Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section A – Biology and Cancer

Chapter 6 – Cancer Immunology

Drew M. Pardoll

SUMMARY OF KEY POINTS

  

   

Cancer cells develop, grow, invade, and metastasize in the context of an organized microenvironment. This is reflected by the fact that for many cancers the majority of cells within the tumor mass are nontransformed.

  

   

The relationship between the tumor cells and the nontransformed cells composing the tumor microenvironment is dynamic and active.

  

   

The immune system is a major component of the tumor microenvironment, and therefore, the tumor must actively organize the immunologic component of its microenvironment.

  

   

Because the immune response—particularly that mediated by killer T cells and cells of the innate immune system—can be a potent enemy to the tumor, a successful cancer must develop mechanisms to instruct the immune system to “tolerate” its existence, particularly when it is invading through tissue barriers and metastasizing to many organ sites.

  

   

If a tumor fails to develop these tolerance induction and immunologic resistance mechanisms, the immune system will eliminate it.

  

   

Oncogenic pathways in the tumor not only mediate cell growth, metabolic activity, and antiapoptotic activity, they also mediate interactions with the immune system.

  

   

Ultimately, tumors do more than merely inhibit effecter functions of the immune system that can be detrimental to them. Tumors can in fact alter immunologic activity to promote tumor growth and development.

  

   

We are now beginning to understand the molecular and cellular basis for tumor–immune system interactions, providing specific molecular targets for immunologic intervention.

OVERVIEW

Historically, interest in cancer immunology stemmed from the perceived potential activity of the immune system as a weapon against cancer cells. In fact, the term “magic bullet,” commonly used to describe many visions of cancer therapy, was coined by Paul Ehrlich in the late 1800s in reference to antibodies targeting both microbes and tumors. 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] [2] [3] [4] Extensive studies by Prehn on the phenomenon of tumor rejection suggested that the most potent tumor rejection antigens were unique to the individual tumor.[5]

As cancer genetics and genomics has exploded over the past decade, it is now quite clear that altered genetic and epigenetic features of tumor cells indeed result in a distinct tumor antigen profile. Overexpression of “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, monoclonal antibodies are the largest growing single class of cancer therapeutics based on successful new U.S. Food and Drug Administration approvals. In striking contrast, cellular immunotherapy of cancer has been quite disappointing in establishing therapeutic success in clinical trials thus far. 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.

Not only is the cancer capable of inducing potent tolerance among tumor-specific T cells, we now know that there are distinct forms of inflammatory and immune responses that are procarcinogenic. Thus, two frontiers in cancer immunology are the elucidation of how the tumor organizes its immune-microenvironment as well as the nature 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.

HOW DO TUMORS DIFFER FROM SELF TISSUES?

Tumors differ fundamentally from their normal cell counterparts in both antigenic composition and biologic behavior. Genetic instability, a basic hallmark of cancer, is a primary generator of tumor-specific antigens. The most common genetic alteration in cancer is mutation, which arises from defects in DNA damage repair systems of the tumor cell. [8] [9] [10] [11] [12] [13] [14] [15] Recent estimates from genome-wide sequencing efforts suggest that every tumor contains a few hundred mutations in coding regions.[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. Based on analysis of sequence motifs, it is estimated that roughly one-third of the mutations identified from genome sequencing of 22 breast and colon cancers[16] were capable of binding to common human lymphocyte antigen (HLA) alleles based on analysis of sequence motifs (J.P. Allison, personal communication).

In accordance with the original findings of Prehn,[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 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 ➙A (found in roughly 40% of colon cancers and >75% of pancreas cancers), the BRAF V599E (found in roughly 70% of melanomas) and the p53 codon 249 G➙T mutation (found in ∼50% of hepatocellular carcinomas). [17] [18] [19] [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 BRAF V599E mutations result in “neopeptides” capable of being recognized by HLA class 1- and class II-restricted T cells. [21] [22] [23] [24]

The other major difference between tumor cells and their normal counterparts derives from epigenetics.[25] Global alterations in DNA methylation as well as 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, 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. This is because, in contrast to most antigens derived from mutation, overexpressed genes are shared among many tumors of a given tissue origin or sometime multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated pancreatic cancer patients,[26] is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers. [27] [28] Whereas 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 gene are the so-called cancer-testis antigens.[29] These genes seem to be highly restricted in their expression in the adult. Many are expressed selectively in the testis of males and are not expressed at all in females. Expression in the testis seems to be restricted to germ cells, and some of these genes actually seem to encode proteins associated with meiosis. [30] [31] [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 cancer patients.[29] From the standpoint of immunotherapeutic targeting, a major drawback of the cancer-testis antigens is that none appear to be necessary for the tumors’ growth or survival. Therefore, their expression seems 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 melanoma patients were found to recognize tyrosinase, a melanocyte-specific protein required for melanin synthesis. [33] [34] In fact, the most commonly generated melanoma-reactive T cells from melanoma patients 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 (e.g., prostate cancer or melanoma).

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 described previously 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. 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 biologic behavior of tumor cells relative to their normal counterparts. Whereas uncontrolled growth is certainly a common biologic feature of all tumors, the major pathophysiologic characteristics of malignant cancer responsible for morbidity and mortality are their ability to invade through natural tissue barriers and ultimately to metastasize. Both of these characteristics, 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 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 tends to be significantly 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 a tumor—so called immune surveillance. However, as will be discussed, oncogenic pathways in the tumor seem to organize the immunologic component of the microenvironment in a fashion that not only protects itself from antitumor immune responses, they can qualitatively shift immune responses to those that actually support and promote tumor growth. Thus, just as with Annekin Skywalker, the 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, first conceived nearly a half-century ago, [36] [37] 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 ( Box 6-1 ). In animal models, carcinogen-induced tumors can be divided into those that grow progressively (termed progresser tumors) and those that are rejected after an initial period of growth (termed regresser tumors). [1] [2] The phenomenon of regresser 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 progresser 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 ).

Box 6-1 

HOW IMPORTANT IS IMMUNE SURVEILLANCE OF CANCER?

Few questions in cancer immunology have been more controversial than the immune surveillance hypothesis. First put forward by Lewis Thomas over a half century ago, the original immune surveillance hypothesis proposed that a natural role for the immune system was to survey the body for tumors similarly to the way the immune system surveys the body for invading foreign pathogens. Tumors were proposed to be distinguished from self-tissues by virtue of expression of tumor-specific antigens (TSAs). One aspect of this hypothesis that has held up to experimental testing has been the existence of TSAs. We now know that the genetic instability of tumor cells generates genetic and epigenetic changes that translate to antigens capable of being recognized by the immune system. However, because tumors seem to have developed mechanisms to subvert immunogenicity and tolerize the immune system, it is generally no longer believed that immune surveillance represents a major endogenous defense against tumorigenesis. It is likely that guardians of the genome that sense DNA damage, such as the ATR/ATM/p53 system, are much more fundamental mechanisms to protect against transformation. However, certain specialized components of the immune system, such as interepithelial lymphocytes activated by stress-induced ligands, may indeed play a complementary role in immune surveillance among specific epithelial tissues that are frequently exposed to carcinogenic stress. The best example is the intraepithelial lymphocytes within cutaneous epithelium that is constantly exposed to carcinogenic ultraviolet irradiation. A more moderate view of immune surveillance has been summarized by the hypothesis of Schreiber and colleagues that tumors “edit” themselves to either become resistant to immunologic surveillance or upregulate pathways (such as the STAT3 pathway) that actively induce tolerance among components of the immune system capable of recognizing TSAs. [57] [58] [59]

 
 

Figure 6-1  The balance among immune surveillance, resistance, and tolerance. Transformation of normal cells to cancer cells involves the creation of true neoantigens resulting from mutation as well as upregulation of self-antigens resulting from epigenetic forces. Successful immune surveillance of tumors based on recognition of these tumor-specific antigens would lead to tumor elimination at early stages. Clinically relevant tumor survival and progression requires that tumors develop resistance mechanisms that inhibit tumor-specific immune responses to kill tumor cells. Alternatively, if the tumor develops mechanisms to induce immune tolerance to its antigens, antitumor effector responses do not develop. Evidence is accumulating that tumors actively develop immune resistance mechanisms as well as immune tolerance mechanisms to survive despite displaying antigens capable of recognition by the immune system.

 

 

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. [38] [39] [40] [41] [42] These studies were taken as 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 that, as in the following discussion, may represent a potent form of antitumor immunity and could contribute to immune surveillance of cancer.

Epidemiologic 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. [43] [44] [45] Many of these cancers are also observed in transplant patients on chronic pharmacologic immune suppression as well as in human immunodeficiency virus/acquired immunodeficiency syndrome patients whose immune system is depressed. The most common cancers in these individuals include lymphoplastic lymphomas as well as Kaposi's sarcoma; however, certain epithelial cancers, such as stomach cancer, were also observed at increased frequency. A unifying theme for the majority of cancers observed in immunodeficient individuals is their microbial origin. The majority of lymphoplastic lymphomas are Epstein-Barr virus-associated lymphomas,[46] and Kaposi's sarcoma is a result of infection with the herpesvirus KSHV (Kaposi's sarcoma herpesvirus).[47] Other virus-associated cancers such as cervical cancer (from human papillomavirus [HPV]) [48] [49] 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. [50] [51] From these studies, the notion emerged that immune surveillance indeed protects individuals against certain pathogen-associated cancers by either preventing infection or altering chronic infection by viruses and other microbes that can eventually induce cancer. These studies were taken to represent evidence that the common non-pathogen-associated cancers most commonly seen in adults in developed countries (e.g., prostate cancer, colon cancer, lung cancer) are not subject to immune surveillance.

Two caveats to this interpretation must be noted, however. First, detailed epidemiologic 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 non-pathogen-associated cancers would have been observed had these individuals lived further into adulthood. Indeed, more recent analyses definitively demonstrate an increased incidence of some non-pathogen-associated cancers, in immunodeficient individuals, particularly melanoma.[52]

In addition to epidemiologic data, dramatic anecdotal examples are difficult to ignore. There have been reports that patients receiving kidneys from a cadaver donor that had been in complete remission from a melanoma before organ donation each rapidly developed metastatic melanoma of donor origin after the transplant. [53] [54] [55] These results indicate that at least for some non-pathogen-associated tumors, the immune system can play a significant role in maintaining the micrometastatic disease in a dormant state. Whether this principle applies to other non-pathogen-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 by Schreiber and colleagues reexamining 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, STAT 1 gene, or the type 1 interferon receptor gene. [56] [57] [58] [59] When these knockout mice are 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 insensitivity by the developing tumors played a significant role in the defect in immune surveillance. Interestingly, in contrast to γ-interferon receptor knockout mice, the mechanism for increased tumor incidence in tumors in type 1 interferon receptor knockout mice did not involve sensitivity by the tumor to type 1 interferons but rather reflected the role of the type 1 interferons 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, γ-interferon, RAG2 double-knockout mice developed a broader spectrum of tumors than RAG2 knockout mice. All of the tumors that arise in these genetically manipulated immunodeficient animals behave as regresser 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, suggest 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. One of the caveats in the interpretation of these studies comes from the work of Enzler and Dranoff, who studied mechanisms of increased tumorigenesis in granulocyte-macrophage colony-stimulating factor (GM-CSF), γ-interferon double-knockout mice.[60] 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 germfree conditions. Nonetheless, although the classic concepts of immune surveillance of cancer remain unsupported by experimental evidence, studies on tumorigenesis in genetically manipulated immunodeficient mice indeed suggest that developing tumors must actively adapt themselves to their immune microenvironment to exist within the context of a competent immune system.

INNATE IMMUNITY, EPITHELIAL IMMUNITY, AND 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. [61] [62] [63] Most NK receptors seem to be inhibitory when engaged; this inhibition is often associated with ITIM (immunoreceptor tyrosine kinase-based inhibitory motif) 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 ITAM (immunoreceptor tyrosine kinase-activating motif) and is associated with an adaptor molecule, DAP 10, which contains neither conventional ITIMs nor ITAMs.[64] Instead, DAP 10 contains a KYXXM motif that seems to bind to phosphatidyl inositol (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 interepithelial lymphocytes (IELs).

IELs represent a distinct population of lymphocytes residing in epithelial tissues that display features of both adaptive and innate immune responses. [65] [66] [67] [68] [69] 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, respiratory tract). Fifty percent of the IELs of the gut express the γδ T-cell receptor (TCR), which is normally expressed by less than 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 a particular VaVb and are thought to recognize a limited subset of microbial or self nonpeptide antigens presented by nonclassical class 1 MHC molecules. Thus, NKG2D expression marks diverse subsets of lymphocytes that, though 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 as well as 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 groove characteristic of most MHC molecules, are stress-induced proteins whose genes contain stress response elements in their promoters. [70] [71] Raulet and colleagues have demonstrated that upregulation of MICA/B is induced through the ATM/ATR/Chk1 pathway of DNA damage recognition.[72] 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, [73] [74] 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, RAE-1α-RAE-1ε, as well as the product of the H60 gene. ULBP3 is an additional NKG2D ligand to be described. [75] [76] These NKG2D ligands seem to be involved in immune recognition and possibly tumor surveillance in mice. [77] [78] [79] Recognition and killing of murine skin keratinocytes or intestinal epithelial cells by γδ IELs require expression of NKG2D ligands and are blocked by anti-NKG2D antibodies. Trasnfection 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.[80] The major initiating event of carcinogenesis in the skin—ultravioleet light—is a potent source of DNA damage that, as mentioned previously, has been shown to induce NKG2D ligands via the 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 ).

 
 

Figure 6-2  Epithelial linings contain intraepithelial lymphocytes that can recognize epithelial cells undergoing genotoxic stress. Intraepithelial lymphocytes (IELs) fall into two categories: those that express the classical αβ T-cell receptor (TCR) that recognizes peptide-MHC complexes on the cell surface and those that express the γδ TCR, whose ligands are less well characterized. IELs also express the NKG2D receptor, which serves as a costimulatory receptor for activation of IELs. The ligands for NKG2D, MICA, and MICB in humans and RAE1α–RAE1e, H60, and ULBP-3 in mice, are induced by genotoxic stress via the ATM/ATR pathways. This is a mechanism by which IELs can survey for damaged epithelial cells due to irradiation (skin) or mutagens that can cause transformation.

 

 

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, Spies and colleagues 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.[81] 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 utilize to evade immune recognition. It also points out straightforward approaches to block these evasion systems. If indeed soluble MICA/B does represent 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.

IMMUNE TOLERANCE AND IMMUNE EVASION—THE HALLMARK OF A SUCCESSFUL TUMOR

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 (see Fig. 6-1 ).

Evidence from both murine tumor systems as well as 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 seems to operate predominantly at the level of T cells; β-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 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, who examined the response of TCR-transgenic T cells specific for the idiotypic immunoglobulin expressed by a murine myeloma tumor. [82] [83] They first demonstrated induction of central tolerance to the myeloma protein followed by peripheral tolerance. Using influenza hemagglutinin as a model tumor antigen, Levitsky and colleagues demonstrated that adoptively transferred hemagglutinin-specific TCR-transgenic T cells were rapidly rendered anergic by hemagglutinin-expressing lymphomas and hemagglutinin-expressing renal carcinomas. [84] [85] 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 seems to be associated with failure of antigen recognition by T cells—that is, the immune system “ignores” the tumor. [86] [87] Beyond studies on transplantable tumors, more recent analyses of immune responses to tumor antigens in tumor-transgenic mice developing 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 associates evaluated CD4 responses to hemagglutinin in double-transgenic animals expressing hemagglutinin and simian virus 40 (SV40) T antigen under control of the prostate-specific probasin promoter.[88] Development and progression of prostate tumors did not result in enhanced activation of adoptively transferred hemagglutinin-specific T cells. Tolerance to hemagglutinin as a normal prostate antigen occurred largely through ignorance, because there was no evidence for antigen recognition by hemagglutinin-specific T cells. 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-interferon production. Analysis of the consequences of transformation in additional tumor-transgenic mouse systems has also been performed.

Willimsky and Blankenstein 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.[89]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 colleagues in the prostate system.

The capacity of spontaneously arising tumors to tolerize T cells has not been uniformly observed. A contrasting result by Ohashi and colleagues 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.[90]These animals develop pancreatic islet cell tumors that express GP33. These investigators found that as tumors progressed in the mice, enhanced T-cell activation occurred. CD8 T-cell activation was demonstrated through bone marrow chimera experiments to occur 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 immunoediting concept put forward by Schreiber and colleagues, 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 (DC) 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 (APCs) 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 from 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.[91] 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 probably central to the capability of tumors to tolerize tumor-specific T cells is the immature or inactive state of tumor-infiltrating DCs. DCs are the major 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 activate DCs to a state whereby they present antigens to T cells together with costimulatory signals that induce T-cell activation and development of effector 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 costimulatory 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. 6-4A ) to one of tumor-specific tolerance ( Fig. 6-4B ).

 
 

Figure 6-3  Dendritic cells (DCs) can either activate adaptive immunity or tolerize T cells depending on their state of maturation. DC progenitors develop from hematopoietic (bone marrow-derived) progenitors under the influence of various cytokines, particularly GM-CSF. Under circumstances of microbial infection, specific pathogen-associated molecular patterns (termed PAMPs) engage pattern recognition receptors (PRRs), leading to release of proinflammatory danger signals that induce DC maturation. DC maturation leads to upregulation of costimulatory molecules, MHC, and chemokines that result in activation of T cells to effector cells (right). In the absence of these “danger signals,” DCs follow a default pathway (left) in which they become “tolerizing DCs” that present antigen (Ag) to T cells in the absence of costimulatory signals. This represents a steady-state pathway for continuous presentation of self-antigens. The consequence is that these T cells are turned off (anergy), inducing tolerance.

 

 

 
 

Figure 6-4  Inhibition of DC activation in the tumor microenvironment can shift tumor-specific immune responses from activation to tolerance. Based on the scenario presented in Figure 6-3 , 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 (A) to one of tumor-specific tolerance (B). LN, lymph nodes.

 

 

REGULATORY T CELLS AND CANCER

Over the past 10 years, regulatory T (Treg) cells have emerged as a central player in maintenance of the tolerant state as well as general downregulation of immune responses to pathogens. [92] [93] Not surprisingly, they seem to play a role in tolerance to tumor antigens as well as in the resistance of tumors to immune-mediated elimination. [94] [95] In contrast to the ephemeral CD8 suppressor cells of the 1970s that failed to withstand experimental scrutiny, the more recently defined CD4+ regulatory T cells are characterized by expression of a central master regulatory transcription factor—FoxP3—whose role in the gene expression programs of regulatory T cells is being actively studied.[96] Although CD4+ regulatory T cells selectively (but not specifically) express several cell membrane molecules, including CD25, neuropilin, GITR, and LAG3, [92] [97] [98] [99] their overall genetic program and inhibitory capacity is absolutely dependent on sustained expression of FoxP3. [100] [101]

Mechanisms of immune suppression by regulatory T cells vary and include production of inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGFβ). [102] [103] [104] In keeping with the emerging appreciation that tumors are by nature highly toleragenic, numerous murine studies have demonstrated that Treg cells expand in animals with cancer and significantly limit the potency of antitumor immune responses—either natural or vaccine induced. For example, in a study by Sutmuller and coworkers a combination of GM-CSF–transduced tumor vaccine plus anti-CTLA4 antibodies was much more effective at eliminating established tumors when animals were treated with anti-IL-2 receptor a antibodies to eliminate CD4+ Treg cells.[105]

It is now appreciated that treatment with low-dose cytoxan is a relatively simple and reasonably effective way to temporarily eliminate cycling Treg cells. [106] [107] [108] [109] This seems to be a major mechanism by which pretreatment with low-dose cytoxan before vaccination can significantly enhance the capacity of vaccines to break tolerance. As new cell membrane molecules that define Treg cells are identified, the capacity to block regulatory T-cell activity with antibodies to these molecules presents new opportunities for immunotherapeutic strategies to break tolerance to tumor antigens.

ONCOGENIC PATHWAYS ACTIVELY MEDIATE TUMOR–IMMUNE SYSTEM INTERACTIONS

The previous sections outline the complex interplay between tumor and host immune system and describe the experimental evidence that the immune system is in general tolerant to tumors and their antigens under circumstances in which a tumor has established and is expanding within the host. Is this tolerance to tumor antigens a passive default pathway, or does the tumor actively manipulate its immune microenvironment in a way to render the immune system tolerant to its antigens. Indeed, evidence is accumulating that activation of oncogenic pathways in the tumor as well as inactivation of tumor suppressor genes have immunologic consequences far beyond the more commonly studied roles in growth regulation and antiapoptosis. Critical signaling pathways whose role has been studied in this context include STAT3, NF-κB, BRAF, and PTEN. Although each of these pathways (either activation or inactivation) has been well studied for its role in “classic” tumor biology such as dysregulated growth, regulation of apoptosis, and resistance to DNA-damaging agents, additional roles in the organization of the immune microenvironment of the tumor have also been elucidated recently.

The best-studied oncogenic pathway to play a role in tumor immune evasion is the STAT3 pathway. STAT3 is one of two STATs (the other being STAT5A) to be constitutively activated in many diverse tumor types. [110] [111] [112] [113] Activation of STAT3 involves tyrosine phosphorylation resulting in homodimerization in the cytosol that leads to nuclear transport where it participates in transcriptional activation (and in some cases repression) of diverse genes. Although synthetic mutations in STAT3 can confer upon it oncogenic activity, constitutive activation of STAT3 in tumors is not a consequence of mutation. Instead, STAT3 is downstream of several important oncogenic tyrosine kinases, both receptor tyrosine kinases and src family tyrosine kinases. Several receptor tyrosine kinases that play important roles in human cancer, including EGFR, HER2/Neu, and cMet, signal in part through STAT3. [114] [115] [116] In addition, src and potentially other src family tyrosine kinases can activate STAT3.[117] In fact, the original association of STAT3 with oncogenesis came from the demonstration that src-dependent transformation required STAT3.[118] Activated STAT3 in tumors participates in transcriptional activation of several genes associated with common cell-autonomous and non-cell-autonomous mechanisms of carcinogenesis and cancer promotion. These include cell cycle regulation (e.g., cyclin D1), antiapoptosis (e.g., BCL-Xl and survivin), and angiogenesis (e.g., vascular endothelial growth factor [VEGF]). [119] [120]

In addition, STAT3 activation in tumors has been shown to repress the production of proinflammatory cytokines and chemokines that could enhance antitumor immune responses.[121] These include proinflammatory cytokines such as type 1 interferons and tumor necrosis factor as well as proinflammatory chemokines such as RANTES and IP-10. Thus, blockade of STAT3 signaling in tumor cells results in the release of multiple proinflammatory mediators and consequent infiltration with cells of both the innate and adaptive immune system that ultimately inhibit tumor growth.

Beyond simply repressing the production and release of molecules that could promote antitumor immune responses, STAT3 signaling also induces the release of factors that inhibit activation of multiple immune cell types in the tumor microenvironment. These include DCs, NK cells, and granulocytes, which, though present in significant numbers within tumors, are generally found in an unactivated state.

Some of the STAT3-regulated factors that induce this “quiescent microenvironment” include IL-10, VEGF, IL-6, and possibly IL-23. As will be described later, some of these cytokines promote distinct forms of immune responses that promote rather than inhibit tumor growth. The receptors for each of these factors are expressed on cells of the hematopoietic system and signal through STAT3. Thus, infiltrating hematopoietic cells within the tumor microenvironment are found to also express constitutively activated STAT3. Blockade of STAT3 in the hematopoietic system (for example, via hematopoietic-specific STAT3 knockout) results in dramatically enhanced activation of DCs and cells in the innate immune system (such as NK cells and granulocytes) and leads to antitumor immune responses. In fact, even aggressive tumors fail to grow when transplanted into animals with hematopoietic STAT3 knockout.[122] Thus, STAT3 seems to be an important global signaling pathway that restrains antitumor immunity.

Another immunologically relevant pathway that is commonly constituently activated in cancer is the NF-κB pathway. [123] [124] Normally, NF-κB is activated in a highly stimulus-dependent fashion, but is constitutively activated in many types of tumors. Multiple NF-κB family members participate in either a canonical or noncanonical NF-κB activation pathway. Common to both pathways is the activation of IκB kinase (IKK), which phosphorylates IκB leading to ubiquitin-dependent degradation and release of NF-κB to traffic from the cytosol to the nucleus and activate gene transcription programs.[125]Alternatively, IKK phosphorylation can result in cleavage of a precursor protein for the activation of the noncanonical NF-κB pathway. The mechanism for constitutive NF-κB activation in tumors is not currently known. Normally, NF-κB plays a central role in the activation of virtually all cells in the immune system—both innate and adaptive. In the case of innate immunity, Toll-like receptors (TLRs) on the surface of cells or intracellular sensors of viral RNA or DNA (the RIGI or MDA5 pathway) result in a signaling cascade that activates NF-κB via TRAF6. [126] [127] Paradoxically, constitutive activation of NF-κB in tumors is associated predominantly with activation of antiapoptotic genes, whereas many of the typical NF-κB-responsive proinflammatory/proimmunity genes are not activated in tumors. Recently, it was demonstrated that the selective NF-κB gene activation program in tumors is dependent on its association with STAT3. Indeed, coactivation of STAT3 and NF-κB is commonly observed in tumors. This coactivation seems in part to be due to a newly defined role for STAT3 in enhancing acetylation of NF-κB p50 subunit, resulting in enhanced retention of active NF-κB in the nucleus of tumor cells. This retention seems to be through the p300 acetyl transferase. The result is a shift in equilibrium toward nuclear retention of NF-κB. In addition, STAT3–NF-κB complexes fail to bind promoters of proinflammatory/proimmunity genes that are typically repressed in tumor cells, whereas STAT3–NF-κB dimers are found associated with promoters driving antiapoptotic genes such as BCL-Xl and survivin. These findings highlight the interactivity between key signaling pathways of tumor cells as well as the interplay between gene expression programs mediating tumor immunity versus tumor survival.

An additional oncogenic pathway that seems to play a role in tumor immune evasion is the BRAF pathway.[128] BRAF is constitutively activated in the majority of human melanomas as a result of a single activating mutation. Kawakami and colleagues[128] have demonstrated that factors produced by melanoma cells that inhibit DC activation are in part driven by Braf. Knockdown of Braf with short interfering RNA abrogates the production by melanoma cells of factors that inhibit DC activation. This inhibition seems to be independent of but complementary to that provided by STAT3 activation in melanoma cells. Thus, it seems that multiple oncogenic pathways active in tumor cells may contribute to the release of factors that inhibit DCs and other components of innate immunity, shifting the balance of immune responses toward tolerance.

In addition to oncogenic pathways, inactivation of tumor suppressor pathways may also play a role in immune evasion by tumors. In one example, Parsa and colleauges demonstrated that expression of a T-cell-inhibitory molecule by tumors, B7-H1 (see later discussion), is linked to inactivation of PTEN. PTEN, an inhibitor of the oncogenic AKT pathway, is emerging as one of the most important tumor suppressor pathways in cancer.[129] More recently, Lowe and colleagues provided evidence that the p53 pathway may play a role in inhibiting innate immune responses to tumors. In a transgenic system in which inactivated p53 is conditionally reexpressed in tumors, they found that the inhibition of tumor growth induced by reactivation of p53 might be in part dependent on induction of innate immune responses mediated by NK cells.[130]

Taken together, these findings strongly suggest that oncogene and tumor suppressor gene pathways in tumors play important roles in orchestrating the interaction between the tumor cell and its immune microenvironment such that immune responses induced by the invasion and metastasis process do not eliminate the tumor cell itself. Whereas most of the focus on the function of oncogenic and tumor suppressor pathways has been on cell-autonomous functions within the tumor such as growth regulation, there is growing appreciation that these pathways additionally affect the tumor microenvironment via nontransformed cells. As an integral part of the tumor microenvironment, the immune system is clearly subject to regulation by these pathways. Understanding of the immunologic consequences of these pathways ultimately provides direct opportunities to develop therapeutic approaches that integrate inhibitors of oncogenic pathways, activators of tumor suppressor pathways, and other immunotherapeutic approaches to cancer.

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 ). This 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.

 
 

Figure 6-5  The hostile immune microenvironment of the tumor. Activation of oncogenic pathways and inactivation of tumor suppressor pathways in the tumor lead to a cascade of molecular and cellular processes in the tumor microenviroment that block the killing function of innate immune effectors such as NK cells and granulocytes and block DC maturation (see Figs. 6-3 and 6-4 [3] [4]). In addition, multiple cell membrane molecules such as IL-10, TGFβ, B7-H1, and B7-H4 are upregulated. These molecules bind to receptors that inhibit T-cell effector function. Immature myeloid cells (iMC) produce NO, which inhibits T cells, and immature plasmacytoid DCs (iPDC) produce indoleamine dioxygenase (IDO), which depletes tryptophan. Regulatory T cells also accumulate in the tumor microenvironment, further blunting antitumor T-cell responses.

 

 

The previous section described how oncogenic pathways in the tumor cell directly affect the immune microenvironment of the tumor. In addition to its role in inhibiting the activation and effector function of DCs, granulocytes, and NK cells in the tumor microenvironment, STAT3 signaling has also been reported to play a role in guiding immature myeloid cells (iMCs) in the tumor microenvironment to differentiate into myeloid suppressor cells (MSCs) rather than DCs with APC activity. iMCs [131] [132] and MSCs [133] [134] [135] [136] represent a cadre of myeloid cell types, including tumor-associatedmacrophages, that share the common feature of inhibiting both the priming and effector function of tumor-reactive T cells. It is still not clear whether these myeloid cell types represent distinct lineages or different states of the same general immune-inhibitory cell subset.

In mice, iMCs and MSCs are characterized by coexpression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker) while expressing low or no MHC class II or the CD86 costimulatory molecule. In humans, they are defined as CD33+ but lack markers of mature macrophages, DCs, or granulocytes and are HLA-DR-. Several molecular species produced by tumors tend to drive iMC/MSC accumulation. These include IL-6, CSF-1, IL-10, and gangliosides. IL-6 and IL-10 are potent inducers of STAT3 signaling. Another cytokine reported to induce iMC/MSC accumulation is GM-CSF.[137] This finding is somewhat paradoxical, in that GM-CSF is a critical inducer of DC differentiation and GM-CSF–transduced tumor vaccines enhance antitumor T-cell immunity via accumulation of DCs at the vaccine site followed by increased DC numbers in vaccine draining lymph nodes. It seems that the paradox is solved based on levels of GM-CSF. High local levels drive DC differentiation at the vaccine site, whereas chronic production of low levels of GM-CSF can promote iMC/MSC accumulation. GM-CSF–transduced vaccines that produce extremely high GM-CSF levels can induce iMC/MSC accumulation at distant sites (i.e., spleen and lymph nodes), because they release enough GM-CSF systemically to drive iMC/MSC accumulation.

Several mechanisms have been proposed to explain how iMC/MSC inhibit T-cell responses within the tumor microenvironment. Most include the production of reactive oxygen species (ROS) and/or reactive nitrogen species. Nitric oxide production by iMC/MSC as a result of arginase activity, which is high in these cells, has been well documented, and inhibition of this pathway with several drugs can mitigate the inhibitory effects of iMC/MSC. ROS, including hydrogen peroxide, have been reported to block T-cell function associated with the downmodulation of the χ chain of the TCR signaling complex,[138] a phenomenon well recognized in T cells from cancer patients and associated with generalized T-cell unresponsiveness.

Another mediator of T-cell unresponsiveness associated with cancer is the production of indolamine-2,3 dioxygenase (IDO).[139] IDO seems to be produced by DCs either within tumors or in tumor-draining lymph nodes. Interestingly, IDO in DCs has been reported to be induced via backward signaling by B7-1/2 upon ligation with CTLA-4. [140] [141] Apparently, the major IDO-producing DC subset is either a plasmacytoid DC (PDC) or a PDC-related cell that is B220+.[142] IDO seems to inhibit T-cell responses through catabolism of tryptophan. Activated T cells are highly dependent on tryptophan and are therefore sensitive to tryptophan depletion. Thus, Munn and Mellor have proposed a bystander mechanism, whereby DCs in the local environment deplete tryptophan via IDO upregulation, thereby inducing metabolic apoptosis in locally activated T cells.[139]

Another inhibitory molecule produced by many cell types that has been implicated in blunting antitumor immune responses is TGFβ, which is produced by a variety of cell types, including tumor cells, and which has pleiotropic physiologic effects. For most normal epithelial cells, TGFβ is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G1 stage.[143] In many cancer cells, however, mutations in the TGFβ pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation. Additionally, in cancer cells the production of TGFβ is increased and may contribute to invasion by promoting the activity of matrix metalloproteinases. In vivo, TGFβ directly stimulates angiogenesis; this stimulation can be blocked by anti-TGFβ antibodies.[144] A bimodal role of TGFβ in cancer has been verified in a transgenic animal model using a keratinocyte-targeted overexpression.[145] 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 TGFβ levels are associated with poor prognosis in several malignancies, including prostate cancer,[146] lung cancer,[147] gastric cancer,[148] and bladder cancer.[149]

From an immunologic perspective, TGFβ possesses broadly immunosuppressive properties and TGFβ knockout mice develop widespread inflammatory pathology and corresponding accelerated mortality.[150] Interestingly, a majority of these effects seem to be T-cell mediated, in that targeted disruption of T-cell TGFβ signaling also results in a similar autoimmune phenotype.[151] Recent experiments by Chen and associates rather convincingly demonstrated a role for TGFβ in Treg-mediated suppression of CD8 T-cell antitumor responses.[152] In these experiments adoptive transfer of CD4+ CD25+regulatory T cells inhibited an antitumor CD8 T-cell effector response, and this inhibition was ameliorated when the CD8 T cells came from animals with a dominant negative TGFβ1 receptor.

One of the unresolved issues in the study of tumor immune evasion relates to the mechanisms by which tumors induce antigen-specific T-cell tolerance. Whereas the many mechanisms described previously, including STAT3 signaling-dependent mechanisms, IDO, ROS, reactive nitrogen species, TGFβ, and others, clearly inhibit priming of T-cell responses and/or tumor killing by activated effector T cells, it remains to be definitively determined which processes actively induce antigen-specific T-cell tolerance that has been documented in transgenic models. Self-tolerance induction for peripheral tissue antigens is now thought to involve specific presentation of tissue-specific antigens to mature T cells in the absence of appropriate costimulatory signals. Similar mechanisms are probably operative in the case of tumor-induced tolerance. Originally, the relevant costimulatory signals were envisioned to be provided by B7 family costimulatory molecules expressed by DCs.[153] It is now becoming clear that additional proinflammatory cytokines such as interferons, IL-12, tumor necrosis factor, and others are critical in the distinction between effector T-cell induction and tolerance induction.

An emerging concept is that immature or not fully matured DCs are critical in presenting self-antigens to induce T-cell tolerance in the absence of TLR-mediated danger signals associated with infection.[154] [155] Unquestionably, DCs found within the tumor microenvironment have a relatively immature, unactivated phenotype characterized by low levels of proinflammatory cytokine production, and CD86 and surface MHC class II expression. As described previously, a major inhibitory signaling pathway induced in tumor-infiltrating DCs is the STAT3 pathway, which, when activated, strongly antagonizes TLR- and CD40-mediated DC activation. As mentioned, tumor-derived factors such as IL-10, IL-6, and VEGF (in part induced by STAT3 signaling in the tumor cell) can induce STAT3 activation in DCs. As described in the previous section, constitutive BRAF signaling in melanoma cells has additionally been shown to induce release of factors that inhibit DC activation.[128] These immature “activation-inhibited” DCs clearly represent a prime candidate for the induction of tumor-specific T-cell tolerance.

It remains an open question whether iMC/MSC represent a distinct intertumoral cell subset capable of presenting antigens to T cells in a toleragenic fashion.[156] A recent report indeed suggested that iMCs loaded with antigen and adoptively transferred into mice can induce antigen-specific T-cell tolerance. Finally, it has been suggested that IDO-expressing DCs can induce antigen-specific T-cell tolerance, because IDO-mediated tryptophan selectively kills or inhibits proliferation of activated T cells.[157] According to this model, IDO-expressing DCs would present antigen to T cells inducing activation followed by activation-associated cell death mediated by depletion of local tryptophan stores by the IDO in the presenting DCs. As described later, regulatory T cells play an additional important role in induction of or maintenance of tumor antigen-specific T-cell tolerance. Whether Treg cells mediate T-cell tolerance independently from immature or toleragenic APCs, or whether the two mechanisms are completely interrelated (i.e., toleragenic DCs inducing a Treg phenotype among antigen-specific T cells and antigen-specific Treg cells acting upon DCs to enhance their toleragenic capacity), remains to be definitely determined.

One of the most important classes of immune-inhibitory molecules shown to be expressed by both tumors and myeloid cells in the tumor microenvironment are members of the growing class of B7 molecules. Originally, B7.1 (also called CD80), and then B7.2 (CD86), were identified as critical costimulatory molecules expressed by APCs (first found on B cells, then macrophages, then DCs). Costimulation of T cells, defined as amplification of activation signals delivered through engagement of the TCR by antigen, was shown to be mediated by binding of B7.1 and B7.2 to CD28, expressed on all naive T cells. Subsequently, feedback inhibition of T-cell activation was shown to be mediated by inhibitory signals delivered by a second receptor for B7.1 and B7.2, termed CTLA-4. CTLA-4 is not expressed on the surface of naive T cells but is rapidly induced after T-cell activation, and, as is the case for TGFβ, CTLA-4 knockout mice develop lymphoproliferative autoimmunity, indicating a tonic role for CTLA-4–B7-1/B7-2 interactions in the prevention of autoimmunity.[158] According to these data, CTLA-4 blockade would be expected to function mostly during T-cell priming events, facilitating or enhancing an immune response. In several murine systems, CTLA-4 blockade exerts a pronounced antitumor effect,[159] generating enthusiasm for translating these observations to the clinic.[160]

A second coinhibitory molecule on T cells is PD1 (programmed death 1), a T-cell surface molecule originally discovered in a T-cell hybridoma undergoing apoptosis.[161] Further studies of PD1 identified expression on activated, but not naive T and B cells, in addition to potential overexpression in anergized CD4 T cells.[162] Recent data show that PD1 is also expressed on the surface of certain CD8 T cells, where it serves as a marker for T cells that have been “exhausted” by exposure to persistent viral antigen in vivo. As is the case for CTLA-4 and TGFβ, PD1 knockout mice develop strain-dependent autoimmune disease.[163] In murine models of experimental autoimmune encephalomyelitis and diabetes, anti-PD1 antagonist antibodies enhance disease progression. [164] [165] [166]

There are currently two known ligands for PD1: B7-H1 (also known as PD-L1) and B7-DC (also known as PD-L2). B7-H1 and B7-DC represent two of the five additional B7 family members identified over the past 10 years. These ligands have very different tissue distribution patterns, with B7-DC expression primarily confined to DCs and macrophages.[167] B7-H1 messenger RNA is widely expressed, but cell surface protein is not detectable in normal tissues other than a subset of macrophages.[168] Interestingly, B7-H1 expression can be detected in several tumor types,[169] and engagement of PD1 by tumor-associated B7-H1 promotes CD8 T-cell apoptosis.

In addition, B7-H1 has been reported to be upregulated on both DCs and macrophages within the tumor microenvironment. Clinically, it has been reported that B7-H1 expression is correlated with poor prognosis in renal cell carcinoma.[170] Interestingly, whereas B7-H1 expression on tumor cells was correlated with poor clinical prognosis, combined expression on tumor cells together with hematopoietic cells within the tumor sections was even more highly correlated with poor clinical outcome. Thus, it seems that PD1/B7-H1 interactions mediate a potent and specific immunoregulatory effect, preventing activated and trafficking CD8 T cells from lysing their targets in vivo. In recent data, this observation has been confirmed in murine tumor models, where blockade of either PD1 or of the PD1 ligand B7-H1 potentiates an antitumor immune response.[171] In contrast, the molecular role of B7-DC ligation in an immune response is complex,[172] and under some circumstances ligation of B7-DC on APCs seems to potentiate a costimulatory interaction with T cells.[173]

Another more recently identified inhibitory B7 family member, B7-H4, also seems to play an important role in the tumor microenvironment.[174] The receptor for B7-H4 has not yet been identified, but this molecule has been definitively shown to play an inhibitory role because treatment of mice with blocking antibodies and gene knockout resulted in increased immune responses. As with B7-H1, B7-H4 is expressed by several tumors and also by macrophages in the tumor microenvironment.[175] B7-H4 is regulated differently than B7-H1 and there seems to be a distinct pattern of tumor-selective expression for the two molecules, with some overlap. Recently, B7-H4 expression in human renal cancer has been shown to correlate with poor clinical prognosis, similarly to B7-H1. Patients whose tumors expressed high levels of both molecules displayed the worst clinical outcome, with almost all developing distant metastases.[176]

PROCARCINOGENIC VERSUS ANTICARCINOGENIC ROLES OF THE IMMUNE RESPONSE

Much to the chagrin of the immunotherapy community, skepticism among the oncology community regarding the capacity to induce therapeutically meaningful antitumor immune responses has been accompanied by increasing focus on the capacity of immune responses to induce cancer and potentially enhance cancer progression. Understanding the paradox between the potential procarcinogenic and anticarcinogenic immunity is arguably the most important frontier in cancer immunology ( Fig. 6-6 ).

 
 

Figure 6-6  Evidence for procarcinogenic and anticarcinogenic roles of the immune system. Ample evidence exists in both animal models and human disease settings that immune responses can promote or inhibit cancer development.

 

 

The capacity of inflammatory (i.e., innate) immune responses to enhance carcinogenesis has become well appreciated on the basis of clinical observations that chronic infections that induce chronic inflammation can lead to cancer. One of the best examples is hepatitis C virus (HCV) infection.[177] HCV infection leads to a chronic persistent state in the majority of infected individuals associated with chronic hepatitis. This chronic hepatitis is associated with development of hepatocellular carcinoma at the rate of roughly 1% per year. In contrast to other procarcinogenic chronic infections with viruses such as HPV that carry their own oncogenes, the HCV genome contains no oncogenes or genes encoding proteins that inactivate tumor suppressor genes. Thus, the evidence is quite strong that the chronic inflammatory response to HCV is responsible for the genesis of hepatocellular carcinoma. Similarly, the inflammatory responses associated with chronic H. pylori infection of the stomach are thought to be central to the genesis of stomach cancer.[178] Further evidence for the procarcinogenic effects of inflammation come from the findings that anti-inflammatory drugs, such as COX-2 inhibitors, can decrease the incidence of colon cancer.[179] Additional evidence includes the propensity of patients with certain forms of chronic colitis (e.g., ulcerative colitis) to develop colon cancer and the association of microinflammatory foci in the prostate with prostate intraepithelial neoplastic lesions.[180]

In animal models, experimental induction of inflammation in both the colon and the liver are associated with increased incidences of cancer. Recently, Karin and colleagues have used conditional knockouts of IKK to demonstrate an important role for NF-κB signaling in experimental models of colonic carcinogenesis.[181] Interestingly, they found that tissue-specific knockout of IKKβ in both colonic epithelial cells as well as in myloid cells resulted in a diminished incidence of dextran sulfate (DSS)-induced colon cancers. Epithelium-specific knockout of IKKβ resulted in a decreased incidence, whereas myeloid-specific knockout of IKKβ resulted in both decreased incidence and decreased progression rate. The effects of myeloid-specific IKK knockout were taken as evidence for an NF-κB-dependent proinflammatory role in carcinogenesis. More recently, carcinogen-induced liver cancer and colon tumor development in mice bearing heterozygous adenomatous polyposis coli gene mutations (Min mice) was shown to be dependent on MyD88, an adapter for TLR signaling that is necessary for TLR-dependent NF-κB activation. [182] [183] In one case, IL-6 production was found to be an important downstream cytokine for liver carcinogenesis.

Although the majority of evidence linking immunity to cancer involves the innate immune system, Coussens and colleagues have provided evidence that components of the adaptive immune response could contribute to carcinogenesis in a positive fashion. In a transgenic model of HPV E6/E7-induced skin carcinogenesis, they demonstrated that elimination of B cells resulted in a decreased incidence of tumorigenesis.[184] Surprisingly, T-cell knockout did not alter the incidence of tumorigenesis. The ultimate mechanism by which B cells contribute to carcinogenesis in this system is not fully defined, although antibody production seems to be involved.

Understanding the paradox between procarcinogenic versus anticarcinogenic effects of the immune response will be critical to ultimately defining successful immunotherapies. Recent studies suggest a solution to this paradox and provide insight into the notion that different forms of immune responsiveness can respectively be anticarcinogenic or procarcinogenic. Much evidence has been accumulated that a certain type of T-cell response, termed Th1, can be potently anticarcinogenic. Th1 responses are characterized by production of γ-interferon by CD4 T cells as well as induction of CTL responses by CD8 T cells. γ-interferon not only can enhance the activity of CD8 CTLs, it can also activate components of innate immunity such as macrophages that can kill tumors. Th1 responses are induced by STAT1 signaling and are significantly enhanced by IL-12 that can be produced by macrophages or DCs. Indeed, IL-12 activates not only Th1 responses but also innate immune responses by NK cells that can additionally kill tumor cells. [185] [186]

Recently, a distinct IL-12 family cytokine, termed IL-23, has been discovered. [187] [188] IL-23 shares the same b chain as IL-12 but has a distinct a chain, termed IL-23 p19. Likewise, the IL-23 receptor shares a common b chain with the IL-12 receptor but also has a distinct IL-23 receptor-specific a chain. Several immunopathologic states related to autoimmune disease that had been attributed to IL-12 and linked Th1 responses have now been shown to be instead attributable to IL-23.[189] Analogous to IL-12 driving both NK-dependent innate as well as Th1-type adaptive immune responses, IL-23 drives distinct innate immune responses from IL-12 that are just now being elucidated (characterized by granulocyte recruitment). In addition, IL-23 promotes the growth of a distinct type of helper T cell, termed Th17. [190] [191] [192] [193] Th17 is applied to this helper T-cell pathway, because it is characterized by production of cytokine IL-17a rather than γ-interferon.

Recently Langowski and coworkers evaluated skin carcinogenesis and tumor growth in mice with either an IL-23 p19 gene knockout or an IL-12 specific p35 gene knockout.[194] As predicted from previous studies on the role of IL-12 in promoting both innate and Th1-dependent antitumor immunity, tumor formation in a carcinogen-induced skin cancer model as well as growth of transplanted tumors was increased in IL-12 p35 knockout mice. In striking contrast, carcinogenesis and tumor growth was decreased in the IL-23 p19 knockout mice. Carcinogenesis and tumor growth was also reduced in knockout mice for p40, the common subunit for IL-12 and IL-23. This result suggests that the procarcinogenic effects of IL-23 production dominate over the anticarcinogenic effects of IL-12 production. Although these initial findings will require extensive follow-up, they support the notion that qualitatively distinct types of immune responses, characterized by distinct cytokines that mediate distinct functions, can be procarcinogenic or anticarcinogenic.

Analysis of signaling pathways involved in the IL-12–Th1 axis and the IL-23–Th17 axis further suggest a model of “competition” between these two immune pathways. Th1 responses depend on signaling through STAT1, which is essential for commitment to the Th1 lineage, and STAT4, which is the major signal transducer for the IL-12 receptor. In contrast, both IL-23 transcription as well as IL-23 receptor transcription and signal transduction require STAT3 signaling. IL-17 production also requires STAT3 signaling, which is generated by IL-6, which, together with TGFβ, is a critical cytokine for Th17 development from naive T cells. [195] [196] This may explain the role of IL-6 in the MyD88-dependent carcinogenesis described previously.[182] At several levels, STAT3 signaling and STAT1 signaling are mutually antagonistic such that increases in STAT3 signaling inhibit STAT1-induced gene expression programs whereas increases in STAT1 signaling inhibit STAT3-dependent gene expression programs.[197] This finding suggests that therapeutic manipulations of STAT signaling could potentially convert procarcinogenic to anticarcinogenic pathways of immune responsiveness. Ultimately it will be critical to evaluate the qualitative nature of immune responses in chronic infections that lead to carcinogenesis to determine whether this “yin-yang” paradigm of procarcinogenic IL-23–Th17 immunity versus anticarcinogenic IL-12–Th1 immunity represents a general principle translatable to human cancer initiation or promotion ( Fig. 6-7 ).

 
 

Figure 6-7  Two mutually inhibitory pathways of immunity may inhibit or promote cancer. The Th1 pathway is promoted by STAT1 and STAT4 signaling, is initiated by type I interferons, and depends on IL-12. Th1 cells are characterized by production of γ-IFN but also produce many other cytokines. The IL-12/Th1 pathway is typically anticarcinogenic. The Th17 pathway is promoted by Stat3 signaling, is initiated by IL-6 and TGFβ, and depends on IL-23. Th1 cells are characterized by production of IL-17a but also produce many other cytokines. Evidence exists that the IL-23-Th17 pathway can promote carcinogenesis. These pathways are mutually inhibitory in a yin-yang fashion.

 

 

CLINICAL IMPLICATIONS FOR MANIPULATION OF THE IMMUNE RESPONSE TO TUMOR CELLS

Fundamentally, we now have clear-cut evidence that antibodies and T cells can selectively recognize and kill cancer cells in patients. Cancer genetics, epigenetics, and genomics have provided us with a far better understanding of the nature and specificity or selectivity of tumor antigens, providing new opportunities for targeted anti-gen-specific immunotherapy. We know much more about the details of antigen recognition by T cells allowing for the opportunity to modify antigens at critical residues to provide for enhanced immune-stimulatory capacity. Finally, we are learning much about the ligands, receptors, and signaling pathways that regulate immune responses and how they are expressed within the tumor microenvironment. Elucidation of these regulatory pathways has demonstrated that the outcome of antigen recognition is in large part determined by the balance between costimulatory signals and inhibitory signals.

These relatively recent insights into the molecular basis of immune regulation are demonstrating profound significance for the development of more potent combinatorial immunotherapy approaches to cancer ( Box 6-2 ). Several consensus have emerged from both preclinical immunotherapy models as well as analysis of cancer patients. First and foremost, the natural state of endogenous tumor-reactive T cells is characterized by general hyporesponsiveness or anergy. This is probably due to several mechanisms that tumors utilize to induce tolerance as they develop. Whereas a number of the newer generation vaccines such as recombinant viral vaccines or GM-CSF gene-modified vaccines can effectively transfer antigen to and activate DCs, T-cell tolerance remains a major barrier that is difficult to overcome by vaccination alone. Preclinical models demonstrate that for poorly immunogenic tumors, once tolerance has been established, therapeutic vaccines alone are ineffective at curing animals with a significant established tumor burden. However, strategies combining vaccination with inhibitors of immunologic checkpoints and agonists for costimulatory pathways are proving capable of overcoming tolerance and generating significant antitumor responses even in cases of established metastatic cancer.

Box 6-2 

CAN THE IMMUNE SYSTEM BE MANIPULATED TO OVERCOME TUMOR-INDUCED TOLERANCE AND IMMUNE EVASION?

One of the most consistent therapeutic failures thus far has come from attempts to treat cancer with tumor vaccines. Evidence has now mounted that a major reason for the lack of efficacy of tumor vaccines and other immunotherapeutic interventions is the barrier of immune tolerance to tumor antigens as well as the upregulation of molecules in the tumor microenvironment that inhibit effector immune responses. The bad news from these insights is that therapeutic efficacy of tumor vaccines used as single agents will probably always be limited, no matter how effective the vaccine is in inducing immunity in a naive host. It is likely that the only viable therapeutic application of cancer vaccines used as single agents will be in the setting of minimal residual disease. However, as the specific molecular pathways of immune tolerance and immune evasion are defined, antibodies as well as small molecule agonists and antagonists are being developed that can either enhance costimulatory pathways that amplify immune responses or specifically block receptors and pathways that inhibit immune effectors such as cytotoxic T cells. Preclinical experiments as well as very early-stage clinical experience suggest that combinations of vaccines that direct immune responses to particular tumor antigens together with agents that either enhance or costimulate immunity, as well as agents that block immune checkpoints, produce strong synergy in enhancing antitumor immune responses. It is likely that these combinatorial approaches will be the most fruitful for developing clinically relevant immune therapies of cancer.

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