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Cancer and the Immune System
CLINICAL IMMUNOLOGY
0031-3955/94 $0.00
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CANCER AND THE IMMUNE SYSTEM Richard T. Smith, MD
From the beginning of the twentieth century, anecdotal evidence accumulated in support of the proposition that active resistance by the endogenous defense systems has a significant role in the course and outcome of cancer in humans.* Such evidence led to the empirical use in the 1920s of "Foley's toxins," which are now recognized as immunostimulants such as are used currently in the active treatment of cancer, with unfortunately inconsistent effects. Substantial progress in this area was not made until the organization, cellular and molecular biology of the immune system, and its role in responding to tumor specific antigens was defined. This began in the early 1950s, when the function of lymphocytes was first perceived. Evidence rapidly accumulated that the immune system was highly diverse and was composed of several types of lymphocytes, generated in the bone marrow (B cells) and the thymus (T cells), each bearing a clonally arrayed antigen specific set of receptors and capable of complex functional differentiation. This basic knowledge has now been applied to experimental and clinical cancer and supports the proposition that most mammalian cancer actively and specifically elicits all of the cellular and humoral components of the immune system. Moreover, evidence suggests a significant surveillance function of the system in completely eliminating nascent tumors and in controlling micrometastases derived from existing ones. "Reviews of details of studies before 1985 may be found in references 2,12,20,22-24. Almost without exception, the principles elucidated therein are bona fide and applicable at the present time. Supported in part by the Whisenant Cancer Research Fund.
From the Departments of Pathology and Pediatrics, University of Florida College of Medicine, Gainesville, Florida
PEDIATRIC CLINICS OF NORTH AMERICA VOLUME 41 • NUMBER 4 • AUGUST 1994
841
842
SMITH
This article briefly reviews the antigens and immune responses involved in human and certain experimental cancer models. Then, recent and current attempts to apply this knowledge to diagnosis and treatment of cancer are evaluated. Lastly, the unresolved problem of why such an otherwise efficient system of defense apparently fails to eliminate most established cancer is delineated. EVIDENCE FOR AN IMMUNE RESPONSE TO TUMORS
To the surgeon or pathologist who reviews the microscopic sections, the cellular infiltrate around the edges of breast, colon, gastric, lung, and other cancers is striking and explicitly that which one would find at the site of an intensive immune reaction. Lymphocytes, plasma cells, and macrophages are present in abundance. More recent analysis has shown that these are B cells in active secretory mode, T cells of both the Thelper and T-killer types, and activated macrophages. Careful quantitative studies of such infiltrates showed a direct positive correlation between the intensity of the infiltrate around the tumor and that in the draining lymph nodes, and the patient's prognosis.s, 11 These data suggested a helpful role of immune reactivity in containing the tumor. In animal cancer models, lymph node and splenic T cells and B cells were found not to be depleted regionally or systemically, but were greatly increased in numbersY Paradoxically, the T cells of the tumor-bearing mouse were hyporesponsive to lectin stimulation.! A significant set of observations was made in advanced, incurable cancer patients, wherein graded numbers of the patients' own tumor cells were injected into the patient subcutaneously (with informed consent) and the outcome of the implanted tumor assessed.25 Patients with widely disseminated cancers were able to reject such autotransplants readily providing the cell numbers were not too large «100 X 106 ). The infiltrates around the rejected tumors were similar to those just mentioned and to those found around allograft rejections. This observation is the clinical corollary of experimental "concomitant immunity" toward chemically induced tumors studied in great detail in micep,20 Further analysis of specific tumor responses to the presence of growing, lethal tumors in both animal models and in humans demonstrate all elements of the immune system, including T cells, B cells, and their products. For example, T cells cytotoxic for the patient's own tumor in vitro are found in the circulation and infiltrating the tumor itself in individuals with melanomas, nephroblastomas, osteosarcomas, neuroblastomas, and many other tumors.lO Proliferative responses of helper T cells eliciting delayed-type hypersensitivity responses in vivo are also reported. In experimental systems, when these T cells are injected together with subthreshold numbers of the specific tumor cells that elicited them, growth is inhibited, and the tumor transplant fails.2 4 Antibodies in the patient's circulation against antigens specific to the tumor have been recognized. It can be reasonably concluded that all of the recognized elements
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of the immune response directed specifically towards antigens of human and animal tumors have been identified both in vivo and in vitro. Moreover, when tumor and T cells are allowed to interact under circumstances avoiding an excess of tumor, they are capable of killing the tumor cells.
TUMOR ANTIGENS
Recognition that tumor-specific antigens (TSA) stimulate the immune system was not possible until development of tumor transplant systems in mice in which genetic identity of donor and recipient allowed separation of transplantation effects (Le., related to major histocompatibility complex [MHC] locus differences) from those specific to the tumor. Two types of tumors were recognized on the basis of transplantation analysis: (1) Those induced by viruses, in which rejection-associated TSA were shared by all tumors induced by the same virus/ and (2) those induced by chemical or physical agents, in which the TSA appeared unique to the tumor.14 These important discoveries were made before the immune system was defined at cell and molecular levels and with no knowledge of the structure and presentation of antigen by MHC components. Nearly 25 years elapsed until TSA were characterized at a molecular level, beginning with the studies of Boon.4 These studies grew out of recent knowledge of principles of antigen recognition by T cells and of molecular cloning technology. First, it is appropriate to review briefly the immunologic principles that are relative to all antigen recognition, including TSA. Protein antigens of all types and origins, including self-components, initiate immune responses after first being internalized in efficient antigen-presenting cells (APC), such as macrophages, dendritic cells, and B cells. Here, in phagocytic vacuoles, enzymatic degradation occurs in which both self and nonself proteins are reduced to peptide fragments of six to nine amino acids. These peptides are transported to the cell surface bound specifically to a complementary ("groove") region of newly constructed MHC molecules. The unbound face of this peptide, together with portions of the MHC molecule surrounding it, is actually what is recognized at the surface of the APC by the specific T-cell receptor. In general, MHC molecules of Class I (i.e., HLA-A, B, C in humans) present antigen specifically to T cells of the T-killer or T-cytotoxic type. Class II MHC (i.e., HLA-D, Dr) present antigen to T-helper cells. All cells express Class I MHC constitutively. However, efficient APC also express MHC Class II constituitively and thereby present antigen in ways that elicit a full immune response of T-helper and T-killer components and antibody. The efficiency of Class II presentation is also related to a capacity of APC to bind strongly to T cells after antigen recognition through specific adhesion molecules such as B7 and B7-2 (or B-70 in humans) ("second signals"). Both MHC types at the cell surface also can bind peptides having structures and size complementary to their groove but generated
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elsewhere. TSA, like viral antigens, are processed in vivo by intracellular degradation. Following efficient antigen recognition, T cells are activated, proliferate, and differentiate. T helper cells "tool up" their intracellular machinery to secrete a congeries of lymphokines. These drive a proliferative process and determine the appropriate route of differentiation of other T cells and B-cell precursors. Why are not all peptides, including those of self origin, immunogenic? In short, several mechanisms of specific immunologic tolerance are evoked. First, an efficient system of eliminating self-reactive clones of T cells works in the thymus throughout life. In the thymus, the developing lymphocyte must first be selected for the ability to recognize self MHC of Class I or II, because this is the context in which the peripheral T cell "sees" antigenic peptides. If a self peptide is also "seen" bound to self MHC in the process, the nascent self-reactive thymic T cell is eliminated by a form of cellular suicide ("apoptosis"). If not, it finishes differentiation and is exported into the peripheral lymphoid system. Two other mechanisms are known to operate outside of the thymus to control self reactivity. One is similar to that which occurs inside the thymus. A T cell presented with an otherwise antigenic peptide in the peripheral lymphoid tissues does not become fully activated, possibly for lack of the second signal from its APC. It is then eliminated by apoptosis. The other peripheral mechanism is termed cellular anergy. Here, the T cell is inappropriately stimulated, simply ceases differentiation, and goes into immunologic limbo. In vitro, such a tolerant cell can be reactivated ("rescued") by adding IL-2 in substantial amounts. Because TSA are of self origin except in virus-induced tumors, these mechanisms are clearly of potential importance in regulating the immune response to cancer cells. TSA defied all attempts at molecular characterization until 1988. Boon4 devised a successful method of cloning and sequencing the TSA genes in antigenic variants of otherwise nonimmunogenic experimental tumors. This involved use of cloned, anti-TSA cytotoxic T cells to screen MHC/peptide complexes presented on target cells in which the recognized peptide was a product of a DNA segment that had been inserted into a cell that lacked the gene. The inserted gene for the TSA, when identified as such, could then be nucleotide sequenced and analyzed in terms of whether it was a mutated normal gene or represented aberrant expression of a developmentally significant normal gene. Boon discovered that the first TSA analyzed was a normal peptide having a single amino acid substitution; i.e., a mutation had occurred in an internal cell component. The T cell could recognize the aberrant peptide as a degradation product delivered to the cell surface in the groove of a Class I MHC molecule because it was now seen as nonself. Boon also showed that experimental tumors having TSA initially could lose the ability to be recognized by TSA specific T cells by selection of nonmutated variants. These could grow and kill the host. Through this approach, two types of TSA were demonstrated. One is the result of a single point mutation in an otherwise normal cell protein, which gives
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rise to the unique TSA such as are found in chemically and physically induced tumors. The other TSA category involves aberrant expression of a protein structure normal at an earlier epoch of development, for which the usual tolerance of self has not developed. These TSA are shared by some similar tumors. This investigator has recently employed a very similar approach to demonstrate at least four TSA in human melanoma. Boon used a patient's own cytotoxic T cells, derived as a cell line, to identify the TSA. Then, Boon identified and sequenced the gene (MAGE-I) coding for one of the melanoma-specific antigens. It turned out that this was a TSA of the second type, an aberrant expression of a normal protein. Boon also found that MAGE-l antigen is a peptide of nine amino acids bound only to the HLA-Al MHC. Boon identified this gene actively expressed in 30% of other melanomas as well as in 15% of breast and lung cancers. The normal function of MAGE-l is not yet known. It might be predicted on the basis of recent oncogene research that it is part of a regulatory gene complex controlling some phase of cell development or proliferation. The immunotherapeutic vistas opened by this information are considered later. These new approaches to defining exactly what TSA are and how they arise in the tumor cell are important. Mutations and aberrant expressions of regulatory genes have been increasingly recognized in progressing cancers. They provide a specific target at which to aim the immune system in a set of strategies being evaluated to enhance those elements most effective in destroying tumors.
IMMUNOLOGIC INTERVENTION IN CANCER
Attempts made thus far to enhance or alter the vigorous immunologic responses to ongoing cancers in favor of the patient are, unfortunately, notable more for promise than concrete results. Two general approaches have been tried. One attempts to enhance or redirect ongoing immunologic reactivity to the tumor. The other involves induction of new or preexisting but unexpressed TSA or modified portions thereof. This is based on the premise that induction of new or preexisting but unexpressed TSA will induce a more effective immune response. Cancer vaccines represent a recurrently active approach that has been in and out of vogue since the 1930s. Most approaches represent some variation of a protocol wherein the patient's own tumor cells, altered or unaltered, are reinjected at sites remote from the primary tumor. In various studies, up to 25% of patients had favorable initial responses, but the protocols were uncontrolled or controlled by historic therapeutic data. These attempts are ongoing with renewed enthusiasm at present. 6 Immunologic enhancement of immune function has taken many other forms. The most venerable, and still in the trial stage, involves the systemic use of adjuvants or immunostimulants. In this approach, sub-
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stances shown in experimental systems to augment the immune response, such as classical Freund's adjuvants, are administered to patients with other modalities of treatment. The key ingredients have been mostly bacterial products of coliforms or acid-fast bacteria. The thrust of research efforts ("biomodulation") was to find a product that enhances responses with minimal local and systemic toxicity. The substances have been designed to inject alone or in combination with a patient's tumor cells as a "vaccine." To date, the results have been unpromising in controlled studies, although anecdotal support for the approach is available, and experimental and clinical studies continue with emphasis on those tumor types that appear to elicit the greatest immune responses, such as melanomas. In this same general category are protocols that employ T-cells extracted from the peripheral blood or from biopsied pieces of the patients' tumors, assuming their TSA specificity is similar to that established in animal systems. The most widely evaluated protocol starts with a substantial number of peripheral lymphocytes, known to contain TSA specific T-killer subpopulations. These cells are activated and expanded by incubating in high concentrations of 11-2. This gives a population of cells that is capable of killing the tumor in vitro but that is not MHC restricted, termed lymphokine-activated killer cells ("LAK" cells). Initial evaluation of the use of LAK cells, with and without IL-2 infusion, in over 200 patients with melanoma or renal cell carcinoma showed three complete remissions in melanoma and suggestive amelioration in a few additional patients. The toxicity observed was formidable. 16,17 The more recent approach involves use of tumor infiltrating lymphocytes (TIL) taken from the patient's tumor. TIL are apparently ineffective in situ. When grown in tissue culture in the presence of low concentrations of IL-2 and in the absence of TSA, however, large numbers are generated that show tumor-specific killing in vitro and the patient's MHC restriction. These cells are presumably resurrected from an anergic state. These are currently being tested by reinjection into the patient, with or without additional recombinant IL-2.3, 16 These intensive trials with well-planned protocols have given a few notable remissions. However, treatments with IL-2, LAK cells, and TIL cells are still considered experimental. The second approach depends upon inducing or giving a tumor new and presumably more immunogenic TSA upon which the immune system can focus more effectively. Most studies involve inserting into the tumor cell a cloned gene known to code for an antigenic moiety, which then acts as a new TSA. Genes can be inserted into other cells by several now established techniques, such as within a virus vector or by electroporation or incorporation into artificial cell-penetrating liposomes. For example, if it were possible to incorporate an immunogenic viral gene selectively into the tumor cells, new targets for effective cytotoxicity would be generated. Alternatively, the gene for a nonself histocompatibility gene such as HLA-A2 might be placed into a substantial portion of tumor cells in an HLA-Al patient; HLA-A2 could present a strong target for rejection. One realistic way to get any gene into all tumor cells in
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vivo would be to add it to the genome of an otherwise harmless virus with affinity for the particular tumor cell type and to give this transformed virus systemically. Early clinical trials are underway in which modified vaccinia virus is being used to introduce genes into human tumors. Virallysates of tumor cells also are being injected on the hypothesis that TSA introduced in this way will be more effective. Also under investigation are methods of incorporating genes for cell interaction molecules and lymphokines, all designed to enhance active but ineffective TSA specific T-cells. For example, genes for IL-2, tumor necrosis factor (TNF), or interferon, three powerfullymphokines, have been inserted into human tumor cells in vitro. The gene for the B7 cell interaction molecule has been inserted into otherwise lethal experimental tumor cells, rendering them ready targets for tumor rejection. Insertion of B-70 into human tumor cells should occur shortly.26 Lastly, the genes for TSA that are targets for rejection by T cells and that are common to a class of tumors, such as MAGE-1 in melanoma, can potentially be used to augment T-cell responses incorporated into vaccine complexes or into viral vectors. Boon4 is currently using MAGEl in vaccines in HLA-A1 patients with melanomas having the MAGE-1 gene expressed (as "antigen E" peptide).
WHY IS AN EFFICIENT IMMUNE SYSTEM SO OFTEN UNSUCCESSFUL IN ELIMINATING TUMOR GROWTH?
The validity of this question depends on the unproved assumption that it most often fails. Unknown at present is the frequency of tumors that never become clinically evident, perhaps due to the efficiency of normal immune surveillance over emerging mutant or dysregulated cells destined to progress toward malignancy. The idea of immune surveillance in control of cancer was first proposed in 1954 by Lewis Thomas. 12 Direct proof that it is a biologically active function of the immune system still escapes rigorous experimental proof. Indirect evidence abounds. This includes the increased malignancy rates in immunologically impaired individuals. The observations of concomitant immunity in mice and humans also can be interpreted in terms of the surveillance hypothesis. As delineated earlier, the immune system is very active in most tumor-bearing individuals. Most major cancers are not totally removed by the surgeon, but the patient frequently recovers. Even in the presence of established and incurable tumor, the system may have a major role in holding local extension and metastasis at bay. The most overlooked factor unique to tumors is a persistent antigen overload. This antigen load has been demonstrated experimentally and clinically. For example, the numbers of individual tumor cells or clusters in the draining veins in cancers of the colon, ovary, stomach and breast are large, on the order of 5 to 50 cells or more per milliliter of blood?' 15 This number is independent of prognosis in the individual patient. Tumor products such as CEA from the gastrointestinal tract and PSA from
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the prostate and products of neuroblastomas are also passed into the blood in large quantities. The observations suggest that large amounts of other cell products, including shared and unique TSA, are being secreted or released by every growing tumor. Logically, the concentration of such products would be in a gradient, with the greatest concentration being at the tumor-interstitial fluid interface. It should not be surprising that some TSA represent complexes of tumor-specific peptide fragments that have been shed by the actively growing tumor and bound to specific antibody or MHC. Indeed, such complexes include the fascinating "blocking factors" that received so much attention in the early 1970s.1O Blocking factor is a phenomenon in which serum from tumor-bearing animals and humans was shown to block specifically either cytotoxic or proliferative responses of tumorspecific T cells. When the tumor was completely removed, blocking factor disappeared from serum.9 The tentative molecular characterization suggested that it consisted of an antigen fragment complexed to an antibody-like moiety/9 although the molecular weight estimates were too low for it to be a complete antibody. This factor should be reevaluated in terms of what is currently known of MHC/ antigen complexing at the cell surface. Another factor that may negatively affect effective immune killing rests in the observation that many tumors progressively lose MHC representation at the surface of the celts Whether this is due to genomic down-regulation or to increased rates of shedding is unknown. Loss of surface Class I MHC could severely limit the possibilities for TSA-specific cytotoxic T-cells finding TSA in the necessary MHC context. Continuous and high antigen load has many other ramifications. For the oncologist, it is a rationale for the long-held conviction that debulking tumors, even if tumor cells persist, has a favorable effect on prognosis. More specifically, if the TSA peptides are circulating bound to MHC fragments, they could bind specifically to tumor-specific T-cell receptors at a site remote from the tumor, defusing any killer effects. Too, a principle of immune tolerance is that inducing tolerance is antigen-dose dependent, and sustaining tolerance requires a continuing source of antigen. 21 In the successful long-term transplant, for example, this source is the transplanted tissue itself. In this context, the tumor-host relationship may be thought of as a chimeric state, the tumor supplying antigen(s) that sustain a volatile state of tolerance at the T-cell level. The high end of the TSA gradient is, of course, in the tumor itself, and this is where one would expect to find the most tolerized or anergic cells. Restoration of tumor reactivity in TIL populations placed in vitro with appropriate lymphokines implies that T-cell tolerance in the form of anergy indeed exists. Like anergy in other contexts, it is potentially reversible in the absence of a continuing antigen load. TIL also have been found to be oligoclonal with respect to melanoma antigens. IS If TSA are considered as a set of self peptides anomalously inducing autoimmunity, it is probable that a very limited repertoire of T-cell receptors recognize other TSA. This would limit the intensity and perhaps the effectiveness of the tumor-specific immune response.
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IMMUNOLOGIC APPROACHES TO DIAGNOSIS OF TUMORS
Applications of advances in immunology to tumor diagnosis have added an exciting new dimension to diagnostic pathology. Tumor antigens in the circulation and in tissue sections are identified using techniques of in situ reaction with monoclonal antibodies, ELISA assays, and other applications. For example, ELISA is widely used to screen for and to follow the progress of prostate cancer (PSA) and colon and liver cancer (CEA). At the tissue level, pathologists are looking at the expression of oncogenes and other anomalous cell products in staging colon cancer; in the identification of puzzling tumors, papilloma virus associated antigens in determining the prognosis of cervical cancer; and many others. The diagnosis and staging of most lymphomas are performed primarily by using specific monoclonal antibodies to determine the phenotype of individual cells for analysis in the fluorescence cell analyzer (FACS). This supplies data that are much more useful to the oncologist in treatment than used to be available through ordinary tissue section examination. It is reasonable to expect more practical screening and diagnostic applications in the future. References 1. Adler WH, Takiguchi T, Smith RT: Phytohemagglutinin unresponsiveness in mouse spleen cells induced by methylcholanthrene sarcomas. Cancer Res 31:864, 1971 2. Amos DB, Stetson CA: Tumor immunity. Ann NY Acad Med 101:1-326, 1962 3. Becker Jc, et al: Tumor infiltrating lymphocytes in primary melanoma: functional consequences of differential 11-2 receptor expression. Clin Exp Immunol 91:121-125, 1993 4. Boon T: Toward a genetic analysis of tumor rejection antigens. Adv Cancer Res 58:179210, 1992 5. Chauvenet PH, Smith RT: Relation of tumor specific antigens and the histocompatibility complex: Dissociation of in vitro alloantigen expression and in vivo immunity, from tumor specific transplantation antigen strength. Int J Cancer 22:79-93,1978 6. Cohen J: Research News: Cancer vaccines get a shot in the arm. Science 262:841-843, 1993 7. Engell H: Cancer cells in the blood: A five to nine year follow up study. Ann Surg 149:457-461,1959 8. Fisher B, Fisher ER: Studies concerning regional lymph nodes. Initiation of immunity. Cancer 27:1001-1004,1971 9. Gainor BJ, et al: Specific antigen stimulated lymphocyte proliferation in osteosarcoma. Cancer 37:743,1976 10. Hellstrom I, et al: Cell mediated immunity against antigens common to human colonic carcinomas and fetal gut epithelium. Int J Cancer 6:346-351,1970 11. Inokuchi K, et al: Stromal reactions around tumor and metastasis and prognosis after surgery for gastric cancer. Cancer 20:1924-1929,1967 12. Klein G: Tumor antigens. Ann Rev Microbio120:223-252, 1966 13. Konda S, Smith RT: The stimulatory effects of tumor bearing upon T-cell and B-cell populations in the mouse spleen. Cancer Res 33:2247,1973 14. Prehn RT, Main JM: Immunity to methylcholanthrene induced sarcomas. J Nat! Cancer Inst 18:769-778, 1957 15. Roberts SS, et al: Prognostic significance of cancer cells in the circulating blood. Am J Surg 113:757-762, 1967
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16. Rosenberg S, et al: Prospective randomized trial of high-dose 11-2 alone or in conjunction with lymphokine activated killer cells (LAK) for the treatment of patients with advanced cancer. J Natl Cancer Inst 85:622-632,1993 17. Schoof OR, et al: Survival characteristics of metastatic renal cell carcinoma patients treated with LAK cells plus 11-2. Urology 41:534-539,1993 18. Sensi M, et al: T-cell receptor structure of autologous melanoma-reactive cytotoxic Tcells clones. J Exp Med 178:1231-1246, 1993 19. Sjogren HO, et al: Suggestive evidence that "blocking antibodies" of tumor bearing individuals may be antigen-antibody complexes. Proc Natl Acad Sci USA 68:1372-1375, 1971 20. Smith RT: Tumor specific immune mechanisms. N Engl J Med 278:1207-1214, 278:12681275,278:1326-1331,1968 21. Smith RT: In Landy M, Braun W (eds): Immunologic Tolerance. New York, Academic, 1969, pp 48-52 22. Smith RT: Potentials for immunologic intervention in cancer. N Engl J Med 287:439, 1973 23. Smith RT, Landy M: Immunobiology of the tumor-host relationship. New York, Academic,1985 24. Smith et al: In Saunders et al (eds): Fundamental Mechanisms of Cancer Immunity. North Holland, Elsevier, 32:449,1981 25. Southern CM, et al: The effects of leukocytes upon the transplantability of human cancer. Cancer 19:1743-1753, 1966 26. Townsend SE, Allison JP: Tumor rejection after direct costimulation of CD8+ T-cells by B7 transfected melanoma cells. Science 259:368-370,1993 Address reprint requests to
Richard T. Smith, MD Box J275 Health Sciences Center University of Florida Gainesville, FL 32610
0031-3955/94 $0.00
+ .20
CANCER AND THE IMMUNE SYSTEM Richard T. Smith, MD
From the beginning of the twentieth century, anecdotal evidence accumulated in support of the proposition that active resistance by the endogenous defense systems has a significant role in the course and outcome of cancer in humans.* Such evidence led to the empirical use in the 1920s of "Foley's toxins," which are now recognized as immunostimulants such as are used currently in the active treatment of cancer, with unfortunately inconsistent effects. Substantial progress in this area was not made until the organization, cellular and molecular biology of the immune system, and its role in responding to tumor specific antigens was defined. This began in the early 1950s, when the function of lymphocytes was first perceived. Evidence rapidly accumulated that the immune system was highly diverse and was composed of several types of lymphocytes, generated in the bone marrow (B cells) and the thymus (T cells), each bearing a clonally arrayed antigen specific set of receptors and capable of complex functional differentiation. This basic knowledge has now been applied to experimental and clinical cancer and supports the proposition that most mammalian cancer actively and specifically elicits all of the cellular and humoral components of the immune system. Moreover, evidence suggests a significant surveillance function of the system in completely eliminating nascent tumors and in controlling micrometastases derived from existing ones. "Reviews of details of studies before 1985 may be found in references 2,12,20,22-24. Almost without exception, the principles elucidated therein are bona fide and applicable at the present time. Supported in part by the Whisenant Cancer Research Fund.
From the Departments of Pathology and Pediatrics, University of Florida College of Medicine, Gainesville, Florida
PEDIATRIC CLINICS OF NORTH AMERICA VOLUME 41 • NUMBER 4 • AUGUST 1994
841
842
SMITH
This article briefly reviews the antigens and immune responses involved in human and certain experimental cancer models. Then, recent and current attempts to apply this knowledge to diagnosis and treatment of cancer are evaluated. Lastly, the unresolved problem of why such an otherwise efficient system of defense apparently fails to eliminate most established cancer is delineated. EVIDENCE FOR AN IMMUNE RESPONSE TO TUMORS
To the surgeon or pathologist who reviews the microscopic sections, the cellular infiltrate around the edges of breast, colon, gastric, lung, and other cancers is striking and explicitly that which one would find at the site of an intensive immune reaction. Lymphocytes, plasma cells, and macrophages are present in abundance. More recent analysis has shown that these are B cells in active secretory mode, T cells of both the Thelper and T-killer types, and activated macrophages. Careful quantitative studies of such infiltrates showed a direct positive correlation between the intensity of the infiltrate around the tumor and that in the draining lymph nodes, and the patient's prognosis.s, 11 These data suggested a helpful role of immune reactivity in containing the tumor. In animal cancer models, lymph node and splenic T cells and B cells were found not to be depleted regionally or systemically, but were greatly increased in numbersY Paradoxically, the T cells of the tumor-bearing mouse were hyporesponsive to lectin stimulation.! A significant set of observations was made in advanced, incurable cancer patients, wherein graded numbers of the patients' own tumor cells were injected into the patient subcutaneously (with informed consent) and the outcome of the implanted tumor assessed.25 Patients with widely disseminated cancers were able to reject such autotransplants readily providing the cell numbers were not too large «100 X 106 ). The infiltrates around the rejected tumors were similar to those just mentioned and to those found around allograft rejections. This observation is the clinical corollary of experimental "concomitant immunity" toward chemically induced tumors studied in great detail in micep,20 Further analysis of specific tumor responses to the presence of growing, lethal tumors in both animal models and in humans demonstrate all elements of the immune system, including T cells, B cells, and their products. For example, T cells cytotoxic for the patient's own tumor in vitro are found in the circulation and infiltrating the tumor itself in individuals with melanomas, nephroblastomas, osteosarcomas, neuroblastomas, and many other tumors.lO Proliferative responses of helper T cells eliciting delayed-type hypersensitivity responses in vivo are also reported. In experimental systems, when these T cells are injected together with subthreshold numbers of the specific tumor cells that elicited them, growth is inhibited, and the tumor transplant fails.2 4 Antibodies in the patient's circulation against antigens specific to the tumor have been recognized. It can be reasonably concluded that all of the recognized elements
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of the immune response directed specifically towards antigens of human and animal tumors have been identified both in vivo and in vitro. Moreover, when tumor and T cells are allowed to interact under circumstances avoiding an excess of tumor, they are capable of killing the tumor cells.
TUMOR ANTIGENS
Recognition that tumor-specific antigens (TSA) stimulate the immune system was not possible until development of tumor transplant systems in mice in which genetic identity of donor and recipient allowed separation of transplantation effects (Le., related to major histocompatibility complex [MHC] locus differences) from those specific to the tumor. Two types of tumors were recognized on the basis of transplantation analysis: (1) Those induced by viruses, in which rejection-associated TSA were shared by all tumors induced by the same virus/ and (2) those induced by chemical or physical agents, in which the TSA appeared unique to the tumor.14 These important discoveries were made before the immune system was defined at cell and molecular levels and with no knowledge of the structure and presentation of antigen by MHC components. Nearly 25 years elapsed until TSA were characterized at a molecular level, beginning with the studies of Boon.4 These studies grew out of recent knowledge of principles of antigen recognition by T cells and of molecular cloning technology. First, it is appropriate to review briefly the immunologic principles that are relative to all antigen recognition, including TSA. Protein antigens of all types and origins, including self-components, initiate immune responses after first being internalized in efficient antigen-presenting cells (APC), such as macrophages, dendritic cells, and B cells. Here, in phagocytic vacuoles, enzymatic degradation occurs in which both self and nonself proteins are reduced to peptide fragments of six to nine amino acids. These peptides are transported to the cell surface bound specifically to a complementary ("groove") region of newly constructed MHC molecules. The unbound face of this peptide, together with portions of the MHC molecule surrounding it, is actually what is recognized at the surface of the APC by the specific T-cell receptor. In general, MHC molecules of Class I (i.e., HLA-A, B, C in humans) present antigen specifically to T cells of the T-killer or T-cytotoxic type. Class II MHC (i.e., HLA-D, Dr) present antigen to T-helper cells. All cells express Class I MHC constitutively. However, efficient APC also express MHC Class II constituitively and thereby present antigen in ways that elicit a full immune response of T-helper and T-killer components and antibody. The efficiency of Class II presentation is also related to a capacity of APC to bind strongly to T cells after antigen recognition through specific adhesion molecules such as B7 and B7-2 (or B-70 in humans) ("second signals"). Both MHC types at the cell surface also can bind peptides having structures and size complementary to their groove but generated
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elsewhere. TSA, like viral antigens, are processed in vivo by intracellular degradation. Following efficient antigen recognition, T cells are activated, proliferate, and differentiate. T helper cells "tool up" their intracellular machinery to secrete a congeries of lymphokines. These drive a proliferative process and determine the appropriate route of differentiation of other T cells and B-cell precursors. Why are not all peptides, including those of self origin, immunogenic? In short, several mechanisms of specific immunologic tolerance are evoked. First, an efficient system of eliminating self-reactive clones of T cells works in the thymus throughout life. In the thymus, the developing lymphocyte must first be selected for the ability to recognize self MHC of Class I or II, because this is the context in which the peripheral T cell "sees" antigenic peptides. If a self peptide is also "seen" bound to self MHC in the process, the nascent self-reactive thymic T cell is eliminated by a form of cellular suicide ("apoptosis"). If not, it finishes differentiation and is exported into the peripheral lymphoid system. Two other mechanisms are known to operate outside of the thymus to control self reactivity. One is similar to that which occurs inside the thymus. A T cell presented with an otherwise antigenic peptide in the peripheral lymphoid tissues does not become fully activated, possibly for lack of the second signal from its APC. It is then eliminated by apoptosis. The other peripheral mechanism is termed cellular anergy. Here, the T cell is inappropriately stimulated, simply ceases differentiation, and goes into immunologic limbo. In vitro, such a tolerant cell can be reactivated ("rescued") by adding IL-2 in substantial amounts. Because TSA are of self origin except in virus-induced tumors, these mechanisms are clearly of potential importance in regulating the immune response to cancer cells. TSA defied all attempts at molecular characterization until 1988. Boon4 devised a successful method of cloning and sequencing the TSA genes in antigenic variants of otherwise nonimmunogenic experimental tumors. This involved use of cloned, anti-TSA cytotoxic T cells to screen MHC/peptide complexes presented on target cells in which the recognized peptide was a product of a DNA segment that had been inserted into a cell that lacked the gene. The inserted gene for the TSA, when identified as such, could then be nucleotide sequenced and analyzed in terms of whether it was a mutated normal gene or represented aberrant expression of a developmentally significant normal gene. Boon discovered that the first TSA analyzed was a normal peptide having a single amino acid substitution; i.e., a mutation had occurred in an internal cell component. The T cell could recognize the aberrant peptide as a degradation product delivered to the cell surface in the groove of a Class I MHC molecule because it was now seen as nonself. Boon also showed that experimental tumors having TSA initially could lose the ability to be recognized by TSA specific T cells by selection of nonmutated variants. These could grow and kill the host. Through this approach, two types of TSA were demonstrated. One is the result of a single point mutation in an otherwise normal cell protein, which gives
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rise to the unique TSA such as are found in chemically and physically induced tumors. The other TSA category involves aberrant expression of a protein structure normal at an earlier epoch of development, for which the usual tolerance of self has not developed. These TSA are shared by some similar tumors. This investigator has recently employed a very similar approach to demonstrate at least four TSA in human melanoma. Boon used a patient's own cytotoxic T cells, derived as a cell line, to identify the TSA. Then, Boon identified and sequenced the gene (MAGE-I) coding for one of the melanoma-specific antigens. It turned out that this was a TSA of the second type, an aberrant expression of a normal protein. Boon also found that MAGE-l antigen is a peptide of nine amino acids bound only to the HLA-Al MHC. Boon identified this gene actively expressed in 30% of other melanomas as well as in 15% of breast and lung cancers. The normal function of MAGE-l is not yet known. It might be predicted on the basis of recent oncogene research that it is part of a regulatory gene complex controlling some phase of cell development or proliferation. The immunotherapeutic vistas opened by this information are considered later. These new approaches to defining exactly what TSA are and how they arise in the tumor cell are important. Mutations and aberrant expressions of regulatory genes have been increasingly recognized in progressing cancers. They provide a specific target at which to aim the immune system in a set of strategies being evaluated to enhance those elements most effective in destroying tumors.
IMMUNOLOGIC INTERVENTION IN CANCER
Attempts made thus far to enhance or alter the vigorous immunologic responses to ongoing cancers in favor of the patient are, unfortunately, notable more for promise than concrete results. Two general approaches have been tried. One attempts to enhance or redirect ongoing immunologic reactivity to the tumor. The other involves induction of new or preexisting but unexpressed TSA or modified portions thereof. This is based on the premise that induction of new or preexisting but unexpressed TSA will induce a more effective immune response. Cancer vaccines represent a recurrently active approach that has been in and out of vogue since the 1930s. Most approaches represent some variation of a protocol wherein the patient's own tumor cells, altered or unaltered, are reinjected at sites remote from the primary tumor. In various studies, up to 25% of patients had favorable initial responses, but the protocols were uncontrolled or controlled by historic therapeutic data. These attempts are ongoing with renewed enthusiasm at present. 6 Immunologic enhancement of immune function has taken many other forms. The most venerable, and still in the trial stage, involves the systemic use of adjuvants or immunostimulants. In this approach, sub-
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stances shown in experimental systems to augment the immune response, such as classical Freund's adjuvants, are administered to patients with other modalities of treatment. The key ingredients have been mostly bacterial products of coliforms or acid-fast bacteria. The thrust of research efforts ("biomodulation") was to find a product that enhances responses with minimal local and systemic toxicity. The substances have been designed to inject alone or in combination with a patient's tumor cells as a "vaccine." To date, the results have been unpromising in controlled studies, although anecdotal support for the approach is available, and experimental and clinical studies continue with emphasis on those tumor types that appear to elicit the greatest immune responses, such as melanomas. In this same general category are protocols that employ T-cells extracted from the peripheral blood or from biopsied pieces of the patients' tumors, assuming their TSA specificity is similar to that established in animal systems. The most widely evaluated protocol starts with a substantial number of peripheral lymphocytes, known to contain TSA specific T-killer subpopulations. These cells are activated and expanded by incubating in high concentrations of 11-2. This gives a population of cells that is capable of killing the tumor in vitro but that is not MHC restricted, termed lymphokine-activated killer cells ("LAK" cells). Initial evaluation of the use of LAK cells, with and without IL-2 infusion, in over 200 patients with melanoma or renal cell carcinoma showed three complete remissions in melanoma and suggestive amelioration in a few additional patients. The toxicity observed was formidable. 16,17 The more recent approach involves use of tumor infiltrating lymphocytes (TIL) taken from the patient's tumor. TIL are apparently ineffective in situ. When grown in tissue culture in the presence of low concentrations of IL-2 and in the absence of TSA, however, large numbers are generated that show tumor-specific killing in vitro and the patient's MHC restriction. These cells are presumably resurrected from an anergic state. These are currently being tested by reinjection into the patient, with or without additional recombinant IL-2.3, 16 These intensive trials with well-planned protocols have given a few notable remissions. However, treatments with IL-2, LAK cells, and TIL cells are still considered experimental. The second approach depends upon inducing or giving a tumor new and presumably more immunogenic TSA upon which the immune system can focus more effectively. Most studies involve inserting into the tumor cell a cloned gene known to code for an antigenic moiety, which then acts as a new TSA. Genes can be inserted into other cells by several now established techniques, such as within a virus vector or by electroporation or incorporation into artificial cell-penetrating liposomes. For example, if it were possible to incorporate an immunogenic viral gene selectively into the tumor cells, new targets for effective cytotoxicity would be generated. Alternatively, the gene for a nonself histocompatibility gene such as HLA-A2 might be placed into a substantial portion of tumor cells in an HLA-Al patient; HLA-A2 could present a strong target for rejection. One realistic way to get any gene into all tumor cells in
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vivo would be to add it to the genome of an otherwise harmless virus with affinity for the particular tumor cell type and to give this transformed virus systemically. Early clinical trials are underway in which modified vaccinia virus is being used to introduce genes into human tumors. Virallysates of tumor cells also are being injected on the hypothesis that TSA introduced in this way will be more effective. Also under investigation are methods of incorporating genes for cell interaction molecules and lymphokines, all designed to enhance active but ineffective TSA specific T-cells. For example, genes for IL-2, tumor necrosis factor (TNF), or interferon, three powerfullymphokines, have been inserted into human tumor cells in vitro. The gene for the B7 cell interaction molecule has been inserted into otherwise lethal experimental tumor cells, rendering them ready targets for tumor rejection. Insertion of B-70 into human tumor cells should occur shortly.26 Lastly, the genes for TSA that are targets for rejection by T cells and that are common to a class of tumors, such as MAGE-1 in melanoma, can potentially be used to augment T-cell responses incorporated into vaccine complexes or into viral vectors. Boon4 is currently using MAGEl in vaccines in HLA-A1 patients with melanomas having the MAGE-1 gene expressed (as "antigen E" peptide).
WHY IS AN EFFICIENT IMMUNE SYSTEM SO OFTEN UNSUCCESSFUL IN ELIMINATING TUMOR GROWTH?
The validity of this question depends on the unproved assumption that it most often fails. Unknown at present is the frequency of tumors that never become clinically evident, perhaps due to the efficiency of normal immune surveillance over emerging mutant or dysregulated cells destined to progress toward malignancy. The idea of immune surveillance in control of cancer was first proposed in 1954 by Lewis Thomas. 12 Direct proof that it is a biologically active function of the immune system still escapes rigorous experimental proof. Indirect evidence abounds. This includes the increased malignancy rates in immunologically impaired individuals. The observations of concomitant immunity in mice and humans also can be interpreted in terms of the surveillance hypothesis. As delineated earlier, the immune system is very active in most tumor-bearing individuals. Most major cancers are not totally removed by the surgeon, but the patient frequently recovers. Even in the presence of established and incurable tumor, the system may have a major role in holding local extension and metastasis at bay. The most overlooked factor unique to tumors is a persistent antigen overload. This antigen load has been demonstrated experimentally and clinically. For example, the numbers of individual tumor cells or clusters in the draining veins in cancers of the colon, ovary, stomach and breast are large, on the order of 5 to 50 cells or more per milliliter of blood?' 15 This number is independent of prognosis in the individual patient. Tumor products such as CEA from the gastrointestinal tract and PSA from
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the prostate and products of neuroblastomas are also passed into the blood in large quantities. The observations suggest that large amounts of other cell products, including shared and unique TSA, are being secreted or released by every growing tumor. Logically, the concentration of such products would be in a gradient, with the greatest concentration being at the tumor-interstitial fluid interface. It should not be surprising that some TSA represent complexes of tumor-specific peptide fragments that have been shed by the actively growing tumor and bound to specific antibody or MHC. Indeed, such complexes include the fascinating "blocking factors" that received so much attention in the early 1970s.1O Blocking factor is a phenomenon in which serum from tumor-bearing animals and humans was shown to block specifically either cytotoxic or proliferative responses of tumorspecific T cells. When the tumor was completely removed, blocking factor disappeared from serum.9 The tentative molecular characterization suggested that it consisted of an antigen fragment complexed to an antibody-like moiety/9 although the molecular weight estimates were too low for it to be a complete antibody. This factor should be reevaluated in terms of what is currently known of MHC/ antigen complexing at the cell surface. Another factor that may negatively affect effective immune killing rests in the observation that many tumors progressively lose MHC representation at the surface of the celts Whether this is due to genomic down-regulation or to increased rates of shedding is unknown. Loss of surface Class I MHC could severely limit the possibilities for TSA-specific cytotoxic T-cells finding TSA in the necessary MHC context. Continuous and high antigen load has many other ramifications. For the oncologist, it is a rationale for the long-held conviction that debulking tumors, even if tumor cells persist, has a favorable effect on prognosis. More specifically, if the TSA peptides are circulating bound to MHC fragments, they could bind specifically to tumor-specific T-cell receptors at a site remote from the tumor, defusing any killer effects. Too, a principle of immune tolerance is that inducing tolerance is antigen-dose dependent, and sustaining tolerance requires a continuing source of antigen. 21 In the successful long-term transplant, for example, this source is the transplanted tissue itself. In this context, the tumor-host relationship may be thought of as a chimeric state, the tumor supplying antigen(s) that sustain a volatile state of tolerance at the T-cell level. The high end of the TSA gradient is, of course, in the tumor itself, and this is where one would expect to find the most tolerized or anergic cells. Restoration of tumor reactivity in TIL populations placed in vitro with appropriate lymphokines implies that T-cell tolerance in the form of anergy indeed exists. Like anergy in other contexts, it is potentially reversible in the absence of a continuing antigen load. TIL also have been found to be oligoclonal with respect to melanoma antigens. IS If TSA are considered as a set of self peptides anomalously inducing autoimmunity, it is probable that a very limited repertoire of T-cell receptors recognize other TSA. This would limit the intensity and perhaps the effectiveness of the tumor-specific immune response.
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IMMUNOLOGIC APPROACHES TO DIAGNOSIS OF TUMORS
Applications of advances in immunology to tumor diagnosis have added an exciting new dimension to diagnostic pathology. Tumor antigens in the circulation and in tissue sections are identified using techniques of in situ reaction with monoclonal antibodies, ELISA assays, and other applications. For example, ELISA is widely used to screen for and to follow the progress of prostate cancer (PSA) and colon and liver cancer (CEA). At the tissue level, pathologists are looking at the expression of oncogenes and other anomalous cell products in staging colon cancer; in the identification of puzzling tumors, papilloma virus associated antigens in determining the prognosis of cervical cancer; and many others. The diagnosis and staging of most lymphomas are performed primarily by using specific monoclonal antibodies to determine the phenotype of individual cells for analysis in the fluorescence cell analyzer (FACS). This supplies data that are much more useful to the oncologist in treatment than used to be available through ordinary tissue section examination. It is reasonable to expect more practical screening and diagnostic applications in the future. References 1. Adler WH, Takiguchi T, Smith RT: Phytohemagglutinin unresponsiveness in mouse spleen cells induced by methylcholanthrene sarcomas. Cancer Res 31:864, 1971 2. Amos DB, Stetson CA: Tumor immunity. Ann NY Acad Med 101:1-326, 1962 3. Becker Jc, et al: Tumor infiltrating lymphocytes in primary melanoma: functional consequences of differential 11-2 receptor expression. Clin Exp Immunol 91:121-125, 1993 4. Boon T: Toward a genetic analysis of tumor rejection antigens. Adv Cancer Res 58:179210, 1992 5. Chauvenet PH, Smith RT: Relation of tumor specific antigens and the histocompatibility complex: Dissociation of in vitro alloantigen expression and in vivo immunity, from tumor specific transplantation antigen strength. Int J Cancer 22:79-93,1978 6. Cohen J: Research News: Cancer vaccines get a shot in the arm. Science 262:841-843, 1993 7. Engell H: Cancer cells in the blood: A five to nine year follow up study. Ann Surg 149:457-461,1959 8. Fisher B, Fisher ER: Studies concerning regional lymph nodes. Initiation of immunity. Cancer 27:1001-1004,1971 9. Gainor BJ, et al: Specific antigen stimulated lymphocyte proliferation in osteosarcoma. Cancer 37:743,1976 10. Hellstrom I, et al: Cell mediated immunity against antigens common to human colonic carcinomas and fetal gut epithelium. Int J Cancer 6:346-351,1970 11. Inokuchi K, et al: Stromal reactions around tumor and metastasis and prognosis after surgery for gastric cancer. Cancer 20:1924-1929,1967 12. Klein G: Tumor antigens. Ann Rev Microbio120:223-252, 1966 13. Konda S, Smith RT: The stimulatory effects of tumor bearing upon T-cell and B-cell populations in the mouse spleen. Cancer Res 33:2247,1973 14. Prehn RT, Main JM: Immunity to methylcholanthrene induced sarcomas. J Nat! Cancer Inst 18:769-778, 1957 15. Roberts SS, et al: Prognostic significance of cancer cells in the circulating blood. Am J Surg 113:757-762, 1967
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16. Rosenberg S, et al: Prospective randomized trial of high-dose 11-2 alone or in conjunction with lymphokine activated killer cells (LAK) for the treatment of patients with advanced cancer. J Natl Cancer Inst 85:622-632,1993 17. Schoof OR, et al: Survival characteristics of metastatic renal cell carcinoma patients treated with LAK cells plus 11-2. Urology 41:534-539,1993 18. Sensi M, et al: T-cell receptor structure of autologous melanoma-reactive cytotoxic Tcells clones. J Exp Med 178:1231-1246, 1993 19. Sjogren HO, et al: Suggestive evidence that "blocking antibodies" of tumor bearing individuals may be antigen-antibody complexes. Proc Natl Acad Sci USA 68:1372-1375, 1971 20. Smith RT: Tumor specific immune mechanisms. N Engl J Med 278:1207-1214, 278:12681275,278:1326-1331,1968 21. Smith RT: In Landy M, Braun W (eds): Immunologic Tolerance. New York, Academic, 1969, pp 48-52 22. Smith RT: Potentials for immunologic intervention in cancer. N Engl J Med 287:439, 1973 23. Smith RT, Landy M: Immunobiology of the tumor-host relationship. New York, Academic,1985 24. Smith et al: In Saunders et al (eds): Fundamental Mechanisms of Cancer Immunity. North Holland, Elsevier, 32:449,1981 25. Southern CM, et al: The effects of leukocytes upon the transplantability of human cancer. Cancer 19:1743-1753, 1966 26. Townsend SE, Allison JP: Tumor rejection after direct costimulation of CD8+ T-cells by B7 transfected melanoma cells. Science 259:368-370,1993 Address reprint requests to
Richard T. Smith, MD Box J275 Health Sciences Center University of Florida Gainesville, FL 32610