22. Immune responses to malignancies

Transcription

1 22. Immune responses to malignancies Theresa L. Whiteside, PhD, ABLMI Pittsburgh, Pa Immune responses to tumor-associated antigens exist in tumor-bearing hosts but are usually not successful in eliminating malignant cells or preventing the development of metastases. Patients with cancer generate robust immune responses to infectious agents (bacteria and viruses) perceived as a danger signal but only ineffective, weak responses to tumor-associated antigens, which are considered as self. This fundamental difference in responses to self versus non-self is further magnified by the ability of tumors to subvert the host immune system. Tumors induce dysfunction, as well as apoptosis in CD8 + antitumor effector cells. The escape of tumors from immune cells is mediated by several distinct molecular mechanisms. Insights into these mechanisms and more effective control of tumor-orchestrated immune dysfunction are needed. Novel strategies for immunotherapy of cancer must address protection and survival of antitumor effector cells in the tumor microenvironment. (J Allergy Clin Immunol 2003;111:S ) Key words: Cancer, immunity, apoptosis, immune suppression, effector T cells In human beings, tumor development from a single transformed cell to a mass of malignant cells is a multistep process. 1 It involves a series of genetic changes, which occur in the progeny of a transformed cell over the period of many years, accumulate, and culminate in the establishment of a tumor characterized by uncontrolled growth. 1 During tumor development, the original transformed clone becomes replaced by multiple genetically altered clones, which expand and make up the heterogenous population of malignant cells found in a tumor. Tumors are genetically unstable, and the emergence of genetic variants ensures that the tumor survives in the face of the host immune system. It is likely that tumor cells recognized by the immune system are eliminated as they arise, and only those tumor cells that manage to foil the immune surveillance escape and survive. Thus, tumor development involves a prolonged series of checks and balances between the host attempting to curtail tumor growth and the tumor, which tries to escape and adapt to the microenvironment, becoming resistant to immune effector cells. As a result of these interactions referred to as immune surveillance and immune From the University of Pittsburgh Cancer Institute and the Departments of Pathology, Immunology, and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Supported in part by NIH grants PO1-DE and RO1-DE Reprint requests: Theresa L. Whiteside, PhD, University of Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, Suite 1.27, 5117 Centre Ave, Pittsburgh, PA by Mosby, Inc. All rights reserved /2003 $ doi: /mai Abbreviations used Anx: Annexin V APC: Antigen presenting cells CTL: Cytotoxic T lymphocytes DC: Dendritic cells DTH: Delayed type hypersensitivity ECM: Extracellular matrix LAK: Lymphokine activated killer MHC: Major histocompatibility complex NK: Natural killer cell NFκB: Nuclear factor kappa B PBL: Peripheral blood lymphocytes RCC: Renal cell carcinoma ROM: Reactive oxygen metabolites RTE: Recent thymic emigrants TADC: Tumor-associated dendritic cell TAM: Tumor-associated macrophages TCR: T-cell receptor TIL: Tumor-infiltrating lymphocytes TNF: Tumor necrosis factor TREC: T-cell receptor excision circle VEGF: Vascular endothelial growth factor ZIP: Zeta inhibitory protein selection, respectively, a successful tumor consists of a mix of many individual clones, all derived from a common precursor but each harboring a different genetic alteration(s) and variously resistant to the host antitumor defense mechanisms. The idea of immune surveillance originated many years ago with F. M. Burnett, and it introduced the concept that tumors are not ignored by the host immune system. In its modern reinterpretation, it not only emphasizes the ability of the host immune system to detect and destroy tumor cells, it also incorporates the premise that tumors are not passive targets for immune intervention and are capable of both escaping and disabling the host immune system. The modern immune surveillance theory recognizes the complexity of interactions between the tumor and immune cells or their products. It surmises that these interactions might be bidirectional, influenced by the local microenvironment and not infrequently might result in a demise not of the tumor but of immune cells. In this article, the nature and components of the immune response against tumors will be discussed, including the reasons for the failure of the immune system to contain tumor growth and metastasis. It is this latter aspect of tumor immunobiology that will be emphasized, largely because of the realization that the existence of multiple ways in which tumors disarm the host immune system might be responsible for the limited success of current immunotherapies for cancer. S677

2 S678 Whiteside J ALLERGY CLIN IMMUNOL FEBRUARY 2003 TUMOR PROGRESSION AND THE HOST IMMUNE SYSTEM There are several lines of evidence that point to an early, as well as late, involvement of the immune system in tumor development. Early lesions, even premalignant foci, such as melanocytic nevi, for example, are frequently infiltrated with hematopoietic cells, including lymphocytes, macrophages, and occasionally, granulocytes. 2,3 The presence of immune cells in the tumor at later stages of their development, for example, the abundance of tumor-infiltrating lymphocytes (TIL) in colon, breast, oral carcinomas, and other human tumors, has been associated with improved patient survival in several studies. 4 In many patients with various types of cancer, it is possible to expand in culture and test antitumor functions of tumor-specific CTL from the peripheral blood or TIL. 4 This finding reproduced in many laboratories suggests that precursors of such CTL exist in the circulation or at the tumor site in patients with cancer and can be induced to proliferate, when autologous dendritic cells (DC) pulsed with relevant tumor epitopes are used as antigen-presenting cells (APC). More recent experiments with tetramers and flow cytometry have directly demonstrated the presence of tumor peptide-specific T cells in the circulation of patients with cancer. 5,6 The frequency of such peptide-specific T cells appears to be higher in the circulation of patients with cancer than in healthy individuals. 7 Finally, the SEREX technology, based on the presence of tumor-specific antibodies in sera of patients with cancer, has been successfully used for tumor-antigen discovery in many laboratories. 8 These findings, as well as recent identification of numerous tumor-associated antigens (TAA), which are able to induce either humoral or cellular immune responses or both in vitro and in vivo, strongly support the notion that the host immune system recognizes the presence of the tumor and responds to it by generating local as well systemic immune responses. 9 If the tumors are not ignored by the immune system, why do they progress? In attempting to answer this important question, it becomes necessary to consider the interplay between the tumor and immune cells in the tumor microenvironment relative to events likely taking place in tissue during infection by an exogenous pathogen. In the latter situation, vigorous cellular and antibody responses are generated to bacterial or viral antigens, presumably because the immune system perceives an infection as a danger signal. 10 In contrast, TAA are perceived as self antigens, and in the absence of the danger signal, immune responses are weak. Thus, whereas the immune system responds vigorously to contain viral or bacterial infections, it responds weakly, if at all, to TAA, which are largely self antigens or altered (eg, overexpressed) self-antigens. The only unique TAA are mutated antigens, and these are strongly immunogenic and elicit robust immune responses. 11 However, only a handful of such mutated TAA are known, and the vast majority of TAA are poorly immunogenic or simply tolerogenic. In this context, cancer can be viewed as an autoimmune phenomenon, in which tolerance to self prevents effective immune responses to TAA. Patients with cancer, who have not been treated with chemotherapy or radiotherapy, generally have normal immune responses to viral or bacterial antigens. Except for late-stage disease, they have normal delayed type hypersensitivity (DTH) responses to recall antigens but are anergic to autologous tumor. Tolerance to self is one but not the only detriment to the generation of antitumor responses in patients with cancer. The second major impediment is the nature of the tumor microenvironment, characterized by the presence of immunosuppressive factors and by the excess of antigens produced and released by the growing tumor. Evidence suggests that tumors produce a broad array of immunoinhibitory factors, which exert either local or systemic effects on the host antitumor immune responses. 12 It is not surprising, therefore, that antitumor immunity may be weak, inefficient, or even absent in patients with cancer, depending on the nature of tumor-host interactions as well as the robustness of regulatory mechanisms in control of immune tolerance. TYPES OF IMMUNE RESPONSES TO MALIGNANCIES Antitumor immune responses can be innate (natural) or acquired (adaptive). Innate immunity is mediated by cells or soluble factors, which naturally exist in tissues or body fluids and can interfere with tumor growth or its well-being. Among hematopoietic cells, macrophages, granulocytes, natural killer cells (CD3 CD56 + ), non MHC-restricted T cells (CD3 + CD56 ) as well as gamma/delta T cells have the natural capability to eliminate tumor cell targets. 13 In addition, natural antibodies with specificities directed at surface components of tumor cells may be present in sera of patients with cancer. Other serum factors, including complement components, C-reactive protein, mannose-binding protein (MBP), and serum amyloid protein, also play a role in innate immunity. 13 Adaptive immune responses to tumors are mediated by T cells when they recognize tumor-derived peptides bound to self-mhc molecules expressed on APC. Tumors can serve as APC, although low levels of expression of MHC molecules on their surface makes this an inefficient process. 14 Recognition of the peptide by the specific TCR complex found on CD4 + or CD8 + T cells and the binding of peptide-mhc ligands to the variable domains of TCR initiates signaling that leads to T-cell activation. 15 For adaptive immune response to occur, T cells expressing correct TCR have to be present. This requirement implies prior sensitization and clonal expansion of memory T cells in response to a cognate tumor epitope (anamnestic or recall responses). Alternatively, precursor T cells expressing the TCR can be primed by the cognate peptide-mhc ligands presented on APC, and the subsequent development of antitumor effector cells is viewed as a primary immune

3 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Whiteside S679 FIG 1. Immune cells directly responsible for death tumors include cytolytic effector cells such as cytotoxic T cells (Tc) and activated natural killer (NK) cells as well as macrophages and granulocytes. In addition, B cells aided by T helper cells (Th) produce tumor-antigen specific antibodies, which may bind complement (C), thus eliciting complement-dependent tumor lysis, mediate antibody-dependent cellular cytotoxicity (ADCC) by NK cells, and form antigen-antibody complexes, which, upon uptake by dendritic cells (DC), are processed, facilitating antigen presentation to Tc and Th lymphocytes. These complex interactions of immune cells include antigen-specific and nonspecific innate responses, which together should result in tumor cell death. Capability of most human tumors to escape or foil the immune mechanisms (see Table V) leads to tumor progression. response. Tumor antigen specific T cells include not only cytolytic effector cells, which are CD8 + and MHC class I restricted, but also helper CD4 + T cells functioning in the context of class II MHC molecules. Figure 1 presents a schematic summary of immune cells involved in antitumor responses. Immune responses to malignant cells can be categorized as local/regional or systemic. In situ or local responses refer mainly to TIL, which accumulate in many human solid tumors and whose role in tumor progression remains highly controversial, as discussed below. Long considered by some an effector arm of antitumor responses, TIL are, in fact, victims of the tumor microenvironment. Their effector functions are impaired, and the failure of local antitumor responses contributes to tumor progression. Systemic immunity to tumors, as measured in the peripheral circulation or by DTH in patients with cancer, is difficult to demonstrate, and tumor-specific responses are particularly elusive. Nonspecific proliferative or cytotoxic responses of peripheral lymphocytes in patients with cancer are variably impaired. 4 More recent data indicate that the same functional impairments seen in TIL are found in circulating, as well as lymph node lymphocytes of patients with cancer. 16 Thus, it appears that tumors have profound suppressive effects on both local and systemic antitumor immunity in these patients. IMMUNE CELLS IN THE TUMOR MICROENVIRONMENT TIL are frequently found in human tumors, and cells mediating innate, as well as adaptive, immunity may be components of these infiltrates. 4 A variety of soluble products, including cytokines and antibodies, might be released by these cells in response to nonspecific or tumor-specific signals in the microenvironment. In theory, antitumor effects of these products combined with direct interactions of infiltrating effector cells with the tumor should result in its demise. In most cases, however, the tumor grows progressively and metastasizes despite prominent TIL infiltrations, largely because it evolves strategies for escape from the immune intervention. 12 Extensive literature exists describing functional impairments of TIL and their inability to contain tumor progression. 4 Among various cells present at the tumor site, T cells (CD3 + TCR + ) have received the most attention. They are by far the largest component of mononuclear tumor infiltrates in all human tumors. 17 The hypothesis that TIL-T represent autotumor-specific CTL has been promoted, although limiting dilution studies performed with TIL-T from various tumor types indicate that the frequency of such CTL is low, compared with peripheral blood lymphocytes (PBL) T. 4 Nevertheless, evidence exists that

4 S680 Whiteside J ALLERGY CLIN IMMUNOL FEBRUARY 2003 TABLE I. Morphologic, phenotypic, and functional characteristics of tumor-infiltrating lymphocytes found in human solid tumors 1. Morphology: Small to large lymphocytes 2. Phenotype: CD3 + TcR α/β + T cells; few (<5%) CD3 CD56 + NK cells Mix of CD4 + and CD8 + cells; high proportions of CD8 + cells reported in some tumors Variable CD4/CD8 ratio Increased proportions of double-negative (CD4 /CD8 ) T cells Largely CD45RO + memory T cells Express activation markers (CD25, HLA-DR) Nearly all are CD Clonality: Oligoclonal as determined by TcR Vb gene expression 4. Specificity: Autotumor-specific T cells detectable in some tumors at a low frequency 5. Functions: Low or absent ζ chain expression: inefficient TcR signaling Suppressed NFκB activation Depressed locomotion, proliferation, cytotoxicity Cytokine profile: no/little IL-2 or IFN-γ production; excess of IL-10 or TGF-β In vitro response to IL-2 variable but more depressed in TIL recovered from metastatic than primary lesions Increased levels of caspase-3 activity Apoptosis of CD8 + T cells (TUNEL + ; Annexin V + ) Vβ-restricted clones of T cells are present in some freshly isolated TIL and that TIL can selectively recognize and kill autologous tumor cells in some cases. 4 In general, however, TIL-T appear to be activated (HLA DR +, CD25 + ) non MHC-restricted lymphocytes containing variably low frequencies of CTL. Their phenotypic and functional characteristics are listed in Table I. Although CD8 + or CD4 + T cells isolated from human tumors express an activation phenotype, they are functionally compromised, although the loss of function is not an all-or-none phenomenon. It appears that TIL obtained from advanced or metastatic lesions are more functionally impaired than those from early lesions, suggesting that tumor burden or the potential of a tumor to suppress immune cells might determine the functional status of TIL. More recent studies of TIL in situ have focused on expression of the ζ chain, a signaling molecule associated with the TCR complex and of nuclear factor kappa B (NFκB), a transcription factor regulating expression of a number of immune and inflammatory genes. 18,19 In a study comprising over 130 cases of human oral cell carcinomas, expression of ζ in TIL-T was found to be an independent and highly statistically significant biomarker of prognosis and survival in patients with stage III and IV disease. 20 The patients with tumors infiltrated by T cells with low or absent ζ expression had significantly shorter 5-year survival compared with the patients with tumors infiltrated by T cells with normal ζ expression. 20 Stimulus-dependent activation of NFκB was found to be impaired in T cells of patients with renal cell carcinoma (RCC). In some patients, the primary defect was the failure of the transactivating complex RelA/NFκB1(p50) to accumulate in the nucleus after T-cell activation as the result of a defect in phosphorylation and degradation of the inhibitor IκBα. 21 In other patients, NFκB activation was defective despite normal stimulus-dependent degradation of IκBα. 22 In both situations, this defective state could be induced by exposure of normal T cells to supernatants of RCC, and the soluble product responsible was identified as an RCC-derived ganglioside. 23 Impaired NFκB activity may contribute to reduced T-cell functions seen in TIL-T in RCC, since this transcription factor controls expression of a number of genes encoding cytokines, their receptors, and other membrane-regulatory molecules essential for T-cell activation. 19 It is important to note that defects in function of the ζ chain and activation of NFκB are observed in TIL-T as well as circulating T cells of patients with various types of cancer. 24 These studies indicate that T cells found in the tumor microenvironment, both CD4 + and CD8 +,are dysfunctional and that the magnitude of their dysfunction may be important in predicting prognosis or survival of patients with cancer. Natural killer cells (CD3 CD56 + CD16 + ), which mediate innate immunity and contain perforin-rich, as well as granzyme-rich granules, are well equipped to mediate lysis of tumor cells. However, most human tumor cells are resistant to perforin-mediated natural killer (NK) cell lysis, and NK cells are rarely found among TIL. 25 There might be several reasons for the paucity of NK cells in tumors: (a) a lack of reliable antibodies for detection of human NK cells in tissues by immunochemistry 25 ; (b) NK cells are present in premalignant or early lesions and absent from advance tumors, which is consistent with their role in immune surveillance rather than killing of cancer cells at the tumor site 25 ; (c) NK cells are dependent on IL-2 for activation and survival, 26 and since human tumors are depleted of type I cytokines, including IL-2, sustaining antitumor NK activity at the tumor site might be particularly difficult; (d) the newest data indicate that the primary biological role of NK cells in tumor-bearing hosts may not be the elimination of tumor targets but rather the facilitation of DC T cell interactions and driving the immune response to TAA. 27 Tumor sites are not likely to be the optimal milieu for this type of immune interaction; hence, the paucity of NK cells at this site. The in vivo role of NK cells in antitumor defense in man is not yet clear, and work continues to define it further.

5 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Whiteside S681 B cells (CD19 +,CD20 + ) are also rare in most human tumors, with the exception of breast cancer and melanoma. 2 The primary function of B cells is differentiation into antibody-producing plasma cells. Although TAA-specific antibodies are frequently detected in the circulation of patients with cancer, these antibodies are made and secreted in the tumor-draining lymph nodes, spleen, or other lymphoid tissues. From these sites, IgG molecules can readily be transported by plasma or lymph to tissue sites. Therefore, the presence of B cells or plasma cells in tumors is not expected a priori, although, it might be that the ability to make antibodies in situ may be an important aspect of host defense. DC, penotypically defined as Lin - CD80 + CD86 + HLA DR +,are commonly present in human tumors. 28 In tumorbearing hosts, DC are responsible for the uptake, processing, and cross-presentation of TAA to naive or memory T cells, thus playing a crucial role in the generation of tumor-specific effector T cells. Tumor-associated DC (TADC) directly exposed to tumor cells and/or tumorderived factors have been shown to readily undergo apoptosis and to have impaired maturation. 29 Specifically, tumor-derived factors, for example, gangliosides, were shown to inhibit DC generation and their function in vitro. 30 This suppressive effect of gangliosides on DC was found to be mediated by tumor-derived vascular endothelial growth factor (VEGF), a known antidendropoietic factor. 29 The data on functional impairments of TADC have to be balanced by numerous reports in the literature, which suggest that the presence of DC in tumors is associated with improved prognosis. 28 DC infiltrations into tumors have been associated with significantly prolonged patient survival and reduced incidence of recurrent or metastatic disease in patients with bladder, lung, laryngeal, oral, gastric, and nasopharyngeal carcinomas. 28 In contrast, patients with lesions reported to be scarcely infiltrated with DC have a relatively poor prognosis. Fewer DC were observed in metastatic than primary lesions. 31 In a recent study, it was demonstrated that the number of S DC present in the tumor was by far the strongest independent predictor of overall survival, as well as disease-free survival and time to recurrence in 132 patients with oral carcinoma, compared with such well established prognostic factors as disease stage or lymph node involvement. 28 Another striking observation concerned the relation between the number of DC in the tumor and expression of the TCR-associated ζ chain in T IL. The paucity of DC in the tumor was significantly related to the loss of ζ expression in TIL, and these two factors had a highly significant effect on patient overall survival. 28 The poorest survival and the greatest risk was observed in patients with tumors that had a small number of DC and little or no ζ expression in TIL (P = ). These data suggest that both the number of DC and the presence of functionally unimpaired T cells in the tumor microenvironment are important for overall survival of patients with cancer. Macrophages (CD14 + ) are also commonly found in human tumors and are referred to as tumor-associated macrophages or TAM. Whereas normal macrophages are APCs that play an important role in control of infections, TAM are reprogrammed to inhibit lymphocyte functions through a release of specific cytokines, prostaglandins or reactive oxygen metabolites (ROM). It is hypothesized that reprogramming of TAM occurs in the tumor microenvironment as a result of tumor-driven activation. 32 Evidence has accumulated indicating that invasiveness of tumors, for example, human primary colon carcinomas, is directly related to the number of TAM detected in the tumor. In invasive breast cancer, an increased TAM count is an independent predictor of reduced relapse-free survival, as well as reduced overall survival. 33 The available data support the active role of TAM in tumor-induced immunosuppression, on the one hand, and in the promotion of tumor growth on the other. The mechanisms that contribute to TAM-mediated inhibition of immune cells are probably numerous, but much attention has been recently devoted to the role of NADPH-dependent ROM, such as superoxide anion or hydrogen peroxide, as potential inhibitors of TIL. 34 T-cell proliferation and NK-mediated antitumor cytotoxicity are profoundly inhibited by macrophage-derived ROM in vitro. 35 The overall conclusion from these studies is that immunoinhibitory activities of TAM, whether due to oxidative stress or to release of inhibitory cytokines, such as IL-10, contribute to making the tumor microenvironment a particularly unfriendly milieu for immune cells. IMMUNE EFFECTOR CELLS IN THE CIRCULATION OF PATIENTS WITH CANCER In human beings, peripheral blood is the major source of cells for studies of their antitumor functions. T lymphocytes, NK cells, macrophages, and B cells and their subsets have all been extensively evaluated in the peripheral circulation of patients with melanoma, breast cancer, oral or renal cell carcinomas, and other cancers by conventional phenotypic and functional in vitro assays. Results indicate that signaling abnormalities, functional impairments, and apoptosis seen in TIL-T are likewise present in PBL-T of many patients with cancer. 36,37 Comparisons of TIL-T and autologous PBL-T showed that concomitant low ζ expression in T cells and increased proportions of apoptotic T cells occurred in a subset of about 40% of patients with melanoma, renal cell, or oral carcinoma. 23,24,36 Most of these patients but not all had advanced stage III or stage IV disease, so that the presence of the local and systemic immune defects could not be correlated with the tumor burden. Nevertheless, the defects were more pronounced and more often found in TIL-T than in PBL-T, an indication that the tumor orchestrated their magnitude. Furthermore, low ζ expression, depressed proliferation in response to anti-cd3 Ab, and apoptosis in TIL-T and PBL-T correlated with high levels of FasL expression on the autologous tumor. 24 These data suggest that functional defects in T cells might be linked to their apoptosis, that these defects are both local and systemic, and that the Fas/FasL pathway contributes to

6 S682 Whiteside J ALLERGY CLIN IMMUNOL FEBRUARY 2003 TABLE II. Characteristics of T lymphocytes in peripheral circulation of patients with cancer Predominant phenotype: T lymphocytes CD3 + CD95 + Anx + (up to 95%) CD3 + CD25 + (increased proportions) CD3 + HLA DR + (increased proportions) CD8 + subset CD8 + CD95 + CD8 + CD95 + Anx + CD8 + memory cells CD8 + CD45RO + Anx + CD8 + effector cells CD8 + CD45RO + CD27 ζ low CD8 + CD28 CD95+ + Anx + CD4 + subset CD4 + CD95 + CD4 + memory cells CD4 + RO45 + CD95 + Regulatory T cells CD4 + CD25 + (increased proportions) Clonality: Polyclonal with various restricted TCRVβ specificities Specificity: Tumor peptide/mhc/tetramer + T cells detectable Functions: Low ζ chain expression in T and NK cells: inefficient TCR signaling Suppressed NFκB activation Depressed proliferation in response to anti-cd3 Ab, PMA/ionomycin, mitogens Depressed cytotoxicity Cytokine profile: highly variable LAK activity in response to IL-2 normal or variably depressed Apoptosis of CD8 + T cells and NK cells (Anx + ) Increased caspase-3 activity in T cells Increased lymphocyte turnover apoptosis of T cells both in situ and in the circulation of patients with different types of cancer. Although the literature suggests that the Fas/FasL pathway contributes to tumor-induced apoptosis of immune cells in patients with cancer, this may not be the only mechanism utilized by tumors to engineer an immune escape. 38,39 At this point, it is possible to speculate that the presence of the constellation of immune defects might allow for the identification of a subset of patients with cancer who have poor prognosis, because their tumors create a particularly immunosuppressive environment. Evidence for spontaneous apoptosis of T cells in the peripheral circulation of patients with cancer has been so far described for melanoma, breast carcinoma and head and neck cancer, including oral carcinoma. 36,40 As indicated in Table II, T cells that undergo apoptosis in the circulation of these patients are CD3 + CD95 +, bind Annexin V (Anx), have elevated levels of caspase-3 activity and decreased expression of the TCR-associated ζ chain. 36,41,42 The proportion of these T cells is significantly elevated (P <.0001) in the circulation of patients with cancer relative to sex- and age-matched normal control patients. Fas + (CD95 + ) T cells not only represent the major population of circulating lymphocytes in patients with cancer, but they also preferentially bind Anx and show decreased expression of ζ. 42 However, the most striking finding that has emerged in the course of these studies has documented that CD8 + T cells, and not CD4 + T cells, bind Anx and are especially sensitive to apoptosis. 42 Thus, Fas +, activated CD8 + T cells that are enriched in the circulation of patients with cancer are primed to die, resulting in a rapid turnover of T lymphocytes and possibly contributing to a loss of antitumor effector cells. The highest proportions of Fas + Anx + CD8 + T cells were generally seen in a subset of patients with active disease. 42 Despite elevated levels of apoptosis of circulating T cells in patients with cancer not treated with chemotherapy or radiation, lymphopenia is infrequent. It is likely, therefore, that a replacement of lost T cells occurs either from the thymus or as a result of peripheral expansion of preexisting memory T cells. Thymic output can be measured in human beings by performing TREC (thymic excision circle) analysis, a PCR-based technique, which allows for quantification of recent thymic emigrants (RTE) in the peripheral circulation. 43 With the use of this approach, it has been determined that patients with cancer had significantly (P =.004) fewer RTE than did healthy age-matched donors. 43 In addition, by using multicolor flow cytometry, it was possible to show that the proportion of naive CD8 + CD45RO CD27 + T cells was significantly lower in patients with cancer than in healthy donors. 43 The results could be interpreted to mean either that the thymic output in patients was lower than that in controls or that peripheral expansion of T cells was greater in patients, diluting TRECs and speeding up maturation of naive T cells, respectively. In any case, these data document that lymphocyte turnover appears to be considerably faster in patients with cancer than in normal control patients. Such rapid turnover of T cells could have detrimental effects on antitumor responses, particularly if effector CD8 + T cells were involved. A possibility was next considered that the observed apoptosis of CD8 + T cells is not a part of global, unselected demise of lymphocytes but is directed at the subsets of T cells responsible for antitumor functions. Using multicolor flow cytometry, two subsets of circulating T cells known to play an important role in antitumor defense were evaluated: CD8 + CD45RO CD27 and CD8 + CD28 effector cells in cohorts of patients with cancer and in normal control patients. 44 The frequency of

7 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Whiteside S683 CD8 + CD45RO CD27 cells was significantly increased in the circulation of patients, regardless of the disease stage (P <.0003). However, expression of the ζ chain in these cells was significantly decreased, and their ability to respond to exogenous stimulation by IFN-γ expression was compromised. 44 Also, Anx was found to bind to a higher proportion of this effector cell subset than to the other subsets within the naive T-cell compartment. Thus, the expanded effector CD8 + CD45RO CD27 T cells appear to be dysfunctional and destined for apoptosis in the circulation of patients with cancer. 44 The second subset of effector cells studied, CD8 + CD28 T cells, was also significantly expanded in the circulation of patients with cancer and, surprisingly, it contained the highest proportion of Anx + cells among the naive or memory CD8 + T cell subsets. 40 This implies that these dying effector T cells were being rapidly replaced, that is, had a rapid turnover rate in patients with cancer. Taken together, these results suggest that the effector subpopulations of CD8 + T cells are targeted for apoptosis in patients with cancer and that a loss of effector function through such targeted apoptosis might compromise antitumor functions and contribute to tumor progression. Studies of apoptosis in tumor-specific CTL populations, using labeled tetramers to identify such T cells by flow cytometry, are currently in progress. Another subset of antitumor effector cells, NK cells, represent only 8% to 10% of lymphocytes in the peripheral circulation. These effector cells have been credited with the ability to eliminate tumor cells in the circulation, thus preventing the establishment of distant metastases. 40 Recent data suggest that in addition to mediating perforin-mediated lysis, NK cells constitutively express several ligands of the TNF family and, therefore, are able to induce apoptosis in a broad variety of tumor cell targets. 45 This apoptotic mechanism of tumor cell elimination may be of a greater biological importance than secretory, granule-mediated killing, largely because most tumor cells express receptors for the TNF family ligands and are sensitive to death by apoptosis. 45 Thus, NK cells appear to be well equipped for interfering with the processes of tumor growth and metastases, and, as indicated above, are considered to play a major role in early stages of tumor development. They are able to discriminate between normal and abnormal cells, mainly because they express receptors which enable them to survey the target for the presence of class I MHC molecules. 46 These receptors are of two types: killer-inhibitory receptors and killer-activating receptors. 47 NK-cell functions and their interactions with other cells or the extracellular matrix (ECM) molecules are regulated through these receptors and FcγRs. In the peripheral circulation of patients with cancer, NK cells, like CD8 + T cells, may also be undergoing spontaneous apoptosis. For example, one recent study suggests that among circulating NK cells in patients with breast cancer, a subset of CD56 bright CD16 dim NK cells, which represents about 95% of all NK cells and is responsible for effector functions, preferentially binds Anx and thus is TABLE III. Characteristics of CD4+CD25+ regulatory T cells Account for 6% of purified human CD4 + T cells Increased proportions of CD4 + CD25 + cells in the circulation and the tumor site in patients with cancer Do not proliferate in response to allogeneic or polyclonal activation Constitutively express intracytoplasmic CTLA 4 and CD122; are CD45RO + ; 50% are DR + Titrated into cultures of T cells, CD4 + CD25 + cells inhibit proliferation in the presence of allogeneic DC CD4 + CD25 + cells predominantly secrete IL-10 and/or TGF-β Inhibit IL-2 production by other T cells primed for apoptosis. 40 These patients also had significantly lower NK activity than the age- and sex-matched, healthy control patients tested in parallel, an indication that Anx-binding NK cells were indeed functionally impaired. These and other recent observations suggest that endogenous circulating NK cells have the potential to play a role in tumor surveillance and control of metastasis dissemination. However, once the tumor is established, it might subvert antitumor functions of NK cells, especially in the subsets of NK cells found at the sites of metastasis and those responsible for cytotoxic functions. In addition to NK cells, another category of nonspecific effector cells, CD3 + CD56 + NK/T cells, which are able to eliminate tumor targets, is present in the circulation and tissues. They represent a very minor subset of circulating lymphocytes in normal individuals, but have been reported to be expanded in patients with cancer, as well as tumor-bearing rodents. 48 NK/T cells are also a minor component of TIL. In the presence of IL-2, NK/T cells readily differentiate into lymphokine-activated killer cells (LAK) characterized by morphology of large granular lymphocytes with ample cytoplasm and numerous granzyme- and perphorin-containing granules. 25 In this respect, NK/T cells resemble CD3 CD56 + NK cells, and together, these lymphocyte subsets are considered to be responsible for LAK activity. Both NK and NK/T cells express receptors for IL-18, and thus are activated in the presence of this cytokine as well. The sensitivity of these effector cells to spontaneous apoptosis has not been studied so far. REGULATORY IMMUNE CELLS IN PATIENTS WITH CANCER The presence in the circulation of patients with cancer of suppressor lymphocytes capable of downregulating functions of other immune cells has been described many years ago. 49 In the modern reincarnation, such cells are phenotypically identified as CD4 + CD25 + T cells and referred to as regulatory T cells. 50 They can be isolated from PBMC by immunoselection on magnetic beads coated with antibodies, and their characteristics are listed in Table III. In mice, depletion of CD4 + CD25 + T cells results in the development of autoimmunity, and in tumorbearing animals, it promotes immune responses to autol-

8 S684 Whiteside J ALLERGY CLIN IMMUNOL FEBRUARY 2003 TABLE IV. Molecularly defined immunoinhibitory factors produced by human tumors * TNF family ligands Induce apoptosis through the TNF family receptors FasL Fas TRAIL TRAIL-R TNF TNF-R1 Cytokines TGF-β Inhibits perforin and granzyme mrna expression; inhibits lymphocyte proliferation IL-10 Inhibits cytokine production, including that of IL-12 GMCSF Promotes expansion of immunosuppressive tumor-associated macrophages ZIP (ζ-inhibitory protein) Mediates degradation of ζ or inhibits its mrna expression 52 Small molecules Prostaglandin E 2 Inhibits leukocyte functions through increased camp Epinephrine Inhibits leukocyte functions via increased camp ROM Inhibits leukocyte functions via superoxide generation Viral-related products p15e (CKS-17, synthetic peptide) Inhibits production of type I cytokines, upregulates IL-10 synthesis EBI-3 (homologue of IL-12 p40) Inhibits IL-12 production Tumor-associated gangliosides Inhibit IL-2 dependent lymphocyte proliferation, induce apoptotic signals, suppress NFκB activation, interfere with DC generation * This partial listing of tumor-associated immunoinhibitory factors has been modified from a review by Whiteside and Rabinowich. 12 It demonstrates the diversity of mechanisms that human tumors are known to have evolved in order to incapacitate the host immune system. TABLE V. Evidence that tumors are not ignored by the immune system Increased frequency of tumor-specific T cells detectable in the circulation of patients with cancer using tetramers, recognizing class I MHC restricted or class II MHC restricted tumor epitopes 5-7 Tumor-specific T cells as well as TIL-T can be cultured in the presence of cytokines, expanded and shown to selectively eliminate tumor cells 8 Tumor-specific T cells have been often used recently as specific and sensitive probes for the identification of T-cell defined tumor epitopes in antigen discovery programs 53 Patients with tumors infiltrated by TIL-T, which have normal expression of the ζ chain and thus are able to signal via the TCR, have better prognosis and longer survival than the patients whose TIL-T are dysfunctional 28 The presence of antitumor antibodies in patient sera has been instrumental in the identification of serologically defined tumor antigens by SEREX 8 ogous tumor. In patients with cancer, tumor-associated lymphocytes are enriched in CD4 + CD25 + T cells. 51 Upon sorting by flow, these T cells have been shown to secrete TGF-β or IL-10. The mechanisms through which these T cells regulate antitumor immune responses is being intensively investigated in many laboratories. Preliminary evidence suggests that DC subsets (ie, plasmacytoid versus myeloid DC) appear to play a key role in inducing regulatory T cells in the tumor microenvironment. CYTOKINE PROFILE IN THE TUMOR MICROENVIRONMENT The presence and type of immune cells at the tumor site, as well as their state of activation determine the cytokine profile that is a unique characteristic of every tumor. Not only immune cells but also tumors produce cytokines and other soluble factors (Table IV). In the tumor milieu, in which tumor cells outnumber lymphocytes, soluble products of tumors or supporting tissue cells such as fibroblasts are present at a relatively high concentration. In turn, immunostimulatory T H 1-type cytokines, such as IL-2, IL-12, and IFN-γ, are rare. In the circulation of patients with advanced cancers, proinflammatory-type cytokines IL-1, IL-6, TNF-γ may be present, reflecting the state of persistent chronic activation presumably induced by circulating TAA or antigenantibody complexes. NEW INSIGHTS INTO ANTITUMOR IMMUNITY For many years, the field of tumor immunity has suffered from several misconceptions, which can be summarized as follows: (1) cancer cells are ignored by the immune system; (2) immune responses are directed only against unique antigens expressed on tumor cells; (3) tumor-specific T cells alone are sufficient for achieving tumor regression; and (4) tumors are passive targets for antitumor responses. Today, it is clear that these tenets are inconsistent with newly available data. Thus, cancer cells are not ignored by the immune system, as indicated in Table V. Most of TAA identified to date are self antigens, which are overexpressed or altered post-transcrip-

9 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Whiteside S685 tionally. 9 Immune responses to these altered self antigens are clearly made, and some of the best known T- cell defined antigens include melanoma differentiation antigens (gp-100, MART-1/Melan A and tyrosinase); the testis antigens (MAGE, BAGE, etc); viral antigens (EBV, HPV, HBV); overexpressed ubiquitous TAA (p-53, HER- 2/neu, htert, PRAME); and overexpressed epithelial antigens (Her-2/neu, MUC-1, CEA). 9 T cell defined unique or mutated epitopes, which generate the strongest immune responses, include ras, p53, cdk4, caspase 8 and others. 9,11 The role of tumor-specific T lymphocytes in tumor immunity has been overly emphasized, because it now appears that their presence in patients with cancer, or their generation as a result of antitumor vaccines, does not usually coincide with tumor regression. 54,55 It appears that in order to break tolerance to self, TAA-specific T cells are not sufficient and innate immunity mediated by nonspecific activated T cells, activated NK cells, and macrophages may be necessary. The crucial role of T H 1-type cytokines in activation of these cells cannot be overemphasized. Most important, however, is the realization that tumors are not inert recipients of immune intervention. The phenomenon of counterattack by the tumor against the host immune system is real and likely to contribute to immune cell dysfunction and their demise. 38,39 Evidence for selective apoptosis of CD8 + antitumor effector cells in patients with cancer has been obtained, as discussed above. However, the molecular mechanisms responsible for effector cell dysfunction or death remain to be elucidated. A broad variety of tumor-derived factors or immune cells induced/activated by the tumor are known to contribute to immune cell dysfunction in tumor-bearing hosts (Table V). Whether all or only some of these factors play a role in tumor escape from the immune system and how these factors influence prognosis or survival of patients with cancer is unknown. CONCLUSIONS The existing evidence for dysfunction and death of antitumor effector cells in tumor-bearing hosts introduces a new paradigm for immunotherapy of cancer. Although previous emphasis has been on activation of immune cells and upregulation of their antitumor functions, the current concept is to consider therapies that might protect immune cells from downregulatory or death-inducing factors present in the tumor microenvironment. Preliminary studies suggest that cytokines or DC-based vaccines might be able to offer such protection from apoptosis to immune effector cells. Other therapeutically promising strategies under current development are listed in Table VI. These novel therapeutic strategies take advantage of a tremendous progress made recently in our basic understanding of interactions between the tumor and the host immune system. 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