Overview

The exquisite capacity of the adaptive immune system to be used as a therapeutic tool against cancer is based on two features. First, the diversity of receptors in the adaptive immune system (T-cell receptor for T cells and antibodies made by B cells) offers unparalleled capacity for target specificity, far greater than any synthetic drug library. Second, diverse cell killing weaponry from both the innate immune system and cytotoxic T cells offer the potential to kill any cell, once it is appropriately recognized. Central to the concept of successful cancer immunotherapy are the dual tenets that tumor cells express an antigenic profile distinct from their normal cellular counterparts and that the immune system is capable of recognizing these antigenic differences.

Support for this notion originally came from animal models of carcinogen-induced cancer in which it was demonstrated that a significant number of experimentally induced tumors could be rejected upon transplantation into syngeneic immunocompetent animals.1–4 Extensive studies by Prehn and Main⁵ on the phenomenon of tumor rejection suggested that the most potent tumor rejection antigens were unique to the individual tumor. The recent wave of cancer genomics and epigenomics illustrates the unique antigenicity of cancer cells. In addition to mutations, deletions, amplifications, and rearrangements—the ultimate neoantigens—aberrant expression of membrane molecules can provide a therapeutic window for tumor-targeted antibodies. For example, overexpression of membrane molecules including “oncogenic” growth factor receptor tyrosine kinases such as HER2/neu and epidermal growth factor receptor (EGFR) via epigenetic mechanisms has provided clinically relevant targets for one arm of the immune system—antibodies.^6,7 Indeed, tumor-targeted monoclonal antibodies are an ever-growing class of cancer therapeutic agents obtaining approval from the U.S. Food and Drug Administration (FDA).

In striking contrast, “cellular immunotherapy” of cancer aimed at activating antitumor T cells to kill tumors showed disappointing results in clinical trials until the past 5 years. Emerging insights about the nature of the interaction between the cancer and the immune system have led us to understand why cell-based cancer immunotherapy approaches such as therapeutic vaccines have been less potent against established cancer than originally imagined. In general, we have learned that tumors use mechanisms of tolerance induction to turn off T cells specific for tumor-associated antigens. Oncogenic pathways in tumors result in the elaboration of factors that organize the tumor microenvironment in ways that are quite hostile to antitumor immune responses. It is now established that cancers possess multiple immune resistance mechanisms related to the complex systems of physiological immune regulation and self-tolerance induction. In the case of T cells, the ultimate amplitude and quality of the T-cell response initiated through antigen recognition by the T-cell receptor (TCR) is regulated by a balance between co-stimulatory and co-inhibitory signals, which is sometimes termed “immune checkpoints.” Under normal physiological conditions, immune checkpoints are critical to the maintenance of self-tolerance (i.e., prevention of autoimmunity) and also to protect tissues from damage when the immune system is responding to pathogenic infection. Their expression can be dysregulated by tumors as an important mechanism to mute effector immune responses that “threaten” them. T cells have been the major focus of efforts to therapeutically manipulate endogenous antitumor immunity because of their capacity for selective recognition of peptides derived from proteins in all cellular compartments, their capacity to directly recognize and kill antigen-expressing cells (CD8 killer T cells), and their ability to orchestrate diverse immune responses (CD4 helper T cells), which integrates adaptive and innate effector mechanisms.

In addition to resistance to potential immune responses used by established tumors, we now know that distinct forms of inflammatory and immune responses exist that are procarcinogenic. Thus two frontiers in cancer immunology are the elucidation of how the tumor organizes its immune microenvironment and the nature of distinct types of immune responses that are anticarcinogenic versus procarcinogenic. As the receptors, ligands, and signaling pathways that mediate immune tolerance and immune-induced procarcinogenic events are elucidated, these factors and pathways can be selectively inhibited by both antibodies and drugs in a way to shift the balance to antitumor immune responses.

This chapter will outline the major features of tumor-immune system interactions and set the stage for molecularly based approaches to manipulate immune responses for successful cancer therapy.

The Antigenic Profile That Distinguished Tumors From Normal Tissues

As previously described, tumors differ fundamentally from their normal cell counterparts in both antigenic composition and biological behavior. Genetic instability, a basic hallmark of cancer, is a primary generator of tumor-specific antigens. Data accrued for more than a thousand human cancers to date demonstrate unequivocally that altered genetic and epigenetic features of tumor cells indeed result in a distinct tumor antigen profile. Cancers express between 50 and 1000 missense mutations in coding regions, roughly 20% of which can potentially create neoantigenic peptides presented by at least one of the individual’s human leukocyte antigen (HLA) alleles and thus recognized by T cells.8–16 Additionally, deletions, amplifications, and chromosomal rearrangements can result in new genetic sequences resulting from juxtaposition of coding sequences not normally contiguous in untransformed cells. The vast majority of these mutations occur in intracellular proteins, and thus the “neoantigens” they encode would not be readily targeted by antibodies. However, the major histocompatibility complex (MHC) presentation system for T-cell recognition makes peptides derived from all cellular proteins available on the cell surface as peptide MHC complexes capable of being recognized by T cells.

In accordance with the original findings of Prehn and Main,⁵ the vast majority of tumor-specific antigens derived from mutation as a consequence of genetic instability are unique to individual tumors. The consequence of this fact is that antigen-specific immunotherapies targeted at most truly tumor-specific antigens would by necessity be patient specific. However, there are a growing number of examples of tumor-specific mutations that are shared. The three best studied examples are the Kras codon 12 G->A (found in roughly 40% of colon cancers and >75% of pancreas cancers), the BrafV600E (found in roughly 50% of melanomas), and the p53 codon 249 G->T mutation (found in ~50% of hepatocellular carcinomas).^17–20 As with nonshared mutations, these common tumor-specific mutations all occur in intracellular proteins and therefore require T-cell recognition of MHC-presented peptides for immune recognition. Indeed, both the Kras codon 12 G->A and the BrafV600E mutations result in “neopeptides” capable of being recognized by HLA class I– and class II–restricted T cells.^21–24

The other major difference between tumor cells and their normal counterparts derives from epigenetics.²⁵ Global alterations in DNA methylation and chromatin structure in tumor cells results in dramatic shifts in gene expression. All tumors overexpress hundreds of genes relative to their normal counterparts, and in many cases, they turn on genes that are normally completely silent in their normal cellular counterparts. Overexpressed genes in tumor cells represent the most commonly targeted tumor antigens by both antibodies and cellular immunotherapies because, in contrast to most antigens derived from mutation, overexpressed genes are shared among many tumors of a given tissue origin or sometimes multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated patients with pancreatic cancer,²⁶ is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers.^27,28 Although mesothelin is expressed at low to moderate levels in the pleural mesothelium, it is not expressed at all in normal pancreatic or ovarian ductal epithelial cells.

The most dramatic examples of tumor-selective expression of epigenetically altered genes are the so-called cancer-testis antigens.²⁹ These genes appear to be highly restricted in their expression in the adult. Typically, they are expressed almost exclusively in germ cells of the testis and ovaries. Expression in the testis appears to be restricted to germ cells, and in fact, some of these genes appear to encode proteins associated with meiosis.^32–32 Cancer-testis antigens therefore represent examples of widely shared tumor selective antigens whose expression is highly restricted to tumors. Many cancer-testis antigens have been shown to be recognized by T cells from nonvaccinated and vaccinated patients with cancer.²⁹ From the standpoint of immunotherapeutic targeting, a major drawback of the cancer-testis antigens is that none appears to be necessary for the tumor’s growth or survival. Therefore their expression appears to be purely the consequence of epigenetic instability rather than selection and antigen-negative variants are easily selected out in the face of immunotherapeutic targeting.

A final category of tumor antigen that has received much attention encompasses tissue-specific antigens shared by tumors of similar histologic origin. Interest in this class of antigen as a tumor-selective antigen arose when melanoma-reactive T cells derived from patients with melanoma were found to recognize tyrosinase, a melanocyte-specific protein required for melanin synthesis.33,34 In fact, commonly generated melanoma-reactive T cells from patients with melanoma recognize melanocyte antigens.³⁵ Although one cannot formally call tissue-specific antigens tumor specific, they are nonetheless potentially viable targets for therapeutic T-cell responses when the tissue is dispensable (i.e., prostate cancer or melanoma). Because melanoma is the easiest cancer from which to grow tumor-specific T cells, Anichini and colleagues³⁶ were able to demonstrate the existence in the same patient of melanoma-specific T cells recognizing tumor-specific (presumably from mutations) and shared antigens.

From the standpoint of T-cell targeting, tumor antigens upregulated as a consequence of epigenetic alterations represent “self-antigens” and are therefore likely to induce some level of immune tolerance. However, it is now clear that the stringencies of immune tolerance against different self-antigens differ according to tissue distribution and normal expression level within normal cells. The mesothelin antigen previously described is such an example. In a recent set of clinical pancreatic cancer vaccine studies, mesothelin-specific T-cell responses were induced by vaccination with genetically modified pancreatic tumor cell vaccines, and induction of mesothelin-specific T cells correlated with ultimate disease outcome.³⁷ Given that the immune system is capable of differential responsiveness determined by antigen levels, it is quite possible to imagine generating tumor-selective immune responses against antigens whose expression level in the tumor is significantly greater within normal cells in the tumor-bearing host. Additionally, upregulated antigens that provide physiologically relevant growth or survival advantages to the tumor are preferred targets for any form of therapy because they are not so readily selected out.

Beyond the antigenic differences between tumor cells and normal cells, there are important immunologic consequences to the distinct biological behavior of tumor cells relative to their normal counterparts. Whereas uncontrolled growth is certainly a common biological feature of all tumors, the major pathophysiological characteristics of malignant cancers responsible for morbidity and mortality are their ability to invade through natural tissue barriers and ultimately to metastasize. Both of these characteristics, which are never observed in nontransformed cells, are associated with dramatic disruption and remodeling of tissue architecture. Indeed, the tumor microenvironment is quite distinct from the microenvironment of normal tissue counterparts. One of the important consequences of tissue disruption, even when it is caused by noninfectious mechanisms, is the elaboration of proinflammatory signals. These signals, generally in the form of cytokines and chemokines, are potentially capable of naturally initiating innate and adaptive immune responses. Indeed, the level of leukocyte infiltration into the microenvironment of tumors is often greater than the leukocyte component of their normal tissue counterparts. Cancers are therefore constantly confronted with inflammatory responses as they invade tissues and metastasize. In some circumstances these inflammatory and immune responses can potentially eliminate so-called tumor immune surveillance. However, as will be discussed, oncogenic pathways in the tumor appear to organize the immunologic component of the microenvironment in a fashion that not only protects itself from antitumor immune responses but also can qualitatively shift immune responses to those that actually support and promote tumor growth. Thus tumors can entice the immune system to the “dark side.” It is these elements of the cancer-immune system interaction that will be the central targets of future immunotherapeutic strategies.

Evidence Pro and Con for Immune Surveillance of Cancer

The fundamental tenet of the immune surveillance hypothesis, which was first conceived nearly a half century ago,38,39 is that a fundamental role of the immune system is to survey the body for tumors as it does for infection with pathogens, recognizing and eliminating them based on their expression of tumor-associated antigens. In animal models, carcinogen-induced tumors can be divided into those that grow progressively (termed “progressor tumors”) and those that are rejected after an initial period of growth (termed “regressor tumors”).^2,3 The phenomenon of regressor tumors was thought to represent an example of the ongoing process of immune surveillance of cancer. A corollary to the original immune surveillance hypothesis is that progressor tumors in animals (presumed to represent clinically progressing cancers in humans) fail to be eliminated because they develop active mechanisms of either immune escape or resistance (Fig. 6-1).

A fundamental prediction of the immune surveillance hypothesis is that immunodeficient individuals would display a dramatic increase in tumor incidence. After an extensive analysis of spontaneous tumor formation in immunodeficient nude mice, which have atrophic thymi and therefore significantly reduced numbers of T cells and T-cell dependent immune responses, no increased incidence of tumors was observed.40–44 The findings of these studies were taken to be a major blow to the immune surveillance hypothesis. However, a caveat to the interpretation of these results is that nude mice still produce diminished numbers of T cells via thymus-independent pathways and therefore can mediate some degree of T-cell–dependent immunity. In addition, nude mice frequently display compensatory increases in innate immunity which, as will be discussed later, may represent a potent form of antitumor immunity and could contribute to immune surveillance of cancer.

Epidemiological studies of patients with heritable immunodeficiencies revealed a significantly increased risk of certain cancers that are distinct from the epithelial cancers commonly observed in normal immunocompetent adults.47–47 Many of these cancers are also observed in patients receiving a transplant who are undergoing chronic pharmacologic immune suppression, as well as in patients with human immunodeficiency virus/acquired immunodeficiency syndrome whose immune system is depressed. The most common cancers in these individuals include lymphoblastic lymphomas and Kaposi sarcoma; however, certain epithelial cancers, such as stomach cancer, are also observed at increased frequency. A common theme for most cancers observed in immunodeficient individuals is a microbial stimulus. Most lymphoplastic lymphomas are Epstein-Barr virus–associated lymphomas,⁴⁸ and Kaposi sarcoma is a result of infection with Kaposi sarcoma herpesvirus.⁴⁹ Other virus-associated cancers such as cervical cancer (from human papillomavirus)^50,51 are also observed at increased frequency. It is now appreciated that stomach cancer is associated with ulcer disease related to infection with the bacterium Helicobacter pylori.⁵² From these studies, the notion emerged that immune surveillance indeed protects persons against certain pathogen-associated cancers by either preventing infection or altering chronic infection by viruses and other microbes that can eventually induce cancer. The findings of these studies were taken to represent evidence that the common nonpathogen-associated cancers most commonly seen in adults in developed countries (e.g., prostate cancer, colon cancer, and lung cancer) are not subject to immune surveillance.

Two caveats to this interpretation should be noted, however. First, detailed epidemiological analyses of immunodeficient individuals were performed at a time when these patients rarely lived beyond their 20s and 30s, when cancer incidence normally increases most significantly. It is therefore possible that a more subtle cumulative increased incidence of common nonpathogen-associated cancers would have been observed had these persons lived further into adulthood. Indeed, more recent analyses definitively demonstrate an increase incidence of some nonpathogen-associated cancers in immunodeficient individuals, particularly melanoma.⁵³ In addition to epidemiological data, dramatic anecdotal examples are difficult to ignore. It has been reported that in patients who received kidneys from a cadaver donor that had been in complete remission from a melanoma prior to organ donation, metastatic melanoma of donor origin developed rapidly after the transplant.^56–56 These results indicate that at least for some nonpathogen-associated tumors, the immune system can play a significant role in maintaining the micrometastatic disease in a dormant state. Whether this principle applies to nonpathogen-associated human tumors besides melanoma remains to be demonstrated.

Several recent studies reevaluating tumor immune surveillance in genetically manipulated mice have revealed clear-cut evidence that various components of the immune system can at least modify, if not eliminate, both carcinogen-induced and spontaneously arising cancers. In a series of studies, Schreiber and colleagues59–59 reexamined cancer incidence in mice rendered immunodeficient via genetic knockout of either the RAG2 gene (deficient in both B and T cells), the γ-interferon receptor gene (IFNGR1), the STAT1 gene, or the type 1 interferon receptor gene (IFNAR1). When these knockout mice were either treated with carcinogens or crossed onto a cancer-prone p53 knockout background, the incidence of cancers was modestly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (longer than 1 year). Transplantation studies demonstrated that direct γ-interferon (γ-IFN) insensitivity by the developing tumors played a significant role in the defect in immune surveillance. Interestingly, in contrast to IFNGR1 knockout mice, the mechanism for increased tumor incidence in tumors in IFNAR1 knockout mice did not involve sensitivity by the tumor to type-1 IFNs but rather reflected the role of the type-1 IFNs in induction of innate and adaptive immunity. Even animals not crossed onto a cancer-prone genetic background or treated with carcinogens developed an increased incidence of invasive adenocarcinomas when observed over their entire life span. Furthermore, a broader spectrum of tumors developed in γ-IFN × RAG2 double-knockout mice than in RAG2 knockout mice. All of the tumors that arise in these genetically manipulated immunodeficient animals behave as regressor tumors when transplanted into immunocompetent animals. These findings indeed suggest that tumors that arise in immunodeficient animals would have been eliminated had they arisen in immunocompetent animals. The relatively subtle effects on tumorigenesis, requiring observation over the life span of the animal, suggests that the original concept of immune surveillance of tumors arising on a daily basis is in fact not correct. Instead, it is clear that the presence of a competent immune system “sculpts” the tumor through a process that has been termed “immunoediting.”

The immunoediting hypothesis has been somewhat controversial, with differing outcomes in different animal models. One of the caveats in the interpretation of these studies comes from the work of Enzler and Dranoff,⁶⁰ who studied mechanisms of increased tumorigenesis in granulocyte-macrophage colony-stimulating factor (GM-CSF) × γ-IFN double-knockout mice. Although they observed an increase in gastrointestinal and pulmonary tumors, they noted that such animals harbored infection with a particular bacterium not normally observed in immunocompetent animals. Maintenance of these double-knockout mice on antibiotics essentially eliminated the increased rate of tumor formation. Thus it is possible that some of the increased tumor rates in genetically immunodeficient animals could be related to unappreciated chronic infections that develop in these animals, which are not housed under germ-free conditions. Nonetheless, although the classic concepts of immune surveillance of cancer remain unsupported by experimental evidence, studies on tumorigenesis in genetically manipulated immunodeficient mice suggest that developing tumors must actively adapt themselves to their immune microenvironment to exist within the context of a competent immune system.

One of the approaches to test the immune surveillance and immunoediting of endogenously arising tumors has been to combine genetically engineered autochthonous tumor models with T-cell receptor transgenic models expressing defined marked T cells specific for a tumor antigen (either the transgenic oncogenic driver protein in the tumors or an antigen co-expressed with the oncogenic driver). In these models, tumor growth can be monitored in immunodeficient versus immunocompetent mice, as well as expression of the cognate tumor antigen recognized by the transgenic T-cell receptor. In such a tumor model driven by Kras and p53 loss, tumors emerging in immunocompetent mice either lost antigen or MHC presentation capacity, unlike tumors emerging in immunodeficient mice.⁶¹. In contrast, in a mouse model of spontaneous random oncogene activation, antigen-specific tolerance was generated in immunocompetent mice without evidence for antigen loss.⁶² In some models, an intermediate result has been observed. For example, under some circumstances, endogenous immune responses can establish an equilibrium state with the tumor in which the tumor is prevented from outgrowth in immunocompetent mice but is not completely eliminated.⁶³ Ultimately, given that experimental outcomes are different in different models (i.e., tolerance vs. surveillance vs. editing vs. equilibrium), it will be important to ascertain which mechanisms are operative in particular human cancers.

Innate Mechanisms of Tumor Immune Surveillance

Although much emphasis has been placed on the role of adaptive immunity, particularly of conventional T cells, in immune surveillance of cancer, a confluence of more recent findings points to innate immunity and epithelial immunity in the immunologic sensing of carcinogenic events in the skin, gut, and possibly other sites. Much of the evidence focuses on the NKG2D receptor. NKG2D was originally defined as an activating natural killer (NK) receptor.66–66 Most NK receptors appear to be inhibitory when engaged; this inhibition is often associated with immunoreceptor tyrosine kinase–based inhibitory motif (ITIM) domains in the cytoplasmic tails. ITIMs provide docking sites for phosphatases that oppose the activity of tyrosine kinases involved in lymphocyte activation. NK activation status is a balance between engagement of activating and inhibitory receptors. NKG2D, the best-studied activating receptor on NK cells, is somewhat unusual in that it does not contain an immunoreceptor tyrosine kinase–based activating motif (ITAM) and is associated with an adaptor molecule, DAP 10, that contains neither conventional ITIMs nor ITAMs.⁶⁷ Instead, DAP 10 contains a KYXXM motif that appears to bind to phosphatidylinositol (PI)-3 kinase upon phosphorylation of the tyrosine in this motif. NKG2D is expressed on all NK cells, as well as on some αβ and γδ T cells. Beyond NK cells, NKG2D is expressed at high levels on a number of subsets of intraepithelial lymphocytes (IELs). IELs represent a distinct population of lymphocytes residing in epithelial tissues that display features of both adaptive and innate immune responses.^68–72 They are thought to represent a major first line of defense against pathogens attempting to invade across epithelial linings exposed to the environment (i.e., skin, gut, and respiratory tract). Fifty percent of the IELs of the gut express the γδ TCR (normally expressed by <3% of circulating T cells), whereas the other 50% express the common αβ TCR. γδ TCR-expressing IELs in different compartments express a very restricted repertoire and are thought to recognize certain types of microbial antigens or potentially self-antigens associated with stress or inflammatory responses to microbial infection. Even the αβ TCR-expressing IELs have an extremely restricted TCR repertoire similar to invariant NK T cells. A significant subset of gut IELs express TCR that utilize a particular V_α and V_β and are thought to recognize a limited subset of microbial or self nonpeptide antigens presented by nonclassical class I MHC molecules. Thus NKG2D expression marks diverse subsets of lymphocytes that, although expressing different families of recognition receptors, act as components of innate immunity in that they recognize a stereotypical set of antigens associated with infection or stress (see later discussion).

The first evidence that the NKG2D receptor might play a role in tumor immune surveillance came from the finding that normal colonic epithelium and a significant proportion of tumors could express the two defined human ligands for NKG2D: MICA and MICB. MICA and MICB, which represent nonclassical MHC class I–type molecules whose structure demonstrates no antigen-binding grove characteristic of most MHC molecules, are stress-induced proteins whose genes contain stress response elements in their promoters.73,74 Gasser and colleagues⁷⁵ have demonstrated that upregulation of MICA/B is induced through the ATM/ATR/Chk1 pathway of DNA damage recognition. An analysis in human cancer suggested a correlation between expression of MICA/B and infiltration of certain subsets of γδ T cells that express NKG2D. Initially it was proposed that MICA and MICB were direct ligands for specific γδ receptors themselves as well as NKG2D,^76,77 but this idea is controversial. MICA and MICB do not have any murine orthologs, but murine NKG2D does bind to products of the retinoic acid–inducible gene family, RAE1αε, as well as the product of the H60 gene. ULBP3 is an additional NKG2D ligand to be described^78,79 and appears to represent the human homologue to the RAE1 molecules in mice. These NKG2D ligands appear to be involved in immune recognition and possibly tumor surveillance of mice.^82–82 Recognition and killing of murine skin keratinocytes or intestinal epithelial cells by γδ IEL require expression of NKG2D ligands and are blocked by anti-NKG2D antibodies. Transfection of murine tumors with genes encoding NKG2D ligands renders them susceptible to NKG2D-dependent killing by NK cells. Emerging data on NKG2D function on IELs together with the potentially stress-induced nature of its ligands suggests that the IEL system of immune surveillance may indeed be relevant to carcinogenesis, as well as infectious challenges.⁶² The major initiating event of carcinogenesis in the skin—ultraviolet light—is a potent source of DNA damage, which, as previously mentioned, has been shown to induce NKG2D ligands via the ataxia telangiectasia (ATM) pathway. Thus, in addition to endogenous killers of genome damaged cells, such as p53, IELs and NK cells may represent an extrinsic sensor of DNA damage and genotoxic stress via recognition of cells that have upregulated NKG2D ligands (Fig. 6-2).

As with the case of classic immune surveillance mediated by classical T cells, the emergence of a clinically evident cancer implies that the tumor has developed a mechanism to circumvent or evade any innate immune surveillance systems. In the case of the NKG2D system, Groh and colleagues⁸³ have provided suggestive evidence that tumors can shed MICA/B in a soluble form as a means of evading NKG2D-dependent recognition. They demonstrated that certain tumors are associated with high levels of shed MICA/B and that soluble MICA/B binds to and downmodulates NKG2D on NK cells, thereby acting as an antagonist to NKG2D activation via cell surface–bound MICA/B. Although this mechanism remains to be proven as a true evasion system for NKG2D-dependent tumor recognition, it points out the diversity of mechanisms that tumors use to evade immune recognition. It also points out straightforward approaches to block these evasion systems. If indeed soluble MICA/B represents a mechanism for tumor immune evasion of innate immune recognition, antibodies that would bind to and clear soluble MICA/B but not block the interaction between cell membrane MICA/B and NKG2D on NK cells could potentially restore the capacity of NK cells to recognize MICA/B-expressing tumors. Further evidence for the importance of soluble MICA/B as a mechanism for escape from innate tumor surveillance comes from the finding that antitumor clinical responses to vaccination of patients with melanoma can be associated with the generation of humoral immune responses that “neutralize” soluble MICA/B.⁸⁴

Immune Tolerance and Immune Evasion—the Immune Hallmarks of Cancer

Although controversy over the ultimate role of immune surveillance in natural modulation of cancer development and progression will undoubtedly continue into the future, one can summarize the current state of knowledge as supporting the notion that natural immune surveillance plays a much smaller role than originally envisioned by Thomas and Burnet. However, developing tumors need to adapt to their immunologic milieu in a manner that either turns off potentially harmful (to the tumor) immune responses or creates a local microenvironment inhibitory to the tumoricidal activity of immune cells that could inadvertently become activated in the context of inflammatory responses associated with tissue invasion by the tumor. These processes—tolerance induction and immune evasion—have become a central focus of cancer immunology efforts and will undoubtedly provide the critical information necessary for development of successful immunotherapies that break tolerance to tumor antigens and break down the resistance mechanisms operative within the tumor microenvironment.

Evidence from both murine tumor systems and human tumors strongly demonstrates the capacity of tumors to induce tolerance to their antigens. This capacity to induce immune tolerance may very well be the single most important strategy that tumors use to protect themselves from elimination by the host’s immune system. Tolerance to tumors appears to operate predominately at the level of T cells; B-cell tolerance to tumors is less certain because there is ample evidence for the induction of antibody responses in animals bearing tumors, as well as in human patients with tumors. However, with the exception of antibodies against members of the EGFR family, there is little evidence that the natural humoral response to tumors provides significant or relevant antitumor immunity. In contrast, numerous adoptive transfer studies have demonstrated the potent capacity of T cells to kill growing tumors, either directly through cytotoxic T lymphocyte (CTL) activity, or indirectly through multiple CD4-dependent effector mechanisms. It is thus likely that induction of antigen-specific tolerance among T cells is of paramount importance for tumor survival.

The first direct evidence for induction of T-cell tolerance by tumors was provided by Bogen and colleagues,85,86 who examined the response of TCR transgenic T cells specific for the idiotypic immunoglobulin expressed by a murine myeloma tumor. They first demonstrated induction of central tolerance to the myeloma protein followed by peripheral tolerance. With use of influenza hemagglutinin (HA) as a model tumor antigen, it was demonstrated that adoptively transferred HA-specific TCR transgenic T cells were rapidly rendered anergic by HA-expressing lymphomas and HA-expressing renal carcinomas.^87,88 Tolerance induction has been demonstrated in both the CD4 and CD8 compartment. In general, initial activation of tumor-specific T cells is commonly observed; however, the activated state of T cells is typically not sustained, with failure of tumor elimination as a frequent consequence. Tolerance induction among tumor antigen–specific T cells is an active process involving direct antigen recognition, although in some murine systems, tolerance to tumors appears to be associated with failure of antigen recognition by T cells; that is, the immune system “ignores” the tumor.^89,90 Beyond studies on transplantable tumors, more recent analyses of immune responses to tumor antigens in tumor transgenic mice that have developed spontaneous cancer have further emphasized the capacity of spontaneously arising tumors to induce tolerance among antigen-specific T lymphocytes. In a model of prostate tumorigenesis, Drake and colleagues⁹¹ evaluated CD4 responses to HA and double transgenic animals expressing HA and SV40 T antigen under control of the prostate-specific probasin promoter. Development and progression of prostate tumors did not result in enhanced activation of adoptively transferred HA-specific T cells. Tolerance to HA as a normal prostate antigen occurred largely through ignorance because no evidence for antigen recognition by HA-specific T cells was found. However, increased recognition was observed upon either androgen ablation (which causes massive apoptosis within the prostate) or development of prostate cancer. Nonetheless, enhanced antigen recognition was not accompanied by activation of effector functions such as γ-IFN production. Analysis of the consequences of transformation in additional tumor transgenic mouse systems has also been performed. Willimsky and Blankenstein⁶² evaluated T-cell responses and rejection in a model of sporadic induction of tumors associated with expression of a tumor-specific antigen only at the time of transformation. They found that preimmunization of mice against the tumor-associated antigen prevented the development of tumors. However, nonimmunized mice developed spontaneous tumors without any significant evidence of natural immune surveillance in the absence of preimmunization. They further demonstrated that an initial antigen-dependent activation of tumor-specific T cells could be observed at the time of spontaneous tumor induction but that this recognition ultimately resulted in an anergic form of T-cell tolerance similar to that observed by Drake and colleagues⁹¹ in the prostate system.

The capacity of spontaneously arising tumors to tolerize T cells has not been uniformly observed. A contrasting result by Nguyen and colleagues⁹² was observed when lymphocytic choriomeningitis virus (LCMV) GP33-specific TCR transgenic CD8 T cells were adoptively transferred into double transgenic mice expressing both SV40 T antigen and LCMV GP33 under control of the rat insulin promoter. Pancreatic islet cell tumors that express GP33 develop in these animals. Nguyen and colleagues⁹² found that as tumors progressed in the mice, enhanced T-cell activation occurred. It was demonstrated through bone marrow chimera experiments that CD8 T-cell activation occurs exclusively via cross presentation in the draining lymph nodes. Despite the activation of tumor-specific T cells, the tumors grew progressively, indicating that the degree of immune activation induced by tumor growth was insufficient to ultimately eliminate the tumors. These results suggest that developing tumors can induce immune responses but may titrate their level of immune activation to one that ultimately does not “keep up” with tumor progression. Such a circumstance is one that is highly susceptible to the immune editing concept put forward by Schreiber and colleagues⁵⁷ in which the tumor edits itself genetically to maintain a sufficient level of resistance to induced immune responses. In the case of the LCMV GP33 T-antigen transgenic mice, because neither anergic nor deletional tolerance was observed, animals treated with the dendritic cell stimulatory anti-CD40 antibody demonstrated significant slowing of tumor growth. Thus it may be possible under some circumstances to shift the balance between tumor immune evasion and tumor immune recognition by agents that affect the overall activation state of either antigen-presenting cells or T cells (see later discussion).

It has been more difficult to obtain definitive evidence that human cancers tolerize tumor-specific T cells because humans cannot be manipulated the way mice are. However, the T cells that are grown out of patients with cancer tend to be either of low affinity for their cognate antigen or recognize antigens that bind poorly to their presenting HLA (human MHC) molecule, resulting in inefficient recognition by T cells. Recently, the first crystal structure of the TCR-peptide-MHC trimolecular complex has been solved for an MHC class II–restricted human tumor antigen.⁹³ Interestingly, the orientation of the TCR, which is of low affinity for the peptide-MHC complex, is distinct from trimolecular complexes for viral (foreign) antigens and is partially similar to trimolecular complexes for a self-antigen. Thus there may be fundamental structural features of tumor antigen recognition that lie between those of foreign antigen and self-antigen recognition.

As will be discussed later, one of the features of the tumor microenvironment that is likely central to the capability of tumors to tolerize tumor-specific T cells is the immature or inactive state of tumor-infiltrating dendritic cells (DCs). DCs are the major antigen-presenting cells (APCs) that present peptides to T cells to initiate adaptive immune responses. In the context of infection, microbial ligands or endogenous “danger signals” associated with tissue destruction activated DCs to a state whereby they present antigens to T cells together with co-stimulatory signals that induce T-cell activation and development of effecter function. However, in the absence of microbial products or danger signals, DCs remain in an immature state in which they can still present antigens to T cells but without co-stimulatory signals. These immature DCs function as “toleragenic” DCs, inducing a state of antigen-specific T-cell unresponsiveness (termed “anergy”; Fig. 6-3). It is thought that steady-state presentation of self-antigens by immature DCs is an important mechanism of peripheral self-tolerance. Thus if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation (Fig. 6-4, A) to one of tumor-specific tolerance (Fig. 6-4, B).

Immunologic Characteristics of the Tumor Microenvironment

Ultimate understanding of the relationship between the tumor and the host immune system requires elucidation of local cross talk at the level of the tumor microenvironment. As mentioned at the outset, the hematopoietic/immune system is a major component of the tumor microenvironment. The systemic tolerance to tumor antigens begins with events that occur in this microenvironment. Beyond mechanisms that skew tumor-specific T cells toward immune tolerance, the tumor microenvironment is replete with mechanisms that dampen antitumor immune responses locally (Fig. 6-5), which represents an important barrier to successful immunotherapy even when activated effector responses can be generated with vaccines. As the specific cells and molecules within the tumor microenvironment that mediate this hostile immune environment are elucidated, inhibitors are being developed and tested to use as adjuncts to vaccination that will allow activated immune cells to function more effectively within the tumor microenvironment.

The immune inhibitory or tolerogenic nature of the tumor microenvironment can be viewed from the perspective of specific signaling pathways in cells, specific molecular interactions between soluble or membrane-bound ligands and their receptors, and via specific immune-inhibitory cell populations. These elements are actually not dissociable, but discussing them separately is useful. It is also critical to point out that none of these inhibitory cells, molecules, and signaling pathways is specific to tumors. They represent physiologic mechanisms to regulate immune responses to self-antigens and also to downmodulate immune/inflammatory responses to foreign antigens so that collateral tissue damage is limited. Tumors co-opt and upregulate them as mechanisms for resistance to immune attack.

TGF-β—A Major Inhibitory Cytokine in the Tumor Microenvironment

A major inhibitory cytokine produced by many cell types that has been implicated in blunting antitumor immune responses is TGF-β, which is produced by a variety of cell types, including tumor cells, and which has pleiotropic physiological effects. For most normal epithelial cells, TGF-β is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G1 stage.¹²⁶ In many cancer cells, however, mutations in the TGF-β pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation. Additionally, in cancer cells, the production of TGF-β is increased and may contribute to invasion by promoting the activity of matrix metalloproteinases. In vivo, TGF-β directly stimulates angiogenesis; this stimulation can be blocked by anti–TGF-β antibodies.¹²⁷ A bimodal role of TGF-β in cancer has been verified in a transgenic animal model using keratinocyte-targeted overexpression.¹²⁸ Initially, these animals are resistant to the development of early-stage or benign skin tumors. However, once tumors form, they progress rapidly to a more aggressive spindle-cell phenotype. Although this clear bimodal pattern of activity is more difficult to identify in a clinical setting, it should be noted that elevated serum TGF-β levels are associated with poor prognosis in a number of malignancies, including prostate cancer,¹²⁹ lung cancer,¹³⁰ gastric cancer,¹³¹ and bladder cancer.¹³²

From an immunologic perspective, TGF-β possesses broadly immunosuppressive properties, and widespread inflammatory pathology and corresponding accelerated mortality develop in TGF-β knockout mice.¹³³ Interestingly, a majority of these effects seem to be T-cell–mediated, because targeted disruption of T-cell TGF-β signaling also results in a similar autoimmune phenotype.¹³⁴ Recent experiments by Chen et al.¹³⁵ rather convincingly demonstrated a role for TGF-β in T_reg-mediated suppression of CD8 T-cell antitumor responses. In these experiments, adoptive transfer of CD4⁺ CD25⁺

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