Tumor-Bound Immunoglobulins:in SituExpressions Of Humoral Immunity1

TU MOR-BOUND IMMUNOGLOBULINS: IN SlTU EXPRESSIONS OF HUMORAL IMMUNITY' Isaac P. Witz Department of Microbiology, The Dr. George S. Wise Center for Life Sciences. Tel Aviv University, Tel Aviv. Israel

I. Introduction

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B. IgG Subclass ... D. The Dynamic State of TAIg

C. Direct Evidence

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B. Available Information

95 97 104 104 105 107 108 115 117 119 119 122

124 127 ..................... 133 133 ............... 137 ............... 139 141

I. Introduction

The information explosion in tumor immunology witnessed by us during the last decade is illustrated by the large number of recent reviews on various aspects of this research field (Baldwin, 1973; Hellstrom and Hellstrom, 1974; Cerottini and Brunner, 1974; Nelson, 1974; Herberman, 1974, 1976; Coggin and Anderson, 1974; Klein, 1975; Stutman, 1975; Prehn, 1976). One of the central issues in this area, namely, the nature of the immune interrelationship between the The research of the author is supported by a grant from the United States-Israel Binational Science Foundation (B.S.F.), Jerusalem, Israel, and by Public Health Service contract No. 1 CB43858 from the Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

95

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host and the tumor he bears, remains, however, largely as terra incognita. An accurate and precise evaluation of tumor-host relations is an essential prerequisite for a rational approach to cancer therapy, in particular immunotherapy, and to a correct assessment of prognosis. It was hoped that with the aid of sophisticated and accurate in vitro assays monitoring antitumor immune reactivity a reliable correlation with in uiuo events will be obtained, thus reflecting the clinical status of the tumor bearer. It seems, however, that some of these hopes were not always fulfilled (Takasugi et al., 1973; Herberman, 1974; Heppner et al., 1975; Bean et al., 1975). At present the host-tumor relationship is assessed mainly by the capacity of immune components, such as lymphocytes, macrophages, or antibodies, to react in vitro against tumor cells. In the majority of the published studies, the immune components originated at sites distant from the tumor: blood of cancer patients or lymphoid organs and blood of laboratory animals. There is no reason to assume that expression of immunity is equal in all sites of the body. This argument holds true also for the tumor site. Certain immunocytes or antibodies expressing efficient antitumor reactivity under laboratory conditions may be unable to reach the tumor site owing to absence of vascularization, to various obstacles, or to lack of the correct signals directing their homing to the site. Other components may encounter no difficulty in reaching the tumor site, or may even be attracted to it. Such a hypothetical imbalance in situ may bring about a completely different outcome in vivo than that suggested by assays utilizing effectors originating from sites distant to the tumor. Furthermore, even if effective immune components reach the site of the tumor in sufficient quantities, there is no guarantee that they can fulfill their function. A complete or a partial inactivation of these components at the tumor site or in the draining lymph node is not unlikely. Inactivation may be immunologically specific by tumor antigen, by antitumor antibodies, or by complexes of the two. Alternatively, or in addition, nonspecific inactivation of immune components may occur. For example, molecules of tumor origin with the capacity to cause a generalized immune suppression (Wonget al., 1974; Fauve et al., 1974; Kamo et al., 1975; Pikovsky et al., 1975) may concentrate at the tumor site and bring about a partial or complete paralysis of certain immune functions. It becomes thus clear that the microenvironment of the tumor site and its effect on the immune response should be studied. The importance of evaluating in situ tumor immunity was stressed by contributions of Black et al. (1954, 1956, 1971, 1975; Black, 1972)

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97

and of others (Hamlin, 1968; Cochran, 1969; Lauder and Aheme, 1972; Hanna et al., 1972; Sarma, 1972; Pomerance, 1972; von Grundmann, 1974) concerning the immune-histology of the malignant area in relation to prognosis or treatment. These studies documented immunocyte infiltration into malignant tumors and concluded that such infiltration is clearly related to prognosis. Other studies, emphasizing, from a different point of view, the importance of analyzing local immunity at the tumor site, demonstrated rather conclusively an anergy of draining lymph nodes compared to seemingly normal functions of other nodes or of peripheral lymphocytes (Alexander and Hall, 1970; Vanky and Stjernsward, 1971; Vanky et al., 1973a; Nind et al., 1973; Flannery et al., 1973a). Recent studies dealing with the presence, characterization, and functions of host immunocytes are especially noteworthy. Thus, T cells (Jondal et al., 1975; Haskill et al., 197513; Edelson et al., 1975), macrophages (Evans, 1972; Van Loveren and Den Otter, 1974; Bartholomaeus et al., 1974; Eccles and Alexander 1974a,b; Haskill et al., 1975a; Wood and Gillespie, 1975; Gauci and Alexander, 1975), histiocytes (Edelson et al., 1975), and lymphocytes or mononuclear cells with Fc receptors (Kerbel et al., 1975; Roubin et al., 1975; Haskill et al., 197513; Tracey et al., 1975; Wood et al., 1975; Muchmore et al., 1975; Braslawsky et al., 1976a,b) have been identified among the host cells residing in malignant tissues. Some of these studies demonstrated that antitumor effector functions were mediated by the tumorderived host cells. This review summarizes the available data on the presence, properties, and functions of humoral immune components, mainly immunoglobulins, at the site of malignant tumors. Most of the studies reviewed deal with nonlymphoid malignancies. Interpretation of data concerned with presence of Ig in leukemia or lymphoma would obviously be very difficult. Reviews on this subject were published previously (Witz, 1971, 1973). II. Presence of Immunoglobulins in Tumors

The following methods were used to detect tumor-associated immunoglobulins (TAIg). 1. Treatment of tumor fragments, single cell suspensions or membrane-rich fractions of tumor tissue with low pH buffers or with salt solutions of high molarity. The resulting eluates are usually analyzed by immunodiffusion with anti-Ig reagents. 2. Direct membrane immunofluorescence of tumor cells using

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fluorescein-conjugated anti-Ig reagents. This technique is relatively insensitive, and most of the investigators who were unable to detect TAIg (see below) used it. 3. Radioimmunofixation using radioiodine-labeled anti-Ig reagents. 4. Radioimmunofixation using radioiodinated protein A from Staphylococcus aureus. This protein has binding activity to the Fc part of most mammalian IgG classes (Sjoquistet al., 1967; Kronvall et al., 1970a,b; Dorval et al., 1974) and can, therefore, serve as a probe in the demonstration of surface-fixed IgG molecules. 5. Mixed hemadsorption, using antibody-sensitized erythrocytes (Fagreus and Espmark, 1961). Tables I and I1 provide some details about the Ig content of some human and animal tumors, respectively. Summarized here are studies that were not reviewed previously (Witz, 1973). In addition to the papers cited in these tables, the following data become available to us. Sulitzeanu et al. (1976b), using direct immunofluorescence, demonstrated that cells in effusions from patients with malignant diseases exhibited membrane-bound Ig. Thus, all 7 tested samples of ovarian carcinoma cells, 1 of 4 breast carcinoma, and 4 of 15 samples of other tumors were stained with the anti-Ig reagent. Von Kleist (1976) reported that low pH eluates of membrane-rich fractions derived from carcinoma of human colon contained Ig. The tumor cells themselves, however, stained weakly with fluoresceinconjugated anti-Ig antibodies. Taken together, all these studies suggest that Ig can be detected in tumors if carefully sought and if sensitive enough methods are used. However, some investigators were unable to demonstrate Ig in tumors. For instance, Fenyo et al. (1973), using a fluorescein-conjugated anti-Ig reagent, did not detect Ig on Moloney lymphoma YAC cells. Witz et al. (1974a), on the other hand, detected TAIg in these tumors. Flannery et al. (197313) did not find, again by membrane immunofluorescence, any TAIg in a transplantable rat squamous cell carcinoma. Lewis et al. (1971) did not detect TAIg in human melanoma although other publications by some of these authors (Phillips and Lewis, 1971; Lewis et al., 1976) as well as by others (Table I) indicated the presence of Ig in these neoplasms. It should be noted that direct immunofluorescence, a relatively insensitive method, was used in these studies. Although the problem whether tissue-bound Ig is a cancerdistinctive phenomenon deserves serious consideration, this question

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99

was never systematically investigated. Various reports do, however, suggest that cells in normal tissues may indeed be associated with Ig (Witz et al., 1967; Witz and Ran, 1970; Roberts et al., 1973; Ablin et al., 1972; Sulitzeanu et al., 1976b), although in lower frequencies and with smaller amounts than cells in neoplastic tissues (Witz et al., 1967; Ran and Witz, 1970; Thunold et al., 1973; Sulitzeanu e t al., 197613). These findings are not surprising, although they may disturb, somewhat, those who search for unique features of cancer cells. It has been demonstrated that natural autoantibodies exist under apparently normal circumstances (Kunkel and Tan, 1964; Winchester et al., 1975; Sulitzeanu et al., 1976a). Voisin (1971) proposed that such antibodies fulfill an important physiological role as contributors to immunological self-integrity. It is, therefore, conceivable that autoantibodies could find their way to the corresponding autoantigen on the appropriate target cells in vivo. Whether the biological activity assigned to these autoantibodies depends on their homing to the corresponding target tissue is still an open question. Many of the animal tumor systems reviewed above were ascites tumors. So were some of the human cancers. It is appropriate to ask whether the free-living cell expresses some surface characteristics involved in attracting and binding of Ig that are not expressed on cells derived from solid tumors; or whether the peritoneal cavity provides conditions especially favorable for Ig-cell interaction. This question is enforced by the results of Isa and Sanders (1975) showing that the ascites form of a mouse teratoma was associated with Ig while solid tumors were not. Robins (1975) dealt with this problem in some detail. He compared the kinetics of antitumor antibody response of rats bearing the ascites and the solid forms of an hepatoma. The ascites cells were found to be coated with Ig. No attempt was made to search for TAIg in the solid tumors. While both cell types gave rise to circulating antibodies reaching peak titers 6 7 days after inoculation, the titer in the ascites variant-bearing rats was much higher than in those bearing the solid tumor. While circulating antibodies could be detected for long periods in the former rats, the level of antibodies dropped completely in the latter once palpable tumors developed. This could be interpreted as an absorption of antibody by the solid tumor mass. Injection of ascites cells into rats bearing the solid hepatoma induced antibody titers as high as in rats injected with ascites cells alone. These results indicated that ascites cells were more immunogenic than cells from solid tumors. The milieu of the peritoneal cavity per se probably did not contribute to the higher immunogenicity of the as-

Y

PRESENCE Type of tumor Melanoma and Hodgkin Acute myelogenous leukemia Various Keratoacanthoma

Method of detection Immunodiffusion of eluates Membrane immunofluorescence Immunodiffusion of eluates Immunofluorescence

TABLE I OF IMMUNOGLOBULIN

Ig-associated cells in tumor Unknown Unknown Unknown Unknown

Breast

Immunodiffusion of extracts

Unknown

Urinary bladder

Immunofluorescence

Host

Various

Immunodiffusion of eluates or radioimmunofixation Radioimmunoassay

Unknown

Immunodiffusion of eluate Immunodiffusion of eluates

Unknown

Various Primary prostatic tumor Melanoma

Unknown

Unknown

0 0

(Ig)

IN

HUMANCANCER Remarks"

Tumor cells originated from PHAtreated patients It is unknown whether cells are coated from the outside or synthesizing Ig

-

Ig correlated with mononuclear cell infiltration Ig levels correlated with plasma and round cell infiltration. Ig may have been produced locally Local production of Ig possible Presence of Ig positively correlated with high malignancy potential of tumor

-

Elution increased the antigenicity of the tumor tissue homogenate. Eluates contained antibodies directed against melanoma antigens

Reference Phillips and Lewis (1971) Gutterman et al. (1973) Thunold et al. (1973) Brown and Tan (1973) Roberts et al. (1973) Johansson and Ljungqvist (1974) Izsak et al. (1974) Jewel1 and Krishnan (1974) Guinan et al. (1974) Gupta and Morton (1975)

*

5

b ?

3 4

Various

Mixed hemadsorption

Various

Radioimmunofixationelntion assay

Ovarian carcinoma Breast

Membrane immunofluorescence Membrane immunofluorescence

Tumor

Membrane immunofluorescence Immunodiffusion of eluates Membrane immunofluorescence

Unknown

Various

Radioimmunoassay

Unknown

Breast

Immunofluorescence

Tumor and host

Melanoma Sarcoma Melanoma

"

Probably tumor Probably tumor

Host

Unknown Host

PHA, phytohemagglutinin; CML, cell-mediated lysis.

Irie et al. (1975) Presence of Ig inversely correlated with capacity to stimulate autologous lymphocytes

Vanky e t al. (1975) Dorsett et al. (1975)

Tumor cells not assayed for Ig. In 5 of 10 primary tumor masses studied, no Ig was detected on infiltrating lymphocytes Eluates and I g isolated from eluates abrogated CML in v i t r o Tumor cells from PHA-treated patients were heavily coated with Ig, however Presence of I g inversely related to expression of F c receptor activity in some of the tumors Synthesis of I g and other serum proteins by cells residing in the tumor

Richters and Kaspersky

(1975) Cornain et al. (1975) Romsdahl and Cox

(1975)

Lewis et al. (1976) Tonder et al. (1976) Hurlimann et al. (1976)

TABLE 11: PRESENCEOF IMMUNOGLOBULIN(Ig) Type of tumor Various ascites tumors Various ascites tumors

Species and strain Mouse, various strains Mouse, various strains

TA3/St, mammary carcinoma (ascites) 6 CBHED, lymphoma (ascites) C-1300, neuroblastoma (solid) 402Ax, teratoma (ascites) D23, heptoma (ascites)

Mouse, A/Sn

MC-D, 3-methylcholanthrene-induced sarcoma PW 13, Polyoma-virusinduced tumor Various ascites tumors

Guinea pig, strain 13

Mouse, C3H Mouse, AIJ Mouse, 129/J and other strains Rat, Wistar

Rat, W/Fu Mouse, various strains

Guinea pig, Line 10, hepatoma strain 2 (ascites) SEYF, a polyoma-virusMouse. AB.Y induced sarcoma (ascites) A-10, Adenocarcinoma Mouse, A/He (ascites) Guinea pig, McD, Methylcholanthrene-induced sarcoma strain 13

Method of detection

IN

ANIMAL TUMORS Ig-associated cells in tumor

Reference

Unknown

Witz et al. (1974a)

Unknown

Ran et al. (1974)

Tumor and host

Witz et al. (1974b)

Unknown

Prager et al. (1974)

Unknown

Terman et al. (1975)

Unknown

Isa and Sanders (1975)

Tumor (host?)

Robins (1975)

Unknown

Huang et al. (1975)

Unknown

Huang et al. (1975)

Tumor. Adherent cells or B cells not involved Unknown

Dorval et al. (1976a)

Tumor (host?)

Braslawsky et al. (1976~)

Radioimmunoassay

Unknown

Radioimmunoassay

Unknown

Tax and Manson (1976) Berczi et al. (1976)

Radioimmunofixation (antiglobulin) Radioimmunofixation (antiglobulin) and immunodihsion of eluates Membrane immunofluorescence (antiglobulin) Membrane immunofluorescence (antiglobulin) Immunodiffusion of eluates Membrane immunofluorescence (antiglobulin) Membrane immunofluorescence (antiglobulin) Radioimmunofixation (antiglobulin) Radioimmunofixation (antiglobulin) Radioimmunofixation (antiglobulin and protein A) Direct and indirect C1 fixation and transfer tests Radioimmunofixation (antiglobulin)

Segerling et al. (1976)

EXPRESSIONS OF HUMORAL IMMUNITY WITHIN TUMORS

103

cites hepatoma cells, since irradiated ascites cells or solid tumors implanted intraperitoneally did not express higher immunogenicity than solid subcutaneous tumors. Although it is probably easier for circulating antibody to reach ascites cells than cells lodging in solid tumors, and although the former may be more immunogenic, it should be remembered that most of the tumors in which TAIg was first detected (Witz et al., 1967; Ran and Witz, 1970) and a high proportion of the tumors listed above were solid tumors. The question whether or not Ig is the only plasma protein found in close association with tumors is a relevant one. Again, not much information is available on this point. It seems, however, that in addition to Ig molecules, other plasma proteins can be occasionally detected within tumors. We have found, for instance (Witz et al., 1964) that transferrin and hemopexin were among the constituents of extracts derived from a transplantable murine mammary carcinoma. Romsdahl and Cox (1975) also found transferrin and hemopexin in eluates from human sarcoma cells as well as other serum proteins, such as albumin. This protein was also detected in tumor eluates by Thunold et al. (1973). On the other hand, some of our other studies (Ran and Witz, 1970) indicated that immunoglobulins were the only serum proteins detected in low-pH eluates of murine sarcoma. This discrepancy can be explained by differences in the working habits of various laboratories; in some, washing of the tumor tissue may be better than in others. Different, less trivial, explanations are also possible. It was reported that the development of tumors is frequently accompanied by changes in the glycoprotein composition of the serum. Such glycoproteins interact with tumor cells and can therefore be detected on their surface (Apffel and Peters, 1969). The data summarized above establish that Ig is found in many, if not all, human or animal neoplasms. However, in some cases the amounts of TAIg may be below the detection threshold of certain assays, SO that more sensitive ones may be required for their detection. Three major sets of questions pose themselves. The first set concerns the identity of the cells in the tumor to which Ig molecules are associated. Are these tumor cells, or, alternatively, are these infiltrating host cells, such as Fc receptor-bearing lymphocytes, or macrophages? Are Ig-synthesizing B cells involved? It is conceivable that some or even all of these alternatives could coexist. The second set of questions concerns the nature of binding of TAIg to the tumor-derived cells. Is it possible to identify among TAIg molecules antitumor antibodies directed against tumor-associated

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antigens (TAA)? Are some of the TAIg molecules serologically unrelated to tumor antigens? In this case, are they bound to tumor cells, to host cells, or to both? Also, in this case, the possible alternatives are not mutually exclusive. The last, and probably the most important, set of questions concerns the biological role, if any, played by TAIg in tumor growth, propagation, and spread. It is important to emphasize that TAIg molecules could be involved in tumor-host relationship even though they may not be antitumor antibody and although the tumor-derived Igassociated cell may not ba a tumor cell. Below we will attempt to summarize the present state of knowledge concerning these questions. 111. Some Properties of Tumor-Associated Immunoglobulins (TAIg)

A. Ig CLASS IgG seems to be the most prominent Ig present in tumors, at least in those of animal origin. Thus, IgG, but apparently no other Ig, were detected in acid eluates of membrane-rich fractions derived from the following autochthonous tumors: acetaminofluorene-induced rat hepatomas (Witz et al., 1967), benzo [ a ]pyrene-induced mouse sarcomas (Witz and Ran, 1970; Ran and Witz, 1970), and spontaneous mammary carcinomas (Ran and Witz, 1970). We have recently carried out an immunochemical analysis of Ig classes and subclasses present in acid eluates from various transplantable ascites mouse tumors (I. P. Witz, unpublished). By the use of monospecific antisera it was seen that IgG was present in all eluates tested. IgM and IgA were in general present only in eluates of plasmacytomas synthesizing these immunoglobulins. Similar results were obtained by Terman et al. (1975) working with a murine neurobl astoma. In human neoplasms, the restriction to IgG is, in most cases, less evident. Thus, Thunold et al. (1973) detected IgG and in some cases also IgA in low-pH eluates of various human malignant tumors. Similar results were obtained by Romsdahl and Cox (1975). They could identify both IgG and IgA in eluates of sarcoma cells as well as many other serum proteins (see above). IgG and IgA were detected in all examined extracts of breast cancer whereas IgM was found in only a third of the tumors (Roberts et al., 1973).In this study, IgM was found in higher concentrations in malignant tissue than in benign tumors and in the noncancerous portion of

EXPRESSIONS OF HUMORAL IMMUNITY WITHIN TUMORS

105

the cancer-bearing breast. The opposite situation existed for IgG and IgA. Predominance of IgM was detected in biopsy material from primary tumors of the urinary bladder (Johansson and Ljungqvist, 1974). Out of eighteen specimens positive for Ig (50% of the specimens analyzed), twelve contained only IgM, three contained only IgG, one contained both IgM and IgG and two contained all three immunoglobulins. Since patients with bladder tumors show a high urinary excretion of IgM, it was interesting to note a highly significant correlation between the presence of IgM in the tumor and its excretion in the urine. Vanky et al. (1975) showed that, if a tumor specimen contained Ig, it contained in most cases both IgG and IgM. Similar results were obtained by Dorsett et al. (1975) with ovarian tumors and by Brown and Tan (1973) analyzing keratoacanthoma, a spontaneously resolving skin tumor. Among the TAIg detected in human cancer by Izsak et al. (1974), IgG was the predominant Ig present. However, IgM and IgA were detected in some of these tumors, but always in association with IgG (M. Ran I. P. Witz, E. Landes, H. J. Brenner, and F. Ch. Izsak, unpublished). In contrast to these results, a study on Ig eluted from a melanoma tumor [originating from a phytohemagglutinin (PHA)-treated patient] revealed the exclusive presence of IgG in the eluate (Phillips and Lewis, 1971). It should be kept in mind that the information provided in this section should not be regarded as complete because in most of the studies cited the analysis of TAIg was not complete. Thus, at best, only the major Ig classes were analyzed, and essentially no information is available as to whether or not the less prominent Ig classes such as IgE or IgD are present within tumors. B. IgG SUBCLASS A large amount of data is available on the biological role of IgG subclasses of murine alloantibodies in relation to rejection or enhancement of allografts or regarding various i n vitro activities of such antibodies. On the other hand, little is known about the function of IgG subclasses in tumor-specific systems with some exceptions. The work of Jose and Skvaril(l974) on the role of human IgG subclasses in the i n vitro blocking of cellular responses against tumors and the study of Pollack and Nelson (1975) on the activity of mouse IgG2 in the induction of antibody-dependent cellular cytotoxicity (ADCC) in a tumor-specific system are especially noteworthy.

106

ISAAC P. WIT2

The available information on the association of IgG subclasses with tumors is limited to very few mouse tumor systems. The results of Ran and Witz ( 1970) using semiquantitative immunodifision dilution assays indicated a preferential presence of IgG2 in spontaneous mammary tumors or primary and transplantable carcinogen-induced tumors as compared to IgG1. Part of their calculations were based on the finding that the levels of IgG2 in the serum of tumor-bearing mice were similar to those in normal mice. E. Eshel, T. Mekori, E. Robinson and I. P. Witz (unpublished), using the quantitative radial immunodiffusion assay of Mancini et al. (1965), found increased titers of IgG2 in the serum of fibrosarcoma-bearing mice at early phases of tumor growth. This finding raised the possibility that the presence of high levels of IgG2 within tumors may actually reflect the increased amounts of this particular Ig in the circulation. Work with three different murine plasmacytomas indicated that Ig molecules other than those produced by the tumor itself were present in eluates ofthese tumors. Thus, IgG2 was eluted from IgM-producing MOPC 104E tumors (Ran and Witz, 1970). Recent experiments in our laboratory confirmed this finding and demonstrated also that low pH eluates from a plasmacytoma producing IgG2a contained, in addition to this particular Ig subclass, also IgG2b. From another plasmacytoma producing Ig2b, both IgG2b as well as IgG2a molecules could be eluted. Recent studies on biological activities of mouse alloantibodies belonging to the IgGl and IgG2 subclasses evoked a renewed interest in the role played by these immunoglobulins in the growth of autochthonous and syngeneic tumors. Harris and Harris (1973) found that noncomplement fixing alloantibodies of the IgGl subclass competed with complement-fixing alloantibodies of the IgG2 subclass for alloantigenic determinants. Thus, depletion of IgGl antibodies from a given alloantiserum augmented considerably the cytotoxic titer of this antiserum. The alloantisera containing high titers of IgGl antibody caused prolonged retention of the appropriate skin grafts while the alloantisera containing low titers of IgG 1 caused accelerated rejection of such grafts. These authors found also that IgG2 alloantibody appeared early after immunization and its level remained constant during the immunization period. The titers of IgGl alloantibody, on the other hand, were relatively low in the early stages of the immunization and increased with further immunization. Thus, whereas early antisera caused accelerated rejections of skin allografts, late antisera caused prolonged retention. Jansen et al. (1975) presented evidence that IgG2 as well as IgGl alloantibodies caused enhancement of allogeneic skin

EXPRESSIONS OF HUMORAL IMMUNITY W I T H I N TUMORS

107

grafts, while only IgG2 alloantibodies brought about an hyperacute destruction of the graft. A possible explanation for these findings was provided by Duc et al. (1975), working on the role of IgGl and IgG2 in enhancement of tumor allografts. These authors found that complexing soluble alloantigen with the corresponding IgG2 alloantibody rendered this Ig enhancing, whereas noncomplexed it was essentially a nonenhancing antibody. Complexing of IgGl alloantibody with the soluble alloantigen preparation did not increase its enhancing activity which was rather pronounced beforehand. In addition, it was found that highly diluted IgG2 alloantibody became strongly enhancing. Diluting IgG 1alloantibody abolished its enhancing effect. These studies may add in solving the dispute concerning the identity of the murine IgG subclasses causing enhancement of tissue allografts. In view of these data, it becomes increasingly important to carry out similar studies on the involvement (if any) of antibody belonging to various IgG subclasses in the growth of syngeneic murine tumors. One should, for instance, compare the sequence of appearance of syngeneic antitumor antibody belonging to the various IgG2 subclasses with the sequence occurring in allogeneic combinations, attempting to define differences, if any, between the antibody response to grafts which are usually rejected and the response to grafts which are retained. In addition, antibody subclasses should be compared in the circulation and at the tumor site. To approach this and related problems, serologically defined syngeneic murine tumor systems are required. Few such systems are available (Ting and Herberman, 1974a,b; Witz et al., 1976), and these could serve as convenient departure points.

c. CHANGES I N THE LEVELO F TAIg I N TRANSPLANTED TUMORS WITH

TIME AFTER IMPLANTATION

There seems to be an increase in the amounts of immunoglobulins within transplanted tumors with time after implantation. Witz et al. (1974a) compared, by a radioimmunoinhibition assay, the amounts of IgG associated with TA3 cells 7 and 10 days after implantation. They found that the average amount of IgG per cell increased during these 3 days by a factor of 3. Segerling et al. (1976) obtained similar results. The amount of Ig bound per guinea pig hepatoma cell increased approximately 2- to 3-fold on cells harvested 10-13 days after implantation compared to 6-day-old cells. The studies of Ran e t al. (1976) on a polyoma-virus induced murine tumor showed a similar pattern. There are several possible explanations for the increased amounts of

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ISAAC P. WITZ

Ig on older tumor cells. It is conceivable that the membrane sites to which Ig is fixed are unsaturated on young cells and that with time such sites become increasingly saturated as the synthesis of cell-fixing Ig increases. Alternatively, or in addition, older cells may express a higher numb& of Ig-fixing sites than younger cells. It has been postulated that cell antigenicity is at its peak during the stationary phase of the cell cycle (Cikes and Klein, 1972). It is possible that a higher proportion of older cells than of younger cells are in the stationary phase, resulting in increased antigenicity and thus in increased coating. D. THEDYNAMIC STATEOF TAIg TAIg disappears from the cell surface after the explantation of in uiuo propagating tumor cells to culture conditions. Thus IgG found to be present in human Burkitt lymphoma or sarcoma biopsies was not detected in cultures of these tumors (E. Klein et al., 1968; G. Klein, 1971; Romsdahl and Cox, 1975). Similar results were obtained in our laboratory using murine ascites tumors. For example, in viuo propagated MDAY cells were associated with high amounts of Ig but when grown in uitro, no such association was detected (Witz et al., 1974a). In view of the potential importance of this phenomenon, experiments aimed at understanding its mechanism were carried out by Ran et al. (1974, 1975) using murine ascites tumors. It was obserded that very soon after their transfer to culture conditions, tumor-derived cells lose some of their Ig. This process was termed uncoating. Rapid uncoating of various murine tumors upon their transfer to culture was confirmed recently by Dorval et al. (1976a). Uncoating could be accelerated somewhat by hourly changes of the medium. On the other hand, changing cell density in the culture (in the range of 1 x lo6 to 20 x lo6 celldml) did not seem to affect this process (Ran et al., 1974). Within the duration of these experiments (up to 6 hours at 37"C), a complete uncoating was never observed. In some tumors only about 50% of the Ig disappeared from the cells within 2 4 hours of in uitro incubation. The levels of Ig on the cells reached then a plateau, and for the next few hours essentially no uncoating was observed. In other tumors, uncoating progressed for longer periods of time and more of the Ig disappeared from the cells. Recent results (Segerling et al., 1976; Huang et a,?., 1975) indicated that TAIg persisted for relatively long periods of time. Thus TAIg was still demonstrable in primary

EXPRESSIONS OF HUMORAL IMMUNITY WITHIN TUMORS

109

cultures of a polyoma virus-induced rat tumor cultivated for 48 hours while no TAIg was present on the same cells cultivated for 14 days (Huang et al., 1975). In the other study, the levels of TAIg present on line-10 guinea pig hepatoma cells remained constant up to 24 hours after explantation of these cells to culture (Segerling et al., 1976). TAIg loss from the tumor cell surface was greater with cells held at 37°C than at 4°C (Ran et al., 1974; Dorval et al., 1976a; Segerling et al., 1976),but uncoating at 4°C did, nevertheless, occur. Uncoating at low temperatures is probably a reflection of an equilibrium reached between Ig molecules on the cells and in the surrounding medium. It is likely to be determined by the affinity of the coating molecules to the cell surface; those with low d n i t y leave the cell surface also at 4°C. The fact that a more efficient uncoating occurs at 37°C may indicate an association with cellular metabolism. Indeed, an excellent correlation existed between the release of surface macromolecules into the medium and uncoating (Ran et al., 1974). However, macromolecule and metabolism inhibitors had no effect on uncoating (Ran et al., 1974; Segerling et al., 1976). Since it was found that some of the inhibitors at the concentrations used did not produce the expected effect (e.g., chloramphenicol and puromycin did not inhibit DNA synthesis in the tumor cells) (Ran et al., 1974), no definite conclusions can be drawn from these experiments. Uncoating could sometimes be prevented when the cells were incubated in ascitic fluid rather than in culture medium (Ran et al., 1974). This result raised the possibility that Ig with tumor-binding capacity is available in some ascitic fluids and maintains the TAIg at a constant level. This conclusion was confirmed by experiments of Fish et al. (1974). By using radioiodinated IgG isolated from the ascitic fluid of TA3 tumors, it was found that the TAIg of these tumors was dynamically exchanged with Ig present in the ascitic fluid. This process apparently required cellular metabolism since it took place at 37°C but not at 4°C.

Ig was detected in culture medium in which freshly explanted murine tumor cells were incubated for a few hours (spent medium) (Ran et al., 1974; Dorval et al., 1976a), but not in spent medium of freshly explanted guinea pig hepatoma cells (Segerling et al., 1976). We have no explanation for this discrepancy. However, even in the former cases there was no correlation between the disappearance of Ig from the surface of tumor cells and its appearance in the spent medium (Fish et al., 1974). While the uncoating process proceeded at a more or less constant rate for a few hours, the Ig concentration in the culture medium reached a plateau within the first hour. This sug-

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gested that some of the Ig molecules were endocytosed whereas others were shed (Dorval et al., 1976a). Experiments were performed to test whether or not the released Ig had the capacity to rebind to fresh indicator cells (Ran et al., 1974; Dorval et al., 1976a). Ig eluted from tumor cell populations by a low-pH buffer was used as positive control. The results indicated that while acid eluted Ig molecules were capable of rebinding to tumor cells, the molecules released spontaneously into the culture medium were devoid of this property. The incapacity of eluted molecules to rebind to tumor cells can be explained by two nonmutually exclusive mechanisms: (1)the eluted Ig was in complex with cellular components; and (2) the eluted Ig was degraded. These possibilities were investigated. As mentioned above, it was found that uncoating correlated with the release of cellular macromolecules into the medium. Some of these macromolecules were apparently cellular antigens either free or in complex with Ig. This tentative conclusion was based on the results of the following experiments (Ran et al., 1974). (1)Incubation of spent medium with radiolabeled acid-eluted Ig molecules inhibited the fixation of the latter onto indicator tumor cells. If the fixation of eluted Ig molecules onto the tumor cells represents an antigen-antibody interaction (see below), the simplest explanation of this inhibition would be that the spent medium contained competing antigen. (2) Freshly explanted tumor cells were incubated at 37°C for a few hours, and the globulin fraction of the spent medium precipitable in 50% ammonium sulfate was radioiodinated. Significantly higher amounts of this globulin were fixed by lymphocytes originating from mice immunized with this particular tumor than by lymphocytes originating from unimmunized donors. Ig released from coated viable murine tumor cells was partially degraded. This was shown by experiments performed by Fish et al. (1974), who measured the capacity of IgG molecules released into short-term culture supernatants, to precipitate either with antisera directed against IgG or at 50% saturated ammonium sulfate. In both cases, released IgG showed a considerable lower precipitability than IgG that was not “processed” by tumor cells. Under experimental conditions provided by Fish et al. (1974), which were deliberately aimed at imitating those existing in uiuo, the binding capacity onto fresh indicator cells of the degraded IgG increased by 10-fold on the average. Thus, at least in the TA3 system studied by these investigators and under these particular experimental conditions, degradation of Ig cannot account for the decreased capacity of Ig isolated from spent medium to bind onto tumor cells. Taken

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together, the studies on TAIg show that it is in dynamic equilibrium with Ig molecules present in the surroundings: TAIg disappearing from tumor cells by endocytosis and/or shedding is replaced by fresh Ig. Exchange of TAIg is apparently connected with cellular metabolism. Some of the Ig molecules shed from tumor cells are degraded while others seem to be complexed with cellular components. The main advantage of experiments using in vivo propagating tumor cell populations coated in situ with Ig is that the research material represents an in vivo reality rather than an artificial situation. The main disadvantage of using such cells is that the nature of the association between the cells and the Ig is, so far, rather obscure. For this reason, we found it necessary to review some studies on the fate of antibodies reacting with defined membrane components of nucleated cells under conditions allowing cellular metabolism. The great majority of investigations dealing with this interaction emphasized, however, the effects of antibody binding on the expression of the corresponding surface antigen rather than the fate of the antibody ligand. Antigenic modulation, capping, or other types of topographical displacement of antigenic determinants were among the more frequent effects obtained (e.g., Old et al., 1968; Bernoco et al., 1971; Takahashi, 1971; Taylor et al., 1971; Edidin and Weiss, 1972; Kourilsky et al., 1972; Looret al., 1972; Sundqvist, 1972,1973; Menne and Flad, 1973; Neauport-Sautes et al., 1973; Raff and De Petris, 1973; Unanue et al., 1973,1974; Stackpole et al., 1974a,b;Yefenof and Klein, 1974; Yu and Cohen, 1974; Hilgers et al., 1975; Rosenthal et al., 1975). Concomitantly with antigenic rearrangements at 37"C, the antibodies causing this effect disappear from the cell surface. The loss of antibody is considerably less prominent when the antibody-coated cells are incubated in the cold. The detachment of antibody from the cells at 4°C is probably due to dissociation of low affinity molecules from the coated cells in an equilibration process. Antibodies directed against the following determinants are lost from the cells following incubation at 37°C: alloantigens (Amos et al., 1970; Chang et al., 1971; Cullen et al., 1973; Fine et nl., 1973; Faanes and Choi, 1974; Lesley and Hyman, 1974; Lesley et al., 1974; Jacot-Guillarmod et al., 1975), TAA (Leonard, 1973; Ran et al., 1975), and surface Ig determinants (Engers and Unanue, 1973; Knopf et al., 1973; Rieber and Reitnmuller, 1974; Antoine and Avrameas, 1974; Yefenof et al., 1976). The disappearance of membrane-bound antibody from cell surfaces as a function of incubation time at 37°C was indicated by the following results: (1) Decreased fixation of labeled anti-Ig reagents (directed against the coating antibody) by the coated cells. (2) Decrease in the

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amounts of elutable antibody. (3) Release of label into the medium when labeled antibody was used to coat the cells. (4) Decreased sensitivity of the coated cells to complement when the sensitizing antibodies had the capacity to mediate CdL. All four methods used showed that antibody did not stay on the surface of the coated cells. Some, possibly important, discrepancies in the kinetics of this process were detected, however, when sensitivity to complement was compared to one of the other parameters. Knopf et al. (1973) studied the interaction between an Ig-producing murine plasmacytoma and xenoantibodies directed against mouse Ig. These antibodies mediated complement-dependent lysis (CdL) of the plasmacytoma cells. The authors observed a different kinetics of loss of sensitivity to complement compared to disappearance of radioactive anti-Ig from the cells. At 37"C, sensitized cells became essentially resistant to CdL after an incubation period of less than 10 minutes, whereas during the same period, 80% of the radioactive antibody was still cell bound. Furthermore, after 80 minutes of incubation, 20% of the initial cell-bound radioactivity was still associated with the cells. A much clearer dissection between loss of sensitivity to complement, a process referred to as desensitization (Cullen et al., 1973; Knopf et al., 1973) and disappearance of radioactive anti-Ig antibodies from the myeloma cell occurred at 24°C. At this temperature, cells became desensitized after 20 minutes of incubation, whereas essentially all the initial radioactivity was still cell bound after 80 minutes. Lesley and Hyman (1974) confirmed these results. They found that a considerable amount of antigenically intact antibody remained associated with desensitized cells. The loss of biological functions of the coating antibodies is therefore not dependent upon their physical dissociation from the cells. A possibly related phenomenon was described by Faanes and Choi (1974). They observed that antibody-sensitized target cells were resistant to cell-mediated lysis. However, when the sensitized cells were incubated at 37°C for 60 minutes, susceptibility to cell-mediated lysis (CML) was reestablished. At this time, 70-80% of the antibody was still cell bound. Association of radioactive antibody with the appropriate target cell does not necessarily mean that the antibody remained bound to the cell membrane. Interiorization of cell-bound antibodies, occurring at temperatures permitting cellular metabolism, is a well-known phenomenon (Raf€ and De Petris, 1973). Since desensitization is not necessarily associated with loss of cell-bound antibody (shedding), it is important to follow its fate. Two main possibilities exist to explain desensitization without

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shedding. (1)Alteration, inactivation, or rearrangement of the antibody on the surface. (2) Interiorization of the antibody, possibly with membrane-antigens. These possibilities are not mutually exclusive. Knopf et al. (1973), using the experimental system described above, namely, Ig-producing mouse plasmacytomas and rabbit antimouse IgG antibody, performed the following experiment. They incubated at 24°C antibody-coated plasmacytoma cells. As indicated above, no shedding of antibody occurred at this temperature. At different intervals after the onset of the incubation they added to the cells an I3'Ilabeled preparation of sheep antibodies against rabbit IgG. An incubation time-dependent decrease in the fixation of the sheep antibody onto the cells was obtained. This indicated that the rabbit antibodies coating the plasmacytoma cells became inaccessible to the sheep reagent or that they progressively lost their antigenicity. The authors did not supply any information as to whether the antibody stayed on the surface (but in altered or degraded form, or in rearranged position) or whether it was interiorized. Lesley and Hyman (1974) used an anti-Ig reagent to detect surface-bound anti H-2 antibody after coated cells were incubated at 37°C for periods up to 3 hours. They also detected a time-dependent decrease in the capacity of the cells to bind the reagent. Similar to the previous results these also could not be interpreted with certainty, since a decreased binding of an Ig reagent onto antibody-coated cells may be due not only to uncoating, but also to changes in the density of the antibody coat. Thus aggregated antibody may fix less anti-Ig antibody than dispersed antibody, although their actual concentration in both cases may be equal. The amounts of membrane-bound antibody at a certain point in time can be assessed more accurately by measuring antibody molecules elutable from the coated cells by a short exposure to a low-pH buffer (R. Ehrlich, Y. Keisari, and I. P. Witz et aZ., unpublished). EL-4 cells coated with an lZ5I-labeledxenoantibody were used. It was found that immediately after exposure to the xenoantibody, followed by washings (all procedures carried out in the cold), about 70% of the cell-bound antibody could be eluted by the low-pH buffer. After about 2 days in culture, when most ofthe antibody had disappeared from the cells, 50% of the remaining cell-bound antibody could be eluted. Inaccurate as it may be, the difference between the amounts of elutable radioactivity and the total cell-bound radioactivity can be regarded as internalized radioactivity (antibody). The question whether shed antibody can rebind to fresh indicator cells has been raised by several authors. Whereas some found that the shed antibody lost its specific reactivity (Fine et aZ., 1973; Jacot-

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Guillarmot et al., 1975), others reported that the shed antibody retained its binding activity, or even exhibited higher binding than unreacted antibody (Lesley and Hyman, 1974; Yefenof et al., 1976; R. Ehrlich, Y. Keisari, and I. P. Witz unpublished). But also according to these investigators, binding activity decreased as a function of incubation time, reaching low levels after a few hours at 37°C. TWOnonmutually exclusive mechanisms can account for the decreased binding activity. The one is a configurational alteration in the binding site of the antibody induced by the interaction with the cell, or degradation of the site by cell-derived proteolytic activity. A second mechanism may be the complexing of antibody with shed cellular antigens resulting in a progressive accumulation of antibodies with saturated binding sites. While some of the studies quoted above implied the presence of immune complexes in conditioned medium of antibody-coated cell cultures (Fine et al., 1973; Rieber and Reithmuller, 1974, Antoine and Avrameas, 1974; Hayami et al., 1974; Jacot-Guillarmod et al., 1975; Yefenof et al., 1976), most of them provided convincing evidence of the existence of degraded antibody in such media. The potential role played by degraded antitumor antibodies in tumor-host relationship will be discussed below in Section VII. Immune complexes between TAA and the corresponding antibodies were postulated to block tumor cell destruction by killer lymphocytes (Baldwin et al., 197313; Hellstrom and Hellstrom, 1974; Jose and Seshardi, 1974). It is thus important to establish how such complexes are formed. Immune complexes can be produced in the circulation, or in situ. Although the vicinity of the tumor is probably very rich in antigen capable of absorbing free antibody molecules, the possibility cannot be excluded that antibody fixes first to the tumor cells and that subsequently antigen-antibody complexes are shed as such from antibody-coated cells. In spite of their importance very little is known about the way complexes are formed and about their concentration in the circulation and in the vicinity of the tumor. A probable contributing factor to this uncertainty is that the molecular size of such complexes, if they exist, may be very similar to that of uncomplexed antibody. This was probably the case in a study performed in ourlaboratory. Ehrlich et al. (1976) did not detect any high-molecular-weight peak when they filtered, through Sephadex columns, radioiodinated rabbit anti E L 4 antibodies shed from such cells into the tissue-culture medium. However, treatment of this conditioned medium with a low-pH buffer significantly increased the binding capacity of the shed antibody onto indicator cells. The discrepancy between the gelfiltration results negating presence of complexes in spent medium and

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the results of increased binding of shed antibody after acid treatment can be explained by a partial degradation of antibody molecules not involving their binding site. The degraded antibody, although complexed with antigen, may not be larger than undegraded-uncomplexed antibody and would therefore not emerge as a separate peak in gel filtration. This possibility has not been tested as yet. The next set of experiments involved the use of EL-4 cells labeled with 1311 by lactoperoxidase. The cells were than coated by '251-labeledrabbit antiEL-4 antibodies, and incubated at 37°C. Attempts were then made to establish whether or not 1311-labeledcellular material would specifically coprecipitate by goat antibodies directed against rabbit IgG or Fc. The results were equivocal; although cellular material did specifically precipitate with the antibody, the amounts precipitated were very small. Based on the studies quoted in this chapter, it is possible to conclude that the behavior and fate of artifically raised antibodies after their interaction with known antigenic specificities on nucleated cells is very similar to the behavior and fate of TAIg. These systems may thus serve as reliable models for the study of tumor-associated Ig molecules. IV. The Nature of Ig-Associated Cells in Tumors

TAIg may reside on neoplastic cells, on host-derived cells, or on both. It may even be produced locally (Charney, 1968; Roberts et al., 1973; Richters and Kaspersky, 1975; Hurlimann et al., 1976). The demonstration that tumor tissue contains large amounts of macrophages and/or lymphocytes expressing Fc receptors (Evans, 1972; Eccles and Alexander, 1974a,b; Haskill et al., 1975a,b; Kerbel et al., 1975; Tracey et al., 1975; Gauci and Alexander, 1975; Szymaniec and James, 1976; Braslawsky et al., 1976a,b) makes it rather likely that at least some of the TAIg is in fact associated with these cells. Even if it were demonstrated in a particular tumor system that TAIg is composed exclusively or mainly of antibodies directed against TAA, the conclusion still cannot be safely drawn that these antibodies are associated with the malignant cells. The antibodies may be associated in a cytophilic mode of binding or as immune complexes to macrophages or to other Fc receptor-bearing cells. Attempts to define Ig-associated cells in tumors by cell separation techniques has little chance of yielding reliable data in view of the rapid uncoating of TAIg i n citro (see Section 111,D). Cell-separation

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techniques are time-consuming and cannot always be performed under conditons that do not favor uncoating. In their study on Ig in breast cancer, Roberts et al. (1973) found that round-cell or plasma-cell infiltration into the tumor correlated positively with IgG levels in these tumors. No such correlation was detected regarding IgA levels. These results may indicate either that IgG is produced locally by the infiltrating cells or that it is adsorbed onto them. The possibility that local production of Ig occurs within mammary cancer was supported by findings that Ig (and other serum proteins) were synthesized in vitro b y explants of human breast cancer tissue (Hurlimann et al., 1976). CarcinQmas with lymphocyte infiltration showed a preferential synthesis of IgG compared to tumors without infiltration. In another study of Ig in breast cancer, Richters and Kaspersky (1975) found only a few Ig-positive lymphocytes in the cancerous mass, whereas Ig-positive lymphocytes were readily demonstrated in the homolateral axillary lymph nodes. No mention was made whether or not tumor cells were associated with Ig. An exclusive restriction of TAIg to host cells occurs in bladder tumors (Johansson and Ljungqvist, 1974). Only plasma cells and lymphocytes, but not tumor cells, were associated with Ig. Identical results were recently obtained by Lewis et al. (1976) in human malignant melanoma. In this study, Ig was found to be associated with small lymphocytes, plasma cells, and macrophages, but not with tumor cells. Since the investigators used membrane immunofluorescence, a method whose sensitivity may not suffice to detect Ig on tumor cells (see Section 11) and since nothing was reported on precautions to prevent uncoating (see Section II1,D) no definite conclusions can be drawn from these experiments. Dorsett et al. (1975), using fluoroisothiocyanate (F1TC)-conjugated antibodies against human Ig in direct membrane immunofluorescence assays, observed that only the tumor cells present in effusions of ovarian carcinomas, but not the normal cellular constituents, were stained. Irie et al. (1975), working with mixed hemadsorption, observed in certain cases positive adsorption of detector-sensitized erythrocytes onto lymphocytes infiltrating the human tumor specimens. This adsorption occurred, however, even in the absence of antibodies against human Ig, whereas adsorption of detector erythrocytes to tumor cells was never detected without the antihuman Ig reagent. The positive reaction with lymphocytes was therefore probably due to the expression of Fc receptors on the infiltrating cells, not to the presence of Ig attached to them.

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Vanky et al. (1975) were rather concerned about the possibility that the Ig they eluted from cancer biopsies was contributed by infiltrating lymphoid cells. They subjected lymphoid cells derived from blood, lymph nodes, spleen, and bone marrow to their elution procedure and determined the amounts of elutable Ig. A biopsy specimen was considered positive for Ig only if at least twice the average amount of Ig elutable from the lymphocyte preparations was eluted from it. The use of iodinated protein A from Staphylococcus aureus to detect TAIg in murine ascites tumors (Dorval et al., 1976a) permitted the conclusion that cells carrying actively produced Ig, such as B cells, are not those that contribute significantly to the presence of TAIg. Protein A does not bind to any significant extent to mouse B cells, but it binds readily to cells with passively adsorbed IgG antibodies (Dorval et al., 1975). Protein A did, however, bind to various in vivo propagated ascites tumors. In the same study, it was also demonstrated that removal of adherent cells from ascitic suspensions did not decrease the capacity of these suspensions to bind a radioiodinated anti-Ig reagent. This indicated that although macrophages within ascitic tumors may be associated with Ig, these cells are not the only ones that bind Ig in viuo. The results of Witz et al. (1974b) showing that 100% of the cells in TA3 tumors were stained by fluorescein-conjugated anti-mouse IgG antibodies also indicated that tumor cells, as well as host cells residing in the tumor, were coated with Ig. Ran et al. (1976) demonstrated that cells in the SEYF-a tumor (a murine polyoma virus-induced sarcoma syngeneic to A.BY mice) lyse after the addition of exogeneous complement. These results (to be discussed in detail in Section V,C) show that tumor cells, sensu strictu, are coated with Ig. Based on the few studies which referred to the problem of the identity of the Ig-associated cells in tumors, one may tentatively conclude that tumor cells as well as tumor-seeking host cells are associated with Ig. V. Antitumor Antibodies as Part of TAlg

Whether or not TAIg is composed entirely or partially of antitumor antibodies localized in vivo on their target antigen is an important as well as a difficult question. Theoretically this problem should be easily solved by determining the activity of Ig eluted from in vivo coated cells against the corresponding and other tumor cells. However, the following factors contribute to the complexity of this issue: (1)Solu-

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tions used to elute Ig may dissociate only low-affinity antibodies while leaving the high affinity ones attached to the cells. (2) These solutions may extract antigens 'from the coated cells which may lead to the formation of immune complexes in the eluate. Such complexes could be fixed by any cell expressing Fc receptors while being incapable of binding onto cells expressing the corresponding specific antigens. (3) The eluting agents may also cause degradation or other types of alterations involving the binding site of the putative antibodies, preventing them from specifically binding to the corresponding cells. (4) Host cells are present within tumors (see Section IV) that may be associated with unrelated Ig. (5) Ig eluted from a certain tumor is reactive with other tumors, possibly owing to the expression of cross-reactive or even common antigens by different tumors. (6) There is nonspecific adherence of proteins (such as immunoglobulins) to tumor cells through electrostatic bonds. In spite of these difficulties, some investigators succeeded in supplying suggestive evidence and sometimes even formal proof that TAIg is composed, at least partially, of antitumor antibodies. The basic assumption underlying the studies reviewed in this section was that antitumor antibody can localize at the tumor site at least in some tumor systems, in spite of less than optimal vascularization and even though circulating tumor antigen reaches, very often, high levels (Thomson et al., 1973a,b; Baldwin et al., 1973a; Kolb et al., 1974; Poskitt et al., 1974; Kim et al., 1975; Bowen et al., 1975; Knight et al., 1975). This assumption is not new and numerous attempts to achieve tumor localization of antitumor antibodies have been reported (for review, see Pressman, 1968).Noteworthy are some recent successful results (Primus et al., 1973; Mach et al., 1974; Bale et al., 1974; Ghose et al., 1975). An argument used often as support for the contention that antitumor antibodies absorb in vivo on tumor cells is the observation that in general no antitumor antibody is demonstrable in tumor bearers and that excision of the tumor or its regression causes the rapid appearance of such antibodies in the circulation (Thomson et al., 1973a; Baldwin et al., 1973c; Basham and Currie, 1974; Harada et al., 1975; Bray and Keast, 1975; Canevari et al., 1975).Basham and Currie (1974) cited the paper of Thomson et al. (1973a) as providing evidence that this argument cannot be used. This paper (Thomson et al., 1973a) was also cited by Lewis et aZ. (1976) as documenting a failure to detect antibody on the surface of rat sarcoma cells. A careful examination of the paper of Thomson et al. (1973a) shows that these investigators did not report on any direct experiments to confirm or negate the presence of

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antitumor antibodies in the rat sarcomas. They did report, however, that the levels of antitumor antibodies increased after excision of the tumors, not only in blood but also in the thoracic duct lymph. A logical argument raised by the authors postulates that, since the pathway of freshly synthesized antibodies is from the draining lymph node to the blood via the thoracic duct, the molecules in the lymph of this duct would not have had an opportunity to be absorbed out by the tumor growing in the leg. This normal pathway, however, does not accommodate deviations possible in cancer, such as local antibody production or lymph-borne metastases. Furthermore, the documentation of a certain mechanism operating in a certain phenomenon does not rule out the possibility that a different mechanism operates also in the same phenomenon. Another argument raised often to negate the presence of antitumor antibodies on tumor cells is the fact that freshly harvested tumor cells are frequently either not stained or stained weakly by direct immunofluorescence using anti-Ig reagents but stained brightly after being preincubated in antibody-containing serum. This phenomenon can be explained as follows: (a) At any point in time only some of the surface antigenic determinants are coated with antibodies. Thus the levels of TAIg may be too low to be detectable b y immunofluorescence. (b) TAIg is uncoated in antibody-free media (see Section 111,D). (c) TAIg is degraded by tumor-derived proteases (see Sections II1,D and VII).

A. EARLYWORK The studies of Sobczak and De Vaux St. Cyr (1971)and of Eilber and Morton (1971) showing serological antitumor activity of elutes from SV40 virus-induced hamster tumors and from human cancer, respectively, have been discussed in a previous review (Witz, 1973).So were the results of Nishioka (1971)on the presence of complement components on tumor cells. B. INDIRECT EVIDENCE

1. Presence of Complement Components on in Vivo Propagating Tumor Cells Brown and Tan (1973) showed that C3 together with IgM and IgG was deposited in human keratoacanthoma. Irie et al. (1975) detected Ig as well as C3 bound to cells originating from all twelve samples of human cancer biopsy or autopsy material assayed. The method used by these investigators was mixed hemadsorption. Only one out of eight

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noncancerous tissues assayed showed a positive reaction. The frequency of cancer cells with attached C3 was equal to or somewhat higher than the frequency of Ig-positive cells. The reason for this was not clear although several explanations were suggested by the authors. Dorsett et al. (1975) and Sulitzeanu et aZ. (197613)found complement components on ovarian carcinoma cells present in malignant effusions. Segerling et al. (1976) showed that guinea pig hepatoma cells were associated with C3 in addition to Ig. The amount of complement components bound to the hepatoma cells increased as a function of propagation time in uiuo. Upon transfer to in uitro culture, the complement components were shed from the surface of the tumor cells into the culture medium. Treatment of the hepatoma cells with metabolic inhibitors did not prevent the shedding of the complement components. The presence of complement components in the tumor does not necessarily mean that these components were fixed by antibodycoated tumor cells. Host cells with receptors for complement may be those that fixed the complement. In addition, complement activated by the alternate pathway rather than by antibody may bind to various cells residing in the tumor. Another issue raised by these findings concerns the reason for the failure of the bound complement components to lyse the coated cells. Nothing is known about this problem but several possible mechanisms exist to explain this failure. Cooper et aZ. (1974) found that MuLVinduced tumor cells were susceptible to CdL only at the G, phase of the cell cycle. During the other phases, the cells were resistant to CdL. However, even during the resistant phases, the cells bound complement components (C5 and C8 were assayed in this particular study) in equivalent amounts (or even higher) to those bound during the G, phase. It is thus possible that those tumor cells remaining viable in spite of complement binding (10-30% of the cells in human tumors according to Irie et al., 1975) were in one of the CdL-resistant phases of the cell cycle. Another mechanism to explain resistance to CdL may be the existence of a C1 inactivator operating at the surface of malignant cells (Osther, 1974). Such an activator could bring about an abortive complement fixation. Other possibilities such as presence of CdL-resistant immunoselected cells within tumors, or a nonspecific fixation of complement components should also be considered. 2 . Masked Antigenicity If antigenic determinants on the membrane of tumor cells are masked by the coating Ig molecules, removal of the latter should increase the antigenicity of the tumor cells. This approach was at-

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tempted by Stjernsward and Vanky (1972) and Vanky et al. (197313). These investigators demonstrated that lymphocyte stimulation could be induced by cell suspensions prepared from autologous human cancer biopsies. It was also shown that a certain percentage of tumor biopsies, being originally incapable of stimulating autologous lymphocytes to synthesize DNA, became stimulatory after being subjected to treatment with a low pH buffer, thus presumably removing a blocking Ig coat from these cells. Actual presence of Ig on the nonstimulating cells prior to treatment with the low pH buffer was not determined. For further discussion of these results see below (Section VII I ,B). A similar approach was attempted by Gupta and Morton (1975) in a carefully executed study. It was demonstrated that an homogenate of melanoma tissue did not react significantly, in a complement-fixation assay, with autologous serum. Treatment of the tissue homogenate with a solution containing a high concentration of salt (15% NaC1) brought about the elution of Ig with complement-fixation properties (see Section V,C). The treated residue expressed a significant increase in its capacity to fix complement with autologous serum. The low-pH buffer-treated melanoma tissue residue reacted also with the corresponding eluates, whereas untreated homogenate showed no reactivity whatsoever. Treatment of tumor tissue, as such, did not seem to bring about a nonspecific capacity to fix complement. This was shown by the fact that treated tissue residues from sarcomas, carcinomas, or normal tissue did not fix complement in the presence of melanoma eluates and vice versa. Robins (1975) demonstrated that ascites rat hepatoma cells coated in vivo with Ig did not absorb appreciable amounts of corresponding antihepatoma antibodies. However, treating the cells with a Ca2+-freemedium, thereby removing TAIg, increased the absorption capacity of the cells. Dorval et al. (1976b) treated in vivo grown Moloney lymphoma YAC cells, for very short durations, with a low pH buffer. This treatment did not affect cell viability as judged by dye exclusion. The treated cells lost some of their Ig coat and concomitantly expressed higher antigenicity toward a syngeneic antiserum recognizing Moloney-virusassociated antigen specificities. The increased antigenicity was manifested by a higher capacity to fix such antibodies, and by a higher sensitivity to CdL mediated by them. An important control revealed that the increased expression of MuLV-associated antigens was unaccompanied by similar alterations in H-2 expression. Short-term cultures of the freshly explanted tumor cells, bringing about spontaneous

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dissociation of Ig from the cell surface (uncoating-see Section III,D), also increased MuLV-associated antigenicity without altering H-2 antigenicity. Similar results were obtained by using a polyoma virusinduced tumor. These cells, after an incubation period of 3 hours at 37"C, bound more antibocfies present in a syngeneic antiserum against polyoma virus-induced tumor cells and less anti-Ig antibodies. Again H-2 antigenicity remained unaffected by this incubation. The so-called unmasking experiments provide only suggestive evidence that antigenic determinants on the tumor cells are masked by antibodies directed against these determinants. Steric hindrance by various molecules, including unrelated Ig molecules, is a possibility to be seriously considered. Moreover, such experiments do not always provide clear-cut results like those presented above. For example, Ran et al. (1975) exposed freshly explanted Moloney-virus-induced YAC lymphoma cells to short-term culture conditions. The sensitivity of the YAC cells to CdL mediated by specific anti-YAC antibodies either increased, decreased, or remained without change during this shortterm culture period, although uncoating occurred in all three instances. These results illustrate that the interpretation of unmasking experiments, especially those involving explantation of cells grown i n viva to short-term culture, is difficult. In addition to uncoating, explanted cells probably undergo physiological alterations and may be driven from one phase of the cell cycle into another. This may affect the antigenic expression of the cells (Cikes and Klein, 1972; Cikes'et al., 1972) or the sensitivity to CdL (Cooper et al., 1974). C. DIRECTEVIDENCE Data showing that TAIg exhibits specific serological activities toward TAA can be considered as reliable proof for the antibody nature of these molecules. The amount of published work concerning this problem is rather small. Previous findings on this subject (Sobczak and De Vaux St. Cyr, 1971) were discussed before (Witz, 1973) and therefore will not be dealt with in this section. Eluates of human melanoma reacted with melanoma antigens in a complement-fixation test (Gupta and Morton, 1975). Since these eluates contained Ig, and since reactivity seemed to be specific for melanoma antigens, it can safely be assumed that the eluates contained antimelanoma antibodies. Von Kleist (1976) eluted Ig from membrane-rich fractions of colon carcinomas by glycine buffer. Eight tumors and adjacent noncancerous mucosa were subjected to elution. All eluates contained Ig (see above). Cells from the HT29 line (derived from a human primary

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carcinoma of the colon) known to express the three principal colon tumor antigens and no Fc receptors (von Kleist et al., 1975)were used as targets to detect the possible presence of antibodies in the eluates. Cells were incubated with eluates and then with fluoresceinconjugated anti-Ig reagents. Cells treated with all eight cancer eluates and with four of the eight nontumor eluates showed fluorescence. The reactivity of the noncancerous eluates with the HT29 cells is not surprising since at least two of the surface antigens expressed on these target cells are normal antigens. None of the eluates stained cells derived from a human fibroblast line. Specific binding of 1251-labeled eluates from a syngeneic rat Moloney sarcoma onto the corresponding cells in uitro was reported by Jones et al. (1974). However, the authors did not indicate whether the tumor-fixing material in these tumor eluates was Ig. Ran et al. (1976) showed that mice bearing a syngeneic polyoma virus-induced sarcoma (SEYF-a) had circulating antitumor antibodies with the capacity to mediate CdL. These antibodies apparently localized in uivo on the tumor cells since the addition of exogenous rabbit complement to these cells caused their lysis. Sensitivity to complement increased as a function of propagation time in uiuo, reaching a maximum at about 3 weeks after tumor inoculation. The sensitivity to lysis mediated by addition of complement decreased thereafter (M. Ran and I. P. Witz, unpublished). In competition experiments, it was found (Ran et al., 1976) that coated cells were appreciably less antigenic toward a syngeneic antiserum derived from hyperimmunized mice than were uncoated cells. Antigenicity was partially restored after the coated cells were incubated at 37°C causing a partial dissociation of the cells and the coating antibody. Results indicating cell lysis following addition of normal heterologous sera as a source of complement should be interpreted with caution. Normal sera often contain natural antibodies cross-reactive with surface antigens of cells from other species (Boyden, 1966).The complement contained in these normal sera may lyse the target cells. Such a lysis could be erroneously interpreted to mean that the cells were coated with potentially cytotoxic antibodies. Absorption of the complement source with the target cells, to remove natural antibodies, should therefore become a standard procedure. A suitable illustration of such a situation is the study of Caspi and Witz (1976) on the MDAY cells, a methylcholanthrene-induced transplantable murine ascites tumor. This tumor behaved like the SEYF-a tumor described above in that freshly explanted tumor cells were lysed after exposure to normal rabbit serum. However, in sharp contrast to the SEYF-a system, the normal serum was no longer toxic to MDAY cells after it was absorbed

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with these tumor cells, a treatment that left its complement activity intact. Antibody-mediating CdL of SEYF-a cells could be eluted from such cells by a low-pH buffer. Antibodies were eluted also from “old” SEYF-a cells (4 weeks after inoculation or older) even though such cells were relatively insensitive to the lytic activity of exogenous complement. The lytic activity of the eluted antibody resided primarily in the IgG2 subclass since it could be completely neutralized by a monospecific anti-IgG2 antiserum (N. Moav and I. P. Witz, unpublished). Another important evidence for the antibody nature of the Ig in the SEYF-a eluates is that the specific activity of SEYF-a eluates, in terms of cytotoxicity toward SEYF-a cells, is higher than that of serum drawn from tumor bearers (N. Moav and I. P. Witz, unpublished). The reactivity spectrum of the eluted anti-SEYF-a antibody is currently under investigation. The analysis of the specificity of the eluted antibody is considerably facilitated by the availability of serologically defined syngeneic antisera directed against ascites SEYF-a cells (Witz et al., 1976). These antisera contained, in addition to antibodies directed at a surface antigen associated primarily with SEYF-a cells, also antibodies against certain other specificities, such as MuLV-associated antigens. In contrast to the polyspecificity of the hyperimmune antisera, low-pH eluates of SEYF-a tumor reacted in most cases only with SEYF-a cells, but rarely with other tumor cells. Berczi et al. (1976) demonstrated that acid eluates of a methylcholathrene-induced guinea pig sarcoma contained IgG molecules with the capacity to fix, in uitro, to cultured cells of the corresponding tumor. Eluates of normal tissue did not contain tumor-fixing IgG. Goldrosen and Dent (personal communication) treated autochthonous and transplantable SV40 virus-induced hamster tumors with a low pH buffer and detected Ig in the eluates. The eluates possessed antibody activity directed to the SV40 nuclear T antigen, but did not contain antibodies directed against cell-surface antigens, and did not block CML of cultured SV40 tumor cells.

VI.

Unrelated lg as Part of TAlg and the Presence of Receptors for Immune Complexes within Tumors

I t is known that tumor tissue contains cells expressing Fc receptors (Milgrom et al., 1968; Tonder and Thunold, 1973; Kerbel et al., 1975; Haskill et al., 197%; Tracey et al., 1975; Wood et al., 1975; Muchmore et al., 1975; Szymaniec and James, 1976). It is thus possible that some of the Ig present in tumors is bound to these receptors. Braslawsky et

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al. (1976a) searched for the presence in TAIg of antibodies clearly

unrelated to any known tumor antigen. They immunized strain A mice

(H-2") either with ovalbumin (OA) or with bovine serum albumin

(BSA). Once the immunized mice developed circulating antibodies against the respective albumins, all mice, as well as unimmunized ones, were inoculated with syngeneic ascites TA3/St tumor cells (a transplanted mammary tumor). After 7 or 9 days of tumor propagation in uiuo, tumor cells were harvested and washed. Radiolabeled BSA or OA were then added to these cells. It was expected that if cells in the tumor were capable of binding unrelated antibody in such a way as would leave the binding site of these antibodies available, then cells originating from BSA-immunized mice should bind higher amounts of BSA than of OA, and vice versa. This was indeed the case. It was thus established that antibodies unrelated to any known tumor cell antigen can comprise at least a part of the total TAIg. The authors did not provide data as to whether free antibody or antigenantibody complexes at antibody excess were bound by the cells. Incubation of the explanted cells at 37°C for a few hours caused dissociation of the passively attached antibody and anti-OA or anti-BSA activity could be recovered in the culture medium. Binding of immune complexes, such as OA-anti-OA (at antigen excess), but not of uncomplexed antibodies by TA3 cells, was obtainedin uitro. The in v i t r o binding of the OA-anti-OA complexes by cells originating from the TA3 tumor could be inhibited by aggregated antibody and by other immune complexes, such as BSA-anti-BSA. Binding of immune complexes was also inhibited by anti H-2" alloantibodies, thus confirming the findings that Fc receptors or Fc-receptor functions could be inhibited by antibodies reactive with surface antigens on the receptor-bearing cells (Dickler and Sachs, 1974; Halloran et al., 1974). By using TA3 cells propagated in F, hybrids and by inhibition assays with alloantibodies directed against both parental strains, it was established that the majority of complex-binding cells within the TA3 tumor were host cells, not tumor cells per se. Some of the complex-binding cells were apparently macroph ages, where as others were nonadherent, nonphagocytic cells. The situation with the SEYF-a tumor seemed to be different in that the tumor cells themselves, in addition to host cells, could apparently bind unrelated immune complexes (Braslawsky et al., 1976b). This conclusion was drawn from inhibition studies by specific antisera. It was found that anti-SEYF-a antibodies originating either from artificially produced, hyperimmune syngeneic antisera (Witz et al., 1976) or from tumor bearers (Ran et al., 1976) inhibited, to a large extent, fixation of OA-anti-OA complexes by cells originating from SEYF-a tumors. These antibodies, which mediated CdL of tumor cells but not

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of normal syngeneic splenocytes or lymph node cells (Witz et al., 1976), did not inhibit the fixation of immune complexes by normal lymphocytes. Enrichment of tumor cells by removal of phagocytic cells from the SEYF-a tumor decreased complex fixation but increased the antibody-madiated inhibition of complex fixation by the phagocyte-depleted tumor-enriched cell population. These results supported the conclusion that tumor cells, and not only infiltrating host cells, can express receptors for immune complexes. Additional experiments excluded the possibility that antibody-coated tumor cells acting as “third party” immune complexes blocked receptors for complexes present on host cells that infiltrated the tumor. It seems that those nonlymphoid tumor cells, such as SEYF-a which express receptors for immune complexes (Fcreceptors?) in vivo do not seem to express such receptors when grown in vitro (Tracey et al., 1975; Szymaniec and James, 1976).This prima facie discrepancy can be explained in several ways, all amenable to experimentation. First, there is no reason to assume that cultured cells, being a selected population, must truly represent the entire spectrum of functions and characteristics of growing in vivo cells. In fact, important differences between cultured cells and their in vivo growing ancestor population, for example, in antigenic composition, have been detected (Franks, 1968; Cikes et al., 1973; Irie et al., 1974; Evans et al., 1975; Cornain et al., 1975). Second, expression of any receptor on cells does not necessarily mean that the receptor is the product of the same cell. For example, Ig binding molecules (possibly Fc receptors) released from activated T lymphocytes (Fridman et al., 1974; Neuport-Sautes et al., 1975), may adhere also to other cells, such as nonlymphoid malignant cells in vivo. Lack of receptor-synthesizing cells in the cultured population will deprive this culture of the Fc receptor function. Third, expression of Fc receptors can be induced by viruses (Yasuda and Milgrom, 1968; Westmorland and Watkins, 1974; Keller et al., 1976) or by other types of stimulation, such as steroid hormones (Lotem and Sachs, 1975). Factors inducing expression of Fc receptors on nonlymphoid cells may exist and operate in vivo but not in uitro. An interesting relationship between TAIg on SEYF-a cells and the expression of immune complex receptors on such cells was indicated (Braslawsky et al., 1976~). As reported above, anti SEYF-a antibodies inhibited fixation of OA-anti-OA complexes by SEYF-a cells (Braslawsky et al., 1976b). In conformity with these results, it was found that as in vivo propagating SEYF-a cells became progressively coated with antibodies, as a function of propagation time, their capacity to bind unrelated immune complexes decreased. Moreover, antitumor antibodies inhibited binding of such complexes by SEYF-a cells harvested from the tumor bearer about 10 days after tumor inoculation but

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not by cells harvested about 30 days after inoculation. Considerably larger quantities of antitumor antibodies could be eluted from the older tumor cells than from the young ones. Incubation of the old cells at 37°C caused dissociation of Ig from the cell surface (see Section II1,D) and restored to some extent the capacity of antitumor antibodies to inhibit complex fixation by these cells. These results indicate that antitumor antibodies accumulating in wivo on the tumor cell surface blocked the complex receptors on these cells either specifically or by steric hindrance. The results on the presence of Fc-receptorlike activity within tumors support and confirm recently published data. Thus, increased K-cell activity within lymphoid organs of mice bearing nonlymphoid tumors has been reported (Calder et al., 1975; Ghaffar et al., 1976). K-cell activity (i.e., cells with F c receptors) was also detected within nonlymphoid tumors (Tracey et al., 1975; Koren and Handwerger, 1975; Kerbel et al., 1975; Haskill et al., 1975b; Wood et al., 1975; Muchmore et al., 1975; Handwerger and Koren, 1976). In view of the possibility that such cells may be involved in antitumor reactivity (Skunak et al., 1972; Pollack et al., 1972; Hellstrom et al., 1973; D e Landazuri et al., 1974; O'Toole et al., 1974; Lamon e t al., 1975), a closer examination of these cells may be of importance. Several questions are open at the present state of knowledge. An identification of the cells within tumors expressing Fc receptors,2 whether exclusively host cells or also tumor cells, must be performed in each tumor system studied. In the latter case, it would be interesting to find out whether tumor cells expressing Fc receptors exert any effects on antitumor immunity. They could, for example, compete with K cells, proper, for the Fc fragment of antitumor antibodies coating other tumor cells. Another point of interest would be to find out if Fc-receptor-bearing cells are blocked in wivo. The biological significance of Fc receptor-bearing cells of tumor origin could be studied by adoptive transfer of such cells to tumor bearers and by in vitro assays. VII. Degradation of Antitumor Antibodies

The microenvironment of the extracellular compartments of the tumor tissue is different from that of nonmalignant tissues (Gullino, 1975). An important factor contributing to the different microenvironmental conditions existing in tumors is cell death or necrosis (Cooper et al., 1975). Cells undergoing autolysis contain increased levels of 'Assuming that such receptors are identical with the receptors for immune complexes reported above and also responsible for the K-cell activity of tumor-derived cells.

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lysosomal enzymes (Niemi and Sylvkn, 1969; Sy1vi.n and Niemi, 1972). Moreover, lysosomal enzymes may even be actively secreted by viable tumor cells (Poole, 1973). Indeed, such enzymes are found in increased amounts in the interstitial fluids of various types of tumors (Sylvhn and Bois-Svensson, 1965; Sylvhn, 1968). The presence of active hydrolases in the tumor may have an important biological significance. The destructive capacities of malignant tumor cells, their invasiveness and their detachment from other tumor cells resulting in the formation of metastases were ascribed to lysosomal enzymes (Poole, 1973). Previously unconsidered effects exerted by such enzymes on immune components residing within the tumor may influence considerably tumor-host relations. In view of the fact that Ig molecules, of which some may be antitumor antibodies (see Section V) localize in the tumor, the tumor environment provides a common interaction ground for tumor-derived proteases and for antitumor antibodies. We investigated the possibility that immunoglobulins are affected by tumor-derived proteases. Mouse immunoglobulins were subjected in vitro at acid conditions to extracts prepared from the lysosomal subcellular fraction of various murine tumors. Degradation of the immunoglobulins was obtained (Keisari and Witz, 1973). The degradation was evaluated by a number of criteria: decreased capacity of the treated Ig molecules to precipitate specifically with anti-Ig antisera and nonspecifically with ammonium sulfate and cold TCA; appearance of low-molecular-weight products in the treated Ig preparations. Similar results were obtained in human systems (Witz et al., 1974b). Next, the effect of lysosomal enzymes on CdL mediated by antitumor antibodies was studied. Ig preparations of xenoantisera and alloantisera mediating CdL of various murine ascites tumors, were subjected to tumor-derived lysosomal extracts. The treated Ig preparations lost their capacity to mediate CdL (Dauphinee et al., 1974; Keisari and Witz, 1975a,b) but retained their capacity to rebind to the appropriate target cells. This was shown by binding experiments (Keisari and Witz, 197513) and by the capacity of the degradation products to specifically block CdL mediated by intact antibodies (Keisari and Witz, 1975a,b) and lymphocyte-mediated lysis (Dauphinee et al., 1974). Keisari and Witz (197%) sequentially precipitated lysosomal enzyme-treated xenogeneic antitumor Ig preparations with 40% and then with 70% saturated ammonium sulfate. Separation was obtained between seemingly undegraded antibody molecules (precipitating at 40% ammonium sulfate) and degraded molecules (not precipitating with 40% ammonium sulfate but precipitating at 70% saturation). These fractions were then filtered through Sephadex G-100 columns. Several subfractions were obtained. It was found that the subfractions of the degraded antibody

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(precipitating at 70% but not at 40% ammonium sulfate) lacked Fc fragments. These degradation products retained, however, their binding activity and blocked specifically, at the target cell level, CdL mediated by untreated antibody. We do not have any data on the cellular source of the proteolytic enzymes used in our studies. Both tumor cells and host cells (especially macrophages) could have been the origin of these enzymes. We know, however, that established tumor cultures lacking host cells yield active preparations of lysosomal proteases. As far as the biological significance of such enzymes is concerned, their cellular source is of no apparent importance. Since these degradation experiments were carried out at a low pH (pH 3.8),the question arose whether or not degradation of antibody by lysosomal enzymes can occur at all in vivo in or around tumors. At least two types of findings on the suitability of the extracellular microenvironment of the tumor for lysosomal enzyme activity support the possibility that such a degradation is not unlikely. The first group of findings concerns the possibility that the pH of the extracellular compartment of the tumor is comparatively low because of the high concentration of lactic acid contained in it (Gullino e t al., 1964). Moreover, there are indications that the pH at the peripheral region of negatively charged cells is significantly lower than the pH at other regions (Weiss, 1967). The second group of findings is that lysosomal proteases, although very active at low pH, are also active at neutral pH (Fell and Dingle, 1963; Poole, 1970). Although the studies summarized so far indicated that extracellular lysosomal proteases can affect the reactivity of antitumor antibodies localized within the in vivo propagating tumor, we turned to another type of degradation experiments taking place at physiological conditions, namely, at short-term tissue culture (Y. Keisari and I. P. Witz, unpublished). It was already shown by quite a few investigators that antibodies directed against various membrane epitopes are degraded by metabolizing cells (see Section 111,D). The design of our experiments was as follows: various murine ascites tumor cells were coated at 4°C with radioiodinated IgG preparations from either xenogeneic or allogeneic antisera directed against the appropriate target cells. The coated cells were placed in the internal chamber of a Marbrook-type culture vessel (Marbrook, 1967) separated by a dialysis membrane from the external chamber which housed the internal one. Both compartments contained culture medium. After various periods of time in culture (usually 1-24 hours), cell-associated radioactivity was determined. At the same time, the radioactivity released into the internal and external chambers was determined. The following results were obtained: (1) after 24 hours of short-term cul-

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ture, only about 1030%of the initial radioactivity was still cell bound. (2) At the same time, about 2030% of the initial radioactivity was found in the internal culture chamber and 4040% of the initial cellbound radioactivity was found in the external chamber, indicating substantial degradation into low-molecular-weight products that could pass through the dialysis membrane. (3) The amounts of radioactive low-molecular-weight products increased in the external compartment as a function of incubation time throughout the entire length of the experiment (24 hours). On the other hand, the amounts of radioactivity shed into the internal compartment reached peak values at about 4 hours after initiation of the experiment and then stayed constant. This may indicate that, during the initial period of incubation, dissociation of apparently intact antibody from the cells is the main event. During the later period of incubation, shedding of the antibody is either accompanied by degradation or, alternatively, most of the antibody shed during this period may have been already degraded. (4) By ammonium sulfate precipitation, it was determined that about 1620% of the radioactivity present in the internal vessel (about 5% of the initial cell-bound radioactivity) was partially degraded. Degradation of antibody can take place outside the cells, i.e., in the medium by proteases released actively or passively from the cells (Rifiin et al., 1974). Since surface-bound proteases have been described to occur on tumor cells (Sylvhn et al., 1974),the possibility also exists that degradation takes place on the cell membrane. The third possibility is that the cell-bound antibody undergoes endocytosis with subsequent degradation (Engers and Unanue, 1973). This problem was also studied by Y. Keisari and I. P. Witz (unpublished). Murine ascites tumor cells were incubated in culture medium containing 1251labeled IgG isolated from a rabbit antiserum and l3II-labeled control IgG (isolated from a rabbit immunized with a non-related antigen). Degradation, if occurring in the medium, should be indiscriminatory and both radiolabeled proteins should have been equally degraded. The results demonstrated, however, that within a period of 24 hours in culture, only the antitumor IgG was degraded, proving that under the experimental conditions employed, degradation took place in the close vicinity of, or inside, the cells. The results also demonstrated that under these conditions, binding of antibody to their target cells is an essential prerequisite for their degradation. An indication that at least some of the antibody is degraded inside the cell was obtained when partially degraded antibody was recovered from detergent-lysed cells. This antibody could not be dissociated from the cells even by low-pH buffers, indicating that most of it was inside the cells (see Section 111,D).

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Analysis of the low-molecular-weight degradation products released into the external vessel of the Marbrook-culture system has revealed that the degradation products were composed of small peptides and amino acids. Preliminary results indicated that shed antibody present in the inner culture compartment lost part of its binding activity, but this point has not been finally established. We were so far unable to detect any blocking activity connected with the shed antibody. This failure may, however, be due to the limited amounts of antibodies that can be used in these experiments (the amounts being limited to those saturating the cells) in contrast to the essentially unlimited amounts of antibodies that can be used in experiments employing cellular lysosomal extracts. Does degradation of Ig by tumor-derived proteases take place in viuo? Although no definite answer to this question can be given, available data suggest that such a process is not unlikely. The findings of Sobczak and De Vaux St. Cyr (1971) showing that Ig fragments were associated with tumor tissue in vivo support the possibility of in viva degradation of Ig by tumor tissue. Similar results were obtained by Cotropia et al. (1975, 1976).These investigators detected IgG and Fab fragments in acid eluates of leukemic blasts, mainly myeloblasts. No Fab fragments were detected in an eluate from a pool of normal leukocytes. All the leukemic eluates contained also the protease inhibitors al-antitrypsin and a-l-antichymotrypsin and some of them contained also the anti protease a-2-macroglobulin. The normal leukocyte eluate contained only the a-l-antitrypsin but none of the other two protease inhibitors. The authors felt that the presence of surface proteases on the malignant cells explains the in uivo binding of the protease inhibitors. Fish et al. ( 1974) showed that the ascitic fluids of two murine tumors contained molecules which had some of the physicochemical properties of IgG but which did not express IgG antigenicity. The fact that tumor cells had the capacity of degrade tumor-binding IgG in vitro led the authors to raise the possibility that the IgG-like molecules detected in vivo were degradation products of IgG. Schedel et al. (personal communication) detected in sera of patients with multiple myeloma circulating (Fab), molecules with anti-Ig antibody activity. These findings can be interpreted to mean that anti-Ig antibodies elicited as a response to antigens of the myeloma protein were specifically fixed to, and subsequently degraded by the malignant cells expressing these antigens. The authors did not test, however, whether or not antibodies with specificities urelated to Ig antigens were also degraded.

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Izzo and Bale (1976) observed a rapid loss of in vivo localized alloantibodies from rat tumors and skin transplants expressing the corresponding antigens. The explanation offered by the authors for this rapid loss was that the localized antibodies were degraded by the target cells, thus depriving shed antibodies from rebinding. No experiments were reported to support this possibility. Keisari and Witz (unpublished) performed preliminary experiments using a slightly different approach. Tumor cells were precoated i n vitro with radiolabeled antibodies and then inoculated into syngeneic mice. Control animals received formaldehyde-fixed cells also precoated with an equal amount of antibodies. The results indicated that more radioactivity was excreted in the urine of mice bearing the viable cells than in urine of the control mice. This may indicate that in the experimental group degradation of Ig was more extensive. The work of Waterhouse (1975) may also be of relevance in connection with Ig degradation by tumor cells. She observed that the total synthesis of IgG in patients with metastatic cancer was increased, whereas the mean survival time of circulating Ig was short, indicating rapid loss from the system. The rapid loss may be explained by degradation. If degradation of antitumor antibodies by tumor-derived proteases does indeed take place i n vivo, the following consequences can be envisaged. The first, most obvious effect would be a specific depletion of antitumor antibodies. Since, as shown above, Ig molecules binding to tumor cells are usually degraded, antitumor antibodies would stand a great risk of being selectively degraded. Such a degradation, if reaching extensive proportions, could bring about a severe specific anergy in antibody-mediated antitumor reactivity. CdL, ADCC, and opsonization would be among the functions affected most severely. Another important, yet untested, consequence of degradation of antitumor antibodies into Fab-like fragments may be specific enhancement of tumor growth. Tumor allografts have indeed been enhanced by Fab fragments of alloantibodies (Chard, 1968; Cruse et al., 1974; Kaliss et al., 1976) and even by Fc fragments of such antibodies (Cruse et al., 1974). The possibility that degraded antitumor antibodies could contribute to tumor enhancement i n vivo is supported by findings mentioned above. Degradation of antitumor antibodies generates fragments that specifically block cellular and humoral cytotoxicity at the target cell level. In addition to these activities, degraded antibody could, most probably, also compete with intact ADCC-mediating antibody for tumor-membrane epitopes. It is not unlikely that tumor-mediated degradation of Ig molecules if occurring in vivo could generate the appearance of hidden antigenic determinants connected with the degradation products. It has been

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known for a long time that determinants undetectable on intact Ig molecules became apparent after proteolytic cleavage (Osterland et al., 1963; Kormeier et al., 1968; Fehr and Lospalluto, 1971; McLaughlin and Solomon, 1973). Antibodies against hidden determinants of Fab fragments are found in some human (Osterland et al., 1963; Harboe et al., 1965; Waller, 1967; Fehr and Lospalluto, 1971) and subhuman primate sera (Litwin, 1970). Anti-Ig antibodies appear also in cancer patients (Lewis et al., 1971), but it is not clear whether these are directed against hidden determinants generated by proteolytic cleavage of Ig. The biological activity of anti-Ig antibodies capable of reacting both with degraded Ig as well as with undegraded molecules is not known at present. It is likely that such antibodies could interfere with various activities connected with humoral immunity. More studies establishing the pattern of anti-Ig antibodies in tumor bearers are, however, required before anything definite can be learned about the role of these antibodies in cancer. Another point that might be of interest in connection with degradation of Ig by tumor-derived proteases is that treatment of various mammalian IgG subclasses with papain rendered them chemotactic to leukocytes (Hayashi, 1975). It may well be that degraded Ig is responsible, at least partially, in attracting inflammatory cells into tumors.

VIII. Biological Functions of TAlg

A. GENERALCONSIDERATIONS Before summarizing the data dealing with the biological significance of TAIg, it should be useful to consider some of the possibilities by which TAIg could intervene in host-mediated antitumor reactivity. As pointed out above, TAIg may be composed of antitumor antibodies as well as of Ig without serological activity toward TAA. In both cases, Ig molecules could be bound either to tumor or to infiltrating host cells. We shall first consider the possible mechanisms connected with a specific binding of Ig to tumor cells. If TAIg contains antibodies capable of activating complement, then local CdL could occur. This possibility is directly supported by the findings summarized in detail in Section V,C on the in vivo coating of a certain murine tumor by antibodies capable of mediating CdL (Ran et al., 1976). Kassel et al. (1973) showed that infusion of normal serum from a variety of species into AKR mice bearing spontaneous leukemia caused destruction of leukemic cells in these mice. Since the active principle in these sera was, most probably, complement, these results

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indirectly support the possibility that antibodies coating in uiuo propagating tumor cells could mediate CdL. Since antitumor antibodies have the capacity to induce ADCC activity in inactive lymphocytes (Pollack et al., 1972; Zighelboim et al., 1973; Lausch et al., 1975; Hakala et al., 1975; Pollack and Nelson, 1975; Lamon et al., 1975; Blair et al., 1976), it is not unlikely that antibodies present in TAIg could perform this function. No data are available to date to support or negate this possibility. Macrophage activation resulting in increased host resistance to a tumor is induced by oposonization of such tumor cells with the corresponding antibodies (Fakhri et al., 1973). The possibility exists that some of the TAIg molecules are oposonins facilitating macrophagetumor cell interaction. Again, nothing is known concerning this question. Specific binding of antibodies to surface antigens could mask these antigens, or some of them. This masking could lower the immunogenicity of the coated cells by interfering with sensitization. In addition, masked antigenic determinants would render the cells less susceptible to CML. Efferent blocking of allogeneic CML in uitro by the corresponding antibody (Moller, 1965; Brunner et al., 1968; Bonavida, 1974; Faanes and Choi, 1974) and antibody-mediated enhancement of tumor growth (Feldman, 1972) support the contention that masking of antigenic determinants may play a role in tumor propagation, although much more direct evidence in tumor-specific systems has to become available in order to prove it conclusively. The coexistence of complement-fixing and noncomplement-fixing antibodies in mouse alloantisera and their competition for the same antigenic determinants was demonstrated (Harris and Harris, 1973). Thus, in uiuo masking of tumor antigens by noncomplement-fixing antibodies will render the coated cells less susceptible to CdL. A possible role of coating antibodies in afferent inhibition of antitumor immunity was suggested by a recent work of Ting and Herberman (1975). They demonstrated that precoating of tumor cells with syngeneic tumor-specific antibodies interfered with the capacity of the cells to evoke antitumor immunity in uiuo. This impaired immunity could be due to the masking of tumor antigens, although it is not the only possible mechanism. It is known that, at least in uitro, antibody does not stay on the cells under conditions allowing intact cellular metabolism (see Section 111,D). I t is thus necessary to postulate that, for an effective in uiuo masking of antigens, either the antibody has to stay fixed on the cells or an active dynamic process has to take place in which antibody molecules shed from the tumor cell are constantly replaced by other (or by the same) molecules. In the latter case, an extensive synthesis of

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antitumor antibodies should occur. It is not clear whether or not the tumor-bearer is at all capable of producing such large amounts of antibody molecules. Moreover, a comparison was made in an allogeneic system (Bonavida, 1974) between the amounts of antibody required to block CML and those required to mediate CdL. Fifty to 200 times more antibody was required to mediate blocking than was necessary for CdL. These facts and considerations raise the question whether biological activities that require large amounts of antibodies such as blocking by masking of antigens are at all possible in viuo. Antigenic modulation (Old et al., 1968) has been proposed as an antibody-mediated escape mechanism from antitumor reactivity (Takahashi, 1971). It is not unlikely that antitumor antibodies may cause the modulation of corresponding antigens in vivo although modulation of TAA by specific antibodies has not been reported so far. In this connection, the results of Sulikeanu et al. (1976b) are most relevant. These authors detected Ig bound to tumor cells present in several types of malignant effusions. In several instances, the Ig on the cells was in a cap shape. Since antibody-mediated capping of membrane determinants may be considered as one of the steps leading toward antigenic modulation, these results suggest that antigenic modulation by TAIg is an in vivo reality. Antigenic modulation may be associated with enhanced shedding of membrane antigens from the tumor cell. Yefenof et al. (1976) investigated the expression of membrane 7 S IgM on Daudi cells (a human B cell line established from an African Burkitt lymphoma), following the in uitro binding of specifically purified antibodies added to the cells at concentrations below saturation levels. They found that concomitantly with shedding of the anti-IgM antibodies during incubation at 37"C, IgM expression on the cell surface decreased compared to uncoated control cells. Some IgM molecules were shed as complexes with antiIgM. 3H-labeled leucine release from antibody-coated cells was considerably enhanced compared to control cells. This might indicate that antigenic modulation involves enhanced membrane catabolism. The shedding of antigen-antibody complexes and the enhanced shedding of membrane components following the binding of antibody may lead to important consequences regarding CML of tumor cells. Antigen and in particular immune complexes of tumor antigen and antitumor antibodies act as potent inhibitors of this immune reactivity (Currie and Basham, 1972; Baldwin et al., 1973b; Hellstrom and Hellstrom, 1974; Jose and Seshardi, 1974; Laux and Lausch, 1974) or as activators for suppressor T cells (Kirkwood and Gershon, 1974; Gershon et al., 1974). In view of these results, one could ask whether or not TAIg is connected with enhanced shedding of tumor antigens or with the in oivo formation of immune complexes.

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Although addition of antibodies directed against membrane antigens to corresponding metabolizing cells results in many cases in a deletion of these antigens, the opposite situation should be considered. Ran et al. (1975) observed that addition of syngeneic antibodies directed against YAC Moloney lymphoma to these cells in vitro, stabilized the expression of the Moloney antigens on the cells. Results compatible with this observation were recently obtained by R. Ehrlich and I. P. Witz (unpublished) using murine EL-4 lymphoma cells. No explanation was offered for this phenomenon. Prehn introduced, a few years ago, the novel theory of immunostimulation of tumor development (Prehn, 1971, 1972; Prehn and Lappe, 1971). Immunological events studied in tumor-bearing animals were compatible with the immunostimulation theory (Fidler, 1974; Fidler et al., 1974; Jeejeebhoy, 1974). Based on i n uitro data showing that interaction of L cells with low concentrations of xenogeneic antibodies resulted in stimulation of DNA synthesis and cell growth in the treated cells (Shearer et al., 1973, 1974), attempts were made to obtain immunostimulation of L cells in uivo (Fink et al., 1975). It was found that tumor growth in antiserum-injected mice was significantly enhanced. It was necessary to provide evidence that the enhanced tumor growth was not due to blocking of cell-mediated immunity. This was achieved by using hosts whose cellular immunity functions were severely depressed by thymectomy and lethal irradiation. These results permit the hypothesis that TAIg may participate in immunostimulation of tumors. This hypothesis is amenable for experimentation. The following biological effects could be obtained by binding of Ig by Fc-receptor-bearing host cells lodging in the tumor. One might expect that Fc receptors on such cells could be completely or partially saturated either by immune complexes unrelated to the tumor system or, more likely, by complexes between tumor antigen and the corresponding antibodies. Such a saturation may eliminate or lower ADCC activity in the vicinity of the tumor, thus affecting the local immunological reactivity against tumor cells. This possibility is, however, not supported by experiments demonstrating considerable ADCC activity of tumor-derived K cells (Tracey et al., 1975). Binding of immune complexes by Fc-receptor-bearing cells may cause activation rather than suppression of ADCC activity. Perlmann et al. (1972) and Greenberg and Shen (1973) demonstrated that binding of antigen-antibody complexes by normal (K?) lymphocytes conferred specific effector functions on these lymphocyte populations. Furthermore, Saksela et al. (1975) showed that antibody alone can induce specific cytotoxicity in normal lymphocytes. Thus, i n uiuo binding of antitumor antibodies complexed with tumor antigen or

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even of antitumor antibody alone onto Fc-receptor-bearing host cells may render them specifically cytotoxic to the tumor cells in the vicinity. It has been reported above (Section VI) that nonlymphoid tumor cells, per se, may express Fc receptors on their membrane. Such tumor cells may compete against Fc-receptor-bearing host cells for Fc fragments of antitumor antibodies, resulting in decreased antitumor reactivity. One of the more basic questions regarding TAIg is whether it plays any biological role in the very initial stages of primary tumor development. No data are available concerning this particular question. However, findings on the occurrence of natural antitumor antibodies in normal individuals (Herberman, 1969; Martin and Martin, 1975; Pierotti and Colnaghi, 1975) are possibly relevant to this problem. The possibility that such antibodies could absorb onto a clone of malignant cells and exert various biological functions such as those summarized above is not ruled out. B. AVAILABLE INFORMATION Most of the available data suggest an inverse relationship between the presence of Ig in tumors and an effective host antitumor reactivity. Sjogren and Bansal (1971), Bansal et al. (1972), and Sjogren et a l . (1972) have shown that acid eluates from polyoma virus-induced tumors in rats and from various human tumors abrogated cytotoxicity of the respective tumor cells by immune lymphocytes or by lymphocytes of tumor bearers. These results, which exhibited the expected specificity, suggested that Ig which might have been present in these tumor eluates, but whose presence was not verified, exhibited the blocking activity. However, results obtained recently in our laboratory suggested that the presence of antigen in acid eluates is not unlikely. The blocking activity reported above may have thus been due to the presence of immune complexes in the eluates or even to uncomplexed antigen. Romsdahl and Cox (1975) showed that low pH eluates of human sarcoma tissue blocked lymphocyte-mediated cytotoxicity of cultured sarcoma cells in microcytotoxicity assays. Purified IgA or IgG preparations from these eluates also had marked blocking activity. These results, although indicating that the blocking molecule is connected with Ig, do not prove that Ig is the only entity involved. IgA or IgG complexed with sarcoma antigen may have been responsible for the blocking activity. Bansal et a l . (1972) and Ran and Witz (1972) demonstrated that acid

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eluates from syngeneic polyoma virus-induced rat tumors and from

methylcholanthrene-induced murine tumors enhanced the in vivo growth of these tumors in the respective hosts. Also in these experiments, as in those reported above, the possibility cannot be excluded that tumor antigen alone or immune complexes were the tumor-

enhancing factor. The unmasking experiments of Stjernswiird and Vanky (1972)and of Vanky et al. (1973b) also supported the hypothesis that TAIg may be involved in abrogation of a successful antitumor resistance. These authors have shown that human cancer cells express in many cases the capacity to specifically stimulate DNA synthesis in autologous lymphocytes. The capacity to induce lymphocyte stimulation is considered to represent an immune reaction. Cells originating from some of the assayed cancer biopsies were incapable of lymphocyte stimulation. Such cancer cells could sometimes be rendered stimulatory by treating them with a low-pH buffer. Exposed antigenicity by removal of a masking antibody coat was postulated to be the responsible mechanism. However, no attempt was made in these reports to verify this hypothesis. A recent work by Vanky et al. (1975) provided further support for the masked-antigenicity hypothesis. These authors demonstrated an inverse relationship between the presence of Ig in the cancer biopsies and the capacity to stimulate DNA synthesis in autologous lymphocytes. Of 18 cancer biopsies that contained Ig, 17 did not stimulate autologous lymphocytes. However, of the 26 Ig-negative biopsies, only about 50% were stimulatory. This might indicate that although absence of TAIg is a necessary prerequisite for the capacity of cancer cells to stimulate lymphocytes, it is not the only one. A prospective study on 25 cancer patients revealed a correlation between the malignant behavior of the tumor and the presence of Ig in the same tumor (Izsak et al., 1974).Clinical grading of malignancy was assessed by examination of 12 measurable criteria belonging to four major parameters: anatomical spread of disease, histopathological grading, rate of tumor growth, and host factors reflecting morbidity and adaptability of the patient. Each of the 12 criteria was given a score of 0 to 4.The maximum score of 48 indicated the most malignant situation. All scores of more than 24 were considered as highly malignant, whereas those of less than 24 were considered as low malignant. Out of 12 highly malignant tumors, 9 were found to be associated with Ig. Out of 13 tumors graded as low-malignant, 8 did not contain detectable amounts of Ig. The positive correlation between the presence of TAIg and malignancy, the negative correlation between presence of Ig and the capacity to stimulate lymphocytes, and the blocking and enhancement experiments support the contention that TAIg may be connected with

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the failure of the immune system in established cancer and with unfavorable prognosis. Such a generalization may, however, not be justified. In acute myelogeneous leukemia, for instance, presence of Ig on the malignant cells correlated with favorable prognosis (Gutterman et al., 1973). Ran et d.(1976) observed that a polyoma virus-induced murine ascites tumor was coated with potentially cytotoxic antibodies. Addition of an exogenous source of complement caused lysis of the cells. Antibody capable of inducing CdL of indicator tumor cells was eluted from the tumor cells propagatingin uiuo. This antibody resided in the IgG2 fraction isolated from low-pH eluates (N. Moav and I. P. Witz, unpublished). Eluates containing high titers of cytotoxic antibodies caused a significant retardation of the growth of the corresponding tumors in uitro, while eluates containing low titers of antibodies had no effect (M. Ran and I. P. Witz, unpublished). Preliminary experiments performed by Witz and Yacubovicz showed that low-pH eluates of TA3 cells amplified the cytotoxic activity (measured by 51Crrelease) of lymphocytes from tumor bearers. Although the unfractionated eluates used in these experiments contained IgG, it is impossible at the present stage of the work to state that these molecules, alone, were responsible for the observed biological activities. Such a conclusion will be possible only if identical results were obtained with purified Ig isolated from these tumor eluates. No discussion of the biological significance of TAIg should be considered complete unless some mention is made of the phenomenon of coating of embryonic cells or trophoblastic basement membrane by Ig, most probably of maternal origin (McCormick et al., 1971; Girardi et al., 1973; Faulk et al., 1974a,b; Voisin and Chaouat, 1974), and of the presence of Fc receptors on the surface of placental cells (Jenkinson et al., 1976). Ig eluted from trophoblastic tissue exhibited several biological effects, such as inhibition of mixed lymphocyte reaction (MLR)and blastogenesis to tuberculin and PHA (Faulk et al., 197413). IgGl eluted from mouse placenta was found to be directed against paternal antigens. Such antibodies enhanced tumor growth of paternal origin in otherwise untreated mice belonging to the maternal strain (Voisin and Chaouat, 1974). Some investigators pointed to similarities between escape mechanisms employed by embryos and cancer cells from the maternal or tumor bearer’s immunity system (Alexander, 1974; Coggin and Anderson, 1974; Hellstrom and Hellstrom, 1975).Association of Ig with these two types of proliferating entities provides additional support for such a similarity. IX. Concluding Remarks

The catalog of immunological data is ever growing. New findings and novel theories alter concepts and replace dogmas that only re-

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cently were considered as classical and fundamental. One such case is the role of cellular and humoral immunity in the resistance against antigenically distinct cancer cells. Cellular immunity is beneficial to the tumor bearer and as such should be stimulated, whereas humoral immunity antagonizes the expression of cellular immunity and should, therefore, be selectively suppressed. This was the conclusion drawn by many cancer immunologists when discussing their own work or that of their colleagues. Although the pendulum does not seem to be swinging to the opposite direction, a more balanced view of cancer immunology is recently evident. Lymphoid cells or macrophages, known to function as efficient antitumor effectors in some tumor systems, at least in uitro, were found to exert suppressor functions (Gorczynski, 1974; Kirchner et al., 1974, 1975). Macrophages reacting with antibody-coated tumor cells protected these tumor cells from CdL or CML (Hershey and MacLennan, 1973). T cells were shown to be involved in the synthesis of blocking factors (Nelson et al., 1975) or even to enhance tumor growth (Umiel and Trainin, 1974; Carnaud et al., 1974; Treves et al., 1974; Rotter and Trainin, 1975).On the other hand, antibodies were sometimes found to contribute positively to antitumor immunity (Currie and Sime, 1973; Shin et al., 1974; Seemayer et al., 1974). All this rapidly accumulating information clearly demonstrates the complexity of antitumor immunity and suggests very strongly that many factors acting in concert or antagonizing each other contribute their share to a delicate balance that is very easily tipped. In order to at least attempt to understand the factors maintaining the balance, one needs a relevant departure point. We agree with those who advocate the tumor site as the most logical choice. Studies on immunoglobulins present within tumors can and should continue in several directions, the most important being the need to gain a deeper insight into their biological significance. One of the feasible approaches to this problem would be to assess the pattern and behavior of TAIg as well as that of tumor-seeking Ig-binding host cells in relation to prognosis, various types of treatments (including immunotherapy using immunomodulators) and clinical status. Such information would also be of great value to the clinician. Another approach involves monitoring the effects on tumor growth produced by passive transfer of TAIg or by altering its levels. Another problem concerned with TAIg molecules is their specificity. A promising approach to this problem is the use of systems in which the serological characteristics of the tumor cells are well analyzed. Studies on TAIg could contribute their share to cancer therapy and detection. Localization of antitumor antibodies in tumors is an impor-

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tant factor in attempts to guide toxic drugs to the malignant cells (Davies and O’Neill, 1973; Davies, 1974; Rowland et al., 1975) and in trials to detect occult tumors. The tumor tissue acting as an in vivo active immunoadsorbent could serve as a source of specific antibodies. A promising and important step in this direction was taken by Terman et al. (1975) demonstrating that IgG isolated from eluates of mouse neuroblastoma and injected into neuroblastoma-bearing mice localized preferentially in the tumor. Ig isolated from human tumors could be used both in immunodiagnosis and immunotherapy if the studies with animal systems are extended and applied to human cancer.

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