Ovarian cancer accounts for only 3% of all cancers in women, but it causes more deaths than any other gynecologic cancer. Treatment with chemotherapy and cytoreductive surgery shows a good response to the therapy. However, in a large proportion of the patients the tumor grows back within a few years. Cancer stem cells, that are less responsive to these treatments, are blamed for this recurrence of disease. Immune therapy either cellular or humoral is a novel concept to treat cancer. It is based on the notice that immune cells invade the tumor. However, the tumor invest heavily to escape from immune elimination by recruiting several immune suppressive mechanisms. These processes are normally in place to limit excessive immune activation and prevent autoimmune phenomena. Here, we discuss current knowledge about the immune (suppressive) status in ovarian cancer. Moreover, we discuss the immunological targets of ovarian cancer stem cells.
Ovarian cancer; Cancer stem cell; Immune therapy; Immune suppression; Tumor microenvironment
Dendritic cell-based immunotherapy is a promising strategy against cancer that appears to be feasible, safe and to induce potent tumor-specific immune responses. The use of naturally circulating dendritic cells (DCs), rather than cultured monocyte-derived DCs, might constitute the next logical step to translate anticancer immune responses into long-lasting clinical benefits.
cancer immunotherapy; dendritic-cell targeting; dendritic-cell vaccination; myeloid dendritic cells; plasmacytoid dendritic cells
The aim of therapeutic dendritic cell (DC) vaccines in cancer immunotherapy is to activate cytotoxic T cells to recognize and attack the tumor. T cell activation requires the interaction of the T cell receptor with a cognate major-histocompatibility complex-peptide complex. Although initiated by antigen engagement, it is the complex balance between co-stimulatory and co-inhibitory signals on DCs that results in T cell activation or tolerance. Even when already activated, tumor-specific T cells can be neutralized by the expression of co-inhibitory molecules on tumor cells. These and other immunosuppressive cues in the tumor microenvironment are major factors currently hampering the application of DC vaccination. In this review, we discuss recent data regarding the essential and complex role of co-inhibitory molecules in regulating the immune response within the tumor microenvironment. In particular, possible therapeutic intervention strategies aimed at reversing or neutralizing suppressive networks within the tumor microenvironment will be emphasized. Importantly, blocking co-inhibitory molecule signaling, often referred to as immune checkpoint blockade, does not necessarily lead to an effective activation of tumor-specific T cells. Therefore, combination of checkpoint blockade with other immune potentiating therapeutic strategies, such as DC vaccination, might serve as a synergistic combination, capable of reversing effector T cells immunosuppression while at the same time increasing the efficacy of T cell-mediated immunotherapies. This will ultimately result in long-term anti-tumor immunity.
DC vaccination; tumor microenvironment; checkpoint blockade; tumor-specific T cells; cancer treatment
•Active immunotherapy is promising for the development of potent cancer therapeutics.•Various types of artificial antigen-presenting cells (aAPCs) may be used as ‘off-the-shelf’ products to induce antigen-specific T cell activation both ex vivo and in vivo.•Size, shape, cytokine delivery mechanism, ligand composition, ligand mobility, and ligand positioning on aAPCs all have significant effects on T cell activation, and therefore should be taken into account when designing novel constructs.
Active anti-cancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells (APCs). Therapy with autologous natural APCs is costly and time-consuming and results in variable outcomes in clinical trials. Therefore, development of artificial APCs (aAPCs) has attracted significant interest as an alternative. We discuss the characteristics of various types of acellular aAPCs, and their clinical potential in cancer immunotherapy. The size, shape, and ligand mobility of aAPCs and their presentation of different immunological signals can all have significant effects on cytotoxic T cell activation. Novel optimized aAPCs, combining carefully tuned properties, may lead to efficient immunomodulation and improved clinical responses in cancer immunotherapy.
artificial antigen-presenting cell; synthetic dendritic cell; immunotherapy; cancer
Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for podosomes of dendritic cells.
Antigen presentation; podosomes; dendritic cells; receptor mediated endocytosis
Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.
Bioassays that predict clinical outcome are essential to optimize cellular anticancer immunotherapy. We have recently developed a robust and simple skin test to evaluate the capacity of tumor-specific T cells to migrate, recognize their targets and exert effector functions. This bioassay detects T cells with an elevated antineoplastic potential and hence rapidly identifies patients responding to immunotherapy.
biomarker; DC-based vaccination; delayed-type hypersensitivity; immunomonitoring; melanoma; multifunctional T cells; skin-infiltrating lymphocytes
Dendritic cell (DC)-based immunotherapy employs the patients’ immune system to fight neoplastic lesions spread over the entire body. This makes it an important therapy option for patients suffering from metastatic melanoma, which is often resistant to chemotherapy. However, conventional cellular vaccination approaches, based on monocyte-derived DCs (moDCs), only achieved modest response rates despite continued optimization of various vaccination parameters. In addition, the generation of moDCs requires extensive ex vivo culturing conceivably hampering the immunogenicity of the vaccine. Recent studies, thus, focused on vaccines that make use of primary DCs. Though rare in the blood, these naturally circulating DCs can be readily isolated and activated thereby circumventing lengthy ex vivo culture periods. The first clinical trials not only showed increased survival rates but also the induction of diversified anti-cancer immune responses. Upcoming treatment paradigms aim to include several primary DC subsets in a single vaccine as pre-clinical studies identified synergistic effects between various antigen-presenting cells.
dendritic cell vaccination; immunotherapy; naturally circulating dendritic cells; melanoma; monocyte-derived dendritic cells; plasmacytoid dendritic cells; myeloid dendritic cells
The physical limits of cell migration in dense porous environments are dependent upon the available space and the deformability of the nucleus and are modulated by matrix metalloproteinases, integrins and actomyosin function.
Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm2; T cells, 4 µm2; neutrophils, 2 µm2). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.
The β2-integrin LFA-1 facilitates extravasation of monocytes (MOs) into the underlying tissues, where MOs can differentiate into dendritic cells (DCs). Although DCs express LFA-1, unlike MOs, they cannot bind to ICAM-1. We hypothesized that an altered integrin organization on the DC plasma membrane might cause this effect and investigated the relationship between membrane organization and function of LFA-1 on MOs and DCs. High-resolution mapping of LFA-1 surface distribution revealed that on MOs LFA-1 function is associated with a distribution in well-defined nanoclusters (100–150-nm diameter). Interestingly, a fraction of these nanoclusters contains primed LFA-1 molecules expressing the specific activation-dependent L16-epitope. Live imaging of MO–T-cell conjugates showed that only these primed nanoclusters are dynamically recruited to the cellular interface forming micrometer-sized assemblies engaged in ligand binding and linked to talin. We conclude that besides affinity regulation, LFA-1 function is controlled by at least three different avidity patterns: random distributed inactive molecules, well-defined ligand-independent proactive nanoclusters, and ligand-triggered micrometer-sized macroclusters.
The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.
pathogen recognition receptor; lectin; electron microscopy; multiprotein assembly; lipid rafts
The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor–deficient mice abrogated CpG-mediated responses. CD8α+ cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8α+ cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs.
Dendritic cell-based anticancer immunotherapy is feasible, safe and results in the induction of tumor-specific immune responses, at least in a fraction of vaccinated patients. The concomitant activation of cytotoxic and helper T cells, by loading DCs with peptides or electroporating them with the corresponding mRNAs, may further enhance vaccine-induced antitumor responses.
cancer immunotherapy; dendritic cell vaccination; helper T cells; metastatic melanoma
Dendritic cell (DC)-based vaccines require the cells to relocate to lymph nodes (LNs). Unfortunately, however, DC migration rates are typically very poor. We investigated strategies to increase the migration efficacy of DC-based vaccines. Surprisingly, a reduction in DC number, but not the conditioning of the injection site, improved LN targeting.
dendritic cell; imaging; migration; immunotherapy; 19F MRI
The identification of growth and differentiation pathways that are responsible for the proliferation and survival of cancer stem cells (CSCs) has opened avenues for the discovery of novel therapeutic targets. In the initial phase of an anticancer immune response, T cells specific for tumor-associated antigens develop in patients and, at least under selected circumstances, are able to eliminate malignant cells. However, it remains unknown whether CSC-specific T cells are also operational. We found naturally occurring multifunctional CD4+ and CD8+ T cells specific for the stem cell marker OCT4 among the peripheral blood mononuclear cells (PBMCs) of both healthy individuals and ovarian cancer patients. Moreover, lymphocytes isolated from the ascites of patients affected by ovarian malignancies also contained OCT4-specific T cells. OCT4-reactive CD4+ T cells did not produce interferon γ (IFNγ) and IFNγ-inducible protein 10 (IP-10) but were capable of proliferation upon stimulation with dendritic cells (DCs) loaded with an OCT4-derived peptide or OCT4 mRNA. OCT4-reactive CD8+ cells did not proliferate in response to a similar challenge, yet produced IP-10 as well as sufficient amounts of IFNγ to induce IP-10 . Furthermore, CD8+ cytotoxic T cells were able to release their lysosomal components, as indicated by the mobilization of CD107a. These results demonstrate the existence of anti-CSC specific T cells in ovarian cancer patients.
CD4; CD8; IP-10; ovarian cancer; proliferation; stem cell markers
Cellular therapy can be defined as the transplantation of living cells for the treatment of medical conditions. Three main objectives of cellular therapy are regeneration of damaged tissue, replacement of function by secretion of biologically active molecules, and redirection of aberrant processes. Given the complex nature of these approaches, in vivo tracking of the transplanted cells is critical to evaluate their potential benefit and to optimize treatment strategies. Recent advances are reviewed that enable in vivo cell tracking as an important adjunct to implement cellular therapy in clinical practice.
in vivo imaging; tracking; cellular therapy; transplantation; regenerative medicine
Dendritic cells (DCs) are central in maintaining the intricate balance between immunity and tolerance by orchestrating adaptive immune responses. Being the most potent antigen presenting cells, DCs are capable of educating naïve T cells into a wide variety of effector cells ranging from immunogenic CD4+ T helper cells and cytotoxic CD8+ T cells to tolerogenic regulatory T cells. This education is based on three fundamental signals. Signal I, which is mediated by antigen/major histocompatibility complexes binding to antigen-specific T cell receptors, guarantees antigen specificity. The co-stimulatory signal II, mediated by B7 family molecules, is crucial for the expansion of the antigen-specific T cells. The final step is T cell polarization by signal III, which is conveyed by DC-derived cytokines and determines the effector functions of the emerging T cell. Although co-stimulation is widely recognized to result from the engagement of T cell-derived CD28 with DC-expressed B7 molecules (CD80/CD86), other co-stimulatory pathways have been identified. These pathways can be divided into two groups based on their impact on primed T cells. Whereas pathways delivering activatory signals to T cells are termed co-stimulatory pathways, pathways delivering tolerogenic signals to T cells are termed co-inhibitory pathways. In this review, we discuss how the nature of DC-derived signal II determines the quality of ensuing T cell responses and eventually promoting either immunity or tolerance. A thorough understanding of this process is instrumental in determining the underlying mechanism of disorders demonstrating distorted immunity/tolerance balance, and would help innovating new therapeutic approaches for such disorders.
activation; tolerance; co-stimulation; co-inhibition; dendritic cells; T cell priming
Plasmacytoid dendritic cells (pDCs) are a specific subset of naturally occurring dendritic cells, that secrete large amounts of Type I interferon and play an important role in the immune response against viral infection. Several studies have highlighted that they are also effective antigen presenting cells, making them an interesting target for immunotherapy against cancer. However, the modes of action of pDCs are not restricted to antigen presentation and IFN secretion alone. In this review we will highlight a selection of cell surface proteins expressed by human pDCs that may facilitate communication with other immune cells, and we will discuss the implications of these molecules for pDC-driven immune responses.
cross talk; surface markers; T lymphocytes; viral infection; pDC migration
Immunotherapy aims to re-engage and revitalize the immune system in the fight against cancer. Research over the past decades has shown that the relationship between the immune system and human cancer is complex, highly dynamic, and variable between individuals. Considering the complexity, enormous effort and costs involved in optimizing immunotherapeutic approaches, clinically applicable tools to monitor therapy-induced immune responses in vivo are most warranted. However, the development of such tools is complicated by the fact that a developing immune response encompasses several body compartments, e.g., peripheral tissues, lymph nodes, lymphatic and vascular systems, as well as the tumor site itself. Moreover, the cells that comprise the immune system are not static but constantly circulate through the vascular and lymphatic system. Molecular imaging is considered the favorite candidate to fulfill this task. The progress in imaging technologies and modalities has provided a versatile toolbox to address these issues. This review focuses on the detection of therapy-induced anticancer immune responses in vivo and provides a comprehensive overview of clinically available imaging techniques as well as perspectives on future developments. In the discussion, we will focus on issues that specifically relate to imaging of the immune system and we will discuss the strengths and limitations of the current clinical imaging techniques. The last section provides future directions that we envision to be crucial for further development.
Immunotherapy; Functional imaging; Dendritic cells; PET; Scintigraphy; MRI
The type of adaptive immune response following host-fungi interaction is largely determined at the level of the antigen-presenting cells, and in particular by dendritic cells (DCs). The extent to which transcriptional regulatory events determine the decision making process in DCs is still an open question. By applying the highly structured DC-ATLAS pathways to analyze DC responses, we classified the various stimuli by revealing the modular nature of the different transcriptional programs governing the recognition of either pathogenic or commensal fungi. Through comparison of the network parts affected by DC stimulation with fungal cells and purified single agonists, we could determine the contribution of each receptor during the recognition process. We observed that initial recognition of a fungus creates a temporal window during which the simultaneous recruitment of cell surface receptors can intensify, complement and sustain the DC activation process. The breakdown of the response to whole live cells, through the purified components, showed how the response to invading fungi uses a set of specific modules. We find that at the start of fungal recognition, DCs rapidly initiate the activation process. Ligand recognition is further enhanced by over-expression of the receptor genes, with a significant correspondence between gene expression and protein levels and function. Then a marked decrease in the receptor levels follows, suggesting that at this moment the DC commits to a specific fate. Overall our pathway based studies show that the temporal window of the fungal recognition process depends on the availability of ligands and is different for pathogens and commensals. Modular analysis of receptor and signalling-adaptor expression changes, in the early phase of pathogen recognition, is a valuable tool for rapid and efficient dissection of the pathogen derived components that determine the phenotype of the DC and thereby the type of immune response initiated.
In order for cellular therapeutics to succeed, comprehensive monitoring of the transplanted cells in vivo is required i.e., their localization, functionality and numbers in a longitudinal manner. Recently, dendritic cell based vaccines have been monitored by their effect on lymphocyte activation using [18F]FLT PET in cancer patients.
cell therapy; cell tracking; [18F]FLT-PET; immunomonitoring; dendritic cell-based vaccines
Monitoring of cell therapeutics in vivo is of major importance to estimate its efficacy. Here, we present a novel intracellular label for 19F magnetic resonance imaging (MRI)-based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of 19F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The 19F label was tested in primary human monocyte-derived dendritic cells. These cells took up label effectively, resulting in a labeling of 1.7 ± 0.1 × 1013 19F atoms per cell, with a viability of 80 ± 6%, without the need for electroporation or transfection agents. This results in a minimum detection sensitivity of about 2,000 cells/voxel at 7 T, comparable with gadolinium-labeled cells. Comparison of the detection sensitivity of cells labeled with 19F, iron oxide and gadolinium over typical tissue background showed that unambiguous detection of the 19F-labeled cells was simpler than with the contrast agents. The effect of the 19F agent on cell function was minimal in the context of cell-based vaccines. From these data, we calculate that detection of 30,000 cells in vivo at 3 T with a reasonable signal to noise ratio for 19F images would require less than 30 min with a conventional fast spin echo sequence, given a coil similar to the one used in this study. This is well within acceptable limits for clinical studies, and thus, we conclude that 19F MRI for quantitative cell tracking in a clinical setting has great potential.
cell tracking; magnetic resonance imaging; dendritic cell vaccines; cell quantification; perfluorocarbon labels; cellular therapy
The plasmacytoid dendritic cell (pDC) constitutes a unique DC subset that links the innate and adaptive arm of the immune system. Whereas the unique capability of pDCs to produce large amounts of type I IFNs in response to pathogen recognition is generally accepted, their antigen-presenting function is often neglected since most studies on antigen presentation are aimed at other DC subsets. Recently, pDCs were demonstrated capable to present antigen leading to protective tumor immunity. In this review, we discuss how pDCs could be exploited in the fight against cancer by analyzing their capacity to capture, process and (cross-) present antigen.
PIVAC 11; Plasmacytoid dendritic cells; Antigen-uptake receptors; Antigen presentation; Targeting
Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E2 (PGE2). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE2-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-011-0908-y) contains supplementary material, which is available to authorized users.
Mechanosensitivity; Podosomes; Dendritic cell; Adhesion