Biochimica et Biophysica Acta, 473 (1977) 121-148 © Elsevier/North-Holland Biomedical Press BBA 87038

SPECIFIC BLOCKING FACTORS -- ARE THEY IMPORTANT? KARL ERIK HELLSTROM, INGEGERD HELLSTROM and JERRY T. NEPOM Division of Tumor Immunology, Fred Hutchinson Cancer Research Center, Departments of Pathology, Microbiology~Immunology and Biochemistry, University of Washington Medical School, Seattle, Wash. (U.S.A.) (Received May 6th, 1977)

CONTENTS I. II. III. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 How the concept of specific blocking factors originated . . . . . . . . . . . . . . 122 What is the nature of specific blocking factors? . . . . . . . . . . . . . . . . . 124 Where are the blocking factors produced? . . . . . . . . . . . . . . . . . . . 130 Specificblocking factors and suppressor cells . . . . . . . . . . . . . . . . . . 132 Where and how do specific blocking factors act? . . . . . . . . . . . . . . . . . 134 Correlation between specific blocking factors detectable in vitro and tumor growth in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 VIII. Local versus systemic effect of specific blocking factors . . . . . . . . . . . . . . 136 IX. Role of specific blocking factors in vivo . . . . . . . . . . . . . . . . . . . . . 137 X. Why are specific blocking factors important? . . . . . . . . . . . . . . . . . . 141 XI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

I. INTRODUCTION M o s t experimentally i n d u c e d a n i m a l t u m o r s a n d m a n y h u m a n neoplasms express t u m o r associated antigens [1]. Some of these are t u m o r specific t r a n s p l a n t a tion antigens (TSTA), defined by their ability to induce a n i m m u n e reaction which will lead to the rejection o f t r a n s p l a n t e d neoplastic cells by the appropriately i m m u n e host [2, 3]. Other antigens do n o t f u n c t i o n as T S T A b u t m a y be d e m o n s t r a t e d by sensitive in vitro techniques [4]. Phase specific (embryonic) t u m o r antigens shared by neoplastic a n d n o r m a l e m b r y o n i c cells are examples of antigens which belong primarily to the latter g r o u p [5]. Still other t u m o r s m a y n o t have any detectable antigens, n o t also present o n the c o r r e s p o n d i n g n o r m a l cells.

Abbreviations: SBF, specific blocking factors; TSTA, tumor specific transplantation antigens; EBV, Epstein-Barr virus.

122 One of the striking observations in tumor immunology is that lymphocytes which can destroy neoplastic cells in vitro are often detected in the lymph nodes of animals carrying small recently established transplantable tumors [4, 6]. Likewise, animals with small tumors often have "concomitant immunity" to cells from the respective neoplasm, detectable by their ability to reject small tumor cell inocula in transplantation tests [7-9]. Furthermore, their lymphocytes can often adoptively transfer tumor immunity in vivo [10]. Taken together, these observations show that neoplastic cells can grow in vivo in the face of an immune response to (at least some of) their antigens. This creates a dilemma. Why does an animal which can react to cells from its growing tumor not regularly do so in a way that leads to rejection of the tumor? Many different mechanisms might explain this phenomenon [1, 2, 6, 11-13]. They need not be mutually exclusive. One such mechanism operates through the formation of soluble factors which can specifically prevent the immune response against the appropriate tumor antigens from being effective in damaging the tumor cells [11]. We refer to these factors as specific blocking factors (SBF), and define SBF as any humoral factors which can, in a specific way, impair an immune response to tumor antigens, independently of their site of action (central, afferent or efferent) and their molecular composition; in this review, SBF affecting primarily cell-mediated tumor immunity will be discussed. By "specific" we mean that an immune response to tumor cells having different tumor antigens is not turned off. SBF may be entirely tumor products (tumor antigens) or the host's lymphoid cells may play a role in their formation (as in the case of antibodies, antigen-antibody complexes and T cell derived suppressor molecules). Several types of SBF probably exist together and play different roles in different systems, or under different conditions. For example, circulating tumor antigens may act as SBF and part of this action may be to induce the host to form immunosuppressive products, which serve as other SBF. We believe that SBF are important for several reasons. In this article we will consider evidence that there is a correlation between SBF detectable in vitro and tumor growth in vivo, indicating that measurements of SBF may ultimately be useful for patient monitoring. We will present recent findings on the molecular nature of some SBF and on the mode of SBF production. We will also discuss evidence that SBF provide a major mechanism by which antigenic neoplastic cells can escape immunological control, suggesting that an understanding of this mechanism may have implications for tumor therapy and prevention. Although our review concerns SBF in tumor systems, the findings discussed may have important implications with respect to immunoregulation in general. II. HOW THE CONCEPT OF SPECIFIC BLOCKING FACTORS ORIGINATED The evidence for SBF in tumor systems has come primarily from in vitro experiments. The colony inhibition and the microcytotoxicity assays have been the tests most frequently used to study cell-mediated anti-tumor immunity in vitro [14].

123 In these assays tumor cells are plated on plastic surfaces. When they have attached, immune or control lymphocyte suspensions are added. In colony inhibition tests the number of colonies formed from the plated cells is determined, while in microcytotoxicity assays the number of target cells remaining attached to the plastic surface is counted. The colony inhibition test takes 3-5 days, while the microcytotoxicity assay takes 30-40 h. Using colony inhibition and microcytotoxicity techniques, Hellstr6m et al. observed that lymphocytes from animals with small to medium sized tumors were commonly cytotoxic to cells from the respective tumor [15]. This finding has been confirmed in many systems [6, 9, 16], and also when tumor cell killing is measured over a 4-6 h time period [17]. On the other hand, lymphocytes from animals with large tumors are often non-reactive when similarly tested. In an attempt to understand why tumors can grow in vivo at a time when lymphocytes are tumor reactive in vitro, studies were initiated to determine whether serum from tumor-bearing animals could abrogate the tumor destructive ability of immune lymphocytes in vitro [19]. Immune lymphocytes were added to plated tumor cells which had been exposed either to serum from an animal carrying the respective tumor, from an animal carrying an antigenically unrelated tumor or from an untreated animal. Sera from animals carrying growing tumors of the same line as the target cells were found to abrogate ("block") the ability of lymphocytes to react against these target cells. It was believed that the "blocking phenomenon", so detected in vitro, was a counterpart to immunological enhancement as seen in vivo after treating animals with hyperimmune serum to tumor antigens [20], and that it provides one of the major mechanisms by which neoplastic cells can escape from immunological destruction [4]. The finding of tumor-specific blocking serum effects was subsequently established in a variety of systems, including chemically induced tumors [21-23], tumors induced by D N A [24] and R N A [25] tumor viruses, and spontaneous neoplasms [26, 27]. Most of this work has been conducted in mice, but analogous findings have been obtained in rats [23, 24], rabbits [18], Japanese quails [28], as well as in human cancer patients [29-31]. Exceptions have been reported when tumor-bearer sera did not block in microcytotoxicity assays [32], but they have been fairly rare. Although the colony inhibition and, particularly, the microcytoxocity assays were used for most of these studies, SBF have been demonstrated in experiments performed with macrophage migration inhibition [33] and leukocyte adherence inhibition [30, 34] techniques. Similar blocking effects have, on the other hand, not been regularly demonstrated in assays measuring the proliferation of lymphocytes exposed to antigen. The occasional reports of SBF detected by such assays have been most commonly on individuals with small tumor burden [35] and may have been due to antigen masking by antibodies rather than to an effect of SBF on the lymphocytes. In the early colony inhibition and microcytotoxicity studies, blocking serum activity was commonly studied at the target cell level by first exposing the tumor cells

124 to tumor-bearer serum, then removing the serum before the lymphocytes were added [25]. In more recent studies [11], however, SBF have been measured by either exposing the lymphocytes to the blocking material, followed by washing, before these are added to the target cells, or by letting it remain with the mixture of lymphocytes and tumor cells for the duration of the assay. The latter procedure is the one most commonly used and is the one used in the studies discussed in this article (unless otherwise stated). In most cases the two procedures have given similar results, except that hyperimmune sera to tumor antigens often block at the target cell level but not at the effector cell level, whereas the blocking effect of free antigens is the reverse [37-39].

IlL WHAT IS THE NATURE OF SPECIFIC BLOCKING FACTORS? Shortly after the "blocking phenomenon" was discovered, when SBF were tested exclusivly at the target cell level, it was believed that SBF in tumor-bearer sera were "blocking antibodies" which protected the tumor cells by masking their cell surface antigens [4]. This view took support from the demonstration that many SBF are present in the 7 S immunoglobulin fraction of sera from tumor-bearing mice ("tumor-bearer sera") and that SBF can be selectively removed by adsorption of serum with tumor cells carrying the appropriate TSTA [41]. As more work was performed, the hypothesis that SBF detected by the microcytotoxicity assay were antibodies became untenable. Since SBF, even when tested at the target cell level, disappeared within 3-7 days following tumor removal [21], it seems likely that at least part of SBF were derived directly from the tumor or through a mechanism which needed a continuous stimulation by a tumor product. For this reason, Hellstr/Sm and Hellstr/Sm suggested that many circulating SBF are complexes between tumor antigens and their antibodies [42]. Experiments were performed to test this hypothesis [43]. Sera from tumorbearing mice were exposed to a glycine buffer at pH 3.1, in order to dissociate the hypothetical antigen-antibody complexes. The sera were subsequently fractionated by ultrafiltration to give one faction with molecular ratio (MR) above 100,000 and another faction with Mx between 10 000 and 100 000. The fractionated material was then tested for its ability to block specific cell-mediated cytotoxicity against the respective tumor. This was done in two ways. First, it was added to target cells, incubated with these for 45 min and subsequently removed before immune lymphocytes were added. Second, it was added to the lymphocytes and allowed to remain with both the lymphocytes and the target cells for the duration of the test. The fraction with a molecular ratio (Mx) above 100 000, in which IgG immunoglobulins could be identified by immunodiffusion assays, did not block cell-mediated cytotoxicity when tested according to either of the two protocols. This argued against the idea [40] that SBF in tumor-bearer sera were "blocking antibodies" which provided an efferent form of enhancement by simply binding to the tumor antigens.

125 The lower M R fraction blocked when allowed to remain with the mixture of lymphocytes and target cells for the duration of the test, while it did not block after incubation with the target cells alone. The blocking activity of this fraction was tentatively attributed to free tumor antigens acting on the effector cells [43]. A mixture of the "small" and the "large" fractions blocked when tested according to either of the two protocols. Blocking occurred only when both fractions came from sera containing the same SBF and not when they were derived from sera containing different SBF. The blocking was ascribed to antigen, antibody complexes which were reformed after mixing. It was postulated that such complexes could bind to both the target cells and the effector cells but that their action was primarily on the effector cells, with the blocking seen at the target cell level playing a less important role [43]. At this time, the observations made in tumor systems suggested a model [ll] similar to one proposed by Diener and Feldman [44] to explain "low zone tolerance": antibodies may cross-link even small amounts of antigens to the surface oflymphocytes in such a way that the lymphocytes become unable to react; a large dose of antigen may be sufficient to block by itself. Brawn [45], Currie and Basham [46], Baldwin et al. [37-39], Thompson et al. [47], and others subsequently reported that extracts which were prepared from tumors, and which most likely contained tumor antigens, could block cell-mediated cytotoxicity in vitro. The data did not prove, however, that antigen per se was responsible for the blocking activity, since some other material with specific blocking activity may have been present in the extracts together with the tumor antigens. Baldwin et al. showed, furthermore, that complexes between artificially prepared tumor antigen extracts and their antibodies blocked at both the target and the effector cell level [48]. The antigen extract itself did not block at the target cell level. The data suggested that free tumor antigen and antigen, antibody complexes, when circulating in the blood stream of tumor-bearing individuals, may serve as SBF. Further support of the view that circulating antigen-antibody complexes can serve as SBF, came from studies by Tamarius et al. [49]. These studies indicated that factors which are present in the sera of multiparous mice, and which are capable of blocking the ability of lymhocytes from such mice to kill tumor cells in vitro [50], are primarily complexes between IgG immunoglobulins (IgGza and IgG2b) and an antigen against which antibodies can be raised by immunizing rabbits with normal mouse embryo cells (followed by absorption); whether the antigen present in the embryonic cells was a phase specific antigen or some other embryonic material against which antibodies can be raised was not studied. Since the first report that circulating SBF may be complexes between tumor antigens and antibodies [43], many studies have been published demonstrating such complexes in animals [51-53] and human patients [54-59] with tumors. For example, in patients with Burkitt's lymphoma, complexes between an Epstein-Barr virus (EBV) determined cell surface antigen and antibodies were detected prior to tumor relapse, while such complexes were not seen in tumor free patients [59].

126 In addition to being found in the serum, complexes have beeen demonstrated in the kidneys of tumor bearing animals [51-53] and humans [60]. Although it has been convincingly demonstrated that individuals with tumors often have both circulating tumor antigen [61-63] and antigen • antibody complexes [43, 55, 56, 59, 64], and that such complexes, as well as free tumor antigens, can act as SBF in vitro, this does not prove that SBF in serum are identical to the antigens and complexes which have been prepared artificially. For this reason, our laboratory set out to further investigate the nature of circulating SBF. Chemically induced mouse sarcomas were selected for the study since such sarcomas have individually distinct TSTA, and since sera from mice carrying them specifically block in vitro with little or no cross-reactivity between different sarcomas [21]. By performing experiments in a "criss-cross patttern", i.e. by testing all combinations of tumor and sera, it was possible to distinguish between specific and non-specific blocking effects of serum fractions. The effector cells tested in the microcytotoxicity assays were primarily T cells [68]. Our strategy was to use immunoadsorbents from immunoglobulin of mice immunized by repeated challenge with the respective tumors [65]. Sera of these mice were "unblocking" when added to sera from mice carrying the respective tumor; i.e. they abolished the blocking effect of the tumor-bearer sera, as previously seen in other systems [25, 66]. They had been prepared by inoculating BALB/c mice with sarcoma cells, excising the sarcomas which grew out and bleeding the mice 7-10 days after a subsequent challenge with the same tumors that had been rejected. Sera from mice carrying one tumor, 1321, blocked the ability of lymphocytes immune to that tumor to destroy 1321 target cells. The same sera did not block when tested with other sets of tumor cells and immune lymphocytes. The blocking activity was removed after the sera had been passed through immunoadsorbent columns prepared from anti-1321 immune serum and it could be recovered by eluting the columns with NaSCN [65]. Passage of tumor-bearer serum through columns constructed using unblocking sera yielded partially purified SBF. In order to obtain better purification of the blocking eluates, an adsorption procedure was used in which material which nonspecifically adsorbed to the columns was first removed by passing sera through columns prepared with normal mouse immunoglobulins [67]. The final effluents and eluates were then studied both for blocking activity in vitro and for their chemical characteristics. Only eluates prepared from the appropriate tumor-bearer serum had blocking activity. It was possible to show that eluates which were prepared by passing tumor bearer sera through the specific immunoadsorent columns, followed by elution, and which had blocking activity in vitro, contained one band on SDS-polyacrylamide gel electrophoresis with a MR of approximately 56 000. These eluates were further purified by gel filtration on Sephadex G-200. One peak was again obtained which had all the detectable blocking activity of the immunoadsorbent purified SBF and which had a MR of approximately 56 000.

127 In order to test the specificity of eluates from tumor-bearer sera, experiments were performed in a criss-cross pattern using two tumor lines, 1321 and 1408, whose major TSTA did not cross-react. The eluates of the affinity columns (anti-1321 and anti-1408, respectively) were found to have specific blocking activity with no crossreactions between the two neoplasms [68]. Thus the eluates behaved similarly to unfractionated tumor-bearer sera, which also gave tumor specific blocking. The next step was to test whether the antibodies used for purifying the blocking factors were tumor specific, or if the specificity seen was a property only of the eluates purified from the tumor-bearer sera. This would be the case if antibodies to different mouse tumors non-specifically removed the blocking activity from tumor-bearers' as compared to control sera from untreated mice. Since hyperimmune sera to chemically induced mouse sarcomas very rarely show tumor specificity when testing with binding assays [68], even if such sera have "unblocking" properties, we tested the immunoadsorbent specificity for SBF directly. The blocking activity of affinity purified SBF from mice carrying sarcoma 1408 was removed by passage through an anti-1408 column, but not through an anti-1321 column, and was recovered in the eluates from the 1408 column, but not from the 1321 column. Reciprocal results were obtained in parallel experiments with tumor 1321. These data demonstrated a high degree of immunologic specificity residing in the antibodies used for the immunoadsorbent columns. Some of these studies are summarized in Fig. 1. The chemical nature of the blocking eluates was then investigated. First, it was shown that the blocking activity of the eluates was sensitive to treatment with trypsin [69]. Second, blocking activity was found to be removed by passage through a Concanavalin A-Sepharose column and to be specifically recovered by subsequent elution with a-methylmannoside [68]. Taken together, these two sets of observations suggest that the blocking eluates are glycoproteins. Preliminary peptide mapping experiments have recently been done in which 1251 labelled tryptic digests were isolated from the affinity purified SBF [69]. Two independently isolated SBF, with different specificities in the blocking tests, were found to have a high degree of identity. Attempts to demonstrate immunological or biological specificity of SBF labelled with 125I have failed, even when very gentle iodination procedures have been used. However, there is preliminary evidence that labelling of the carbohydrate moiety does not interfere with the ability of SBF to bind to the appropriate columns [70]. If these observations are confirmed in further studies, they may lead to the development of radio-immunoassays for circulating SBF. Experiments were recently performed to test whether the blocking activity of eluates from affinity immunoadsorbents could also be specifically removed by adsorption with the appropriate tumor cells [68]. This was done because studies reported as early as 1970 [41], and subsequently confirmed [28], had shown that the blocking activity of tumor-bearer sera could be specifically removed by adsorption with tumor cells of the appropriate type and that it could be recovered by elution

128 Specific

blocking

Tumor type

A

activity

Tumor

type

B

Sera :

NMGG

A

+

--

B

--

-I-

C

--

--

Column fractions: column A unadsorbed : A

--

--

B

--

+

column B unadsorbed : A

+

--

adsorbed, then eluted : column A A

4-

-

--

--

column B A

--

B

--

-+

I MGG ( A o r B)

I-.=o I

B

I

Fig. 1. Schematic representation of the affinity-purification of serum blocking factors. Adapted from ref. 68. Methylcholanthrene-induced fibrosarcomas with different TSTA and the sera from mice carrying such tumors are indicated as "A" (1321 fibrosarcoma) or "B" (1408 fibrosarcoma). Normal sera from age and sex matched control mice are noted as "C". Such sera were fractionated on immunoadsorbent columns prepared by coupling either normal mouse immunoglobulin (NMGG) or tumor-immune-antibodies from sera of mice immune to either tumor A or tumor B ([MGG) to Sepharose 4B. The blocking activities measured in MCT assays are summarized for these sera as well as for the column fractions generated from them. "Specific blocking activity" refers to the blocking activity of sera or fractions when tested with target tumor cells and effector lymphocytes with the same tumor specificity as the indicated type. For example, when looking for type A blocking, fractions, A, B, and C were tested for ability to block the killing of type A tumor cells by lymphocytes immune to A as compared to control lymphocytes (immune to B, non-immune, normal, or both). The eluted fractions with specific blocking activity indicate affinity-purified fractions which were further purified by Sephadex G-200 chromatography and which were characterized after radiolabelling by SDS-polyacrylamide gel electrophoresis and peptide mapping of tryptic digests. f r o m the t u m o r cells used for the a d s o r p t i o n [43]. T w o different affinity-purified S B F were a d s o r b e d with either the a p p r o p r i a t e o r i n a p p r o p r i a t e t u m o r cells and subseq u e n t l y tested for b l o c k i n g activity. T h e b l o c k i n g activity o f c o l u m n eluates, consisting p r i m a r i l y o f the 56 000 MR c o m p o n e n t , c o u l d be specifically r e m o v e d by a d s o r p t i o n with the a p p r o p r i a t e cells [68]. The b l o c k i n g activity o f whole sera could be similarly r e m o v e d , in a g r e e m e n t with previous findings. T h r e e possible e x p l a n a t i o n s o f this o b s e r v a t i o n s h o u l d be considered. First, the b l o c k i n g eluates m a y c o n t a i n f r a g m e n t s o f i m m u n o g l o b u l i n enabling t h e m to b i n d to the t u m o r cells via the antigen c o m b i n i n g sites o f the i m m u n o g l o b u l i n molecules. Studies on further purified b l o c k i n g eluates should exclude or s u p p o r t this view. Second, S B F m a y be t u m o r antigens with the interesting p r o p e r t y o f

129 combining better with cells having the same antigens than with cells having different ones. Although it is considered utterly unlikely that tumor antigens should have this property, experiments are in progress to test for it, studying antigens released from tumor cells in culture. Third, the 56 000 MR SBF may be an immunosuppressive molecule, different from both tumor antigens and antibodies. The experiments which have now been summarized indicate that sera from tumor-bearing mice contain a factor which is both necessary and sufficient for their blocking activity, and which is a glycoprotein with an apparent MR of 56 000. In order to reconcile these observations with the evidence for antigen • antibody complexes with blocking properties in similar sera [43], we postulate that the 56 000 MR factor may exist in the form of a complex with immunoglobulin molecules, or that it may be formed by host ceils (e.g. T lymphocytes) stimulated by tumor-associated immune complexes. One possibility is that the 56 000 MR component represents free tumor antigen and thus is similar to antigens with blocking properties which have been isolated directly from tumors [37-39, 45-47]. This is probably the simplest interpretation of the data. If further work shows it to be correct, it will then be crucial to find out whether there are molecular differences between antigens which act as SBF and antigens which, instead, evoke tumor rejection. Alternatively, the mode of presentation of the antigen to the host (e.g. in the form of circulating molecules as compared to cell-bound ones) may be responsible for the difference. The present data can also be interpreted as evidence for a host cell derived molecule, e.g. formed by suppressor T cells in response to the appropriate tumor antigen. The specific adsorption of the factor to tumor cells having the "right" antigens [68] would support this view. This interpretation can also be reconciled with the failure to detect antibodies to TSTA in sera of mice with transplanted, methylcholanthrene induced sarcomas. The "unblocking" antibodies used for purification of the 56 000 MR factor would then be antibodies to an immunosuppressive substance formed during the initial phase of tumor growth in those immunized mice from which the unblocking antibodies were derived. It is interesting that Greene et al. have isolated a factor from tumor-bearers' T lymphocytes which is immunosuppressive in vivo, has a size smaller than immunoglobulins, and can be removed by adsorption with tumor cells having the appropriate antigens [71]. There are, however, differences between that factor and the one demonstrated by our group. The former is obtained by breaking up T cells and is not detected as circulating SBF. It also does not cross-react with immunoglobulins, while "our" factor is at least partially retained by immunoadsorbents prepared from goat anti-sera to mouse IgG [70]. It remains an open question as to whether the two factors are related, and if an immunoglobulin (-like) component is added as a cell-bound factor becomes soluble. Before concluding this section we went to emphasize that the 56 000 MR SBF is not likely to be the only SBF responsible for specific blocking serum effects in tumor systems. As already pointed out, there is ample evidence that tumor anti-

130 gens, and antigen • antibody complexes, can be detected in the circulation of tumorbearing hosts, and that they can block the immune response to tumor antigens, in vitro. One question which needs to be answered by further studies is, then, to what extent free antigens and/or complexes block directly and to what extent they block by stimulating the formation of other SBF (like T cell derived immunosuppressive molecules), and which SBF are the most important ones in various systems, with respect to their usefulness for monitoring individuals with tumor as well as with respect to providing an escape mechanism for tumors from immunological control.

IV. WHERE ARE THE BLOCKING FACTORS PRODUCED? Knowledge about where and how SBF are produced is crucial for understanding their role in regulating tumor growth and for knowing how to manipulate their formation. To what extent are they tumor components (antigens), and to what extent does the host play a major role in their synthesis (as for antibodies or T cell derived immunoregulatory molecules) and/or in their release from tumor cells? In this section, we will discuss data from mouse tumor experiments, suggesting that host immune responses are important for the formation of SBF. These data suggest that antibodies can facilitate the release of tumor antigens from the cell surface and they also indicate that at least some SBF are partially or completely host derived immunoregulatory products. Thompson et al. [47] were among the first to suggest that an immune response to tumor cell surface antigens facilitated the release of such antigens from cells. Tumors were transplanted to rats which had been sublethally X-irradiated. Sera from such rats were found to have a much lower blocking activity than did sera from tumor-bearing controls, whose immunological reactivity had not been depressed by radiation. It remains an open question whether the effect of X-irradiation was to depress humoral antibody formation by B lymphocytes or, e.g. to depress some mechanism involving suppressor T cells. Experimental support for the view that humoral antibodies to tumor antigens may facilitate the formation of SBF came from Hayami et al. These authors showed that if antibodies to antigens c o m m o n to Rous sarcomas were added to cultures of Rous sarcoma cells, SBF were released into the culture medium [72]. They further demonstrated that tumor-bearing Japanese quails which had been bursectomized and, therefore, could not form humoral antibodies, had lower amounts of SBF in serum than did controls. These findings remind us of the demonstration by Amos et al. [73] and by Klein [74] that factors facilitating the growth of an allogeneic tumor in vivo can be formed upon contact between tumor cells and "enhancing" antibodies to their H-2 antigens. The strongest evidence that the immune response plays a role in the formation of SBF has probably come from studies by Nelson et al. on cultivated spleen cells

131 from tumor-bearing mice [75-78]. This evidence has implicated T lymphocytes in playing a crucial role in SBF production. Nelson et al. studied cell cultures prepared from the spleens of BALB/c mice carrying chemically induced sarcomas. They showed that such cultures produced blocking factors which could abrogate T cell mediated killing of tumor cells in vitro, and which had a high degree of specificity for the tumor borne by the spleen donors. The SBF were detected in the culture supernatants after a 24-72 h incubation in vitro [75], and could be still demonstrated following 3 weeks culturing of the spleen cells. They were produced de novo and not just released by the spleen cells, since the addition of cycloheximide to the cultures reversibly inhibited protein synthesis as well as the production of SBF [76]. When tritiated leucine was added to the culture medium, the label was incorporated into molecules which were retained by antiimmunoglobulin immunoadsorbents [76]. All detectable blocking activity of the culture supernatants could be recovered in low pH eluates prepared from such immunoadsorbents. Cultures prepared from the spleens of mice whose tumors had been removed did not produce SBF [75]. The failure to do so was seen within 5-8 days of tumor removal. Cultures from spleens of both tumor-bearing and tumor excised mice did, however, make tumor specific lymphocyte dependent antibodies [77]. These antibodies were detected in much higher titers in the culture supernatants than were SBF. These findings indicate that continuous presence of tumor antigen is needed for SBF production, while it is not needed for continued production of lymphocyte dependent antibodies. Lymphocytes carrying the Thy-1 marker appeared to be responsible for production of the SBF in this system, since antibodies to Thy-1 (in the presence of complement) could inhibit SBF production [78]. This production was restored by adding Thy-1 positive cells from mice carrying the same tumor. Somewhat surprisingly, it was found that Thy-1 positive cells from the spleens of untreated mice could also produce the appropriate SBF as long as they were incubated in the presence of anti Thy-1 treated spleen cells from tumor-bearing mice (which most likely provided the needed antigen stimulus). Thus an uncommitted T cell population may quickly acquire the ability to make SBF, in the presence of the proper antigens. It will be interesting to find out whether, in vivo, T cells from normal spleen, given together with appropriate tumor antigen, would enhance tumor growth, and whether the enhancement seen would be approximately as large as that obtained if the "normal" spleen cells are replaced by spleen cells from mice carrying the same tumor; these will be the expected results, if the in vivo data reflect the in vitro observations. Removal of immunoglobulin-carrying B cells, of plasma cells, or of macrophages, did not interfere with blocking factor production by the spleen cultures [78]. It did, on the other hand, eliminate the ability of spleen cultures from tumor-bearing or tumor excised mice to form lymphocyte dependent antibodies. Such antibodies could still be formed, in high titers, by cultures whose T cells had been removed. Thus production of the immunoglobulin-like molecules responsible for the blocking

132 activity of culture supernatants appeared to be different from that of antibodies responsible for antibody dependent cell-mediated cytotoxicity. The simplest interpretation of these findings is that the blocking molecules were, at least partially produced by the T cells themselves. If they were formed by B cells as a T dependent (or independent) event, removal of B cells and plasma cells should have abolished SBF production, as it abolished production of the lymphocyte dependent antibodies. In agreement with these data, it has recently been demonstrated that spleen cells from tumor-bearing "nude" (nu/nu) mice do not form SBF in vitro, independently of whether the SBF are tested at the effector or the target cell level [79]. The mechanisms leading to the SBF production by tumor-bearer spleen cultures are poorly known. The observed facts are consistent with the following scheme, which is speculative and offered only to stimulate discussion. Some cells within spleen cultures from tumor-bearing mice contain tumor specific antigens. These antigens interact with T lymphocytes. As a result of this interaction, molecules are produced which we retained on anti-immunoglobulin columns and which block in microcytoxicity test assays, either alone, as T suppressor molecules, or after combination with tumor antigen. In the latter case, the SBF may be complexes between tumor antigens and immunoglobulin-like molecules. Such complexes may be produced by the shedding of T cell surface receptors to which tumor antigens have bound. They may also result from the release of tumor antigen from some non-T cell, either before or after the antigen has combined with T cell derived immunoglobulin-like molecules. The fact that removal of most macrophages, null cells and immunoglobulin-carrying B cells from spleen cultures does not abolish the ability of such cultures to form SBF, indicates that the antigen most likely interacts directly with the T cells. It remains unclear whether the immunoglobulin-like part of the SBF is derived from T cells to which the tumor antigen has originally bound, or from T cells to which the antigen has not bound. The latter possibility is supported by the observation that non-immune T cells will restore production of SBF by T depleted spleen cultures.

V. SPECIFIC BLOCKING FACTORS AND SUPPRESSOR CELLS The Thy-1 positive cells responsible for the production of SBF by spleen cultures may be operationally defined as suppressor T cells, since they are T cells making a product which suppresses a specific immunological function (target cell killing by immune lymphocytes). Treves et al. have further demonstrated that T cells from the spleens of tumor-bearing mice can enhance tumor growth in vivo, and they have attributed this to a suppressor cell activity [80]. However, it is unclear how any of these cells compare to suppressor cells seen in other systems [81, 82]. In most of these systems, the major role of suppressor cells appears to be to suppress an earlier event in the immune response than that leading to the killing of target cells by immune

133 lymphocytes. That does not exclude, however, that T cells involved in SBF production represent a (slightly different type of) suppressor cells. Perhaps the best evidence for suppressor cells in tumor-bearing animals has been presented by Fujimoto et al. [83, 84], who found that spleen and thymus cells from tumor-bearing mice could specifically enhance the growth of the respective tumor in vivo. Although there were no detectable circulating SBF, a tumor enhancing factor could be extracted from spleen and thymus cells [71]. A suppressor cell effect, which may involve SBF (Kall, unpublished observations), has been seen when lymphoid cells from tumor-bearing mice were co-cultivated with tumor cells of the appropriate antigenic specificity for a 5-6 day period. Such cells could abrogate the cytotoxic effect of tumor immune lymphoid cells when admixed with them and tested in microtoxicity test assays [85]. Part of this suppressor effect appeared to be specific. A nonspecific component was also seen, however, in agreement with observations in other systems [86]. Lymphoid cells from Japanese quails with growing Rous virus induced sarcomas were shown to inhibit the in vitro cytotoxicity of lymphoid cells from quails whose sarcomas had regressed [87]. It was not determined whether this suppression was specific. Likewise, lymphoid cells from mice with growing tumors could abrogate the response of admixed lymphoid cells from tumor immune mice as demonstrated by macrophage migration inhibition assays [33, 88]. This abrogation was specific for the given tumor antigens. Furthermore, lymphocytes from tumor-bearing mice have been shown to block the in vitro sensitization of lymphocytes to the specific antigens of the neoplasm [89]. To what extent the suppression seen in these systems is mediated by SBF is unclear; there is, however, evidence that supernatants from those lymphoid cells, which are suppressive in macrophage migration inhibition assays, contain SBF [33]. It is interesting in this context that experiments of Gershon et al. have indicated that suppressor T ceils may be activated by tumor cells inoculated together with specific antibodies [90], suggesting that (part of) the blocking effect of antigens and complexes may be mediated by suppressor cells. There may thus be at least two types of relationship between SBF and suppressor cells. First, some SBF (as discussed in preceding sections) may be T cell derived immunosuppressive substances. Second, antigen and antigen-antibody complexes may activate suppressor cells, whose suppressive function may, or may not, be due to the formation of humoral substances (detectable as another type of SBF). In addition to the specific suppressor effects discussed here, many examples of nonspecific suppression have been reported from tests with tumor-bearer lymphocytes (see e.g. refs. 86, 91), and ascribed to both macrophages and T cells. Such nonspecific suppression will not, however, be considered in this review of specific blocking factors and cells producing them.

134 vI. WHERE AND HOW DO SPECIFIC BLOCKING FACTORS ACT? Most evidence for the presence of circulating SBF has come from studies performed by the microtoxicity test technique. The blocking effect has been shown as an abrogation of the ability of immune lymphocytes to cause detachment of plated tumor target cells during a 30-36 h incubation period [11]. In some systems, it has been demonstrated that the effector cells whose cytotoxic effect was specifically inhibited were T lymphocytes [78, 92-94]. It is unclear to what extent SBF can specifically inhibit tumor cell killing by K cells acting together with lymphocyte dependent antibodies; by neutralizing antigen combining sites on the lymphocyte dependent antibodies, probably both free antigen and complexes would be able to block specifically. A nonspecific blocking of K cell killing by antigen, antibody complexes has been well established [94, 95]. As discussed above, blocking effects of tumor-bearer sera have also been reported in studies performed using macrophage migration inhibition assays [33] and leukocyte adherence inhibition tests [34]. In the latter case, circulating SBF have been shown to inhibit the in vitro production of a lymphokine whose activity is measured by its ability to cause the detachment of plated indicator cells (including macrophages) from glass surface. It has generally not been possible to block tumor cell destruction in short term cytotoxicity assays (such as 4-6 h SlCr release tests) by exposing them to SBF which are active in microcytotoxicity tests [96]. Only when the target cells are exposed to antibodies has blocking been detected in such tests, probably as an efferent form of enhancement by antibodies masking target cell antigens [96]. To what extent blocking by antibodies occurs in vivo is unclear. One should, however, not ignore the possibility that blocking at the target cell level, corresponding to a classical type of efferent enhancement [97], may be induced by circulating antitumor antibodies in vivo. Under certain conditions, such a mechanism may prevent tumor destruction even in a host with strong tumor immunity. The failure to detect blocking effects with tumor-bearer sera in short term cytotoxicity assays indicates that although such sera block in microcytotoxicity tests (and can suppress the ability of lymphocytes to form certain lymphokines), they do not interfere with the cytotoxic effect of overt killer cells. For this reason, we postulate that SBF from tumor-bearer sera have to exert their blocking effect on a (differentiation) process by which ("pre-killer") lymphocytes, belonging to the respective antigen specific clone, become active killer cells which are capable of lysing their targets. We further postulate that some SBF, in the form of immunosuppressor substances, act directly on the "pre-killer" lymphocytes, while SBF existing as antigens and complexes may block by first activating suppressor cells or, possibly, by directly interacting with the pre-killer cells. Although the killer cells would not be sensitive to blockade by SBF, their cytotoxicity would be abrogated by humoral antibodies, if such are present in sufficient amounts to mask the surface antigens of the target cells.

135 Studies by Biesecker et al. [98] are interesting in this context. They demonstrated that rats treated with enhancing antibodies and subsequently accepting an allogeneic kidney graft, had lymphocytes cytotoxic in vitro to cells from the graft donors, as well as blocking factors specifically inhibiting this effect. However, the cytotoxic lymphocyte effect, as well as the blocking serum activity, could be detected only with the microcytotoxicity technique. Sera from the transplanted rats did not block the ability of immune lymphocytes to kill in 4-6 h 5~Cr release tests, and lymphocytes from the same rats did not kill in the short term assay. We want to end this section by emphasizing the needs for analyses of defined SBF on lymphocyte populations whose surface markers and functions have been well characterized. Such studies are mandatory for understanding the ways by which antigen, complexes, and immunosuppressive T cell products can block.

vii. CORRELATION BETWEEN SPECIFIC BLOCKING FACTORS DETECTABLE IN VITRO AND TUMOR GROWTH IN VIVO A major question concerning the significance of SBF is whether an in vitro measurement of SBF shows any correlation with tumor growth in vivo. Since, as we have repeatedly pointed out, there is more than one type of SBF, the degree of correlation observed may depend on what type of SBF is being studied. Many reports have been published showing that blocking serum activity, as directed with microcytotoxicity assays, disappears quickly upon tumor removal (within 5-8 days) as well as upon tumor regression [11, 22]. This has been seen in mice with Moloney sarcomas [25], rabbits with Shope papillomas [18], mice and rats with chemically induced sarcomas [21], rats with polyoma virus induced sarcomas [99], and rats with chemically induced hepatomas [23]. Can the presence of blocking serum activity be prognostically useful, then, by predicting that an animal has a small growing tumor which would not otherwise be detected? Among the best experiments testing this were those reported by Steele et al., who studied chemically induced colonic carcinomas of rats [100]. Such tumors have common tumor associated antigens, against which immunity can be detected by testing lymphocytes from rats which either have a colonic carcinoma or from which such a carcinoma has been removed [101]. A group of W/Fu rats was given a dose of chemical carcinogen which could induce colon carcinomas in almost all the inoculated animals. Rats given the carcinogen were then tested for the presence of serum factors blocking lymphocyte mediated cytotoxicity to the common tumor antigens. They were also monitored by a radiological technique by which tumor as small as 1 mm in diameter could be detected. Steele et al. found that those rats whose sera blocked either had a small, colonic tumor or developed such a tumor shortly thereafter. Rats whose sera did not block did not have growing tumor and did not develop any within the next few weeks. It thus became clear that, in'this system, the in vitro demonstration of blocking serum activity was indicative of the presence of tumor in vivo [100].

136 For obvious reasons, studies on human cancer patients are more difficult to control than are those on experimental animals. This is particularly since "natural killer cells", occurring in both tumor patients and healthy individuals, are more difficult to control for in human than in animal experiments, so that assessment of tumor specific cell-mediated immune reactions becomes more difficult [102]. Nevertheless, attempts have been made to search in human patients for correlations between blocking serum activity in vitro and tumor growth in vivo. A few years ago, Hellstr6m et al. reported on tests of blocking serum activity in patients at a time point following surgery when the patients were considered to be clinically tumor free. Patients whose sera blocked relapsed more frequently within three to six months than did patients whose sera did not block [103, 104]. The correspondence between the presence of SBF and tumor relapse was not absolute, however. Our conclusion is that measurements of blocking serum activity in animals and human patients provide information which shows a fairly good correlation with the clinical course. More quantitative, as well as less cumbersome, techniques to measure SBF are, however, needed to extend this work, e.g. radioimmunoassays. Were such techniques available, it would be feasible to test, using a large patient sample, whether measurements of SBF in patient sera will have substantial clinical value, as compared to other ways of monitoring cancer patients. VIII. LOCAL VERSUS SYSTEMIC EFFECT OF SPECIFIC BLOCKING FACTORS Locally active blocking factors might explain the phenomenon of concomitant tumor immunity, i.e. the finding that established tumors grow progressively in animals which, at the same time, can often reject a small transplant from the same tumor [7-9]. One would expect at least some SBF (like antigens) to be present in the area of a growing tumor in higher titers than are present systemically. A high local concentration of antigens from tumor cells and serving as SBF (alone or after interacting with the host), may also explain why the ability of immunized hosts to reject cells of the appropriate neoplasm can be overcome by inoculating a relatively large number of tumor cells [3]. Ran and Witz were among the first to show that material capable of enhancing tumor growth in vivo can be eluted from tumors at low pH [105]. They also demonstrated that such material can have antibody activity against the given neoplasm. Bansal et al. performed similar experiments with polyoma virus induced sarcomas in rats [106]. They could elute material at pH 3.1 which specifically blocked cell-mediated cytotoxicity to polyoma tumors in vitro. This material was also capable of enhancing the growth of polyoma tumors in vivo. Similar elution of SBF (active in vitro) was demonstrated by exposing human tumor cells to a buffer with pH 3.1 [1071. As pointed out above, the major effect of SBF is likely to be on the lymphoid cells rather than on the tumor cells [43]. To be effective in turning off the local immune response to tumor antigens, SBF, therefore, must be present in a form capable

137 of interacting with lymphoid cells. We have recently performed experiments to test this. Tissue fluids were obtained from methylcholanthrene induced mouse sarcomas by pressing approximately 1 g tumors through stainless steel screens into 5 ml of culture medium, followed by centrifugation. Such fluids were found to block cellmediated cytotoxicity in vitro in a tumor specific way except at the highest concentrations of the fluids, when they were toxic to the lymphocytes and target cells [108]. In contrast to serum blocking activity, which generally titers out at around 1 : 40, the tumor fluids remained blocking after they had been diluted 1 : 1 000 to 1 : 10 000. The next step was to pass the blocking tumor fluid through immunoadsorbent columns prepared from "unblocking" anti-tumor antibodies, as described previously. Such columns were found to remove the blocking activity, which was subsequently recovered in the column eluates [108]. Thus material obtained directly from tumors (without preceding elution) was found to be similar to SBF derived from serum, as far as tested. In addition to acting at the tumor site, SBF may be present in the lymph nodes and spleens. Cells from lymph nodes and spleens of animals with large tumors often react less to antigens of the respective tumor than do lymphoid cells from animals where tumors are small. This is perhaps best seen in experiments performed in vivo, e.g. by mixing tumor cells and lymphocytes and studying tumor outgrowth [10, 109]. It is, however, also observed in vitro. Barski et al. were among the first to point this out, and introduced the term "eclipse" for the decrease of cell-mediated reactivity in animals with large tumors [16, 110, 111]. Hattler and Soenlehn subsequently reported that antigen • antibody complexes were bound to the surface of lymphocytes from tumor-bearing human patients and prevented these from proliferating upon exposure to tumor antigen [111 ]. Hellstr6m et al. have eluted blocking material from lymph nodes and spleens of tumor-bearing animals [113]. The experiments were conducted in a criss-cross pattern with tumors of different antigenic specificities. The lymphoid tissues were either exposed to a pH 3.1 glycine buffer or were treated with urea. Material so obtained could specifically block cell-mediated cytotoxicity to the respective tumor in vitro. The finding that blocking material can be eluted from lymphoid tissues is compatible with the demonstration that lymphoid cells from tumor-bearing animals often gain in cytotoxic acitivity [ll3, 114], as well as in the ability to react to tumor antigens in macrophage migration inhibition tests [88], if they are incubated in vitro for a 24 h period before testing. Blocking factors have, under such circumstances, been detected in the culture medium [33]. Quite likely, they may have been released from the surface of the lymphocyes. IX. ROLE OF SPECIFIC BLOCKING FACTORS IN VIVO The in vitro demonstration of SBF in sera from animals and human patients with tumors does not prove that such factors can facilitate tumor growth in vivo,

138 since an increase in serum SBF may occur as a result, rather than a cause, of tumor growth. Thus experiments are needed to test in vivo the role of SBF detected in vitro. There is, indeed, evidence that SBF can facilitate tumor growth in vivo. Suggestive data were first presented by Bansel et al. [115]. They showed that rats which had growing polyoma tumors and whose sera blocked in vitro, accepted small grafts of polyoma tumor cells. Animals whose polyoma tumors had been removed and whose sera did not block, needed larger inocula for tumor outgrowth. Although provocative, these findings can have alternative explanations, since the presence of a growing tumor may suppress tumor rejection without the necessary participation of SBF. More direct evidence that SBF play a role in vivo comes from experiments showing that material containing such factors, when inoculated into syngeneic animals, can enhance the growth of tumor cells having the respective antigenic specificity. Such enhancement was first shown by Pierce for sera from mice bearing Moloney virus induced sarcomas [117], and by Ran and Witz who studied material eluted from chemically induced mouse sarcomas [105]. Enhancement was subsequently demonstrated by Bansal et al. with polyoma virus induced rat sarcomas [106]. However, the specificity of the enhancement was not fully established in any of these systems. There have been several subsequent reports in which tumor-bearer sera were not found to enhance tumor growth in vivo [109, 118, 119]. One possible explanation for the lack of such enhancement is that the dose of blocking serum inoculated was too low to be effective, as would be the case if SBF were rapidly eliminated. Such elimination is, indeed, to be expected on the basis of the demonstration that blocking serum activity disappears within 5 days of removal of a growing tumor [21]. Small amounts of remaining immune complexes in the tumor-bearer sera may then "arm" K cells [120] and inhibit tumor growth rather than enhancing it. Data by Proctor et al. [119] may be explained this way. These authors found that sera from tumorbearing rats inhibitited (rather than enhanced) the formation of tung metastases when inoculated into syngeneic animals. Vaage reported analogous findings with sera from tumor-bearing mice [119]. Parenjpee et al. did, on the other hand, see a relatively weak, but statistically significant, enhancement with tumor-bearer sera when sttl~lying a different mouse tumor system [ 121 ]. Antigen preparations have more commonly enhanced tumor growth in vivo than have sera from tumor-bearing animals. Thus, Vaage could enhance the growth of transplanted syngeneic mouse tumors in both immunized and unimmunized mice by giving tumor antigen extracts [122]. This effect was specific for a given neoplasm. It may have represented a case of active immunological enhancement [97], or it may have been mediated by some other mechanism. Paranjpee and Boone [123] could depress delayed hypersensitivity reactions to tumor antigens, as measured by a footpad assay, by giving antigens from the respective tumors. The fact that free tumor antigen can enhance tumor growth may explain why animals immunized to the TSTA of syngeneic tumor cells are generally unable to

139 reject large inocula of the respective neoplasm, a phenomenon which has been regularly demonstrated [3]. It may also explain why effective tumor immunity is less well transferred to animals bearing an antigenically related neoplasm than to animals which are tumor free [124]. We have begun to investigate whether tumor enhancement by antigen is due to an interaction between the antigen and the effector cells or whether the antigen enhances in a more indirect way, first interaction with some host component, e.g. with suppressor cells or cells capable of forming the antibody part of antigen. antibody complexes [125]. The experiments are performed on several transplanted methylcholanthrene induced BALB/c sarcomas which have individually unique TSTA. As source of antigen, sarcoma cells are used which had been X-irradiated with 15 000 rads. It was first observed that tumor growth was significantly enhanced when untreated mice, or mice immunized to the TSTA of the given sarcoma, were inoculated with tumor cells which had been mixed with irradiated cells from the same tumor (as source of antigen). This effect was abolished if the mice were given 450 rads prior to challenge, suggesting that a radiosensitive cell population, different from that mediating tumor rejection, could interfere with the rejection process in the presence of an excess of tumor antigen of the "right" type. Experiments were made to test this assumption, using mice which had been pre-irradiated with 450 rads [125]. All mice got mixtures of non-irradiated tumor cells, and lymphocytes from mice immunized to the given syngeneic tumor (as source of immune cells). In addition, groups were included in which X-irradiated cells of the same or a different sarcoma (as source of antigen), and spleen cells from normal, untreated mice (as source of suppressor cells and/or cells producing antibodies), untreated or X-irradiated (450 rads), were added to the mixtures before they were inoculated to the mice. Tumor growth was then followed. Mice receiving only sarcoma cells (and the immune lymphocytes) developed few tumors (approximately 25 %), as did mice which also got X-irradiated spleen cells together with antigen from either the same or another tumor time. Mice getting tumor cells, immune lymphocytes and spleen cells which had not been pre-irradiated, together with anitigen from different sarcoma (or no antigen) also developed few tumors. If, however, the mixtures inoculated did include non-irradiated spleen cells, and antigen from the sarcoma, many tumors developed (approximately 85 ~ ) ; the difference between this group and the various controls was highly statistically significant. The findings indicate that the tumor enhancing effect of antigen in vivo is mediated by a radiosensitive host cell population, which is different from that constituting the effector cells. The nature of this cell population and its mechanisms (including the possible production of measurable levels of SBF) is presently being investigated. The most likely explanation, in view of previous findings, is that the antigen enhances by activating a suppressor cell population. As one way of testing the relevance, in vivo, of SBF detected in vitro, we have used "tumor fluids" prepared from chemically induced mouse sarcomas. Untreated

140 mice, and mice which had been previously immunized against the respective tumor, were inoculated with the tumor fluids and challenged subcutaneously with tumor cells [109]. The experiments were conducted in a criss-cross pattern using several transplanted methylcholanthrene induced BALB/c sarcomas of different antigenic specificities. In addition to using tumor fluids from the various neoplasms tested, culture medium was included as a further control. Tumor fluids from any of the tested neoplasms could enhance the growth of small tumor cell inocula, as compared to controls receiving only culture medium. In addition, a tumor specific effect was detected, i.e. tumor growth was enhanced more when the animal was inoculated with the "right" than with the "wrong" fluid. The enhancing effects were seen only when relatively large quantities of blocking material were inoculated. According to in vitro blocking assays, these quantities were at least ten-fold in excess of those amounts present in tumor-bearer sera tested in the past. The enhancing effects were more clear-cut when the tumor cells were mixed with the fluids before they were inoculated than when the tumor cells were given subcutaneously and the fluids intraperitoneally. This implies that a high SBF concentration is needed to enhance tumor growth, and that the effect of SBF is to a large extent local, rather than systemic, in keeping with the finding of concomitant tumor immunity. In preliminary experiments it has been possible to show tumor enhancement also by using purified eluates from tumor fluids passed through immunoadsorbent columns [108]. The same eluates were capable of blocking specific lymphocyte mediated cytotoxicity in vitro. These data support the view that SBF detected in vitro have an in vivo effect. If SBF are important for tumor growth in vivo, one would expect that procedures counteracting SBF formation and/or action would have an antitumor effect. If these procedures were effective, the anti-tumor effect observed may be expected to be remarkable. Surgical removal of a growing tumor [21] or its destruction by radiotherapy [22] or chemotherapy may provide the simplest means of abolishing SBF production by removing the source of tumor antigen. In addition, there is some evidence that antisera with "unblocking" activity are tumor suppressive in vivo. Thus Hellstr~Sm et al. found that about 30 ~ of mice with progressively growing Moloney sarcomas underwent regression after receiving sera from mice whose Moloney sarcomas had regressed [126]. Similarly, Bansal et al. demonstrated that sera which were unblocking in the rat polyoma tumor system could suppress tumor growth in vivo. This was seen both in rats in which a small dose of polyoma tumor cells had been transplanted [127] and in rats with primary polyoma tumors [128]. The effects observed may have been due to neutralization of tumor antigen, to inhibition of suppressor cells and/or their products, and/or to a cytotoxic or lymphocyte dependent antibody effect. This needs to be elucidated. Studies are also needed on both chemically induced and spontaneous tumors, since Moloney sarcomas and polyoma virus induced neoplasms are not good representatives of tumors in nature.

141 An important question is whether any of the anti-tumor effect of chemotherapeutic agents is due to inactivation of suppressor cells and/or interference with SBF formation and action, as sometimes suggested [129, 130]. One may speculate that certain chemotherapeutic agents (e.g. cyclophosphamide) could improve antitumor immunity in at least two ways. First, by killing tumor cells, they would decrease the antigen load, thereby decreasing SBF production. Second, by inhibiting suppressor lymphocytes and cells producing the antibody part of antigen-antibody complexes, they may interfere with the ability of antigens and complexes to thwart cell-mediated tumor destruction. Research in this area may lead to better ways of tumor therapy. X. WHY ARE SPECIFIC BLOCKING FACTORS IMPORTANT?. SBF (as defined in the Introduction to this review) are important for at least three reasons: They may be used for monitoring animals and patients with neoplasia, and establishing specificity of cell-mediated immune reactions to tumor antigens. They may facilitate tumor growth in vivo, so that procedures by which they may be counteracted can be therapeutically important. They may also provide models of certain types of immunoregulation. The roles of different SBF may, however, be different in different systems and under different conditions. Let us first discuss the use of SBF for patient monitoring. As we have pointed out in a preceding section, there is evidence that SBF are seen not only in individuals with overt tumor but also in those who are clinically tumor free but whose tumors subsequently recur within a 3-6 month period. Characterization of the SBF existing in various types of patients followed by the development of more precise methods for measuring SBF might make it possible to monitor subjects at risk and obtain information useful for the clinician. The demonstration that immunoadsorbent purified SBF from sera of tumor-bearing mice will bind to antibodies of the right specificity may act as a starting point towards developing such methods, in the form of radioimmunoassays, to replace the less quantitative techniques presently available. The presence of SBF may also be used for establishing the specificity of cellmediated immune reactions to tumor antigens. Reactions which can be blocked by SBF are more likely to be specific than those which cannot [1], and one would not expect SBF to be able to inhibit target cell destruction by "natural killer cells" although this needs to be investigated. This may be potentially useful since many of the cell-mediated immune reactions detected against tumor antigens are hampered by lack of specificity [102], due to a variety of circumstances. Second, if SBF play a major role in vivo for the escape of tumors from immunological control, as we believe, approaches by which SBF formation can be selectively depressed or by which efficient immune responses can be induced in their presence, would be likely to be therapeutically useful. In addition to interfering with cell-mediated immune responses to tumor antigens, SBF circulating as antigen • antibody complexes, may have pathological

142 effects. They may have the ability to cause tissue injury by binding complement, e.g., when localized in the kidneys [51-53, 60], thus causing certain autoimmune reactions. Antigen • antibody complexes may also be able to nonspecifically inhibit various immune functions, e.g. by interfering with macrophage activity or by being anti-complementary. Third, study of SBF in tumor systems may have broad implications for our understanding of immunoregulation in general. To stimulate discussion, we are presenting three tentative models of the formation of SBF as the result of interactions between tumor cell antigens and host immune cells. The three models are presented in Fig. 2. Host response

SBF

Tumor

ag

ab

1

•

~,

o ° . ° . ° . . , ,

-p. ag

-] o H . ° t T cell

2a

3 t IZ >-t ab 2b

Fig. 2. Mechanisms of SBF production (see text for details). In all three models, the initial event is the expression of tumor-specific antigens in the growing tumor. Corresponding immunologically specific components are contributed by either T or B cells. As outlined in the models, many interacting components in the immune response to tumor are potentially immunoregulatory. Furthermore, as pointed out repeatedly in this article, several types of SBF probably exist and their mode of action may differ between systems. Thus, more than one of the three models may ultimately be experimentally supported as a "correct" one. In part I we depict free tumor antigens and tumor-specific antibodies as soluble components, along with their corresponding complexes. As discussed above, there is ample evidence for the existence of such complexes, as well as of free antigen, in tumor-bearer sera, although different systems may differ from each other in this respect. Presumably, the antigens are released in three ways: spontaneously by the tumor, upon stimulation by antibody, and when dead cells in the tumor mass lyse. There is evidence that antigens and immune complexes can block T cell killing [95], although it is unclear whether they block the effector cells directly or act via

143 suppressor cells, the effect of which may or may not be mediated by humoral factors. Furthermore, immune complexes may specifically and nonspecifically block K cell killing [95], and antigen would be expected to block K cell killing by neutralizing lymphocyte dependent antibodies. Part 2a presents a model that parallels part 1 but which incorporates the increasing evidence for a T cell requirement for production of some SBF. In this model, the signal leading to SBF production is, as before, tumor antigen (alone or after combination with antibody in the form of a complex) but this signal is received by host T cells, which subsequently form the effective blocking factor, which is postulated to interact with the (pre-killer) effector cells. Thus, instead of blocking effector cells directly, the antigen blocks by triggering a suppressor cell function. The antigen (or complex) may be bound to the surface of T cells or of some other lymphoid cells, or it may be harbored internally. In the former case, it should be possible to demonstrate its presence at the surface of lymphoid cells. Evidence discussed above has identified the host component involved in SBF synthesis as a Thy-1 positive cell present in the spleen. Whether this cell is analogous to suppressor T cells in other systems will remain unknown until further analyses of its surface markers have been made. According to this model, the T cells release upon stimulation (by antigens and/or complexes) an antigen-specific molecule, perhaps a receptor structure. There is evidence for the existence of such molecules with suppressive properties in vitro, both from tumor systems [71] and from systems in which immune responses have been studied to defined antigens [131]. Although most T cell factors reported have been tightly associated with the intact cells and not easily released, the model postulates that similar molecules can be released by antigen stimulation in a soluble form and account for the properties of SBF of the type seen in mice with chemically induced sarcomas. The immunosuppressive molecules described in the literature [71,131], as well as the SBF isolated from sera of mice with chemically induced sarcomas, are smaller than immunoglobulins but still retain antigen binding properties. Whether a T cell suppressor molecule would act alone or as a complex with tumor antigen is not known. An element of antigen-specificity is present in either case, contributed by either the host or the tumor. As discussed in a preceding section, one possibility is that some SBF are formed by the shedding of T cell receptor molecules to which the specific tumor antigens have bound. The fact that two independently isolated SBF with different immunological specificities showed a high degree of molecular similarity in peptide mapping experiments is of extreme interest, since it points towards the existence of a group of related immunosuppressive molecules which can act as SBF. The model depicted in part 2b is an extension of model 2a, but is, at this time, even more speculative. Since reactive Y ceils express antigen-specific receptors, the possibility exists that the host can respond to these receptors by the formation of antibodies against their idiotypes, and that such antibodies are immunosuppressive. While there is relatively little direct evidence for model 2b [132], in allogeneic immune

144 responses in the rat, non-responsiveness can, indeed, be induced with anti-idiotypic antibodies [133]. The 56 000MR SBF described in a preceding section could not be an anti-idiotypic antibody, while the "unblocking" serum used for its isolation possibly could. It is very likely that the nature of SBF differs between tumor systems and at various stages in tumor development. With the definition of SBF we have given, circulating tumor antigens and antigen • antibody complexes would qualify as SBF, independently of whether their action is directly on the effector cells or via a mechanism involving suppressor cells. Measurements of circulating such antigens and complexes may serve as clinical predictors of tumor recurrence, as soon as reliable serologic assays become available. Other forms of SBF, including immunoregulatory T cell products, formed by the host in response to antigens and/or complexes, may play the more important roles in vivo. Their formation may be more amenable to therapeutic manipulation than the release of antigens from tumor cells. The relative role of complexes, as compared to antigen, in activating suppressor mechanisms and the relative abilities of antigens and complexes to directly block effector cells needs to be elucidated; until experiments have been carried out on well characterized cell populations, we do not know what type of molecule is actually effecting what T cell population. The in vivo experiments cited in a previous section do, however, suggest that tumor enhancement is not the outcome of direct interaction between antigen and effector cells but involves an additional, radiosentive (suppressor?) cell population. If these observations will be confirmed by further work, they will lend support to the model outlined under 2b, as far as indicating that a suppressor cell mechanism is involved. To what extent such suppressor cells then form measurable amounts of soluble products in vivo with specific blocking activity needs to be investigated. XI. CONCLUSIONS Sera from animals and human patients contain various factors which can specifically block (inhibit) lymphocyte mediated cytotoxicity to tumor cells in vitro, as assayed by a 30-40 h microcytotoxicity test. These specific blocking factors (SBF) are of several types, including free tumor antigen, antigen ° antibody complexes and, perhaps, specific immunosuppressive molecules formed upon contact between suppressor T lymphocytes and tumor antigen (or complexes); different such factors may be involved to a different extent in different systems. Lymphocytes which are capable of immediately killing their targets as assayed by short term (4-6 h) tests, are, on the other hand, not affected by SBF in tumor-bearer sera; the action of such lymphocytes can, however, be blocked by antibodies "masking" the antigens of the target cells. This has led us to postulate that SBF act upon a differentiation process by which immune but non-cytotoxic ("pre-killer") T lymphocytes become capable of specificially destroying tumor cells carrying the appropriate antigens. Transplantation experiments in mice indicate that an effect of tumor antigen is

145 more likely the result of interaction between the antigen and a radio-sensitive spleen cell population, than a direct interaction between the antigen and the effector cells. A factor necessary and sufficient for the blocking activity of sera from mice with chemically induced sarcomas has been shown to be a glycoprotein with an approximate MR of 56 000, which can be retained by appropriate immunoadsorbent columns and which can be removed by adsorption to tumor cells having the appropriate antigens. This molecule could possibly be a tumor antigen but is (in view of its antigen binding abilities) more likely a specific, immunosuppressive molecule, which is formed by lymphocytes upon contact with free tumor antigen and/or antigen ° antibody complexes. SBF are important for at least three reasons. Their presence in serum correlates with tumor growth in vivo, so that measurements of SBF may by useful from the prognostic point of view, particularly if defined types of SBF (antigens, versus complexes, versus immunosuppressive T cell products) are measured. Manipulation of SBF production and action, using immunological techniques, perhaps in combination with chemotherapeutic agents capable of modifying the immune response, may point towards new ways for tumor therapy (and prevention). Studies on SBF may also provide models of immunoregulation in general. Our knowledge about the roles of various SBF and about their mode of action is far from complete. Analyses of the molecular nature of SBF are mandatory for further progress in this area. ACKNOWLEDGEMENTS The work of the authors in this area was supported by grants CA 19148 and CA 19149 from the National Institutes of Health, grant I M 43H from the American Cancer Society, by contract CP 53570 within the Virus Cancer Program of the National Cancer Institute, by contract NO1 CB 64018 from the National Cancer Institute and by SO7 RRO5220 from the National Institutes of Health. The third author has been supported as a Medical Scientist Trainee ( G M 02103), National Institutes of Health. REFERENCES 10ettgen, H. F. and Hellstr~Sm,K. E. (1972) in Cancer Medicine, (Frei, E. and Holland, J. eds.), pp. 951-990, Lea and Febiger, Philadelphia, Pennsylvania 2 Old, L. J. and Boyse, E. (1964) Annu. Rev. Med. 15, 167-186 3 Sj6gren, H. O. (1965) Progr. Exp. Res. 6, 289-322 4 Hellstr6m, K. E. and Hellstr6m, I. (1969) Adv. in Cancer Res. 12, 167-223 5 Alexander, P. (1972) Nature 235, 137-140 6 Baldwin, R. W. (1973) Adv. Cancer Res. 18, 1-75 7 Southam, C. M. (1967) Progr. Exp. Tumor Res. 9, 1-39 8 Gershon, R. K., Carter, R. L. and Kondo, K. (1967) Nature 213, 674-676 9 Lausch, R. M. and Rapp, F. (1971) Int. J. Cancer 7, 322-330 10 Mikulska, Z. B., Smith, C. and Alexander, P. (1966) J. Natl. Cancer Inst. 36, 29-35 11 Hellstr/Sm, K. E. and Hellstr/Sm, I. (1974) Adv. in Immunol. 18, 209-277

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