Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen^1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response³.DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment⁴. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens⁵. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma^6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.     

ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells

Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against cancer. It follows, therefore, that the capacity of emerging immunotherapeutic agents to orchestrate tumour eradication may depend on their ability to induce antigen cross-presentation. ImmTACs [immune-mobilising monoclonal TCRs (T cell receptors) against cancer] are a new class of soluble bi-specific anti-cancer agents that combine pico-molar affinity TCR-based antigen recognition with T cell activation via a CD3-specific antibody fragment. ImmTACs specifically recognise human leucocyte antigen (HLA)-restricted tumour-associated antigens, presented by cancer cells, leading to T cell redirection and a potent anti-tumour response. Using an ImmTAC specific for a HLA-A*02-restricted peptide derived from the melanoma antigen gp100 (termed IMCgp100), we here observe that ImmTAC-driven melanoma-cell death leads to cross-presentation of melanoma antigens by DCs. These, in turn, can activate both melanoma-specific T cells and polyclonal T cells redirected by IMCgp100. Moreover, activation of melanoma-specific T cells by cross-presenting DCs is enhanced in the presence of IMCgp100; a feature that serves to increase the prospect of breaking tolerance in the tumour microenvironment. The mechanism of DC cross-presentation occurs via ‘cross-dressing’ which involves the rapid and direct capture by DCs of membrane fragments from dying tumour cells. DC cross-presentation of gp100-peptide-HLA complexes was visualised and quantified using a fluorescently labelled soluble TCR. These data demonstrate how ImmTACs engage with the innate and adaptive components of the immune system enhancing the prospect of mediating an effective and durable anti-tumour response in patients.     

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.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Effects of ceftriaxone‐induced intestinal dysbacteriosis on dendritic cells of small intestine in mice

Intestinal microflora plays a pivotal role in the development of the innate immune system and is essential in shaping adaptive immunity. Dysbacteriosis of intestinal microflora induces altered immune responses and results in disease susceptibility. Dendritic cells (DCs), the professional antigen‐presenting cells, have gained increasing attention because they connect innate and adaptive immunity. They generate both immunity in response to stimulation by pathogenic bacteria and immune tolerance in the presence of commensal bacteria. However, few studies have examined the effects of intestinal dysbacteriosis on DCs. In this study, changes of DCs in the small intestine of mice under the condition of dysbacteriosis induced by ceftriaxone sodium were investigated. It was found that intragastric administration of ceftriaxone sodium caused severe dysteriosis in mice. Compared with controls, numbers of DCs in mice with dysbacteriosis increased significantly (P = 0.0001). However, the maturity and antigen‐presenting ability of DCs were greatly reduced. In addition, there was a significant difference in secretion of IL‐10 and IL‐12 between DCs from mice with dysbacteriosis and controls. To conclude, ceftriaxone‐induced intestinal dysbacteriosis strongly affected the numbers and functions of DCs. The present data suggest that intestinal microflora plays an important role in inducing and maintaining the functions of DCs and thus is essential for the connection between innate and adaptive immune responses.     

Tolerance and immunity in a mathematical model of T-cell mediated suppression

Regulatory CD4+CD25+ T cells play a major role in natural tolerance to body components and therefore are relevant to understand the self-non-self discrimination by the immune system. The most pressing theoretical question, regarding the fact that these regulatory cells perform their function through linked recognition of the APCs, is how this "non-specific" mechanism permits a proper balance between tolerance and immunity that is compatible with an effective self-non-self discrimination. To tackle this issue, we develop a numerical simulation, which extends a previous mathematical model of T-cell-mediated suppression to include the thymic generation and the peripheral dynamics of many T cell clones. This simulation can mimic the capacity of the immune system to establish natural tolerance to self-antigens and reliably mount immune responses to foreign antigens. Natural tolerance is based on ubiquitous and constitutive self-antigens, which select and sustain clones of specific regulatory cells. Immune responses to foreign antigens are only achieved if they displace the self-antigens from the APCs, leading to a loss of the regulatory cells, and/or if the foreign antigen introduction entails a sharp increase in the total number of APCs. Meaningful behavior is obtained even if differentiation of regulatory cells in the thymus is antigen non-specific, but requires that a minimum number of new T cells enter the periphery per unit of time, and that the repertoire is selected so that anti-self-affinities are within a proper interval. We conclude that positive selection is required to generate a sufficiently high frequency of self-antigen specific regulatory cells that reliably mediate natural tolerance. Negative selection is required to avoid the emergence at the periphery of very high affinity anti-self-regulatory cells that will make the tolerant state so robust that it could no be broken by the introduction of a foreign antigen. This result highlights the importance of repertoire selection in dominant tolerance proposing a novel role for the processes of positive and negative selection within this framework.     

树突状细胞在抗感染免疫研究中的最新进展

树突状细胞(Dendritic cell,DC)是体内功能最强的抗原提呈细胞,也是介导机体固有免疫应答和适应性免疫应答的桥梁,其作用也越来越受到科研工作者的关注,而树突状细胞体外培养技术的发展成熟,为设计和发展DC依赖性疫苗提供了科学依据,也为感染性和肿瘤性疾病的预防和治疗展示了很好的应用前景。因此,对树突状细胞抗感染免疫方面研究的最新进展做一综述。     

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4CD25Foxp3 regulatory T cells and down-regulates cardiac allograft rejection

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4⁺CD25^highFoxp3⁺ regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.     

Ret transgenic mouse model of spontaneous skin melanoma: focus on regulatory T cells

Ret transgenic mouse model of skin malignant melanoma is characterized by the overexpression of the human ret transgene in melanin‐containing cells. Transgenic mice spontaneously develop skin tumors with metastases in lymph nodes, lungs, liver, brain, and the bone marrow. Tumor lesions show typical melanoma morphology and express melanoma‐associated antigens. Although transgenic mice demonstrate an accumulation of melanoma antigen‐specific memory and effector T cells, their anti‐tumor effects could be blocked by highly immunosuppressive leukocytes enriched in the tumor microenvironment and in the periphery. Here, we discuss the role of one of the most potent immunosuppressive subset, regulatory T cells, in the melanoma progression in this model.     

MAGE-3原核表达载体的构建和表达

通过RT-PCR扩增957bp的MAGE-3全长编码序列,将该片段克隆至Pgex-4T-2原核表达载体,转化大肠杆菌BL-21,经IPTG诱导表达,并经12%SDSPAGE凝胶电泳,考马斯亮蓝染色及Western blot鉴定,证明了目的基因的有效表达,目的蛋白高达细菌总蛋白的32%。表达产物经Glutathione Sepharose 4B 纯化后,每100mL菌液最终可获得3mg的目的蛋白,蛋白纯度在90%以上。纯化的GST-MAGE-3蛋白在体外冲击树突状细胞,能诱导特异性CTL杀伤肿瘤细胞活性。     

The aged microenvironment contributes to the age-related functional defects of CD4 T cells in mice

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T‐cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T‐cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T‐cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T‐cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations.     

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN- for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN- for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.     

Role of dendritic cells in the induction of regulatory T cells

Dendritic cells (DCs) play a key role in initiating immune responses and maintaining immune tolerance. In addition to playing a role in thymic selection, DCs play an active role in tolerance under steady state conditions through several mechanisms which are dependent on IL-10, TGF-β, retinoic acid, indoleamine-2,3,-dioxygenase along with vitamin D. Several of these mechanisms are employed by DCs in induction of regulatory T cells which are comprised of Tr1 regulatory T cells, natural and inducible foxp3+ regulatory T cells, Th3 regulatory T cells and double negative regulatory T cells. It appears that certain DC subsets are highly specialized in inducing regulatory T cell differentiation and in some tissues the local microenvironment plays a role in driving DCs towards a tolerogenic response. In this review we discuss the recent advances in our understanding of the mechanisms underlying DC driven regulatory T cell induction.     

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD70 on dendritic cells

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus‐derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70‐dependent Th1 priming, while leaving the IL‐12‐dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN‐γ‐secreting CD4⁺ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27‐dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

记忆性T细胞的形成、维持和功能

免疫记忆是指机体在对某一抗原产生特异性识别及应答的同时,记住该抗原,当再次遭遇同一抗原时,能发生快速和强烈的免疫应答。树突状细胞吞噬病原微生物后,通过主要组织相容性复合体分子提呈抗原短肽段,与T细胞相互作用。在T细胞抗原受体信号和共刺激信号的协同作用下,抗原特异性T细胞增殖,收缩,小部分细胞作为记忆细胞长期存活。免疫记忆T细胞在表型特征和功能上都存在多样性。深入研究机体记忆性T细胞的特征,不仅能指导新型疫苗的设计,而且可望帮助治疗疾病。     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

The antibody response against MART‐1 differs in patients with melanoma‐associated leucoderma and vitiligo

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma‐associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T‐cell immune responses directed against the melanocyte‐differentiation‐antigens MART‐1 (Melan‐A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART‐1 antibodies were only present in patients with MAL. Melanocyte antigen‐specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART‐1 differ between these diseases, which can help to differentiate MAL from vitiligo.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.