Dendritic cells as promoters of transplant tolerance

Dendritic cells (DCs) play a crucial role during the initiation of immune responses against non-self antigens. Following organ transplantation, activated donor- and recipient-derived DCs participate actively in graft rejection by sensitising recipient T cells via the direct or indirect pathways of allorecognition, respectively. There is increasing evidence that immature/semi-mature DCs induce antigen-specific unresponsiveness or tolerance to self antigens, both in central lymphoid tissue and in the periphery, through a variety of mechanisms (deletion, anergy and regulation). In the past few years, DC-based therapy of experimental allograft rejection has focused on ex vivo biological, pharmacological and genetic engineering of DCs to mimic/enhance their natural tolerogenicity. Successful outcomes in rodent models have built the case that DC-based therapy may provide a novel approach to transplant tolerance. Ongoing research into the role that DCs play in the induction of tolerance should allow for its clinical application in the near future.     

Potential approaches for more successful dendritic cell-based immunotherapy

Introduction: Dendritic cells (DCs) are the most important antigen-presenting cell population for activating antitumor T-cell responses; therefore, they offer a unique opportunity for specific targeting of tumors.

Areas covered: We will discuss the critical factors for the enhancement of DC vaccine efficacy: different DC subsets, types of in vitro DC manufacturing protocol, types of tumor antigen to be loaded and finally different adjuvants for activating them. We will cover potential combinatorial strategies with immunomodulatory therapies: depleting T-regulatory (Treg) cells, blocking VEGF and blocking inhibitory signals. Furthermore, recommendations to incorporate these criteria into DC-based tumor immunotherapy will be suggested.

Expert opinion: Monocyte-derived DCs are the most widely used DC subset in the clinic, whereas Langerhans cells and plasmacytoid DCs are two emerging DC subsets that are highly effective in eliciting cytotoxic T lymphocyte responses. Depending on the type of tumor antigens selected for loading DCs, it is important to optimize a protocol that will generate highly potent DCs. The future aim of DC-based immunotherapy is to combine it with one or more immunomodulatory therapies, for example, Treg cell depletion, VEGF blockage and T-cell checkpoint blockage, to elicit the most optimal antitumor immunity to induce long-term remission or even cure cancer patients.     

Genetically modified dendritic cells in cancer immunotherapy: a better tomorrow?

Importance of the field: Dendritic cells (DC) are powerful antigen-presenting cells that induce and maintain primary cytotoxic T lymphocyte (CTL) responses directed against tumor antigens. Consequently, there has been much interest in their application as antitumor vaccines.

Areas covered in this review: A large number of DC-based vaccine trials targeting a variety of cancers have been conducted; however, the rate of reported clinically significant responses remains low. Modification of DC to express tumor antigens or immunostimulatory molecules through the transfer of genes or mRNA transfection offers a logical alternative with potential advantages over peptide- or protein antigen-loaded DC. In this article, we review the current results and future prospects for genetically modified DC vaccines for the treatment of cancer.

What the reader will gain: Genetically-modified dendritic cell-based vaccines represent a powerful tool for cancer therapy. Numerous preclinical and clinical studies have demonstrated the potential of dendritic cell vaccines alone or in combination with other therapeutic modalities.

Take home message: Genetically modified DC-based anti-cancer vaccination holds promise, perhaps being best employed in the adjuvant setting with minimal residual disease after primary therapy, or in combination with other antitumor or immune-enhancing therapies.     

Current issues in dendritic cell cancer immunotherapy

Dendritic cells (DCs) initiate and direct the immune response. Their inability to detect danger signals from transformed cells and to generate an effective immunological response may allow cells with a malignant phenotype to evolve into cancers. This defect can be corrected for many cancer types and the immune response boosted to eliminate malignant cells by means of DC-based vaccines/therapies. Rapid advances in our understanding of basic DC physiology and improved methods for DC isolation have made clinical application of DC therapy practical, and encouraging phase I/II results are emerging.     

Engineering Dendritic Cells to Enhance Cancer Immunotherapy

Cancer immunotherapy aims to establish immune-mediated control of tumor growth by priming T-cell responses to target tumor-associated antigens. Three signals are required for T-cell activation: (i) presentation of cognate antigen in self MHC molecules; (ii) costimulation by membrane-bound receptor-ligand pairs; and (iii) soluble factors to direct polarization of the ensuing immune response. The ability of dendritic cells (DCs) to provide all three signals required for T-cell activation makes them an ideal cancer vaccine platform. Several strategies have been developed to enhance and control antigen presentation, costimulation, and cytokine production. In this review, we discuss progress toward developing DC-based cancer vaccines by genetic modification using RNA, DNA, and recombinant viruses. Furthermore, the ability of DC-based vaccines to activate natural killer (NK) and B-cells, and the impact of gene modification strategies on these populations is described. Clinical trials using gene-modified DCs have shown modest results, therefore, further considerations for DC manipulation to enhance their clinical efficacy are also discussed.     

Bone Marrow–generated Dendritic Cells Pulsed with Tumor Extracts or Tumor RNA Induce Antitumor Immunity against Central Nervous System Tumors

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene–modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow–generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.     

Dendritic cell therapy with interferon-alpha synergistically suppresses outgrowth of established tumors in a murine colorectal cancer model

Both dendritic cell (DC)-based immunotherapy and interferon (IFN)-alpha therapy have been proved to have potent long-lasting antitumor effects. In anticipation of synergistic antitumor effects, we performed combination therapy with DCs and IFN-alpha gene-transduced murine colorectal cancer MC38 cells (MC38-IFN-alpha). DCs incubated with MC38-IFN-alpha, but not neomycin-resistance gene-transduced MC38 cells (MC38-Neo), effectively enhanced proliferation of allogeneic splenocytes in vitro. In 12 of 17 mice, DCs in combination with MC38-IFN-alpha prevented the development of a parental tumor, while DCs and MC38-Neo did in only three of 17 mice (P=0.008). In a therapeutic model of an established parental tumor, inoculation of DCs and MC38-IFN-alpha suppressed the growth of the established parental tumors significantly compared with the administration of DCs with MC38-Neo or naive splenocytes with MC38-IFN-alpha (P=0.016 and 0.024, respectively). Analyses of immunohistochemistry and tumor-infiltrating mononuclear cells showed that CD8(+), CD11c(+), and NK1.1(+) cells markedly infiltrated the established tumors of mice treated with DCs and MC38-IFN-alpha. From the results of observation of parental tumor outgrowth in immune cell-depleted mice, CD8(+) cells, and asialo-GM-1(+) cells were thought to contribute to the antitumor effects induced by the combination therapy. Furthermore, MC38-specific cytolysis was detected when splenocytes of mice inoculated with DCs and MC38-IFN-alpha cells were stimulated with MC38-IFN-alpha cells in vitro. Since DC-based immunotherapy in combination with IFN-alpha-expressing tumor cells induces potent antitumor cellular immune responses, it should be considered for clinical application.     

树突状细胞疫苗治疗多发性骨髓瘤的研究进展

近年来多发性骨髓瘤的发病率不断上升,但目前对于多发性骨髓瘤仍然没有很好的治疗方法。树突状细胞是一类抗原呈递细胞,具有启动和调控免疫反应的能力。利用肿瘤抗原冲击树突状细胞的免疫治疗策略已被证明是安全的并且对许多肿瘤具有一定的治疗效果。本文综述了多发性骨髓瘤相关的各种肿瘤抗原类型及树突状细胞免疫治疗多发性骨髓瘤的临床实验,并且分析了树突状细胞免疫治疗在未来实验研究方面的前景。     

Genetically modified dendritic cells in cancer therapy: implications for transfusion medicine

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) identified in various tissues, including the skin (Langerhans cells), lymph nodes (interdigitating and follicular DCs), spleen, and thymus. Properties of DCs include the ability to (1) capture, process, and present foreign antigens; (2) migrate to lymphoid-rich tissue; and (3) stimulate innate and adaptive antigen-specific immune responses. Until recently, the ability to study DCs has been limited by their absence in most culture systems. It is now known that specific cytokines can be used to expand DCs to numbers sufficient for their in vitro evaluation and for their use in human immunotherapy trials. Human DCs can be derived from hematopoietic progenitors (CD34+-derived DCs) or from adherent peripheral blood monocytes (monocyte-derived DCs). Cultured DCs can be recognized by a typical veiled morphologic appearance and expression of surface markers that include major histocompatibility complex class II, CD86/B7.2, CD80/B7.1, CD83, and CD1a. DCs are susceptible to a variety of gene transfer protocols, which can be used to enhance biological function in vivo. Transduction of DCs with genes for defined tumor antigens results in sustained protein expression and presentation of multiple tumor peptides to host T cells. Alternatively, DCs may be transduced with genes for chemokines or immunostimulatory cytokines. Although the combination of ex vivo DC expansion and gene transfer is relatively new, preliminary studies suggest that injection of genetically modified autologous DCs may be capable of generating anti-tumor immune responses in patients with cancer. Preclinical animal studies showing potent antigen-specific tumor immunity after DC-based vaccination support this hypothesis and provide rationale to further evaluate this approach in patients. Preliminary human studies are now required to evaluate optimal DC dose, schedule of vaccination, route of delivery, and maturational state of cultured cells. Initiation of these phase I/II cell therapy-based studies will occur in collaboration with hospital-based transfusion facilities. Issues relating to cell harvesting, storage, culture methodology, and administration require the collaborative efforts of basic scientists, immunologists, clinical investigators, and transfusion medicine staff to ensure strict quality control of injected cellular products. This review is intended to provide a brief overview of clinical DC-based gene transfer.     

Gene-modified dendritic cells for use in tumor vaccines

Dendritic cells (DCs) are potent antigen-presenting cells capable of priming activation of naive T cells. Because of their immunostimulatory capacity, immunization with DCs presenting tumor antigens has been proposed as a treatment regimen for cancer. The results from translational research studies and early clinical trials point to the need for improvement of DC-based tumor vaccines before they become a more broadly applicable treatment modality. In this regard, studies suggest that genetic modification of DCs to express tumor antigens and/or immunomodulatory proteins may improve their capacity to promote an antitumor response. Because the DC phenotype is relatively unstable, nonperturbing methods of gene transfer must be employed that do not compromise viability or immunostimulatory capacity. DCs expressing transgenes encoding tumor antigens have been shown to be more potent primers of antitumor immunity both in vitro and in animal models of disease; in some measures of immune priming, gene-modified DCs exceeded their soluble antigen-pulsed counterparts. Cytokine gene modification of DCs has improved their capacity to prime tumor antigen-specific T cell responses and promote antitumor immunity in vivo. Here, we review the current status of gene-modified DCs in both human and murine studies. Although successful results have been obtained to date in experimental systems, we discuss potential problems that have already arisen and may yet be encountered before gene-modified DCs are more widely applicable for use in human clinical trials.     

Dendritic cells as promoters of transplant tolerance

Dendritic cells (DCs) play a crucial role during the initiation of immune responses against non-self antigens. Following organ transplantation, activated donor- and recipient-derived DCs participate actively in graft rejection by sensitising recipient T cells via the direct or indirect pathways of allorecognition, respectively. There is increasing evidence that immature/semi-mature DCs induce antigen-specific unresponsiveness or tolerance to self antigens, both in central lymphoid tissue and in the periphery, through a variety of mechanisms (deletion, anergy and regulation). In the past few years, DC-based therapy of experimental allograft rejection has focused on ex vivo biological, pharmacological and genetic engineering of DCs to mimic/enhance their natural tolerogenicity. Successful outcomes in rodent models have built the case that DC-based therapy may provide a novel approach to transplant tolerance. Ongoing research into the role that DCs play in the induction of tolerance should allow for its clinical application in the near future.     

RNA as performance-enhancers for dendritic cells

Importance of the field: Although studies have demonstrated that antigen-loaded dendritic cells (DC) elicit antigen-specific immune responses, the clinical benefit from DC-based cancer immunotherapy remains low. RNA, in the form of mRNA, has not only been used as a source of antigen but more recently as a way to stimulate DC to produce immunostimulatory molecules. As siRNA it has allowed researchers to modify DC to produce a favorable cytokine profile or to present antigen that may generate the desired immune response.

Areas covered in this review: When loading DC with RNA that encodes immunostimulatory protein, rather than a source of antigen, optimal translation and efficient transfection into DC are critical. Studies addressing these issues and the functional consequences of modulating DC function are reviewed.

What the reader will gain: RNA can be used to load DC with antigen and to encode proteins that will enhance the immune response. Co-transfection with multiple mRNAs or mRNA plus siRNA can significantly improve vaccine efficacy.

Take home message: One conclusion from Phase I clinical trials with DC loaded with tumor antigen is that tumor-specific induction of immune responses is not sufficient to destroy pre-established tumors. The advantage of transfection with mRNA is the ability to load DC with antigen-encoding mRNA and immunostimulatory protein-encoding mRNA to achieve the desired clinical response.     

The use of molecular imaging of gene expression by radiotracers in gene therapy

Introduction: Progress with gene-based therapies has been hampered by difficulties in monitoring the biodistribution and kinetics of vector-mediated gene expression. Recent developments in non-invasive imaging have allowed researchers and clinicians to assess the location, magnitude and persistence of gene expression in animals and humans. Such advances should eventually lead to improvement in the efficacy and safety of current clinical protocols for future treatments.

Areas covered: The molecular imaging techniques for monitoring gene therapy in the living subject, with a specific highlight on the key reporter gene approaches that have been developed and validated in preclinical models using the latest imaging modalities. The applications of molecular imaging to biotherapy, with a particular emphasis on monitoring of gene and vector biodistribution and on image-guided radiotherapy.

Expert opinion: Among the reporter gene/probe combinations that have been described so far, one stands out, in our view, as the most versatile and easy to implement: the Na/I symporter. This strategy, exploiting more than 50 years of experience in the treatment of differentiated thyroid carcinomas, has been validated in different types of experimental cancers and with different types of oncolytic viruses and is likely to become a key tool in the implementation of human gene therapy.     

不同分子分型乳腺癌超声弹性特征

目的 应用声触诊组织量化成像（VTIQ）技术观察不同分子分型乳腺癌超声弹性特征。方法 对101例术前乳腺癌患者行常规超声及VTIQ检查，观察肿块边缘带、中央区及周围组织弹性特征，测量其剪切波速度最大值（SWV_max），分析不同分子分型乳腺癌的超声弹性特征。结果 Luminal型弹性特征多为边缘优势型，人类表皮生长因子受体2（HER-2）过表达型主为中央优势型，三阴性多为均匀型（P<0.05）。各分子分型肿瘤边缘带SWV_max Luminal A > Luminal B > HER-2过表达 > 三阴性（P<0.05），中央区SWV_max差异无统计学意义（P>0.05）。肿瘤周围组织SWV_max差异无统计学意义（P>0.05）。结论 乳腺癌弹性特征及SWV_max与其分子分型相关，肿瘤边缘带弹性特征尤为重要，可在一定程度上反映不同分子分型乳腺癌的生物学特点。术前VTIQ有助于制定治疗方案及评估预后。     

Adenoviral gene therapy of gastrointestinal tumour metastases in the liver

Summary

Gastrointestinal cancers are the second most common cause of cancer death. Once metastasised, 5 year survival is < 5% in gastrointestinal cancer. Because the liver is the preferred site for distant organ metastasis of colon cancer, treatment of hepatic metastases remains a challenge for experimental cancer therapy approaches. Gene therapy provides tools to combat cancer on a molecular level. In contrast to conventional chemotherapy, vectors are used to insert DNA into tumour cells, neighbouring parenchymal cells, or cells involved in the cellular immune defense. The shuttle vectors are of nonviral or viral origin. Adenoviral vectors have been developed for high efficiency in vivo gene transfer and expression. The incorporation of foreign DNA can result in direct tumour cell killing, using suicide genes. Cytokine genes, or genes encoding for tumour-specific antigens recognised by the cellular host immune system, can result in anti-tumoral immune stimulation. Experimental suicide-gene expression in hepatic metastasis of gastrointestinal tumours, utilising thymidine kinase and cytosine deaminase, results in significant tumour necrosis and regression. Intratumoral interleukin-2 and interleukin-12 gene expression can induce a systemic cellular antitumoral immune response, with long-term survival demonstrating the potential of this new therapeutic approach in cancer therapy.     

Ganoderma lucidum mycelium and spore extracts as natural adjuvants for immunotherapy

OBJECTIVES: Ganoderma lucidum (GL) is one of the most commonly used Chinese herbs in the oriental community, with more than 30% of pediatric cancer patients taking GL. The immunomodulating and anticancer effects exerted by GL extracts have been demonstrated by in vitro and in vivo studies. There was, however, no comparison between the immunomodulating effects of GL mycelium extract (GL-M) and spore extracts on human immune cells. Dendritic cells (DCs) are professional antigen-presenting cells and their role in DC-based tumor vaccine has been well defined. The possibility of GL as natural adjuvant for human DCs remains unknown. DESIGN: This study explored the differential effect of GL-M and GL spore extract (GL-S) on proliferation and Th1/Th2 cytokine mRNA expression of human peripheral blood mononuclear cells (PBMCs) and monocytes. Their effects on the phenotypic and functional maturation of human monocyte-derived DCs were also investigated. RESULTS: GL-M induced the proliferation of PBMCs and monocytes, whereas GL-S showed a mild suppressive effect. Both extracts could stimulate Th1 and Th2 cytokine mRNA expression, but GL-M was a relatively stronger Th1 stimulator. Different from GL-S, GL-M enhanced maturation of DCs in terms of upregulation of CD40, CD80, and CD86, and also reduced fluorescein isothiocyanate-dextran endocytosis. Interestingly, GLM- treated DCs only modestly enhanced lymphocyte proliferation in allogenic mixed lymphocyte culture with mild enhancement in Th development. CONCLUSION: These findings provide evidences that GL-M has immunomodulating effects on human immune cells and therefore can be used as a natural adjuvant for cancer immunotherapy with DCs.     

Progress of dendritic cell-based cancer vaccines for patients with hematological malignancies

ABSTRACT

Introduction: Dendritic cells (DCs) are the most professional antigen-presenting cells eliciting cellular and humoral immune responses against cancer cells by expressing these antigens on MHC class I/II complexes to T cells. Therefore, they have been employed in many clinical trials as cancer vaccines for patients with cancer. This review focuses on the use of DCs in leukemia patients expressing leukemia-associated antigens (LAAs).

Areas covered: The contribution of both stimulating vs. tolerogenic DCs as well as of other factors to the milieu of anti-leukemia immune responses are discussed. Several DC vaccination strategies like leukemia lysate, proteins and peptides have been developed. Next generation DC vaccines comprise transduction of DCs with retroviral vectors encoding for LAAs, cytokines and costimulatory molecules as well as transfection of DCs with naked RNA encoding for LAAs. Published as well as ongoing clinical trials are reported and critically reviewed.

Expert opinion: Future results will demonstrate whether next-generation DCs are really superior to conventional pulsing with peptide, protein or tumor lysate. However, currently available methods based on nucleic acid transfection/transduction are tempting in terms of material production costs and time for clinical application according to good manufacturing practice (GMP).     

Persistent, antigen-specific, therapeutic antitumor immunity by dendritic cells genetically modified with an adenoviral vector to express a model tumor antigen

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of cellular immune responses. Using a BALB/c syngeneic colon carcinoma cell line expressing a model tumor antigen beta-galactosidase (betagal), we previously reported (Song et al, J Exp Med 1997; 186: 1247-1256) that immunization of mice with a single injection of DCs genetically modified with an adenovirus vector expressing betagal confers potent protection against a lethal intravenous tumor challenge, as well as suppression of pre-established lung tumors, resulting in a significant survival advantage. In the present study, we have addressed the question: how long does the memory of tumor antigen- specific immunity persists after DC priming in vivo using this genetically modified DC-based cancer vaccination strategy? To accomplish this, two groups of mice were evaluated: (1) mice surviving >400 days following protection from an initial intravenous tumor challenge after immunization with DC genetically modified to express betagal; and (2) mice surviving >300 days that had previously demonstrated regression of pre-established lung tumors after treatment with DC immunization. By analyzing the antigen-specific cytotoxic T lymphocyte response and challenging these long-term survival mice with a second subcutaneous tumor administration, the data demonstrate that a single administration of DC genetically modified to express a model antigen induces long-lasting, antigen-specific antitumor immunity in both naive and tumor-bearing hosts, observations that have important implications in the development of genetically modified DC-based antitumor vaccination strategies. Gene Therapy (2000) 7, 2080-2086.     

Genetically modified dendritic cells – a new,promising cancer treatment strategy?

Dendritic cells (DCs), the most potent antigen-presenting cells (APCs), were discovered almost 30 years ago. Due to the priming of antigen-specific immune responses mediated by CD4+ and CD8+ lymphocytes, DCs are crucial for the induction of adaptive immunity against cancer. Therefore, vaccination of cancer patients with DCs presenting tumour-associated antigens (TAAs) have been believed to be a promising anticancer strategy. Multiple clinical trials have been carried out in order to evaluate the safety and efficacy of cancer vaccines based on antigen-pulsed DCs. However, pulsing of DCs with particular peptides has several disadvantages: i) short-time duration of antigen–major histocompatability complex (MHC) complexes, ii) a requirement for matching defined peptides with MHC complexes and iii) exclusive presentation of single antigen epitopes. Application of gene transfer technologies in the field of DC-based vaccines made possible the development of novel, anticancer immunisation strategies. In several animal models, DCs modified with genes encoding TAA or immunostimulatory proteins have been shown to be effective in the induction of antitumour immune responses. Based on these encouraging results, a first clinical trial of prostate cancer patients vaccinated with gene modified DCs has recently been initiated. In this article, methods used for genetic modification of DCs and anticancer vaccination strategies based on genetically modified DCs are reviewed.