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Specific suppressor cells in mice bearing a syngeneic mastocytoma Takei, Fumio 1976

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SPECIFIC SUPPRESSOR CELLS IN MICE BEARING A SYNGENEIC MASTOCYTOMA by FUMIO TAKEI B. Agr. University df Tokyo 1968 M. Sc. University of B r i t i s h Columbia 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUBMIA June, 1976 0 Fumio Takei, 1976 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree tha t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t ten pe rm i ss i on . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date JyUsnS? ^ \' //^/ ! _ B P 7 4 - 4 4 6 C i i ABSTRACT P815 X2 mastocytoma c e l l s , when injected subcutaneously into syngeneic DBA/2 mice, induced T lymphocyte mediated cy t o t o x i c i t y i n the mice. During the course of tumor growth t h i s cytotoxic a c t i v i t y decreased and ultimately the tumor k i l l e d the mice. General immunological r e a c t i v i t y of lymphocytes from tumor bearing mice measured by their p r o l i f e r a t i v e response to mitogens remained unaffected. In order to elucidate the mechanism of the decrease i n cytotoxicity i n the l a t e r stages of tumor growth, an i n v i t r o method to generate cytotoxic c e l l s against syngeneic P815 c e l l s was developed. When spleen c e l l s from mice with tumors i n early stages of growth were incubated i n v i t r o with mitomycin C-treated tumor c e l l s , s p e c i f i c c y t o t o x i c i t y mediated by T lymphocytes was greatly enhanced. In contrast, spleen c e l l s , taken from mice with tumors at a la t e r stage i n their growth or from normal untreated mice, did not develop cytotoxic a c t i v i t y . Serum from P815 tumor bearing mice did not have a direct i n h i b i t o r y effect on the cyt o t o x i c i t y . The unresponsive-ness of spleen c e l l s from mice with tumors i n the l a t e r stages of growth seemed to be due to the presence of suppressor c e l l s since i n ' v i t r o generation of cytot o x i c i t y by spleen c e l l s from early tumor-bearing mice was inhibited by the addition of spleen c e l l s or thymocytes from mice with progressively growing tumors. Normal spleen c e l l s or thymocytes did not affect the response. Suppressive lymphoid c e l l s from tumor bearing mice did not i n h i b i t the mitogen responses of normal spleen c e l l s . The suppressor c e l l s i n P815 tumor bearing DBA/2 mice were further characterized. Suppressive a c t i v i t y was almost completely eliminated by treating these c e l l s with a n t i 6 serum and complement. Treatment with i i i a n t i mouse Ig serum and complement or with carbonyl iron did not affect their suppressive a c t i v i t y indicating that the suppressor c e l l s are T lymphocytes. I t was found that c y t o t o x i c i t y against L1210 leukemia l i n e i n DBA/2 mice could also be generated i n v i t r o by the same method as described for the P815 tumor hy incubating spleen c e l l s from mice with L1210 tumors i n an early stage of their growth with mitomycin C-treated L1210 c e l l s . When suppressive thymocytes from P815 tumor bearing mice were tested for the i r capacity to i n h i b i t the generation of anti-L1210 c y t o t o x i c i t y , they did not affect the a c t i v i t y , indicating that the suppressor c e l l s i n P815 tumor bearing mice are s p e c i f i c to the tumor. When ficoll-hypaque density c e l l separation was carried out using cytotoxic spleen c e l l s and suppressive spleen c e l l s from P815 tumor bearing mice, the dense fr a c t i o n was enriched for k i l l e r c e l l s while the suppressive a c t i v i t y was mainly recovered i n the l i g h t f r a c t i o n . Therefore, k i l l e r c e l l s and suppressor c e l l s i n P815 tumor bearing mice are thought to be d i s t i n c t populations although they both belong to the T lymphocyte group. Lymphoid c e l l s from P815 tumor bearing mice were tested for suppressive a c t i v i t y at various stages of tumor growth. Suppressive a c t i v i t y was f i r s t detected i n thymuses i n the early stages of tumor growth when spleens and lymph nodes had some cytotoxic a c t i v i t y . The suppressive a c t i v i t y of thymocytes persisted during the stage of slowed tumor growth when highly cytotoxic a c t i v i t y could be detected i n the spleens and lymph nodes. After the tumors resumed accelerated growth, lymph node c e l l s became suppressive. In the late stage of tumor growth spleen c e l l s as well as lymph node c e l l s and thymocytes were suppressive. A c e l l - f r e e extract, prepared by freeze-thawing suppressive thymocytes from P815 tumor bearing mice, was also suppressive and i t s a c t i v i t y was s p e c i f i c , i . e . i t i n h i b i t e d the generation of anti-P815 c y t o t o x i c i t y but not anti-L1210 c y t o t o x i c i t y . A p o s s i b l e r o l e of the suppressor c e l l s i n the present study i n the escape of tumors from the h o s t ' s immune system and i n the r e g u l a t i o n of the c e l l u l a r immune response i s d i s c u s s e d . V TABLE OF CONTENTS Page INTRODUCTION 1 Immune responses against tumors 1 Failure of the immune response to reject tumors 2 Object of present work 5 Suppressor c e l l s i n tumor bearing host 5 Suppressor T c e l l s i n immune regulation 6 MATERIALS AND METHODS 10 Mice 10 Tumor 10 Preparation of lymphoid c e l l s and tumor c e l l s 11 Cytotoxicity assay 11 In v i t r o generation of cytotoxic c e l l s 12 Suppression experiments 13 Mitogen stimulation of spleen c e l l s 13 Carbonyl iron treatment 14 Anti 9 serum and complement treatment 14 Anti mouse Ig serum and complement treatment 14 Nylon wool column 15 Ficoll-hypaque density c e l l separation 15 Preparation of thymocyte extracts 16 RESULTS 17 Growth of tumor and cytotoxicity of spleen c e l l s 17 In v i t r o generation of cyto t o x i c i t y 21 Effect of serum on cytotoxicity 26 Suppressor c e l l s i n mice with progressively growing tumor 30 Quantitative analysis of suppression 33 Characterization of suppressor c e l l s 36 S p e c i f i c i t y of suppressor c e l l s 36 Ficoll-hypaque density c e l l separation 39 v i Page Relationship between tumor growth and suppressive a c t i v i t y i n lymphoid organs from tumor bearing mice 39 Suppression by thymocyte extract 43 DISCUSSION 44 REFERENCES 52 v i i Page LIST OF FIGURES F i g u r e 1 Growth of P815 tumor and c y t o t o x i c i t y of spleen c e l l s from 4 P815 tumor-bearing mice ( i n j e c t e d w i t h 5 x 10 tumor c e l l s ) 18 F i g u r e 2 Growth of P815 tumor c o r r e l a t e d w i t h c y t o t o x i c i t y of spleen c e l l s from P815 tumor-bearing mice ( i n j e c t e d with 10 tumor c e l l s ) 19 F i g u r e 3 E f f e c t of dose of mitomycin C - t r e a t e d tumor c e l l s on i n v i t r o i n d u c t i o n of c y t o t o x i c i t y 23 F i g u r e 4 Incubation p e r i o d for i n v i t r o generation of anti-P815 c y t o t o x i c i t y 24 F i g u r e 5 L i n e a r r e g r e s s i o n a n a l y s i s of the suppression of anti-P815 c y t o t o x i c i t y . 34 F i g u r e 6 E f f e c t of dose of suppressive lymphoid c e l l s on the degree of suppression 35 F i g u r e 7 C h a r a c t e r i z a t i o n of suppressor c e l l s i n spleen from P815 tumor b e a r i n g mice 37 v i i i Page LIST OF TABLES Table I Characterization of cytotoxic c e l l s i n spleens from P815 tumor bearing mice 20 Table I I P r o l i f e r a t i v e response of spleen c e l l s from P815 tumor bearing mice to mitogens 22 Table I I I Effector c e l l s mediating i n v i t r o generated cy t o t o x i c i t y 25 Table IV S p e c i f i c i t y of c y t o t o x i c i t y generated i n v i t r o 27 Table V Effect of serum on anti-P815 c y t o t o x i c i t y 28 Table VI Effect of serum on i n v i t r o generation of anti-P815 c y t o t o x i c i t y 29 Table VII Suppression of i n v i t r o generation of cy t o t o x i c i t y 31 Table VIII Lack of suppression i n p r o l i f e r a t i v e response of tumor bearing spleen c e l l s against mitogens 32 Table IX S p e c i f i c i t y of suppressor c e l l s 38 Table X Ficoll-hypaque density c e l l separation of k i l l e r c e l l s 40 Table XI Ficoll-hypaque density c e l l separation of suppressor c e l l s 41 Table XII Suppressive a c t i v i t y i n lymphoid organs from tumor bearing mice... 42 Table XIII Suppression by thymocyte extracts 44 ACKNOWLEDGEMENT I would l i k e to thank Dr. J. Levy and Dr. D.G. Kilburn for their many suggestions, helpful c r i t i c i s m , and invaluable encouragement during the research and writing of this thesis. I also wishsto thank Dr. G. Weeks, Dr. A. Tingle and Dr. J.J.R. Campbell for their suggestions concerning the f i n a l draft. 1 INTRODUCTION 1. IMMUNE RESPONSES AGAINST TUMORS It i s now well recognized that most tumors i n experimental animals as well as humans are antigenic to the hosts. Animals can be immunized against syngeneic tumors by various methods. Tumor c e l l s treated with mitomycin C or i r r a d i a t i o n to i n h i b i t their c e l l d i v i s i o n , when injected into syngeneic animals, induce s p e c i f i c immunity i n some cases (Klein et al., 1960; Revesz, 1960). Chemical modification of tumor c e l l s surface by glutaraldehyde (Sanderson and Frost, 1974), iodoacetate (Prager e_t al. , 1971), or neuraminidase (Currie and Bagshawe, 1969) seems to enhance the antigenicity of tumor c e l l s , and animals that received the modified tumor c e l l s developed immunity to the or i g i n a l tumors. Surgical removal of certain type of tumors also induced s p e c i f i c immunity (Barski and Youn, 1969). Animals immunized against tumors by these methods may reject subsequent challenge of the same tumors but not unrelated tumors implying the presence on tumor c e l l s of s p e c i f i c antigens. I t seems that this tumor immunity i s mediated mainly by immune lymphocytes and macrophages and that humoral immunity plays l i t t l e role i n the Rejection of tumors (Burnet, 1971). C e l l mediated immune responses against tumors have been also detected i n tumor bearing animals or human patients by various i n v i t r o assays such as the lymphocyte p r o l i f e r a t i o n assay (Vanky e_t a l . , 1971), macrophage migration i n h i b i t i o n (Bloom, 1971), cytotoxicity (Takasugi and K l e i n , 1970), and cytostasis assay (Chia and Festenstein, 1973). These assays can generally be correlated to i n vivo measurement of tumor immunity such as i n f i l t r a t i o n of lymphoid c e l l s into the tumor mass or tumor neutralization upon adoptive transfer (Winn, 1959). 2 2. FAILURE OF THE IMMUNE RESPONSE TO REJECT TUMORS Although immune responses against tumors have been detected i n the tumor bearing host without preimmunization, tumors normally grow progressively and f i n a l l y k i l l the host. To explain t h i s apparent paradox, a number of mechanisms by which tumors escape from the host's immune system have been proposed: (a) Immunosurveillance The immunosurveillance theory (Burnet, 1971) suggests that tumor c e l l s appear i n hosts very often, but most of them are k i l l e d o f f by the immune system. Therefore, only weakly antigenic tumors escape from the immune system and the immunity induced i n hosts i s simply too weak to r e j e c t r a p i d l y growing tumors. This theory was supported by the observations that human patients with immunological d e f i c i e n c y diseases have a higher incidence of cancer. S i m i l a r l y , immunosuppression of the r e c i p i e n t s with kidney transplants resulted i n a higher than normal incidence of cancer. However, cancer•in 1 these patients i s r e s t r i c t e d to mainly lymphoid o r i g i n . 1 Recent studies on the tumor incidence i n immunosuppressed mice (Simpson and Nehlsen, 1971) and nude mice (Rygaard and Polusen, 1975) which have no c e l l u l a r immunity have shown that spontaneous tumors did not develop i n these mice, while a higher incidence of v i r u s induced tumors d i d , presumably due to th e i r d e f e c t i n a n t i - v i r u s immunity (Houghton and Whitmore, 1975). These observations are s u f f i c i e n t : to throw some doubt on the immunosurveillance theory. (b) Immunostimulation The immunostimulation theory by Prehn (Prehn and Lappe, 1971) suggests that the immune responses against spontaneous tumors are normally very weak and that a weak immune reaction, rather than i n h i b i t n g tumor growth, may 3 a c t u a l l y stimulate i t . Faster tumor growth i n the presence of very weak immune reactions has been observed i n some spontaneous tumor systems, but i t s mechanisms are unknown. It i s not yet clear i f the 'immunostimulation' e f f e c t i s r e a l l y involved i n the escape of tumors from the immune response e s p e c i a l l y i n the case of vi r u s or chemically induced tumors which normally induce stronger immune responses. (c) Modulation The TL antigen on mouse lymphoma c e l l s i s l o s t when the tumors grow i n mice which have been immunized against TL antigen, but the c e l l s regain i t when transferred to non-immune mice (Old et_ j i l . , 1968). Many other c e l l surface antigens are known to move on the surface or to disappear from i t altogether under the influence of antibody. This i s att r i b u t e d to the two dimensional agglutination of surface antigen by antibody and subsequent gathering of antigens i n t o patches which lead to endocytosis (Taylor et^ a l . , 1971) . It was suggested that the same mechanisms might enable tumor c e l l s to escape from the immune response to tumor c e l l surface antigens. However, i t i s well known that at le a s t some surface antigens on tumor c e l l s p e r s i s t upon i n vivo transplantations of tumor c e l l s as well as i n t i s s u e cultures. (d) Blocking f a c t o r s It has been reported that serum from tumor bearing animals or human patients contains factors which s p e c i f i c a l l y block c y t o t o x i c i t y of lymphocytes from tumor bearing hosts (Hellstrom and Hellstrom, 1974). The factors are thought to be antibodies that bind to antigenic s i t e s on the tumor c e l l surface (Hellstrom and Hellstrom, 1974), or antigen-antibody complexes (Baldwin et a l . , 1972). I t has been suggested that immune responses detected i n v i t r o may be blocked i n vivo by these factors and, therefore, are not e f f e c t i v e to r e j e c t tumors. However, anti-tumor responses determined 4 i n v i t r o , presumably i n the absence of blocking factors, are progressively decreased as the tumor grows (LeFrancois_et_al., 1971; Whitney _ e t _ a l . , 1974). It has become apparent recently that none of these rather s i m p l i s t i c theories adequately explain the relationship of the immune response to tumor growth, and further data are being sought to c l a r i f y t his obviously complex relationship, (e) Suppression C e l l mediated immunity i s often impaired i n tumor bearing animals (Adler, 1971; Whitney, et / a l . .yrigJxA^orahumanspatientStr (Krant et_„ a l . , 1968; Gatti ejt _al. , 1970; Eil b e r _et _al. , 1970). In v i t r o studies have shown that the lymphocytes of tumor bearers undergo an impaired p r o l i f e r a t i v e response to mitogens or antigens (Adler_et _ a l . , 1971; Whitney et a l . , 1974). The i n vivo correlate to these observations has involved the flii.se of skin testing. Human studies have shown that patients with progressively growing tumors frequently lack the a b i l i t y to respond to r e c a l l antigens which e l i c i t cell-mediated immunity (Eilber e_t _al. , 1970; Sugi-Foca ejz al. , 1973). I t has been reported that the serum of both cancer patients (Nimberg et^ a l . , 1975) and experimental animals bearing tumors (Whitney and Levy, 1974, 1975, a, b) contains factors which non-specifically block the response of normal lymphocytes in v i t r o to a number of immunological reactions including: mitogen responsivess, mixed leukocyte reaction and s p e c i f i c antigen responses. However, i t i s not yet clear i f t h i s non-specific suppression of general immunity i s the cause of the suppression of s p e c i f i c anti-tumor immunity or the result of progressive tumor growth caused by the suppression of s p e c i f i c anti-tumor immunity which involves some other mechanisms. 5 3. OBJECT OF PRESENT WORK It i s clear that the relationship between the immune system and tumor c e l l s i s highly complex and may involve a number of separable mechanisms. It i s also clear that the o r i g i n a l hypotheses designed to explain t h i s relationship are no longer acceptable i n the l i g h t of accumulating data i n this f i e l d of study. The work reported i n th i s thesis was undertaken to investigate status of the immune system during the progressive growth of a tumor with pa r t i c u l a r reference to recent data implicating a role for suppressor c e l l s i n the f a i l u r e of the immune system to combat tumor growth. 4. SUPPRESSOR CELLS IN TUMOR BEARING HOST Recently i t has been reported that suppressor c e l l s which i n h i b i t various immune responses i n a non-specific manner are present i n tumor bearing animals. Suppressor c e l l s i n mice bearing murine sarcoma virus induced sarcomas (Kirchner et a l . , 1974) and methylcholanthrene induced sarcomas (Eggers and Wunderlich, 1975; Pope et a l . , 1976) were described as the lymphoid c e l l s that i n h i b i t general immune responses such as the response to mitogens (Kirchner _et _ a l . , 1974) or to alloantigens i n mixed leukocyte culture (Eggers and Wunderlich, 1975). Suppressor c e l l s i n mice bearing murine sarcoma virus induced sarcomas have been i d e n t i f i e d as B lymphocytes and have been shown to i n h i b i t s p e c i f i c anti-tumor cyt o t o x i c i t y as well as general immune responses by means of antigen-antibody complexes (Gorczynski ^ t _ a l . , 1975). Another group of investigators found that the suppressor c e l l s i n the same system may be c e l l s of the monocyte/macrophage series and i n h i b i t DNA synthesis of T lymphocytes (Kirchner et a l . , 1975). Suppressor c e l l s i n mice bearing a methycholanthrene induced sarcoma have also been i d e n t i f i e d 6 as adherent c e l l s of the macrophage/monocyte series (Pope et a l . , 1976). While the foregoing are examples of generalized non-specific i n h i b i t i o n of immune responsiveness suppressor c e l l s that i n h i b i t only s p e c i f i c a n t i -tumor immune responses have also been reported, eg. i n Japanese quails bearing Rous v i r u s induced sarcomas (Hayami et_ al_. , 1972) , i n mice bearing sarcomas induced by Moloney sarcoma v i r u s (Halliday,11971, 1972) or methyl-cholanthrene (Fujimoto e_t_al_.', 1976.. a, b) and i n rats bearing mammary-adenocarcinomas (Kuperman et a l . , 1975). This type of suppressor c e l l i n mice bearing sarcomas induced by Moloney sarcoma v i r u s or methylcholanthrene were found to be s p e c i f i c for the given tumor studied (Halliday, 1972; Fujimoto et a l . , 1976 a) and belong to the T lymphocyte population (Fujimoto et a l . , 1976 a, b). Although the r e l a t i o n s h i p between nonspecific suppressor c e l l s and the suppressor c e l l s that i n h i b i t s p e c i f i c anti-tumor immune responses i s unknown, i t i s thought that both of them can i n h i b i t s p e c i f i c anti-tumor immunity which may r e s u l t i n progressive tumor growth. 5. SUPPRESSOR T CELLS IN IMMUNE REGULATION T lymphocytes are known to have various functions. Upon stimulation by antigens or mitogens, they produce various factors such as migration i n h i b i t i o n f a c t o r , etc., d i f f e r e n t i a t e into cytotoxic k i l l e r c e l l s , and cooperate with B lymphocytes as helper c e l l s i n antibody production. More recently another function of T lymphocytes — the suppressor that i n h i b i t s immune responses — has been recognized and thought to be involved i n regulation of immune responses (Gershon, 1975). Suppressor T c e l l s have been extensively studied i n various systems involving antibody formation. 7 When animals were t o l e r i z e d to sheep red blood c e l l s , thymocytes from tolerant animals s p e c i f i c a l l y suppressed the antibody response to the same antigen i n normal syngeneic animals (Gershon and Kondo, 1971). The same e f f e c t s of T lymphocytes have been confirmed i n many other systems of immunological tolerance and have been reviewed extensively elsewhere (Nachtigal et a l . , 1975). It has been suggested that suppressor T c e l l s are involved i n se l f - t o l e r a n c e and that a deficiency of suppressor c e l l s may therefore contribute to the etiology of autoimmunity (Gerber e_t a l . , 1974) . Suppressor T c e l l s were also found i n rats immunized with d i n i t r o -phenylated Ascaris-extract (DNP-Asc) (Tada et^ a l . , 1975). When thymocytes or spleen c e l l s from these rats were transferred to r e c i p i e n t rats which were producing high and per s i s t e n t Ig E antibody against DNP-Asc, they s p e c i f i c a l l y suppressed the ongoing Ig E antibody response against DNP-Asc. Suppression of antibody allotype also seems to involve suppressor T c e l l s . When male SJL/L mice which carry one allotype and female BALB/c mice carrying another allotype were mated, immunization of BALB/c mice with SJL/L a l l o t y p e before mating completely or c h r o n i c a l l y suppressed the production of SJL/E allotype i n more than h a l f the progeny by the time they were s i x months o l d (Jacobson et aJ_. , 1972). Subsequently i t was found that an active factor associated with T lymphocytes i s responsible for chronic allotype suppression, because T lymphocytes from c h r o n i c a l l y suppressed progeny suppressed production of paternal allotype by unsuppressed hybrid spleen c e l l s (tHer.zenber^ et a l . , 1975). Histocompatibility linked genetic unresponsiveness of some s t r a i n s of mice to c e r t a i n synthetic peptide antigens seem to be another immunological phenomenon that involves the a c t i v i t i e s of suppressor T c e l l s . It was found 8 that B lymphocytes s p e c i f i c for the peptide f a i l e d to cooperate with T lymphocytes and that this f a i l u r e was due to the presence of suppressor T c e l l s at least i n some of the non-responder mice (Benacerraf et a l . , 1975). The role of suppressor T c e l l s i n the c e l l u l a r immune response i s not yet clear, although some i n v i t r o studies suggest they may be involved. When splenic T lymphocytes were stimulated by Concanavalin A, the stimulated c e l l s were found to i n h i b i t the a b i l i t y of T lymphocytes to d i f f e r e n t i a t e into cytotoxic c e l l s . (Peavy and Pierce, 1974). Similar non-specific suppressor T c e l l s have been spontaneously generated when normal spleen c e l l s were incubated i n v i t r o for 3 - 4 days without antigens or mitogens (Burns et a l . , 1975). However, the immunological significance of these suppressor c e l l s i s unknown, and their analogues i n vivo have yet to be demonstrated. On the other hand, the regulatory role of thymocytes i n the c e l l u l a r immune response has been suggested by various observations i r i vivo. Gershon et a l . (1974) reported such effects of thymocytes i n graft-versus-host reactions induced by the inoculation of parental thymocytes into mice. When the spleen l o c a l i z i n g f r a c t i o n of parental thymocytes were removed by splenectomy after the inoculation of parentalccells, the response of parental c e l l s against F^ was either enhanced or suppressed depending on the timing of the splenectomy. This observation suggests that the effect of regulatory c e l l s i n the thymus i s b i d i r e c t i o n a l . The regulatory role of thymocytes has been also suggested i n the generation of cyt o t o x i c i t y against allogeneic c e l l s . Simpson and Cantor (1975) reported that adult thymectomy increased the a b i l i t y of T lymphocytes to generate primary cytotoxic responses, but had l i t t l e effect on the develop-ment of cytotoxic T memory a c t i v i t y . Similar effects of thymectomy were observed i n autosensitization 9 against syngeneic c e l l s (Carnaud et a l . , 1975). When mouse lymphocytes were exposed to syngeneic fibroblasts i n M i l l i p o r e chambers inserted into the peritoneal cavity of thymectomized mice, cytotoxic c e l l s directed against syngeneic fibroblasts were induced. This effect was inhibited by thymic extract suggesting a possible role of suppressor T c e l l s i n the maintenance of se l f tolerance. These results support the contention that suppressor T c e l l s are also involved i n the regulation of the c e l l u l a r immune response. Direct evidence may i n fact be provided by studies i n tumor systems. MATERIALS AND METHODS Mice Female DBA/2, CBA and BALB/C mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). Tumor P815X2 mastocytoma and L1210 lymphocytic leukemia i n DBA/2 mice were obtained from Dr. J.B. Smith (Institute for Cancer Research, P h i l -adelphia, Pennsylvania). P388 lymphocytic leukemia i n DBA/2 mice and S49A lymphoma i n BALB/C mice were obtained from the Salk I n s t i t u t e for B i o l o g i c a l Studies (San Diego, C a l i f o r n i a ) . P815 and P388 tumors were maintained i n the ascites form by s e r i a l transplantation by intraperitoneal i n j e c t i o n of the tumor c e l l s from ascites f l u i d . The S49A tumor prepared o r i g i n a l l y from a subcutaneously growing tumor i n BALB/C mice and the L1210 tumor from ascites f l u i d i n DBA/2 mice were maintained i n tissue cultures using RPMI 1640 medium (Grand Island B i o l o g i c a l Company, Grand Island, N.Y.) containing 10% heat inactivated f e t a l calf serum (FCS, Grand Island B i o l o g i c a l Company) and supplemented with 100 units/ml p e n i c i l l i n and 10 ug/ml streptomycin. This medium was used for c e l l cultures throughout the present study and i s designated as RPMI 1640 culture medium. CBA mice, which d i f f e r at the H-2 locus from DBA/2 mice, rejected 10^ P815 c e l l s injected intraperitoneally. BALB/C mice which are H-2 id e n t i c a l but d i f f e r at the M locus and the minor histocompatibility antigens rejected 10^ P815 c e l l s injected subcutaneously. Intraperitoneal 2 3 in j e c t i o n of 10 P815 c e l l s or subcutaneous i n j e c t i o n of 2 X 10 c e l l s into DBA/2 mice always induced tumors which grew progressively and k i l l e d the animals. 11 Preparation of lymphoid c e l l s and tumor c e l l s Mice were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n , their spleens, thymuses or lymph nodes removed asce p t i c a l l y , teased apart i n phosphate buffered saline (PBS) containing 5% FCS and the c e l l clumps disaggregated by either expulsion through tuberculin syringe with a #26 gauge needle or by passing small pieces through a 60 mesh stainless s t e e l mesh. Red blood c e l l s were lysed by treating spleen c e l l s with 0.14M NH^Cl solution i n PBS for two minutes. The c e l l s were then washed twice with PBS containing 5% FCS. Cells from ascites tumors or tissue cultures were washed twice with PBS containing 5% FCS. C e l l counts were carried out by direct microscopic examination and the i r v i a b i l i t y assessed by the trypan blue exclusion method. Cytotoxicity assay Target c e l l s for the c y t o t o x i c i t y test were labeled with "^Cr-sodium chromate (New England Nuclear, Boston, Mass.) as described by Brunner et a l . (1968) with s l i g h t modifications. Two m i l l i o n target c e l l s were incubated with 100-200 uCi ^^"Cr-sodium chromate i n 0.5 ml. RPMI 1640 culture medium for one hour at 37°C. The labeled c e l l s were washed four times with PBS, resuspended i n RPMI 1640 culture medium and incubated for one hour. Immediately before the assay the target c e l l s were harvested by centrifugation, resuspended i n the medium and counted. The effector c e l l s to be tested and 4 10 labeled target c e l l s were mixed at various ratios i n either 12 X 75 mm round bottom p l a s t i c tubes (Falcon, #2003, Oxnard, California) or i n the well of microculture plates (Linbro Chemical, IS-FB-96-TC, New Haven, Conn.). The f i n a l volume was adjusted to 0.4 ml. i n case of p l a s t i c tubes and 0.25 ml for microculture plates. The c e l l s were incubated at 37°C for either 12 6 hours or 18 hours i n a humidified incubator with 5% CC^, 95% a i r . After incubation the c e l l s were sedimented by centrifugation (200 x g, 5 min.) and 0.2 ml > (for p l a s t i c tubes) or 0.1 ml (for microculture plates) of the 51 supernatant was removed for counting of Cr on a gamma counter (Beckman Biogamma). Percent s p e c i f i c c y t o t o x i c i t y was calculated as follows: -„ ^ ^ . test release (cpm) -spontaneous release (cpm) X 100 % s p e c i f i c c y t o t o x i c i t y = : :: *—r : ;— r / v maximum release (cpm) - spontaneous release(cpm) 4 Spontaneous release was measured by incubating 10 target c e l l s alone, 4 and maximum release was obtained by lysing 10 target c e l l s by either freeze-thawing three times or treating with 5% t r i t o n X 100 (Sigma Chemical Company, St. Louis, Missouri). In some cases the control release measured by incubating 4 10 target c e l l s with normal spleen c e l l s was used i n place of the spontaneous release. However, the control release was never s i g n i f i c a n t l y higher than the spontaneous release. In v i t r o generation of cytotoxic c e l l s 6 ' Tumor c e l l s (5 X 10 /ml;,.) were incubated with 50 u^g/ml. mitomycin C (Sigma Chemical Company) i n RPMI 1640 culture medium at 37 C for one hour, then washed with PBS three times and resuspended i n medium. Mitomycin C treated tumor c e l l s and 10^ lymphoid c e l l s i n RPMI 1640 culture medium supplemented with 5 X 10 M^ 2-mercaptoethanol were dispensed into 17 X 100 mm p l a s t i c tubes (Falcon, #2001) and the f i n a l volume was adjusted to 2.5 ml. The tubes were incubated at 37°C for various times, then the c e l l s were harvested by centrifugation, washed and resuspended i n medium, after which the viable c e l l s were counted and tested for cy t o t o x i c i t y . The mitomycin C treated tumor c e l l s did not survive more than 48 hours and, therefore, were not apparent i n the cultures at the time of harvesting cytotoxic c e l l s . 13 Suppression experiments DBA/2 mice were injected subcutaneously with a low dose of P815 c e l l s 4 3 3 (10 or 2 x 10 ) or L1210 c e l l s (10 ). After 13-16 days for P815 tumors or 10-12 days for L1210 tumors, spleens were used as a source of immune c e l l s . Lymphoid c e l l s to be tested for suppressive a c t i v i t y from mice bearing large tumors were mixed with immune spleen c e l l s i n culture to generate c y t o t o x i c i t y against P815 or L1210 c e l l s . The suppressive a c t i v i t y was estimated from the decrease i n the c y t o t o x i c i t y as compared to control cultures i n which normal lymphoid c e l l s were added. The t o t a l number of lymphoid c e l l s were always adjusted to 10^. Per cent suppression was calculated from the decrease i n cyt o t o x i c i t y at a fixed effector/target c e l l r a t i o . When the degree of sup-pression had to be quantitated more precisely, c y t o t o x i c i t y was tested at various effector/target c e l l r a t i o s and the decrease i n the t o t a l number of l y t i c units was assessed. One l y t i c u n i t , which was defined as the number of 4 effector c e l l s required to lyse 50% of 10 target c e l l s i n 18 h incubation was estimated by l i n e a r regression analysis of per cent c y t o t o x i c i t y versus log-, arithm of effector/target c e l l r a t i o . Total number of l y t i c units were c a l -culated from c e l l recovery i n cultures. Mitogen stimulation of spleen c e l l s Spleen c e l l s were tested for the p r o l i f e r a t i v e response to concanavalin A (Con A), phytohemagglutinin (PHA) and lipopolysaccharide (LPS) as described elsewhere (Whitney and Levy, 1974). In short, 5 x 10^ spleen c e l l s were cultured with 4 ug/ml Con A, 0.5% PHA or 10 yg/ml LPS i n 0.25 ml RPMI 1640 culture medium (5% FCS). After 2 days for LPS stimulation or 3 days for Con A and PHA stimulation, 1.0 uCi of t r i t i a t e d thymidine was added to each culture. Sixteen hours after isotope addition the c e l l s were harvested, washed and counted with a Nuclear Chicago Unilux 1 s c i n t i l l a t i o n counter. 14 Carbonyl Iron Treatment 5 - 10 X 10 spleen c e l l s were suspended i n 2.0 ml of medium and 200 mg of carbonyl iron powder (General Aniline and Film Company, New York, New York) was added. The c e l l s were incubated for 1 hour at 37°C i n 17 X 100mm p l a s t i c tubes(Falcon). The tubes were then placed on top of a magnet and the supernate was removed by pasteur pipette. This procedure was repeated three times. Anti 0 serum and Complement Treatment Antiserum against brain associated Q antigen was raised i n rabbit by i n j e c t i n g DBA/2 brain i n complete Freund's adjuvant as described elsewhere (Kelly et a l . , 1974). The antiserum was extensively absorbed with DBA/2 mouse l i v e r . Five m i l l i o n c e l l s to be,treated were suspended i n 0.5 ml of a 1/4 d i l u t i o n of a n t i 9 serum and 0.5 ml of a 1/5 d i l u t i o n of guinea pig complement. The mixture was incubated for 1 hour at 37°C, washed with PBS three times, resuspended i n the medium and viable c e l l s were counted. This treatment was cytotoxic to T lymphocytes but not to B lymphocytes as indicated by the elim-ination of responses to Con A and PHA without a concomitant reduction i n the LPS response or numbers of plaque forming c e l l s . Anti mouse Ig serum and complement treatment Antiserum against mouse Ig was raised i n sheep by injecting the Ig fra c t i o n prepared from mouse serum as described elsewhere (Whitney e t a a l . , g 1974). The antiserum was absorbed with DBA/2 mouse thymocytes (10 thymocytes for 1 ml of antiserum ). Spleen c e l l s were treated with a n t i Ig serum and complement i n an i d e n t i c a l manner to that described for a n t i 0 serum and complement treatment. This treatment was cytotoxic to B lymphocytes but not to T lymphocytes as indicated by the elimination of response to LPS or plaque forming c e l l s without concomitant reduction i n Con A and PHA responses. Nylon Wool column Spleen c e l l s were depleted of B lymphocytes by adherence to nylon wool 8 as described by Ju l i u s et a l . (1973). In short, 10 spleen c e l l s were put onto a column containing 0.6 g nylon wool which had been washed extensively with d i s t i l l e d water, dried, autoclaved and washed again with PBS containing 5% FCS. The column was..incubated for 45 minutes at 37°C, then the c e l l s which did not adhere to the nylon wool were eluted from the column with PBS containing 5% FCS. This fraction of c e l l s showed a high mitogenic response to Con A and PHA but did not respond to LPS. Ficoll-hypaque density c e l l separation Ficoll-hypaque was prepared from stock solutions of f i c o l l 400 (Pharmacia Fine Chemicals, Uppsala, Sweden) and sodium hypaque (Winthrop Laboratories, Aurora, Ontario) by the method described elsewhere (Pope et a l . , 1976). Spleen c e l l s were suspended at a concentration of 4.0 x 10^ c e l l s per ml i n 2.5 ml of PBS. This suspension was ca r e f u l l y layered onto 2.5 ml of ficoll-hypaque (denr s i t y 1.06) i n a 17 x 100 mm p l a s t i c tube (Falcon, #2001). The tube was cent-rifuged for 30 minutes at 400 x g. The c e l l s found i n the supernate were c o l -lected with a pasteur pipette, washed with PBS and resuspended i n medium. This fraction was designated as the l i g h t f r a c t i o n (d <1.06). The c e l l s sedimenting to the bottom of the tube were washed and resuspended i n PBS, then further sep-arated by repeating the same procedure as described above using ficoll-hypaque with a higher density (density 1.08). The c e l l s i n the supernatant f r a c t i o n , designated as the medium fraction (density between 1.06 and 1.08), and those sedimenting to the bottom of the tube, designated as dense fraction (d >1.08), were washed with PBS, resuspended i n medium and viable c e l l s were counted by trypan blue exclusion. In some experiments spleen c e l l s were separated i n one step using f i c o l l -hypaque with a density of 1.08 only. This gives two frac t i o n s , i . e . the dense (d >1.08) and the l i g h t (d <1.08) fractions. 16 Preparation of thymocyte extracts A single c e l l suspension was prepared from the thymuses of normal or P815 tumor bearing DBA/2 mice. The c e l l suspension at the concentration of 5 X 10^/ml i n PBS was freeze-thawed three times, centrifuged for 20 minutes at 400 x g and the supernatant was collected. This c e l l free extract was s t e r i l i z e d by f i l t r a t i o n through m i l l i p o r e membrane (pore size 0.2 \i) before use. 17 RESULTS Growth of Tumor and Cytotoxicity of Spleen C e l l s 4 DBA/2 mice were injected subcutaneously with 5 x 10 syngeneic P815 mastocytoma c e l l s . At various times following i n j e c t i o n of tumor c e l l s , the mice were s a c r i f i c e d , tumor weight was measured and spleen c e l l s were tested for c y t o t o x i c i t y against tumor c e l l s and thei r p r o l i f e r a t i v e response to Con A, PHA, and LPS was assessed. Solid tumors were f i r s t observed on day 8 and the tumor continued growing u n t i l day 12. After day 12 tumor growth slowed. Specific c y t o t o x i c i t y of the tumor-bearer spleen c e l l s increased markedly during t h i s period of slowed tumor growth (days 12-16). Figure 1 represents the average of data obtained from 6 animals for each data point. Tumor growth i n l i v i n g animals was measured with c a l i p e r s , and during t h i s period of high c y t o t o x i c i t y , tumor growth frequently stopped completely and often showed some regression. However at about day 16 to 18, s p e c i f i c cyto-t o x i c i t y dropped, and t h i s coincided with accelerated tumor growth. The mice were usually k i l l e d by the tumor 20 - 28 days after i n j e c t i o n . The cytotoxi-c i t y detected i n t h i s system appears to be mediated by T lymphocytes, because i t was abolished by a n t i 9 and complement treatment but was not affected by nyloon wool column separation (Table I ) . 4 I f a lower dose of tumor c e l l s (10 ) was injected, the phase during which tumor growth slowed and regressed was accentuated. In these animals, also, there was a peak of s p e c i f i c c y t o t o x i c i t y followed by a decline. As with 4 those animals receiving 5 x 10 c e l l s , tumor growth eventually accelerated and k i l l e d the animals. A t y p i c a l set of data are presented i n figure 2. 18 m g I4 00 r TUMOR SIZE 1200 10 0 0 800 600 40 0 200 o/ /o 12 1 0 8 6 4 2 CYTOTOXICITY 8 10 12 14 D A Y S 16 1 8 20 F i g u r e 1. Growth of P815 tumor and c y t o t o x i c i t y of spleen c e l l s from P815 tumor-bearing mice. The mice r e c e i v e d 5 X 1 0 4 P815 c e l l s subcutaneously. Tumor s i z e was measured by the weight of the e x c i s e d tumors. The c y t o t o x i c i t y of spleen c e l l s were t e s t e d by Cr r e l e a s e assay. The spleen c e l l to target c e l l r a t i o was 200:1 and the c e l l s were incubated f o r 18 h r . Each data p o i n t r e p r e s e n t s the average of data obtained from 6 animals and v e r t i c a l bars show standard e r r o r of the mean. mg 6 0 01 400 200r T U M O R S I Z E F i g u r e 2. Growth of P815 tumor c o r r e l a t e d with c y t o t o x i c i t y of spleen c e l l s from tumor-bearing mice. The mice r e c e i v e d 10 P815 c e l l s subcutaneously. Tumor s i z e was measured by weight of the e x c i s e d tumors. The c y t o t o x i c i t y of spleen c e l l s were t e s t e d by -'-'-Cr r e l e a s e assay. The spleen c e l l to t a r g e t c e l l r a t i o was 200:1 and the c e l l s were incubated f o r 18 h r . Each data point r e p r e s e n t s the average of d a t a obtained from 5 animals and b a r s show standard e r r o r of the mean. TABLE I. CHARACTERIZATION OF CYTOTOXIC CELLS IN SPLEENS FROM P815 TUMOR BEARING MICE Specific c y t o t o x i c i t y f.oSEM (b) (%) Exp. I. none 10.5 ±0.3 anti 9 + C ' 0.2±0.2 Exp. I I . none 6.6 ± 0.4 nylon wool column 17.6 ± 0.9 (a) Spleen c e l l s from mice bearing small P815 tumors (14 days after subcutaneous in j e c t i o n of 5 X 10^ P815 c e l l s ) were used as a source of cytotoxic c e l l s . (b) Cytotoxicity against P815 c e l l s was tested by 5 1 C r release assay. The spleen c e l l to target c e l l r a t i o was 200:1 and the c e l l s were incubated for 18 hr. The figures are the means of t r i p l i c a t e tests. :(»a) Treatment of c e l l s The p r o l i f e r a t i v e response of spleen c e l l s to Con A, PHA and LPS d i d not change s i g n i f i c a n t l y up to day 19 (Table I I ) . A f t e r day 19 the tumor o f t e n metastasized to spleen or p e r i t o n e a l c a v i t y and the c y t o t o x i c i t y and the mitogenic responses could not be p r o p e r l y tes t e d because of the high backgrounds due to the tumor c e l l s . I n v i t r o Generation of C y t o t o x i c i t y In an attempt to e l u c i d a t e the cause of the decrease i n c y t o t o x i c i t y which was observed a f t e r 14 days of tumor growth an i n v i t r o method to generate c y t o t o x i c c e l l s against syngeneic mastocytoma c e l l s was developed. When spleen c e l l s from mice w i t h small tumors (10-12 days a f t e r i n j e c t i o n of tumor c e l l s ) were incubated i n v i t r o w i t h mitomycin C t r e a t e d P815 c e l l s , s i g n i f i c a n t l e v e l s of c y t o t o x i c i t y could be demonstrated. The a c t i v i t y was dependent on the i n c u b a t i o n time and the dose of mitomycin C t r e a t e d tumor c e l l s ; the highest c y t o t o x i c i t y being obtained w i t h 10^ spleen c e l l s were incubated w i t h 5 X 10^ mitomycin C t r e a t e d tumor c e l l s f o r 4 days ( F i g . 3,4). S i g n i f i c a n t l e v e l s of c y t o t o x i c i t y could be detected a f t e r 6 hours of i n c u b a t i o n although i n most cases we used 18 hours i n c u b a t i o n f o r the ^ C r r e l e a s e assay. Under the c o n d i t i o n s used i n these experiments, normal spleen c e l l s d i d not develop s i g n i f i c a n t c y t o t o x i c i t y . Under the c o n d i t i o n s described here, no i n t a c t tumor c e l l s remained i n the c u l t u r e a f t e r 4 days i n c u b a t i o n ; t h e r e f o r e no measures needed to be taken to remove them p r i o r to the mixing of v i a b l e spleen c e l l s w i t h "^Cr l a b e l e d tumor c e l l s . To e s t a b l i s h which c e l l s were the c y t o t o x i c e f f e c t o r s c u l t u r e d spleen c e l l s were t r e a t e d i n v a r i o u s ways, p r i o r to t e s t i n g . C y t o t o x i c i t y was completely abolished by a n t i 9 and complement treatment, while n e i t h e r B 22 TABLE I I . PROLIFERATIVE RESPONSE OF SPLEEN CELLS FROM P815 TUMOR BEARING MICE TO MITOGENS Stimulation (b) (cpm ± SEM) Days after tumor c e l l i n j e c t i o n (a) LPS (10 ug/ml) Con A (4 ug/ml) PHA ((5%) )0 8 12 14 16 19 47467 ± 5425 47962 ± 2691 59987 ± 3491 60516 ± 3369 51439 ± 8540 41970 ± 3565 190467 ± 25881 139204 ± 7004 241806 ± 15726 233127 ± 20820 159188 + 26737 175236 ± 26769 64537 ± 7311 46455 ± 1219 67594 ± 11125 79658 ± 5385 47262 ± 7875 56982 ± 8755 (a) DBA/2 mice were injected subcutaneously with 5 X 10 P815 c e l l s (b) Mitogen stimulation was calculated f rom the mean r a d i o a c t i v i t y of t r i p l i c a t e test cultures with mitogens minus that of control cultures without mitogens. The figures are the average of data obtained from 6 animals i n each group. 2 4 MITOMYCIN C 6 8 10 TREATED P 8 1 5 CELLS (X10 5) F i g u r e 3. E f f e c t of dose of mitomycin C - t r e a t e d tumor c e l l s on i n v i t r o i n d u c t i o n of c y t o t o x i c i t y . Spleen c e l l s from mice b e a r i n g s m a l l tumors (10 days a f t e r subcutaneous i n j e c t i o n of 5 X 10 P815 c e l l s ) were incubated with v a r i o u s numbers of mitomycin C - t r e a t e d P815 c e l l s f o r 4 days, then t e s t e d for the c y t o t o x i c i t y a g a i n s t P815 c e l l s by - ^ C r r e l e a s e assay. The e f f e c t o r c e l l to target c e l l r a t i o was 50:1 and the c e l l s were incubated for 18 h r . 7 In 1 0 0 Figure 4. Incubation period for i n v i t r o generation of anti-P815 cyt o t o x i c i t y . Spleen c e l l s from mice bearing small P815 tumors (10 days after subcutaneous i n j e c t i o n of 5 X 10 P815 c e l l s ) were incubated with 5 X 10^ mitomycin C treated P815 c e l l s for various periods of time, then tested for anti-P815 c y t o t o x i c i t y by -'•'-Cr release assay. The effector c e l l to target c e l l r a t i o was 25:1 and the c e l l s were incubated for 18 hr. 25 TABLE I I I . EFFECTOR CELLS MEDIATING IN VITRO GENERATED CYTOTOXICITY % Cytotoxicity Exp l ( b ) Exp 2 ( C ) non-treated 12.3 35.1 36.8 a n t i 6 + C' 0 0.2 0 carbonyl iron 10.0 38.1 34.2 nylon wool column 11.1 36.3 31.2 (a) Cytotoxicity was induced i n v i t r o using spleen c e l l s from small tumor bearing mice (10 days after subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s ) . Effector c e l l to target c e l l r a t i o i n -'•'•Cr release assay was 50:1. (b) Incubation time with target c e l l s was 6 hr. (c) Incubation time with target c e l l s was 18 hr. 26 lymphocyte d e p l e t i o n by adherence to nylon wool nor removal of phagocytic c e l l s by carbonyl i r o n treatment a f f e c t e d the c y t o t o x i c i t y (Table I I I ) . Therefore i t was concluded that the c y t o t o x i c i t y was mediated by T lymphocytes. In v i t r o incubated c e l l s which were h i g h l y c y t o t o x i c to P815 c e l l s d i d not show any c y t o t o x i c i t y to un r e l a t e d P388 and S49A tumor c e l l s . When normal DBA/2 spleen c e l l s were s e n s i t i z e d i n v i t r o against CBA spleen c e l l s •byi the same c u l t u r e method, the s e n s i t i z e d c e l l s , which were c y t o t o x i c to PHA st i m u l a t e d CBA spleen c e l l s , d i d not show s i g n i f i c a n t c y t o t o x i c i t y against.P815 c e l l s (Table I V ) . Therefore, the a c t i v i t y appears to be s p e c i f i c . E f f e c t of serum on c y t o t o x i c i t y Since i t has been reported that serum from tumor bearing hosts sometimes contains 'blocking f a c t o r ' which s p e c i f i c a l l y i n h i b i t s anti-tumor c y t o t o x i c i t y ( Hellstrom and H e l l s t r o m , 1974) or f a c t o r s which n o n s p e c i f i c a l l y i n h i b i t general lymphocyte a c t i v i t i e s (Whitney and Levy, 1974), serum from DBA/2 mice bearing p r o g r e s s i v e l y growing P815 tumors (17 - 19 days a f t e r subcutaneous 4 i n j e c t i o n of 5 X 10 P815 c e l l s ) was te s t e d f o r the i n h i b i t i o n of anti-P815 c y t o t o x i c i t y . Spleen c e l l s from mice bearing small P815 tumors (16 days a f t e r subcutaneous 4 i n j e c t i o n of 10 P815 c e l l s ) were t e s t e d f o r anti-P815 c y t o t o x i c i t y i n the presence of serum from normal DBA/2 mice or P815 tumor bearing mice (Table V ) . The serum was a l s o t e s t e d f o r the i n h i b i t i o n of the i n v i t r o generation of anti-P815 c y t o t o x i c i t y at v a r i o u s concentrations (Table V I ) . In both experiments the serum from P815 tumor bearing DBA/2 mice, as compared to normal DBA/2 mice serum, had no i n h i b i t o r y e f f e c t . Therefore, i t i s u n l i k e l y that the d e c l i n e of c y t o t o x i c i t y observed as the tumor progressed i s due to the presence of 'blocking f a c t o r ' or n o n - s p e c i f i c i n h i b i t o r y f a c t o r s i n the serum. 27 TABLE IV. SPECIFICITY OF CYTOTOXICITY GENERATED IN VITRO In V i t r o culture (a) Cytotoxicity Test ' Responding Cells Stimulating c e l l s Target c e l l s % cyt o t o x i c i t y P815 bearing spleen P815 P815 19.5***(°) P815 bearing spleen P815 P388 0.3 N S ( d ) P815 bearing spleen P815 S49A 0.4 N S normal DBA spleen CBA spleen P815 i . o N S normal DBA spleen CBA spleen CBA s p l e e n ^ 25.5*** (a) "'"''Cr release after 6 hr incubation, effector to target r a t i o 50:1. (b) GBA spleen c e l l s were incubated for 3 days with 1% PHA before labeling with 51cr. (c) S t a t i s t i c a l l y s i g n i f i c a n t P< 0.005. (d) S t a t i s t i c a l l y not s i g n i f i c a n t 2 8 TABLE V. EFFECT OF SERUM ON ANTI-P815 CYTOTOXICITY Serum concentration Specific cytotoxicity ± SEM 1 1 . 3 ± 1 . 5 5% 3 . A ± 0 . 6 5% 6 . 2 ± 0 . 3 (a) JJ"Cr release assay: effector c e l l to target c e l l ratio was 200:1, incubation period was 18 hr. The figures are the means of tr i p l i c a t e tests. (b) Serum from normal DBA/2 mice. (c) Serum from DBA/2 mice with big P815 tumors. (16-19 days after injection subcutaneously wi th 5 X 104 P815 c e l l s ) . . (b) normal serum (c) tumor serum 29 TABLE VI. EFFECT OF SERUM ON IN VITRO GENERATION OF ANTI-P815 CYTOTOXICITY Serum concentration S p e c i f i c cytotoxicity^± SEM 42.6 ± 0.7 5.6 ± 0.7 7.2 ± 0.8 10.9 ± 0.9 4.5 ± 0.4 4.6 ± 0.8 17.4 ± 1.1 (a) Anti-P815 c y t o t o x i c i t y was generated i n v i t r o using spleen c e l l s from mice bearing small P815 tumors (14 days a f t e r subcutaneous i n j e c t i o n of 10 P815 c e l l s ) , "^Cr release assay: E f f e c t o r c e l l to target c e l l r a t i o was 50:1, incubation period was 18 hr. The figures are the means of t r i p l i c a t e t e s t s . (b) Serum from normal DBA/2 mice (c) Serum from big P815 tumor bearing mice (16-19 days a f t e r subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s ) . none normal serum 4% 2% 1% (c) tumor serum 4% 2% 1% 30 Suppressor Cells In Mice with Progressively Growing Tumor In contrast to spleen c e l l s from small tumor bearing mice, spleen c e l l s from mice with progressively growing tumors(16 - 19 days after i n j e c t i o n of tumor c e l l s ) did not develop cyt o t o x i c i t y after incubation with mitomycin C treated tumor c e l l s . Moreover, addition of these spleen c e l l s to cultures of spleen c e l l s from mice bearing small tumors in h i b i t e d the development of cyt o t o x i c i t y (Table VII). This was not due simply to the d i l u t i o n of reactive with unreactive c e l l s , because the addition of normal spleen c e l l s , which were also unreactive i n culture did not diminish the response as much as did c e l l s from progressors. The suppressive a c t i v i t y was also found i n thymus from the same mice. However, the same suppressive spleen c e l l s or thymocytes showed normal p r o l i f e r a t i v e responses to Con A and PHA and did not affect the response of normal spleen c e l l s to mitogens when mixed together (Table V I I I ) . I t seems unlikely that the suppression was due to metastatic tumor c e l l s i n spleen or thymus. Cells to be tested were carefully checked for the presence of tumor c e l l s by microscopic examination before and after culture. No tumor c e l l s were found i n spleens or thymuses used i n the suppression experiments, while as few as 200 tumor c e l l s (untreated with mitomycin C) added to the incubation mixture proliferated and were easily differentiated under the microscope after the incubation. Low background counts i n mitogenic response tests also support the conclusion that tumor c e l l s were not present i n spleens and thymuses used i n these tests because spleen c e l l s had very high background counts i f they were contaminated with tumor c e l l s . Therefore i t seems most l i k e l y that the unresponsiveness of spleen c e l l s from mice with progressively growing P815 tumors was due to the presence of suppressor 31 TABLE VII. SUPPRESSION OF IN VITRO GENERATION OF CYTOTOXICITY % C y t o t o x i c i t y v ' normal spleen normal thymus late P815 s p l e e n ( d ) late P815 thymus early P815 s p l e e n ^ early P815 s p l e e n ^ + normal spleen early P815 spleen + late P815 spleen early P815 spleen + normal thymus early P815 spleen + late P815 thymus Cells cultured Exp l ^ Exp 2 ( b ) E x p ( c ) Exp 4 ( c ) 0 1.2 0.6 0 0 0 0 -0 0 10.1 0 0 0 0 -22.8 11.6 46.8 89 17.4 8.8 21.5 84 6.2 0.7 13.4 48 22.7 10.2 31.7 84 3.5 5.4 4.1 74 (a) Effector to target r a t i o was 50:1 (b) 6 hour incubation for "**"Cr release assay (c) Overnight incubation for ^ C r release assay (d) Spleen or thymus c e l l s from mice 16-19 days after subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s (e) Spleen c e l l s from mice 10 days after subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s (f) For the mixing experiments 5 X 10 each c e l l population was mixed 3 2 TABLE VIII. LACK OF SUPPRESSION IN PROLIFERATIVE RESPONSE OF TUMOR BEARING SPLEEN CELLS AGAINST MITOGENS CPM ± SEM Cells Control Con A PHA normal spleen 18975 + 442 170758 + 3057 91415 + 4416 P815 s p l e e n ( a ) 23881 + 965 163218 + 4926 83374 + 4516 normal thymus 207 + 23 4164 + 381 656 + 209 P815 thymus 241 + 22 7755 + 491 641 + 228 normal s p l e e n ^ + P815 spleen 23507 + 1964 163392 + 6160 80749 + 4038 normal spleen^) + normal thymus 6012 + 602 89898 + 3148 37084 + 768 normal s p l e e n ^ 4+ P815 thymus 5799 + 315 104923 + 6358 38045 + 572 (a) Spleen c e l l s from mice bearing progressively growing P815 tumors (16-19 days after subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s ) (b) 2.5 X 10^ each c e l l population was mixed (c) The figures are the mean r a d i o a c t i v i t i e s of t r i p l i c a t e test cultures. c e l l s which inhibited i n v i t r o generation of cytotoxicity against P815 c e l l s . Preliminary studies have indicated that suppressor c e l l s of this type may develop even i n those animals i n whom tumors have been resected between days 10-14 after implantation. Such animals were s a c r i f i c e d at varying time intervals after tumor resection, and the i r spleen c e l l s tested for c y t o t o x i c i t y . At no time after resection were appreciable levels of cytotoxicity detected, and a l l animals so treated developed metastatic disease after a short time. Quantitative analysis of suppression Suppression of the i n v i t r o generation of cytotoxicity against P815 tumor c e l l s by the addition of spleen c e l l s from DBA/2 mice bearing progressively growing P815 tumors was quantitatively analyzed. When the per cent cyto-t o x i c i t y was plotted against the logarithm of effector/target c e l l ratio*?., a li n e a r relationship was, observed i f the cyt o t o x i c i t y was higher than 20% (Fig. 5). One l y t i c u n i t , which was defined as the number of effector 4 c e l l s to cause 50% l y s i s of 10 target c e l l s i n 18 hours incubation, was calculated by linear regression analysis of the cytotoxicity versus the logarithm of c e l l r a t i o . The t o t a l l y t i c units i n test cultures and control cultures were obtained from c e l l recovery i n the cultures. Then the degree of suppression was quantitated by the decrease i n t o t a l l y t i c units i n test cultures as compared to controls. No signigicant difference i n c e l l recovery between test cultures and controls were normally observed. Using this method the dose response of suppressor c e l l s i n the cultures to generate anti-P815 cytotoxicity was tested (Fig. 6). The degree of suppression was dependent on the dose of suppressive lymphoid c e l l s and a higher dose of suppressive spleen c e l l s or thymocytes caused higher suppression. In the following suppression experiments 5 X 10 suppressive spleen or thymus c e l l s were mixed with 5 X 10 immune spleen c e l l s . 100 2.5 5 1 0 20 40 80 EFFECTOR/TARGET CELL RATIO Figure 5. Linear regression analysis of the suppression of a n t i -P815 c y t o t o x i c i t y . Spleen c e l l s from DBA/2 mice with small P815 tumors (14 days after subcutaneous i n j e c t i o n of 2 X 10-^  P815 c e l l s ) were used as immune c e l l s to generate anti-P815 c y t o t o x i c i t y i n i n v i t r o cultures. Normal spleen c e l l s or P815 tumor^bearing spleen c e l l s (19 days after subcutaneous in j e c t i o n of 5 X 10 P815 c e l l s ) were mixed with an equal number of immune c e l l s and the t o t a l lymphoid c e l l number adjusted to 10 i n each culture. The cy t o t o x i c i t y test was carried out at various effector/target c e l l r a t i o s and per cent c y t o t o x i c i t y was plotted against logarithm of the r a t i o . Closed c i r c l e s represent the results of control cultures con-t a i n i n g normal spleen c e l l s and open c i r c l e s represent the r e s u l t s of test cultures containing tumor bearing spleen c e l l s . Per cent c y t o t o x i c i t y i s the mean of t r i p l i c a t e tests. V e r t i c a l bars show standard error of the mean. 60 40 2 0 -2 0 1 0 20 50 4 01 DOSE OF SUPPRESSIVE CELLS (%) Figure 6. Effect of dose of suppressive lymphoid c e l l s on the degree of suppression. Spleen c e l l s (open bars) or thymocytes (hatched bars) from mice with progressively growing P815 tumors (18 days after subcutaneous i n j e c t i o n of 5 X 10 P815 c e l l s ) were used for the suppression of the anti-P815 c y t o t o x i c i t y . Total lymphoid c e l l number was always adjusted to 10^. Per cent suppression was estimated from the decrease i n t o t a l l y t i c unit of the test cultures as compared to that of control cultures containing normal spleen c e l l s or thymocytes. 36 Characterization of suppressor c e l l s Since suppressor c e l l s were demonstrated intthymuses as well as spleens from P815 tumor bearing mice (16 - 19 days a f t e r subcutaneous i n j e c t i o n of 4 5 X 10 P815 c e l l s ) , i t was considered l i k e l y that they were T lymphocytes. In order to confirm t h i s , suppressive spleen c e l l s were treated i n various ways following which t h e i r suppressive a c t i v i t y was tested (Fig. 7). The a c t i v i t y was almost eliminated by a n t i 0 serum and complement treatment. B lymphocyte depletion by a n t i mouse Ig serum and complement, or removal of phagocytic c e l l s by carbonyl iron/magnet treatment did not a f f e c t the suppressive a c t i v i t y . It was concluded that the suppressor c e l l s i n P815 tumor bearing mice were T lymphocytes. S p e c i f i c i t y of suppressor c e l l s In order to determine the s p e c i f i c i t y of the suppressor c e l l s from P815 tumor bearing mice, t h e i r e f f e c t on the generation c y t o t o x i c i t y against another syngeneic tumor i n DBA/2 mice was tested. DBA/2 mice were 3 injected subcutaneously with 10 syngeneic L1210 leukemia c e l l s . When spleen c e l l s from these mice, taken 10 - 12 days a f t e r tumor c e l l i n j e c t i o n , were incubated with mitomycin C treated L1210 c e l l s i n the same way as i n the P815 tumor system, strong c y t o t o x i c i t y against the L1210 tumor c e l l s redeveloped. When thymocytes from P815 bearing mice, which suppressed the generation of anti-P815 c y t o t o x i c i t y , were added to the spleen c e l l s from L1210 leukemia bearing mice, no suppression of anti-Ll210 c y t o t o x i c i t y was observed (Table IX). This observation supports the contention that the suppressor c e l l s observed i n t h i s system are s p e c i f i c for c e l l s e x h i b i t i n g s p e c i f i c c y t o t o x i c i t y f o r the P815 mastocytoma. 37 I 60r 5 OF Z o LU o. ft. 40 30 20 10 CONTROL ANTI - %• ANTI-lg CARBONYL + + IRON Figure 7. Characterization of suppressor c e l l s i n spleen from P815 tumor bearing mice. Spleen c e l l s from mice with progressively growing P815 tumors (17-19 days a f t e r subcutaneous i n j e c t i o n of 5 X 10 4 P815 c e l l s ) were tested for the suppressive a c t i v i t y . a f t e r various treatments. Non-treated (control) or treated c e l l s were added to the i n v i t r o cultures to generate anti-P815 c y t o t o x i c i t y to consist 50% of the lymphoid c e l l s i n the cultures. Per cent suppression was estimated from the decrease i n c y t o t o x i c i t y of the test cultures at a f i x e d e f f e c t o r / t a r g e t c e l l r a t i o as compared to that of the cultures containing normal spleen c e l l s treated i n i d e n t i c a l manners. Results are the average of three experiments and v e r t i c a l bars show standard error of the mean. 38 TABLE IX. SPECIFICITY OF SUPPRESSOR CELLS (a) % Specific Cytotoxicity ' ± SEM Target Cells cultured Exp. 1 Exp. 2 Exp. 3 Exp. 4 P815 immune ( b ) (10 7) 66.1 ±2.9 52.4 ± 1.5 67.0 ±2.0 31.6 ±0.5 P815 immune (5X106) p815 + 62.9 ±1.0 49.8 ± 2.6 31.3 ±0.8 24.7 ±0.3 normal thymocytes (5X10 ) P815 immune (5X106) + 54.1 ± 0.5 34.7 ± 1.5 25.4 ±2.6 18.2 ±1.6 P815 thymocytes w(5X10 ) L1210 immune ( d )(10 7) 15.8 ± 1.5 44.4 ± 1.0 36.5 ± 3.9 77.7 ± 2.8 L1210 immune (5X106) L 1 2 i o + i 4 - 4 - i - 8 2 2- 9 ± °-4 31.2 ± 1,9 67.1 ± 1.5 normal thymocytes (5X10 ) L1210 immune (5X106) + , 15.8 ± 0.9 26.11 ± 0.5 33.6 ± 4.0 79.9 ± 1.5 P815 thymocytes (5X10 ) (a) J X C r release assay; effector c e l l to target c e l l r a t i o was 20:1, incubation period was 18 hours. Spontaneous release of P815 c e l l s was 16-20%, L1210 c e l l s 13-17%. The figures are the means of t r i p l i c a t e tests. (b) Spleen c e l l s from DBA/2 mice with small P815 tumors (14 days after subcutaneous in j e c t i o n of 10 4 P815 c e l l s ) . (c) Thymocytes from DBA/2 mice with progressively growing P815 tumors (16-19 days after subcutaneous i n j e c t i o n of 5X10 P815 c e l l s ) . (d) Spleen c e l l s from DBA/2 mice with small L1210 tumors (10-12 days after subcutaneous inj e c t i o n of IO 3 L1210 c e l l s ) . 39 Ficoll-hypaque density c e l l separation Since both cytotoxic k i l l e r c e l l s and suppressor c e l l s i n P815 tumor bearing mice appeared to belong to the T lymphocyte population, attempts were made to p h y s i o l o g i c a l l y d i f f e r e n t i a t e one population from the other. For t h i s purpose ficoll-hypaque density c e l l separation was ca r r i e d out using spleen c e l l s from P815 tumor bearing mice, following which each f r a c t i o n was tested for c y t o t o x i c i t y against P815 c e l l s or suppressive a c t i v i t y . When spleen c e l l s from mice bearing small P815 tumors (14 days 4 a f t e r subcutaneous i n j e c t i o n of 5 X 10 P815 c e l l s ) were used, the cytotoxic c e l l s were enriched i n the dense f r a c t i o n (Table X), while the suppressor a c t i v i t y i n spleen c e l l s from big P815 tumor bearing mice (18 - 19 days 4 a f t e r subcutaneous i n j e c t i o n of 5 X 10 P815 c e l l s ) was mainly recovered i n the l i g h t f r a c t i o n (Table XI). These r e s u l t s suggest that the k i l l e r c e l l s and the suppressor c e l l s i n t h i s tumor system are not p h y s i o l o g i c a l l y i d e n t i c a l . However, i t may be possible that the suppressor c e l l s i n big tumor bearing mice r e s u l t from a further d i f f e r e n t i a t i o n step of k i l l e r c e l l s . Relationship between tumor growth and suppressive a c t i v i t y i n lymphoid organs from tumor bearing mice Since suppressive a c t i v i t y was detected i n thymuses as well as i n spleens from mice bearing big P815 tumors, further experiments were c a r r i e d out to investigate the suppressive a c t i v i t i e s i n spleens, thymuses and lymph nodes at various stages of tumor growth. DBA/2 mice were injected subcutan-4 eously with 5 X 10 P815 c e l l s . At various times following i n j e c t i o n of tumor c e l l s , three mice i n each group were s a c r i f i c e d , t h e i r spleen c e l l s , thymocytes and lymph node c e l l s were pooled separately and tested for suppressive a c t i v i t y (Table XII). Suppressive a c t i v i t y was f i r s t detected i n the thymus 8 days a f t e r 40 TABLE X. FICOLL-HYPAQUE DENSITY .CELL SEPARATION OF KILLER CELLS C e l l f r action ( c e l l density) (a) C e l l recovery C y t o t o x i c i t y ^ (%) ± SEM unseparated l i g h t (d<1.06) medium (1.06<d<1.08) dense (1.08 <d) 15.4 17.4 67.2 17.0 ± 1.0 14.5 ± 0.8 10.3 ± 1.2 19.0 ± 0.7 unseparated l i g h t (d <1.06) medium (1.06 <d<1.08) dense (1.08 <d) 22.2 13.0 64.9 6.8 ± 2.1 3.6 ± 1.2 1.4 ± 0.8 13.2 ± 1.0 (a) Ficoll-hypaque density c e l l separation was carried out i n two steps using ficoll-hypaque with density of 1.06 and 1.08. (b) ^ xCr release assay: effector to target c e l l r a t i o was 200:1, incubation period was 18 hours. The results are average of quadruplicate incubation mixtures. (c) Spleen c e l l s from mice with small P815 tumors (14-16 days after subcutaneous in j e c t i o n o f 2X103 P815 c e l l s ) were used as a source of k i l l e r c e l l s . 41 TABLE XI. FICOLL-HYPAQUE DENSITY CELL SEPARATION OF SUPPRESSOR CELLS C e l l F r a c t i o n Suppression (density) (%) Exp. 1 ^ u n s e p a r a t e d ^ 17.2 l i g h t (d <1.08) 58.4 dense (d> 1.08) -46.3 Exp. 2^b^ u n s e p a r a t e d ^ 40.6 l i g h t (d<1.06) 57.4 medium (1.06 < d <1.08) 25.5 dense (1.08 <d) 34.6 (a) C e l l separation was c a r r i e d out i n one step using ficoll-hypaque with density of 1.08. (b) C e l l separation was c a r r i e d out i n two steps using ficoll-hypaque with density of 1.06 and 1.08. (c) Per cent suppression was estimated from the decrease i n t o t a l l y t i c units of the test cultures as compared to those of control cultures containing normal spleen c e l l s treated i n i d e n t i c a l manners. (d) Spleen c e l l s from mice with progressively growing P815 tumors (17-19 days a f t e r subcutaneous i n j e c t i o n of 5X10 4 P815 c e l l s ) were used as a source of suppressor c e l l s . TABLE XII. SUPPRESSIVE ACTIVITY IN LYMPHOID ORGANS FROM TUMOR BEARING MICE (a) Days after tumor in j ection Percent . (b) Suppression Spleen Thymus Lymph Node 4 18.7 N S 7.0 N S 8 8 -46.0<d) 61.2 -48.6 12 -595.1 53.9 - 5.0 N S 16 -389.8 4.3 N S 40.2 19 17.2 38.4 36.0 (a) DBA/2 mice received 5X10H P815 c e l l s subcutaneously. Spleens, thymuses or lymph noeds from 3 mice i n each group were pooled and tested. (b) Per cent suppression was calculated from the per cent decrease i n t o t a l l y t i c units i n the test cultures as compared to the controls. (c) Difference i n cyto t o x i c i t y i s not s t a t i s t i c a l l y s i g n i f i c a n t by t- t e s t . (d) Negative numbers show the enhancement of the cyt o t o x i c i t y by tumor bearing lymphoid c e l l s . 43 tumor c e l l i n j e c t i o n , when the tumors were very small and spleen c e l l s and lymph node c e l l s showed some cytotoxic responses. The suppressive a c t i v i t y i n thymus persisted i n the stage of slow tumor growth (day 12) while spleen c e l l s showed a strong immune response. Lymph.node c e l l s became suppressive (day 16) before the appearance of suppressor c e l l s i n spleens. When tumors grew progressively (day 19), suppressor c e l l s were detected i n spleens as well as i n thymuses and lymph nodes. Suppression by thymocyte extracts C e l l free extracts, prepared by freeze-thawing single c e l l suspension of normal or P815 tumor bearing thymocytes i n PBS, were tested for suppressive a c t i v i t y . The thymocyte extracts were added to the i n v i t r o cultures to generate s p e c i f i c c y t o t o x i c i t y against P815 or L1210 c e l l s . The extract prepared from thymocytes from P815 tumor bearing mice inhibited the generation of anti-P815 cytotoxicity as compared to the normal thymocyte extract (Table X I I I ) . The same extract did not affect the generation of anti-L1210 c y t o t o x i c i t y . Therefore the suppression of anti-P815 cyt o t o x i c i t y by the thymocyte extract i s s p e c i f i c and i s not due to the presence of non-specifically toxic substances i n the extract. 44 TABLE XIII. SUPPRESSION BY THYMOCYTE EXTRACTS (k\ C y t o t o x i c i t y v '(%) ± SEM C e l l Extracts ' anti P815 ( c ) a n t i L1210 - 60.5 + 2.6 38.2 ± 2.5 normal thymus 54.8 + 2.7 47.1 ± 1.8 P815 thymus 43.6 + 3.3 49.4 ± 1.3 (a) Thymocyte suspension i n PBS (SXlo'/ml) was freeze-thawed three times aand centrifuged 20 min at 400 x g.. Supernatant was f i l t r a t e d through mil l i p o r e membrane (pore size 0.2 u) . (b) "^Cr. release assay: Effector to target r a t i o was 40:1, incubation period was 18 hours. The figures are the means of t r i p l i c a t e tests. (c) Spleen c e l l s from DBA/2 mice bearing small P815 tumors (14 days after subcutaneous i n j e c t i o n of 2 X 10^ P815 c e l l s ) were used as P815 tumor immune c e l l s to generate anti-P815 c y t o t o x i c i t y . (d) Spleen c e l l s from DBA/2 mice bearing small L1210 tumors (10 days after subcutaneous i n j e c t i o n of L1210 c e l l s ) were used as L1210 tumor immune c e l l s to generate anti-L1210 c y t o t o x i c i t y . DISCUSSION In preliminary studies, i t was observed that spleen c e l l s of DBA/2 mice, injected subcutaneously with a syngeneic mastocytoma c e l l l i n e , developed s p e c i f i c cytotoxicity to their tumors during the f i r s t two weeks of tumor growth. The immunity could be measured by a direct assay. Cytotoxic c e l l s were i d e n t i f i e d as T lymphocytes. Maximum cytotoxic a c t i v i t y coincided, i n tumor bearing animals, with a period of slowed tumor growth, as assessed by direct measurement of the tumor siz e . Following this period, tumor growth resumed at an accelerated rate, and th i s occurrence coincided with a loss of s p e c i f i c cytotoxicity. The spleen c e l l s of tumor bearing animals, at the time of loss of s p e c i f i c c y t o t o x i c i t y , appeared to be normal i n other functions such as their i n v i t r o responses to mitogens. This observation was unlike previous observations made i n th i s laboratory that the lymphocytes of DBA/2 mice with progressively growing transplantable methylcholanthrene induced sarcomas l o s t both tumor s p e c i f i c immunity and other immunologic functions such as mitogen responsiveness and the a b i l i t y to respond In v i t r o to unrelated antigens (Whitney et_ a l . , 1974). In order to investigate this apparent suppression of s p e c i f i c cytotoxicity we established an i n v i t r o method to induce cytotoxicity against the tumor. Unlike the methods reported by Wagner and Rollinghoff (1973) i n which normal spleen c e l l s were sensitized against syngeneic tumors, we used spleen c e l l s from mice bearing small tumors which already demonstrated some cytotoxic a c t i v i t y against the tumor c e l l s . Therefore, the response i n these cultures was probably a secondary response, which f a c i l i t a t e d detection, obviating the need for a complicated culture method. Similar methods to generate cytotoxicity against syngeneic tumors i n v i t r o have been reported recently i n other tumor systems including a mammary adenocarcinoma i n rats (Kuperman et a l . , 1975a), a lymphoma (Glaser et_ a l . , 1976) and a methylcholanthrene induced sarcoma (Kail and Hellstrom, 1975) i n mice. The cytotoxicity induced i n the present studies was sp e c i f i c to the tumor and mediated by T lymphocytes. Using t h i s method, c e l l s capable of i n h i b i t i n g the i n v i t r o generation of cytotoxicity were found i n the spleen and thymus of mice at a l a t e r stage of the progressive growth of their tumors. Serum from mice with progressively growing P815 tumors inhibited neither the a c t i v i t y of cytotoxic c e l l s nor the generation of them i n v i t r o . I t appears that the decline of cytotoxicity observed as the tumors progress can be attributed to the presence of suppressor c e l l s which i n h i b i t the generation of cytotoxic c e l l s . Characterization of these suppressor c e l l s i n P815 tumor bearing DBA/2 mice indicates that: (1) . Suppressor c e l l s were sensitive to ant i 9 serum and complement treatment, but insensitive to anti mouse Ig serum and complement treatment or carbonyl iron and magnet treatment. Therefore, they seem to be T lymphocytes. (2) . Suppressor c e l l s were s p e c i f i c to the tumor, i . e . , suppressor c e l l s from P815 tumor bearing mice did not i n h i b i t the i n v i t r o generation of cytotoxicity against another tumor l i n e (L1210 leukemia i n DBA/2 mice), nor did they i n h i b i t the p r o l i f e r a t i v e response of T lymphocytes to mitogens. (3) . Suppressor c e l l s and k i l l e r c e l l s migrate i n separate fractions i n ficoll-hypaque density c e l l separation. Therefore, suppressor T c e l l s and k i l l e r T c e l l s i n th i s system appear to be d i s t i n c t populations. Suppressor c e l l s that i n h i b i t s p e c i f i c anti-tumor immunerresponses have been reported i n other tumor systems. Suppressor c e l l s from Japanese quails bearing Rous virus induced sarcoma (Hayami et a l . , 1972) and rats bearing a mammary adenocarcinoma (Kuperman et_ a l . t 1975b) inhibited the generation of s p e c i f i c c y t o t o x i c i t y against the i r respective tumors. The s p e c i f i c i t y and the nature of the c e l l s involved was not tested i n these studies. Suppressive a c t i v i t y of peritoneal c e l l s from mice bearing sarcomas induced either by Moloney sarcoma.virus or methylcholanthrene were reported by Halliday (1972). Using the macrophage migration i n h i b i t i o n assay, he found that peritoneal c e l l s from mice with progressively growing sarcomas did not show an. anti-tumor response. When these progressor c e l l s were mixed with the c e l l s from mice whose tumors had regressed spontaneously or had been sur g i c a l l y removed, they inhibited the anti-tumor response normally observed with the regressor c e l l s . This effect was s p e c i f i c for the given tumor studied. A similar phenomenon has been reported by Fujimoto et a l . (1976 a, b) i n mice bearing methylcholanthrene induced sarcomas. Suppressor c e l l s from these animals or t h e i r extracts, when injected into immune mice, decreased s p e c i f i c anti-tumor a c t i v i t y as determined by direct measurement of the tumor size i n vivo. The suppressor c e l l s were found i n the thymus, spleen, lymph nodes and bone marrow of tumor bearing mice and were thought to be T lymphocytes because of their s e n s i t i v i t y to treatment with anti 0 serum and complement. Suppressor c e l l s which i n h i b i t general immune responses i n a nonspecific way have been also reported i n mice with sarcomas induced by murine sarcoma virus (Kirchner et a l . , 1974) or methylcholanthrene (Eggers and Wunderlich, 1975; Pope _et _al. , 1976). Gorczynski et^ al. (1975) proposed that the suppressor c e l l s i n the former tumor system were B lymphcoytes which inhibited the generation of cy t o t o x i c i effector c e l l s against tumor as well as the PHA response of T lymphocytes by means of antigen-antibody complexes. In contrast, Kirchner _et _al. "(1975) suggested that the suppressor c e l l i n the same tumor system were c e l l s of the monocyte/macrophage series and that they inhibited 48 general immunity by i n h i b i t i n g DNA synthesis i n T lymphocytes. The nonspecific suppressor c e l l s i n a methylcholanthrene induced sarcoma system have also been i d e n t i f i e d as adherent c e l l s of the macrophage/monocyte series (Pope et a l . . 1976) and i n h i b i t general immune responses of normal lymphocytes such as the p r o l i f e r a t i v e response to mitogens (Pope et_ a l . , 1976) or the response to allo-antigens i n mixed leukocyte cultures (Eggers and Wunderlich, 1975). Although the relationship between nonspecific and s p e c i f i c suppressor c e l l s i n tumor bearing animals i s not yet clear, i t i s thought that both of them can i n h i b i t s p e c i f i c a n t i tumor immunity which may result i n progressive tumor growth. In the present study s p e c i f i c suppressor c e l l s were detected i n mice during the early stages of tumor growth when tumors,were very small and some cytotoxic reaction was detected. Similar observations i n a methylcholanthrene induced sarcoma were reported by Fujimoto et a l . (1976 a, b) who detected s p e c i f i c suppressor c e l l s i n animals within 24 hours after tumor c e l l i n j e c t i o n . On the other hand nonspecific suppressor c e l l s i n sarcoma bearing mice were normally detected i n the l a t e r stages of tumor growth when the tumors grew progressively (Popeet a l . , 1976; Eggers and Wunderlich, 1975). Moreover, the c e l l s involved i n s p e c i f i c and nonspecific suppression seem to be di f f e r e n t . Specific suppressor c e l l s are thought to be T lymphocytes while nonspecific suppressor c e l l s are not T lymphocytes (Kirchner et a l . , 1974; Pope et a l . , 1976). Therefore, these two types of suppression i n tumor bearing animals may involve d i s t i n c t mechanisms. I t i s possible that both types of suppression occur i n many tumor systems, presumably i n different stages of tumor growth. In f a c t , both types of suppressor c e l l s have been detected i n very similar tumor systems such as sarcomas induced by methylcholanthrene (Eggers and Wunderlich, 1975; Fujimoto et a l . , 1976) or murine sarcoma virus (Kirchner et a l . , 1974; Halliday, 1972). The f a i l u r e to detect non-specific suppression i n P815 mastocytoma tumor bearing mice may be due to the fast growth and metastasis oftthe tumors. Antigen s p e c i f i c suppressor T c e l l s have been widely studied i n various immunological systems (Gershon, 1975). I t i s generally assumed that these suppressor c e l l s are d i r e c t l y involved i n the regulation of immune responses. However, i t i s not known how suppressor a c t i v i t y i s induced and i n what way they i n h i b i t immune responses. One of the approaches to these questions i s to study relationships between various functions of T lymphocytes, i . e . helper, k i l l e r and suppressor. Recent studies on Ly antigens on subpopulations of T lymphocytes suggested that helper c e l l s and k i l l e r c e l l s are d i s t i n c t populations, but k i l l e r c e l l s and suppressor c e l l s share the same Ly antigens (Medawar and Simpson, 1975). However, the present study suggests that suppressor c e l l s and k i l l e r c e l l s i n P815 tumor bearing mice are not physiologically i d e n t i c a l . When P815 tumor bearing spleen c e l l s were fractionated by f i c o l l -hypaque density c e l l separation, k i l l e r c e l l s were enriched i n the fr a c t i o n bf heavier density while suppressor c e l l s were found mainly i n the l i g h t e r f r a c t i o n . I t i s possible that they are i n the same subpopulation of T lymphocytes but are i n a different stage of c e l l d i f f e r e n t i a t i o n . The present studies on the development of suppressor c e l l s at various stages of tumor growth indicate that suppressor c e l l s develop i n the thymuses of P815 tumor bearing animals at an early stage i n tumor growth, while spleen c e l l s are s t i l l demonstrating s i g n i f i c a n t levels of cytotoxicity toward P815 c e l l s . Since cytotoxic c e l l s are never apparent i n the thymus at any time during tumor growth, i t seems unlikely that suppressor c e l l s result from a further d i f f e r e n t i a t i o n step of the s p e c i f i c k i l l e r c e l l s . These results also suggest a possible role of the thymus i n immune responses i n adult l i f e . Although i t has been well known that the thymus plays an essential role i n T lymphocyte d i f f e r e n t i a t i o n i n neonatal l i f e , the role of thymus i n adult l i f e has been unknown. Recent studies on the subclasses of T lymphocytes have shown that a population of T lymphocytes i n the peripheral lymphoid system which seem to have a short l i f e span are p a r t i a l l y diminished by adult thymectomy.(Cantor et a l . , 1975). I t has also been shown that adult thymectomy enhances various immune responses such as antibody formation against T lymphocyte independent antigens (Kerbel and Eidinger, 1972), primary cytotoxic responses to alloantigens (Simpson arid Cantor, 1975) and the s e n s i t i z a t i o n against syngeneic fibroblasts (Carnaud et a l . , 1975), suggesting that the short l i v e d T lymphocytes that are sensitive to adult thymectomy are involved i n the regulation of these immune reactions. Although the relationship between suppressor c e l l s i n thymuses, spleens and lymph nodes i n P815 tumor bearing mice i s not clear at this point, the present studies suggest that the thymus plays an important role i n the suppression of anti-tumor immunity. Although antigen s p e c i f i c suppressor c e l l s have been studied i n various immunological systems at the c e l l u l a r l e v e l , their mechanisms are s t i l l l argely unknown. Since s p e c i f i c suppressor c e l l s seem to recognize antigens i n a s p e c i f i c way, i t i s generally assumed that they also carry surface receptors for antigens and that the receptors are i n some way involved i n suppression. In the present study c e l l free extracts prepared from suppressive thymocytes had the same suppressive a c t i v i t y as the intact c e l l s . Similar results have been reported by Fujimoto et a l . (1976)' using thymocytes from mice bearing sarcomas. Tada et a l . (1975) also detected antigen s p e c i f i c suppressive factors i n c e l l free extracts prepared from lymphoid c e l l s which suppressed antibody formation both i n vivo and i n v i t r o . By using s p e c i f i c antiserum against h i s t o c o m p a t i b i l i t y a n t i g e n s , they demonstrated that the suppressive f a c t o r s were a s s o c i a t e d w i t h some h i s t o c o m p a t i b i l i t y a n t i g e n s , most l i k e l y the products of I r e g i o n genes. F u r t h e r s t u d i e s u s i n g c e l l free e x t r a c t s prepared from suppressive thymo-cytes i n the P815 tumor system w i l l enable us to study not only the tumor-host r e l a t i o n s h i p i n tumor b e a r i n g hosts but the r e g u l a t i o n of the c e l l u l a r immune response at the molecular l e v e l . REFERENCES Alder, W.H., Takiguchi, T., and Smith, R.T. 1 9 7 1 . 'Phytohemagglutinin unresponsiveness i n mouse spleen c e l l s induced by methylcholanthrene. 1 Cancer Res., 3 JL , 8 6 4 . Baldwin, R.W., Price, M.R. and Robins, R.A. 1 9 7 2 . 'Blocking of lymphocyte mediated cytotoxicity for rat hepatoma by tumor-specific antigen-antibody complexes.' Nature New B i o l . , 2 3 8 , 1 8 5 . Barski, G. and Youn, J.K. 1 9 6 9 . 'Evolution of cell-mediated immunity i n mice bearing an antigenic tumor. Influence of tumor growth and surgical removal.' J. Natl. Cancer Inst., 4 3 _ , 1 1 1 . Beneceraff, B., Kapp, J.A., Debre, P., Pierce, C.W. and De La Croix, F. 1 9 7 5 . 'The stimulation of s p e c i f i c suppressor T c e l l s i n genetic non-responder mice by line a r random copolymers of L-amino acids.' Transplant. Rev., 2 6 , 2 1 . Burnet, F.M. 1 9 7 1 . 'Immunological surveillance i n neoplasia.' Transplant Rev. , ]_, 3 . Burns, F.D., Marrack, P.C. , .Kappler, J.W. and Janeway, CA. 1 9 7 5 . 'Functional heterogeneity among the T-derived lymphocytes of the mouse. IV. Nature of spontaneously induced suppressor c e l l s . ' J . Immunol., 1 1 4 , 1 3 4 5 . Cantor, H., Simpson, E., Sato, V.L., Fathman, CG, and Hertzenberg, L.A. 1 9 7 5 . 'Characterization of subpopulation of T lymphocytes. I. Separation and functional studies of peripheral T-cells binding different amounts of fluorescent anti-Thy 1 . 2 . (theta) antibody using a fluorescence-activated c e l l sorter (FACS).' C e l l . Immunol., 15_ , 1 8 0 . 8. Carnaud, C , I l f e l d , D. , Petranyi, G. and K l e i n , E. 1975. 'The role of thymus on autosensitization against syngeneic normal and malignant c e l l s . ' Eur. J. Immunol., 5_, 575. 9. Chia, E. and Festenstein, ,H. 1974. 'Specific cytostatic effect of lymph node c e l l s from normal and T c e l l - d e f i c i e n t mice on syngeneic tumor target c e l l s i n v i t r o and i t s s p e c i f i c abrogation by body f l u i d s from syngeneic tumor-bearing mice.' Eur. J. Immunol., 3_, 483. 10. Currie, G.A. and Bagshawe, K.D. 1969. 'Tumor-specific immunogenicity of methylcholanthrene-induced sarcoma c e l l s after incubation i n neuraminidase.' B r i t . J . Cancer, 2_3, 141. 11. Eggers, A.E.,and Wunderlich, J.R. 1975. 'Suppressor c e l l s i n tumor-bearing mice capable of non-specific blocking of i n , v i t r o immunization.' J . Immunol., 114, 1554. 12. E i l b e r t , F.R. and Morton, D.L. 1970. 'Impaired immunologic r e a c t i v i t y and recurrence following cancer surgery.' Cancer, 25_, 362. 13. Fujimoto, S., Greene, M.I. and Sehon, A.H. 1976a.' 'Regulation of the immune response to tumor antigens. I. Immunosuppressor c e l l s i n tumor-bearing hosts.' J. Immunol., 116, 791. 14. Fujimoto, S. , Greene, M.I7., and Sehon, A.H. 1976b. 'Regulation of the immune response to tumor antigens. I I . The nature of immunosuppressor c e l l s i n tumor-bearing hosts.' J.- Immunol., 116, 800. 15. Gerber, N.L., Hardin, J.A., Chused, T.M. and Steinberg, A.D. 1974. 'Loss with age i n NZB/W mice of thymic suppressor c e l l s i n graft-vs-host reaction.' J. Immunol., 113, 1618. 16. Gershon, R.K. and Kondo, K. 1971. 'Infectious immunological tolerance.' Immunology, TL_, 903. 17. Gershon, R.K., Lance, E.M. and Kondo, K. 1974. 'Immuno-regulatory role of spleen l o c a l i z i n g thymocytes.' J* Immunol., 112, 546. 54 18. Gershon, R.K. 1975. 'A d i s q u i s i t i o n on suppressor T c e l l s . ' Transplant., Rev., 26, 170. 19. Gorczynski, R.M., Kilburn, D.G., Knight, R.A., Norbury, C., Parker, D.C. and Smith, J.B. • 1975. 'Non-specific and s p e c i f i c immunosuppression i n tumor bearing mice by soluble immune complexes.' Nature, 254, 141. 20. Glaser, M., Bonnard, G.D. and Herberman, R.B. 1976. 'In v i t r o generation of secondary cell-mediated cytotoxic response against a syngeneic Gross virus-induced lymphoma i n rats.' J. Immunol., 116, 430. 21. Halliday, W.J. 1971. 'Blocking effect of serum from tumor-bearing animals on macrophage migration i n h i b i t i o n with tumor antigens.' J . Immunol., 106, 855. 22. Halliday, W.J. 1972. 'Macrophage migration i n h i b i t i o n with mouse tumor antigens: properties of serum and peritoneal c e l l s during tumor growth and after tumor loss.' C e l l . Immunol., _3, 113. 23. Hayami, M., Hellstrom, I . , Hellstrom, K.E. and Yamanouchi, K. 1972. 'Cell-mediated destruction of Rous sarcomas i n Japanese quails.' Inst. J. Cancer, J-0, 507. 24. Hellstrom, K.E. and Hellstrom, I. 1974. 'Lymphocyte mediated cyto t o x i c i t y arid blocking serum a c t i v i t y to tumor antigens.' Advances i n Immunology, 18, 209. 25. lHer,z;enb.er,gf„ L.A. , Okumura, K. and Metzler, CM. 1975. 'Regulation of immunoglobulin and antibody production by allotype suppressor T c e l l s i n mice.' Transplant. Rev., 27_, 57. 26. Houghton, G. and Whitmore, A.C. 1976. 'Genetics, the immune response and oncogenesis.' Transplant. Rev., 2_8, 75. 55 27. Jacobson, E.B. and Hertzenberg, L.A. 1972. 'Active suppression of immuno-globulin allotype. I. Chronic suppression after peritoneal exposure to maternal antibody to paternal allotype i n (SJL X BALB/C) F^ mice.' J . Exp. Med., 135, 1151. 28. J u l i u s , M .Hi , Simpson, E. and Hertzenberg, L.A. 1973. 'A rapid method for the i s o l a t i o n of functional thymus-derived murine lymphocytes.' Eur. J. Immunol., _3, 645. 29. K a i l , M.A. and Hellstrom, I. 1975. 'Specific stimulatory and cytotoxic effects of lymphocytes sensitized i n v i t r o to either alloantigen or tumor antigens.' J. Immunol., 114, 1083. 30. K e l l y , B., Kaye, B., Yoshizawa, W, Levy, J.G. and Kilburn, D.G. 1974. 'Selective binding of chemically defined antigenic peptides to mouse lymphocytes.' Eur. J. Immunol., k_, 356. 31. Kerbel, R.S. and Eidinger, D. 1972. 'Enhanced immune responsiveness to a thymus independent antigen early after adult thymectomy: Evidence for short-lived i n h i b i t o r y thymus-derived c e l l s . ' Eur. J. Immunol., _2,- 114. 32. Kirchner, H., Chused, T.M., Herberman, R.B., Holden, H.T. and Laurin, H. 1974. 'Evidence of suppressor c e l l a c t i v i t y i n spleens of mice bearing primary tumors induced by Moloney sarcoma v i r u s . ' J. Exp. Med., 39_, 1473. 33. Kirchner, H., Muchmore, A.V., Chused, T.M., Holden, H.T. and Herberman, R.B. 1975. 'Inhibition of p r o l i f e r a t i o n of lymphoma c e l l s and T lymphocytes by suppressor c e l l s from spleens of tumor-bearing mice.' J. Immunol., 114, 206. 34. K l e i n , G., Sjogren, H.O., K l e i n , E. and Hellstrom, K.E. 1960. 'Demonstration of resistance against methylcholanthrene-induced sarcomas i n the primary autochthonous host.' Cancer Res., 20_, 1561. 56 35. Krant, M.J., Manskopl, G. and Brandrup, CS. 1968. 'Immunologic al t e r a t i o n i n bronchgenic cancer.' Sequential Studies. Cancer, 2_1_, 623. 36. Kuperman, 0., Fortner, G.W. and Lucas, Z.J. 1975a. 'Immune response to a syngeneic mammary adenocarcinoma. I I . In v i t r o generation of cytotoxic lymphocytes.' J. Immunol., 115, 1277. 37. Kuperman, 0., Fortner, G.W., Lucan, Z.J. 1975b. 'Immune response to a syngeneic mammary adenocarcinoma. I I I . Development of memory and suppressor functions modulating c e l l u l a r c y t o t o x i c i t y . ' J. Immunol., 115, 1282. 38. Le Francois, D., Youn, J.K. and Belehradek, J. 1971. 'Evolution of cell-mediated immunity i n mice bearing tumors produced by a mammary carcinoma c e l l l i n e . Influence of tumor growth, surgical removal, and treatment with irradiated tumor c e l l s . ' J . N a t l . Cancer. Inst., 46_, 981. 39. Medawar, P.B. and Simpson, E. 1976. 'Thymus dependent lymphocytes.' Nature, 258, 106. 40. Nachtigal, D., Zan-Bar, I. and Feldman, M. 1975. 'The role of s p e c i f i c suppressor T c e l l s i n immune tolerance.' Transplant. Rev., 26_, 87. 41. Nimberg, R.B., Glasgow, A.H., Menzoian, J.O., Constania, M.B., Cooperland, S.R., Mannick, J.A. and Schmid, K. 1975. 'Isolation of a immunosuppressive peptide fr a c t i o n from the serum of cancer patients.' Cancer Res., 35, 1489. 42. Old, L.J., Stockert, E., Boyse, E.A. and Kim, J.H. 1968. 'Antigen modulation. Loss of TL antigen from c e l l s exposed to TL antibody. Study of the phenomenon i n v i t r o . ' J. Exp. Med., 127, 523. 43. Peavy, D.L. and Pierce, C.W. 1974. 'Ce l l mediated immune responses i n  v i t r o . I. Suppression of the generation of cytotoxic lymphocytes by concanavalin A and concanavalin A-activated spleen c e l l s . ' J . Exp. Med., 140, 357. 57 44. Pope, B.L., Whitney, R?B., Levy, J.L. and Kilburn, D.G. 1976. 'Suppressor c e l l s i n the spleens of tumor-bearing mice. Enrichment by centrifugation on hypaque-ficpll and characterization of the suppressor population.' J. Immunol., i n press. 45. Prager, M.D., Derr, I . , Swann, A. and Catropia, J. 1971. 'Immunization with chemically modified lymphoma c e l l s . ' Cancer Res., 31_, 1488. 46. Prehn, R.T. and Lappe, M.A. 1971. 'An immunostimulation theory of tumor development.' Transplant. Rev. , 7_, 26. 47. Revesz, L. 1960. 'Detection of antigenic differences i n isologous host-tumor system by pretreatment with heavily irradiated tumor c e l l s . ' Cancer Res., 20_, 443. 48. Rygaard, J. and Povlsen, C.Of' 1976. 'The nude mouse vs. the hypothesis of immunological surveillance.' Transplant. Rev., 2_8, 43. 49. Sanderson, C.J. and Frost, P. 1974. 'The induction of tumor immunity i n mice using glutaraldehyde-treated tumor c e l l s . ' Nature, 248, 690. 50. Simpson, E. and Cantor, H. 1975. 'Regulation of the immune response by subclass of T lymphocytes. I I . The effect of adult thymectomy upon humoral and c e l l u l a r response i n mice.' Eur. J. Immunol., 5_, 330. 51; Simpson, E. and Nehlsen, S.L. 1971. 'Prolonged administration of a n t i -thymocyte serum i n mice. I I . Histopathological investigation.' C l i n . Exp. Immunol., 9_; 79. 52. Stjernsward, J . , Almgard, L.E., Franzen, S., von Schreeb, T. and Wadstrom, L.B. 1970. 'Tumor-distinctive c e l l u l a r immunity to renal carcinoma.' C l i n . Exp. Immunol., J3, 963. 58 53. Sugui-Foca, N., Buda, J . , McManus, J . , Thiem, J . , and Reemtsma, K. 1973. 'Impaired responsiveness and serum in h i b i t o r y factors i n patients with cancer.' Cancer Res., 33, 3473. 54. Tada, T., Taniguchi, M. and Takemori, T. 1975. 'Properties of primed suppressor T c e l l s and thei r products.' Transplant. Rev., _26, 106. 55. Takasugi, M. and K l e i n , E. 1970. 'A microassay for cell-mediated immunity.' Transplantation, 9^, 219. 56. Taylor, R.B., Duffus, W.P.H., Raff, M.C. and De P e t r i s , S. 1971. 'Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody.' Nature New B i o l . , 233, 225. 57. Vanky, F., Stjernsward, J . , K l e i n , G. and Nilsonne, V. 1971. 'Serum-mediated i n h i b i t i o n of lymphocyte stimulation by autochthonous humor tumors.' J. Natl. Cancer Inst., 47_, 95. 58. Wagner, H. and Rollinghoff. 1973. 'In v i t r o induction of tumor-specific immunity. I. Parameters of activation and cytotoxic r e a c t i v i t y of mouse lymphoid c e l l s immunized i n v i t r o against syngeneic and allogeneic plasma c e l l tumors.' J . Exp. Med., .138, 1. 59. Whitney, R.B., Levy, J.G. and Smith, A.G. 1974. 'Influence of tumor size and surgical resection on cell-mediated immunity i n mice.' J. Natl. Cancer Inst., 53_, 111. 60. Whitney, R.B. and Levy, J.G. 1975a. 'Effects of sera from tumor-bearing mice on mitogen and allogenic c e l l stimulation of normal lymphoid c e l l . ' J. Natl. Cancer Inst;, 54_, 733. 61. Whitney, R.B. and Levy, J.G. 1975b. 'Studies on the mode of action of immunosuppressive substances i n the serum of tumor-bearing mice.' J. Natl. Cancer Inst., 55_, 1447. Winn, H.J. 1959; 'The immune response and the homograft reaction. Natl. Cancer Inst. Monogr. , 2_, 113. 

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