Abeloff’s Clinical Oncology Issue 1 - 2014 The Immune System in Melanoma Initiation and Progression R. Todd Reilly, PhD,* James O. Armitage, MD,† and John E. Niederhuber, MD* Historical Context of Immunotherapy Although the notion of leveraging host immunity in the fight against cancer was first conceived over a century ago, technological advances in the past 20 years have both broadened our understanding of the functioning of the immune system and spurred the development of novel immunologically based treatment options for a range of cancers. In this review, we focus specifically on summarizing the current understanding of the complex interplay between the initiation and progression of cutaneous melanoma and the host immune system, highlighting the key features of tumor growth and immune function that determine the balance between suppression and activation of tumor-specific immunity. William Coley, in the late 1800s, is widely recognized as the first to postulate the potential for host immunity to serve as a mechanism for cancer treatment when, as a surgeon at what was then known as Memorial Hospital in New York City (now Memorial Sloan-Kettering Cancer Center), he observed the spontaneous regression of “sarcomas” in patients who had a concomitant bacterial infection.1-3 Coley went on to treat several cancer patients through direct injection of the tumor with bacterial extracts (Coley’s Toxins), noting the (temporary) growth-inhibitory action of the treatment but also the considerable “risks of inoculation, when successful.”1 Although Paul Erlich in 1909 first proposed that the immune system may play a role in eliminating nascent tumors,4 the dangers associated with inoculating patients with bacterial extracts combined with the inconsistent outcomes reported resulted in a generally unfavorable view of immunotherapy within the clinical community. Following Paul Erlich’s introduction of the notion of a relationship between cancer development and the host immune system, including the concept of immunosurveillance,4 the hypothesis was largely ignored for almost 50 years until the convergence of a growing *Inova Translational Medicine Institute, Inova Health System, Falls Church, Virginia †University of Nebraska Medical Center, Omaha, Nebraska Copyright © 2014 Elsevier, Inc. understanding of allograft rejection and the recognition of tumor-associated neoantigens in mouse models of chemically induced tumors. The identification of unique tumor specific antigens led to separate (and subtly different) reassertions by Thomas and Burnet that a critical function of the immune system is to patrol for and eliminate nascent tumor cells.5-9 Support for the immunosurveillance hypothesis dimmed again in the 1970s when immunocompromised mouse models were used as a basis to study spontaneous and chemically induced tumor incidence.10-13 This line of investigation culminated in a series of studies in which immunocompromised athymic nude mice were shown to have the same tumor incidence as immunocompetent animals,14-16 leading many to seriously question the concept of immunosurveillance. Not appreciated at the time was the fact that athymic mice are not completely immunocompromised; they possess natural killer (NK) cells and maintain a small population of T cells as well.17-19 Over the subsequent 30 years, as our understanding of the diverse range of immune cells has evolved, so too has the immunosurveillance hypothesis. The current understanding, which is still being refined as further knowledge is acquired, encompasses not only immunosurveillance but also immunoediting, whereby an equilibrium process exists in which genetic instability within an incipient tumor results in the outgrowth of individual Share your opinion and get a free Echapter from Abeloff’s Clinical Oncology This material was supported by an educational grant from Bristol-Myers Squibb. 1 2 tumor cells expressing novel antigenic determinants (reviewed20-22). These immunoedited tumor cells are then eliminated by innate immune effector cells (primarily NK cells) and by adaptive immune effector cells (primarily T cells). Ultimately, this equilibrium process is thought to result in either the elimination (or control) of the incipient tumor, or the outgrowth (escape) of tumor cell variants that evade the immunoediting process and progress to become clinically detectable tumors.20-22 It wasn’t until the late 1980s and 1990s, when the development of transgenic mouse models and other new technologies made it possible to (relatively) quickly and easily study the behavior of immune cells at the genetic level as well as at the population level, that investigators were able to develop strategies to orchestrate perturbations in the balance between immune activation and suppression. In this review, we discuss a variety of interventions developed to promote immune-mediated mechanisms for melanoma treatment, with emphasis on our evolving understanding of the immunologic pathways and mechanisms underlying those interventions. Basic Tumor Immunology Opening Because opportunities for immune intervention in cancer therapy have been identified at virtually every stage of the immune response, reviewing those targets and interventions within the context of a general review of basic processes underlying immune recognition, activation, and effector function is useful. The mammalian immune system has evolved to encompass the integrated actions of a range of cells together with both soluble and membrane bound ligands and their respective receptors to protect the host primarily against pathogenic microbes while remaining generally nonresponsive to host cells and tissues. In humans, immunity to foreign pathogens generally involves the sequential engagement of both innate and adaptive immune functions; the former characterized by a more immediate, less specific, and shorter duration response that gives way to the more focused and longer lasting effects of the latter. Effector functions of the innate and adaptive immune responses are very tightly controlled to prevent both collateral and inappropriately targeted damage to normal host tissues. It is within this dynamic equilibrium between the opposing influences of immune activation and tolerance that the opportunities to induce effective antitumor immunity lie. The Role of Innate Immunity Innate immunity serves as a first line of host defense and, as such, is composed of epithelial and mucosal barriers (skin, epithelia, and mucosa), a cadre of soluble antimicrobial factors (complement, cytokines, chemokines), pattern recognition receptors that allow rapid identification of pathogen-associated molecular patterns (PAMPs), and a range of effector cells (including dendritic cells, eosinophils, macrophages, mast cells, monocytes, neutrophils, and NK cells). The innate immune response is The Immune System in Melanoma Initiation and Progression characterized by rapid initiation (minutes to hours), a low specificity of response relative to the adaptive immune response, and localized inflammation. The first innate effector cells at the site of infection typically are macrophages and mast cells, which upon activation secrete a range of cytokines and chemokines that mediate the physiologic hallmarks of the innate immune response: the dilation and increased permeability of nearby blood vessels and resultant accumulation of fluid and blood proteins and the recruitment of neutrophils, lymphocytes, and monocytes (precursors of macrophages) into the inflamed site. Antigen Presentation and AntigenPresenting Cells Dendritic cells (DCs) are among the innate effector cells recruited to sites of inflammation. DCs, referred to as professional antigen-presenting cells (APCs), play a critical role in bridging innate and adaptive immune responses by acquiring antigens from the site of an infection and presenting those antigens to effector cells to initiate the adaptive immune response (Figure 1). An antigen can be any macromolecule (protein, polysaccharide, or lipid conjugate thereof) that elicits an immune response. DCs that are recruited to an inflammatory site take up protein antigens, processing and degrading them internally into short peptides (or determinants), and packaging and presenting those peptides in association with major histocompatibility complex (MHC) molecules on their surface. If DCs receive the appropriate stimulation through the engagement of pattern recognition receptors or other “danger signals” (e.g., proinflammatory cytokines or certain kinds of cellular debris), they undergo a process of maturation that results in the increased expression of cell surface molecules (MHC Class I [MHC-I], MHC-II, CD80 and CD86 (a.k.a. B7-1 and B7-2), and other receptors and ligands that play a role in modulating the adaptive immune response) and soluble factors (cytokines and chemokines). Activated DCs also upregulate specific cell surface adhesion molecules that facilitate their migration to the lymph node, where they encounter adaptive immune effector cells. Rudolph Virchow, in 1863, first observed immune infiltrates within tumors, leading him to hypothesize that inflammation played a role in tumor development.23 It is now widely accepted that chronic inflammation–resulting from chronic infections (e.g., Helicobacter pylori/gastritis or viral hepatitis) or autoimmunity (e.g., inflammatory bowel disease)–can promote carcinogenesis (reviewed24). Chronically activated leukocytes continue to secrete proinflammatory factors, notably tumor necrosis factor (TNF), interleukin (IL)-1, and IL-8/CXCL-8, which promote tumor growth and development.25-28 Notably, these factors are also involved in stimulating the secretion of transforming growth factor-beta (TGF-β), IL-10, and IL-1β by melanoma (reviewed24,29,30), which play an important role in influencing the outcome of the adaptive antitumor immune response. The Immune System in Melanoma Initiation and Progression 3 B cell) by CD40L (on the T cell). The fully activated B cell then proliferates (clonal expansion) and begins to secrete soluble antibody in the form of GM-CSF (pentameric) IgM. After about a week, the plasma cells undergo class switchIL-4,FLT-3L ing, resulting in the production of solBone marrow Dendritic cell uble IgG antibodies, followed by a proprogenitor progenitor cess called affinity maturation, which Ag uptake/Processing further refines the genetic constructs Microbial infection encoding the antibody’s antigen bindNo danger danger signals ing site and leads to the production of signals Exogenous Endogenous “Tolerizing DC” higher-affinity binding of the BCR/ LPS TNF antibody to its target antigen. Upon CpG CD40L clearance of the target antigen, a small Activated DC proportion of the plasma cells differentiate into memory B cells, a very long-lived B cell type capable of proModerate MHC II High MHC II Chemokines Chemokines ducing high-affinity antigen-specific Adhesion molecules Adhesion molecules IgG much more rapidly than in a priCostimulatory molecules Costimulatory molecules mary immune response. Antibodies can mediate phagocytosis of cells expressing their cognate antigen through Figure 1. Dendritic cells (DCs) can either activate adaptive immunity or opsinization of the target cell and can tolerize T cells depending on their state of maturation. also facilitate target cell lysis either diFrom Pardoll D. Cancer immunology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE, eds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia: Churchill Livingstone; 2014:85. rectly, through compliment activation, or indirectly, by activating antibodydependent cell cytotoxicity (ADCC) Adaptive Immunity mechanisms of innate effector cells. Whereas tumorIn contrast to the rapid responses achieved in the innate specific antibodies have been generated (e.g., trastuzumab immune response, which is believed to be evolutionariand rituximab), such antibodies are predominantly used to ly older, the adaptive immune response can require 7 to block proliferative signaling pathways within cancer cells 14 days to become fully activated, is highly specific for rather than as a platform for immune-mediate tumor rejecdistinct antigens and antigenic epitopes (peptide detertion, although there are some examples of the latter. minants derived from antigen processing by APCs), and The cognate receptor on T cells, unlike its B-cell counresults in the formation of long-lasting memory effector terpart, does not recognize soluble antigen; TCRs recogcells that are able to rapidly reactivate in the event that nize peptide antigens in the context of MHC I and II molethe specific pathogen (antigen) is encountered again. The cules. After APCs (DCs) take up and process the antigenic exquisite specificity of the adaptive immune response is proteins, the derived peptides are bound by MHC I and even more impressive when the incredible diversity of anMHC II molecules, which are then transported to the surtigens that can potentially be “recognized” by the adaptive face of the APC for inspection by T cells. The TCR of CD4+ effector cells is considered. Both the diversity and specificT cells interact with MHC II-peptide complexes, which ity of antigen recognition are enabled through a series of are expressed primarily by APCs, while the TCR of CD8+ unique genetic recombination steps during cell differentiaT cells interact with antigenic peptides complexed with tion and activation that give rise to the antigen receptors MHC I molecules, which involves nearly all cells includon the surface of B and T cells, the B-cell receptor (BCR) ing APCs. TCR engagement with MHC bearing its cognate and T-cell receptor (TCR). peptide provides signal 1 for T-cell activation. If the APC is The BCR is a membrane-bound form of the same improperly activated, it will also express CD80/CD86 on its munoglobulin (IgM) (antibody) that ultimately is secreted cell surface. Engagement of CD80/CD86 with CD28 on the by the fully activated B cell (plasma cell) and is capable T-cell surface provides signal 2, leading to T-cell proliferaof recognizing soluble antigens. Engagement of the BCR tion (clonal expansion) and differentiation into an effector to its cognate antigen is the initial step in B-cell activacell (CD4+ helper T cell or CD8+ cytotoxic T cell). tion (referred to as signal 1). The BCR-antigen complex is Activated CD8+ T cells undergo rapid, IL-2–dependent then internalized and degraded, and antigenic epitopes are proliferation and upregulation of surface receptors, such presented on MHC II on the B-cell surface for inspection as the cytokine receptor CXCR-3, to facilitate trafficking by (activated) CD4+ T cells, which provide signal 2 to the to the peripheral tissues and secretion of proinflammatory and antiviral cytokines such as TNF-α and interferon B cell through engagement of the CD40 coreceptor (on the 4 (IFN)-γ. When activated CD8+ T cells encounter cognate peptide in the context of MHC I expressed on the surface of somatic cells, they release perforin and granzyme from lytic granules. Perforin polymerizes within the cell membrane of the target cell, forming a pore through which granzyme enters. The granzymes, which are serine proteases, then trigger apoptosis in the target cell. Some CD8+ T cells also express Fas, which can activate Fas-L on target cells to trigger apoptosis; however, this pathway is primarily used to terminate lymphocytes upon elimination of a pathogen. In addition to Fas-mediated elimination of lymphocytes, some CD8+ T cells also secrete IL-10 in the effector phase as a means to attenuate cytolytic effector function, thereby minimizing damage to uninfected bystander cells. Effector cell function by CD4+ helper T cells, as their name implies, involves providing secondary signals to other innate and adaptive effector cells; however, the phenotype of activated CD4+ T cells can vary depending on a number of factors, including the subtype of DC encountered by the naïve CD4+ T cell, the levels of cognate antigen-MHC encountered at activation, and the cytokine milieu present at activation. Th1 CD4+ helper T cells tend to secrete IFN-γ and IL-2, and are primarily involved in facilitating innate and adaptive immune responses to intracellular pathogens as well as some autoimmune responses. Th2 CD4+ helper T cells secrete a more diverse array of cytokines (including IL-4, IL-5, IL-9, and IL-10) and are involved primarily in facilitating innate and adaptive immunity to extracellular parasites as well as allergy and asthma. The Th17 phenotype is associated with the secretion of IL-17, IL-21, and IL-22 and is involved with facilitating immunity to extracellular bacteria and fungi as well as some autoimmune responses. Finally, a subset of CD4+ regulatory T cells (Treg) can arise in the thymus or can be induced peripherally under certain conditions (e.g., stimulation in the presence of TGF-β and the absence of proinflammatory cytokines). Treg cells, identified in the mid 1990s by Sakaguchi and colleagues,31 play a vital role in limiting T-cell proliferation and attenuating effector function32,33 (reviewed34). Because of the specificity and potent effector function of the adaptive immune response, we have evolved mechanisms of immunologic tolerance to protect from the inappropriate activation of immunity against self-antigens (i.e., autoimmunity). Immunologic tolerance arises as the sum of two distinct processes, central tolerance and peripheral tolerance. Central tolerance involves the elimination of potentially self-reactive T cells in the thymus during the early phases of their development. Because central tolerance cannot reliably eliminate all potentially selfreactive T cells, we have also evolved processes termed peripheral tolerance. For example, T cells that receive signal 1, cognate antigen in the context of MHC, in the absence of signal 2, appropriate costimulation, undergo a process termed anergy, in which the T cells become refractory to activation or effector function on subsequent encounters with cognate antigen. Similarly, T cells that repeatedly receive signal 1, even with appropriate costimulation, can The Immune System in Melanoma Initiation and Progression undergo clonal exhaustion, in which further responses to antigenic stimulation are highly attenuated. Immune Modulation Although mechanisms of central and peripheral tolerance play critical roles in shaping the immune repertoire, in many ways the more important processes of immune regulation–in particular, where clinical interventions for tumor immunotherapy and autoimmunity are concerned–lie in the pathways activated by the costimulatory and coinhibitory receptors that serve to modify the response to signal 1 plus signal 2 for T-cell activation. In fact, because APCs and T cells express on the surfaces not only CD28-B7 but an array of costimulatory and coinhibitory ligand/receptor pairs, signal 2 is viewed more accurately as the net sum of the activating and attenuating signaling cascades that are initiated in conjunction with TCR engagement with cognate peptide-MHC complexes. The requirement for activating signals beyond TCR engagement was first recognized in the 1980s and 1990s when the CD28/B7-1 costimulatory receptor/ligand pair were first cloned (with B7-2 discovered shortly thereafter) and their role in stimulating IL-2 secretion by Th1 CD4+ Helper T cells was identified.35 During the same time period, another receptor expressed on the T-cell surface was discovered that had high sequence homology with CD28. That receptor, cytotoxic T-lymphocyteassociated protein 4 (CTLA-4), was subsequently shown to bind to B7-1 with much greater affinity than CD2836. An inhibitory role for CTLA-4 was first postulated separately by Bluestone37 and Allison,38 but that role was not confirmed definitively until the mid-1990s when CTLA-4 knockout mice were shown to develop a generalized lymphoproliferation with massive lymphatic infiltration of all organs.39,40 In fact, this time from the mid 1980s through the late 1990s represents a period of rapid progress in identifying and characterizing the myriad of accessory receptors that are either constitutively expressed or up/downregulated on T cells on TCR ligation and that play a role in determining the outcome of that encounter with antigen. Costimulatory receptors such as CD27, CD134 (OX40), CD137 (4-1BB), glucocorticoid-induced tumor necrosis factor receptor (GITR), herpes virus entry mediator (HVEM), and inducible costimulator (ICOS) enhance T-cell proliferation, cytokine secretion, and survival. Their ligands (CD70, CD1334-L, CD137-L, GITRL, LIGHT, and ICOS-L, respectively) generally are expressed by APCs (B cells, DCs, macrophages). Each of these costimulatory molecules has been targeted in animal models and has shown promise for the augmentation of antitumor immunity and, in some cases, has progressed to clinical evaluation (reviewed41). Although somewhat smaller in number, the coinhibitory receptors have come to play a much larger role than costimulators in preclinical and clinical investigations of immune modulation. Unlike CD28, CTLA-4 is not expressed constitutively on the T-cell surface but is upregulated and expressed on the surface on TCR ligation.37,42 Because of its higher affinity for B7-1 and B7-2, CTLA-4 effectively 5 The Immune System in Melanoma Initiation and Progression outcompetes CD28 for ligand binding and acts to block Tcell activation and proliferation36 (reviewed41,43). Preclinical studies of anti-CTLA-4 blocking monoclonal antibodies (mAbs) demonstrate that CTLA-4 blockade can enhance CD8+ T-cell responses44 and mediate the rejection of established tumors in mice.45,46 In addition to CTLA-4, programmed cell death protein-1 (PD-1)47 has also emerged as an important target for the modulation of antitumor immunity (Figure 2). PD-1 is found on the surface of activated T cells, as well as on B cells, DCs, NK T cells, and monocytes. PD-1 ligation initiates a series of events that result in the attenuation of signals stemming from TCR ligation. Unlike the massive and generalized lymphoproliferation seen in CTLA-4 knockout mice, PD-1 knockout mice show milder and antigen-restricted autoimmunity that varies in a strain-dependent manner.48,49 Expression of the ligands for PD-1, PD-L1 (B7H1) and PD-L2 (B7-DC) is not restricted to lymphocytes. Importantly, PD-1 ligands are upregulated in hematopoietic, epithelial, and endothelial cells in response to inflammatory cytokines (e.g., type I and type II interferons).50-52 PD-L1 expression has also been shown in a variety of tumor types,53-56 including melanoma.57 Although less fully characterized, PD-L2 expression is also upregulated on a variety of normal and malignant cells in response to certain proinflammatory signals. Because of the timing and localization of PD-1 expression and its ligands, PD-1 is hypothesized to help attenuate peripheral T-cell responses during the latter stages of pathogen clearance during infections and to prevent autoimmunity, but also to limit Tcell responses to persistent antigens.58-61 Blocking PD-1 signaling on T cells in animal models can restore CD8+ T-cell responses in models of chronic infection 61 and mediate tumor rejection.62,63 Conversely, the engineered expression of PD-L1 by tumor cell lines was shown to limit effective CD8+ T-cell–mediated tumor rejection.53 Other coinhibitory receptor/ligand interactions are also potential targets for immunomodulation. These include the B7-H4 ligand (B7S1, B7x), which is found on activated B and T cells as well as monocytes.64,65 Although a T-cell– expressed receptor for B7-H4 has not yet been definitively identified, the blockade of B7-H4 using mAbs has been shown to enhance T-cell activation and effector function in vitro and in vivo.64-66 B7-H4 expression has been demonstrated in a range of cancers.66-69 In addition, expression of B7-H4 by tumor-associated macrophages (TAMs) has been shown to play a role in suppressing antitumor immunity.70 Expression of lymphocyte activation gene-3 (LAG-3) is upregulated on T cells after activation and competes with CD4 for binding to MHC II, delivering a signal that inhibits T-cell proliferation and cytokine secretion.71,72 In addition to its expression on activated CD4+ T cells, LAG-3 expression is also seen in some Treg cells. Thus, blockade of LAG-3 signaling is believed to promote antitumor effector function both by restoring T-cell activation and inhibiting Treg-mediated suppression.73,74 The Unique Relationship Between Melanoma and Host Immunity Although various mechanisms of immune activation against cancer have been targeted for virtually all tumor types, melanoma historically has drawn the most attention for the development of immunologically based therapeutic regimens. The relative breadth of melanoma-specific immunotherapies has its roots in three primary B7.1/2 CD28 B7.1/2 CD28 observations. T cell still in + + First is the observation secondary APC APC lymphoid tissue that a significant proportion of individual melanoma tuSignal 1 Signal 1 mors undergo spontaneous – regression, and there is evCTLA-4 idence to suggest that reActivation of naïve Antigen experienced or resting T cells T cell gression is associated with an immunologic tumor infiltrate.75-77 Whether a simiB7.1/2 CD28 lar rate of (presumably) Tissue + Traffic to or immune-mediated tumor DC periphery tumor rejection occurs in other Signal 1 Signal 1 tumor types is currently unknown; however, the lo– calization and pigmentaPD-L1 PD-1 tion of melanomas make Activation of naïve Antigen experienced these spontaneous regresor resting T cells T cell Inflammation sions more apparent. Figure 2. CTLA4 and PD1 checkpoints act to regulate different elements of the T-cell Second, the observation response. of spontaneous melanoFrom Pardoll D. Cancer immunology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE, ma regression is often aseds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia: Churchill Livingstone; 2014:88. sociated with vitiligo, an 6 autoimmune-mediated depigmentation of the skin. Vitiligo is often accompanied by the presence of autoantibodies against self-antigens expressed by both melanocytes and melanoma cells.78,79 In addition, tumor-associated antigens (TAAs) from melanoma were among the first tumor-specific antigens to be identified. In melanoma, TAAs tend to fall into three main categories: lineage specific or differentiation antigens (those expressed by both normal and malignant melanocytes), cancer-testis antigens (those expressed during tissue development, but absent from adult tissues–except the testis and placenta), and overexpressed/mutated proteins (those expressed at higher levels or mutated in tumor cells relative to normal cells). Melanocyte lineage-specific TAAs (reviewed80-82) include gp75, gp100, Melan A/MART1, tyrosinase, and TRP-1. Cancer-testis antigens relevant to melanoma include the BAGE, GAGE, and MAGE family proteins as well as NY-ESO-1. Other important melanoma TAAs include mutated forms of β-catenin and CDK4. Although our understanding of the factors mediating spontaneous regression of melanoma is still very limited, including what is effectively only the assumption of a role for the immune system in mediating that regression, the relative wealth of melanoma-specific TAA and the availability of effective animal models of melanoma historically have favored the development of melanoma-specific immunotherapies. The Complex Balance Between Immune Activation and Suppression in Melanoma Nonspecific T-Cell Activation The advances that promoted the re-emergence of immunosurveillance-immunoediting along with the studies identifying melanoma TAAs and the characterization of melanoma-specific immune responses set the stage for the development of immunotherapies designed to activate pre-existing melanoma-specific immunity. Among the first such immunotherapies was the use of IFN-α and IL-2 as adjuvant therapy for melanoma. INF-α is known to activate NK cell-mediated cytotoxicity and to enhance antigen presentation to T cells. Similarly, IL-2 promotes T-cell proliferation and survival and mediates T-cell differentiation into effector cells. Both are presumed to activate innate effector responses to tumor cells as well as the generation of tumor-specific adaptive immunity. Clinical trials of adjuvant therapy with IFN-α and IL-2 in the 1980s and 1990s showed sufficient improvements in relapse-free survival (IFN-α) and overall survival and durable regression (IL-2) to merit FDA approval. Given the role of each of these cytokines in the nonspecific activation of immunity, it is not surprising, however, that both cytokines have significant associated toxicities related to the activation of immunity to auto-antigens (in addition to melanoma-specific antigens) and nonspecific inflammation leading to capillary leak syndrome and, thus, require careful monitoring and compensatory actions in the clinical therapeutic setting (reviewed83). The Immune System in Melanoma Initiation and Progression Modulating the summative coreceptor signaling (signal 2) during T-cell activation also provides an opportunity to expand endogenous melanoma-specific T cells. As the first and best characterized T-cell coinhibitory receptor, CTLA-4 has been an important target for immunotherapeutic intervention. In the late 1990s, CTLA-4 blocking was shown to mediate the spontaneous rejection of immunogenic tumors in mice45 as well as the enhancement of vaccine-induced antitumor immunity in less immunogenic models84-86 without the generalized lymphoproliferation seen in CTLA-4 knockout mice. Clinical testing of a humanized CTLA-4-blocking mAb was initiated in 2008, and FDA approval was eventually granted in 2011.87-90 Like IL-2 therapy, treatment with anti-CTLA-4 provides some clinical benefit; however, it also elicits the induction of autoinflammation (colitis and hypophysitis)89,91-93 that must be carefully managed. Active (Antigen-Specific) Vaccination and Adoptive Transfer Following the advent of systemic cytokine immunotherapies for melanoma, a variety of approaches aimed at activating adaptive immunity (primarily focused on the development of CD8+ cytotoxic T cells) targeting specific melanoma TAAs or multiple TAAs have developed. Given that melanoma antigens were among the first human TAAs identified in the early 1990s,80,81,94 it is not surprising that these became the focal point for initial vaccination efforts. Further characterization of TAAs led to the discovery of individual epitopes known to bind to human MHC I and MHC II molecules (human leukocyte antigens [HLAs]). Using peripheral T cells from melanoma patients, peptides derived from Melan A/Mart-1 and gp100 were shown to activate T-cell–specific immune responses both in vitro and in vivo95-97; however, there was no direct correlation between ex vivo T-cell activation and objective clinical response.98,99 In many of these early clinical studies, putative immunogenic peptides were delivered in the absence of adjuvant (which provides the immunologic “danger” signals necessary to evoke signal 2 during T-cell activation), potentially leading to tolerization of endogenous melanoma-specific T-cell populations rather than activation. Overall, vaccination with melanoma-specific peptides has been proven safe but has not shown significant clinical benefit. Using current technologies, immunogenic peptides for clinical use can be produced relatively inexpensively; however, the use of individual peptides (alone or even in combination) restricts the potential pool of activated melanoma-specific T cells. Moreover, peptide vaccines are, by definition, restricted to individual HLAtypes, thereby limiting their application to only those patients who express the relevant HLA molecule(s). To provide more potent immunostimulation with peptide vaccines, many have turned to pulsing peptide antigens directly onto ex vivo activated DCs. Initial studies using animal models in the early 1990s demonstrated that DCs derived from peripheral blood could be used to 7 The Immune System in Melanoma Initiation and Progression elicit peptide-specific T-cell responses.100,101 The identification of culture methods leading to the generation of significant quantities of activated DCs from peripheral blood monocytes in the mid 1990s further advanced the field,102 ushering in clinical trials of DC-based immunotherapies for melanoma103-106 and a range of other cancer types and leading to FDA approval of sipuleucel-T (an autologous DC vaccine preparation) for prostate cancer in 2010.107 With the advent of more careful characterization of DC subtypes, investigators currently are characterizing the differences in T-cell responses elicited by plasmacytoid DCs and myeloid DCs compared with monocyte-derived DCs and their implications for melanoma immunotherapy.108,109 To overcome the limited scope of antigenic targets associated with peptide-based vaccines, various vaccine platforms have been developed to target whole-protein TAAs and multicomponent TAAs, allowing for endogenous processing and presentation of potentially multiple antigenic epitopes on endogenous HLA alleles. DNA-based vaccines can encode one or more TAAs and also contain CpG motifs within the DNA vaccine that activate a specific pattern recognition receptors on innate effector cells (toll-like receptor 9), providing a strong adjuvant effect.110 Preclinical studies of DNA vaccines targeting a range of melanoma antigens in the B16 mouse model have shown protective effects.111-113 DNAbased vaccines generally are delivered intradermally or intramuscularly and are thought to result in transcription and translation of the encoded antigen directly by APCs (DCs, in particular) and/or indirectly through the transfection of bystander cells in the area of vaccination.114 DNA vaccines have shown safety and efficacy for the prevention of certain infectious diseases; however, clinical trials of a melanomaspecific DNA vaccine were discontinued when the drug did not meet expectations.115-121 Other vector-based methods to evoke melanoma-specific immunity include the use of recombinant viruses122 and bacteria.123-125 The use of recombinant pathogens to deliver TAAs provides the advantage of introducing one or more antigenic melanoma proteins in the context of PAMPs, providing a potent adjuvant effect for the generation of melanoma-specific immunity; however, these approaches also induce highly potent neutralizing antibodies to the vector (virus or bacteria), which can diminish the effectiveness of subsequent vaccinations. The use of whole-cell vaccination (reviewed126-128), which includes autologous vaccine preparations derived from patient tumor samples as well as allogeneic vaccines comprised of well-defined cell lines, similarly provide the opportunity to elicit immunity across a range of potential melanoma antigens. In particular, whole-cell vaccines can include a range of known and as yet undefined melanoma antigens. As with peptide-based vaccines, vector-based vaccines have shown promise in preclinical models and are able to elicit melanoma-specific T-cell responses129,130 but have shown only limited efficacy in clinical application.131,132 To bypass altogether the difficulties inherent in eliciting potent antitumor immunity in vivo, many have turned to the ex vivo activation and expansion of tumor-specific CD8+ cytotoxic T cells. Melanoma in particular has been associated historically with the presence of tumorinfiltrating lymphocytes (TILs). Adoptive T-cell therapy (ACT) using expanded, patient-derived TIL preparations was first developed by the Rosenberg lab in the 1980s.133 This approach has evolved to include not only the isolation of tumor-specific T cells from TILs (and peripheral blood), their ex vivo expansion and readministration to the patient, but also two methods through which patient lymphocytes are modified for more potent antitumor effect. One approach addresses the relatively low proportion of tumor-specific T cells in patient-derived samples by modifying peripheral blood lymphocytes (PBLs) to express a melanoma-specific TCR. The recombinant TCRexpressing T cells are then adoptively transferred back to the patient. The use of chimeric antigen receptors (CARs) takes this approach a step further, bypassing the need for TCR-peptide/MHC engagement by modifying T cells to express an engineered receptor composed of a single-chain antibody against a melanoma-specific antigen fused with an intracellular signaling domain (e.g., CD3 or Fc receptor) to elicit T-cell–mediated cytotoxicity when the singlechain antibody engages its cognate antigen on tumor cells. Of the three approaches, ACT has been studied far more extensively and has shown promising results,134,135 whereas TCRs and CARs have been less well studied to date.136-139 Overcoming of Treg-Mediated Suppression The challenges associated with activating melanomaspecific T cells (those that have survived central tolerance mechanisms) are many; however, the challenges do not end with activation. Whether activated spontaneously, through active vaccination (by various means), or through ex vivo manipulation, tumor-specific CD8+ cytotoxic T cells must persist, proliferate, and carry out effector functions in vivo to exert a productive antitumor effect. Animal studies using the B16 melanoma model have shown that CD4+ CD25+ Treg cells actively suppress the effector function of tumor-specific CD8+ T cells in tumor-bearing mice.140 Similar studies in separate mouse melanoma models showed that depletion of Treg was critical for successful tumor eradication after adoptive transfer of tumorspecific CD8+ T cells and IL-2.141,142 Simply depleting all CD4+ CD25+ T cells is problematic, however, because CD25+ is also expressed on effector T-cell populations. Moreover, Treg populations in humans appear to uniformly express CD25 or Foxp3, another surface receptor associated with the Treg phenotype.143 Early studies of Treg populations isolated from melanoma patients indicated antigen-specific suppression associated with CD4+ CD25+ T cells expressing IL-10.144 Treg cells expressing the immunosuppressive cytokines IL-10 and TGF-β have been isolated from patients with metastatic melanoma with various surface phenotypes (CD4+ CD25+, CD4+ CD25+, and CD4+ CD25hi Foxp3+).145-147 There currently are no clear mechanisms to specifically deplete Treg populations in humans, although more generalized approaches to achieve 8 nonmyeloablative lymphodepletion have been used.148,149 In addition, further characterization of putative Treg surface markers such at LAG-3 and GITR may lead to the development of Treg-depleting therapeutics.41 Immune suppression within the tumor microenvironment is also mediated through the action of myeloidderived suppressor cells (MDSCs), which are believed to be derived in conditions of chronic inflammation.150,151 MDSCs can suppress immune effector function (both innate and adaptive) by depleting arginine and tryptophan from the local environment152 or through the production of immunosuppressive cytokines such as IL-10 and TGF-β.153 Because the presence of MDSCs within some tumors is correlated with a poor prognosis,154,155 countering their suppressive effects is an area of intense effort.156-158 Modulation of the Effector Response In addition to the indirect immunosuppressive mechanisms at work in the tumor microenvironment described previously, tumor cells can directly suppress T-cell effector function through their surface expression of PD-L1. Although autoimmunity in PD-1 knockout mice was mild compared with that seen in CTLA-4 knockout mice,48,49 subsequent studies of PD-L1/PD-L2 knockout mice predisposed to autoimmunity showed rapid, organ-specific autoimmunity,159 suggesting a peripheral role for PD-1 in limiting T-cell effector function. Consistent with this hypothesis, preventing PD-1 mediated signaling was shown to enhance vaccine response and even reverse aspects of clonal exhaustion in T cells.61 A role for PD-1 blockade in tumor immunotherapy was first demonstrated in 2002, when PD-L1 expression was shown in melanoma as well as several other human cancers and its expression on tumor cells was shown to suppress tumor-specific T-cell responses in mice.53,160 Clinical trials of several anti-PD-1 biologics have been initiated, and the approach has shown promise (reviewed60). There are currently four PD-1-targeted therapeutics under clinical evaluation; three of these are mAbs, and one is a fusion protein composed of the ligand PD-L2 on a human IgG1 backbone. In addition to evaluating the safety and efficacy of anti-PD-1 therapeutics in the clinical management of melanoma, these agents also are being evaluated for the treatment of other solid and hematologic malignancies as well as for the reversal of T-cell exhaustion associated with chronic infection. By comparison with anti-CTLA-4, which is thought to nonspecifically promote T-cell activation/proliferation and thereby predispose the patient to autoinflammation, anti-PD-1 has not shown the same severity of autoinflammation (pneumonitis).60 Conclusion Beginning in the late 1980s, technological advances in gene manipulation significantly accelerated our understanding of the basic immunologic principals underlying the generation of immune response, particularly with respect to T-cell activation and effector function. This paved the way for the first truly productive forays into the generation of The Immune System in Melanoma Initiation and Progression immunotherapies for cancer, with particular emphasis on melanoma, in which broad promotion of endogenous antitumor immune responses was sought. The subsequent discovery of a range of T-cell costimulatory and coinhibitory receptors in the early 1990s sparked the development of the next generation of improved approaches or melanoma immunotherapy and also directed our attention not only to the complexities of T-cell activation, but also the myriad of ways in which peripheral tolerance mechanisms–particularly within the tumor microenvironment–serve to further limit antitumor effector function. These advances sought to elicit more specific antitumor immunity through vaccination and also to promote T-cell expansion through the elimination of coinhibitory signaling. As we move towards the third generation of immunotherapeutic development, we must now expand our perspective once again, taking into account our evolving understanding of T-cell activation to more carefully orchestrate and integrate the multiple immunomodulatory signaling pathways to achieve a more effective and durable antitumor immune response. 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