Immunosuppressive Factors in Human Cancer

IMMUNOSUPPRESSIVE FACTORS IN HUMAN CANCER Dov Sulitzeanu The Lautenberg Center for General and Tumor Immunology, The Hebrew University, Hadassah Medical School, Jerusalem

I. Introduction

11. Immunosuppression in Cancer-Recent

Evidence 111. Immunosuppressive Factors Produced by Tumor Cells/Cell Lines IV. Well-Characterized Immunosuppressive Molecules A. Transforming Growth Factor p B. Lymphocyte Blastogenesis Inhibitory Factor C. P15E D. Suppressive E-Receptor V. Partially Characterized Immunosuppressive Factors VI. Immunosuppressive Factors in Sera and Effusions VII. Other Immunosuppressive Factors A. Colony-Stimulating Factors B. Acute-Phase Proteins C. Miscellaneous Molecules VIII. Concluding Remarks: Immunosuppressor or Growth-Regulatory Cytokines? References

I. Introduction Patients with advanced cancer are frequently found to exhibit impaired immune responses, as demonstrated by a variety of in vitro reactions (Stutman, 1975; Kamo and Friedman, 1977; Cianciolo and Snyderman, 1983; Aune, 1987; Nelson and Nelson, 1987; Schulof et al., 1987; Cianciolo, 1988; Brunson and Goldfarb, 1989). Early research on immunosuppression in cancer was focused mainly on defective responses at the cellular level: depressed macrophage and polymorphonuclear cell functions (migration, phagocytosis, bactericidal activity); depressed Ig production by B cells; reduced natural killer (NK) cytotoxicity; and, most often, the inability of the patients’ lymphocytes to respond to mitogenic stimuli (lectins, antigens, or alloantigens). More recently, attention has shifted to defects in cytokine release [particularly interleukin-2 (IL2)] and cytokine receptors. The exact mechanism of the immunosuppression is still uncertain, but a multiplicity of factors have been postulated to contribute to its occurrence: suppressor T cells and

247 ADVANCES IN CANCER RESEARCH, VOL. 60

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suppressor macrophages, immune complexes, acute phase proteins, tumor-derived suppressor molecules, suppressor substances in sera and effusion fluids of patients, and chemotherapy and irradiation. In this article, I propose to review succinctly recent studies (published mainly during the last 5 years) on immunosuppressive molecules (mostly of human origin) elaborated by tumor cells or found in sera and effusions. It is true that the active molecules have only rarely been purified to homogeneity or identified with certainty. However, progress is being made and we believe that an assessment of the present state of the art may be instructive and also useful in charting further directions of research. II. Immunosuppression in Cancer-Recent

Evidence As mentioned above, a great deal of the early research on immunosuppression in cancer has dealt with the poor proliferative response obtained after treatment of lymphocytes with mitogenic stimuli. The advent of the lymphokine activated killer (LAK) cell era (Rosenberg, 1988) presented immunologists with a new target and prompted a flurry of activity aimed at showing that peripheral blood lymphocytes (PBL) of patients with advanced cancer or of tumor-bearing animals were unable to develop into fully effective LAK (Monson et al., 1987; Shu et al., 1987; Ting et al., 1987; Maccubbin et al., 1989; Favrot et al., 1990) or cytotoxic lymphocytes (CTL) (Shu et al., 1987; Maccubbin et al., 1989). Lymphocytes isolated from tumors [tumor-infiltrating lymphocytes (TIL)] were also found to proliferate poorly and to acquire only limited cytotoxicity (Whiteside et nl., 1988). Interest now appears to have switched to the impaired production of cytokines or of their receptors by PBL of cancer patients (Herman et al., 1985; Monson et al., 1986; Elliot et al., 1987; Mantovani et ul., 1987; Hakim, 1988). However, reports of normal I L 2 production by PBL of patients with malignant disease (Hargett et al., 1985; Wanebo et al., 1986; Eskinasi et al., 1989) and of normal IL-1 production by cells of tumor-bearing mice (Holan and Lipoldova, 1990) may indicate that such impairment does not always account for the observed immunosuppression.

I l l . Immunosuppressive Factors Produced by Tumor Cells/Cell Lines

Culture supernatants of tumor cells of all types and of all species have been found by numerous investigators to contain strongly immunosuppressive factors, as evidenced by their capacity to inhibit a variety of immune reactions (Spector and Friedman, 1983; Aune, 1987; Nelson

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and Nelson, 1987; Cianciolo, 1988; Brunson and Goldfarb, 1989): delayed-type hypersensitivity; macrophage accumulation at sites of inflammation; macrophage chemotaxis, phagocytosis, and cytotoxicity; skin graft rejection (Gresser et al., 1975); lymphocyte proliferation in response to mitogenic stimuli; antibody synthesis; generation of LAK, TIL, and CTL cells; and lymphokine production (see details and references in Table I). A multitude of substances derived from human or animal tumors, capable of suppressing immunocyte functions, have been described in the literature. Unfortunately, in most cases, the characterization of the active molecules has been only partial at best; therefore it is not possible to determine to what extent these publications might be overlapping. Only a few of the immunosuppressive factors have been studied in sufficient detail and have been characterized at the molecular level; these will be reviewed first. Other factors, described sporadically in recent years, will then be listed. For references to older work, dating in fact to the beginning of tumor immunology, the reader is referred to the earlier reviews cited at the beginning of this section.

IV. Well-Characterized Immunosuppressive Molecules A. TRANSFORMING GROWTHFACTORp Transforming Growth Factor p (TGF-P) was identified originally by its ability to impart a transformed phenotype to normal fibroblasts. Several closely related molecules constituting the TGF-P family were discovered subsequently (three in mammals). TGF-P is produced by most cells (Wakefield et al., 1987) and has an extraordinarily wide range of biological activities (for recent reviews, see Bascom et al., 1989; Sporn and Roberts, 1989; Barnard et al., 1990; Roberts et al., 1990; Moses et al., 1991). Of particular relevance to this review are its strong inhibitory capacity for epithelial cells of both normal and malignant origin (Barnard et al., 1990) and its potent immunosuppressive activity (Palladino et al., 1990; Lucas et al., 1991). TGF-P inhibits T- and B-cell proliferation (Kehrl et al., 1986a,b, 1989; Wahl et al., 1988a) and expression of B-cell activation markers (Cross and Cambier, 1990); it inhibits LAK and CTL generation (Ranges et al., 1987; Espevik et al., 1988; Grimm et al., 1988; Kasid et al., 1988; Mule et al., 1988; Fontana et al., 1989; Jin et al., 1989; Geller et al., 1991; Smyth et al., 1991; Tada et al., 1991), NK cytolytic activity (Rook et al., 1986; Lucas et al., 1991), and macrophage oxygen metabolism (Tsunawaki et al., 1988). It downregulates Human Leukocyte Antigen (HLA) DR (Czarniecki et al., 1988; Zuber et al., 1988) and

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TABLE I IMMUNOSUPPRESSIVE FACTORSPRODUCED BY TUMOR CELLS/CELL LINES

Tumor Chlorectat cancer; other tumor extracts Various tumor cell supernatants r-cell leukemia

Factor size (kD,) 4.5 ND 88

>SO0

Characteristics and biological activity Acid extract inhibits response to mitogen, alloantigen; PI ( 3 . 0 ; sensitive to trypsin Inhibit mitogenic response of PBL

Hoagland et al. (1986)

Inhibits proliferation response of normal T cells to mitogens, alloantigens; inhibits proliferation of malignant, hemopoietic cells, normal myelomonocytic progenitor cells Inhibits response to Con A and to IL-2; suppress IL-2 production; inhibits response to B-cell mitogen; stable to heat, trypsin; not stable to acid PI 7.9; inhibits response to mitogen; IL-2 production; sensitive to heat at 56°C. pH extremes Inhibits response to mitogen, antigen, alloantigen Inhibits response to mitogens, DTH; delays rejection of syngeneic tumor Inhibits response to mitogen, IL-2; sensitive to heating at 56°C

Santoli et al. (1986)

T-cell leukemia

50,70

Colon cancer

56

Promyelocytic leukemia (;olon carcinoma

ND

H ydatidiform mole

35-50

T-cell leukemia

80,11.5

Suppress T-cell proliferation; highly purified preparations with PIS 3.5 and 7.4, respectively

T-cell leukemia

66

B CLL

<5

Melanoma

225

Stable at 56°C; cytostatic; inhibits response to PHA Inhibits response to PHA, IL-2 production; susceptible to neuraminidase Heat stable (56°C); inhibits LAK cell generation; produced also by gastrointestinal cancer cell line

ND

Reference

Miescher et al. ( 1986)

Shirawaka et al. (1986); Tanaka et a/. (1987) Ebert et 01 ( 1 987) Paietta et al. (1987) Pommier et al. (1987) Bennett et al. (1988); Cowan et al. ( 1989) Montaldo et al. (1988); Ponzoni et al. ( 1988) Abolhassani el al. (1989) Burton P t al. ( 1989) Guillou et al. (1989a,b)

I

(continued)

IMMUNOSUPPRESSIVE FACTORS IN CANCER

25 1

TABLE I (Continued)

Tumor

Factor size (kD,)

Choriocarcinoma

ND

Renal cancer

ND

T-cell line (Jurkat) Lung cancer

ND >150

Characteristics and biological activity

Reference

Inhibits response of T cells to phorbol esther, calcium ionophore, IL-2; also response to lectins, alloantigens; inhibits generation of alloreactive CTL Inhibits response to mitogen, IL-2 production; nondialyzable; sensitive to 56°C Inhibits response to mitogen, alloantigen; inhibits Ig production Inhibits response to PHA, IL-2 production; inhibits IL-2-dependent proliferation of activated lymphocytes; sensitive to trypsin, 60°C

Matsuzaki et al. (1989a,b)

Muraki et al. (1989) Telerman et al. (1989) Wang et al. ( 1989)

Note. ND: no data.

IL2R (Kehrl et al., 1986a), and it delays rejection of heart allografts (F’alladino et al., 1990). TGF-P was identified as a tumor-associated immunosuppressive molecule following a series of studies of immunosuppression in glioblastoma. It had been known for some time that patients with glioblastoma responded poorly in T-cell-mediated immune reactions (see Siepl et al., 1988, for brief summary of early work). Subsequent investigations prompted by these observations led to the demonstration that sera of glioblastoma patients contain an immunosuppressive factor (Brooks et al., 1972). A similar factor, which inhibited IL2-induced proliferation and CTL generation, was found in glioblastoma cell culture supernatants (Fontana et al., 1984) as well as in freshly explanted malignant glioma cells (Roszman et al., 1987). The factor was identified as TGF-P2 (Wrann et al., 1987; Siepl et al., 1988; Bodmer et al., 1989). Further work showed that TGF-P mRNA was found in all tumors tested (Derynk et al., 1987) and that the protein was made by many tumor cells lines: rhabdomyosarcoma (Romeo and Mizel, 1989), colorectal carcinoma (Coffey et al., 1986), breast carcinoma (Knabbe et al., 1987; Arteaga et al., 1990), endometrial carcinoma (Boyd and Kaufman, 1990), prostatic adenocarcinoma (Ikeda et al., 1987), and thyroid carcinoma (Jasani et al., 1990). TGF-P was also demonstrated in ascitic fluids of patients with ovarian

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(Hirte and Clark 1991; Wilson et al., 1991) and breast cancer (Hatzubai and Sulitzeanu, 1992), and in ascitic fluids of mice bearing plasmacytoma (Berg and Lynch, 1991) or hepatoma (Tada et al., 1991). TGF-P is probably responsible for many of the immunosuppressive activities attributed to “suppressor” lymphocytes and macrophages. The role of TGF-P as a mediator of suppressor cell activity is nicely demonstrated by the work of Wahl et al. (1988b), who showed that the immunosuppression induced in rats by injection of GpA streptococcal cell walls was due to TGF-P released by macrophages. B. LYMPHOCYTE BLASTOGENESIS INHIBITORY FACTOR Lymphocyte blastogenesis inhibiting factor (LBIF), a newly identified cytokine produced by the U937 macrophage cell line (Fujiwara and Ellner, 1986; Fujiwara et al., 1987), was purified to homogeneity by Sugimura et al. (1988, 1989). It is a 45-kDa polypeptide chain, with a PI of approximately 4.5, possessing a variety of immunosuppressive activities (Sugimura et al., 1990); it inhibits I L 1 , IL-2, antigen- and lectininduced proliferation, and expression of the IL-2 p75 receptor; it arrests lectin-stimulated T cells at early G1 phase; and it acts on both human and murine lymphocytes. Furthermore, it is cytostatic or cytotoxic for numerous tumor cell lines of various lineages, at very low concentrations-of the order of 10-40 ng/ml. In fact, its growth-inhibitory activity was manifested against a wider range of targets than that of other cytokines tested (TGF-P, I L 1-p, TNF-a, IFN--y, IFN-a). Cytokine LBIF appears to share with TGF-P the ability to act both on lymphocytes and on epithelial cells. These cytokines should therefore be considered both immunosuppressors and growth inhibitors. C. p15E It is well known that infection with retroviruses may cause a marked impairment of immune responsiveness (Snyderman and Cianciolo, 1984). Mathes et al. (1978, 1979) were the first to publish evidence implicating p15E, a transmembrane retroviral envelope protein, as a major mediator of retroviral immunosuppression. Subsequent studies revealed that retroviral structural components and the p15E protein possess a remarkable range of immunosuppressive activities (Cianciolo and Snyderman, 1983; Cianciolo, 1988; Brunson and Goldfarb, 1989): they inhibited lymphocyte blastogenesis, tumor immunity in uiuo, macrophage accumulation in vivo, and monocyte chemotactic response and erythroid colony formation in vitro. Substances crossreacting with p 15E

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253

were demonstrated in murine and human cell lines, in plasma of patients with hematologic disorders (Jacquemin et d., 1984), and also in human malignant effusions. Addition of effusion fluid inhibited the monocyte response to chemoattractants and this inhibition could be reversed by anti-p15E antibodies. Cianciolo et al. (1985) prepared a synthetic, 17 amino acid peptide, homologous to a highly conserved region of the p15E proteins of murine and feline retroviruses, of HTLV I, 11, and 111 and also of a putative protein encoded by an endogenous C-type human retroviral DNA. Conjugates of this peptide inhibited the proliferation of an IL2dependent murine CTL line and of alloantigen-stimulated murine and human lymphocytes. It also inhibited the cytocidal activity of recombinant I L l - a and -p against A 375 melanoma cells (Kleinerman et al., 1987) and the cytotoxicity of human N K cells (Harris et al., 1987). It suppressed the respiratory burst of human monocytes (Harrell et al., 1986) and their ability to produce interferon-y in response to stimulation by staphylococcal enterotoxin A (Ogasawara et al., 1988). Antibodies to CKS- 17 neutralized the capacity of tumor cell culture supernatants to inhibit the production of I L 2 by EL-4 cells stimulated by mitogen (Nelson and Nelson, 1990). Conjugates of CKS-I 7 depressed delayedtype hypersensitivity (DTH) reactions in mice (Nelson et al., 1989) and this effect could be prevented by actively immunizing the mice with conjugated CKS- 17 or by injecting the animals with immunoglobulin of immunized mice. Such immunization could protect not only against the effects of CKS-17 but also against similar effects of tumor products (Nelson et al., 1985, 1989). The CKS-17-like factor produced by tumor cells is reported to be a molecule of -18 kDa (Nelson and Nelson, 1990), but, regrettably this molecule has not been characterized any further. Its relation to the 74kDa protein (Jacquemin et al., 1984) and to the other crossreacting molecule found in dividing cells (Snyderman and Cianciolo, 1984) is not known. Its origin as an endogenous human retrovirus product (Lindvall and Sjogren, 1991) cannot be considered proven, although this possibility must be taken into account, in view of the reported presence of such viruses in human cells (Larsson et al., 1989; Wilkinson et al., 1990). D. SUPPRESSIVE E-RECEPTOR T h e suppressive E-receptor (SER) was isolated by Oh and co-workers (1987a) from malignant effusions derived from patients with several types of cancer (ovarian, lung, head and neck). This immunosuppressive molecule inhibited E rosette formation (hence the name) as well as sever-

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a1 cellular immune responses, both in nitro and in vivo (Oh et al., 1987b, 1988, 1990). These included T-cell proliferation in response to mitogen and alloantigen, Ig synthesis induced in zdro by PWM + PMA, antibody response to T-dependent antigen in nivo, N K function, and polyrnorphonuclear phagocytosis. T h e molecule was identified as a polymeric form of haptoglobin, related immunochemically to neonatal haploglobin. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, SER yielded two polypeptides (38-42 kDa and 17-19 kDa), with N-terminal amino acid sequences identical to those of the f3 and a-2 subunits of adult haptoglobin (Oh et al., 1987a). T h e SER is secreted by macrophages, which, however, do not synthesize haptoglobin. The authors propose that SER may be an oxidized form of plasma haptoglobin, generated by macrophages (Oh et al., 1989). Thus, SER is not a tumor-derived, but rather a tumor-associated, molecule. V. Partially Characterized Immunosuppressive Factors

A list of publications describing recently studied immunosuppressive factors is given in Table I. Despite the meager data available, it seems that a large number of molecules with immunosuppressive activity may be produced by tumor cells, judging from the considerable variation in physicochemical properties reported by the different investigators. Unfortunately, little information is available on the production of analogous substances by nonmalignant cells (Brunson and Goldfarb, 1989). Several such substances, tested mainly for growth-inhibitory activity, are listed in Table 11.

VI. Immunosuppressive Factors in Sera and Effusions

Immunosuppressive factors can be found in normal sea, but their level is increased in cancer sera (Wile, 1989).These factors may contribute to the defective cellular responses of' patients. Thus, cells of patients with lung cancer, which exhibited low reactivity to rnitogens, responded normally when tested in the presence of sera of healthy donors (Hadjipetrou-Kourounakis et af., 1985). Numerous immunosuppressive factors (generally poorly characterized) have been described in sera and effusions of patients (see Table 111 for a list of recent publications). Of these, only SER and the pl5E-related molecule have been studied in greater detail (Section IV).

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IMMUNOSUPPRESSIVE FACTORS I N CANCER

TABLE I1 INHIBITORY FACTORSPRODUCED BY NORMAL TISSUES Human trophoblast cells Human lung cells

Suppression of NK activity

Saji et al. (1987)

Suppression of lympyhocyte blastogenesis, NK activity

Rat liver

Growth inhibitor for several cell types

Human, rat liver cells Normal mouse lung Human fetal liver cells

Inhibition of liver cell proliferation

Baughman and Strohofer (1989) Chapekar et al. (1989) Chen et al. ( 1989) Ikuta et al. ( 1989) Wu et al. (1989)

Growth inhibitor of mouse monocytic leukemia cells Inhibitor of HL-60 cell proliferation

TGF-P (Section IV,A) has been identified in ascitic fluids and further work is likely to show it to be a major contributor to the immunosuppressive activity of effusions. In the course of a systematic fractionation of effusions from patients with breast cancers, aimed at identifying immunosuppressive and growth-inhibitory factors contained therein, several of the fractions were found to contain TGF-P-like molecules, as determined by inhibition tests with anti-TGF-P serum (Hatzubai and Sulitzeanu, 1992). T h e multitude of seemingly different factors described in the literature seem to indicate that effusion fluids (and presumably, also patients’ sera) may be teeming with a variety of cytokines, with immunosuppressive and probably other biological activities. The results of our fractionation studies, which show immunosuppressive and growth-inhibitory activity in many fractions, differing greatly in physicochemical properties, suggest that this assumption is probably correct. VII. Other Immunosuppressive Factors A. COLONY-STIMULATING FACTORS

Tumor cell lines, particularly of mouse, but also of human origin, have often been shown to secrete appreciable amounts of colony-stimulating factors (CSF) (Hardy and Balducci, 1985; see also Ralph et al., 1986: Tweardy et al., 1987’;Evans et al., 1989; Kacinski et al., 1989). This apparently also occurs in vivo, since increased CSF-1 levels can be demonstrated in patients’ sera (Hardy and Balducci, 1985; Kacinsky et al.,

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TABLE 111

I M M U N O S U P P R E S S I V E SUBSTANCES IN SERA A N D

ASCITES

Source

Type of cancer

Serum

Stomach

Inhibition of PBL response

Stomach

Inhibition of PHA cytotoxicity

Ly mphocytic leu kemia (rats) Leu kosis, bovine B-CLL

Inhibition of PHA, Con A response

Melanoma Various Lung, esophageal cancer H&N cancer Ascites

Various Gastrointestinal

P-8 I5 niastocytoma (mouse) Walker 256 carcinoma (rat) Various

Characteristics and biological activity

Reference Kanayarna et al. (1985) Sugiyama et al. (1987) Strumberg et ul. (1988)

Inhibition of mitogen stimulation, phagocytosis, other immune fuctions Reduction of PHA-induced IL-2 production Inhibition of LAK activation and of PHAinduced cytotoxicity; factor production dependent o n nionocytes Inhibition of lymphocyte blastogenesissmall molecules Suppressor of macrophage phagocytosis

Takamatsu et al. (1988) Burton et al. (1989) Itoh et al. (1989)

Inhibitor of LAK generation

Bugis et al. ( 1990) Medoff et al. ( I 986)

.iO-kDa molecule; pl 3.4; resistant to proteolysis; suppressed response to PHA Inhibition of PHA response and of DTH a 1-acid glycoprotein Molecule smaller than 10 kDa; lipid-like; resistant to proteolysis; inhibited response to Con A Eight hands of suppresssor substances, causing suppression of PHA induced splenocyte proliferation, obtained by preparative PAGE Inhibitor of LAK cell induction

Wile (1989) Zhou et al. (1989)

Fujii et a/. (1987) Cornelius and Normann (1988) Cohen et al. (198s) Pelton et al. (1991)

1989). Hemopoietic alterations have indeed been seen in both human and experimental cancer, including increased production of granulocytes and macrophages, and decreased B cells, thymocytes, and NK cells (Fu et al., 1991). Although the colony-stimulating factors are not immunosuppressive by themselves, they stimulate proliferation and differentiation of hemopoietic cells and induce the appearance of mac-

IMMUNOSUPPRESSIVE FACTORS I N CANCER

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rophages possessing immunosuppressive activity (Hardy and Balducci, 1985; Tsuchiya et al., 1988; Young et al., 1989; Fu et al., 1990, 1991). Daily injections of granulocyte macrophage-CSF into mice caused immunologic alterations similar to those noted in tumor-bearing animals. The macrophage-associated suppression was attributed to cell-to-cell contact as well as to release of PGE,, since the effect could be partially reversed by indomethacin (Fu et al., 1991). B. ACUTE-PHASE PROTEINS

Acute-phase proteins (haptoglobin, al-acid glycoprotein, ceruloplasmin, C-reactive protein, and others) are produced by the liver in response to inflammation and are found in increased amounts in sera of patients with cancer (briefly reviewed by Samak et al., 1982). Some of them were found to depress lymphocyte proliferation. Synthesisof acute phase proteins is mediated by a number of cytokines, which may be produced directly by the tumor cells (e.g., hepatocyte-stimulatingfactor 111, Baumann and Wong, 1989) or indirectly, by tumor products acting on macrophages (Evans et al., 1991). C. MISCELLANEOUS MOLECULES

A number of additional molecules have been studied as potentially immunosuppressive: gangliosides (Krishnaraj et al., 1982; Ladisch et al., 1983; Robb, 1986), prostaglandins (Aune, 1987; also see Droller et al., 1978; Chouaib et al., 1985; Kicza and Szkaradkiewicz, 1986; Leung, 1989), 1-methyl-adenosine(Takano et al., 1986),and uric acid and uracyl (Sami et al., 1986). However, interest in such molecules has been rather marginal and therefore they are mentioned only briefly here. VIII. Concluding Remarks: lmmunosuppressor or Growth-Regulatory Cytokines?

There can be no doubt that tumor cells release into the culture medium (and most likely also in vim) a fairly large number of cytokines with immunosuppressive activity. Unfortunately, in the vast majority of cases, the identification of these cytokines (some of which might be as yet unknown) has lagged far behind the description of their immunosuppressive properties. Given the technical difficulties involved in isolating the tiny amounts likely to be present in the culture supernatants or body fluids and the apparent multitude of factors, this is not unexpected. However, this is a challenge which, one hopes, will be met, since the

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presence of these cytokines in body fluids cannot fail to affect the hosttumor relationships. A tumor-derived cytokine likely to draw increasing attention as a cancer-associated immunosuppressive molecule is TGF-P. Many tumors have already been shown to secrete this highly active immunosuppressive molecule in vitro and although in uivo secretion has been little studied so far, one can expect it to be as widespread as in vitro secretion. TGF-P is a major suppressor factor in ovarian cancer ascites (Hirte and Clark, 1991), where it is quite likely to be derived, at least in part, from malignant cells. TGF-f3 may well be a major contributor to the impairment of immune functions (decreased DTH and reduced proliferation on phytohemagglutinin (PHA) or alloantigen stimulation-Wiebke et al., 1988; Hank et al., 1990; Favrot et al., 1990) seen to develop in patients undergoing IL-2 therapy. Interleukin-2 is a potent inducer of LAK cells and these have been shown to release appreciable amounts of TGF-P (Kasid et al., 1988; Larisch-Bloch et al., 1990), which most probably inhibits further LAK cell development as well as other immune reactivities. Interestingly, unlike most other cells, which secrete latent TGF-P, the LAK cells appear to secrete a biologically active form of the molecule, which nevertheless is attached to a large carrier protein (LarischBloch et af., 1990). Activation could occur, however, in the culture supernatant, through the action of proteolytic enzymes (Kehrl et al., 1989). Many immunologists appear to incriminate tumor-derived (or tumorinduced) immunosuppression as a major contributor to the failure of immunotherapy to affect the course of the disease (Mule et al., 1988; Guillou et al., 1989,; Barba et a/., 1989). Immunosuppression has indeed been proposed as the reason why LAK cell treatment is so effective in eliminating clinical metastases in mice, yet so disappointingly ineffective in man. T h e difference, according to Guillou et at. (1989b), is due to the source of the LAK cells, which are prepared from healthy syngeneic cells in animal experiments, but are prepared from autologous (and presumably suppressed) lymphocytes, for use in patients. Yet, Hirte and Clark (1991) suggest that LAK cells can be induced by IL-2 in patients with cancer-associated effusions, despite the presence of inhibitors in the effusions. A dif€icult, though highly important, question is the extent to which immunosuppression might be responsible for the escape of the developing tumor from immune control and, ultimately, for the growth and spread of the tumor. Experimental evidence implicating tumor-induced immunosuppression in the development of the malignant process is not lacking. Thus, Mullen et al. (1985) showed that tumor-induced immu-

IMMUNOSUPPRESSIVE FACTORS IN CANCER

259

nosuppression may permit growth of a secondary tumor, whereas TorreAmione et al. (1990) demonstrated that transfection with TGF-P cDNA enabled an otherwise immunogenic tumor to grow progressively in mice. Yet, it is unlikely that these elegant experimental models (particularly the highly immunogenic UV-induced tumor employed by TorreAmione et al.) resemble the vast majority of human cancers. There is still a great deal of uncertainty regarding the actual role immunity plays in the control of human malignancy (Sulitzeanu, 1985; Brunson and Goldfarb, 1989), and until this point is settled, the role of immunosuppression will also remain in doubt. Individual opinions will vary and they are more likely to rest on beliefs than on hard scientific data. Nevertheless, the immunosuppression that accompanies tumor development is bound to affect the patient at least indirectly, by the predisposition to infections, which complicate the treatment and can be a major cause of mortality. A well-studied and illuminating example of the likely relationship among malignancy, immunosuppression, and infection can be found in multiple myeloma. Patients with multiple myeloma are aWicted with a severe Bcell deficiency, resulting in an inability to make primary antibody responses, which, in turn, is responsible for the morbidity and mortality due to recurrent bacterial infections. A similar deficiency is exhibited by mice with the related malignancy plasmacytoma (Jacobson and ZollaF’azner, 1986). A very interesting work (Berg and Lynch, 1991) has shown recently that B cells of mice with plasmacytoma differ from normal B cells in the expression of several surface markers (e.g., decreased surface IgM, transferrin receptor, and CD23 receptor). This difference was traced to TGF-P, which is secreted in very large amounts by the plasmacytoma cells. It now remains to be seen whether an analogous situation exists in the human disease, namely excess TGF-P secretion by the multiple myeloma cells, resulting in immunosuppression and consequent susceptibility to infection. It must be stressed that, although investigators appear to assume that the immunosuppressive factors in sera and effusions are actually tumor products, this assumption seems unwarranted. Thus, TGF-j3 is produced by all cells, whereas SER and the factor described by Sheid and Boyce (1984) are macrophage products. The 74-kDa molecule that crossreacts with p15E is also present, albeit in smaller amounts, in normal sera (Jacquemin et al., 1984). An inhibitor of LAK cell generation demonstrated in sera of patients with head and neck cancer was also found in control (normal) sera (Bugis etal., 1990).There is no evidence, in fact, that any of the factors studied thus far are exclusively tumor products.

Isolation p r o c e d u r e A m m o n i u m sulfate precipitation (% SdtLl~dtlOll)

DEAE-sephacel Chromatography (mM T r i s bufier)

1

25-50

100-200

2

29-50

250-500

5

250-500

1.5 5 1.5

Fraction No.

3

50-75

Growth inhibition

Concentration

(mg/rnl) 5" 2.5 0.5

Immunosuppression

++

++ ++ +++ ++

++ +

A315

HT29

+++

++

+++ ++ +++ ++

++ ++

+ + ++ ++ +++ ++

Nule. Fractions were prepared by precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-sephacel. They were tested for immunosuppressive activity (ability to inhibit PHA-induced lymphocyte proliferation) and for growth inhibition (inhibition of A375 nielanoma and HTP9 colon carcinoma cells). The differences in the isolation procedure and the differences in biological activity suggest that the activity of the three fractions was probably due to different cytokines. Percentage immunosuppression/growth inhibition is indicated as follows: + + +, >60%; + +, 30-60%; +, 20-30%; -, less than 20%. a Growth-inhibitory activity of this fraction against A375 (but not against HTPL))cells was abolished by anti-TGF-P I serum, suggesting that the fraction contained at least t w o different inhibitors.

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26 1

At a more theoretical level, one might ask whether tumors can be expected to produce substances that specifically downregulate the immune system? One might invoke, perhaps, selection pressures leading, in the course of a minievolutionary process, to the survival of cells capable of neutralizing the immune defenses, an argument that has, of course, a strong teleological flavor. It appears more plausible, on the other hand, to assume that the “immunosuppressive cytokines” produced by the tumor may be simply growth-regulatory substances, which, while inhibiting growth and differentiation of lymphocytes, may act similarly on many other cell types. One such cytokine is already well known-TGF-P, a classical growth inhibitor that is also a strong immunosuppressor molecule. The less well-known lymphocyte inhibitor, LBIF, was similarly found to possess wide-ranging growth-inhibitory activity. Work in progress in our laboratory (Hatzubai and Sulitzeanu, 1992) suggests that effusions from patients with breast cancer contain a rather large number of cytokines (as judged from the activity of fractions obtained by a variety of techniques) with both growth-inhibitory and immunosuppressive activity (Table IV). Not surprisingly, one of these is TGF-P. Finally, the release of growth-inhibitory substances by tumors may provide a ready explanation for what has been a rather enigmatic phenomenon thus far, namely the outbreak of metastases that sometimes follow the surgical removal of a primary tumor (Prehn, 1991). Removal of the primary tumor could simply free susceptible metastatic foci from the growth control exerted by growth-inhibiting cytokines derived from the primary tumor, thus allowing the metastases to grow. Examples of autocrine growth control abound in the literature. Thus, growth of chronic myeloid leukemia cells is inhibited by endogenous tumor necrosis factor (Duncombe et al., 1989), whereas growth of breast cancer cells is inhibited by the TGF-f3 they secrete (Knabbe et al., 1987; Arteaga et al., 1990). More complex growth-regulatory loops may originate in the colony-stimulating factors released by tumor cells, which induce the appearance of suppressor macrophages. Cytokines released by the latter have been demonstrated to be toxic for tumor cells, singly (Lachman et al., 1986; Lovett et al., 1986), or in various combinations (Morinaga et al., 1989; Maekawa et al., 1990).Thus, all that would be required to account for the outbreak of metastases would be a difference in susceptibility to negative growth control, between the primary and the metastatic cell. T h e possibility that metastatic foci might be controlled in this fashion seems worth investigating, since positive results might have therapeutic implications.

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ACKNOWLEDGMENTS I gratefully acknowledge the support of The Society of Research Associates of the Lautenberg Center, the Concern Foundation of Los Angeles, and the WakefernlShoprite Endowment for Basic Research in Cancer Biology and Immunology. Special thanks are due to Ms. Marcella Wachtel for assistance in the preparation of this manuscript.

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