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 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