Immunologic Mechanisms in UV Radiation Carcinogenesis

IMMUNOLOGIC MECHANISMS IN UV RADIATION CARCINOGENESIS Margaret L. Kripke Cancer Biology Program, NCI-Frederick Cancer Research Center, Frederick, Maryland

I. Introduction

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11. Carcinogene 111. Immunosuppression an

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IV. Antigenicity of UV-Induced Tumors ..................................... A. Transplantation Studies ............................................. B. Mechanisms in the Rejection of UV-Induced Tumors ....... ...... C. Specificity of the Immune Responses to UV-Induced Tumors . . . . . . . . . . V. Immunologic Reactivity of Mice during UV Carcinogenesis . . . . . . . . . . . . . . . A. Response to UV-Induced Tumors .................................... B. Immune Responses to Other Antigens ................................ C. Immunologic Basis for Tumor Susceptibility ......................... D. Properties of UV-Induced Suppressor Cells ........................... VI. Mechanisms in UV-Induced Immunosuppression ........................ A. Pathway for Induction of UV-Induced Suppressor Cells ............... B. Antigens Involved in the Induction of Suppressor Cells by 96 UV Exposure ................................................. 97 C. Suppression Mediated by Other Mechanisms . . . . . . . . . . . 98 VII. Conclusions ............................................. 103 References .................... ....................................

I. Introduction

The question of whether the immune system is involved in cancer induction and pathogenesis has received considerable attention over the past few decades. This interest was generated by early work on the mechanisms of rejection of foreign tissues. Both Burnet (1957, 1964) and Thomas (1959) postulated that the elaborate immunologic mechanisms for recognizing and destroying tissue allografts evolved as a means of eliminating cancer cells that arose within the host. This attractive hypothesis of “immune surveillance” provided the basis for a large body of experimental work that addressed the predictions of the theory. These studies included searches for new or altered antigens on cancer cells (Klein, 1966); tests of cancer induction and cancer antigenicity in immunosuppressed hosts (Kripke and Borsos, 1974a); and an examination of the immunosuppressive potency of various carcinogens (Stjernsward, 1969). These approaches have led to the conclusion that, in cancers associated with oncogenic viruses, the immune system 69 ADVANCES IN CANCER RESEARCH, VOL. 34

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can play a decisive role in both cancer development and subsequent growth. This has been demonstrated in experimental animal systems (Law, 1969; Huebner et al., 1976) and with some naturally occurring lymphoid tumors, e.g., feline leukemia (Essex et al., 1973), avian leukosis (Hanafusa, 1975), and Burkitt’s lymphoma (Klein, 1971). The situation with cancers of nonviral etiology is less clear. The diversity among such tumors with regard to antigenic specificity and antigenic strength (Old et d.,1962; Basombrio, 1970; Klein, 1966) makes it difficult to generalize about the role of the immune system in their development. Even in the case of the more antigenic, chemically induced murine tumors, the nature of the involvement of the immune system in their evolution is controversial (Kripke and Borsos, 197413). One obstacle to investigating this issue directly is the fact that each chemically induced tumor is antigenically unique, as assessed by transplantation tests with tumors of recent origin (Basombrio, 1970; Globerson and Feldman, 1964; Klein, 1966; Prehn, 1962; Price and Baldwin, 1975).For this reason, it is extremely difficult to evaluate the immune response to a particular developing tumor until it reaches a rather large size. The most important period, that before the tumor can be detected grossly, has been inaccessible for a direct study of the specific immune response to a developing tumor. This problem has been circumvented to some extent in the investigation of murine skin cancers induced by UV radiation. Various peculiarities in the antigenicity of these tumors and in the immunologic effects of irradiation with UV rays have permitted an examination of some of the immunologic events that occur before the emergence of these tumors. Some of these events are likely to be unique features of this tumor system because of the unusual immunologic consequences of UV irradiation. However, this system provides a dramatic illustration of the complexity of the relationship between the immune system and developing cancers, and it also permits us to draw some general conclusions about the nature of tumor antigenicity. II. Carcinogenesis by UV Radiation

The importance of sunlight in the etiology of human skin cancer was recognized as early as 1894 (Unna, 1894). Shortly thereafter, studies with mice and rats confirmed the carcinogenic activity of sunlight (Findlay, 1928) and demonstrated that the UV region of the sun’s spectrum was responsible for this effect (Roffo, 1934). Since that time, research in this area has been directed toward three major themes: attempts to define the relationships between UV radiation exposure and skin cancer induction in man and in laboratory rodents, descrip-

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tions of the pathogenesis.of skin cancer, and attempts to determine the mechanism by which UV radiation causes neoplastic transformation. Although a detailed review of these topics is beyond the scope of this article, some of the conclusions from this work are important for understanding and interpreting the immunologic phenomena. The pioneering work of Blum and his colleagues in the 1940s provided an invaluable body of information on the quantitative relationships between UV exposure and murine skin cancer (Blum, 1959). From these experiments, it was possible to conclude that cancer induction was related to the cumulative dose of UV radiation, and that certain steps of the carcinogenic process were irreversible. An important characteristic of experimental UV carcinogenesis is that there is little reciprocity with regard to time and dose. Blum’s experiments demonstrated that giving the same total dose of radiation at different dose rates does not necessarily produce the same number of tumors on the ears of strain A mice (Blum, 1959). Recent studies have demonstrated, in addition, that giving a dose of UV radiation in different patterns of fractionation produces different tumor incidences and induction times. In hairless mice, multiple, small doses of radiation are more effective in producing skin cancers than are larger, less frequent doses (Forbes, Blum, and Davies, personal communication; Forbes et al., 1978), whereas in the rat, the opposite result has been reported (Strickland et

al., 1979).

Human epidemiologic data support the concept that UV radiation, or at least sunlight, is a causative agent in the production of some skin cancers. Studies by Urbach demonstrate that the parts of the body most heavily exposed to sunlight are the most frequent sites of occurrence of basal and squamous cell carcinomas (Urbach, 1969). Other epidemiologic work reveals that human skin cancer incidence increases with increasing solar exposure caused by geographic and climatic factors (National Research Council, 1975) and that its probability of occurrence increases with increasing age of the population (Fears et al., 1977). These findings suggest that certain skin cancers in susceptible human populations are induced by direct exposure to sunlight and that the carcinogenic process involves the cumulative effects of multiple exposures to radiation from the sun. The role of sunlight in the etiology of human melanoma is much less clear. Although there seems to be come association of melanoma with sun exposure, this relationship is not the direct one observed for other types of skin cancer (Fears et al., 1977; National Research Council, 1975). Although it is most likely that the carcinogenic effect of sunlight in humans is mediated by the mid-UV, or UV-B, portion of the sun’s spectrum (-280-315 nm), there is little direct evidence supporting

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this possibility. Evidence relating skin cancer to sunlight exposure generally does not distinguish between total spectral irradiance and the UV portion of the spectrum. The involvement of UV-B radiation in skin cancer induction is inferred from studies with laboratory animals (Roffo, 1934; Epstein, 1978; Blum, 1943), and from the assumptions that the wavelengths inducing an erythema1 response are similar to those inducing skin cancer (National Research Council, 1975). Although there is some evidence from studies on mice that supports this possibility that erythema-inducing wavelengths are important for carcinogenesis (Freeman, 1975), this remains unsubstantiated for human skin cancer. This problem eventually may be resolved by studies on the long-term effects on skin cancer incidence of using sunscreens known to reduce erythema. Descriptive studies on the development of UV-induced skin cancer have revealed both similarities and differences in its pathogenesis in different strains and species of animals. An important unifying feature of skin cancer induction is its association with precancerous lesions, or actinic keratoses (Epstein, 1970). The existence of such preneoplastic lesions suggests that skin cancer develops by multiple, sequential stages, rather than as a result of a single, infrequent event. A second generalization that emerges from the descriptive studies is that sunlight- or UV-associated skin cancers are among the least aggressive neoplasms. Reports of metastasis of human nonmelanoma skin cancer are rare, and metastasis appears to be a late occurrence in the evolution of these tumors (National Research Council, 1975; Mikhail et al., 1977). Metastasis of primary UV-induced skin cancers in mice is also infrequent (Spikes et al., 1977; Grady et al., 1941), although many UV-induced fibrosarcomas metastasize after transplantation (Lill and Kripke, 1978; Kripke et al., 1978). There are many differences in UV-induced skin cancer development among various laboratory animals. Susceptibility to tumor induction is quite variable in different rodent species: mice are highly susceptible, and guinea pigs are relatively resistant (Stenback, 1975). Even within various mouse strains, there are wide variations in susceptibility to tumor induction (Kripke, 1977). Some of these variations can be attributed to characteristics of the epidermis, such as the presence of hair, thickness of the epidermis, or pigmentation (Epstein, 1970), others remain unexplained and are loosely attributed to “genetic” or “host factors” (Kripke, 1977). In addition to differences in tumor incidence, there are differences in the sites of tumor occurrence in different mouse strains and in the histologic types of the resulting tumors. For example, under defined conditions of UV exposure, skin cancer occurs

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predominantly on the ears of shaved BALB/c mice, on the backs of shaved C3H mice (Kripke, 1977), and on the eye and surrounding structures of shaved DBN2 mice (M. Kripke, unpublished data). Squamous cell carcinoma and fibrosarcoma are the main histologic types of tumor produced in mice by UV exposure, but the proportions of these differ in various strains (Kripke, 1977) and within a strain under different conditions of exposure (Blum, 1959; Spikes et al., 1977). One important conclusion to be drawn from these observations is that cancer develops only in cells that are exposed directly to UV radiation. This implies that direct interaction between the carcinogen and the target cell is required for carcinogenesis, and that cells in tissue layers below the penetration range of the radiation are not at risk for UV carcinogenesis. One of the most active areas of investigation in UV carcinogenesis at present concerns the effects of UV radiation on DNA. Since the transformed phenotype is transmitted to successive generations of cancer cells, it is likely that neoplastic transformation results from changes in DNA structure or expression. Studies with the DNA of prokaryotic and eukaryotic cells have revealed that UV radiation induces several types of lesions in DNA, and that there are various repair mechanisms within these cells that can remove or repair these damaged sites (reviewed in Smith, 1978). Interest in DNA damage and repair in relation to photocarcinogenesis has been stimulated by work on two evolutionarily diverse subjects-man and a tropical fish. In a series of elegant experiments, Setlow and Hart demonstrated the importance of pyrimidine dimers, induced in DNA by UV radiation, in the formation of thyroid carcinoma in the Amazon molly. The formation of tumors was proportional to the number of such dimers in DNA, and the specific repair of these lesions by photoreactivating enzyme prevented tumor induction (Hart et aZ., 1977). The second source of information on the role of pyrimidine dimers in photocarcinogenesis comes from the studies of Cleaver (1968; Cleaver and Bootsma, 1975) and Robbins (Robbins et al., 1974) on patients with the rare genetic disease, xeroderma pigmentosum. This disease is associated with a variety of manifestations, one of which is a marked propensity for developing skin cancer on sun-exposed parts of the body. Cells from such individuals exhibit defects in repair pathways that ordinarily would remove pyrimidine dimers produced in DNA by exposure to UV radiation. Thus, an inability to repair these lesions in DNA is associated with a high risk of skin cancer development. A second mechanism in the induction of skin cancers by UV radiation has been suggested by the experiments of Zajdela and Latar-

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jet (1978). After each UV exposure, they painted the ears of mice with caffeine, an inhibitor of postreplication repair, and found that fewer tumors were produced. They interpret this finding as suggesting that repair of UV-damaged DNA by the excision repair pathway is inaccurate and that misrepair of the damage is actually responsible for the production of some tumors. Most of the studies to date on UV-induced damage and repair of DNA have been concerned with the effects of short wavelength, or UV-C (-200-280 nm) radiation, because it is efficiently absorbed by DNA where it produces mainly pyrimidine dimers. One problem with this work is the fact that these wavelengths normally are not present in the sunlight that reaches the earth’s surface, due to their absorption by the ozone layer of the upper atmosphere (Freeman et aZ., 1970). Furthermore, because of the low energy of radiation in the UV-C region, it penetrates the germinative layer of the epidermis inefficiently, and is, therefore, relatively ineffective in producing experimental skin cancers compared with longer wavelengths (Kirby-Smith et al., 1942; Strickland et aZ., 1979). Thus, the radiation that is most efficient in producing pyrimidine dimers seems to have the least relevance for solar carcinogenesis. On the other hand, pyrimidine dimers are formed in mouse skin irradiated with UV-B radiation (Ley et aZ., 1977), and there is some work suggesting that the proportion of dimers produced by different light sources correlates with their ability to induce skin tumor formation in rats (Strickland, 1978). Thus, in spite of the absence of UV-C radiation in sunlight, pyrimidine dimers still could be the primary carcinogenic lesion produced in DNA by sunlight exposure (Setlow, 1974). One point that remains to be addressed is whether there could be other lesions in DNA besides pyrimidine dimers that also contribute to photocarcinogenesis by inducing neoplastic transformation. Recent work with bacterial systems demonstrates that the relative proportion of pyrimidine dimers decreases with increasing wavelength, whereas the proportion of single-strand breaks increases (Webb, 1977). At present, there is no way to exclude the possibility that solar photocarcinogenesis represents a collection of tumors induced by quite different mechanisms, by radiation from different regions of the spectrum. Another question concerns the multistage character of skin cancer development. It is possible that pyrimidine dimers in DNA represent only one of several changes that must occur in a cell during its progression to a final transformed state. Other alterations in DNA produced by longer wavelengths could participate in carcinogenesis by mediating subsequent steps in tumor development.

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Thus, investigations of the induction and repair of UV-damaged DNA have provided some important clues to the mechanisms involved in photocarcinogenesis. However, the relative importance of specific DNA lesions and of particular wavelengths has not been established. An additional hypothesis for a chemical intermediate in UV carcinogenesis also has been proposed. Work b y Black and his colleagues has shown that cholesterol a-oxide is formed in human and mouse skin after UV irradiation (Lo and Black, 1972; Chan and Black, 1974). This compound was thought to have carcinogenic activity, based on work by Bischoff (1963), and thus, Black and his colleagues suggested that this could represent one pathway toward photocarcinogenesis. However, recent studies have not confirmed Bischoff s report that cholesterol epoxide produces skin cancer (Black and Chan, 1977), making it uncertain whether this represents a mechanism of primary importance in photocarcinogenesis. A recent advance that could help to clarify the molecular mechanisms in UV carcinogenesis is the demonstration that rodent cells in culture can be transformed with UV radiation. Tumorigenic cells have been produced by UV-C treatment of a murine fibroblast cell line, both in the presence (Mondal and Heidelberger, 1976) and the absence (Chan and Little, 1976, 1979) of chemical promoters, by UV-C treatment of freshly explanted fetal hamster fibroblasts (DiPaolo and Donovan, 1976), and by UV-B treatment of neonatal mouse epidermal cells (Ananthaswamy, 1979). Morphologic transformation of human dermal fibroblasts with UV-B radiation also has been achieved (Sutherland et al., 1980), although the tumorigenic potential of these transformed lines has not been assessed. The fact that transformation of cells in culture can be achieved with UV-C and UV-B radiation provides a means of comparing the mechanisms of transformation resulting from irradiation with various wavelengths in the UV region of the spectrum. Ill. Immunosuppression and UV Carcinogenesis

Interest in the contribution of host factors to photocarcinogenesis has been aroused only very recently. It originated from the astute clinical observation of Penn (1970) that renal transplant patients were at an increased risk of developing certain types of cancer. Concerned that the long-term use of immunosuppressive drugs to prevent rejection of a grafted kidney also could depress an immunologic surveillance mechanism, Penn collected cancer incidence data on these patients. These data indicated that the transplant recipients had an increased

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risk of developing tumors of the lymphoreticular system and skin cancers on sun-exposed areas of the body, although the incidence of cancer in other organs and tissues was not elevated (Koranda et al., 1974). This observation, subsequently confirmed by others (Marshall, 1974; Hoxtell et al., 1977),provided the groundwork for experimental sthdies on the effects of immunosuppressive agents on photocarcinogenesis. To date, five independent studies of this issue have been reported. Koranda et a1. (1975) examined the effects of chronic, low doses of prednisone, azathioprine, and a combination of these drugs on UVinduced skin cancer development in hairless mice and found an increase in skin cancer with azathioprine treatment, but no effect with prednisone administration. Nathanson et al. (1976) reported that photocarcinogenesis in hairless mice was enhanced by chronic administration of antilymphocyte serum, and decreased by 6-mercaptopurine treatment. A decrease in tumor incidence following administration of hydroxyurea and no effect of methotrexate on skin cancer development in hairless mice were observed by Epstein (1978).A fundamental difficulty in interpreting these studies is that the extent of immunosuppression produced b y the various treatments was not tested. Thus, whether their effects on photocarcinogenesis were mediated by imniunologic factors is uncertain. This problem was addressed in the most extensive study of this kind, carried out by Eichwald Bnd his associates at the University of Utah over a period of several years. In this study, the immunosuppressive activity of the various drugs was monitored throughout the course of the carcinogenesis experiments. An unexpected obstacle to this approach was encountered early in these investigations when it was found that the doses of the “immunosuppressive” drugs that could be administered chronically over long periods of time without morbidity were not immunosuppressive (Kripke et a1., 1973). In spite of this limitation, the effects of these agents and antilymphocyte globulin (ALG) on photocarcinogenesis in C3H mice were studied. The results were similar to those of Korandaet al. (1975) and Nathanson et al. (1976) in that ALG, cyclophosphamide, and cortisone acetate accelerated tumor induction b y UV radiation, whereas methotrexate had no effect (Daynes et al., 1979b). Recently, we have approached this problem by examining UV carcinogenesis in mice depleted of thymus-derived (T) lymphocytes by adult thymectomy, lethal X-irradiation, and reconstitution with neonatal liver cells, rather than by chronic administration of cytotoxic drugs (Norbury and Kripke, 1978).Tests of immunologic function were carried out to confirm the long-term immunosuppression in T-

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cell-depleted mice and restoration of immune function in control animals that were T cell depleted and then restored with thymus grafts. Skin tumor induction by UV radiation was accelerated in Tcell-depleted mice, compared to untreated animals. However, the T-cell-depleted, thymus-grafted mice were even more susceptible to tumor induction, even though they had nearly normal immunologic function. This result is confounded further by the observation that the tumors that arose in untreated mice were of a different histological type (fibrosarcoma) than those that developed in mice that had been subjected to X-irradiation as part of the T-cell-depletion procedure (squamous carcinoma). Two important points emerge from these and the earlier results of others. First, the assumption that the “immunosuppressive’’ treatments affect only the immune system is incorrect. This is most clearly illustrated by the change in histologic type of the tumors observed after immunosuppression by X-irradiation. It is apparent that the target of the carcinogen, the skin, has been altered by the “immunosuppressive” procedure. This change in histologic type of skin tumor was also observed in the studies with cyclophosphamide (Daynes et al., 1979b), indicating that cytotoxic drugs, as well as X-rays, can directly affect the carcinogenic process, aside from their immunologic effects. Second, the diverse effects of immunosuppression on photocarcinogenesis suggest that the immune system is more sophisticated and complex than our ability to manipulate it at present. The possibility of analyzing the relationship between host immunity and carcinogenesis by altering immunologic processes is dependent on our ability to control immunologic reactivity in predictable ways. The fact that the immune system represents an efficient and complex homeostatic mechanism, the regulation of which is only partially understood, severely limits the application of this approach for the moment. However, another avenue for investigating the relationship between host immunity and UV carcinogenesis has been provided by studies on the antigenicity of UV radiation-induced tumors. IV. Antigenicity of UV-Induced Tumors

A. TRANSPLANTATION STUDIES

At a time when tumors induced in inbred animals b y various oncogens were being examined for the presence of tumor-specific transplantation antigens (TSTA), Graffi and his associates published the first studies on the antigenicity of UV-induced sarcomas (Graffi et al.,

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1964). These tumors, produced in Strain XVII albino mice, induced a high level of transplantation resistance. Furthermore, this resistance was specific for the individual tumor used for immunization (Pasternak et al., 1964), as had been found previously for tumors induced by polycyclic hydrocarbons (Prehn, 1962). These findings might have gone unremarked except for a comparison made by these investigators between the antigenicity of UVinduced tumors and that of sarcomas induced by other carcinogens (Graffi et al., 1967). The UV-induced sarcomas, in general, were as antigenic as those induced by potent chemical carcinogens. This was somewhat surprising because of the generalization formulated by several investigators that tumor antigenicity seemed to be inversely related to the latency period for tumor induction (Stjernsward, 1969; Bartlett, 1972; Prehn, 1969). Because the UV-induced tumors appeared after very long latencies, they might have been expected to be only very weakly antigenic. The fact that they were not prompted me to reinvestigate this issue when the opportunity arose in conjunction with the studies on immunosuppression and UV carcinogenesis. What I rediscovered was a phenomenon noted in 1939 by Rusch and Baumann (1939) that UV-induced skin cancers are difficult to transplant, even in strictly isogenic recipients. In 1939, this phenomenon was attributed to bacterial contamination of the superficial, frequently ulcerated tumors. Nearly 40 years later, an immunologic explanation seemed more plausible, and, in fact, immunologic rejection turned out to be responsible for most of the transplantation failures (Kripke, 1974). The finding that many UV-induced tumors, including both fibrosarcomas and carcinomas, fail to grow progressively in normal syngeneic mice but will grow in immunosuppressed recipients (Kripke, 1974, 1977) provides a useful tool for comparing the antigenicity of these tumors with that of tumors induced b y other agents. This measure of TSTA differs from the more conventional one involving implantation of tumor cells, excision, and rechallenge, because it relies on a primary immune response for tumor rejection, as opposed to the secondary, or memory response that is invoked in conventional procedures. However, it provides a simple approximation of the relative antigenicity of groups of tumors within a given mouse strain. Among different mouse strains, there are large differences in the proportions of UV-induced tumors that regress in normal recipients (“regressor” tumors) upon transplantation. For example, in one study, about 75% of the tumors induced by UV radiation in C3H mice regressed upon transplantation of the primary tumor, whereas only about 30% of those induced in the BALB/c strain were regressors (Kripke, 1977). Here, however, it is not

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possible to distinguish between the intrinsic antigenicity of the tumor and the immunologic capabilities of the host strain, because tumor rejection is a function of both factors. With these transplantation tests, it is possible to compare certain antigenic characteristics of UV-induced tumors with those of chemical carcinogen-induced tumors. The results of two types of comparisons indicate that factors influencing the antigenicity of methylcholanthrene (MCA)-induced tumors do not seem to modulate the antigenicity of UV-induced tumors. The relationship between antigenicity and latency that is so striking with MCA-induced tumors (Bartlett, 1972) is not apparent among UV-induced tumors of either histologic type. The UV-induced tumors appear to be uniformly of high antigenicity, and the occurrence of transplantable versus regressor tumors is independent of latency (Kripke, 1974). Thus, the clustering of highly antigenic tumors early in the course of tumor induction with MCA (Bartlett, 1972) does not seem to occur in UV carcinogenesis. The second comparison concerns antigenicity and carcinogen dose, Prehn (1975) reported that higher doses of MCA generally produced tumors of higher antigenicity than that of tumors produced with lower doses. A similar comparison among UV-induced tumors suggested that the antigenicity of these tumors is independent of carcinogen dose (M. Kripke, unpublished data). Both of these influences on the antigenicity of MCA-induced tumors have been attributed to alterations in host immunity resulting from immunosuppressive properties of the carcinogen (Bartlett, 1972; Prehn, 1975). If this is correct, it implies that the immune system exerts a certain degree of selection against highly antigenic transformed cells, and that this selection can be modified by agents that affect the immune system. Because there appears to be little or no modulation of tumor antigenicity in W carcinogenesis, nor any selection against tumors that are highly antigenic, the relationships among the carcinogen, the tumor antigens, and the immune response must be quite unusual in this tumor system. B. MECHANISMSI N

THE REJECTION O F

UV-INDUCED TUMORS

At present, there is little detailed information on the effector mechanisms responsible for the regression of UV-induced tumors in normal syngeneic recipients. The regression is T-lymphocyte-dependent, because tumor growth will occur in mice depleted of T lymphocytes by various means including adult thymectomy and sublethal X-irradiation (Kripke, 1974), antilymphocyte serum treatment (Kripke, 1974; Daynes et al. 1977), congenital absence of the thymus (Kripke,

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1977), and treatment with cytotoxic drugs (M. Kripke, unpublished data). Restoration of T-cell-depleted mice with thymus grafts reverses this susceptibility to tumor transplantation (Norbury and Kripke, 1978). These findings support the contention that the regression of these tumors is mediated by immunological processes, in particular those dependent on T lymphocytes. Although tumor regression is T cell dependent, this does not necessarily mean that T cells are the ultimate effectors of tumor cell destruction. Because T cells play a regulatory role in the formation of antibody by B cells and produce lymphokines that recruit and activate other cells that can destroy tumor cells, in addition to acting as cytotoxic effector cells, it is not possible to infer from these experiments that the T lymphocytes are actually involved directly in the killing of the tumor cells. Circumstantial evidence for the participation of cytotoxic T cells in tumor regression was obtained by Lill and Fortner (1978), however. They recovered the host cells infiltrating a tumor during its regression in a normal syngeneic recipient. The inflammatory infiltrate consisted of mainly T lymphocytes and polymorphonuclear leukocytes, with a smaller proportion of macrophages and few or no B lymphocytes. The T lymphocytes recovered from regressing tumor fragments were highly cytotoxic for the specific tumor in uitro, even at ratios of effector to target cells as low as 1 : 1. Glass-adherent cells in the infiltrate also were cytotoxic for tumor cells in culture, but this cytotoxicity was not specific for the implanted tumor. The largest numbers of cytotoxic T lymphocytes were present in the tumor between 4 and 6 days after tumor implantation, a time that just precedes the beginning of morphologic degeneration of the tumor tissue. This association, along with the absence of cytotoxic T lymphocytes in tumors that do not undergo regression, suggests that the cytotoxic T lymphocyte is at least one of the mediators of tumor regression. It is interesting in this regard that, in one study, substantial numbers of T lymphocytes could be recovered from human cutaneous basal and squamous cell carcinomas that had low malignant potential (Viac et uZ., 1977),and in another, the size of these tumors was inversely related to the degree of lymphocytic infiltrate (Dellon et ul., 1975). Additional evidence that the regression of UV-induced tumors in normal recipients is immunologically mediated is that tumor regression is accompanied by the development of heightened resistance, or immunity, to the regressing tumor (Kripke, 1974). This secondary immune response, which is specific for the regressing tumor (Kripke, 1974), can be demonstrated in a variety of in viuo and in uitro assays.

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The in uiuo assays include sublethal X-irradiation of mice in which a tumor has regressed, followed by tumor challenge (Kripke, 1974), passive transfer of immunity to immunosuppressed recipients (Fisher, 1978),and a modified Winn assay in which tumor and lymphoid cells are mixed and injected subcutaneously into immunosuppressed recipients (J. Mattes and M. Kripke, unpublished data). In all tests, lymphoid cells from “regressor” mice are more active in tumor rejection than those from normal animals. The passive transfer of immunity to a secondary host is also dependent on T lymphocytes (Daynes et al., 1 9 7 9 ~but ) ~ the precise subclasses involved have not been determined. Tumor regression in a syngeneic host also can be accompanied by the activation of a suppressor cell pathway. This is suggested by experiments of Perry et al. (1980) in which C3H mice were injected repeatedly with anti-1-Jk serum following implantation of a UV-induced regressor tumor. Accelerated regression of the tumor was observed in animals treated with anti-I-Jk serum, relative to that in mice treated with normal mouse serum. This is interpreted as indicating that IJk-bearing suppressor T cells are activated following tumor challenge, and their elimination with antiserum results in accelerated tumor rejection. The in uiuo immune response against UV-induced fibrosarcomas can be prevented entirely by inducing an anti-idiotypic immune response against tumor-immune lymphocytes (Flood et al., 1980).However, the role of such anti-idiotypic responses in the regulation of immunity to these tumors during tumor regression in uiuo is undetermined. Various aspects of the immune response to UV-induced tumors have been studied in in uitro systems. Preparations of lymphoid cells fiom tumor-immunized syngeneic mice have cytotoxic activity in a variety of in uitro assays (Kripke et al., 1977a; Fortner and Kripke, 1977; Thorn, 1978; Daynes et al., 1979a);they exhibit proliferative responses in uitro (Flood et al., 1980; Woodward et al., 1979),and they produce macrophage-activating factors upon cocultivation with the sensitizing tumor (Kripke et al., 1977a). In uitro restimulation of lymphocytes from tumor-immunized mice produces a secondary cytotoxic response (Thorn, 1978), and recent experiments suggest that this differentiation of lymphocytes in uitro into cytotoxic effectors requires an Ia+ macrophage in the cultures for this secondary stimulation to occur (Woodward et al., 1979). Antibodies also are formed against UV-induced fibrosarcomas by syngeneic mice (DeLuca et al., 1979); however, the antisera are generally of low titer, and their significance for tumor rejection is not known.

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C. SPECIFICITYOF TUMORS

MARGARET L. KRIPKE

THE

IMMUNE RESPONSESTO UV-INDUCED

In assessing the specificity of the immune response to tumor-specific transplantation antigens in uiuo, it is essential to minimize any possibility of obtaining artifactual cross-reactions. This can be done by including controls for histocompatibility of the host and tumor, by using only tumors in their first few transplant generations, and by ruling out the possibility that cross-reactions stem from contamination of the tumor or host with infectious agents (Basombrio, 1978). Studies with UV-induced tumors in which these criteria have been met have demonstrated that UV-induced tumors, like chemically induced fibrosarcomas, have individually distinct tumor-specific antigens (Pasternak et al., 1964; Graffi et al., 1967; Kripke, 1974). Other transplantation studies have suggested that UV-induced tumors also can have crossreactive antigens (Spellman and Daynes, 1978a). In these studies, however, the criteria for defining TSTA were not followed closely, so artifactual causes for these cross-reactions cannot be ruled out. For example, some in uioo transplantation tests demonstrating crossreactivity among UV-induced tumors were performed with tumors induced in one C3H subline (Daynes et al., 1977) and tested in a different subline (Spellman and Daynes, 1978a). No negative controls for immunization (i.e., normal tissue or non-UV-induced tumors from the original subline) were included in these experiments, so the crossreactivity observed could be attributed to histoincompatibility between the sublines, rather than to common TSTA. This does not rule out the possibility that UV-induced tumors could actually share some tumor-specific antigens, but thus far, their existence has not been demonstrated unequivocally in transplantation tests. A search for common antigens among UV-induced tumors using serologic techniques also failed to detect ubiquitous antigens on UVinduced fibrosarcomas. An extensive serologic analysis revealed that there are cross-reacting antigens among UV-induced tumors, but most of them could be attributed to the presence of contaminating antiviral antibodies in the antisera (DeLuca et al., 1979). Many of the tumors induced in mouse strains carrying the endogenous murine leukemia virus will produce the virus during growth of the cells in uiuo or in uitro (Ihle et al., 1977), and antibodies against this virus are normally present in the sera of mice that harbor endogenous viruses (Ihle et al., 1980). After removal of the antiviral activity from the antisera, antibodies specific for individual tumors could be detected. A few instances of cross-reactions between pairs of allogeneic UV-induced

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tumors were noted, but there was no definitive serologic evidence for an antigen that was shared widely among UV-induced tumors (DeLuca et al., 1979).In uitro cytotoxicity tests of cell-mediated immunity to these tumors have also demonstrated mainly individually specific tumor antigens (Fortner and Kripke, 1977; Thorn, 1978).

V. Immunologic Reactivity of Mice during UV Carcinogenesis

A.

RESPONSE TO

UV-INDUCED TUMORS

The finding that many UV-induced tumors were immunologically rejected upon transplantation to normal syngeneic mice raised the question of how these tumors could survive immunologic destruction in the primary host. Studies addressed to this question revealed the unexpected finding that mice exposed to UV radiation lost their ability to reject UV-induced tumors (Kripke and Fisher, 1976). This inability to reject UV-induced tumors occurred long before there was any visible evidence of primary tumors on the UV-treated mice (Kripke and Fisher, 1976), and it was induced by radiation absorbed by the shaved skin of the animals (Kripke, 1976). Recently, we have investigated some of the photobiologic characteristics of this unresponsiveness (DeFabo and Kripke, 1979, 1980). Experiments with BALB/c mice showed that the proportion of animals susceptible to tumor challenge was linearly related to the log,, of the UV dose delivered to the animals. In addition, a given dose of radiation produced a certain percentage of susceptible recipients, regardless of the manner in which the dose was fractionated. Thus, twelve 1-hour exposures to the sunlamps over a 4-week period resulted in the same degree of tumor susceptibility as a single 12-hour exposure; furthermore, changing the dose rate of the radiation by as much as 10fold, while keeping the total dose constant, also did not affect the proportion of mice susceptible to tumor challenge. This reciprocity of time and dose contrasts markedly with the lack of reciprocity observed in the induction of primary tumors in mice by UV radiation (Blum, 1959; Forbes et al., 1978), where multiple, fractionated exposures are much more efficient in inducing tumors than single or infrequent doses of UV radiation. This suggests that, although certain steps in the carcinogenic process can be repairable or reversible, the changes leading to immunologic unresponsiveness are irreversible and probably involve different mechanisms.

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The portion of the UV spectrum responsible for inducing susceptibility to tumor challenge has been narrowed to wavelengths within the UV-B region. Using FS40 sunlamps, which emit a continuous spectrum from about 260 nm to 340 nm, with peak output at 313 nm, we were able to remove certain wavebands by inserting plastic filters between the lamps and the animals. Removing all wavelengths below 270 nm with a polystyrene filter did not alter the susceptibility of mice to tumor challenge; in contrast, exposure of the animals with equal UV doses through mylar, which removes wavelengths below 315 nm, eliminated the effect. This places the “immunosuppressive” activity of UV radiation in approximately the same waveband as the carcinogenic activity (Freeman et al., 1970). The systemic character of this immunologic unresponsiveness is evident from two types of experiments. In one, the mice were shaved and W-irradiated dorsally, and the challenge tumor was implanted subcutaneously on the ventral (unirradiated) side of the animals (Kripke and Fisher, 1976; DeFabo and Kripke, 1979). In the other, UV-exposed animals were injected intravenously with a tumor cell suspension, and the number of pulmonary tumor foci was counted 3 weeks later (Kripke and Fidler, 1980). In both protocols, UV-treated mice were susceptible to tumor growth, whereas unirradiated animals were resistant. This demonstrates that the immunologic unresponsiveness is systemic and not restricted to areas of the mice that are exposed directly to UV radiation. In addition, once induced, this susceptibility remains for very long periods in the absence of further UV irradiation (Kripke and Fisher, 1976; Kripke et aZ., 1977b). The finding that UV-irradiated mice are susceptible to challenge with any UV-induced syngeneic tumor is curious in light of the individual antigenic specificity of these tumors. It is this finding that makes the idea of additional shared antigens on these tumors particularly attractive. On the other hand, it raises the possibility that UV radiation could be acting as a general immunosuppressive agent that could prevent other immunologic reactions as well. B. IMMUNE

RESPONSES T O

OTHER ANTIGENS

The first factor we considered in this regard was the ability of UVirradiated mice to reject skin and tumor allografts (Kripke and Fisher, 1976). We found that tissue allograft rejection across a major histocompatibility barrier occurred equally well in normal and UV-treated animals. Subsequent investigations have demonstrated that rejection of grafts with minor histocompatibility differences is also normal in UV-

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treated mice, as is antibody formation to sheep erythrocytes and polyvinylpyrrolidone, the inflammatory response, the primary in uitro cytotoxic reaction to trinitrophenyl (TNP)-modified syngeneic cells, graft-versus-host reactivity, the allogeneic mixed leukocyte reaction, the proliferative response to lymphocyte mitogens, and the growth and regression of non-UV-induced syngeneic tumors (Norbury et al., 1977; Spellman et al., 1977; Kripke et al., 1977b, 1979).These results indicate that the inability of UV-treated mice to reject UV-induced tumors is a selective unresponsiveness and is not attributable to a generalized deficiency in the immune response to other antigens. There are, however, other immunologic reactions that are affected by UV irradiation. We found that early in the course of UV treatment, the animals functioned poorly as recipients for a local graft-versus-host reaction, and they failed to respond to sensitization with the contact allergen, dinitrochlorobenzene (DNCB) (Kripke et al., 197713). Although it was not apparent initially, this finding has provided a clue to the mechanism underlying the unresponsiveness of UV-treated mice to the UV-induced tumors.

c. IMMUNOLOGIC BASISFOR TUMORSUSCEPTIBILITY In order to determine whether the susceptibility of UV-irradiated mice to challenge with UV-induced tumors is mediated immunologically, three types of experiments were carried out (Fisher and Kripke, 1977). The first was parabiosis, in which UV-irradiated mice were surgically anastamosed to normal animals, and each partner was challenged with a UV-induced fibrosarcoma. Although pairs of normal parabiotic mice were resistant to challenge, normal mice parabiotically joined to UV-treated animals became susceptible to tumor challenge. This prompted us to attempt the transfer of susceptibility to normal animals using serum and lymphoid cells from UV-irradiated mice. Numerous attempts to transfer susceptibility or reactivity with serum or plasma from UV-treated mice have been unsuccessful to date (Fisher, 1978; Daynes and Spellman, 1977). However, when lymphoid cells from UV-treated donors were used to reconstitute lethally X-irradiated syngeneic mice, these recipients were susceptible to tumor challenge, whereas those given normal lymphoid cells remained resistant. Mixing the lymphoid cells from normal and UVtreated mice prior to injection also rendered the recipients susceptible to challenge, suggesting that a suppressor mechanism might be involved (Fisher and Kripke, 1977). The third type of experiment involved in uiuo challenge of normal or UV-irradiated mice with tumor

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cells, followed by i n uitro tests of lymphocyte cytotoxicity (Fortner and Kripke, 1977). In this approach also, the UV-treated mice failed to respond to UV-induced tumors under conditions in which non-UVexposed animals could generate cytotoxic lymphocytes. Using a different protocol, Spellman and Daynes (1977) also demonstrated that lymphoid cells from UV-irradiated donors contained suppressive activity. They injected lymphoid cells from UV-treated mice intravenously into normal syngeneic recipients and showed that the recipients were converted to a state of tumor susceptibility. In addition, they demonstrated that removing the theta-bearing lymphocytes from this lymphoid cell population eliminated its suppressive activity. CELLS D. PROPERTIESOF UV-INDUCEDSUPPRESSOR 1. Characteristics and Origin Based on their susceptibility to antitheta serum and complement (Spellman and Daynes, 1977; Fisher and Kripke, 1978) and their enrichment by nylon wool purification (Fisher and Kripke, 1978; Spellman and Daynes, 1978b), the UV-induced suppressor cells seem to belong in the category of regulatory T lymphocytes. The Ly phenotype of these T cells has not been determined, but they express Ia antigen, and they are extremely sensitive to the toxic effects of y radiation (Daynes et al., 1979~).Attempts to eliminate these cells completely by in uivo treatment with anti-I-Jk serum were unsuccessful (Perry et al., 1980). These suppressor cells can be obtained from the spleen and lymph nodes of UV-treated animals (Spellman and Daynes, 1977; Daynes et al., 1979c), but their presence in the thymus is still equivocal (Spellman and Daynes, 1977). Removal of the spleen after UV irradiation does not eliminate tumor susceptibility (Fisher and Kripke, 1978), a finding that also suggests that the suppressor cells are distributed throughout the lymphoid tissues. Recent experiments have demonstrated that the generation of these suppressor cells does not require the thymus or the spleen. As illustrated in Table I, removal of either organ prior to UV exposure did not prevent the development of suppressor cells in the remaining lymphoid organs (Thorn, Fisher, and Kripke, unpublished data). Because it is possible to induce tumor susceptibility in BALB/c mice by a single exposure to UV radiation (DeFabo and Kripke, 1979), we have attempted to transfer this susceptibility with lymphoid cells also. We found that lymphoid cells removed within 24 hours after a single

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TABLE I EFFECTOF THYMECTOMY (T,) OR SPLENECTOMY (s,)ON THE UV-INDUCED SUPPRESSOR CELLS"

a7

INDUCTION OF

Mice reconstituted with lymphoid cells from*

Final incidence of challenge tumof

Normal mice Normal and UV (Sham T,) mice Normal and UV (T,) mice

019 49

Normal mice Normal and UV (Sham S,) mice Normal and UV (S,) mice

0110 10110

5/9

15/15

R. T. Thorn, M. S. Fisher, and M. L . Kripke (unpublished data). mice were given 800 R 24 hours before intravenous injection of spleen

* Recipient

and/or lymph node cells. Cells ( 5 x 10') from each donor type were injected. ' Mice were challenged subcutaneously with tumor fragments of a UV-induced syngeneic fibrosarcoma 24 hours after reconstitution.

UV exposure do not transfer susceptibility to X-irradiated recipients, but cells removed 4 weeks after UV treatment transferred susceptibility to tumor challenge. This implies that some period of time between these extremes is required for the generation of suppressor cells in UV-irradiated mice (DeFabo and Kripke, 1980). The suppressor cells induced by UV radiation seem to be quite long-lived relative to the tumor-specific suppressor cells described b y Fujimoto et al. (1976). When the suppressor cells are mixed with normal lymphoid cells and injected into lethally X-irradiated mice, these recipients remain tumor-susceptible for at least 6 weeks after reconstitution (Fisher and Kripke, 1978). Suppressor cells obtained from mice given lower doses of UV radiation were able to suppress tumor rejection in normal recipients for a period of 3 to 4 weeks (Spellman and Daynes, 1978b). However, in these protocols, it is not possible to distinguish whether the suppressor cells themselves are long-lived or whether they are short-lived, but the effects mediated by them are long-lasting. 2. Mechanism of Action How these suppressor cells function to prevent tumor rejection has not been determined in any detail. A few isolated findings related to this issue have been made, but the complete sequence of events involved in this regulation of immunity to UV-induced tumors is far from being delineated.

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The most elementary question that must be addressed in this context is whether the lymphocytes from UV-irradiated mice actually function by regulating the immune response or by directly stimulating tumor growth. Both in uiuo and in uitro studies of tumor-lymphocyte interactions have shown that lymphocytes can stimulate tumor growth under some circumstances (Prehn, 1977). Two types of in uiuo experiments suggest that the lymphocytes from UV-treated mice do not stimulate tumor cell growth but act by another mechanism. Using the lung colony assay, we showed that injection of immunosuppressed mice with UV-induced suppressor cells did not increase the number of pulmonary metastases over that produced by injection of tumor cells alone (Kripke and Fidler, 1980). More recently, we have used a modified version of the Winn test to analyze suppressor cell activity (Thorn, Fisher, and Kripke, unpublished data). In these experiments, immunosuppressed mice are injected subcutaneously with tumor cells alone, or with mixtures of tumor cells and T lymphocytes from normal and/or UV-irradiated mice. A summary of five such experiments using two different UV-induced tumors is presented in Table 11. These results show that the UV-induced suppressor cells can function in this local immune reaction. They also suggest that tumor growth is not stimulated directly by these cells, because the tumor incidence is the same in groups challenged with tumor cells alone (82%) and those receiving mixtures of tumor cells and UV-induced suppressor cells (80%).These studies make it most likely that the T lymphocytes from UV-irradiated mice function by interfering with the immune response of other lymphoid cells against the antigens of UV-induced tumors. The stage at which the immune response to tumor antigens is blocked by these suppressor cells appears to be an early one in the TABLE I1 ACTIVITY OF UV-INDUCED SUPPRESSOR T CELLS IN A WINN-TYPETEST^ Cells injected subcutaneously into immunosuppressedb syngeneic recipients (ratio)

Number of mice with progressive tumodnumber challenged

Tumor cells alone Tumor + UV T cells (1: 100) Tumor + normal T cells (1: 100) Tumor + UV + normal T cells (1: 100:100) Tumor + normal T cells (1:200)

32/39 (82%) 39/49 (80%) 5/41 (12%) 37/56 (60%) 3/22 (14%)

R. T. Thorn, M. S. Fisher, and M. L. Kripke (unpublished data). Mice were immunosuppressed by adult thymedomy, lethal X-irradiation,and reconstitution with neonatal liver cells.

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sequence of events leading to the elimination of the tumor cells. This tentative conclusion comes from a variety of experiments that imply that the UV-induced suppressor cells do not depress the activity of mature effector cells. First, mice that are immunized with a tumor before they are treated with UV radiation remain resistant to challenge with that tumor after UV irradiation, although they are susceptible to the growth of other UV-induced tumors (Kripke and Fisher, 1976). This demonstrates that antitumor effector cells can function in a UVirradiated host. However, it is not clear from this experiment whether the suppressor cells are ineffective against preformed immune cells, or whether a specific subpopulation of suppressor cells is absent in these animals as a result of the immunization before UV exposure. Second, the addition of UV-induced suppressor cells to in uitro cytotoxicity reactions of immune lymphocytes against tumor cells does not reduce the amount of cytotoxicity obtained (R. Thorn, unpublished data). Finally, the intravenous injection of lymphocytes from tumorimmune donors into UV-irradiated mice can suppress tumor growth in these recipients (Fisher, 1978; Daynes et al., 1979~). Based on these approaches, it appears that the UV-induced suppressors do not affect the activity of the effector cells themselves. However, this evidence cannot be considered definitive, because it can be argued that the difference in the effect of the suppressor cells on normal and immune cells is merely quantitative and does not represent a qualitative difference. Attempts to analyze the step at which the immune response is blocked by UV-induced suppressor cells have been made using an in uitro lymphocyte cytotoxicity assay. The limitation of this approach is, of course, that it assumes that lymphocyte cytotoxicity in uitro accurately represents the process of tumor rejection in uiuo. Thus, the conclusions drawn from these studies depend heavily on the validity of this assumption. Nevertheless, this approach permits the dissection of this particular immune reaction into several stages that can be examined individually for their ability to be influenced by suppressor cells. Using a combination ofin uiuo and in uitro procedures, Thorn (1978) has analyzed the role of UV-induced suppressor cells in the generation of cytotoxic T lymphocytes. He found that primary sensitization, either in uitro or in uiuo, against UV-induced fibrosarcoma cells occurred equally well with lymphocytes from normal and UV-irradiated donors. Thus, the magnitude of the primary cytotoxic response appears to be unaffected by the presence of the UV-induced suppressor cells. In contrast, in uitro restimulation of lymphocytes from in uiuo-sensitized donors resulted in lower cytotoxic activity of the cells from UV-

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irradiated donors, compared to that of cells from unirradiated mice. Based on these results, Thorn concluded that the cytotoxic memory response against UV-induced tumors was deficient in UV-irradiated animals, and that the UV-induced suppressor T cells functioned by inhibiting the generation of cytotoxic memory cells. In addition, he suggested that in this system tumor rejection is dependent not on the primary cytotoxic response but on its amplification by differentiation of cytotoxic memory cells and/or on rejection mechanisms other than that of the cytotoxic lymphocyte. One point that remains to be resolved from these studies concerns the specificity of the cytotoxic reactions being investigated. Although the T-cell-mediated cytotoxicity detected is specific for an individual tumor, it is not clear whether the same antigenic determinants are being recognized by the lymphoid cells from UV-irradiated and normal donors. Consistent with the primary in uitro sensitization data, it is possible to immunize UV-irradiated mice in uiuo against UV-induced tumors so that they can resist a homologous tumor challenge (Daynes et al., 1977; Thorn and Kripke, 1979). However, preliminary studies using anti-idiotypic reagents suggest that the lymphocyte clones stimulated by tumor immunization of UV-treated mice are not of the same specificity as those activated in normal animals (H. Schreiber, personal communication). Thus, the finding of equivalent levels of cytotoxicity in primary in uitro sensitization with lymphocytes from normal and UV-irradiated animals does not necessarily mean that the responses are identical, because they still can differ in specificity. The lack of detailed information about the specificity of the lymphocyte clones that react against UV-induced tumors makes it difficult to draw conclusions about the specificity of the UV-induced suppressor cells. At a gross level, it seems that these suppressors lack specificity for the individually specific tumor antigens, as they prevent the rejection of many different UV-induced tumors (Fisher and Kripke, 1977, 1978; Spellman and Daynes, 1977). This situation has led Spellman and his colleagues to postulate that the suppressor cells are directed against a common antigen that is expressed on all UV-induced tumors and that is separate from the individual antigen (Spellman and Daynes, 1978a). We have suggested a similar model in which the suppressor cells are directed against a common determinant and the effector cells are directed against an individual determinant on the same antigenic molecule (Kripke et al., 1979). However, there are no experimental data to date that can rule out a third possibility, which is that the UV-induced suppressor cells consist of a mixed population in which each subpopulation is specific for an individual tumor antigen. Thus, the suppressor population, induced during the course of UV

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irradiation, would prevent immunologic rejection of all UV-induced tumors, but an individual suppressor cell would have specificity for a single tumor. It is extremely important to be able to distinguish between this latter possibility and the others involving shared antigens or determinants, because this information could reveal the nature of the antigens by which regulation of the immune response against autochthonous tumors is controlled. 3. Role in Carcinogenesis It is clear that UV-induced suppressor T cells can prevent the rejection of transplanted UV-induced tumors. However, whether these cells actually play a role in carcinogenesis is still undetermined. One preliminary experiment addressed to this question has been completed, and it suggests that suppressor cells can, in fact, influence the development of primary UV-induced skin cancers (Kripke, unpublished data). In this study, mice were lethally X-irradiated and then reconstituted with lymphoid cells from normal and/or UV-treated donors; 2 weeks later they were grafted with large, circumferential strips of UVirradiated skin. In this way it was possible to determine the influence of suppressor cells on the development of tumors in skin that had been UV-irradiated, but not subjected to X-irradiation. The rate of tumor development in these animals is illustrated in Fig. 1, and these results suggest that the presence of UV-induced suppressor cells serves to accelerate tumor induction. However, this conclusion is tentative because few animals were used in this experiment, and additional studies clearly are required to establish this point. VI.

Mechanisms in UV-Induced Immunosuppression

A. PATHWAY FOR INDUCTION OF UV-INDUCED SUPPRESSOR CELLS 1. Studies on Contact Hypersensitivity

Having identified suppressor T cells as a likely participant in the failure of tumor rejection in UV-treated mice, the next logical question was, how are cells generated following exposure of the skin to UV radiation? The first clue to the solution of this problem came from studies in our laboratory initiated by J. M. Jessup on the response of UV-treated mice to the contact allergen, 2,4-dinitrochlorobenzene (DNCB). He found that after only a few hours of UV irradiation, mice became anergic to sensitization with DNCB in dimethyl sulfoxide administered by subcutaneous injection (Kripke et al., 197%). To pursue

92

MARGARET L. KRIPKE LYMPHOID CELL DONORS Normal -----UV-Treated- - - - Normal + UV-Treated-

d

r-I

0.75-

C

1 u) .c

0

0.50-

n

10

20

30

40

50

60

70

Week After Grafting

FIG. 1. Development of primary tumors in UV-irradiated skin grafted onto syngeneic recipients. Recipients had been lethally X-irradiated and reconstituted with 5 x lo7 lymphoid cells from normal and/or UV-irradiated donors 4 weeks before grafting. Grafted skin was removed from donor mice that had been treated with FS40 sunlamps for 1 hour, three times per week for 4 months. Each group consisted of 10-15 mice (M. L. Kripke, unpublished data).

this finding, he performed a series of cell transfer experiments, which demonstrated that the reactivity of lymphocytes to DNCB was unaffected by UV irradiation. We, therefore, concluded that UV exposure was affecting this immune response by interfering with antigen processing or antigen presentation, a function normally carried out by macrophages or Langerhans cells, rather than lymphocytes (Jessup et al., 1978). Although we speculated that this alteration in antigen presentation might be a route leading to the development of the UVinduced suppressor cells (Kripke, 1980), we had no evidence to support this possibility. Recently, such evidence was provided in a series of elegant experiments carried out at the Harvard Medical School by Drs. B. Benacerraf, M. Greene, and M. S. Sy. Based on the approach of immunizing with trinitrophenol (TNP)-conjugated antigen-presenting cells (APC) to induce contact hypersensitivity, these experiments provided direct evidence that the APC from UV-irradiated mice are functionally defi-

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cient. In addition, it was found that injection of UV-irradiated mice with TNP-conjugated APC from UV-treated donors resulted in the development of TNP-specific suppressor T lymphocytes (Greene et al., 1979). These findings could provide the link between the altered antigen presentation in UV-irradiated mice and the occurrence of UVinduced suppressor T cells, as it is quite plausible that these suppressors of tumor rejection also are generated as a result of altered APC function. For this reason, we are beginning to investigate some of the photobiologic characteristics of the systemic suppression of contact hypersensitivity by UV radiation to see if they resemble those involved in the suppression of tumor rejection. Thus far, we have found that suppression of contact hypersensitivity following skin painting with 2,4,6trinitrochlorobenzene (TNCB) or l-fluoro-2,4-dinitrobenzene (DNFB) is linearly related to the log,, dose of UV radiation (Noonan, Kripke, Pedersen, and Greene, submitted), as was suppression of tumor rejection (DeFabo and Kripke, 1979). Other similarities demonstrated to date include dependence on wavelengths below 315 nm and reciprocity with regard to dose rate (Noonan, Kripke, and DeFabo, unpublished data; DeFabo and Kripke, 1980). These experiments, and studies comparing the phenotypic characteristics and functional activity of the tumor suppressor cells induced by UV irradiation and the suppressors generated by skin painting with contact allergens following UV exposure, should eventually resolve the question of whether these are produced by the same pathway of altered antigen presentation.

2. Nature of the Antigen-Presenting Cell Alteration Because the UV-induced alteration in APC function and its significance in suppressor cell induction have been recognized only very recently (Greene et al., 1979), information on the nature of the alteration and its induction by UV radiation is fragmentary, at best. What has been observed, thus far, is that either a single or fractionated dose of UV to the shaved dorsum of mice renders them systemically unresponsive to sensitization by skin painting on the ventral (unirradiated) side 3 or more days later. This unresponsiveness is not related to erythema and skin damage, as monochromatic radiation at wavelengths below those inducing erythema induce maximal suppression (E. De Fabo and F. P. Noonan, unpublished data). Plastic-adherent spleen cells removed from mice 3 or more days after UV exposure will not present antigen in a way that leads to contact sensitization. Furthermore, attempts to sensitize UV-treated mice by skin painting or by injection with TNP-conjugated APC from UV-irradiated donors resulted in the

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appearance of TNP-specific suppressor T cells in the spleen (Greene et al., 1979; Noonan, Kripke, Pedersen, and Greene, submitted). The splenic-adherent cell is presumably a member of the macrophage series, but this has not been demonstrated, nor is it known whether the TNP that is conjugated to the APC of UV-irradiated or normal donors differs in quantity or distribution on the cell surface. Although this APC alteration prevents the development of delayed hypersensitivity to all three of the contact sensitizers tested so far (DNCB, TNCB, and DNFB), the extent of its influence in inhibiting other immune responses has not been established. From earlier studies, it is clear that the immune response to histocompatibility antigens is unaffected by this alteration (Kripke and Fisher, 1976; Kripke et al., 1977b; Thorn, 1978; Spellman et d., 1977), including that carried by the Y chromosome (H-Y antigen) of the C57BU6 strain (Kripke et al., 1979). Also, antibody production against sheep red blood cells (SRBC) (Kripke et al., 1977b; Spellman et al., 1977) and transplantation resistance against non-UV-induced syngeneic tumors (Kripke et al., 1979) appear to proceed normally in the face of this APC alteration. On the other hand, recent work b y Greene and his co-workers demonstrates that this UV-induced alteration in APC function affects antibody production against TNP-GAT, a random copolymer of L-glutamic acid, L-alanine, and L-tyrosine conjugated with TNP (Letvin et al., 1980), Also, the in uitro proliferative response of lymphocytes from UVirradiated mice primed in viuo with GAT or. with TNP- or p-azobenzenearsonate-conjugatedAPC is reduced, and the proliferative response of normal T lymphocytes cannot be triggered b y antigen-pulsed splenic adherent cells from UV-irradiated donors (N. L. Letvin, 1. J. Fox, M.I. Greene, B. Benacerraf, and R. Germain, unpublished data). In the guinea pig also, UV-B irradiation prevents the induction of delayed hypersensitivity to dinitrophenyl-conjugated bovine y-globulin (W. Morison, personal communication). Thus, it remains to be determined whether the UV-induced failure of effective antigen presentation in some immune responses, but not in others, is due to the complexity of the antigen, its association with recognition molecules, its physical form, or some other undefined property. What is suggested by these studies, however, is that different antigens, or classes of antigens, are processed or presented for triggering of lymphocytes by different pathways, only some of which are affected by UV irradiation of the host. One of the more interesting unresolved questions concerning altered antigen presentation is the mechanism by which topical UV irradiation

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results in an alteration in splenic adherent cells. It is clear from studies on the penetration of mouse skin by UV-B radiation that these wavelengths do not penetrate more than a few millimeters below the skin surface (Hansen, 1969). Thus, the splenic adherent cells have not been exposed directly to UV radiation in situ. There are at least four possible pathways that might lead to an alteration in the activity of these cells. (1)The adherent spleen cell population could be derived from circulating cells that have been exposed to UV during their passage through superficial capillaries in the skin. (2) An acellular photoproduct could be produced in the skin by UV radiation and reach the spleen by way of the circulatory system. Its effect would then be mediated through a direct interaction with resident or circulating splenic adherent cells. (3) Langerhans cells, thought to be the epidermal APC (Sting1et id.,1978),could be altered by UV radiation and migrate to the spleen where they are recovered as altered splenic adherent cells. (4) In response to UV-mediated damage of the skin, APC normally residing in the spleen emigrate, either to participate in the removal of damaged tissue or to replace the Langerhans cells destroyed by irradiation. This emigration would result in a requirement for new APC in the spleen, and the influx from the bone marrow of functionally immature, but adherent, APC in the splenic population could lead to the observed alteration in antigen presentation. At the moment, it is not possible to distinguish among these alternatives with any degree of assurance. However, it is intriguing in this context that topical UV-B irradiation of mice leads to a rapid disappearance of epidermal Langerhans cells, and that this depletion of Langerhans cells is associated with an inability to induce contact hypersensitivity in the irradiated site (Toews et al., 1980). Thus, the Langerhans cell is a promising candidate for the site of the initial interaction between UV radiation and the skin. If the alteration in APC function in UV-exposed animals results from a direct effect of particular wavelengths on the APC themselves, this might account for the peculiar phenomena observed with lymphoid cells exposed to UV radiation in uitro. It has been known for some time that sublethal doses of 254 nm (UV-C) radiation selectively alter some of the activities of lymphoid cells, possibly through the functional inactivation of particular cell surface recognition molecules (Lindahl-Kiessling and Safwenberg, 1971; Rollinghoff and Wagner, 1975; Horowitz et a1 ., 1974; Wagner and Rollinghoff, 1976). A functional analysis of various purified lymphoid cell populations following exposure to various UV wavelengths in vitro could resolve the question of whether the im-

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munologic alterations exhibited by lymphoid cells following UV irradiation in uiuo or in uitro are manifestations of the same photobiologic events.

B.

ANTIGENS INVOLVED IN THE INDUCTION OF SUPPRESSOR CELLS BY EXPOSURE

uv

Based on our current understanding of immunologic processes, we must assume that the induction of suppressor cells by UV radiation requires an antigenic stimulus. Although the precursors of antigenspecific suppressor cells are undoubtedly present in the host at the time of UV irradiation, their expansion into an active and detectable population probably depends on the introduction of an appropriate antigen at a critical time after exposure to UV radiation. This is certainly the case in the production of suppressor T cells against contact allergens in UV-irradiated mice. Spleen cells taken from unsensitized, UV-irradiated mice do not suppress contact hypersensitivity upon transfer to an unirradiated recipient. Suppression of the response in the recipient is achieved only with lymphocytes from UV-irradiated donors that were contact-sensitized at least 3 days after UV exposure (Noonan, Kripke, Pedersen, and Greene, submitted). Because the UV-induced suppressors of tumor rejection occur following UV exposure, without the deliberate introduction of exogenous tumor antigens, the antigenic stimulus for their induction must arise endogenously as a result of UV exposure. The nature of this antigenic stimulus is not at all apparent, but it must be related in some way to antigens that occur on the surface of UV-transformed cells, because it is the immune response against these antigens that is inhibited by the resulting suppressor cells. One possible way in which this could come about is that antigens are produced in the skin as a result of UV-induced cellular damage, and these antigens, by chance, crossreact with the TSTA that arise subsequently during transformation by UV. Alternatively, the antigenic stimulus for the induction of suppressor cells may be the TSTA themselves, or some part of the TSTA; however, this would require that there be occult neoplastic or UVinitiated cells in the skin after only a few hours of UV exposure. A third possibility is that a small amount of UV radiation induces a stably inherited antigenic change in exposed cells that is not associated with transformation, and subsequently, one such cell undergoes neoplastic transformation and acquires, in addition, an individually specific TSTA. At present, there is no experimental evidence that rules out any of

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these possibilities, and there are probably other alternatives that are equally compelling. However, it seems quite likely that the induction of endogenous suppressor cells by UV radiation is the result of at least two distinct photobiologic events: one leading to the alteration of APC function, the other leading to the production of new antigens in the skin. If these two events are mediated by different wavelengths of radiation, it should be possible to separate them by using monochromatic radiation. Experiments currently in progress have shown that suppression of contact hypersensitivity occurs following exposure to monochromatic radiation, particularly in the 260-270 nm region ( D e Fabo and Noonan, unpublished data), but no information on the formation of UV-induced antigens is available as yet.

c. SUPPRESSION MEDIATEDBY OTHER MECHANISMS Work involving several different immunologic systems has raised the question of the significance of suppressor T cells in specific immunologic tolerance. Studies by Sy et al. (1977) demonstrated that, although suppressor cells are associated with unresponsiveness to DNFB, their elimination by splenectomy did not lead to the recovery of reactivity. These and other studies (Fujiwara and Karyone, 1978) suggest that mechanisms other than those associated with suppressor T cells are important in inducing and maintaining the tolerant state to some antigens. Thus far, it has been difficult to examine this situation directly in the UV-induced tumor system, using approaches that were successful in other systems. We have attempted to prevent the appearance of UVinduced suppressor cells in order to test whether animals lacking these cells were tumor-resistant. However, neither thymectomy, splenectomy, nor cyclophosphamide treatment of mice prior to UV irradiation prevented the appearance of the UV-induced suppressor cells (R. T. Thorn, M. S. Fisher, and M . L. Kripke, unpublished data). Work by Daynes et aZ. ( 1 9 7 9 ~has ) suggested that these suppressor cells may be extremely sensitive to y irradiation. However, attempts to eliminate them by exposing UV-irradiated mice to low doses of y rays are still inconclusive. One finding that may be of relevance here is that reconstitution of lethally X-irradiated, UV-irradiated mice with normal lymphoid cells does not restore their ability to reject syngeneic UV-induced tumors (Fisher and Kripke, 1977; Daynes et al., 1 9 7 9 ~ )Although . one would expect that this failure to reject tumor challenge was caused by the induction of new suppressor cells in the transferred population, at-

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tempts to demonstrate this have been unsuccessful so far (M. S. Fisher, unpublished data). Thus, the possibility that other nonsuppressor cell-mediated mechanisms contribute to the unresponsiveness of UVirradiated mice to UV-induced tumors cannot be ruled out entirely at the present time. VII. Conclusions

It is clear, at least in this tumor system, that the immune response is intimately involved in cancer development. The type and extent of this involvement may be extreme in the case of UV carcinogenesis because of the peculiar immunologic consequences of UV exposure and the unusual antigenic characteristics of the resulting tumors. However, several general principles are illustrated quite dramatically by this system. The first concerns the definition of tumor antigenicity. In experimental animals, antigenicity is defined, in functional terms, by the ability of a tumor to evoke a detectable immunologic response in a histocompatible host. From these experiments, it is clear that antigenicity is a relative term that depends on the particular host used to test it. Although W-induced tumors are exceptionally antigenic in normal syngeneic recipients, they fail to provoke an immune response in the primary host that is detectable by any conventional procedure. Thus, if the only measure of antigenicity available were the immune response induced by the tumor in its primary host, we would conclude incorrectly that the tumors were not antigenic. This is, of course, precisely the situation with human cancers, in which only the responses of the primary host can be used to define antigenicity. It is quite possible that much of the difficulty in finding evidence for the existence of human tumor antigens stems from this limitation and is caused by the inability to perform the same syngeneic transplantation tests used in inbred animals to define these antigens. Similarly, this tumor system provides a very dramatic illustration of the fact that the primary host is not necessarily equivalent to the normal recipient of a transplanted syngeneic tumor. In the primary host, UV-induced tumors grow progressively, whereas the opposite (immunologic rejection) occurs in normal recipients. The primary host and even a carcinogen-treated, precancerous animal behave differently from normal animals with regard to tumor challenge. Recent experiments indicate that in MCA-induced carcinogenesis also, the carcinogen-treated host differs from a normal animal in its response to syngeneic tumor challenge (Lill et al., 1978)a finding suggested some years ago by the experiments of Stjernsward (1968)and Basombrio and

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Prehn (1972). Thus, an altered immunologic response of the primary host to neoplasia could be a rather general phenomenon, but one that is illustrated much more strikingly in the UV system than in others because of the high degree of antigenicity of the UV-induced tumors. These immunologic studies of UV carcinogenesis also clearly demonstrate that the regulatory pathways described for exogenous antigens also control the immune response against TSTA, even in the autochthonous host. The failure to view the immune system as a complex homeostatic mechanism, regulated by positive and negative controls, has undoubtedly hindered our progress in the area of cancer immunotherapy. In fact, this deficiency may deserve more responsibility for lack of progress in this area than the more convenient argument that human tumors are only weakly antigenic. Whether or not these findings in the UV system are broadly representative of experimental cancer induction, they have provided a number of findings with practical significance. The finding that some, but not all, immunologic responses are affected by a UV-induced alteration in antigen presentation provides an approach for analyzing the different pathways by which various antigens can trigger the response of lymphocytes. In addition, it is now possible to induce at will, by a brief external exposure of mice to UV radiation, antigen-specific T cells that suppress contact hypersensitivity. This finding could be used to help define the conditions leading to activation of suppressor rather than helper cell regulatory pathways. Eventually, this approach might be exploited in the induction of suppressor cells that could prevent undesirable immune reactions, such as allograft rejection, contact allergy, or autoimmunity. Another promising avenue for exploration that could be of practical value is based on the suggestion that regulation of the immune response to individually specific tumor antigens might be carried out through antigenic determinants that are shared among tumors of the same etiology. The idea that immunologic regulation occurs by way of determinants that differ from those recognized by the effector response is suggested by the work of Turkin and Sercarz et al. (1977) on the immune response to isolated portions of the lysozyme molecule. In this system, regulation of antibody formation occurs through a portion of the molecule that does not, itself, induce antibody formation. If this model recurs in tumor immunity, then immunity to sets of individually specific tumor antigens could be controlled by means of a common, regulatory determinant that does not, itself, evoke an effector response. The finding that UV-irradiated mice can distinguish between UVinduced tumors and those induced in syngeneic mice by other agents,

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such as MCA and oncogenic viruses (Kripke et al., 1979),also raises an interesting and potentially useful possibility that, even in the case of chemically and physically induced tumors, antigenicity can somehow reflect the etiologic agent. Thus, tumor antigenicity could be determined by a particular carcinogen in such a way that all tumors induced by a single carcinogen belong to a common antigenic class. Based on the existing data, this could occur in either of two ways. First, tumors could have both group- or carcinogen-specific antigens and individually specific determinants, in which the shared specificities are responsible for regulation and the individual ones are responsible for effector reactivity. In this model, each carcinogen would produce tumors with a particular shared specificity. Alternatively, each carcinogen could produce tumors with a unique set of individual specificities, none of which would be expressed on tumors induced by other carcinogens. In both models, the carcinogen would dictate the antigenic category to which a particular tumor belonged, and tumor antigenicity would not result entirely from some random molecular alteration. There are several lines of evidence that lend some credence to this hypothesis that antigenicity is dictated in some orderly way by the carcinogen. First, the UV-irradiated animal is unresponsive to UVinduced tumors of different histologic types, suggesting that this recognition involves the etiologic agent, rather than the embryologic origin of the tissue, Second, the high degree of antigenicity of these tumors seems to be a unique and unifying characteristic of tumors induced by UV radiation, including those transformed by in uitro exposure of murine cells to UV radiation. In fact, preliminary experiments suggest that some murine cells transformed in uitro by UV-B radiation exhibit preferential growth in UV-irradiated syngeneic recipients, relative to their growth in normal mice (Fig. 2; Ananthaswamy and Kripke, unpublished data). If this proves to be true generally, then it would suggest that there are antigenic similarities between cells transformed in uiuo or in uitro by UV radiation that are recognized by the UVirradiated host, Third, even tumors that are produced by 4nitroquinoline-N-oxide or by 8-methoxypsoralen plus UV-A radiation, carcinogens that damage DNA directly and evoke repair mechanisms similar to those induced by UV-B radiation, do not exhibit the high degree of antigenicity that characterizes UV-B-induced tumors (M. S. Fisher, M. L. Kripke, W. L. Morison, and J. R.Parrish, unpublished data). Furthermore, preliminary evidence indicates that the skin tumors induced by 8-methoxypsoralen and UV-A radiation are not “perceived” by UV-B irradiated mice as belonging to the same an-

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I-

$

Weeks After Inoculation UVB-15b (5 x 105)

F'Ic. 2. Growth of a tumor induced in oitro by UV radiation, following subcutaneous injection of 5 x 10.' cells into immunosuppressed (ATX), UV-irradiated, or untreated syngeneic BALB/c mice (H. Ananthaswamy and M. L. Kripke, unpublished data).

tigenic category as UV-B-induced tumors (Table 111, W.Morison, M. Kripke, and J. Parrish, unpublished data). If this hypothesis is correct, that antigenicity in some way reflects etiology, this could prove to be of epidemiologic significance. Since the UV-irradiated mouse can discriminate between UV-induced tumors and those induced by other agents, it might b e possible to use this form of recognition to determine whether UV radiation was the agent that induced a tumor of unknown etiology. Recently, we have attempted to use the UV-irradiated mouse as an indicator of tumor etiology in the case of a malignant melanoma that arose in a C3H mouse. The animal had been given 10 1-hour exposures to UV-B radiation over a 2-week period, and then was painted with croton oil twice weekly for nearly 2 years (Kripke, 1979). The growth of a cloned tissue culture cell line derived from this melanoma has been compared in normal and UV-irradiated C3H mice, and the cells were found to grow preferentially in UV-irradiated hosts (Kripke and Fidler, unpublished data). As this is the only C3H tumor that has exhibited this characteristic, aside from those known to be induced by chronic UV-B exposure, it seems quite likely that the UV radiation was of major etiologic significance in the development of this melanoma. If other carcinogens also

102 GROWTH OF

MARGARET L. KFUPKE TABLE 111 PSORALEN-UV-A-INDUCED SKIN TUMORS IN UV-B-IRRADIATED MICE"

Tumor (dose)'

P-2524(6 x 10') P-327(3 X 10')

Treatment of recipienr UV- B Normal UV-B Normal Immunosuppressed

Number of mice with palpable tum or/n um ber challenged

2 weeks

4 weeks

12 weeks

u10 w9 0/10 0/10

5/10 719 1/10 w10

7/10 7/10 2/10 2/10

015

45

5/5

' M. L. Kripke, W. L. Morison, and J. R. Parrish (unpublished data).

'Single cell suspension of tumors from the second transplant generation in immunosuppressed mice were prepared by collagenase-DNase digestion for subcutaneous injection. ' Syngeneic C3H mice were exposed to UV radiation for 1 hour, three times per week for 12 weeks prior to injection of tumor cells. Immunosuppressed mice were adult thymectomized and sublethally X-irradiated (450 R).

induce shared regulatory specificities, then it is at least theoretically possible to envision a battery of tests that would help to identify the inducing agent of a variety of tumors. In addition to these theoretical implications, the strong antigenicity of UV-induced tumors can be used to ask some fundamental questions about the nature of tumor antigens and about mechanisms in radiation carcinogenesis. For example, if tumors induced by UV-C radiation were also highly antigenic and shared with UV-B induced tumors the capacity to grow preferentially in UV-B-irradiated mice, this could suggest that the mechanisms of neoplastic transformation by these two wavebands of UV radiation were the same. Also, if skin tumors induced by a two-stage process involving initiation by W radiation and promotion with phorbol ester were highly antigenic like those induced by chronic UV irradiation, this would suggest that tumor antigenicity is established at an early and irreversible step in the process of transformation. Thus, the UV tumor system has provided us with a unique opportunity to explore many facets of the complex relationship between a developing cancer and the host immune system. It is clear, at least in this system of highly antigenic tumors, that host immunologic factors are as important in cancer pathogenesis as the initial transforming

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events. The unusual combination of carcinogenic and immunologic changes mediated by UV radiation has permitted us to observe the extent to which carcinogen-induced tumors can be antigenic; it has also provided an illustration of the capability of normal immunologic mechanisms to eliminate highly antigenic syngeneic and autochthonous tumors.

ACKNOWLEDCMENTS The assistance of Ms. Janet Jenkins in preparing this manuscript and the editorial guidance of Ms. Elynor Sass is acknowledged with thanks. Research by the author was sponsored by the National Cancer Institue (NCI) under Contract Number NOl-CO75380 with Litton Bionetics, Inc.

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