Immunoediting: evidence of the multifaceted role of the immune system in self-metastatic tumor growth

ABSTRACT: BACKGROUND: The role of the immune system in tumor progression has been subject to discussion for many decades. Numerous studies suggest that a low immune response might be beneficial, if not necessary, for tumor growth, and only a strong immune response can counter tumor growth and thus inhibit progression. METHODS: We implement a cellular automaton model previously described that captures the dynamical interactions between the cancer stem and non-stem cell populations of a tumor through a process of self-metastasis. By overlaying on this model the diffusion of immune reactants into the tumor from a peripheral source to target cells, we simulate the process of immune-system-induced cell kill on tumor progression. RESULTS: A low cytotoxic immune reaction continuously kills cancer cells and, although at a low rate, thereby causes the liberation of space-constrained cancer stem cells to drive self-metastatic progression and continued tumor growth. With increasing immune system strength, however, tumor growth peaks, and then eventually falls below the intrinsic tumor sizes observed without an immune response. With this increasing immune response the number and proportion of cancer stem cells monotonically increases, implicating an additional unexpected consequence, that of cancer stem cell selection, to the immune response. CONCLUSIONS: Cancer stem cells and immune cytotoxicity alone are sufficient to explain the three-step "immunoediting" concept - the modulation of tumor growth through inhibition, selection and promotion.     

Interactions between macrophages and T-lymphocytes: tumor sneaking through intrinsic to helper T cell dynamics

In a mathematical model of the cellular immune response we investigate immune reactions to tumors that are introduced in various doses. The model represents macrophage T-lymphocyte interactions that generate cytotoxic macrophages and cytotoxic T-lymphocytes. In this model antigens (tumors) can induce infinitely large T-lymphocyte effector populations because effector T-lymphocytes are capable of repeated proliferation and we have omitted immunosuppression. In this (proliferative) model small doses of weakly antigenic tumors grow infinitely large (i.e. sneak through) eliciting an immune response of limited magnitude. Intermediate doses of the same tumor induce larger immune responses and are hence rejected. Large doses of the tumor break through, but their progressive growth is accompanied by a strong immune response involving extensive lymphocyte proliferation. Similarly a more antigenic tumor is rejected in intermediate doses and breaks through in large doses. Initially small doses however lead to tumor dormancy. Thus although the model is devoid of explicit regulatory mechanisms that limit the magnitude of its response (immunosuppression is such a mechanism), the immune response to large increasing tumors may either be a stable reaction of limited magnitude (experimentally known as tolerance or unresponsiveness) or a strong and ever increasing reaction. Unresponsiveness can evolve because in this model net T-lymphocyte proliferation requires the presence of a minimum number of helper T cells (i.e. a proliferation threshold). Unresponsiveness is caused by depletion of helper T cell precursors.     

Macrophage T lymphocyte interactions in the anti-tumor immune response: a mathematical model

In this paper we present a model of the macrophage T lymphocyte interactions that generate an anti-tumor immune response. The model specifies i) induction of cytotoxic T lymphocytes, ii) antigen presentation by macrophages, which leads to iii) activation of helper T cells, and iv) production of lymphoid factors, which induce a) cytotoxic macrophages, b) T lymphocyte proliferation, and c) an inflammation reaction. Tumor escape mechanisms (suppression, antigenic heterogeneity) have been deliberately omitted from the model. This research combines hitherto unrelated or even contradictory data within the range of behavior of one model. In the model behavior, helper T cells play a crucial role: Tumors that differ minimally in antigenicity (i.e., helper reactivity) can differ markedly in rejectability. Immunization yields protection against tumor doses that would otherwise be lethal, because it increases the number of helper T cells. The magnitude of the cytotoxic effector cell response depends on the time at which helper T cells become activated: early helper activity steeply increases the magnitude of the immune response. The type of cytotoxic effector cells that eradicates the tumor depends on tumor antigenicity: lowly antigenic tumors are attacked mainly by macrophages, whereas large highly antigenic tumors can be eradicated by cytotoxic T lymphocytes only.     

The intrinsic immunogenic properties of cancer cell lines,immunogenic cell death,and how these influence host antitumor immune responses

Modern cancer therapies often involve the combination of tumor-directed cytotoxic strategies and generation of a host antitumor immune response. The latter is unleashed by immunotherapies that activate the immune system generating a more immunostimulatory tumor microenvironment and a stronger tumor antigen-specific immune response. Studying the interaction between antitumor cytotoxic therapies, dying cancer cells, and the innate and adaptive immune system requires appropriate experimental tumor models in mice. In this review, we discuss the immunostimulatory and immunosuppressive properties of cancer cell lines commonly used in immunogenic cell death (ICD) studies being apoptosis or necroptosis. We will especially focus on the antigenic component of immunogenicity. While in several cancer cell lines the epitopes of endogenously expressed tumor antigens are known, these intrinsic epitopes are rarely determined in experimental apoptotic or necroptotic ICD settings. Instead by far the most ICD research studies investigate the antigenic response against exogenously expressed model antigens such as ovalbumin or retroviral epitopes (e.g., AH1). In this review, we will argue that the immune response against endogenous tumor antigens and the immunopeptidome profile of cancer cell lines affect the eventual biological readouts in the typical prophylactic tumor vaccination type of experiments used in ICD research, and we will propose additional methods involving immunopeptidome profiling, major histocompatibility complex molecule expression, and identification of tumor-infiltrating immune cells to document intrinsic immunogenicity following different cell death modalities.Subject terms: Cancer models, Antigen-presenting cells, Immune cell death     

IgG-recognizing shed tumor-associated antigens can promote tumor invasion and metastasis

Tumors secreting glycoproteins that act as tumor-associated antigens have been described as highly invasive and metastatic. In this study, the consequences of the humoral immune response (HIR) against these antigens were investigated. Using an in vitro model of tumor cell invasion, results indicated that the invasiveness of tumor cells secreting antigenic secreted/shed tumor glycoproteins (STGP) increases in the presence of specific anti-STGP IgG, polymorphonuclear cells and monocytes. This in vitro model showed that the coincidental presence in the matrix of both STGP and specific anti-STGP IgG increases the local release of IL-1beta, IL-6 and vascular endothelial growth factor (VEGF) by stromal cells, but not by tumor cells. Using an in vivo model, the experiments show that immune-competent mice develop an anti-tumor HIR with anti-STGP IgG production. In this model, tumor growth was increased in parallel with the serum concentration of specific anti-STGP IgG. In athymic nude (nu/nu)-beige mice the same trend was observed, suggesting a T-cell-independent tumor-promoting effect induced by anti-STGP IgG. Tumor histology showed intense infiltration of IgG-positive plasma cells and lymphocytes. A severe combined immunodeficient-beige mouse-based in vivo model of tumors, experimentally infiltrated with monoclonal IgG plasmocytoma cells, showed that only specific anti-STGP-IgG-secreting cells could exacerbate tumor invasion, angiogenesis and metastasis. These results suggest that tumors shedding/secreting antigenic STGP can induce a host IgG immune response that can promote invasion and metastasis by inducing tumor infiltrating stromal cells to release proinflammatory cytokines and VEGF.     

Multi-Stability and Multi-Instability Phenomena in a Mathematical Model of Tumor-Immune-Virus Interactions

Recent advances in virology, gene therapy, and molecular and cell biology have provided insight into the mechanisms through which viruses can boost the anti-tumor immune response, or can infect and directly kill tumor cells. A recent experimental report (Bridle et al. in Molec. Ther. 18(8):1430–1439, 2010) showed that a sequential treatment approach that involves two viruses that carry the same tumor antigen leads to an improved anti-tumor response compared to the effect of each virus alone. In this article, we derive a mathematical model to investigate the anti-tumor effect of two viruses, and their interactions with the immune cells. We discuss the conditions necessary for permanent tumor elimination and, in this context, we stress the importance of investigating the long-term effect of non-linear interactions. In particular, we discuss multi-stability and multi-instability, two complex phenomena that can cause abrupt transitions between different states in biological and physical systems. In the context of cancer immunotherapies, the transitions between a tumor-free and a tumor-present state have so far been associated with the multi-stability phenomenon. Here, we show that multi-instability can also cause the system to switch from one state to the other. In addition, we show that the multi-stability is driven by the immune response, while the multi-instability is driven by the presence of the virus.     

Effects of thermal stress on tumor antigenicity and recognition by immune effector cells

The primary rationale for the application of clinical hyperthermia in the therapy of cancer is based on the direct cytotoxic effect of heat and the radio-chemosensitization of tumor cells. More recently, additional attention is given to the observation that heat and heat-shock proteins can activate the host’s immune system. The expression of heat-shock genes and proteins provides an adaptive mechanism for stress tolerance, allowing cells to survive non-physiologic conditions. However, the same adaptive mechanism can ultimately favor malignant transformation by interfering with pathways that regulate cell growth and apoptosis. Cytoprotection and thermotolerance raised the concern that heat-treated tumor cells might also be resistant to attack by immune effector mechanisms. Many studies, including those from our group, address this concern and document that heat-exposure, although transiently modulating sensitivity to CTL, do not hinder CTL attack. Moreover, there are promising reports of heat-related upregulation of NK-activating ligands, rendering those tumors which have lost MHC class I molecules target for NK cell attack. Heat-induced cytoprotection, therefore, does not necessarily extend protection from cytotoxic immune mechanisms. When interpreting the effects of heat, it is important to keep in mind that thermal effects on cell physiology are strongly dependent on the thermal dose, which is a function of the magnitude of change in temperature and the duration of heat exposure. The thermal dose required to induce cell death in vitro strongly varies from cell type to cell type and depends on microenvironmental factors (Dewey 1994). Therefore, to dissect the immunological behaviour of a given tumor and its micro-environment at different thermal doses, it is essential to characterize the thermosensitivity of every single tumor type and assess the proportion of cells surviving a given heat treatment. In this review, we summarize the pleiotropic effects that heat exposure has on tumor cells. In particular, we focus on the effects of heat on the antigen presentation of tumor cells and their susceptibility to immune effector mechanisms. We emphasize that the response to thermal stress is not a one-time point event, but rather a time period starting with the heat exposure and extending over several days of recovery. In addition, the response of tumor cells and their susceptibility to immune effector cells is strongly dependent on the model system, on the magnitude and duration of the thermal stress and on the time of recovery after heat exposure. Consideration of these aspects might help to explain some of the conflicting results that are reported in the field of thermal stress response. This article forms part of the Symposium in Writing "Thermal stress-related modulation of tumor cell physiology and immune responses", edited by Elfriede Noessner.     

T cell immunity induced by live, necrotic, and apoptotic tumor cells

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.     

Not all cancers are created equal: Tissue specificity in cancer genes and pathways

Tumors arise through waves of genetic alterations and clonal expansion that allow tumor cells to acquire cancer hallmarks, such as genome instability and immune evasion. Recent genomic analyses showed that the vast majority of cancer driver genes are mutated in a tissue-dependent manner, that is, are altered in some cancers but not others. Often the tumor type also affects the likelihood of therapy response. What is the origin of tissue specificity in cancer? Recent studies suggest that both cell-intrinsic and cell-extrinsic factors play a role. On one hand, cell type–specific wiring of the cell signaling network determines the outcome of cancer driver gene mutations. On the other hand, the tumor cells’ exposure to tissue-specific microenvironments (e.g. immune cells) also contributes to shape the tissue specificity of driver genes and of therapy response. In the future, a more complete understanding of tissue specificity in cancer may inform methods to better predict and improve therapeutic outcomes.     

Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.     

Regulatory B cells and T cell Regulation in Cancer

Recent researches shed light on B cell role on various autoimmune diseases, including autoantibody-mediated diseases as well as T cell-mediated autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. B cells play a critical role in the immune response beyond the production of antibodies through mechanisms such as antigen presentation and cytokine production. Furthermore, B cells have recently been recognized to play a role in promoting tumor immunity against cancer. However, not all B cells positively regulate immune responses. Regulatory B cells negatively regulate immune responses by the production of anti-inflammatory cytokines such as interleukin (IL)-10, IL-35, and transforming growth factor-beta. Thus, a balance between effector and regulatory B cells regulates the immune response through the release of cytokines. In this review, we highlight the main emerging roles of B cells in tumor immunity with a focus on the T cell response. These findings can guide a protocol for selectively depleting regulatory B cells as a potential therapeutic strategy for patients with cancer.     

肿瘤患者CD8+T细胞中CD28表达的研究进展

CD28与B7结合形成的共刺激信号是T细胞激活的第二信号,肿瘤患者CD8^＋T细胞上CD28分子在肿瘤免疫中发挥着重要作用。人体抗肿瘤免疫主要由CD8^＋T细胞介导,根据CD28的表达与否可将CD8^＋T细胞分为细胞毒T细胞（CD8^＋CD28^＋,CTL）和抑制性T细胞（CD8^＋C28^-,Ts）。CTL是体内杀伤肿瘤细胞的主要功能性细胞之一,当该细胞与肿瘤接触时,通过共刺激信号而被激活,发挥其对肿瘤细胞的杀伤作用;Ts在机体的免疫耐受中发挥作用。现就肿瘤患者CD8^＋T细胞上CD28的表达作一综述。     

Role of neutrophil extracellular traps in regulation of lung cancer invasion and metastasis: Structural insights from a computational model

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

Summary A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described.With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

The Tumor Growth Paradox and Immune System-Mediated Selection for Cancer Stem Cells

Cancer stem cells (CSCs) drive tumor progression, metastases, treatment resistance, and recurrence. Understanding CSC kinetics and interaction with their nonstem counterparts (called tumor cells, TCs) is still sparse, and theoretical models may help elucidate their role in cancer progression. Here, we develop a mathematical model of a heterogeneous population of CSCs and TCs to investigate the proposed “tumor growth paradox”—accelerated tumor growth with increased cell death as, for example, can result from the immune response or from cytotoxic treatments. We show that if TCs compete with CSCs for space and resources they can prevent CSC division and drive tumors into dormancy. Conversely, if this competition is reduced by death of TCs, the result is a liberation of CSCs and their renewed proliferation, which ultimately results in larger tumor growth. Here, we present an analytical proof for this tumor growth paradox. We show how numerical results from the model also further our understanding of how the fraction of cancer stem cells in a solid tumor evolves. Using the immune system as an example, we show that induction of cell death can lead to selection of cancer stem cells from a minor subpopulation to become the dominant and asymptotically the entire cell type in tumors.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described. With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

The interferons and their receptors--distribution and regulation

The interferons (IFNs) were originally described over 50 years ago, identified by their ability to confer viral resistance to cells. We now know that they are much more than just anti-viral cytokines collectively having roles in both innate and adaptive immune responses, in tumor surveillance and defense, and modulation of immune cell function. Three types of IFN have now been described, simply referred to as type I, II and III. Distinguishable by the unique receptors that they rely on for signal transduction, the three types of IFN have specific and varied roles in the maintenance of human health and defense against pathogens. In mounting an IFN-mediated immune response, the human body has developed the ability to regulate IFN-mediated signal transduction. Like all cytokines, the ability of a cell to respond to IFN is completely dependent on the presence of its cognate receptor on the surface of the target cell. Thus, one of the major mechanisms used by the human body to regulate the strength and duration of the IFN response is through regulation of receptor levels, thereby altering the cytokine-specific responsiveness of the target cell. This review will discuss the receptor system utilized by the type I IFNs and compare it with that of the type II and III IFNs, which also regulate immune responses through controlling receptor level on the cell surface.     

A novel immunoregulatory axis of NKT cell subsets regulating tumor immunity

There are many mechanisms that regulate and dampen the immune response to cancers, including several types of regulatory T cells. Besides the T reg cell, we have identified another immunoregulatory circuit initiated by NKT cells that produce IL-13 in response to tumor growth and this IL-13 then induces myeloid cells to make TGF-beta that inhibits cytotoxic T cell-mediated tumor immunosurveillance in several mouse tumor models. This finding created a paradox in the role of NKT cells in tumor immunity, in that they can also contribute to protection. We resolve this paradox by the finding that the suppressive NKT cell is a type II NKT cell that lacks the canonical invariant T cell receptor, whereas the protective cell is a type I NKT cell that expresses the invariant receptor. Further, we see that these two subsets of NKT cells counter-regulate each other, defining a new immunoregulatory axis. The balance along this axis may determine the outcome of tumor immunosurveillance as well as influence the efficacy of anti-cancer vaccines and immunotherapy.     

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.     

Chemotherapy induces ATP release from tumor cells

Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.