Biochimica et Biophysica Acta, 473 (1977) 93-119 © Elsevier/North-Holland Biomedical Press BBA 87037

IMMUNOGENICITY

OF

TUMOR

ANTIGENS

R O N A L D B. H E R B E R M A N

Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Md. 20014 (U.S,A.) (Received May 10th, 1977)

CONTENTS I.

Introduction

II.

Types of t u m o r antigens

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A. T u m o r s induced by chemical carcinogens

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B. T u m o r s induced by oncogenic viruses . . . . . . . . . . . . . . . . . . . . .

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C. E m b r y o n i c antigens

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D. S p o n t a n e o u s t u m o r s in experimental animals . . . . . . . . . . . . . . . . . .

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E. H u m a n t u m o r s

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III. I m m u n i z a t i o n against t u m o r antigens by various types o f materials . . . . . . . . . . A. Intact t u m o r cells

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B. Extracts of t u m o r cells C. Viruses

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D. Other i m m u n o g e n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4.

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Allogeneic cell immunity . . . . I m m u n i z a t i o n by i m m u n e R N A . I m m u n i z a t i o n by soluble products I m m u n i z a t i o n by microorganisms

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IV. Mechanisms for the immunogenicity of t u m o r antigens . . . . . . . . . . . . . . .

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A. Role of the M H C and other factors in immunogenicity . . . . . . . . . . . . . .

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B. Effector mechanisms involved in the response to t u m o r antigens . . . . . . . . . .

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1. 2. 3. 4. V.

T cells . . . B cells . . . Natural killer Macrophages

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Possible m e c h a n i s m s for insuffkient or undetectable immunogenicity A. Immunoresistance

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B. Interference with immunogenicity or effector functions . . . . . . . . . . . . . .

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VI. Discussion References

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Abbreviations: TASA, tumor-associated cell surface antigen; T A T A , tumor-associated transplantation antigen ; SV40, simian virus 40; BCG, Bacillus calmette Gu6rin, Mycobacterium boris; M H C , m a j o r histocompatibility complex; A D C C , antibody-dependent cell-mediated cytotoxicity.

94 I. INTRODUCTION The immunogenicity of tumors is a central issue in tumor immunology, particularly in experimental animal systems where immune responses against the tumors are usually induced by immunization procedures. In fact, because of the frequent close association between antigenicity of a tumor and immunogenicity, the terms are often used interchangeably. However, it is important at the outset of this review to point out some subtle but essential differences between these words. Any material in a tumor may be considered an antigen if it can be shown to react specifically with antibodies or lymphocytes, regardless of whether these immune reactants occur naturally or were produced in response to the particular tumor material or to other tumor-derived or even non-tumor-related materials. A tumor antigen can only be considered immunogenic when it has been shown to elicit in vivo or in vitro a specific immune response. In the course of this review, several examples of tumor antigens which have not been demonstrated to be immunogenic will be given. This apparently paradoxical situation arises most frequently when an immune response to a tumor antigen is detected without deliberate immunization. For example, with most natural humoral and cell-mediated immunity to tumors, and with most immune reactions detected in cancer patients, the antigens and mechanisms leading to the responses are quite difficult to determine. Some of the observed immune reactions could be the result of sensitization to cross-reactive antigens, even microbial, or could be due to a nonspecific, polyclonal activation. In this review, I will attempt to briefly summarize some of the available information on immunogenicity of various types of tumor antigens, the procedures used to induce immunity to tumors, and some of the factors determining the immunogenicity of tumor-derived materials. In addition, some attention will be given to recent information on the mechanisms involved in the recognition and response to tumor antigens, and on possible mechanisms for the apparent lack of immunogenicity of some tumors. In such a discussion of immunogenicity of tumor antigens, it has been necessary to consider much of the information and advances related to immunogenicity of other types of antigens. Information acquired about histocompatibility antigens is particularly relevant, since in transplantation and tumor immunology there are special problems related to the induction of in vivo resistance to allografts or tumors, rather than just a need for detecting immune responses in a particular in vitro assay. Studies on the immunogenicity of tumor antigens are quite important for understanding of the mechanisms involved in the immune responses to tumors. Careful dissection of factors influencing immunogenicity of tumor antigens should help to provide practical solutions to problems related to immunoprevention and immunotherapy of cancer, which have not been solved by empirical studies.

95 II. TYPES OF TUMOR ANTIGENS Tumor cells possess many antigens, which vary in specificity, location in the cell, and biological role. In order to keep the differences between various tumor antigens in focus during the course of this review, it may be helpful to define and discuss some terms. Antigens which are uniquely present on tumor cells and qualitatively different from antigens on any normal cells may properly be called tumor specific. As previously discussed [1], such complete restriction of antigens to tumors is quite difficult to prove, and therefore the term tumor-associated antigen is used much more frequently. A tumor-associated antigen is an antigen that is found on a tumor cell and is undetectable in the cells of an adult normal individual, but may also be found on normal cells under special circumstances (e.g. embryonic antigens which are normally expressed during fetal development). In this review, most of the attention will be directed toward tumor-associated antigens on the cell surface (TASA). It must be stressed that the term tumor antigen is actually too vague, since tumors contain many normal antigens and these need to be distinguished from TASAs or other tumor-associated antigens. This is particularly important since, unless sufficient controls are included in a study, some normal antigens might be mistakenly considered to be tumor associated. A wide variety of other types of antigens may be expressed on the tumor cell surface and often can only be excluded if they are thought of. These include normal histocompatibility antigens, organ or tissue antigens, and microbial antigens which may be present because of contamination or latent infection of the tumor cells. A particular problem arises in the studies of in vitro cultures of tumor cells, which are usually maintained in medium containing fetal bovine serum. It is frequently rediscovered that heterologous serum proteins can bind avidly to tumor cell surfaces and thereby mimic TASAs [e.g. 2 4 ] . Fetal bovine serum antigens on tumor cells can react with natural antibodies which are present in most human and other sera [3], or of particular relevance to this review, immunization with cultured tumor cells can produce immunity directed at least in part against fetal bovine serum antigens [4]. Tumor-associated cell surface components may vary widely in their immunogenicity. Some TASAs may not be immunogenic in the tumor-bearing host or even in the same species, but may be quite immunogenic in heterologous species. The best known example of such an antigen is carcinoembryonic antigen [5], which is present on many human tumors. Carcinoembryonic antigen has not been conclusively shown to induce an immune response in cancer patients, but it regularly produces high titers of antibodies in rabbits and goats. It is not entirely clear why some TASAs are not immunogenic in the host. One possible explanation is that materials like carcinoembryonic antigens are not truly tumor associated, since they can be detected in the cells and sera of normal individuals [6,7], and therefore humans may be tolerant to it. Most of the attention here will be devoted to TASAs which are potentially immunogenie in the host. The most important type of immunogenicity of TASAs to the host is that which

96 leads to resistance against tumor growth. TASAs which have such properties are defined as tumor-associated transplantation antigens (TATAs). Immunogenicity is virtually a part of the definition of a TATA, since it is usually demonstrated by a protocol involving immunization and then challenge. There are very few instances of immunosensitive tumors, i.e., tumors which can be reacted against by immune individuals, which are not immunogenic. In general, immunogenicity appears to be a more sensitive indicator of TATAs than immunosensitivity. There are many instances of tumors which can be shown to be immunogenic but are immunoresistent, i.e., they grow equally well in immune and normal recipients [e.g. 8,9]. Some TASAs, which do not produce resistance against tumor growth and thus cannot be considered TATAs, may still be quite immunogenic in the host as evidenced by a humoral and/or cell-mediated immune response. The majority of studies in tumor immunology are concerned with measurements of immune reactivity in vitro, and it is frequently assumed that such studies reflect immunity to TATAs. However, since not all TASAs can be shown to be TATAs, evidence for an in vivo role of each particular TASA and immune reaction would need to be obtained before such a conclusion is drawn. However, few detailed studies have been performed to relate results obtained in vitro to in vivo protection against tumor challenge. As noted above for TATAs, immunogenicity of TASAs appears to be a more sensitive and stable characteristic of tumor cells than is immunosensitivity, i.e., reactivity with antibodies or immune lymphocytes [8]. Tumors and their TASAs are usually classified according to the method of tumor induction, and since this is useful for many antigens on tumors in experimental animals, the discussion below will be organized in that way. However, embryonic antigens and TASAs of human tumors do not fit this classification and will be discussed separately.

IIA. Tumors induced by chemical carcinogens TATAs were first clearly demonstrated on sarcomas of mice, which had been induced by methylcholanthrene [10-12]. An integral part of the experimental design was the ability to immunize syngeneic mice with the tumors. Intradermal inoculation of tumor cells, or ligation or removal of tumor transplants resulted in resistance against subsequent challenge with the same tumor. Klein et al. [13] later obtained the same result in mice bearing primary tumors, proving conclusively that the phenomenon could not be due to histocompatibility differences between the tumor and the host. It has generally been found that the TATAs of chemically induced tumors are individually distinct. Animals immunized against one tumor will subsequently resist challenge by the same tumor, but not by another tumor induced by the same chemical [12,13]. These antigens are thought to be the result of new genetic information related to somatic mutation rather than selection of a clone of cells with an idiotypic cell surface specificity, since even tumors arising from the same clone of cells had individually specific antigens [14]. This lack of common antigenicity among tumors

97 requires that the same tumor be used for immunization and challenge. Therefore, only tumors with effective immunogenicity and immunosensitivity would be found to have TATAs. This restriction of antigenicity also makes it very difficult to prove the specificity of transplantation antigens on tumors which have been maintained in serial passage for several years or more. Since genetic drift of histocompatibility antigens can occur in inbred strains [15], it is quite possible that an antigenic difference between tumor and the present hosts could be due to divergent histocompatibility antigens. It is therefore important to avoid long transplanted, chemically induced tumors and to do experiments, whenever possible, with primary tumors or tumors in early transplant generations. Although this has been recognized by many tumor immunologists for a long time, it is regrettable that many immunologic studies are still performed on transplantable tumors which were induced over 20 years ago. The immunogenicity of the TATAs of chemically induced tumors had been found to vary widely, and this has been related in part to the type [16] and dose [17] of carcinogen used for induction. Much information has been gathered on this point, and this has been discussed in detail in a review by Baldwin [18]. Contrary to the general rule of individually specific TATAs on chemically induced tumors, some tumors have been found to contain common TATAs [19-22], i.e. immunization with one tumor led to some protection against challenge by other tumors. In addition, common TATAs in rat urinary bladder papillomas were suggested by the finding that immunization of rats with tumor cells prior to the administration of the carcinogen resulted in a significant increase in the latent period for development of tumors [23]. There are several possible explanations for this cross-reactivity among some tumors: (1) In some experiments, the protective effects of immunization were not shown to be immunologically specific and may have been due to nonspecific stimulation of the animals by the immunization procedure. In this regard, it might have been helpful to sublethally irradiate the animals before challenge, since this has been empirically found to suppress some nonspecific effects of prior immunization but not to interfere with specific immunity [24]. (2) Common antigens may have been induced by expression of viruses in the tumors. This might result from infection during passage, or more likely, by activation of endogenous type C viruses. Many murine tumors induced by chemicals, particularly those which have been maintained in tissue culture or by transplantation for many passages, have been found to contain type C viruses and their associated antigens [e.g. 25-28] and some mouse endogenous virus-associated antigens appear to function as TATAs [29]. (3) Embryonic antigens may be expressed on the cell surface of chemically induced tumors [30-34] and, as will be discussed in more detail below (Section IIC), they have sometimes been found to function, at least weakly, as TATAs [34,35]. The immunogenicity of TASAs of chemically induced tumors has been studied in a variety of assays of humoral and cell-mediated immunity (for review see ref. 18). As in the studies of TATAs, both individual specific and common TASAs have been found and the same explanations for the latter are relevant. The frequency of common antigens has been higher in the in vitro assays, at least partially due to the

98 relative ease in detecting reactivity to embryonic antigens in the in vitro studies. In one study showing cross-reactions between methylcholanthrene-induced sarcomas [36], some evidence for the presence of both embryonic and type C virus-associated antigens was obtained.

lIB. Tumors induced by oncogenic viruses The TATAs and other TASAs of virus-induced tumors and their immunogenicity have been discussed at length in previous reviews in this series [37-39] and elsewhere [40-43]. Therefore only a few summary statements and other remarks will be made here. The most important feature to mention about these systems is that tumors induced by the same virus have been shown to share common TATAs and other TASAs. This feature has allowed more detailed and independent assessment of antigenicity and immunogenicity. For example, it has been possible to immunize with virus or some other strongly immunogenic preparation and then test for immune reactivity against a variety of tumors. Also, by selecting a very immunosensitive tumor for in vivo challenge or as a target cell in in vitro assays, it has been possible to more sensitively detect the immunogenicity of various tumors. An example of the usefulness in being able to dissociate immunogenicity from immunosensitivity for detection of T A T A has come from studies of antigens associated with murine sarcoma virus. Stephenson and Aaronson [44] failed to detect a T A T A on a transformed nonproducer cell line when immune mice were challenged with these cells. However, McCoy et al. [45] were able to hyperimmunize with an immunogenic tumor and then show protection against this tumor. In contrast, the nonproducer tumor could not be shown to be immunogenic itself. In addition to the common virus-associated antigens, some virus-induced tumors may also contain individually specific antigens. Individually distinct T A T A s and other TASAs have been particularly associated with mouse m a m m a r y tumors, induced by the mouse m a m m a r y tumor virus [46,47]. However, these antigens do not appear to be directly related to the virus. The individually specific antigens have mainly been demonstrated in virus-infected mice, probably because the responses to the stronger virus-associated antigens were suppressed or undetectable in such mice. In fact, based on the inability to induce resistance against tumor challenge, it was thought for some time that mice neonatally infected with m a m m a r y tumor virus or other oncogenic viruses were immunologically tolerant to the virus-associated antigens [48-51]. However, subsequent studies have indicated that virus-associated TATAs and other TASAs can be quite immunogenic in rodents with vertically or neonatally transmitted virus infections [52-55].

IIC. Embryonic antigens Embryonic antigens are antigens which are normally expressed during embryonic development, usually during the first half of the gestation period, and then become undetectable after birth and are not found in normal adult cells. Embryonic antigens have been found to be expressed, often in large quantities, on the cell surface

99 of many tumor cells and this expression appears to be independent of the method used for tumor induction [30,56]. It was initially suggested that TATAs and other TASAs on virus-induced tumors were actually embryonic antigens [57]. It was then shown that embryonic antigens were distinct from virus-assocated TASA's [56] and from individually specific TATAs on chemically induced tumors [30], and that the embryonic antigens usually did not function as TATAs [30,58]. Further studies have tended to put the role of embryonic antigens into perspective, with the data placing them somewhere between the two extremes mentioned above. Embryonic antigens seem to be quite immunogenic in the host, producing antibodies and cell-mediated immune reactions in multiparous females and in individuals immunized against tumors [30,56,59]. There have been several instances in which embryonic antigens have been shown to function as TATAs and cause protection against tumor challenge [e.g. 34,35,59]. However, the overall experience has been that these antigens only infrequently produce in vivo resistance to tumor growth and that this is usually weaker than that induced by other TATAs. Ting and Grant [60] have presented evidence that the degree of responsiveness of the host and the amount of embryonic antigen expressed on tumor cells are two factors which influence the effectiveness of fetal tissue immunization in producing resistance to tumor challenge. As discussed earlier (Section IIA), embryonic antigens represent one possible explanation for common TASAs on chemically induced tumors. Embryonic antigens associated with development of a particular tissue or organ might be expected to account for some of the common tissue or organ-related TASAs. Most immunization studies have employed preparations from whole fetuses, and the distribution of the embryonic antigens among different organs of the fetus has not been extensively studied. However, there have been several indications of embryonic organ-specific antigens which cross-react with tumors arising in that organ [33,61,62]. IID. Spontaneous tumors in experimental animals Almost all of the tumors discussed above were deliberately induced by chemicals or viruses in experimental, usually inbred, animals. Such models are frequently criticized as artificial and quite different from spontaneous tumors arising in random bred populations [e.g. 63]. Many such spontaneous tumors have been shown to lack detectable TATAs [63, reviewed in 64] and the generalization is often made that spontaneous tumors are nonantigenic or nonimmunogenic. However, this is by no means entirely correct. Some spontaneous tumors in random bred animals are caused by viruses, e.g. feline leukemia and Marek's disease; these tumors have TATAs and other TASAs, and immunization of susceptible individuals by virusassociated antigens has been shown to protect well against spontaneous tumor occurrence [65-67]. In addition, Baldwin et al. [30] found that almost all spontaneous tumors in rats had embryonic cell surface antigens which can be readily detected in in vitro immunologic assays.

100 l i E . H u m a n tumors

The evidence for the existence of TASAs in man has recently been reviewed [68]. Most of the supporting data have come from immunization of heterologous species or from in vivo and in vitro studies of immunity in cancer patients. The most frequently observed pattern has been that of antigens associated with cancer of a particular organ or histologic type. As mentioned above (Section IIC), this pattern might be attributed to embryonic antigens, and this has been found to be the case in several instances [5,62,69-71]. In addition to the common TASAs, some individually specific cell surface antigens have been recently described [e.g. 72]. There is very little direct evidence for immunogenicity of human TASAs in cancer patients or for human TATAs. It has generally been assumed that the presence of antibodies or immune lymphocytes in cancer patients is due to immunization by the TASAs. This is probably correct, but as pointed out in the Introduction, other explanations might be offered. Despite this lack of documentation ofimunogenicity, a number of human immunotherapy trials have been initiated with intact tumor cells or fractions, and success in these trials is predicated on the presence of TATAs in the inocula. Although therapeutic efficacy in such trials has yet to be proven, some studies of immunologic reactivity of the treated patients have provided evidence for immunogenicity. Powles et al. [73] observed that inoculation of patients wich acute leukemia with autologous irradiated blast cells produced a transient increase in their in vitro lymphoproliferative response to the tumor cells. In some other trials employing allogeneic tumor cells or extracts, antibodies or cell-mediated immune reactions were detected but they were not clearly shown to be directed against TASAs [74-77]. More studies of immunologic responses to TASAs of cancer patients receiving "specific" immunotherapy are needed to provide more information on the immunogenicity of different forms and schedules of therapy. Hopefully, such information should help to design more rational immunotherapy trials.

III. IMMUNIZATION AGAINST TUMOR ANTIGENS BY VARIOUS TYPES OF MATERIALS Over the years, there have been performed a very large number of studies which involved immunization of syngeneic recipients with tumor cells or subcellular fractions of tumor cells. Although it will not be possible to review the details of even a substantial proportion of these investigations, it seems worthwhile to attempt to make some generalizations about the factors which may have determined the effectiveness of the immunization procedures. One principle which seems to underlie many of the results is that exposure of the recipients to relatively large doses of TASAs, often over a substantial period of time, is necessary to induce a vigorous immune response and, particularly, to induce resistance against tumor challenge. In the discussion below, we will repeatedly point to evidence which supports this principle.

101 I l i A . Intact tumor cells

Immunizations have been performed most frequently with intact, viable tumor cells. In many of the early studies on TATAs, immunity was induced by ligation or removal of an actively growing tumor, after it had reached an appreciable size [11,12]. This is a quite effective method when the tumor does not metastasize rapidly, but may be unsuccessful with tumors with early dissemination [78]. A similar method, which has been very successful for mouse plasma cell tumors, is to eradizate the growing tumor with an effective chemotherapeutic agent [78]. Some tumors grow locally and then spontaneously regress, and this usually results in strong immunity and transplantation resistance [79]. Intradermal inoculation of a number of different tumor cells results in transient tumor growth and then regression, and this has been shown to be a very effective means of immunization [10,80,81]. Again, the main limitation to this approach is that with some tumors, inoculation intradermally with a dose large enough to produce tumors leads to progressive tumor growth rather than regression. Use of a subthreshold intradermal dose, or of a subcutaneous dose of a regressing tumor, which does not produce any detectable tumors, has usually not been effective in inducing transplantation immunity or detectable immune reactivity [78,79,81,82]. It has been possible in some tumor systems to immunize with subthreshold doses of viable tumor cells, but then multiple inoculations over an extended period of time have usually been needed [e.g. 83]. To avoid the problems of possible progressive tumor growth from immunizations with viable tumor cells, many investigators have used X-irradiated cells. In human immunotherapy trials with intact tumor cells, X-irradiation is almost always performed. Some studies have found large doses of irradiated cells to be effective immunogens, but many others have shown that irradiated cells are much less immunogenic than untreated, viable tumor cells [79,84]. Recently, Campbell et al. [84] performed a study to determine the possible mechanism for loss of immunogenicity after X-irradiation of a highly antigenic rat tumor and of a mouse tumor. No evidence for a direct deleterious effect on TASAs was obtained, and although the irradiated cells had an impaired ability to induce primary immunization, they were as effective as unirradiated cells in eliciting in vivo secondary responses in previously immunized recipients. The most likely explanation for these results is that primary induction of immunity to many TASAs requires a large and persisting dose of antigen, and X-irradiation, by inhibiting proliferation of the tumor cell inocula, acts to decrease the amount of TASA presented to the immune system. In secondary immunizations, in which immune memory cells may play an important role, the amount of antigen on the non-proliferating irradiated cells may be a sufficient stimulus. In accord with this interpretation, mitomycin C-inactivated tumor cells have been shown to elicit a strong secondary cytotoxic response in vitro but were relatively ineffective in generating a primary response in vitro [85]. As a further indication of the need to exceed a certain threshold dose in primary immunization, Stillstr6m [86] found that a ten-fold increase in the dose of irradiated tumor cells led to a thousandfold increase in protection against tumor challenge.

102 A number of other procedures have been used to inactivate tumor cells and to modify their antigenicity. In many of these studies, it is difficult to evaluate the effects of the treatment on immunogenicity since controls with unmodified but inactivated (e.g. by X-irradiation) tumor cells were not included. Some investigators have found that modification of some tumor cells with iodoacetamide increased their immunogenicity [87]. Chemical coupling of a hapten to X-irradiated tumor cells [88] or neuraminidase treatment of tumor cells [89] has been found to increase immunogenicity. In studies with neuraminidase treatment, it is of interest to note that Rios and Simmons [89] found the immunogenicity of treated mouse m a m m a r y tumors was restricted to the individually specific T A T A s rather than the common virus-associated antigens. As will be discussed below (Section IIIC), infection of tumor cells with viruses which bud from the cell surface can produce TASAs with increased immunogenicity. In a recent study, A1-Ghazzouli et al. [90] showed that infection with murine leukemia virus of a spontaneous mouse sarcoma, with no detectable immunogenicity, resulted in strong immunogenicity. Immunized mice were resistant to challenge with the uninfected tumor cells and displayed cell-mediated cytotoxicity against the uninfected target cells. As discussed earlier (Section IID), this experiment indicates that spontaneous tumors may have TATAs, but their immunogenicity may be relatively poor. Exposure of some mouse tumors to certain chemotherapeutic agents led to sublines with increased immunogenicity [91,92]. This phenomenon is substantially different from the modifications described above, since the increased immunogenicity of the treated cells was due to new antigens not found on the untreated cells, and these antigens persisted upon further passage of the tumor in the absence of the drug. IIIB. Extracts o f tumor cells Crude extracts and soluble antigens from tumors have been used very extensively in studies of TASAs. However, there have been relatively few reports on the immunogenicity of such preparations. In general, it has been rather difficult to obtain effective immunization with disrupted tumor cells or their fractions. It has been suggested that the surface membrane needs to be intact or at least in large fragments for its antigens to be potent immunogens [93,94], but other studies have failed to support this contention [95]. Alternatively, as discussed above for immunization with intact cells, effective immunogenicity of TASAs may often depend on prolonged exposure to large amounts of antigen and the doses and schedules used for soluble antigens may often have been inadequate. One would anticipate that if tumor antigens were purified without appreciable loss in activity, lower doses of antigens would be required for immunization. However, documentation of progressive increases in specific activity of TASA with purification has usually not been provided. Despite the problems in this area, some immunogenic extracts have been prepared from virus-induced and carcinogen-induced tumor cells. Antigens from adenovirus-induced tumor cells were prepared by sonication and separation by Sephadex G-200 chromatography [96,97]. As little as 60 ~g of soluble antigen,

103 without adjuvant, produced specific resistance to tumor challenge. Although these procedures were unsuccessful in obtaining tumor specific transplantation antigen from simian virus 40 (SV40) tumors, immunogenic soluble antigens from SV40 tumors have been preparaed by papain digestion of crude membranes [98] and by detergents [99]. In one of these studies [98], all of the tumor specific transplantation antigen activity was included on G-150 Sephadex and some could be physically separated from H-2 histocompatibility antigen activity; however, there was an actual decrease in specific activity during purification, despite the use of complete Freund's adjuvant along with the soluble antigen for immunization. In the other study [99], TATA and an in vitro detected TASA were detected in a similar size range on G-150, but also in a high molecular weight fraction; good yields of TASA were obtained by this procedure but the specific activity of the separated TATA was not determined. Using similar procedures as those used in their studies on SV40 antigens, Law and his colleagues [98] have prepared soluble TATA from a murine leukemia virus-induced tumor. Similar procedures as those described above have been utilized to prepare soluble TATAs from tumors induced by chemical carcinogens [100-102]. Very little progress has been reported thus far in purification of the antigens from crude soluble extracts. In most of the studies, large amounts of material were required for immunization and the degree of immunity induced was rather weak. IIIC. Viruses

A variety of oncogenic DNA and RNA viruses are highly immunogenic, producing resistance to challenge by tumors induced by the same virus and producing humoral and cell-mediated immune reactions against TASAs. With the oncogenic DNA viruses, which usually do not persist in the transformed cells, Habel [103] postulated that inoculation of virus resulted in in vivo transformation of cells with resultant expression of immunogenic TATA. However, by studying the effects of ultraviolet irradiation on polyoma virus, it was found that irradiated virus could still produce transplantation resistance in hamsters, even after it had lost its transforming ability and other viral functions [104]. It appears likely that one of the genes in the DNA viral genome codes for TATA and that persistence of that genetic function is sufficient to induce transplantation resistance. With the oncogenic RNA viruses, the relationship of the TATAs and other TASAs on tumor cells to the virion antigen is not clear, since virus-induced tumor cells contain infectious virus. These antigens and their immunogenicity have been reviewed recently [37-43]. It bears noting here, however, that murine sarcoma virus is particularly immunogenic and therefore this tumor system has been adopted by many tumor immunologists for studies of the mechanisms of resistance to tumor growth (see Section IV). The particularly strong immunogenicity of murine sarcoma virus may be related to the fact that it induces local tumor growth and then regression. As noted above with intact tumor cells (Section IIIA), this large, transient antigenic load may produce efficient immunization. Myxoviruses unrelated to the tumors, when harvested from virus-infected

104 tumor cells, have been found to be quite effective in inducing resistance to subsequent challenge by uninfected tumor cells [105-107]. This procedure, which has been termed heterogenization [105], appears to depend on the incorpoaration of TATAs into the membrane of the virus. The augmentation of immunogenicity has been ascribed to a helper effect of the virus antigens in close proximity to TATA [108], but the precise mechanism has not yet been proven. It has been pointed out that it may be quite difficult to generalize the application of this procedure from the results obtained in a few experimental tumor systems [43,107].

IIID. Other immunogens Although most immunizations against TATAs and other TASAs have been performed with intact tumor cells or extracts, or with oncogenic viruses, it is important to note that inoculation of other types of materials may lead to specific or nonspecific altered resistance against tumor challenge or to detectable immune responses against TASAs. The types of preparations used and the mechanisms responsible for their effects have been quite heterogeneous and in each case it is necessary to carefully determine whether generation of specific immunity to TASAs is involved and whether this is due to cross-reacting antigens on the immunogen and the tumor cell surface. 1. Allogeneic cell immunity. Kobayashi and his colleages have described a phenomenon called "allogeneic cell immunity", in which animals immunized with allogeneic tumor cells or normal tissues, particularly skin, have increased resistance to challenge with syngeneic, antigenically unrelated tumors [109]. The mechanisms underlying these effects, which can be rather strong, remain to be determined. It has been attributed to nonspecific immunostimulation, being easily abrogated by low doses of irradiation which do not affect tumor specific immunity [24]. However, it has been possible to locally transfer this resistance with lymphoid cells [110]. Particularly since resistance is induced by 3 days after immunization with allogeneic cells, the possible role of augmented natural cell-mediated immunity needs to be considered (see Section IVB,3). 2. Immunization by immune RNA. There have been a number of reports on induction of immunity against tumors by in vitro or in vivo exposure to RNA extracted from immunized animals [e.g. 111]. In most studies, xenogeneic immune R N A was used, but similar effects have been induced by RNA from immunized, syngeneic rats. The results have appeared to be specific, but complete criss-cross specificity experiments with two unrelated syngeneic tumors have not been reported. Based on the promising results in experimental tumor systems, this approach is being investigated as a form of human immunotherapy [11 l]. The mechanism underlying this phenomenon has not been clearly determined. Digestion of the RNA with RNAase abrogates activity and this has been suggested to be a specific transfer of immunologic information. However, the possibility of transfer of an antigen • RNA complex with augmented immunogenicity has not been ruled out. In a recent study on the role of antigen in the immune RNA-mediated induction of immunity to nontumor-related antigen (human gamma globulin) [112], mixture of the RNA with spe-

105 cific antibody abrogated its immunogenicity, providing rather strong evidence for the involvement of antigen. This approach might be very useful in analyzing the mechanism of induction by immune RNA of anti-tumor immunity. 3. Immunization by soluble products of tumor cells. Soluble products which are actively produced and secreted from tumor cells, e.g., hormones, carcinoembryonic antigen, a-fetoprotein, have usually been found to be nonimmunogenic in the host. However, by chemical alteration, even some of these antigens have been rendered immunogenic, presumably by breaking tolerance [113], have altered a-fetoprotein and made it immunogenic in syngeneic mice; however, this immunization did not induce resistance to tumor challenge. Some myeloma proteins, which are produced by mouse plasma cell tumors and which have immunogenic idiotypic determinants, have been shown to induce specific resistance against challenge with the homologous tumor and not against other plasma cell tumors [114]. This is apparently due to localization of some myeloma protein in the cell surface membrane, which can function as a TATA. It is of interest that growth of tumor cells after immunization with myeloma protein may lead to a selection of stable variants which produce only light chains which lack the idiotypic determinants [114]. 4. Immunization by microorganisms. Some microorganisms, particularly BCG (Mycobacterium bovis) and Corynebacterium parvum, have been used extensively for immunotherapy or immunoprevention of experimental and human tumors. Their anti-tumor effects have usually been assumed to be due to the ability of these microbial agents to stimulate the reticuloendothelial system and to act as adjuvants. However, recent studies have indicated that other mechanisms need to be considered. It has been known for many years that some microorganisms may share crossreacting antigens with mammalian cells [e.g. see 115]. Recently several investigators have found that BCG may have antigens common to some rodent and human tumor cells. In most cases, this has been shown by reactivity with tumor cells by antibodies raised in a heterologous species against BCG. However, it has been possible to show that antibodies produced by immunization of guinea pigs with BCG react specifically with a TASA on a syngeneic hepatoma as well as with human melanomas [116]. Reciprocally, it was found that antibodies to the hepatoma reacted with BCG; however, this cross-reactive antigen appeared to be distinct from other TASAs on the tumor cell. Minden et al. [117] found antibodies to BCG and to melamona antigens in human sera and suggested that some immune reactions to melanoma antigens are due to stimulation by mycobacteria. However, the specific cross-reactivity between BCG and melanoma was not clearly demonstrated in this study. BCG [118] and some other microorganisms [119] have been shown to augment natural cell-mediated cytotoxicity (see below, Section IVB,3). Therefore, this is yet another mechanism which may account for the anti-tumor efficacy of these microbial agents.

106 IV. MECHANISMS FOR THE IMMUNOGENICITY OF TUMOR ANTIGENS Recently there has been rapid progress in the understanding of many of the factors involved in the immune response to antigens, particularly in regard to the role of the major histocompatibility complex (MHC) in immunity involving T cells [120]. There already are a number of indications that this basic information will help considerably in the understanding of mechanisms of the immune response to tumor antigens. A major challenge to tumor immunologists is to obtain detailed information on the nature of the TASAs recognized by the host, their relationship to the MHC, and the types of effector cells involved in the immune response. As will be discussed below, some of the data already obtained have provided important information about some of the basic concepts of antigenic recognition and regulation of the immune response.

IVA. Role of the M H C and other factors in immunogenicity Genetic factors, particularly related to the MHC, have been found to play important roles in determining the immunogenicity of tumor antigens. Two major types of roles can be identified: (a) effects on the responsiveness of the host to a particular antigen; and (b) effects on the antigenicity of TASAs and their recognition by the host. It has been well documented in a number of species that the ability to react to a variety of synthetic and natural T cell-dependent antigens is controlled by the immune response (Ir) region of the M H C [121]. The role of genetic factors in the response to tumor antigens has been studied mainly in oncornavirus-induced tumor systems [42]. So far, only one genetic locus, influencing susceptibility of mice to spontaneous leukemia and to infection by oncornaviruses, has been clearly shown to be linked to the MHC [122]. This genetic locus, termed Rgv-1, maps in, or close to, the I region of the H-2 complex. However, the mechanism of this resistance remains to be worked out. It is probably related to regulation of the immune response to some viral antigen or virus-induced TASA, but this has not been defined. With the ability to sensitively measure, by a variety of immunologic assays, the immune responses to TASAs, this area of genetic control of host responsiveness to tumor antigens seems ready for detailed investigation and there have been a few recent studies on this [reviewed in 43]. The relationship of TASAs to MHC products has been investigated much more extensively. It has been known for several years that the quantitative expression of some TASAs on chemically induced [123] and virus-induced [124] mouse tumors is inversely related to the amounts of detectable H-2 antigens. A few recent studies have indicated that some TATAs and other TASAs may be altered histocompatibility antigens or alloantigens of strains different from that of the host from which the tumor arose [125-128]. In the initial study of Invernizzi and Parmiani [125], it was shown that immunization of BALB/c mice with normal tissues from C57BL/6 or C3Hf origin induced resistance to challenge with BALB/c chemically induced

107 sarcomas. From this study, it was thought that foreign H-2 antigens might be expressed in tumors and that these represented the TATAs. However, Klein and Klein [129] analyzed the chromosomal markers and immunogenicity of hybridized tumor cells and concluded that the TATAs were not coded for by genes on the chromosome bearing the H-2 locus. In line with this finding, Parmiani and Invernizzi [127] showed that non-H-2 alloantigens on skin or other normal tissues could produce resistance to tumor challenge. They further showed that mice syngeneic to the tumor, when immunized against the tumor, had accelerated rejection of skin allografts from the strain in which the tumor-associated alloantigen was found. They postulated that the TATAs of chemically induced sarcomas were the result of mutations, induced by the carcinogen, in the structural genes coding for some histocompatibility antigens. Since there are many histocompatibility loci and many possible alleles for each, this explanation might account for the apparent individual specificities of the TATAs on these tumors. However, this interpretation still needs to be further documented. One concern, as discussed earlier, is that inoculation of some tissues may nonspecifically augment resistance to tumor growth. Against this possibility is that some specificity in the resistance induced by tissues of different strains was demonstrated [127]. However, it would be very helpful if evidence could be provided for specific humoral or cell-mediated immune responses of the host against these apparent, shared antigens. Another line of evidence for the possible role of histocompatibility antigens in the immune response to cell surface has come from studies of T cell-mediated cytotoxicity against virus-infected or hapten-modified target cells [130,131 ]. These studies have led to the conclusion that immune T cells can only lyse syngeneic target cells or those which have the same H-2K or H-2D alloantigens as the cells used for immunization. Several reports on investigations with syngeneic tumor systems have supported this conclusion [132-136]. However, Holden et al. [137] have found that, under some conditions, T cells from mice immunized by murine sarcoma virus can react well in several assays with some allogeneic leukemia cells. Similarly, Burton et al. [138] found that T cells immunized against plasma cell tumors could react against allogeneic plasma cell tumors. Therefore it appears that the association of tumor antigens with the M H C is not a universal phenomenon and this makes unlikely the hypothesis that all TASAs and other surface antigens recognized by immune T cells are modified or altered H-2 antigens. It seems more likely that the presence of compatible M H C antigens with adjacent TASAs leads to more effective interactions between the immune T cells and tumor cells. This model would account for the H-2 restrictions which have been described in some systems and for the preference, but not complete restriction, for syngeneic targets in the system of Holden et al. [137]. Similar to the demonstration of reactivity in vitro of immune lymphocytes against both allogeneic and syngeneic targets, in vivo immunization with allogeneic tumor cells has been shown repeatedly to produce protection against challenge with syngeneic tumors [e.g. 139]. Further indications of the role of the M H C in immunogenicity and antigenicity

108 of tumor antigens have been provided by the study of tumors which tack some or all of the M H C antigens. Forni et al. [140] studied five sublines of the L2C guinea pig leukemia and found that the one subline which lacked Ia antigen was unable to induce resistance against challenge by the same or other sublines. However, despite this lack of detectable immunogenicity, the Ia-deficient subline was sensitive to the immunity induced by the other sublines. It also was able to induce an in vitro proliferative response in lymphocytes from immunized guinea pigs. These data therefore seem to indicate that the TATA is independent of the Ia antigens, but that the presence of Ia antigens helps to induce immunity of the TATA. It is quite possible that, as with the role of H-2 antigens in susceptibility of target cells to lysis, the requirement for these antigens is relative and not absolute. Only one means of immunization, injection of tumor cells in complete Freund's adjuvant, was attempted and some of the other methods discussed above might have been more effective. Most of the evidence for a physical association between virus or tumor antigens with MHC antigens has been indirect. One direct method for analyzing this is to determine whether the antigens can be separated during the course of purification. Davies et al. [141,142] found that TL antigen and an antigen associated with the EL-4 mouse leukemia could be physically separated from H-2 antigens. Similarly, Law et al. [98] showed that SV40 TATA could be separated from H-2 antigens by lectin immunoadsorbent or Sephadex columns, in some other studies, partially purified soluble fractions contained both TASAs and H-2 antigens [143-145]. However, it is quite possible that further purification, or alternate methods of isolation, would separate the two types of antigens. In vitro systems have been very useful for detailed analysis of the various cells and other factors involved in the induction of immune repsonses to tumor antigens. It has been possible to generate cytotoxic effector cells in vitro, which are specifically directed against TASAs, and macrophages have been shown to play an important role in presenting soluble TASAs to the lymphocytes [146,147]. Similarly, macrophages have been found to be required for in vitro production of migration inhibitory factor by T cells from mice immunized by murine sarcoma virus [148]. In detailed studies on the role of macrophages in this system, kandolfo et al. [148,149] showed that histocompatible macrophages, either from syngeneic normal mice or from mice with the same H-2 haplotype, were required for stimulation of immune T ceils by crude soluble tumor extracts. The role of the MHC in this phenomenon is not clear. The data are compatible with either the physiological interaction model discussed earlier in this section, in which MHC histocompatible cells interact more effectively, or with the complex antigenic determinant model [150], whereby T cells only recognize complexes of antigens with MHC determined cell surface structures. In contrast to these findings with soluble tumor antigens, Landolfo et al. [151] observed that when intact tumor cells were used to stimulate migration inhibitory factor production by immune T cells, there was not an absolute requirement for macrophages and allogeneic tumor cells as well as syngeneic or H-2 compatible tumor cells could stimulate. These data suggested that soluble TASAs and intact tumor cells may

109 stimulate migration inhibiting factor production by two different mechanisms. IVB. Effeetor mechanisms involved in the response to tumor antigens

One important feature of the immune response to tumor antigens, which has become increasingly clear in the past few years, is that a variety of effector mechanisms are often involved. Since this has been extensively reviewed elsewhere [e.g. 1,37], in the discussion below only some recent findings on each of the main categories of effector mechanisms will be summarized. 1. T cells. In terms of immune factors of importance to in vivo resistance to tumor growth, T cells appear to play the predominant role. Several investigators have found that T cells were required for adoptively transferring resistance against challenge with tumor cells or with oncogenic virus [152-155]. One of the principal in vitro assays for study of T cell immunity to TASAs has been the s 1Cr release cytotoxicity assay. A main point of apparent lack of correlation between in vitro reactivity in this assay and in vivo resistance against tumor growth was the more transient detection of cytotoxicity after immunization. However, recent studies have provided a simple answer to this. In in vivo experiments to demonstrate resistance against tumor growth, the challenge with tumor cells actually represents the second exposure of the immune host to tumor-associated antigens. A key factor in resistance to challenge may be the ability of the host to mount a rapid secondary immune response, particularly in the region of the challenge. In three different tumor systems, rapid development of T cell-dependent cytotoxic reactivity after secondary tumor challenge has been demonstrated [156-158]. Similar results have also been obtained in vitro by incubation of immune lymphocytes with tumor cells for 5-9 days [85,159-161]. These data clearly indicate that immune individuals have memory cells for cytotoxicity, but re-exposure to antigen is needed for generation. It appears, at least in some systems, that proliferation of lymphocytes is needed for generation of cytotoxic T cells against TASAs [162,163]. Direct evidence for lymphoproliferative responses to TASAs has been obtained in a variety of tumor systems [e.g. 1,160,164]. Even at times when proliferative responses have been difficult to detect, some proliferative response has been required for generation of cytotoxic T cells [163]. Recently, Oehler et al. [165] used a more sensitive technique for the mixed lymphocyte-tumor cell interaction and found that the kinetics of the proliferative response and of the generation of cytotoxicity were similar. As noted earlier in this section (IVA), immune T cells also respond to TASAs by production of migration inhibiting factor. Almost all studies of cell-mediated immunity to tumors involve the use of cells from spleen, lymph nodes or peripheral blood. However, it would seem most relevant to examine the nature and function of cells in the tumors themselves. This has been done most extensively in murine sarcoma virus-induced tumors. These tumors contain large numbers of T ceils and some of these T cells have specific cytotoxic reactivity against antigenically related tumor cells [166,167]. 2. B cells. The main manifestation of B cell responses to TASAs is production

110 of specific antibodies. Immunization with tumor antigens usually leads to the formation of antibodies, as well as to cell-mediated immune responses. The extensive literature on antibodies to tumors has been reviewed recently in detail [168,169]. For the purposes of the present discussion, only a few selected points will be emphasized. It is important to note that in addition to the induction of antibodies by immunization with tumor cells, natural or spontaneously appearing antibodies may be seen, with reactivity against a variety of tumor-associated or virus-associated antigens [reviewed recently in 42]. In addition to their direct effects on tumor cells, antibodies to TASAs may act in cooperation with certain lymphocytes bearing receptors for the Fc portion of immunoglobulin, to produce antibody-dependent cell-mediated cytotoxicity (ADCC). Such antibodies have been found to play a role in the immune response against tumors induced by murine sarcoma virus [170-173] and by mouse mammary tumor virus [174]. ADCC antibodies of some human tumor target cells have also been detected [175]. B cells have also been reported to play a direct role in cell-mediated cytotoxicity against tumor cells. In visual microcytotoxicity assays, at certain times after immunization, B cells and not T cells appeared to be the effector cells [176,177]. However, the B cell nature of these effector cells was not conclusively demonstrated, and other cells with Fc receptors may have been responsible. 3. Natural killer cells. Another effector cell mechanism, which is of considerable interest and potential significance for in vivo resistance to tumor growth, is natural cell-mediated cytotoxicity [reviewed in 178]. Mice and rats develop this reactivity for a transient period, and humans have apparent long-term persistence of this reactivity. High levels ofcytotoxicity were found in nude mice, and although the effector cells were initially found to have no detectable cell surface markers, recent studies have indicated that natural killer cells in mice may be pre-T cells, with a low density of 0 antigen and Fc receptors. Similarly, human natural killer cells have been found to have low affinity receptors for sheep erythrocytes and to have Fc receptors. A feature of this mechanism which is directly relevant to considerations of immunogenicity is that inoculation of mice with various tumor cells led to a rapid, transient increase in natural killer cell activity, with a peak at three days. Similarly, a variety of murine viruses, and BCG and Corynebacterium parvum, were quite effective in augmenting natural killer cell activity. After these inoculations, the natural killer cell activity had the same specificity as found in uninoculated mice. Therefore, these treatments cannot be considered to be specifically immunogenic for a particular tumor cell but rather a triggering of the general mechanisms. As discussed in [178], some previous observations on immunogenicity of some manipulations, manifested by early development of cytotoxic reactivity, may have been due to augmentation of natural killer cell activity. There are several preliminary indications that natural killer cells may play a significant in vivo role. Kiessling et al. [179] found a correlation between levels of natural reactivity in different strains of mice and the relative resistance to growth of

111 transplantable leukemia cells. We have recently found that some leukemia cells which are very sensitive to natural killer cell activity grew less well in nude mice than in conventional mice of the same strain. As discussed in detail in [178], these findings on natural killer activity may have important implications for immune surveillance. A principal challenge to the concept of immune surveillance has been that spontaneous tumors frequently lack detectable TATAs and therefore might not be susceptible to control by the immune system. However, as discussed earlier, almost all of the negative evidence has been obtained by procedures designed to detect transplantation resistance and other immune responses which have generally been associated with immune T cell activity. With the new recognition of the possible role of natural killer cells, the questions of antigenicity and resistance to tumor growth need to be asked by protocols designed to detect this function as well as that of immune T cell-mediated cytotoxicity. For example, to detect increased resistance to challenge by tumor cells which might be induced by augmentation of natural killer cell activity, the time of challenge would probably have to be much sooner after immunization than the 1-2 week interval usually employed and attempts at hyperimmunization might be counterproductive. In addition, the antigens associated with natural killer cell activity appear to be distinct from those recognized by other effector mechanisms. Therefore the entire question of antigenicity of tumors, or lack thereof, will need to be reexamined. 4. Macrophages. Macrophages represent yet another effector cell mechanism for reactivity against tumor cells. Some of the methods used for immunization against tumors, and growth of a variety of tumors, may lead to increased cytotoxic and cytostatic effects of macrophages against tumor cells [e.g. 180,181 ]. As discussed above for natural killer cells, macrophages may have significant in vivo antitumor effects, and nonimmunogenic tumors may be able to stimulate this mechanism or might be susceptible to the effects of macrophages.

v. POSSIBLE MECHANISMS FOR INSUFFICIENT OR UNDETECTABLE

IMMUNOGENICITY Throughout this review, emphasis has been placed on the variety of mechanisms for immunogenicity of tumors and for immune responses against tumors. However, it is important to point out that in most cases, TATAs are not sufficiently immunogenic. Unfortunately, the usual outcome is progressive tumor growth and death of the host. Much attention needs to be given to the mechanisms for the failure of the immune system, albeit multifaceted in its antitumor responses, to adequately control tumor growth. A variety of mechanisms have been observed or suggested to account for insufficient or undetectable immunogenicity, and these will be briefly discussed below.

I12

VA. Immunoresistance A significant part of this problem appears to be immunoresistance of tumor cells to the immune response, rather than actual lack of immunogenicity. It has been found in a number of studies that for tumor cells to be able to react with antibodies or immune cells, they need to have sufficient quantities or densities of the relevant TASAs [e.g. 8,83]. T u m o r cells with subthreshold amounts of TASAs are immunoresistant. The loss or rapid shedding of TASAs from tumor cells has been suggested as a possible explanation for the failure of such antigens to function as effective TATAs [35]. The labile association of embryonic antigens with the tumor cell surface may account for their inefficient role in resistance to tumor growth [35]. One well-studied mechanism in this area is antigenic modulation [182]. This mechanism was originally described with TL antigen. This TASA is quite immunogenic in some strains of mice but does not function as a TATA, since exposure of tumor cells to anti-TL antibody causes a reversible loss of antigen. Some embryonic antigens have been shown to undergo antigenic modulation [183] and this may explain why they usually fail to be involved in resistance against tumor growth. Aoki and Johnson [184] found that antigens associated with a Gross virus-induced mouse leukemia underwent antigenic modulation. Ioachim and his colleagues have found that Gross virus-induced rat tumors lost detectable antigenicity and developed increased malignancy when passaged in rats given virus at birth [185] or in sublethally irradiated rats [186]. Since TASAs returned upon in vitro cultivation of these tumor cells, these interesting effects have been interpreted as a form of antigenic modulation. From the above discussion, it should be obvious that the nonantigenicity of some tumors may only be apparent, due either to the inability of the particular assay procedure to detect low amounts of antigen, to the physiologic state of the cells at the time of testing, or to the failure of the particular effector mechanism under study to react with the antigens. Therefore very extensive studies are needed before a tumor should be considered nonantigenic. VB. Interference with immunogenicity or effector functions TASAs may be nonimmunogenic or insufficiently immunogenic because of defects in the immune system of the host or the presence of suppressor cells or other factors which interfere with the immune response. In addition, it has recently been found that the tumor cells themselves may contain various suppressive factors which can interfere with the immunogenicity of their TASAs. Depressed immunologic competence in tumor-bearing hosts has been noted by many investigators. Defects in humoral antibody responses and in the ability to mount cell-mediated immune responses have been described. A generally depressed ability to respond immunologically would be expected to have an important bearing on the development of effective immunity to TASAs and on effective resistance to progressive tumor growth. Although responses can develop despite the depression, they may be too delayed or quantitatively inadequate for protection. The general subject of immune depression in cancer has been reviewed recently in detail [187].

113 Until recently, there were no adequate explanations for the depressed immunity in tumor-bearing individuals. One mechanism which has been studied extensively and which can account for the depressed lymphoproliferative responses in tumorbearing individuals to mitogens and TASAs is the presence of suppressor cells [188, 189]. In at least some tumor systems, the suppressor cells have the characteristics of macrophages. Suppressor macrophages have been found within tumors themselves, as well as in the lymphoid organs of tumor-bearers [166]. These suppressor macrophages only inhibited some phases of cell-mediated immune responses. They had no effect on the activity of cytotoxic effector cells, but they could interfere with the secondary generation, either in vivo or in vitro, of cytotoxic effector cells. However, other types of suppressor cells have been described and some of these have been found to inhibit humoral immune responses rather than, or in addition to, cell-mediated immunity [e.g. 190,191]. Another factor to be considered for the depressed immunity and poor immunogenicity of some tumors is immunosuppression by the tumors themselves or by products of the tumors. Only low concentrations of the rat lymphoma (C58NT)D caused proliferation of normal allogeneic or syntgeneic immune lymphocytes, and this could be attributed to inhibitory properties of higher doses of the tumor cells [192]. The active factor has been shown to be a contaminating parvovirus in the tumor, the Kitham rat virus [79]. This virus has been a consistent contaminant of several rat tumors, it appears that it can grow preferentially in actively dividing cells with resultant cytopathic effects on the stimulated lymphoblasts. A similar inhibitory phenomenon has been seen with some mouse tumors, due to contamination by another parvovirus, minute virus of mice. Other suppressive factors in tumors, which have not been directly associated with viruses, have been found to inhibit immune responses [e.g. 193]. In considering factors which may interfere with the cell-mediated immune response of tumor-bearing individuals, serum-blocking factors are most frequently thought of [reviewed recently in 194]. In a number of tumor systems, serum factors have been shown to specifically or nonspecifically interfere with immune reactions to TASAs. With leukemias, serum antibodies have been shown to frequently cause immunologic enhancement [195]. One possible mechanism for this is interference with in vivo immunogenicity [196]. Although serum blocking factors were initially all thought to be antibodies, evidence for the role of circulating antigens and antigen • antibody complexes has also been obtained [e.g. 197-199]. In addition to specific blocking factors, sera from tumor-bearing individuals have also been shown to nonspecifically inhibit some cell-mediated immune responses [200,201].

vI. DISCUSSION Immunogenicity of tumor-associated antigens is a very complex topic, and many factors can influence the recognition of these antigens by the host and the magnitude

114 of the immune responses. The amount and form of the antigens presented, the presence of the appropriate M H C antigens, the method and route of antigen administration, and the persistence of the antigens in vivo all appear to be important factors in immunogenicity. The immune responses are also very dependent on the reactivity of the host. In analyzing these responses, it is necessary to consider the genetic background of the individual, the reactivity of different types of effector cells, including natural killer cells, and a variety of cell-surface antigens on the tumors. In addition, the immune response is not homogeneous within the various lymphoid organs. The level of immunity detected in the spleen or peripheral blood may not be as relevant to the in vivo immune status as the immune reactivity in the region of tumor growth or within the tumor itself. Reexposure of the host to tumor antigens may evoke a rapid and rigorous secondary immune response of lymphoid cells in the region and may also cause a migration of memory cells to the site of challenge. Of central importance in tumor immunology are the mechanisms by which tumors can grow progressively despite the potential for a vigorous and effective immune response against TASAs. Some phases of the immune response, proliferation of lymphocytes and the generation of cytotoxic effector cells, seem to be particularly sensitive to inhibition, and a variety of factors can cause inhibition of immune responses. The depressive factors include suppressor macrophages and other cells, specific and nonspecific serum factors, and viruses and probably other factors within the tumor cells themselves. It will be important, in each tumor system, to determine which, if any, of these inhibitory factors play the predominent role in promoting tumor growth. Practical means might then be found to eliminate or inhibit the particular inhibitory factor and thereby improve the effective immune response against the tumor. Many of the issues discussed in this review appear to have important implications for studies of immunoprevention and immunotherapy of tumors. We need methods for demonstrating that the materials used for immunization are antigenic, and that the route and mode of immunization are effective. It seems unlikely that many of the practical problems now being faced in immunoprevention or immunotherapy trials will be solved solely by more empirical in vivo studies. A careful dissection of factors influencing immunogenicity of TASAs within each system may be essential for successful results.

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