptors (TLR). SREC-I may thus play a key role in APC function by coordinating immune responses to internal and external antigens in APC.     

Mycobacterial heat shock proteins as vaccines - a model of facilitated antigen presentation

Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their upregulated expression in response to host exposure to raised temperatures. Further study has revealed that they have numerous functions in the cell, primarily as chaperones mediating both the correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The energy requirement for this chaperone activity is provided by the ATPase activity found in most families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs). Much previous work has implicated hsps in the immune response to pathogens and recent studies have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs), mediates the integration of the innate and acquired immune responses. This central role for hspCs in immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking of antigens chaperoned in hspCs for antigen presentation by the mature DCs.     

Characteristics of human dendritic cells generated in a microgravity analog culture system

Summary Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected, to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR⁺ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytoseAspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of Dc expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.     

Targeting dendritic cells for priming cellular immune responses

The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immune responses against all classes of pathogens and also against tumors is well established. Their unique potential both to maintain self-tolerance and to initiate protective immune responses against foreign and/or dangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC lining antigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compounds including proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carrying such structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with high endocytic capacity, which provides potential targets for delivering designed molecules. The best route for targeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, which reside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolic products, which induce mobilization of cells to draining lymph nodes where they act as highly potent professional antigen presenting cells. This is brought about by the ability to present their accumulated intracellular content for both CD4+ helper (Th) and CD8+ cytotoxic/cytolytic T lymphocytes (Tc/CTL). Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cell surface in the context of major histocompatibility complex encoded class I and II molecules for presentation to Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigen presentation but also on other features of DC. Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting and T cell-activating functions of DC at their immature and activated differentiation states. These biological activities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interaction of DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that monocyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptive immune responses against pathogens and also against tumors.     

Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.     

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.     

The ‘Co-Delivery’ Approach to Liposomal Vaccines: Application to the Development of influenza-A and hepatitis-B Vaccine Candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination ‘co-delivery’ and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell – a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

The 'co-delivery' approach to liposomal vaccines: application to the development of influenza-A and hepatitis-B vaccine candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination 'co-delivery' and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell--a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

Melanoma cancer vaccines and anti-tumor T cell responses

Melanoma is a disease which has been shown to be responsive to immune intervention. This has been suggested by reports of spontaneous responses of metastatic disease with strong immune infiltrates, and supported by recent data correlating clinical response after IFNalpha treatment with development of generalized autoimmunity. Since the identification of melanoma-associated tumor antigens, many groups have performed clinical trials to take advantage of this discovery with melanoma-specific cancer vaccines. These trials, in which multiple antigen delivery strategies have been tested in hundreds of patients, have demonstrated that these vaccines are safe, immunogenic, and yield a low frequency of objective clinical responses. The ability to perform careful immunological monitoring has allowed important insights into the nature of the anti-tumor immunity generated by these vaccinations. While many trials have found that the absolute frequency of T cells specific for a vaccine-encoded antigen are a marker of immunization, it does not correlate with objective clinical response. Induction of broad immunity to multiple tumor antigens, taking advantage of cross-reactive T cells and activation of persistent T cells may be more important. Harnessing additional modes of amplifying immune responses (lymphodepletion, cytokine support, inhibition of negative immune self-regulation) are now being tested and should improve clinical responses from 5% to 10% complete response seen currently.