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P815 tumor-specific T suppressor cell and suppressor factor Maier, Tom 1981

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P815 TUMOR-SPECIFIC T SUPPRESSOR CELL AND SUPPRESSOR FACTOR by TOM MAIER B.S., The University of Washington, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1981 © Tom Maier I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f I C ̂  0 ia T c? ^ y The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date b*( . V- , DE-6 (2/79) i i ABSTRACT The work reported here involves studies of suppressor T c e l l s (T gC) and th e i r suppressor factor (SF) which s p e c i f i c a l l y suppress the i n v i t r o generation of c e l l s cytotoxic for a syngeneic tumor, P815, i n DBA/2 mice. This work can be divided into three sections: a) the immunogenetic properties and requirements of t h i s T gC and SF, b) the Lyt phenotype of the T gC as well as that of the c e l l s involved i n the cytotoxic response to the syngeneic tumor, c) the properties of syngeneic and allogeneic a n t i s e r a r a i s e d to the P815 s p e c i f i c SF. a) P815-antigen s p e c i f i c T gC and suppressive extracts . ' obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 tumors, were compared for t h e i r immunogenetic properties and require- ments. I t was shown that pretreatment of T gC populations with a n t i - l a ^ antiserum plus rab b i t complement removed the suppressive a c t i v i t y . S i m i l a r l y , absorption of the SF with a n t i - l a ^ antiserum removed the suppressive properties of the material. It was found that the TjC and SF were capable of s p e c i f i c a l l y suppressing the anti-P815 response of B6D2F^ r a d i a t i o n chimeras possessing lymphoid c e l l s of the b 12 H-2 or H-2 haplotype equally as well as they could suppress the response of H-2^ bearing c e l l s . This indicates that the T gC and SF are not H-2 r e s t r i c t e d with respect to K or D markers on responder c e l l s i n t h i s system. b) T C were also i d e n t i f i e d i n the spleens of DBA/2 mice injec t e d s i n t r a p e r i t o n e a l l y with membrane extracts of the P815 tumor. The Lyt phenotypes of va r i o u s e f f e c t o r c e l l s was determined. DBA/2 a l l o g e n e i c k i l l e r c e l l s were i d e n t i f i e d as L y t - l + 2 + , whereas the syngeneic e f f e c t o r c e l l s were found to be predominantly Lyt-1 2 +. The suppressor c e l l p o p u l a t i o n l o s t i t s a b i l i t y to suppress the i n v i t r o c y t o t o x i c anti-P815 response a f t e r treatment w i t h a n t i - L y t - 1 serum p l u s complement but not a f t e r treatment w i t h a n t i - L y t - 2 serum, i n d i c a t i n g that an L y t - l + 2 c e l l i s e s s e n t i a l i n t h i s suppression. c) P815 tu m o r - s p e c i f i c SF was p a r t i a l l y p u r i f i e d by passage of suppressive spleen e x t r a c t s through an immunoadsorbent c o n t a i n i n g P815 membrane components. A n t i s e r a r a i s e d i n syngeneic DBA/2 and a l l o g e n e i c , C57BL/6, mice were t e s t e d . I t was found that these a n t i s e r a , but not t h e i r c o n t r o l s were capable of absorbing out the SF. The a n t i s e r a were a l s o capable, i n the presence of complement, of e l i m i n a t i n g T gC from suppressive spleen c e l l p o p u l a t i o n s . However, the a n t i s e r a were not capable of e l i m i n a t i n g syngeneic tumor s p e c i f i c i n v i t r o generated k i l l e r c e l l s , i n d i c a t i n g that the receptor molecules on suppressor and e f f e c t o r c e l l s i n t h i s system are d i s t i n c t from each other. Only the a n t i s e r a "raised i n syngeneic DBA/2 mice had any observable e f f e c t on P815 tumor growth i n v i v o . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF FIGURES v LIST OF TABLES v i l ACKNOWLEDGEMENTS v l i l CHAPTER I - INTRODUCTION 2 CHAPTER II - CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIRMENTS OF P815 TUMOR-SPECIFIC SUPPRESSOR T CELLS AND THEIR SOLUBLE FACTOR 8 A. Introduction 8 B. Materials and Methods 10 C. Results 1 8 D. Discussion CHAPTER I I I - THE LYT PHENOTYPE OF CELLS INVOLVED IN THE CYTOTOXIC RESPONSE TO SYNGENEIC TUMOR AND OF TUMOR-SPECIFIC SUPPRESSOR CELLS AND IMPROVED PROCEDURES IN THE GENERATION OF TUMOR-SPECIFIC SUPPRESSOR CELLS 3 3 A. Introduction 3 3 B. Materials and Methods C. Results 43 D. Discussion ^ CHAPTER IV - IN VITRO AND IN VIVO EFFECTS OF SYNGENEIC AND ALLOGENEIC ANTISERA RAISED TO TUMOR-SPECIFIC SUPPRESSOR FACTOR FROM DBA/2 MICE 5 5 A. Introduction B. Materials and Methods ^ C. Results 6 8 oo D. Discussion CHAPTER V - SUMMARY DISCUSSION Lit e r a t u r e Cited 94 100 V LIST OF FIGURES Figure T i t l e Page 1 The a b i l i t y of P815 suppressor c e l l s to suppress the i n v i t r o primary c y t o t o x i c response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated P815 tumor c e l l s 19 2 The a b i l i t y of a n t i - l a ^ a l l o a n t i s e r u m p l u s r a b b i t C to e l i m i n a t e P815 suppressor c e l l s 20 3 The a b i l i t y of DBA/2 P815 suppressor c e l l s to suppress the c y t o t o x i c response of splenocytes of H-2 r a d i a t i o n chimeras to P815 22 4 The a b i l i t y of DBA/2 P815 suppressor c e l l s to suppress the c y t o t o x i c response of splenocytes from H-2 (A.TH) r a d i a t i o n chimeras to P815. 23 5 The a b i l i t y of the suppressor f a c t o r i s o l a t e d from P815 suppressor thymocyte populations to suppress the i n v i t r o primary c y t o t o x i c response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated tumor c e l l s . 24 6. The e f f e c t of a n t i - l a antiserum adsorption on the suppressive a c t i v i t y of the suppressor f a c t o r i s o l a t e d from P815 suppressor thymocyte populations... 26 7 The a b i l i t y of P815 suppressor f a c t o r to suppress the c y t o t o x i c response of splenocytes from H~2 r a d i a t i o n chimeras to P815 27 8 Flow diagram i l l u s t r a t i n g the p r e p a r a t i o n of P815 tum o r - s p e c i f i c suppressor c e l l s . . 40 9 The a b i l i t y of s p l e n i c P815 suppressor c e l l s to suppress the i n v i t r o primary c y t o t o x i c response of normal DBA/2 splenocytes to syngeneic mitomycin-C t r e a t e d P815 tumor c e l l s 44 10 The a b i l i t y of nylon wool-passed s p l e n i c P815 suppressor c e l l s to suppress the i r i v i t r o primary c y t o t o x i c response of normal DBA/2 splenocytes to syngeneic mitomycin-C t r e a t e d P815 tumor c e l l s 45 11 Time course of appearance and disappearance of antigen- s p e c i f i c suppressor c e l l s i n the spleen of DBA/2 mice i n j e c t e d i n t r a p e r i t o n e a l l y w i t h P815 membrane e x t r a c t s on day 0 46 v i LIST OF FIGURES Continued Figure T i t l e Page 12 The e f f e c t of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rab b i t C on DBA/2 allogeneic e f f e c t o r c e l l s r a i s e d against C57BL/6, on a EL4 target 48 13 The e f f e c t of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rab b i t C on DBA/2 e f f e c t o r c e l l s primed against the syngeneic P815 mastocytoma 49 14 The e f f e c t of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rab b i t C on P815-primed nylon wool-passed DBA/2 splenic suppressor c e l l s 50 15 Flow diagram i l l u s t r a t i n g the preparation of P815 tumor-specific suppressor factor (P815-SF) 60 16 The a b i l i t y of P815-specific suppressor factor to suppress the i n v i t r o primary cytotoxic response of normal DBA/2J splenocytes to. syngeneic mitomycin C treated P815 tumor c e l l s 69 17 T i t r a t i o n of immunoadsorbent p u r i f i e d P815-specific suppressor factor 70 18 Characterization of P815-SF by ELISA assay 7 5 19 The a b i l i t y of anti-P815-SF a n t i s e r a plus complement to eliminate P815-specific suppressor c e l l s 77 20 E f f e c t of anti--P815-SF antisera i n vivo on P815 tumor growth i n syngeneic DBA/2 mice. . 80 21 E f f e c t of anti-P815*-SF a n t i s e r a i n vivo on L1210 tumor growth i n syngeneic DBA/2 mice 8 3 22 The i n a b i l i t y of anti-P815-SF an t i s e r a , i n the presence of complement, to lyse 5 1 C r labeled P815 tumor c e l l s . ... 85 23 Does anti-SF antisera have any a c t i v i t y d irected towards P815 tumor membrane determinants? v i i LIST OF TABLES Table T i t l e Page I 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 37 I I Anti-P815 a n t i s e r a i s able to re a c t and bind to P815- s p e c i f i c suppressor f a c t o r 73 I I I R e s u l t s of Students' t - t e s t run on 3 separate e x p e r i - ments t e s t i n g 3 i n d i v i d u a l bleeds of mice immunized w i t h P815-SF or c o n t r o l B a l e n t l - " S F " 74 IV Anti-P815-SF a n t i s e r a plus complement ( C ) treatment has no e f f e c t on the a b i l i t y of s p e c i f i c c y t o t o x i c e f f e c t o r T c e l l s to l y s e the appropriate target 79 V E f f e c t of anti-P815-SF a n t i s e r a jm v i v o on s u r v i v a l time of DBA/2 mice a f t e r P815 tumor i n j e c t i o n 81 VI E f f e c t of anti-P815-SF a n t i s e r a jLn v i v o on s u r v i v a l time of DBA/2 mice a f t e r L1210 tumor i n j e c t i o n 84 ACKNOWLEDGEMENTS I am very g r a t e f u l to Dr. Levy f or her valuable i n s t r u c t i o n s , guidance, and enthusiasm throughout the course of my research. I thank Dr. Doug Kilburn for h i s very useful discussions of th i s work. I would l i k e to thank Primrose Gontier f o r helping f i n i s h the typing of t h i s t h e s i s . 1 would also l i k e to extend a s p e c i a l thanks to everyone i n the lab f o r helping to make my stay i n Vancouver so very enjoyable. i X FOR PE8BI 1 CHAPTER I INTRODUCTION 2 CHAPTER I Introduction It i s now apparent that the immune response i s the r e s u l t of a complex s e r i e s of ongoing interconnecting events which i s only i n i - t i a t e d by antigenic stimulation (1). I n i t i a l l y , the immune response was thought of as a p h y s i o l o g i c a l response of the body to antigen, i n which the sole end product was antibody. Later, the two main limbs of the immune response were recognized (humoral and cell-mediated), and within the past two decades, the two lymphocyte subpopulations mediating these responses were defined. Thus i t was r e a l i z e d that at.', l e a s t two types of inte r a c t i o n s occurred between antigen and the two responding c e l l types, the T and B lymphocytes. This was followed by the r e a l i z a t i o n that the immune response i s regulated v i a T c e l l - B c e l l i n t r e a c t i o n s , and also T cell-macrophage, and T c e l l - T c e l l i n t e r - actions. I t i s now recognized that the T lymphocyte population i s made up of d i s t i n c t subsets which i n t e r a c t with each other as well as B c e l l s and macrophages, to both amplify and i n h i b i t a v a r i e t y of immunological responses. T c e l l s have demonstrated enhancing e f f e c t s on the immune response by way of helper T c e l l s (T u) (2-10). rl T c e l l s have also been found to i n h i b i t the immune response by way of suppressor T c e l l s (T g) (11-44). Although some of these studies have demonstrated the existance of non-specific regulatory T c e l l s , the antigen s p e c i f i c regulatory T c e l l s are ex p e c i a l l y i n t r i g u i n g because they appear to possess the s p e c i f i c i t y that usually marks the immune response. 3 One of the f i r s t observations of a n t i g e n - s p e c i f i c T g were made regarding antibody formation i n r a t s (45-47). This was q u i c k l y expanded to i n c l u d e s e v e r a l d i f f e r e n t a n t i g e n i c systems (il-17,22-44, 48-58). From t h i s and other work some general c h a r a c t e r i s t i c s of a n t i g e n - s p e c i f i c T g begin to emerge. F i r s t they show s p e c i f i c antigen or i d i o t y p e b i n d i n g p o t e n t i a l . Antigen s p e c i f i c T g a l s o express l a determinants. This i s u s u a l l y I - J coded. The i n h i b i t o r y a c t i v i t y of these c e l l s may or may not by g e n e t i c a l l y r e s t r i c t e d to s e l f H-2 type. The a n t i g e n - s p e c i f i c T^ i n many experimental systems have a surface phenotype of Lyt- 1 2"*". However, there are a l s o some r e p o r t s of T g having the L y t - l + 2 phenotype or even L y t - l + 2 + . In many of the i n v e s t i g a t i o n s concerning a n t i g e n - s p e c i f i c r e g - u l a t o r y T c e l l s , i t has been shown that s o l u b l e f a c t o r s derived from these c e l l s can apparently d u p l i c a t e t h e i r f u n c t i o n (59-61). Thus, the a n t i g e n - s p e c i f i c f a c t o r s once produced may work independantly of the T c e l l s which produced them. This would have the e f f e c t of i n c r e a s i n g the e f f e c t i v e r a d i u s of the r e g u l a t o r y T c e l l , much i n the same way that antibody f u n c t i o n s w i t h respect to the B c e l l . These .factors may a l s o i n t e r a c t v i a network type i n t e r a c t i o n s to induce the a c t i v a t i o n of other lymphocytes, which e v e n t u a l l y leads to the f i n a l net e f f e c t on the immune response. This has been best studied i n the a n t i g e n - s p e c i f i c suppression systems (60). As i n the a n t i g e n - s p e c i f i c T g i n v e s t i g a t i o n s , the i n i t i a l d i s c o v e r y of a n t i g e n - s p e c i f i c suppressor f a c t o r (SF) was made by Tada and c o l - leagues (62-64). Again, t h i s observation was q u i c k l y broadened to many other experimental systems (47,59-61,65-80). The a n t i g e n - s p e c i f i c 4 suppressor f a c t o r s e x h i b i t s e v e r a l p h y s i c a l and f u n c t i o n a l c h a r a c t e r - i s t i c s i n common. They are produced or extr a c t e d from antigen primed T g. They are pr o t e i n s w i t h s p e c i f i c antigen (or i d i o t y p e ) b i n d i n g p o t e n t i a l . Their molecular weight i s u s u a l l y between 35-75,000. They l a c k constant r e g i o n immunoglobulin determinants but may c a r r y v a r i a b l e ( i d i o t y p i c ) determinants of the immunoglobulin heavy chain. They have determinants which are encoded by the I r reg i o n ( u s u a l l y I - J ) , and l i k e the a n t i g e n - s p e c i f i c T g they may or may not be g e n e t i c a l l y r e s t r i c t e d i n t h e i r e f f e c t to a c t i n g on c e l l s of the same H-2 type. F i n a l l y , s i n c e T c e l l s would be u n l i k e l y to express a n t i g e n - s p e c i f i c molecules unless they were re c e p t o r s , a n t i g e n - s p e c i f i c suppressor f a c t o r s are an obvious candidate f o r the antigen receptor of T g. Several reviews have been published r e c e n t l y on a n t i g e n - s p e c i f i c T c e l l f a c t o r s , and one i s d i r e c t e d there f o r a more comprehensive examination of anti g e n - s p e c i f i c suppressor f a c t o r s (59-61). The e a r l y i n v e s t i g a t i o n s c a r r i e d out by Takei and colleagues i n t h i s l a b o r a t o r y provided p r e l i m i n a r y data on which the work i n t h i s t h e s i s i s based. They found that DBA/2 mice i n j e c t e d w i t h syngeneic P815 mastocytoma c e l l s 8 days p r e v i o u s l y , contained i n t h e i r thymus P8 1 5 - s p e c i f i c T g (P815-T g), which could i n h i b i t the i n v i t r o generation of P 8 1 5 - s p e c i f i c c y t o t o x i c T c e l l s (P815-T c) (11,12). The experimental design f o r demonstrating t h i s was as f o l l o w s . The i n v i t r o assay f o r c y t o t o x i c T c e l l s i n v o l v e d the c u l t u r i n g of "immune" DBA/2 splenocytes (taken 12 days f o l l o w i n g subcutaneous i n j e c t i o n of a small number of P815 c e l l s i n t o the DBA/2 mice) w i t h mitomycin-C tre a t e d P815 c e l l s f o r 4 days p r i o r to c e l l h a r v e s t i n g . S p e c i f i c c y t o t o x i c i t y was measured by a 5 standard 18 h Cr release assay. When thymocytes from DBA/2 mice, inoculated 8 days previously with P815, were cocultured in this system, the generation of cytotoxic T cel ls was greatly reduced as assessed by the reduction in "̂*"Cr release mediated by these cultures in comparison to appropriate.controls. This suppressive effect was shown to be antigen-specific. It was later shown that a P815-specific suppressor factor (P815-SF) could be isolated from sonicated P815-T "extract (65). This SF could replace the P815-Ts in the in vi t ro functional assay. The P815-SF was capable of suppressing the in vi t ro generation of syngeneic ce l ls cytotoxic for P815 tumor ce l l s i f i t was added during the f i r s t 30 h of culture. It was found to have a molecular weight in the range of 40-60,000, with an isoelectr ic point in the range of 4.6-4.9. The P815-SF could be removed by passage through immunoadsorbent columns prepared from membrane fragments of P815 but not by analogous columns prepared with L1210 (syngeneic leukemia of DBA/2) membrane fragments. The P815-SF was also not removed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin antibody. The work reported here involved continued studies of these suppressor T cel ls and the suppressor factor extracted from them. Both the c e l l and factor are P815 specific and obtained from DBA/2 mice primed with P815 tumor ce l ls or P815 membrane fragments. The function- a l assay involves the a b i l i t y of either ce l ls or their extracts to speci f ica l ly inhibit a primary in vi t ro cytotoxic response of normal DBA/2 splenocytes to P815 tumor c e l l s . The work in this report has been divided into three self-contained chapters for ease of reading. 6 Chapter II examines some of the immunogenetic properties and require- ments of the P815-T . This work was c a r r i e d out between 6/77 and s 9/78. Chapter III examines the Lyt phenotype of the P815-T g as well as that of the c e l l s involved i n the cytotoxic response to the syngeneic P815 tumor. This i n v e s t i g a t i o n was c a r r i e d out between 6/78 and 2/79. The work reported i n Chapter IV examines the i n v i t r o and i n vivo e f f e c t s of syngeneic DBA/2 and allogeneic C57BL/6 an t i s e r a raised to P815-SF. This work was done between 10/78 and 5/81. Chapter V i s a summary discussion of the studies reported i n the preceeding three chapters. 7 CHAPTER II CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIREMENTS OF P815 TUMOR-SPECIFIC SUPPRESSOR T CELLS AND THEIR SOLUBLE FACTOR 8 CHAPTER II Introduction There have been a number of reports showing that the lymphoid organs of tumor-bearing mice contain T c e l l s that are capable of s p e c i f i c a l l y suppressing the response of normal c e l l s to that tumor (12,15,36,83-85). I t has also been shown that, i n some cases, i t i s possible to i s o l a t e from such populations of suppressor c e l l s , soluble factor(s) also capable of i n h i b i t i n g the s p e c i f i c anti-tumor response of normal lymphoid c e l l s (65,76,86,87). Studies on the c h a r a c t e r i z a t i o n of soluble s p e c i f i c suppressor factors i n tumor-bearing (65,76,86) animals have shown that they do not appear to d i f f e r s i g n i f i c a n t l y from a n t i g e n - s p e c i f i c suppressive factors demonstrated i n animals immunized with a v a r i e t y of antigenic s t i m u l i (59-61,77,81,82,88). Generally, suppressive factors have been found to have m.w. i n the range of 30-75,000. Moreover, they have been shown to exhibit antigen s p e c i f i c i t y (65,75-78,87) and not to share antigenic s i m i l a r i t i e s with the constant regions of immuno- glo b u l i n (65,77,78,87). Studies with these factors have also shown that they can be removed by absorption with antisera raised to products of the major h i s t o c o m p a t i b i l i t y complex (MHC) excluding the K and the D regions ( a n t i - l a serum). Further analyses of these absorptions have shown that the subregion of the l a that i s expressed on the suppressor factor i s encoded by the I-J region (77,81,82,86). A recent observation that treatment of tumor bearing animals with small amounts of a n t i - I - J antiserum caused a s i g n i f i c a n t slowing i n the growth of syngeneic tumor (89) indicate that elimination of I - J - 9 bearing c e l l s i n vivo may be of a c l i n i c a l s i g n i f i c a n c e . The present study was undertaken to determine whether suppressor c e l l s e l i c i t e d i n tumor-bearing animals were capable of suppressing the primary i n v i t r o cytotoxic response of normal c e l l s to tumor antigens, and to compare the suppressor c e l l s , thus generated, with the equivalent soluble factors i n terms of la-bearing c h a r a c t e r i s t i c s and genetic r e s t r i c t i o n s regarding t h e i r suppressive q u a l i t i e s . M a t e r i a l s and Methods Mice and tumors. Female DBA/2, B6D2F 1 > B10.D2, BIO and C57BL/6 mice (6 to 10 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, Maine). A.TH animals were obtained from the Banting and Best I n s t i t u t e from Dr. T. D e l o v i t c h and were bred i n our own animal u n i t . P815 mastocytoma and L1210 leukemia were obtained from Dr. Bruce Smith ( I n s t i t u t e f o r Cancer Research, P h i l a d e l p h i a , Pennsylvania) and maintained as an a s c i t e s tumor as described p r e v i o u s l y (11,12). C u l t u r e system f o r generation of c y t o t o x i c e f f e c t o r c e l l s . S i n g l e - c e l l suspensions were prepared from spleens of normal DBA/2 mice by p r e s s i n g the t i s s u e through a s t a i n l e s s s t e e l 60- gauge mesh i n RPMI 1640 (Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , N.Y.) medium c o n t a i n i n g 10% h e a t - i n a c t i v a t e d f e t a l c a l f serum (FCS), 10 mM HEPES and 5 x 10~ 5 mM of 2-mercaptoethanol. Gentamycin was a l s o added to a f i n a l c o n c e n t r a t i o n of 50 g/ml. Suspended c e l l s were washed through 3.0 ml of FCS, resuspended i n medium, and counted f o r v i a b l e c e l l s by u s i n g trypan blue. Tumor c e l l s were washed twice i n medium before resuspension i n complete medium f o r counting. S p e c i f i c c y t o t o x i c i t y against e i t h e r P815 or L1210 was generated i n v i t r o by i n c u b a t i n g 10 7 s p l e n i c lymphoid c e l l s w i t h 5 x 10^ mitomycin C-treated tumor c e l l s i n Linbro m u l t i w e l l p l a t e s c o n t a i n i n g 24 f l a t bottom w e l l s (No. 76-033-05) at 37°C i n a h u m i d i f i e d incubator w i t h 5% C0 2 f o r 5 days. The t o t a l volume of each c u l t u r e was 2.5 ml. A f t e r 11 5 days, c e l l s were harvested by ce n t r i f u g a t i o n , washed, and v i a b l e c e l l s were counted by trypan blue exclusion and tested for c y t o t o x i c i t y . Assay for suppressor c e l l s and suppressor fa c t o r s . When the assay was used for measuring suppressor c e l l a c t i v i t y , 5 x 10 normal splenic lymphocytes were incubated with 6 6 5 x 10 suppressor thymocytes; a con t r o l contained 5 x 10 normal splenic lymphocytes and 5 x 10^ normal thymocytes. On a l l occa- sions, experiments were run at l e a s t i n duplicate with lymphocytes from i n d i v i d u a l pools of c e l l s i n order to minimize errors incurred by inaccurate counts. Assay for thymic suppressor factor involved incubation of 10 7 splenic lymphocytes with various con- centrations of the factor under the same conditions as stated above. When the degree of suppression was quantitated, c y t o t o x i c i t y was tested at various e f f e c t o r to target c e l l r a t i o s and the decrease i n t o t a l l y t i c u nits was assessed. One l y t i c u n i t , which was defined as the number of ef f e c t o r c e l l s required to lyse 4 50% of the 10 target c e l l s i n 18 hr incubation, was estimated by l i n e a r regression analysis of percentage of c y t o t o x i c i t y vs logarithm of e f f e c t o r to target c e l l r a t i o . T o t a l l y t i c u nits were calculated from c e l l recovery i n cultures and percentage of suppression was estimated from the decrease i n t o t a l l y t i c u n i t s . No s i g n i f i c a n t differences i n c e l l recovery between test cultures and controls were normally observed. 12 When thymocytes on t h e i r own, at a concentration of 10 7 per well were cultured with micomycin-treated P815, no detectable k i l l e r c e l l s were generated, i n d i c a t i n g that i n the syngeneic k i l l i n g system, thymocytes do not contain s i g n i f i c a n t l e v e l s of precur- sors. This was also found to be the case when 5 x 10 thymocytes were cultured with 5 x 10, mitomycin-treated DBA/2 spleen c e l l s or C57BL/6 chimeric c e l l s . C y t o t o x i c i t y assay. Target tumor c e l l s were labeled with "'"'"Cr-sodium chromate (New England Nuclear, Boston, Mass.) as described previously (11). 4 51 Various numbers of lymphocytes and 10 Cr-labeled target c e l l s i n RPMI 1640 cult u r e medium were dispensed i n the wells of m u l t i - d i s h microculture plates (Linbro Chemical, 1S-FB-96-TC, New Haven, Conn.) and the f i n a l volume was adjusted to 0.20 ml. The plates were incubated for 18 hr i n a CO2 enriched, humidified incubator at 37°C. The c e l l s were then sedimented by cen t r i f u g i n g the plates at 200 x G for 5 min, 0.10 ml of the supernatant was removed, and i t s r a d i o a c t i v i t y was measured on a gamma counter (Beckman Biogamma). Percentage of s p e c i f i c c y t o t o x i c i t y was c a l - culated as follows: S p e c i f i c c y t o t o x i c i t y (%) test release (CPM) - spontaneous release (CPM) x maximum.release (CPM) - spontaneous release (CPM) 4 Spontaneous release was measured by incubating 10 target c e l l s 4 alone, and maximum release was tested by l y s i n g 10 target c e l l s 13 with 5% TRITON X 100 (Sigma Chemical Company, St. Louis, Mo.). Spontaneous release of P815 c e l l s was 20 to 25% and that of L1210 c e l l s was 15 to 20% of maximum release. Generation of suppressor c e l l s . 3 DBA/2 mice were injected subcutaneously with 2 x 10 P815 c e l l s i n the r i g h t flank. They were s a c r i f i c e d 8 days l a t e r and th e i r thymocytes used as. a source of suppressor c e l l s . This pro- t o c o l had been shown previously (65) to.be r e l i a b l e and reproducible for the generation of T lymphocytes s p e c i f i c a l l y suppressive i n the assay system. Preparation of suppressor f a c t o r . This method has been described i n d e t a i l previously <(65). B r i e f l y , suppressive thymocytes prepared as stated above, were disrupted by sonication (Biosonic 80) at 0°C for three 1-min bursts and the soluble material was subjected to preparative i s o e l e c t r i c focusing a f t e r u l t r a c e n t r i f u g a t i o n at 60,000 x G for 60 min. By t i t r a t i o n of i n d i v i d u a l f r a c t i o n s , from both normal and tumor bearer ex- t r a c t s , the s p e c i f i c a l l y suppressive f r a c t i o n was i s o l a t e d . This material was stable at 4°C and active up to d i l u t i o n s equivalent to 1/125 of a sing l e thymus. Assays for t h i s material were always run against controls containing equal concentrations of the equivalent f r a c t i o n s from normal thymocytes. I s o e l e c t r i c focusing. Preparative i s o e l e c t i r c focusing was run on a 110 ml LKB- focusing column through a 5 to 50% l i n e a r sucrose gradient and a 14 t o t a l of 1.6% LKB ampholines (1.2%, pH 4 to 6, and 0.4%, pH 5 to 7). Crude thymic e x t r a c t s i n volumes of 2.5 to 3.0 ml w i t h 280 nm absorbance of about 10.0/ml were used. The el e c t r o d e s o l u t i o n s c o n s i s t e d of 7.0 ml H 20 and 3.0 ml 1 M NaOH ( l i g h t electrode) and 15 g sucrose, 13 ml ^ 0 and 3 ml 1 M H^PO^ (dense e l e c t r o d e ) . Focusing was c a r r i e d out at 4°C f o r 24 hr at 950 v o l t s and 0.8 mA. A f t e r the focusing was complete, d i s t i l l e d water was pumped slowly i n t o the column and 1.0 ml f r a c t i o n s of the focused m a t e r i a l s were c o l l e c t e d . These f r a c t i o n s were monitored f o r pH and absorbance at 280 nm. P r e l i m i n a r y t e s t i n g had i n d i c a t e d that the suppressive m a t e r i a l focused at between pH 4.6 and 4.7. Therefore, when f r a c t i o n s were being pooled f o r subsequent t e s t i n g , both pH and absorbance at 280 nm were used as i n d i c e s f o r c u t t i n g the f r a c t i o n s . Pooled samples were d i a l y z e d against p h y s i o l o g i c s a l i n e to e l i m i n a t e the sucrose and ampholines. They were then concentrated down to t h e i r o r i g i n a l volume w i t h an Amicon u l t r a f i l t e r w i t h a UM10 membrane. F r a c t i o n s were then d i a l y z e d against RPMI 1640 a f t e r which t h e i r absorbance at 280 nm was recorded. They were then s t e r i l i z e d by M i l l i p o r e f i l t r a t i o n and stored at 4°C u n t i l they were t i t r a t e d or used f o r f u r t h e r c h a r a c t e r i z a t i o n s t u d i e s . P r e p a r a t i o n of a l l o a n t i s e r a . The a n t i - l a ^ antiserum used here was prepared by immunizing B10 mice repeatedly w i t h B10-D2 splenocytes. Animals were i n j e c t e d i . p . every week w i t h 10 7 B10.D2 splenocytes f o r 8 weeks. Subsequently, they were bled from the r e t r o o r b i t a l sinus on 15 a l t e r n a t e weeks and c o n t i n u a l l y immunized over a period of 2 months. The anti-H-2^ antiserum thus prepared was 100% c y t o - t o x i c to P815 c e l l s i n the presence of r a b b i t complement (C) up to a f i n a l d i l u t i o n of 1/1200. In order to render the a n t i - serum s p e c i f i c f o r the l a re g i o n of the H-2^ MHC i t was absorbed e x h a u s t i v e l y against P815 c e l l s u n t i l i t was no longer c y t o t o x i c i n the presence of r a b b i t C f o r these t a r g e t s . The absorbed antiserum (anti-la'*) , when test e d f o r d i r e c t k i l l i n g of DBA/2 s p l e n i c lymphocytes k i l l e d 60 to 65% of these c e l l s up to a f i n a l d i l u t i o n of 1/80 but had no appreciable c y t o t o x i c i t y f o r thymocytes at a d i l u t i o n of 1/4. When te s t e d by ^ C r r e l e a s e assay w i t h LPS b l a s t s , i t caused 75% "^Cr r e l e a s e up to a 1/100 d i l u t i o n . In t e s t s reported here the a n t i - l a ' * a n t i s e r a was used at a f i n a l c o n c e n t r a t i o n of 1/40 i n the presence of a 1/15 d i l u t i o n of r a b b i t C (Lowtox Cedar Lane Labs, Hornby, O n t a r i o ) . t2 A n t i s e r a s p e c i f i c f o r H-2 (A.TH a n t i A.TL) was obtained from Dr. T. D e l o v i t c h (Banting and Best I n s t i t u t e ) . Antiserum to H-2^ haplotype was prepared by immunizing B10.D2 mice w i t h B10 splenocytes as described above. R a d i a t i o n chimeras. B6D2F^ mice were l e t h a l l y i r r a d i a t e d w i t h 900 R and recon- s t i t u t e d w i t h 10 7 bone marrow c e l l s from e i t h e r DBA/2, C57BL/6 or A.TH donors. The donor c e l l s had been t r e a t e d w i t h r a b b i t a n t i - p u r i f i e d b r a i n a s s o c i a t e d thy-1 antigen. The antiserum was obtained from M. L e t a r t e (Ontario Cancer I n s t i t u t e ) and 16 was used at a d i l u t i o n of 1/200 with a 1/15 d i l u t i o n of rabbit C. Animals were kept for 8 weeks before they were used i n any experiments. When these animals were s a c r i f i c e d , t h e i r spleen c e l l s were tested with the appropriate a l l o a n t i s e r a plus C to ascer t a i n that t h e i r c e l l s were those c h a r a c t e r i s t i c of the donor a l l o t y p e . A n t i - l a ^ k i l l i n g of suppressor c e l l s . In experiments t e s t i n g for the presence of l a markers on suppressor c e l l s , 4 x 10 7 tumor bearer or normal thymocytes were suspended i n 1.0 ml of a 1/40 d i l u t i o n of antiserum i n complete medium and incubated for 45 min at 37°C af t e r which 0.5 ml of a 1/5 d i l u t i o n of rabbit C i n the same medium was added and incubation was continued for another 45 min. Appropriate controls of C and medium only were run concurrently. C e l l s were washed, resuspended i n medium, and counted for v i a b i l i t y . In no instance were v i a b l e counts i n c e l l suspensions containing the a n t i - l a ^ antiserum s i g n i f i c a n t l y d i f f e r e n t from those of the controls and i n no instance were counts lower than 90% of the number of c e l l s before treatment. These c e l l s were sub- sequently used i n suppressor c e l l assays. Immunoadsorbent assays. The a b i l i t y of a n t i - l a ^ to remove the suppressive factor from thymic extracts was tested by using an immunoadsorbent assay described previously (65). " B r i e f l y , both normal.and suppressor i s o e l e c t r i c focused f r a c t i o n s were mixed with either the a n t i - 17 l a antiserum or a control of anti-H-2 antiserum, and incubated overnight at 4°C. These materials were then passed over immuno- adsorbent Sepharose 4B columns to which goat anti-mouse Ig had been attached by cyanogen bromide. The capacity of the column was s u f f i c i e n t to remove at le a s t twice the amount of the mouse Ig added to the f r a c t i o n s . The material e l u t i n g from the columns was t i t r a t e d for suppressive a c t i v i t y over d i l u t i o n s ranging from 1/5 to 1/125 (these d i l u t i o n s of eluted materials represent between 0.2 and 0.04 of a thymus equivalent). 18 Results The a b i l i t y of suppressor c e l l s generated as previously described (12,65) to suppress s p e c i f i c a l l y the primary i n v i t r o generation of DBA/2 c e l l s cytotoxic for the syngeneic P815 mastocytoma i s shown i n Figure 1. A primary cytotoxic response was generated against either P815 or L1210 mitomycin-treated c e l l s i n the presence of either normal or suppressor thymocytes. As can be seen i n Figure l a , the response to P815 i n the presence of suppressor thymocytes was s i g n i f - i c a n t l y suppressed whereas the response to L1210 was not. I t was thus shown that these suppressor c e l l s that had been found previously (12) to suppress the generation of P815 cytotoxic c e l l s i n populations of splenocytes primed i n vivo by exposure to P815, could also suppress the primary response i n an analogous manner. These r e s u l t s are e n t i r e l y reproducible, the only d i f f e r e n c e being i n the r e l a t i v e numbers of l y t i c u nits between cultures. The data shown i n Figure 1 are representative i n that c o n t r o l cultures contain roughly twice the number of l y t i c u n i t s as do cultures containing suppressor c e l l s . It has been reported by others that s p e c i f i c T suppressor c e l l s bear detectable l a antigens on t h e i r surface (81,90-94). Experiments were run to determine whether anti-la** alloantiserum plus rabbit C could eliminate suppressor c e l l s i n t h i s system. The r e s u l t s of a repre- sentative experiment are shown i n Figure 2. Although the C control showed that suppressor c e l l s were present i n the treated population of thymocytes (Fig. 2b), the a n t i - l a treatment removed suppressive a c t i v i t y so that the c y t o t o x i c i t y generated i n the test population was not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l population contain- 19 Figure 1. The a b i l i t y of P815 suppressor c e l l s to suppress the i n v i t r o primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated P815 tumor c e l l s . Anti-P815: the cytotoxic response of c e l l s primed i n v i t r o with P815 c e l l s on Cr labeled P815 c e l l s i n an 18 hr-assay. • • , primary culture of normal DBA/2 splenocytes and normal DBA/2 thymocytes ( l y t i c u nits were 33.3); o o, primary culture of normal DBA/2 splenocytes and P815 suppressor thymocytes ( l y t i c u n i t s were 15.4). Anti-L1210: the cytotoxjLj response of c e l l s primed i n v i t r o with L1210 c e l l s on Cr labeled L1210 c e l l s i n an 18 hr assay. • 1 , primary cu l t u r e of normal DBA/2 spleno- cytes and normal DBA/2 thymocytes; 0 0 , primary culture of normal DBA/2 splenocytes and P815 suppressor thymocytes. L y t i c u n i t s for both cultures were 11.1. 20 Figure 2. The a b i l i t y of a n t i - l a " alloantiserum plus rabbit C to eliminate P815 suppressor c e l l s . A n t i - l a : the cytotoxic response of c e l l s primed in_ v i t r o with P815 aft e r treatment of thymocytes with a n t i - l a a n t i - serum plus C. 0 0 , primary culture of normal DBA/2 splenocytes plus a n t i - l a - treated normal thymocytes; • — - j * , primary culture of normal DBA/2 splenocytes plus a n t i - l a treated suppressor thymocytes. L y t i c units i n both cultures were 19.2. C co n t r o l : C control of the a n t i - l a experiment 0 0 , primary culture of normal DBA/2 splenocytes and C-treated normal thymocytes ( l y t i c u nits were 24.4); • • , primary culture of normal DBA/2 splenocytes and C-treated P815 suppressor thymocytes. L y t i c units for suppressor c e l l populations i n t h i s instance could not be calculated because 50% l y s i s of c e l l s was not achieved i n the assay. 21 ing anti-la-treated normal thymocytes (Fig. 2a). The potential of suppressor cells to operate across a histo- compatibility barrier was tested by using spleen cells from B6D2 radiation chimeras that had been reconstituted with DBA/2, C57BL/6, or A.TH bone marrow cells, thus providing cells of various H-2 haplo- types rendered tolerant to H-2̂  alloantigens. These were then cultured with suppressor cells generated in vivo in DBA/2 mice plus mitomycin C-treated P815 cells. The results are shown in Figures 3 and 4. In Figure 3, the suppressive activity of these cells when cocultured with spleen cells of either C57BL/6 or DBA/2 allotype was essentially the same, indicating that suppressor cells do not require K or D identity in order to carry out their suppressive effect. These experiments have been repeated a number of times and are totally reproducible. Similar experiments were run with chimeras reconstituted with A.TH cells (Fig. 4). This haplotype shares the D locus with DBA/2 t2 but differs at a l l other regions of the MHC (H-2 ). As can be seen, again there was no difference in the suppressive activity of the DBA/2 t2 d suppressor cells with either H-2 or H-2 splenocytes. All the spleen cells from the chimeras were tested with appropriate antisera to ensure that they were the haplotype of the donor cells. The comparative properties of the suppressor cells and the suppressive factor separated from the suppressor cell populations were studied. Figure 5 shows the ability of suppressor factor to suppress specifically the primary in vitro generation of DBA/2 cells cytotoxic for the syngeneic P815 mastocytoma. As can be seen in Figure 5a, the response to P815 in the presence of suppressor factor 22 F i g u r e 3 . The a b i l i t y of DBA/2 P815 suppressor c e L l s to suppress the c y t o t o x i c response of s p l e n o c y t e s of H-2 r a d i a t i o n chimeras to P8T .5. b H-2 : the c y t o t o x i c response of H-2 (C57BL/6 r e c o n s t i - tuted) r a d i a t i o n chimera splenocytes^primed i_n v i t r o w i t h P815. 0 0 , pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and normal DBA/2 thymocytes ( l y t i c u n i t s were 6 9 . 0 ) ; • 1 pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and P815 sup- p r e s s g r thymocytes ( l y t i c u n i t s were 3 5 . 7 ) . H-2 : c o n t r g l assay f o r suppressor c e l l s . 0 0 , pr imary c u l t u r e of H-2 (DBA/2) chimera s p l e n o c y t e s and normal DBA/2 thymocytes ( l y t i c u n i t s were 4 1 . 7 ) ; 9 — : — f , pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and P815 suppressor thymocytes ( l y t i c u n i t s were 2 7 . 4 ) . 23 ol I 1 • — i — " 1 1 • ' 12-5:1 25:1 50:1 100:1 12-5:1 25:1 50:1 100:1 E F F E C T O R : T A R G E T RATIO J F i g u r e 4. The a b i l i t y o f DBA/2 P815 suppressor c e l l s to s u p p r e s s the c y t o t o x i c response of s p l e n o c y t e s from H-2 (A.TH) radiajj^on chimeras to P815. ^2 H-2 : the c y t o t o x i c response of H-2 r a d i a t i o n chimera s p l e n o c y t e s primed i n v i t r o w i t h P815. 0 0 , pr imary c u l t u r e o f H-2 chimera s p l e n o c y t e s and normal DBA/2 thymocytes ( l y | £ c u n i t s were 10.9) ; * pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and P815 suppressor thymocytes ( l y t i c u n i t s c o u l d not be c a l c u l a t e d because of low^ l e v e l s of c y t o t o x i c i t y ) . H-2 : c o n t r o l assay^for suppressor c e l l s . 0 0 , pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and^normal DBA/2 thymocytes; • • , pr imary c u l t u r e of H-2 chimera s p l e n o c y t e s and P815 suppressor thymocytes ( L y t i c u n i t s c o u l d not be measured i n t h i s e x p e r i m e n t ) . 24 I 12.5:1 25 .1 5 0 . 1 1 0 0 : i 1 2 . 5 M 2 5 : 1 5 0 M 100 :1 E f f e c t o r : T a r g e t R a t i o Figure 5. The a b i l i t y o f the suppressor factor i s o l a t e d from P815 suppressor thymocyte populations to suppress the i n v i t r o primary cytotoxic response of normal DBA/2 splenocytes to syngeneic mitomycin C-treated tumor c e l l s . The d i l u t i o n s used i n t h i s assay represent approximately 1/25 of a sing l e thymus. Anti-P815: the c^Jotoxic response of c e l l s primed i n v i t r o with P815 c e l l s on Cr labeled P815 c e l l s i n an 18 hr-assay. • • , primary culture of normal DBA/2 splenocytes and . . normal DBA/2 thymocyte extract ( l y t i c u n i t s were 59.1); 0 0 , primary culture of normal DBA/2 splenocytes and P815 suppressor thymocyte extract ( l y t i c units were 31.8). Anti-Ll210: the c y t o t o x j L j response of c e l l s primed i n v i t r o with L1210 c e l l s on Cr labeled L1210 c e l l s i n an 18 hr-assay. • • , primary culture of normal DBA/2 spleno- cytes and normal DBA/2 thymocyte extract ( l y t i c units were 45.6) 0 0 , primary culture of normal DBA/2 splenocytes and P815 suppressor thymocyte extract ( l y t i c units were 44.8). 25 was s i g n i f i c a n t l y suppressed whereas the response the L1210 was n o t . Experiments were r u n to determine whether a d s o r b t i o n w i t h a n t i - l a ^ a l l o a n t i s e r u m c o u l d e l i m i n a t e suppressor f a c t o r a c t i v i t y i n our system. P a r t i a l l y p u r i f i e d f a c t o r and the e q u i v a l e n t f r a c t i o n of normal thymocyte e x t r a c t s were subjected to treatment w i t h a n t i - l a ^ a n t i s e r u m or a n t i - H - 2 ^ a n t i s e r u m and adsorbed on immunoadsorbent columns c o n t a i n i n g anti-mouse immunoglobul in. The e l u t e d m a t e r i a l s were t i t r a t e d f o r s u p p r e s s i v e a c t i v i t y . The r e s u l t s are shown i n F i g u r e 6. Whi le s u p p r e s s i v e a c t i v i t y was present i n the a n t i - H - 2 suppressor f a c t o r e l u a t e s , the s u p p r e s s i v e m a t e r i a l t r e a t e d w i t h a n t i - l a ^ was n o t , showing t h a t i t expresses determinants encoded by the l a r e g i o n of the MHC. The a b i l i t y of the f a c t o r , when added to B6D2F^ C57BL/6 c h i m e r i c s p l e n o c y t e s , to suppress the g e n e r a t i o n of c e l l s c y t o t o x i c to P815, was a l s o t e s t e d . The r e s u l t s ( F i g . 7) show, as w i t h the suppressor c e l l s , t h a t the s u p p r e s s i v e a c t i v i t y of the f a c t o r w i t h H-2^ c e l l s was s i m i l a r to t h a t observed w i t h the DBA/2 c e l l s t e s t e d a t the same t i m e . 26 Figure 6. The e f f e c t of a n t i - l a antiserum adsorption on the suppres- s i v e a c t i v i t y of the suppressor f a c t o r i s o l a t e d from P.815 suppressor -thymocyte populations. The d i l u t i o n s used i n t h i s assav represent approximately 1/25 of a s i n g l e thymus. A n t i - l a : the e f f e c t of adsorption of the suppressive f a c t o r w i t h a n t i - l a antiserum, o—-^o, primary c u l t u r e of normal DBA/2 splenocytes and. a n t i - l a adsorbed normal thymocyte e x t r a c t s ; • — p r i m a r y c u l t u r e of normal DBA/2 splenocytes and a n t i - l a adsorbed suppressor thymocyte e x t r a c t s . ^ L y t i c u n i t s i n both cases were 57.1. Anti-H-2 : the e f f e c t of adsorption of the suppressive f a c t o r w i t h a n t i - l a antiserum, o —^o, primary c u l t u r e of normal DBA/2 splenocytes and a n t i - l a adsorbed normal thymocyte e x t r a c t ( l y t i c u n i t s were 76.9); • g, primary c u l t u r e of normal DBA/2 splenocytes and a n t i - l a adsorbed suppressor thymocyte e x t r a c t ( l y t i c u n i t s were 38.5). 27 pi • i i 1—i ' 1 12.5n 25:1 5CM 1OO0 1250 25;1 5CM 1000 EFFECTOR :TARGET RATIO Figure 7. The a b i l i t y of P815 suppressor factor t g suppress the cyto- toxic response o f splenocytes from H-2 r a d i a t i o n chimeras to P815. The amount of factor used i n these experiments was approximately 1/25 thymus equivalent. H-2 : the cytotoxic response of H-2 r a d i a t i o n chimera splenocytes primed i n v i t r o with P815. 0 0, primary cul t u r e of H-2 chimera splenocytes and normal thymocyte e x t r a c t ( l y t i c u n i t s were 62.5); • •, primary culture of H-2 chimera splenocytes and suppressor factor ( l y t i c units^were 16.1). H-2 : con t r o l assay^for suppressor factor, o o, primary culture of H-2 chimera splenocytes and normal thymocyte extract ( l y t i c u n i t s were 48.8); • • , primary culture of H-2 chimera splenocytes and suppressor factor ( l y t i c u nits were 33.9). 28 D i s c u s s i o n In previous s t u d i e s on suppressor c e l l s and the s p e c i f i c f a c t o r s p o s s i b l y released from them, i t was demonstrated that such suppressive elements were capable of s p e c i f i c a l l y suppressing the generation of c y t o t o x i c c e l l s i n v i t r o by spleen c e l l s from P815-primed animals (11,12,65). In t h i s chapter, i t was shown that P815-specific sup- pressor c e l l s and P815 s p e c i f i c suppressor f a c t o r are a l s o capable of suppressing a primary i n v i t r o c y t o t o x i c response to P815 ( F i g . 1,5). Thus, the s t a t e (primed or v i r g i n ) of the c e l l p o pulation on which i t a cts appears to be unimportant f o r the P815-specific suppressor c e l l or P815-specific suppressor f a c t o r to express t h e i r a c t i v i t y . The a b i l i t y of a l l o a n t i s e r a d i r e c t e d to the l a r e g i o n (the I - J subregion i n most studies) to k i l l suppressor T c e l l s has been reported by others (81,90-93). These st u d i e s i n v o l v e d mainly the c h a r a c t e r - i z a t i o n of T suppressor c e l l s p a r t i c i p a t i n g i n the r e g u l a t i o n of the antibody response to s o l u b l e antigens. The suppressor c e l l s studied here are those involved i n r e g u l a t i n g the generation of c y t o t o x i c c e l l s . However, they a l s o appear to possess surface markers recognized by anti-haplotype sera to the l a r e g i o n , so i n t h i s respect they do not d i f f e r from other populations of a n t i g e n - s p e c i f i c T suppressor c e l l s . Since at t h i s time i t i s not p o s s i b l e to develop an antiserum s p e c i f i c f o r the I - J subregion of the I a ^ haplotype, i t was not p o s s i b l e to t e s t the c e l l s f o r t h i s s p e c i f i c i t y although many other s t u d i e s have shown that t h i s subregion of the l a i s expressed on a n t i g e n - s p e c i f i c suppressor T c e l l s . 29 In t h i s chapter i t was undertaken to determine whether the popu- l a t i o n of T suppressor c e l l s under study was H-2 r e s t r i c t e d . Because the immune response being assessed f o r suppression involved the development of c e l l s c y t o t o x i c f o r a syngeneic tumor c e l l l i n e , i t was necessary to use spleen c e l l s from r a d i a t i o n chimeras that were d b t o l e r a n t to normal H-2 a l l o a n t i g e n s . Thus B6D2F^ animals (H-2 x H-2** F^) were i r r a d i a t e d and r e c o n s t i t u t e d w i t h e i t h e r p a r e n t a l bone marrow c e l l s (C57BL/6 or DBA/2) or w i t h A.TH c e l l s (H-2't'2) that shares only the D locus w i t h H-2^. Suppressor c e l l s were r a i s e d i n DBA/2 tumor bearers and then c u l t u r e d i n v i t r o w i t h mitomycin C-treated P815 c e l l s and chimeric spleen c e l l s of e i t h e r DBA/2, C57BL/6, or A.TH o r i g i n . The experiments, which were repeated three times showed une q u i v o c a l l y that the suppressor c e l l s were capable of suppressing the anti-P815 c y t o t o x i c response of spleen c e l l s d i f f e r i n g at the K (A.TH) or both the K and D locus (C57BL/6) and thus d i d not e x h i b i t the c l a s s i c a l form of H-2 r e s t r i c t i o n . This does not exclude the p o s s i b i l i t y that some form of l a r e c o g n i t i o n was required s i n c e these chimeric c e l l s had been in f l u e n c e d by the thymus of the i r r a d i a t e d B6D2 r e c i p i e n t s , but does show that K or D i d e n t i t y i s not e s s e n t i a l f o r t h i s type of i n t e r a c t i o n . The suppressor f a c t o r ( s ) i s o l a t e d from the suppressor c e l l thymocyte population and p a r t i a l l y p u r i f i e d by pr e p a r a t i v e i s o e l e c t r i c f o c using appeared to share the same p r o p e r t i e s as the suppressor c e l l s i n that i t could s p e c i f i c a l l y suppress the primary i n v i t r o c y t o t o x i c response of DBA/2 spleen c e l l s ( F i g . 5) and could be s p e c i f i c a l l y removed by a n t i - l a * * a l l o a n t i s e r u m ( F i g . 6) . In t h i s 30 respect t h i s factor i s not d i f f e r e n t from factors described by others (77,81,82,86,89). As mentioned above, i t was not possible to test the subregion s p e c i f i c i t y with the H-2^ system. The data reported so far on the H-2 r e s t r i c t i o n s observed i n suppressive factors are somewhat equivocal. In a seri e s of reports by Tada and h i s associates (78,95,96) involving suppressive factor(s) that c o n t r o l the antibody response, i t was found that t h i s factor required a syngeneic target c e l l (presumably a helper T c e l l ) before i t could e f f e c t i v e l y suppress the antibody response. Moorhead, who produced a s p e c i f i c suppressor factor i n v i t r o that could suppress the devel- opment of delayed-type h y p e r s e n s i t i v i t y to DNFB i n mice, found that t h i s m a t erial was H-2 r e s t r i c t e d and that i d e n t i t y at either the K or D end of the MHC was s u f f i c i e n t to permit suppression i n r e c i p i e n t mice (79,80,97). A I^E c l a s s s p e c i f i c suppressor factor also shows syngeneic MHC r e s t r i c t i o n of a c t i o n (66). A l t e r n a t e l y , Waltenbaugh and h i s colleagues (98) i n studying a soluble factor that suppresses the antibody response to the synthetic copolymer L-glutamyl^^-L- . tyrosine"^ (GT) showed that a suppressive f a c t o r , e l i c t e d i n mice by i n j e c t i o n of GT alone, could e f f e c t i v e l y suppress the response to GT coupled to c a r r i e r protein across allogeneic b a r r i e r s . Similar MHC u n r e s t r i c t e d a c t i v i t y i s found i n the GAT system (99). A SRBC s p e c i f i c suppressor factor of a T-hybrid l i n e has also recently been shown to act across allogeneic b a r r i e r s (100). Kontiainen and collegues also observed no H-2 r e s t r i c t i o n of t h e i r suppressor factors (101). The r e s u l t s here show that the suppressor c e l l s under study (and the presumed equivalent f a c t o r ) , are capable of suppressing the 31 generation of c y t o t o x i c i t y to P815 by h i s t o i n c o m p a t i b l e c e l l s . The r e s u l t s show c l e a r l y that the K or D gene products do not have to be shared by the suppressor c e l l (or i t s f a c t o r ) and i t s t a r g e t . Because r a d i a t i o n chimeras, i n which the r e c i p i e n t s were B6D2F^ animals, were used f o r t e s t i n g r e s t r i c t i o n , the conclusions can only extend to a l a c k of r e s t r i c t i o n at the K and D l o c i of the MHC si n c e thymic i n f l u e n c e by the r e c i p i e n t may have in f l u e n c e d r e c o g n i t i o n mechanisms on the part of the chimeric c e l l s . CHAPTER I I I THE LYT PHENOTYPE OF CELLS INVOLVED IN THE CYTOTOXIC RESPONSE TO SYNGENEIC TUMOR AND OF TUMOR-SPECIFIC SUPPRESSOR CELLS AND IMPROVED PROCEDURES IN THE GENERATION OF TUMOR-SPECIFIC SUPPRESSOR CELLS 33 CHAPTER I I I I n t r o d u c t i o n A n t i g e n i c determinants found on lymphocyte c e l l surface molecules can be used as markers f o r studying the r e l a t i o n s h i p of the expression of these determinants and c e l l u l a r f u n c t i o n . Thus, i n the l a s t few years, many s t u d i e s have examined c e l l surface a n t i g e n i c determinants which c o u l d be used to study and c h a r a c t e r i z e d i s t i n c t subsets of T c e l l s . One of the most wi d e l y s t u d i e d a n t i g e n i c systems of T c e l l s has been the one determined by the Ly-1 and Ly-2 l o c i i n the mouse (102). Since these antigens appear to be expressed s e l e c t i v e l y on v a r i o u s sub- sets of u n d i f f e r e n t i a t e d and d i f f e r e n t i a t e d T c e l l s i t has been suggested that they be c a l l e d L y t antigen (103). The gene c o n t r o l l i n g the express- i o n of Lyt-1 antigen i s found on chromosome 19, whereas the gene c o n t r o l l - ing Lyt-2 expression i s found on chromosome 6 (104). Two a l l e l i c forms, L y t 3 and Lyt* 5, of the Lyt-1 and Lyt-2 antigens have been found and are designated Lyt-1.1, -2.1 and Lyt-1.2, -2.2 r e s p e c t i v e l y (104). A major p a r t of the L y t work has been c a r r i e d out i n mice bearing the Lyt* 3 a l l e l e . In such animals a number of b a s i c observations have been made. K i l l e r c e l l s r a i s e d against a l l o g e n e i c c e l l s ( a l l o g e n e i c k i l l e r c e l l s ) have been designated as Lyt-1 2 + (105-107). Recently, i t has been reported that a l l o g e n e i c e f f e c t o r c e l l s w i t h t h i s a l l e l e may + + be of the L y t - 1 2 phenotype (108-110). This apparent discrepancy may be accounted f o r p o s s i b l y on a q u a n t i t a t i v e r a t h e r than q u a l i t a t i v e b a s i s . K i l l e r c e l l s r a i s e d a gainst syngeneic tumor c e l l s (syngeneic k i l l e r c e l l s ) have a l s o been reported to express the L y t - l + 2 + phenotype (107,111,112). Helper cells have been described by a number of investigators as expressing the Lyt-l +2 phenotype (114-116). Antigen-specific suppress- or cells isolated from antigen-primed mice have been identified as Lyt-1 2 + (115). Suppressor cells generated in vitro by concanavalin A were also found to be Lyt-1 2 +, while the helper cells thus generated were Lyt-l +2~ (114,117). There have been fewer studies carried out on mice bearing the Lyt al l e l e . In these studies allogeneic k i l l e r cells have been identified as L y t - l + 2 + (108,118). K i l l e r s of syngeneic tumor cells in C3H mice were found to be predominantly Lyt-1 2 + although i t was indicated that a population of Lyt-1 2 cells might also be involved (119). In the case of suppressor c e l l s , i t was found that two distinct populations of cells were present, those which suppressed the delayed type hypersensi- t i v i t y reaction and were Lyt-l +2 and those which suppressed a humoral response and were Lyt-1 2 + (120). It has also been reported that in vitro-induced suppressor cells in CBA mice are Lyt-1 2+ (118), while the helper cells are L y t - l + 2 + (118,121). The present study was carried out in DBA/2 mice (Lyt-1.1, -2.1) and involved the response of their cells to the syngeneic mastocytoma P815. The Lyt phenotypes of in vitro-generated allogeneic k i l l e r s , syngeneic k i l l e r c e l l s , and in vivo-generated T suppressor cells were characterized using anti-Lyt-1.1 and anti-Lyt-2.1 serum. M a t e r i a l s and Methods Experimental Animals and Tumors Female DBA/2J mice (H-2 d, Lyt-1.1, -2.1) and C57BL/6 mice (H-2*3, Lyt-1.2, -2.2) (The Jackson L a b o r a t o r i e s , Bar Harbor, Maine) between the ages of 2 and 5 months of age were used e x c l u s i v e l y . The tumor l i n e s used were the P815 mastocytoma and L1210 leukemia, both syngeneic f o r DBA/2J mice, and both maintained and transplanted as a s c i t e s tumors i n DBA/2J mice, or as f r o z e n c u l t u r e s maintained at -70°C i n l i q u i d n i t r o g e n . The methods of tumor maintenance have been described elsewhere (11). EL-4 lymphoma, syngeneic f o r C57BL/6 mice was maintained s i m i l a r l y . C e l l s S i n g l e - c e l l suspensions were prepared from spleens of DBA/2 mice by p r e s s i n g the t i s s u e through a s t a i n l e s s s t e e l 60-gauge mesh i n RPMT 1640 (Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , N.Y.) medium c o n t a i n i n g 10% h e a t - i n a c t i v a t e d f e t a l c a l f serum (FCS), 10 mM Hepes b u f f e r , and 5 x 10 ^ mM of 2-mercaptoethanol. Gentamycin was a l s o added to a f i n a l c o ncentration of 50 yg/ml. Suspended c e l l s were washed through 3.0 ml of FCS, resuspended i n complete medium, and counted f o r v i a b l e c e l l s by using trypan blue. Tumor c e l l s were washed twice-: i n medium before resuspen- s i o n i n complete medium f o r counting. Cult u r e system f o r the generation of c y t o t o x i c e f f e c t o r c e l l s A l l o g e n e i c e f f e c t o r s The method f o r the jLn v i t r o generation of a l l o g e n e i c k i l l e r c e l l s have been described i n d e t a i l elsewhere (122). 36 B r i e f l y , 5 x 10 spleen c e l l s from DBA/2J mice were cultured, f o r 4 days i n the presence of.5 x 10 mitomycin-treated spleen c e l l s from C57BL/6 mice i n 24-well L i n b r o t r a y s i n 2.5 ml RPMI 1640 medium supplemented w i t h 10% FCS, 10 mM Hepes b u f f e r , 5 x 10 ^ M 2-mercaptoethanol, and 50 g/ml genta- mycin. C e l l s were harvested at 4 days, counted by trypan blue e x c l u s i o n , and assayed f o r t h e i r a b i l i t y to k i l l "^Cr- l a b e l e d EL4 c e l l s i n a standard 4 hr assay, us i n g t a r g e t : e f f e c t o r c e l l s from 30:1 to 3.75:1. Syngeneic e f f e c t o r s The method by which a primary i n v i t r o c y t o t o x i c response to syngeneic tumor c e l l s can be generated has been described e l s e - where (113).The method i s b a s i c a l l y that described above w i t h the exception that 5 x 10^ DBA/2 spleen c e l l s were c u l t u r e d f o r 5 days w i t h 5 x 10"* mitomycin-treated P815 c e l l s . Harvested c e l l s were run i n the standard "'"'"Cr r e l e a s e assay f o r 18 hr on l a b e l e d P815 c e l l s w i t h e f f e c t o r : . target r a t i o s of between 100:1 and 12.5:1. Previous work i n t h i s l a b o r a t o r y has demonstrated that the k i l l e r c e l l s generated i n t h i s way are s p e c i f i c f o r the s t i m u l a t o r c e l l s (P815 i n t h i s instance) (123,124). (Also see Table I ) . C y t o t o x i c i t y t e s t This has been described i n d e t a i l i n Chapter I I . Generation of suppressor c e l l s In most of the previous work c a r r i e d out i n t h i s l a b o r a t o r y , a n t i g e n - s p e c i f i c T suppressor c e l l s involved i n r e g u l a t i o n of the c y t o t o x i c response of syngeneic mice to the P815 tumor l i n e (123,124). 37 Table I. 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 In v i t r o culture ,. Cytotoxocity Assay Responding C e l l s Stimulating c e l l s Target c e l l s % c y t o t o x i c i t y Normal DBA Spleen P815 P815 56.4 + 2.5 Normal DBA Spleen P815 L1210 6.8 + 2.6 Normal DBA Spleen P815 EL-4 5.7 + 3.0 Normal DBA Spleen C57BL/6 P815 0.7 + 0.6 Normal DBA Spleen C57BL/6 L1210 5.8 + 3.2 Normal DBA Spleen C57BL/6 EL-4 61.2 + 2.9 a51 Cr release a f t e r 18 hr incubation^ e f f e c t o r : target r a t i o of 100:1 for P815 stimulated c e l l s . Cr release a f t e r 4 hr incubation, e f f e c t o r : target r a t i o of 715:1 for EL-4 stimulated c e l l s . 38 have been obtained from the thymuses of animals into which 3 x 3 10 tumor c e l l s had been injected subcutaneously 8-9 days previous- l y (65). The suppressor c e l l s used i n t h i s study were generated by the i n t r a p e r i t o n e a l i n j e c t i o n of soluble membrane extracts of P815 c e l l s . P815 c e l l s , from mice bearing the tumor as a s c i t e s , were drawn from the peritoneal cavity, washed i n PBS, and lysed by freezing and thawing three times i n d i s t i l l e d water. Large membrane fragments and other c e l l debris were removed by ce n t r i f u g a t i o n at 12,000g for 30 min. Membrane components were then pelleted by c e n t r i f u a t i o n at 105,000g for 120 min. The membrane p e l l e t was resuspended i n PBS and subjected to three 1-min bursts of ultrasound (Biosonic probe, 30 setting) at 0°C. The material was again centrifuged and the supernatant presumably containing s o l u b i l i z e d membrane components and small membrane fragments was used. The protein content of t h i s material was determined by the standard Lowry te s t . Suppressor c e l l s i n the spleens of DBA/2J mice were r e a d i l y induced by i n t r a p e r i t o n e a l i n j e c t i o n of between 80 and 200 yg protein per animal. Animals were s a c r i f i c e d 4 to 5 days af t e r antigen in e c t i o n , and t h e i r spleens used as a source of suppressor c e l l s . This method i s an adaptation of that described recently for the generation of suppressor T c e l l s to methylcholanthrene-induced sarcomas 0-25). Because the spleens of these animals contained both antigen- s p e c i f i c and nonspecific suppressor c e l l s (see below), i t was necessary to separate these two populations. Spleen c e l l s were passed through nylon wool columns according to the method of J u l i u s el: a l (126). The m o d i f i c a t i o n s used here were that c e l l s , a f t e r a p p l i c a t i o n to the column, were incubated f o r 30 min at 37°C and then e l u t e d at 37°C. The columns were then incubated f o r a f u r t h e r 30 min at 4°C before a second e l u t i o n i n co l d medium. The c e l l s from both e l u t i o n s were pooled and used as a source of antigen- s p e c i f i c suppressor c e l l s . This procedure u t i l i z e d columns which were f a i r l y t i g h t l y packed, c o n t a i n i n g about 12-15 ml of nylon wool through which 20 ml of supplemented medium at 37°C had been passed 8 before use. A t o t a l of 2 x 10 spleen c e l l s were a p p l i e d to columns of t h i s s i z e and approximately 20% of the c e l l s were recovered, by e l u t i o n . The p r o p e r t i e s of the e l u t e d c e l l s were c h a r a c t e r i s t i c of T - c e l l - e n r i c h e d populations i n that they were 95% s e n s i t i v e to a n t i - t h y - 1 serum plus complement and contained very few phagocytic c e l l s (<1.0%). The generation of suppressor c e l l s i s i l l u s t r a t e d i n Figure 8. Assay f o r suppressor c e l l s The method used to assay f o r suppressor c e l l s has been described 6 i n d e t a i l elsewhere.(123). B r i e f l y , 5 x 10 normal spleen c e l l s were cocultured w i t h 5 x 10^ (or fewer i n some cases) suppressor c e l l popu- l a t i o n s i n the presence of 5 x 10^ mitomycin-treated P815 c e l l s i n 24-well L i n b r o t r a y s i n 2.5 ml of medium. C e l l s were c u l t u r e d f o r 5 days before assay f o r generation of c e l l s c y t o t o x i c f o r P815. Controls i n these s t u d i e s c o n s t i t u t e d the use of 5 x 10 normal spleen c e l l s plus 5 x 10 normal spleen or spleen-derived c e l l s which had been tr e a t e d i n a manner analogous to the suppressor p o p u l a t i o n . When the degree of suppression was q u a n t i t a t e d , c y t o t o x i c i t y 40 Preparation of P815 Tumor-Specific Suppressor C e l l s (P815-T ) Injection of 80-200 vg of Soluble P815 Tumor Membrane Extract IP DBA/2J Mice 4 Days Splenocytes - Containing both S p e c i f i c Suppressor C e l l s ( T - C e l l s ) : And Non-Specific Suppressor C e l l s (Probably M0) Passed Through Nylon Wool Column (30' at 37°) Eluted with 37° media Further 30' at 4° Eluted with 4° media Eluted Enriched T - c e l l Population Containing P815~Specif i c T .. Figure 8. Flow diagram i l l u s t r a t i n g the preparation of P815 tumor-specific suppressor c e l l s . 41 was tested at various e f f e c t o r to target r a t i o s and the decrease i n t o t a l l y t i c u n i t s was assessed. One l y t i c u n i t , which was defined as the number of e f f e c t o r c e l l s required to lyse 50% 4 of the 10 target c e l l s i n 18 hr incubation, was estimated by l i n e a r regression analysis of percentage of c y t o t o x i c i t y vs . logarithm of e f f e c t o r to target c e l l r a t i o . T o t a l l y t i c u nits were caluclated from c e l l recovery i n cultures, and percentage of suppression was estimated from the decrease i n t o t a l l y t i c u n i t s . In a l l experiments, no s i g n i f i c a n t differences i n c e l l recovery between test cultures and controls were observed. In a l l suppression experiments reported here, except the time course one (Fig- H ) , a minimum of 37% suppression was seen. Anti-Lyt treatment of c e l l s The c e l l s tested f or t h e i r Lyt phenotype i n t h i s study were: allogeneic k i l l e r c e l l s , syngeneic k i l l e r c e l l s , and syngeneic tumor s p e c i f i e d suppressor c e l l s . DBA/2J mice are of the L y t - 1.1 and Lyt-2.1 phenotype. The antisera used here were the kind g i f t of Dr. R. Nowinski (The Fred Hutchinson Cancer Research Center, Seattle, Washington) who had raised and characterized the a n t i sera according the previously described procedures (127). The anti-Lyt-1.1 serum t i t e r e d at 1:640 and the anti-Lyt-2.1 t i t e r e d at 1:160 when t i t r a t e d i n the presence of rabb i t comple- ment on B 6 ^ L y t - l . l and B6Lyt-2.1,-3.1 thymocytes, r e s p e c t i v e l y . The anti-Lyt-1.1 serum had no e f f e c t on B6Lyt-2.1, -3.1 thymocytes, likewise, the anti-Lyt-2.1 serum had no e f f e c t on B 6 . L y t - l . l thymocytes. In the experiments run here, higher l e v e l s of the 42 ant i s e r a were found to be necessary i n order to k i l l the appropriate c e l l s e f f e c t i v e l y . For both the allogeneic and syngeneic k i l l e r c e l l s , the procedure was i d e n t i c a l . E f f e c t o r c e l l s were harvested, counted, and d i s t r i b u t e d to small p l a s t i c test tubes at a t o t a l concentration of 3.0 x 10 7 c e l l s per tube. These c e l l s were centrifuged and the supernatant removed. Three hundred m i c r o l i t e r s of medium was added to each tube and 50 y l of either anti-Lyt-1.1, Lyt-2.1, or medium was added to the appropriate tube. The c e l l s were mixed and incubated at room temperature for 45 min following which 50 y l of neat rabbit complement (Low-tox, Cedar Lane, Ontario, Canada) was added to each tube except the c o n t r o l , to which 50 y l of medium was added. Incubation was continued for another 45 min. C e l l s were washed three times i n supplemented medium, counted by trypan blue exclusion, and subsequently tested for t h e i r cytotoxic a c t i v i t y . One population of a 1:1 mixture of anti-Lyt-1 and anti-Lyt-2-treated c e l l s were also reconstituted and tested for c y t o t o x i c i t y . In the assay of suppressor c e l l s , larger c e l l numbers had to be treated because the c e l l s were subsequently going into the large cultures for 5 days. For t h i s reason, 6 x 10^ c e l l s were treated, and double the amounts of a l l reagents were used. A protocol as described above was used, except that a f t e r treatment, washing, and counting, the c e l l s were put into c u l t u r e at a concentration of 5 x 10 6 with 5 x 10 6 normal spleen c e l l s plus mitomycin-C treated P815 c e l l s . 43 R e s u l t s The spleen c e l l s of DBA/2J mice, 4 days f o l l o w i n g i n t r a p e r i t o n e a l i n j e c t i o n of P815 membrane e x t r a c t s , were very suppressive when they were c o c u l t u r e d w i t h 5 x 10^ DBA/2 spleen c e l l s i n the presence of mitomycin-treated P815 c e l l s . The generation of c y t o t o x i c c e l l s i n these c u l t u r e s was markedly below that seen i n c o n t r o l c u l t u r e s ( F i g . 9a). When these suppressor c e l l s were assayed i n c u l t u r e w i t h normal spleen c e l l s plus mitomycin-treated L1210 c e l l s , the generation of c e l l s c y t o t o x i c f o r t h i s tumor were al s o markedly suppressed ( F i g . 9b). Since these two systems do not cross r e a c t at t h i s l e v e l , (see Fig.T) ,(12) i t was concluded that i n t r a p e r i t o n e a l i n j e c t i o n of membrane e x t r a c t s of P815 c e l l s could generate both s p e c i f i c and n o n s p e c i f i c suppressor c e l l s . Since the most common n o n s p e c i f i c suppressor c e l l i s an adherent cell.,- pro- bably of the macrophage-monocyte s e r i e s (18,128,129), an attempt to achieve s p e c i f i c i t y i n the system by passing the spleen c e l l s from antigen- i n j e c t e d animals through nylon wool columns was attempted. The c e l l s e l u t e d from these columns at e i t h e r 37 or 4°C were test e d f o r t h e i r suppressive a c t i v i t y ' . Since both populations were found to c o n t a i n s p e c i f i c suppressor c e l l s , subsequent s t u d i e s i n v o l v e d a pool of the two populations ( F i g . 10). The time course of appearance and disappearance of the antigen- s p e c i f i c suppressor c e l l s was examined. As can be seen i n Figure l i , DBA/2 splenocytes show a n t i g e n - s p e c i f i c suppression w i t h i n 2 days f o l l o w i n g i n t r a p e r i t o n e a l i n j e c t i o n of P815 membrane e x t r a c t s . This suppression peaks at 4-5 days, then f a l l s o f f r a p i d l y to c o n t r o l l e v e l s by 7-9 days post i n j e c t i o n . 44 SOI 100=1 125=1 EFFECTOR ̂ TARGET RATIO Figure 9. C y t o t o x i c i t y t i t r a t i o n s of DBA/2 spleen c e l l s primed against e i t h e r P815 stimulator ^a) or L1210 stimulators (b). (•) Control containing 10 normal DBA/2 splenocyjzes i n the priming culture; (o) cultureg containing 5 x 10 normal DBA/2 splenocytes and 5 x 10 splenocytes from mice inoculated 4 days previously with P815 membrane extracts (80 yg per mouse). 45 125 :1 50 :1 l O O ' l 125:1 EFFECTOR;TARGET RATIO Figure 10. C y t o t o x i c i t y t i t r a t i o n s of DBA/2 spleen c e l l s primed against either P815 (a) or L1210 £b) stimulators. (•) Control culture gontaining 5 x 10 normal DBA/2 spleno- cytes and 5 x 10 normal DBA/2 nylon wool-pgssed splenic lymphocytes; (o) c u l t u r e s containing 5 x 10 normal DBA/2 splenocytes and 5 x 10 P815 primed DBA/2 nylon wool- passed splenic lymphocytes ( c e l l s eluted at 37 and 4 C were pooled). i i \ \ \ 2 4 6 8 10 Days a f t a r Lp. i n j e c t i o n of P815 m e m b r a n e e x t r a c t Figure 11. Time course of a p p e a r a n c e and disappearance of antigen- s p e c i f i c s u p p r e s s o r c e l l s i n t h e s p l e e n o f DBA/2 m i c e i n j e c t e d i n t r a p e r i t o n e a l l y w i t h P815 membrang e x t r a c t s on day 0. Control cultureg c o n t a i n e d 5 x 10 normal DBA s p l e n o c y t e s a n d 5 x 10 n o r m a l DBA/2 nylon w o o l - p a s s e d g S p l e n i c l y m p h o c y t e s . Test c u l t u r e s g o n t a i n e d 5 x 10 n o r m a l DBA/2 s p l e n o c y t e s a n d 5 x 10 P815 p r i m e d DBA/2 n y l o n w o o l - p a s s e d s p l e n i c l y m p h o c y t e s . Results a r e plotted as MEAN % s u p p r e s s i o n c a u s e d b y P815 i n j e c t e d s p l e e n a s c o m p a r e d t o c o n t r o l s . 47 The L y t phenotypes of a v a r i e t y of e f f e c t o r c e l l s i n DBA/2 mice was i n v e s t i g a t e d . Since previous f i n d i n g s by others had c h a r a c t e r i z e d a + + a l l o g e n e i c k i l l e r s i n mice of the Lyt a l l e l e as Lyt-1 2 , i t was considered important to t e s t the a n t i s e r a by determining i t s e f f e c t on a l l o g e n e i c k i l l e r c e l l s . The r e s u l t s are shown i n Fi g u r e 12, i n which i t can be seen that both a n t i - L y t - 1 . 1 and a n t i - L y t - 2 . 1 plus complement e f f e c t i v e l y abrogated the a l l o g e n e i c k i l l e r c e l l s . M i xing of a n t i - L y t - 1 . 1 and a n t i - L y t - 2 . 1 - t r e a t e d c e l l s p r i o r to the c y t o t o x i c i t y assay d i d not r e c o n s t i t u t e the response (data not shown). These r e s u l t s , t h e r e f o r e , are i n agreement w i t h the observations of others and i n d i c a t e that the a l l o g e n e i c k i l l e r c e l l i n the DBA/2J animal + + expresses the Lyt-1 2 phenotype.(108,118). An experiment was run co n c u r r e n t l y to determine the Lyt phenotype of the c y t o t o x i c e f f e c t o r to syngeneic tumor c e l l s . The r e s u l t s are shown i n Figure 13. In t h i s case complement plus a n t i - L y t - 2 . 1 but not a n t i - L y t - 1 . 1 reduced the k i l l i n g e f f i e n c y of t h i s p o p u l a t i o n , i n d i c a t i n g t h a t the Lyt phenotype of t h i s c e l l i s L y t - 1 2 +. Mix i n g of the two tr e a t e d populations d i d not r e s t o r e the response. F i n a l l y , the Lyt phenotype of the DBA/2 a n t i g e n - s p e c i f i c suppressor c e l l was i n v e s t i g a t e d . The r e s u l t s are shown i n Figure 14. In t h i s i n s t a n c e , the a n t i - L y t - 1 antiserum e l i m i n a t e d suppressor c e l l s , or t h e i r development, whereas the a n t i - L y t - 2 serum had no e f f e c t . In t h i s case a l s o , mixtures of a n t i - L y t - 1 and L y t - 2 - t r e a t e d c e l l s d i d not r e c o n s t i t u t e the response. I t would t h e r e f o r e appear, i n t h i s system, that .the suppressor c e l l , i t s p r o g e n i t o r , or a c e l l v i t a l i n i t s development expresses the Lyt- 1 2 phenotype. 48 i 3-75:1 75:1 15:1 30:1 EFFECTOR : TARGET RATIO I ! I - Figure 12. The e f f e c t of anti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C' on DBA/2 allogeneic e f f e c t o r . c e l l s raised against C57BL/6, on a EL4 target. (o) Control culture; (t) anti-Lyt-1.1-treated e f f e c t o r c e l l s ; (A) a n t i - L y t - 2.1-treated e f f e c t o r c e l l s . 49 100 - 80 Control A ll 60 C Y T O T O X IC  40 ' a n t i Ly-1-1 < anti Ly-2-7 ^? ON 20 - • I 1X5:1 25:1 50:1 100:1 EFFECTOR: TARGET RATIO Figure 13. The e f f e c t of ariti-Lyt-1.1 and anti-Lyt-2.1 serum plus rabbit C' on DBA/2 ef f e c t o r c e l l s primed against the syngeneic P815 mastocytoma. (o) Control; (•) a n t i - L y t - 1.1-treated e f f e c t o r c e l l s ; (A) anti-Lyt-2.1-treated e f f e c t o r c e l l s . 50 1001 80 O I->• u QN 60r- 40 20 • A Control 0 Suppressor Cell Control • Anti-Lyl-1 • A Anti-Ly 2~1 i • 12.5 1 25 1 50--1 100--1 EFFECTOR '. TARGET RATIO F i g u r e 14. The e f f e c t of a n t i - L y t - 1 . 1 and a n t i - L y t - 2 . 1 serum p l u s r a b b i t C' on P815-primed n y l o n wool-passed DBA/2 s p l e n i c ^ suppressor c e l l s . (A) C o n t r o l g C u l t u r e s c o n t a i n i n g 5 x 10 normal DBA/2 c e l l s p l u s 5 x 10 n y l o n wool-passed normal DBA/2 g p l e n o c y t e s ; (o) p o s i t i v e c o n t r o l c u l t u r e c o n t a i n i n g 5 x 10 normal DBA/2 sp lenocytes p l u s 5 x 10 n y l o n w o o l - passedgP815 suppressors c e l l s ; (•) c u l t u r e c g n t a i n i n g 5 x 10 normal DBA/2 s p l e n o c y t e s p l u s 5 x 10 a n t i - L y t - 1 . 1 - treated^ P815 suppressor c e l l s ; (A) c u l t u r e c g n t a i n i n g 5 x 10 normal DBA/2 s p l e n o c y t e s p l u s 5 x 10 a n t i - L y t - 2 . 1 - t r e a t e d P815 suppressor c e l l s . The SEM i n t h i s e x p e r i - ment was always <3.0% and t h e r e f o r e not shown. C e l l r e c o v e r i e s of a n t i s e r u m - t r e a t e d suppressor T c e l l p o p u l a t - i o n s were as f o l l o w s : a n t i - L y t - 1 . 1 , 68.7%;" a n t i - L y t - 2 . 1 , 70.3%. . These data are based on "estimates from the comple- m e n t - t r e a t e d c o n t r o l c e l l s which were regarded as 100% r e c o v e r y . L y t i c u n i t s were: (A) 84 . 3 , (o) 46 . 1 , (•) 78.6, (A) 41 .7 . 51 D i s c u s s i o n In t h i s chapter, a method f o r the i n v i v o i n d u c t i o n of s p e c i f i c suppressor c e l l s which i n h i b i t the hi v i t r o generation of c e l l s c y t o - t o x i c f o r a syngeneic tumor has been described. The method i s s i m i l a r to the method described by Fujimoto f o r the i n d u c t i o n of the same type of suppressor c e l l s w i t h a d i f f e r e n t tumor (125). However, i n the work shown here, the suppression was not s p e c i f i c when whole spleen c e l l p o pulations of tumor a n t i g e n - i n j e c t e d mice were used, as i n d i c a t e d by t h e i r a b i l i t y to suppress the jm v i t r o c y t o t o x i c response to L1210 leukemia c e l l s as w e l l as P815 mastocytoma c e l l s . Since i t i s w e l l documented that a n t i g e n - s t i m u l a t e d spleen c e l l s may c o n t a i n a populat- ion of n o n s p e c i f i c suppressor c e l l s which e x h i b i t the c h a r a c t e r i s t i c s of adherence to p l a s t i c or nylon wool and phagocytic a c t i v i t y (18,128, 129), i t was f e l t that the passage of the stimulated spleen c e l l s through nylon wool might remove the n o n s p e c i f i c suppressive a c t i v i t y . This proved to be the case. That the a n t i g e n - s p e c i f i c suppressor c e l l was of thymic o r i g i n has been shown p r e v i o u s l y (12) and i s i m p l i c i t i n the f i n d i n g that the nylon wool nonadherent pop u l a t i o n (which i s > 9 5 % thy-1 p o s i t i v e ) contained the a n t i g e n - s p e c i f i c suppressor c e l l . The s t u d i e s on the Lyt phenotypes of the va r i o u s e f f e c t o r c e l l s i n t h i s i n v i t r o system were i n f o r m a t i v e . The f i n d i n g that the a l l o - geneic k i l l e r c e l l i n the DBA/2J mouse was Lyt-l +2"*" was not s u r p r i s i n g , since i t confirms the observations of others t h a t , i n mice bearing the Lyt-1.1 and 2.1 a l l e l e , the a l l o g e n e i c k i l l e r c e l l i s indeed L y t - l + 2 + (108,118). Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e regarding syngenic k i l l e r and suppressor c e l l s i n mice expressing t h i s a l l e l e . 52 The f i n d i n g that the syngeneic k i l l e r d i f f e r s from the allogeneic k i l l e r c e l l i n expressing the Lyt-1 2 + phenotype i s somewhat s u r p r i s i n g . However, other investigators have reported that anti-tumor syngeneic k i l l e r c e l l s i n C3H mice were predominantly of the Lyt-1 2 + phenotype. These workers also noted the probable involvement of L y t - l + 2 + c e l l s i n the syngeneic system. In a recent report very applicable to t h i s study, M i l l s and colleagues found that i n DBA/2 mice, syngeneic tumor k i l l e r s directed towards P815 tumor c e l l s expressed'Lyt-1 antigen.(130). They however did not look at Lyt-2 antigen expression on these c e l l s . Therefore, as far as expression of Lyt-1 on syngeneic k i l l e r s goes, that study and the one reported here seem to be i n c o n f l i c t . One reason for t h i s may be the d i f f e r e n t methods of generation of cytotoxic c e l l s to P815. Syngeneic cytotoxic c e l l s i n the M i l l s study were generated i n v i t r o ( i n the presence of IL-2) from P815 tumor bearing DBA/2 mice. Therefore, they were probably generating secondary cytotoxic k i l l e r s as opposed to primary cytotoxic c e l l s i n t h i s i n v e s t i g a t i o n . Thus, there may be a dif f e r e n c e i n the Lyt-1 antigen expression on primary vs secondary syngeneic k i l l e r s i n the DBA/2-P815 system. This of course leads to the recent findings that a l l T c e l l s probably express Lyt-1 and Lyt-2, although i n varying amounts (131). Therefore, i t i s impossible to conclude at t h i s time that the syngeneic k i l l e r c e l l i s Lyt-1 2 +, since i t may only be quantitative differences between i t and the allogeneic k i l l e r i n expression of the Lyt-1 a l l o a n t i g e n which appear to render them as d i s t i n c t populations. The observation made here that anti-Lyt-1 antiserum e f f e c t i v e l y abrogated suppressor c e l l function, i n the in v i t r o system used, i s explicable i n at least two possible ways. F i r s t , i t has been reported previously that suppressor c e l l s which lower DTH reactions i n mice of a + -the Lyt a l l e l e are of the Lyt-1 2 phenotype (120). Second, the c e l l that was i s o l a t e d from immune spleens may not be the e f f e c t o r c e l l for suppression but may induce the suppressors i n cu l t u r e . Evidence f o r a mechanism such as t h i s has been presented for both humoral and c e l l - mediated responses (42-44, 132,133). In the model presented by Germain, Benacaraf and colleagues (22,23,44,60,134-136), the f i r s t c e l l i n immune suppression i s a Lyt -l"*", I - J + , antigen-binding "inducer" of suppression. This c e l l may "induce", i n a cascade-like mechanism, other T c e l l s to a c t i v e l y suppress i n the same system. The 5-day culture period of the assay here might allow the induction of suppressors in s i t u under the influence of th i s L y t - l + 2 c e l l . At present, i t can only be concluded that i n a population of suppressor c e l l s , or t h e i r precursors, an L y t - l + 2 c e l l i s e s s e n t i a l for the suppressive e f f e c t to the observed i n i n v i t r o cytotoxic assays. CHAPTER IV IN VITRO AND IN VIVO EFFECTS OF SYNGENEIC AND ALLOGENEIC ANTISERA RAISED TO TUMOR- SPECIFIC SUPPRESSOR FACTOR FROM DBA/2 MICE 55 CHAPTER IV Introduction It i s now recognized that T lymphocytes are made up of d i s t i n c t sub- sets which i n t e r a c t with each other as well as B lymphocytes and macro- phages to both amplify and i n h i b i t a v a r i e t y of immunological responses. Thus T c e l l s have demonstrated enhancing e f f e c t s on the immune response by way of T helper c e l l s (2-10). T c e l l s have also been found to i n - h i b i t the immune response by way of T suppressor c e l l s (11-17,19~44,65). In many of the investigations concerning ant i g e n - s p e c i f i c regu- lat o r y T c e l l s , i t has been shown that soluble factors derived from these c e l l s can apparently duplicate t h e i r functions (14,59-61,67-74,76,77,82, 137-139). L i t t l e i s known of these factors but, as indicated i n Chap- ter I I , what i s known shows they e x h i b i t c e r t a i n p h y s i c a l , chemical and antigenic properties i n common. They are proteins with s p e c i f i c antigen binding p o t e n t i a l , have molecular weights of about 50,000, have deter- minants which are coded by the Ir region, lack constant region immuno- glo b u l i n determinants, but may carry variable ( i d i o t y p i c ) determinants of the I heavy chain. A l l t h i s leads to the i n t r i g u i n g p o s s i b i l i t y that these " f a c t o r s " may i n fa c t be the antigen receptors of these s p e c i f i c regulatory T c e l l s . Yamauchi e_t al_ (125) have recently shown that i n j e c t i o n of soluble S1509a tumor extracts into syngeneic A/J mice act i v a t e s , i n the spleen tumor-specific T suppressor c e l l s . This has recently been extended to the syngeneic P815 tumor system i n DBA/2J mice (Chapther I I I , 13). In t h i s chapter, i t i s shown that by passing extracts of t h i s suppressive population over P815 membrane - Sepharose columns, P815-specific suppressor factor can be eluted. This P815-specific suppressor factor was then injected into syngeneic and allogeneic groups of mice. The hope being that i n the syngeneic mice the- umique d e t e r m i n a n t s ( i d i o t y p i c ? ) , found i n or near the antigen binding s i t e of the P815-specific sup- pressor f a c t o r , would e l i c i t an antibody response i n at l e a s t some of the animals. While both these and the ©onstant d e t e r m i n a n t s on the suppressor factor might induce an antibody response i n the allogeneic mice. Both groups of mice produced antisera which was capable of reacting i n v i t r o s p e c i f i c a l l y with the P815-specific suppressor f a c t o r , and with complement was able to abrogate the function of P815-specific T suppressor c e l l s , but was unable to e f f e c t the action of P815-specific T cytotoxic e f f e c t o r c e l l s . The an t i s e r a produced i n syngeneic mice was also shown to be e f f e c t i v e i n vivo i n slowing P815-tumor growth and prolonging s u r v i v a l times of DBA/2 mice injected with t h i s tumor. 57 M a t e r i a l s and Methods Mice and Tumors. Female DBA/2J and C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). Except f o r the prepa- r a t i o n of anti-suppressor f a c t o r a n t i s e r a , a l l mice were used between the ages of 2 and 5 months of age. The tumor l i n e s used were the P815 mastocytoma and L1210 leukemia, both syngeneic f o r DBA/2J mice. They have both been maintained and t r a n s - planted as a s c i t e s tumors i n DBA/2J mice (11), or as frozen c u l t u r e s maintained i n l i q u i d n i t r o g e n i n t h i s l a b f o r the past 6 years. B a l e n t l tumor, syngeneic to Balb/c mice, was a l s o used and maintained as above. C e l l s . This has been described i n d e t a i l i n Chapter I I I . C u l t u r e system f o r generation of c y t o t o x i c e f f e c t o r c e l l s . The method by which a primary i n v i t r o c y t o t o x i c response to syngeneic tumor c e l l s can be generated has been described pre- v i o u s l y • (123):. " B r i e f l y , s p e c i f i c . c y t o t o x i c i t y .against e i t h e r P815 or L1210 was generated i n v i t r o by i n c u b a t i n g 5 x 10 spleen c e l l s w i t h 5 x 10^ mitomycin tr e a t e d tumor c e l l s i n Linbro m u l t i w e l l p l a t e s c o n t a i n i n g 24 f l a t bottom w e l l s , at 37°C i n a h u m i d i f i e d incubator w i t h 5% CO2 f o r 5 days. The t o t a l volume of each c u l t u r e w e l l was made up 2.5 ml w i t h complete 1640 medium. A f t e r i n c u b a t i o n , c e l l s were harvested, counted,-resuspended'in complete: 1640 medium'at 'a concentration 58 of 10 /ml and t i t r a t e d i n quadruplicate with doubling d i l u t i o n s s t a r t i n g at an e f f e c t o r : t a r g e t r a t i o of 100:1 i n a standard 18 h ^ C r release assay which has been described i n previous chapters (II and I I I ) . The s p e c i f i c e f f e c t o r c e l l s i n t h i s syngeneic tumor system have previously been shown to be T lymphocytes (11,13). Generation of suppressor c e l l s . The method, for generation of tumor-spcific T suppressor c e l l s , involved i n the regulation of cytotoxic response of DBA/2J mice to the syngeneic P815 tumor, has been described previously (Chapter I I I ) . B r i e f l y , suppressor c e l l s used i n t h i s study were generated by the i n t r a p e r i t o n e a l i n j e c t i o n of soluble membrane extracts of P815 c e l l s . Suppressor c e l l s were r e a d i l y induced i n the spleens of DBA/2J mice by i n t r a p e r i t o n e a l i n - j e c t i o n of 150 Ug of protein per animal 4-5 days before s a c r i f i c e . Because the spleens of these animals contained both antigen- s p e c i f i c and non-specific suppressor c e l l s (13) i t was necessary to separate the two populations. Spleen c e l l s were passed through nylon wool columns by a modified method of J u l i u s et a l (126). The method i s b a s i c a l l y that described before (Chapter III, 13) with a few modifications outlined below. In short, the c e l l s , a f t e r a p p l i c a t i o n to the nylon wool column, were incubated for 60 min at room temperature (20°C) and then eluted with warmed 37°C complete 1640 medium. Approximately 15% of the c e l l s were recovered by t h i s e l u t i o n process. The properties of the eluted c e l l s were c h a r a c t e r i s t i c of T c e l l enriched populations i n that 59 they were >95% s e n s i t i v e to anti-Thy 1 serum plus complement and contained very few phagocytic c e l l s (<1.0%). This popu- l a t i o n was a n t i g e n - s p e c i f i c i n i t s suppressive a c t i v i t y , and has been found repeatedly to co n t a i n c e l l s , which under the c o n d i t i o n s used here, were capable of suppressing the i n v i t r o primary c y t o t o x i c .response 30-70%. Pr e p a r a t i o n of suppressor f a c t o r . A n t i g e n - s p e c i f i c T suppressor c e l l s , obtained from spleens of DBA/2J mice i n j e c t e d i n t r a p e r i t o n e a l l y 4-5 days p r e v i o u s l y w i t h P815 tumor membrane fragments were used f o r the prep a r a t i o n of P815 a n t i g e n - s p e c i f i c suppressor f a c t o r (P815-SF). C o n t r o l f a c t o r preparations were made from normal DBA/2 splenocytes, as w e l l as from mice i n j e c t e d w i t h B a l e n t l tumor membrane fragments ( t h i s p r e p a r a t i o n was termed B a l e n t l - " S F " even though i t had no demonstratable e f f e c t , suppressive or otherwise). The spleno- cytes were sonicated f o r 3 one minute b u r s t s (Biosonic 80, 60 watt s e t t i n g ) at 0°C. T h i s m a t e r i a l was t h e n c e n t r i f u g e d f o r 30 min at 15,000 x G at 0°C. The supernatant was then taken and mixed w i t h normal DBA/2J membrane fragments l i n k e d to Sepharose 4B. This step was taken to ensure that any m a t e r i a l which n o n - s p e c i f i c a l l y bound to DBA/2J membrane would be removed here. A f t e r 60 min at 0°C any non-bound m a t e r i a l was el u t e d w i t h i c e c o l d phosphate buffered s a l i n e (PBS). This m a t e r i a l was then mixed w i t h r a b b i t anti-mouse immunoglobulin l i n k e d to Sepharose 4B. This was to remove any anti-body molecules which Preparation of P815 Tumor-Specific Suppressor Factor (P815-SF) 60 IP Injection of 100-200 ug of Soluble P815 Membrane Extract into DBA/2J Mice 4 Days Splenocytes (Contain P815-specific T suppressor c e l l s ) Sonication + Centrifugation Supernate (Crude Extract) v Normal DBA/2 Membrane: Sepharose Column (60', 0°) Elute with PBS Rabbit Anti-MIg: Sepharose Column (60', 0°) Elute with PBS P815 Membrane: Sepharose Column (60', 0°) Wash with PBS Elute Absorbed Material with 2M NaCl si/ P815-Specific SF Figure 15. Flow diagram i l l u s t r a t i n g the preparation of P815 tumor-specific suppressor factor (P815-SF) 61 might bind i n the following step. The capacity of the a n t i - mouse immunoglobulin column was s u f f i c i e n t to remove at l e a s t twice the amount of Ig found i n the extracts. After 60 min at 0°C any non-bound material, was eluted with i c e cold PBS. This material was then mixed with P815 membranes linked to Sepharose 4B. A f t e r 60 min at 0°C the beads were washed thoroughly with i c e cold PBS. Bound material was eluted under midly d i s s o c i a t i n g conditions with 2M NaCl. The absorbance at 280 nm of t h i s material was i n a l l cases O.01; therefore, i t has been d i f f i c u l t to quantify i t other than by i t s b i o l o g i c a l properties. I t was f i l t e r s t e r i l i z e d and stored at -70°C. This material which was eluted from the P815 column was then t i t r a t e d for suppressive a c t i v i t y . Concentration when mentioned i s quoted as spleen equivalents based on the volume of o r i g i n a l spleen homogenates corrected for the change i n volume following passage through the columns. Values for the eluted f r a c t i o n s are based on the assumption that a l l the a c t i v i t y was recovered. A l l the above- mentioned columns were prepared as described previously (65,140) by the cyanogen bromide method (141). The preparation of suppress- or factor i s i l l u s t r a t e d i n Figure 15. Assay for suppressor c e l l s and suppressor f a c t o r s . The method used to assay for suppressor c e l l s has been described i n d e t a i l elsewhere (123). B r i e f l y , 5 x 10u normal spleen c e l l s were cocultured with 5 x 10^ (or fewer i n some cases) suppressor c e l l populations i n the presence of 5 x 10^ mitomycin 62 C treated tumor c e l l s , i n 24 well Linbro trays i n 2.5 ml of complete 1640 medium. C e l l s were cultured for 5 days before being assayed for c e l l s cytotoxic to the tumor. Controls i n these studies constituted the use of 5 x 10 normal spleen c e l l s plus 5 x 10 normal spleen c e l l s or spleen c e l l s which had been treated i n a manner analogous to the suppressor popu- l a t i o n . Assay for the splenic suppressor factor a c t i v i t y involved incubation of 5 x 10^ normal spleen c e l l s with various concen- t r a t i o n s of the factor preparation, under the same conditions as outlined above. A l l experiments reported herein, using these assays were repeated at l e a s t three times on separate occasions (therefore separate preparations of both suppressor c e l l s and f a c t o r ) . Preparation of antisera against P815-specific suppressor f a c t o r . The anti-P815-specific suppressor factor antisera ( a n t i - P815-SF antisera) used i n t h i s study was prepared by immunizing syngeneic DBA/2J mice and allogeneic C57BL/6 mice repeatedly with P815-specific immunoadsorbent eluted suppressor factor (see above). Control material (Balentl-"SF") was prepared i n p a r a l l e l from DBA/2J mice i n j e c t e d 4-5 days previously with B a l e n t l tumor membrane extracts. This material was injected into control groups of mice from the same batch. Animals were injected subcutaneously every 2 weeks with 0.1 ml (0.036 spleen equivalent) of either P815-specific suppressor factor or c o n t r o l Balentl-"suppressor f a c t o r " i n complete Freund's adjuvant. A f t e r 63 4 months, the mice were bled from the r e t r o o r b i t a l sinus once a month (one week a f t e r the l a s t immunization) while m a i n t a i n i n g the immunization schedule. The r e s u l t i n g a n t i s e r a was i n a c t i v a t e d at 56°C f o r 30 min, then tes t e d f o r i t s a b i l i t y to abrogate i n the presence of complement the f u n c t i o n of P815 s p e c i f i c T suppressor c e l l s . In a l l experiments reported here the a n t i s e r a from the mice w i t h i n each group were pooled before t e s t i n g . Each of the four groups contained at l e a s t 15 mice each. Anti-P815-SF a n t i s e r a k i l l i n g of c e l l s . The c e l l s tested f o r t h e i r expression of P 8 1 5 - s p e c i f i c suppressor f a c t o r determinants i n t h i s study were: syngeneic P815 t u m o r - s p e c i f i c suppressor T c e l l s and syngeneic P815 tumor- c y t o t o x i c T c e l l s . In p r e l i m i n a r y t e s t s the two anti-P815-SF a n t i s e r a pools were found to be e f f e c t i v e , i n con j u n c t i o n w i t h r a b b i t complement (Rabbit Low Tox Complement, Cedar Lane Labs, Hornby, O n t a r i o ) , i n e l i m i n a t i n g suppressor c e l l f u n c t i o n , at d i l u t i o n s of up to 1:40. In experiments run here a l l the a n t i s e r a was used at a f i n a l c o ncentration of 1:10, i n the presence of a 1:9 f i n a l c o n c e n t r a t i o n of complement. At t h i s c o n c e n t r a t i o n no anti-normal DBA/2J or anti-tumor a c t i v i t y was detected i n the anti-P815-SF a n t i s e r a . In experiments t e s t i n g f o r the presence of P 8 1 5 - s p e c i f i c suppressor f a c t o r determinants on suppressor c e l l s , 2 x 10 7 s p l e n i c T suppressor c e l l s , prepared from DBA/2J mice i n j e c t e d w i t h tumor membrane e x t r a c t s and 2 x 10^ splenocytes from normal 64 DBA/2 controls, were suspended i n 0.40 ml of a 1:10 d i l u t i o n of antisera and incubated for 45 min at room temperature. Then 50 y l of neat rabbit complement was added and incubation was continued for another 45 min. Appropriate controls of complement and medium only were run concurrently. Following incubation c e l l s were washed twice i n complete 1640 medium, and counted by trypan blue exclusion. In no instance were viable counts i n c e l l suspensions containing the anti-P815-SF antisera s i g n i f i c a n t l y d ifferent from those of the controls, and i n no instance were counts lower than 90% of the number of c e l l s before treatment. These c e l l s were subsequently used i n suppressor c e l l assays. In experiments testing for the presence of P815-specific suppressor factor determinants on cytotoxic c e l l s , the protocol was as described above except 1.6 x 10^ c e l l s , harvested from 5 day i n v i t r o cultures with mitomycin C treated tumor c e l l s , were treated and subsequently tested i n the cyt o t o x i c i t y assay. Since th i s experiment es s e n t i a l l y resulted i n negative r e s u l t s , cyto- toxic c e l l s were also treated with monoclonal anti-Thy 1 antisera (kindly supplied by Dr. H.-S. Teh, U.B.C., and used at a d i l u t i o n of 1:100) plus complement. Anti-SF antisera absorption of suppressor factor a c t i v i t y . The a b i l i t y of anti-P815-SF antisera to remove the suppressive a c t i v i t y from P815-specific suppressor factor preparations was tested by an immunoadsorbent assay similar to the one described previously (Chapter 11,65). - I n short, P815-specific suppressor 65 factor preparations or normal co n t r o l material prepared i n p a r a l l e l were mixed with either the anti-P815-SF antisera or the c o n t r o l anti-Balentl-"SF" antisera, and incubated for 120 min at 0°C. These materials were then passed through immuno- adsorbent Sepharose 4B columns to which rabbit anti-mouse immuno- glo b u l i n had been attached by cyanogen bromide treatment. The capacity of the column was s u f f i c i e n t to remove at l e a s t twice the amount of mouse immunoglobulin added to the suppressor factor preparations. The material eluted from the columns was t i t r a t e d for suppressive a c t i v i t y over d i l u t i o n s ranging from 1:25 to 1:300 (these d i l u t i o n s of eluted material represent between 0.036 and 0.0033 spleen equivalents). ELISA assay. This test was c a r r i e d out as described before.(142). B r i e f l y , 0.2 ml of antigen (e.g. P815-SF i n F i g 18) i n pH 9.6 carbonate buffer was attached to substrate m i c r o t i t e r plates (Cooke Engineering Co., Alexandia, Va., No. 1-220-295) for 18 hr at 4°C. After washing with PBS-tween buffer, a n t i s e r a to be tested for t h e i r a c t i v i t y against the coated antigens were added to the wells i n 0.2 ml a l i q u o t s . Following incubation for 2 hr at room temperature and subsequent washing, the developing a l k a l i n e phosphatase-linked rabbit anti-mouse Ig (RaMI ) or goat a n t i - r a b b i t (GoRI ), at a d i l u t i o n of 1:600 or 1:400 r e s p e c t i v e l y , was added i n 0.2 ml a l i q u o t . A f t e r a further 2 h incubation and f i n a l washing with buffer, 0.2 ml of the enzyme substrate so l u t i o n (Sigma-104-105, p-nitrophenyl phosphate 66 disodium) was added to each well and the enzyme substrate reaction was allowed to continue while color developed i n the wells. The color change was followed on a T i t e r t e k Muliskan (Flow Laboratories). A l l tests were done at l e a s t i n t r i p l i c a t e . Gontrols of normal mouse serum (NMS), normal rabbit serum (NRS) or antigen alone with no added serum were always run and the appropriate control response was subtracted to get the s p e c i f i c response. Antisera plus complement k i l l i n g of P815 tumor c e l l s . P815 tumor c e l l s were labeled with "^Cr as described pre- v i o u s l y (Chapter II) . The "'"'"Cr labeled P815 tumor c e l l s were resuspended at 2 x 10^/ml i n complete medium. 100 u l (2 x 10^) of these c e l l s were dispensed into the wells of multi-dish microculture plates. 50 u l of antisera plus 50 u l of 1:2 d i l u t i o n of rabbit complement was then added to each w e l l . Appropriate controls of complement and medium only were run concurrently. They were then incubated for 90 minutes at room temperature. 0.10 ml of the supernatant was removed, and i t s r a d i o a c t i v i t y was measured.on a gamma counter. In vivo e f f e c t s of anti-P815-SF antisera. The i n vivo experiments of anti-P815-SF antisera were ca r r i e d out to determine t h e i r e f f e c t on P815 tumor growth i n DBA/2 mice. Two days p r i o r to i n j e c t i o n of P815 tumor c e l l s 50 u l of a n t i - P815-SF ant i s e r a , anti-Balentl-"SF" a n t i s e r a , or PBS were i n - 3 jected i . v . into DBA/2 mice. Two-days l a t e r 2 .x 10. P815 tumor 67 c e l l s were injected s.c. into these mice. Tumor growth was followed by two dimensional measurements with c a l i p e r s . There were 7 mice/ groups, and these experiments were c a r r i e d out on three separate occasions. Therefore, a t o t a l of 21 mice i n each of the four groups. The combined r e s u l t s are reported here. Analysis of Data. On a l l occasions, experiments were repeated a minimum of three times. Within each experiment, a l l treatment groups were tested f o r c y t o t o x i c i t y i n quadruplicate over an e f f e c t o r : t a r g e t r a t i o range of 100:1 to 12.5:1 i n the ^ C r release assay. Q u a n t i f i c a t i o n of cyto- t o x i c i t y i n l y t i c units was calculated as described previously (Chapters II and III) and t o t a l l y t i c units from recovered c e l l s a f t e r i n v i t r o culture i s recorded i n the fig u r e or table legends. S t a t i s t i - c a l analysis of data from repeated experiments was done by Students' t - t e s t . These r e s u l t s are shown i n tables where appropriate. 68 Results Since i t had been found (Chapter 111,13,125) that i n t r a p e r i t o n e a l i n - j e c t i o n of s o l u b i l i z e d tumor membrane antigens induce, i n the spleen, T suppressor c e l l s s p e c i f i c f o r that tumor, an attempt was made to prepare from these c e l l s r e l a t i v e l y pure s p e c i f i c suppressor f a c t o r . The r e l a t i v e p u r i f i c a t i o n of the P 8 1 5 - s p e c i f i c suppressor f a c t o r was achieved by passing crude spleen e x t r a c t s , from DBA/2J mice p r e v i o u s l y i n j e c t e d i n t r a p e r i t o n e a l l y w i t h s o l u b i l i z e d P815 tumor e x t r a c t , over immunoadsorbent columns made up of membrane e x t r a c t s of P815 c e l l s , and subsequently e l u t i n g from the column the P815- s p e c i f i c suppressor f a c t o r w i t h 2M NaCl. Figure 16a shows,, that when added to i n v i t r o c u l t u r e s , the P815-specifIc suppressor f a c t o r s i g n i f i c a n t l y i n h i b i t s the generation of c y t o t o x i c c e l l s to P815 tumor c e l l s when.compared to untreated c o n t r o l c u l t u r e s . The s p e c i f i c i t y of the P 8 1 5 - s p e c i f i c suppressor f a c t o r i s shown by i t s i n a b i l i t y to a f f e c t the generation of c y t o - t o x i c c e l l s to L1210, another tumor syngeneic to DBA/2J mice ( F i g . 16b) . The P 8 1 5 - s p e c i f i c suppressor f a c t o r was found to be r e l a t i v e l y s t a b l e when stored at -70°C, and was a c t i v e at very high d i l u t i o n s . Figure 17 shows a t y p i c a l t i t r a t i o n curve f o r t h i s suppressor f a c t o r . While the suppressor f a c t o r was very i n h i b i t o r y at high d i l u t i o n s , at high concentrations i t was not, i n f a c t sometimes i t was some- what s t i m u l a t o r y . The reason f o r t h i s i s not understood, but t h i s was a r e p r o d u c i b l e observation. The t i t r a t i o n curve (Figure 17) may p o s s i b l y be accounted f o r by the presence of P 8 1 5 - s p e c i f i c helper 69 Figure 16. The a b i l i t y of P 815-speeific suppressor f a c t o r to suppress the i n v i t r o primary c y t o t o x i c response of normal DBA/2J splenocytes to syngeneic mitomycin C t r e a t e d P815 tumor c e l l s . ,(a) Anti-P815: the c y t o t o x i c a c t i v i t y of c e l l s primed i n v i t r o w i t h P815 c e l l s on Cr l a b e l l e d P815 c e l l s i n an 18 h assay. (b) Anti-L1210: the c y t o t o x i c a c t i v i t y of c e l l s primed i n v i t r o w i t h L1210 c e l l s on Cr l a b e l l e d L1210 c e l l s i n an 18 h assay. • •, primary c u l t u r e of normal DBA/2J splenocytes and 1/150 d i l u t i o n (0.0071. spleen equivalent) of P 8 1 5 - s p e c i f i c suppressor f a c t o r . T o t a l l y t i c u n i t s : (a) 7.35 (b) 29.30. • • , primary c u l t u r e of normal DBA/2J splenocytes, no suppressor f a c t o r added. T o t a l l y t i c u n i t s (a) 31.32 (b) 27.17 70 - 4 0 \- 1:150 1:600 1:1200 D I L U T I O N Figure 17. T i t r a t i o n of immunoadsorbent p u r i f i e d P815-specific sup- pressor factor. Cytotoxic values were calculated at an eff e c t o r : t a r g e t r a t i o of 50:1 and converted to % sup- pression by comparison with equivalent cultures incubated i n the presence of a control suppressor factor i s o l a t e d i n an analogous fashion from L1210 primed mice. 71 factor which could co-purify with the suppressor material. However, other i n v e s t i g a t o r s , working with suppressor factor producing h y b r i - domas have also found that high concentrations of hybridoma super- natants are sometimes much less suppressive than are higher d i l u t i o n s (143), so t h i s may not be the explanation. While the methods used for the p u r i f i c a t i o n of the P815-specific suppressor factor were c a r r i e d out to maximize the purity of the f a c t o r , i t i s quite probable that the material eluted from P815 columns contained a number of contaminants. Because of the exceedingly small amounts of protein i n our factor preparations (<0.01 absorbance at 280 nm), at t h i s time i t i s d i f f i c u l t to e s t a b l i s h i t s degree of p u r i t y by conventional biochemical means. However, i t appears that t h i s material has properties analogous to those described previously (Chapter 11,65,123) for a suppressor factor i s o l a t e d by s i m i l a r methods from thymocytes of P815-bearing mice (Chapter 11,65). Taken together, these r e s u l t s show that a very i n h i b i t o r y and highly s p e c i f i c suppressor factor can be prepared from spleens of DBA/2J mice previously injected i n t r a p e r i t o n e a l l y with soluble tumor membrane extracts. Since we had a r e l a t i v e l y pure, s p e c i f i c suppressor f a c t o r , a program was set up to immunize groups of both syngeneic DBA/2J and allogeneic C57BL/6 mice with P815-specific suppressor f a c t o r . The hope was that i n the syngeneic mice the u n i q u e d e t e r m i n a n t s ( i d i o t y p i c ? ) , found i n or around the antigen binding s i t e of the P815- s p e c i f i c suppressor f a c t o r , would e l i c i t an antibody response i n at l e a s t some of the animals. While both these and the c o n s t a n t 72 determinants on the suppressor factor might induce an antibody response i n the allogeneic mice. Control groups of mice were injected with material (Balentl-"SF") prepared i n an i d e n t i c a l fashion ( i . e . f i n a l d i l u t i o n o f f of P815 membrane columns) from DBA/2J mice, inje c t e d i n t r a p e r i t o n e a l l y with B a l e n t l tumor extracts. A l l the r e s u l t s reported here are from antisera pooled within each of the four groups. The fact that the DBA/2J and C57BL/6 antisera thus prepared contained anti-P815-SF a c t i v i t y was shown by t h e i r a b i l i t y to absorb out the i n h i b i t o r y a c t i v i t y of the P815-specific suppressor material. A representative set of data i s shown i n Table I I . P815-specific suppressor factor was mixed with either the anti-P815-SF antisera or anti-Balentl-"SF" antisera, and subsequently absorbed on immuno- adsorbent columns containing anti-mouse immunoglobulin. The unattached eluted material was then tested for suppressive a c t i v i t y . While suppressive a c t i v i t y was s t i l l present i n the con t r o l a n t i - B a l e n t l - "SF" a n t i s e r a treated eluates, the suppressive material treated with anti-P815-SF antisera l o s t i t s a c t i v i t y , thus showing that both DBA/2J anti-P815-SF antisera and C57BL/56 anti-P815-SF antisera recognize and can bind determinants expressed on the P815-specific suppressor f a c t o r . This experiment was run on three separate occasions and, the diff e r e n c e s were a l l s i m i l a r and s i g n i f i c a n t i n each case. The a b i l i t y of the anti-P815-SF a n t i s e r a and not the c o n t r o l anti-Balentl-"SF" a n t i s e r a to bind to determinants expressed on P815-SF i s shown using a d i f f e r e n t method i n Figure 18. The ELISA assay was used to show that both.the DBA/2 and the C57BL/6 a n t i - P815-SF an t i s e r a reacted p o s i t i v e l y with P815-SF, while neither of Table I I . A n t i - P 8 1 5 a n t i s e r a i s ab le to r e a c t and b ind to P 8 1 5 - s p e c i f i c suppressor f a c t o r . Suppressor f a c t o r p reparat ions were t e s t e d a f t e r r e a c t i o n w i t h v a r i o u s mouse a n t i s e r a and a b s o r p t i o n on i n s o l u b i l i z e d r a b b i t ant i -mouse I g . P 8 1 5 - S p e c i f i c a SF added P 8 1 5 - S p e c i f i c S F b Absorbed With . . % C y t o t o x i c i t y , 100:1 5 0 : 1 T o t a l L y t i c 6 U n i t s P f + DBA a n t i - P 8 1 5 - S F 53.4± 3 . 1 33 .8 ± 2 .6 36.4 N .S . + DBA a n t i - B a l e n t l - " S F " 43.0± 3 . 1 23 .5 ± 0 .9 2 3 . 1 <.005 + C57BL/6 a n t i - P 8 1 5 - S F 74.8 ± 5 .8 35 .3 ± 2 .2 39.7 N .S . + C57BL/6 a n t i - B a l e n t l - " S F " 49 .1± 1.9 20.9 ± 1 . 1 25.8 <.005 + - 4 1 . 1 ± 3 . 9 17 .5 ± 1 . 4 21.7 <.005 — — 56.9 ± 0 . 9 36.7 ± 1 . 3 39.6 lSF added at the s t a r t of 5 day i n v i t r o c u l t u r e s fo r the generat ion of c y t o t o x i c c e l l s as d e s c r i b e d i n M a t e r i a l s and Methods. SF was t r e a t e d as o u t l i n e d i n next column p r i o r to a d d i t i o n . A 1/150 d i l u t i o n (0 .0071 sp leen e q u i v a l e n t ) of SF was used. 'SF was mixed w i t h the a n t i s e r a then passed through an ant i -mouse Ig column. M a t e r i a l which passed through was t e s t e d f o r suppress i ve a b i l i t y . C o n t r o l SF p repara t ions were on ly passed through ant i -mouse Ig column then t e s t e d . C y t o t o x i c i t y was t e s t e d by 18 h ~^Cr r e l e a s e assay . Numbers are % s p e c i f i c r e l e a s e ± S . E . M . E f f e c t o r : T a r g e t r a t i o . 4 One l y t i c u n i t was d e f i n e d as the number of e f f e c t o r c e l l s r e q u i r e d to l y s e 50% of 10 t a r g e t c e l l s . T o t a l l y t i c u n i t s were the number of l y t i c u n i t s found i n the recovered c e l l p o p u l a t i o n . Data were ana lysed by S t u d e n t s ' t - t e s t . N .S . - not s i g n i f i c a n t . P va lues of <.05 were cons idered s i g n i f i c a n t . Table I I I . Results of Students' t - t e s t run on 3 separate experiments testing 3 i n d i v i d u a l bleeds of mice immunized with P815-SF or control B a l e n t l - "SF'^ Differences between groups were calculated from data obtained i n Cr release assay at effector-target r a t i o s of 50:1 (levels at which k i l l i n g i s i n the l i n e a r part of the curve). A l l groups were compared to the k i l l i n g i n cultures containing suppressor c e l l s which had been treated only with C. Treatment of Suppressor C e l l s P a Expt. 1 Expt. 2 Expt. 3 DBA anti-P815-SF <.025 <.005 <.005 DBA anti-Balentl-"SF" N.S. N.S. N.S. C57BL/6 anti-P815-SF <.05 <.05 <.005 C57BL/6 anti-Balentl-"SF" ' <.025 N.S. N.S. P values of <.05 are considered s i g n i f i c a n t . 75 o J3 0 M < 0.8 0.6 0.4 0.2 '/10 V40 Vo40 A n t i s e r a D i l u t i o n Figure 18. C h a r a c t e r i z a t i o n of P815-SF by ELISA assay. P815-SF (0.0023 spleen equivalent) was used as antigen i n the ELISA. Various a n t i s e r a were added to determine what c h a r a c t e r i s t i c s were expressed on the P815-SF. • - — • , DBA/2 anti-P815-SF; o o, DBA/2 a n t i - B a l e n t l - " S F " ; • •, C57BL/6'anti-P815- SF, • •, C57BL/6 a n t i - B a l e n t l - " S F " , k 4, anti-H-2 ; A A, a n t i - l a ; x x, anti-MIg. Incubation of p l a t e a f t e r a d d i t i o n of enzyme substrate was f o r 45 minutes. 76 the anti-Balentl-"SF" antisera reacted above background l e v e l s . These r e s u l t s confirm the r e s u l t s of the absorbtion experiments (Table I I ) . Another important feature to note i s which of the other antisera preparations reacts with the P815-SF. While both anti-HZ^ and anti-la** a n t isera react with P815-SF, anti-mouse Ig antisera does not. This shows that P815-SF has determinants coded by the l a region of the H-2 major h i s t o c o m p a t i b i l i t y complex, but does not have "common" mouse Ig constant region determinants. These properties are the same as those described previously for P815-specific sup- pressor factor i s o l a t e d from thymocytes of P815 bearing mice (Chapter I I , 65,21). The p o s s i b i l i t y that the a c t i v i t y of the P815-specific T suppressor c e l l s could be abrogated by anti-P815-SF a n t i s e r a plus complement was investigated. P r i o r to addition to normal DBA/2J spleen c e l l s plus mitomycin C treated P815 c e l l s at the s t a r t of i n v i t r o culture, the s p e c i f i c T suppressor c e l l population was treated with complement plus either anti-P815-SF a n t i s e r a or a n t i - B a l e n t l - "SF" a n t i s e r a . Both of the anti-P815-SF a n t i s e r a produced i n the syngeneic and allogeneic mice were e f f e c t i v e i n eliminating the sup- pressive e f f e c t normally produced by these c e l l s (Fig. 19). Again, as with the adsorption of the P815-specific suppressor f a c t o r , neither of the anti-Balentl-"SF" antisera controls had an e f f e c t . With a l l the bleeds tested the DBA/2J anti-P815-SF a n t i s e r a and the C57BL/6 anti-P815-SF antisera were approximately equally e f f e c t i v e (Table I I I ) . The data presented i n Figure 19 i s from experiment 2. Therefore, i t appears that i n t h i s system the suppressor c e l l , i t s progenator, 77 1? 5 1 25:1 50-1 12.5:1 251 50:1 E F F E C T O R : T A R G E T R A T I O •Figure 19. The a b i l i t y of anti-P815-SF a n t i s e r a p l u s complement to e l i m i n a t e P 8 1 5 - s p e c i f i c suppressor c e l l s . (a) DBA/2J a n t i - S F : - the c y t o t o x i c response of c e l l s primed i n v i t r o w i t h P815 a f t e r treatment of the suppress- i v e sp lenocytes w i t h DBA/2 a n t i - S F a n t i s e r a p l u s complement. (b) C57BL/6 a n t i - S F : - the c y t o t o x i c response of c e l l s primed i n v i t r o w i t h P815 a f t e r treatment of the suppress- i v e sp lenocytes w i t h C57BL/6 a n t i - S F a n t i s e r a p l u s comple- ment . • ^ , pr imary c u l t u r e of normal DBA/2J sp lenocytes p l u s a n t i - P 8 1 5 - S F and complement t r e a t e d P 8 1 5 - s p e c i f i c suppressor s p l e n o c y t e s . T o t a l l y t i c u n i t s (a) 101.02 (b) 90.13 • • , pr imary c u l t u r e of normal DBA/2J sp lenocytes p l u s a n t i - B a l e n t l - " S F " and complement t r e a t e d P815 s p e c i f i c suppressor s p l e n o c y t e s . T o t a l l y t i c u n i t s (a) 63.19 (b) 52.25 0 0 , pr imary c u l t u r e of normal DBA/2J sp lenocytes p l u s complement only t r e a t e d P 8 1 5 - s p c i f i c suppressor s p l e n o - c y t e s . T o t a l l y t i c u n i t s (a) and (b) 57 .54. 78 or a c e l l v i t a l for i t s development, expresses on i t s surface, sup- pressor f a c t o r , or at l e a s t determinants common to i t . The p o s s i b i l i t y that i n v i t r o generated syngeneic c e l l s cyto- toxic for P815 could also be k i l l e d by these a n t i s e r a was also i n - vestigated. In v i t r o generated cytotoxic T c e l l s s p e c i f i c for either P815 or L1210 were treated with complement plus either anti-P815-SF antisera or anti-Balentl-"SF" antisera. As can be seen i n Table IV, neither the syngeneic DBA/2J anti-P815-SF antisera nor the allogeneic C57BL/6 anti-P815-SF antisera had any e f f e c t on the k i l l i n g c a r r i e d out by the cytotoxic T c e l l s . Therefore, showing that i n the syngeneic P815 tumor system of DBA/2J mice the s p e c i f i c cytotoxic T c e l l s lack determinants i n common with those found on ei t h e r the s p e c i f i c T suppressor c e l l s or the s p e c i f i c suppressor factor as detected by the anti-SF antisera. In repeats of these experiments, no s i g n i f i c a n t d i f f e r e n c e s were found between groups on any occasions. F i n a l l y the i n vivo e f f e c t of the anti-P815-SF antisera on P815 tumor growth i n DBA/2 mice was assessed. Two days p r i o r to i n j e c t i o n with P815 tumor c e l l s , DBA/2 mice were injec t e d intravenously with 50 y l of one of the four antisera preparations on PBS. Figure 20 shows the growth of the tumor a f t e r i n j e c t i o n subcutaneously. As can be seen the DBA/2 anti-P815-SF antiserum greatly slowed the growth of the P815 tumor. The other antisera preparations had no e f f e c t on tumor growth. These r e s u l t s are also born-out i n the improved s u r v i v a l time of animals injected with DBA/2 anti-P815-SF antisera (Table V). The mice injected with DBA/2 anti-P815-SF a n t i s e r a had a 48% increased s u r v i v a l time compared to those injected with PBS 79 Table IV. Anti-P815-SF antisera plus complement (C') treatment has no effect on the a b i l i t y of specific cytotoxic effector T c e l l s to lyse the appropriate target. Effectors Generated Cells Treated With b % Cytotoxicity 0 p e Against Antisera C 100:l d 5 0 : l d P815 Anti-Thy 1 + 6.6 ± 3.3 2.5 ± 1.4 <.005 DBA anti-P815-SF + 40.5 ± 2.5 28.5 ± 4.7 N.S. DBA anti-Balentl-"SF" + 36.9 ± 4.7 29.8 ± 3.0 N.S. C57BL/6 anti-P815-SF + 45.9 ± 4.8 32.4 + 3.5 N.S. C57BL/6 anti-Balentl-"SF" + 45.0 ± 4.0 32.2 ± 3.8 N.S. - + 44.0 ± 5.2 31.7 ± 3.1 - - 45.2 ± 3.8 34.9 ± 2.3 2 5 : l d 12.5:l d L1210 Anti-Thy 1 + 9.8 ± 2.7 1.2 ± 0.8 <.005 DBA anti-P815-SF + 40.5 + 3.9 24.1 ± 1.6 N.S. DBA anti-Balentl-"SF" + 42.7 ± 2.3 26.6 ± 2.1 N.S. C57BL/6 anti-P815-SF + 39.2 + 4.1 27.7 ± 2.7 N.S. C57BL/6 anti-Balentl-"SF" + 47.9 ± 3.2 21.5 ± 3.2 N.S. - + 39.2 ± 3.8 27.2 ± 1.0 - 39.8 ± 0.8 26.2 ± 2.2 a E f f e c t o r s were generated in v i t r o by mixing DBA/2J spleen c e l l s with the appropriate mitomycin C treated tumor c e l l s and incubating them for 5 days as described in the Materials and Methods. ^The effector c e l l s were treated with antisera plus complement then washed and tested for their a b i l i t y to lyse the appropriate ^ l c rlabelled targets as described in Materials and Methods. Control effector populations were treated with only complement or media alone. c 51 . Cytotoxicity was tested by 18 h Cr release assay. Numbers are % specific release ± S.E.M. dEffector:Target r a t i o . eData were analysedby Students, t-test. N.S. - not signif i c a n t . In three experiments, no significant differences were found between any of the anti-SF groups. 80 255 S IZE O F 195 T U M O R 135 75 - 2 0 11 15 19 21 / \ ant isera iv 2 M 0 3 D A Y S P815 sc Figure 20. E f f e c t of anti-P815-SF antisera i n vivo on P815 tumor growth^in syngeneic DBA/2 mice. Mice were injected with 2 x .10 P815 subcutaneously on day 0. Two days p r i o r 50 y l of antisera had been injected intraveneously. Tumor si z e was measured i n two dimensions with c a l i p e r s . This figure shows the combined r e s u l t s of three separate experiments with a t o t a l of 21 mice/group. A A, DBA/2 anti-P815-SF; • •, C57BL/6 anti-P815-SF; A A, DBA/2 anti-Balentl-"SF"; • • , C57BL/6 anti-Balentl-"SF"; • • , PBS. 81 Table V. E f f e c t of anti-P815-SF an t i s e r a i n vivo on s u r v i v a l time of DBA/2 mice af t e r P815 . . . . . a tumor i n j e c t i o n . DBA/2 mice injected i . v . with 10 mean s u r v i v a l time (days) c 20 30 40 DBA/2 anti-P815-SF anitsera DBA/2 anti-Balentl-"SF" antisera C57BL/6 anti-P815-SF antisera C57BL/6 anti-Balentl-"SF" a n t i s e r a PBS 38.1 ± 4.4 p<.005 29.0 ± 2.3 N.S. 27.2 ± 3.5 N.S. 29.5 ± 2.4 N.S. 25.7 ± 2.5 *Three separate experiments were run and the r e s u l t s were combined for t h i s table. "T)BA/2 mice were injected i . v . wij^h 50 u l of antisera two days p r i o r to P815 tumor challenge. 2 x 10 P815 tumor c e l l s were injected s.c. on day zero. "The mean s u r v i v a l time o f ^ i n d i v i d u a l groups (21 mice/group) of mice a f t e r i n j e c t i o n of 2 x 10 P815 tumor c e l l s s.c. 82 alone. Therefore, even though both DBA anti-P815-SF a n t i s e r a and C57BL/6 anti-P815-SF antisera were reactive i n v i t r o only the syngeneic DBA/2 preparation shows any e f f e c t i n vivo. Experiments were also c a r r i e d out i n vivo with the anfc.i-P815-SF antisera to assess i t s e f f e c t on L1210 tumor growth i n DBA/2 mice. The r e s u l t s shown i n Figure 21 and Table VI show that neither the syngeneic DBA/2 anti-P815-SF nor the C57BL/6 anti-P815-SF a n t i s e r a had a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on L1210 tumor growth i n vivo. Since the preparation of the P815-SF used to induce the a r i t i - P815-SF an t i s e r a e n t a i l e d a f i n a l e l u t i o n step o f f a P815 membrane: Sepharose column, there i s a p o s s i b i l i t y that the P815-SF material could also contain small amounts of P815 antigen which was shed o f f the column along with the bound suppressor f a c t o r . I t i s there- fore possible that the antisera may contain anti-P815 antigen a c t i v i t y , which could i n t e r f e r e with the i n t e r p r e t a t i o n of some of the r e s u l t s reported here. Two d i f f e r e n t experiments were set up to explore t h i s p o s s i b i l i t y . The f i r s t i s shown i n Figure 22. Here the two anti-P815-SF antisera were tested for th e i r a b i l i t y to lyse "^Cr labeled P815 tumor c e l l s i n the presence of complement. As can be seen neither the DBA/2 anti-P815-SF a n t i s e r a nor the C57BL/6 anti-P815-SF antisera had any anti-P815 l y t i c a b i l i t y . The second experiment, to determine i f the two anti-P815-SF antisera contain any anti-P815 a c t i v i t y , was to react these antisera with P815 membrane extracts i n an ELISA assay. I t can be seen i n Figure 23 that both DBA/2 and C57BL/6 anti-P815-SF antiserum contain 83 - 2 0 8 9 10 11 12 13 antisera iv 2 x 1 0 3 D A Y S j P815sc , ! i Figure 21. E f f e c t of anti-P815-SF a n t i s e r a i n v i v o on L1210 tumor growth^in syngeneic DBA/2 mice. Mice were i n j e c t e d w i t h 2 x 10 L1210 subcutaneously on day 0. Two days p r i o r 50 y l of a n t i s e r a had been i n j e c t e d intraveneously. Tumor s i z e was measured i n two dimensions w i t h c a l i p e r s . This f i g u r e shows the combined r e s u l t s of two separate experiments w i t h a t o t a l of 18 mice/group. A A, DBA/2 anti-P815-SF; •- •, C57BL/6 anti-P815-SF; • -A, DBA/2 a n t i - B a l e n t l - " S F " ; • • , C57BL/6 a n t i - B a l e n t l - " S F " ; • • , PBS. 84 Table VI. E f f e c t of anti-P815-SF an t i s e r a i n vivo om' s u r v i v a l time of DBA/2 mice a f t e r L1210 tumor i n j e c t i o n . *• b DBA/2 mice injected i . v . with DBA/2 anti-P815-SF an t i s e r a DBA/2 anti-Balentl-"SF" antisera C57BL/6 anti-P815-SF antisera C57BL/6 anti-Balentl-"SF" antisera PBS mean s u r v i v a l time (days) 5 10 15 20 17.4 ± 1.9 N.S. 16.7 ± 1.3 N.S. 15.7 + 1.5 N.S. 16.5 ± 2.2 N.S. 16.7 + 2.1 Two separate experiments were run and the r e s u l t s were combined for t h i s table. 'DBA/2 mice were injected i . v . witb^ 50 y l of an t i s e r a two days p r i o r to L1210 tumor challenge. 2 x 10 L1210 tumor c e l l s were injected s.c. on day zero. The mean s u r v i v a l time o f ^ i n d i v i d u a l groups (18 mice/group) of mice a f t e r i n j e c t i o n of 2 x 10 L1210 tumor c e l l s s.c. 85 80 6 0 4 0 20 Vt J/64 '/256 D i l u t i o n of A n t i s e r a Figure 22. The i n a b i l i t y of anti-P^15-SF an t i s e r a , i n the presence g£ complement, to l y s e Cr labeled P815 tumor c e l l s . Cr labeled P815 tumor c e l l s were incubated with various d i l u t i o n s of an t i s e r a plus complement anti-P815-SF; o H-2 ; • •, a n t i - l a o,dC57BL/6 anti-P815-SF; DBA/2 Hi, a n t i - 86 '4 '/20 '/80 '/320 '̂280 I I Dilution of Antisera Figure 23. Does anti-SF antisera have any a c t i v i t y d i r e c t e d towards P815 tumor membrane determinants? Crude P815 membrane extract was used as antigen i n the ELISA assay to determine i f anti-SF antisera has any a c t i v i t y directed agasint P815. • • , DBA/2 anti-P815-SF, 0 o, C57BL/6 a n t i - P815-SF, • •, rabbit anti-P815; P — r a b b i t anti-DBA/2. Incubation of plates a f t e r addition of enzyme substrate was for 90 minutes. only a very small amount of anti-P815 r e a c t i v i t y . It should be noted that the ELISA assay i s a very s e n s i t i v e method of detecting antibody r e a c t i v i t y , with only very small amounts (as l i t t l e as 50 ng/ml) of s p e c i f i c antibody necessary to show a p o s i t i v e reaction i n a very short time (142). Therefore the amount of anti-P815 r e a c t i v i t y found i n the anti-P815-SF antisera preparations i s a very small percentage of the t o t a l r e a c t i v i t y and probably plays no ro l e i n any of the experiments reported here, e s p e c i a l l y the ones mediated through comple- ment . 88 Discussion The r e s u l t s presented previously (Chapter II) show that i n t r a p e r i - toneal i n j e c t i o n of s o l u b i l i z e d P815 tumor membrane antigen into syn- geneic DBA/2J mice induces, i n the spleen, T suppressor c e l l s s p e c i f i c for P815. I t was shown i n t h i s chapter that a very i n h i b i t o r y and r e l a t i v e l y p u r i f i e d P815-specific suppressor factor could be prepared from these suppressive spleen c e l l s . This P815-specific suppressor factor was injected into syngeneic DBA/2J and allogeneic C57BL/6 mice and the r e s u l t i n g a n tisera was shown to (a) i n t e r a c t with P815- s p e c i f i c suppressor factor by absorption studies and ELISA assays, (b) i n t e r a c t with P815-specific T suppressor c e l l s by complement mediated k i l l i n g studies, and (c) not to i n t e r a c t with P815-specific T cytotoxic c e l l s by complement mediated k i l l i n g studies, while (d) only the syngeneic antisera was shown to be e f f e c t i v e i n vivo. The r e s u l t s shown i n Figure 16 ind i c a t e that a suppressor factor extracted from a suppressive c e l l population can duplicate the b i o l - o g i c a l function of these c e l l s . In t h i s case both the T suppressor c e l l and the suppressor factor presumed derived from i t are able to s p e c i f i c a l l y suppress the primary i n v i t r o generation of cytotoxic T c e l l s to P815 (8, Chapter I I ) . These r e s u l t s plus those i n Figure 18 show that t h i s suppressor factor has properties analogous to those described previously for a suppressor factor i s o l a t e d by s i m i l a r methods from thymocytes of P815-bearing DBA/2 mice (9,21,65,123, Chapter I I ) . It was then possible to use the P815-suppressor factor to prepare anti-P815-SF antisera. This was done by i n j e c t i n g both syngeneic 89 DBA/2 and allogeneic C57BL/6 mice with P815-SF. As Table II and Figures 18 and 19 show the r e s u l t i n g anti-P815-SF antisera could react with determinants expressed on the suppressor factor and the suppressor c e l l i t i s presumed derived from. The r e s u l t s shown i n Table IV indicate that determinants expressed on P815 s p e c i f i c suppressor factor and P815-specific T suppressor c e l l s are not ex- pressed on P815-specific T cytotoxic c e l l s . Since the preparation of the P815-SF used to induce the a n t i - P815-SF antisera entailed a f i n a l e l u t i o n step o f f of a P815 membrane column, there i s a p o s s i b i l i t y that the P815-SF material could also contain small amounts of P815 antigen which was shed o f f the column along with the bound suppressor factor. I t i s therefore possible that the antisera possibly contained anti-P815 antigen a c t i v i t y , which could i n t e r f e r e with the i n t e r p r e t a t i o n of some of the r e s u l t s reported here. The r e s u l t s shown i n Figure 22 and 23 show that the amount of anti-P815 r e a c t i v i t y found i n the anti-P815-SF antisera i s at most a very small percentage of the t o t a l r e a c t i v i t y and probably plays no r o l e i n any of the experiments reported here, e s p e c i a l l y the ones mediated through complement. And of course, since the B a l e n t l - "SF" was prepared i n an i d e n t i c a l fashion to P815-SF ( i . e . f i n a l e l u t i o n o f f of a P815 membrane column), the antisera prepared to i t serves as an excellent i n t e r n a l c o n t r o l for these experiments. I t should be restated here that at the concentration used i n t h i s work there was no detectable anti-normal DBA/2J a c t i v i t y found i n any of the antisera preparations. Also i t should be noted that the suppressor factor was prepared from DBA/2J c e l l s primed i n vivo, and during 90 preparation and p r i o r to immunization into the appropriate syngeneic and allogeneic mice, was only suspended i n PBS without f e t a l c a l f serum. Therefore, i t i s reasonable to assume that any antibodies being formed i n immunized animals would be directed e x c l u s i v e l y to antigens of DBA/2J o r i g i n . Thus, i t seems probable, that the a n t i - P815-SF antisera from the syngeneic DBA/2J mice i s directed to unique determinants ( i d i o t y p i c ? ) expressed at or near the antigen binding s i t e of the suppressor factor. The anti-P815-SF antisera from the allogeneic C57BL/6 mice would probably be dir e c t e d to both receptor s i t e determinants as well as the constant determinants. Similar conclusions were drawn by others using very s i m i l a r protocols (26, 144-146). The observation that these antisera do not k i l l anti-P815 cyto- toxic T c e l l s but do k i l l P815-specific suppressor T c e l l s , implies that these two c e l l types bear unrelated determinants. As mentioned i n the introduction, i t i s very possible that the s p e c i f i c suppressor factors may be the receptors of s p e c i f i c suppressor T c e l l s . There- fore, since the antisera used contained anti-SF a c t i v i t y (Table II and Figure 18), i t i s possible that the determinants recognized on the suppressor T c e l l s are associated with the receptor molecules of these c e l l s . The fa c t that these antisera did not k i l l a n t i - P815 cytotoxic T c e l l s implies the p o s s i b i l i t y that these two P815- s p e c i f i c T c e l l types may bear unrelated receptor determinants at l e a s t at the antigen binding s i t e . A l t e r n a t i v e l y , the s e n s i t i v i t y of T cytotoxic c e l l s to the a n t i - sera plus complement treatment may r e f l e c t a lower density or d i f - 91 ferent arrangement of receptors on t h e i r surface when compared to T suppressor c e l l s . I t i s becoming very apparent that d i f f e r e n t i a l a c t i v a t i o n of the various T c e l l subsets can be accomplished by varying the mode of immunization (30,125,147-152). I t also seems there may be deter- minants which s e l e c t i v e l y react with T helper c e l l s and other deter- minants which react with T suppressor•cells. This can be demonstrated most d e f i n i t i v e l y with small chemically defined molecules i n which various fragments of the whole molecule have been shown to s e l e c t i v e l y a c t i v a t e either T suppressor c e l l s or T helper c e l l s i n c e r t a i n s t r a i n s of mice (20,153,157). Furthermore, Yamauchi et a l . , i n experiments in v o l v i n g varying immunization protocols and blocking studies, have very recently shown that i n one tumor system T sup- pressor c e l l s and T cytotoxic c e l l s recognize d i f f e r e n t antigeneic determinants (125). In t h i s chapter, these findings are extended by showng that antisera, from both syngeneic DBA/2J and allogeneic C57BL/6 mice, directed against P815-specific suppressor factor reacts with P815-specific T suppressor c e l l s but not s p e c i f i c T cytotoxic c e l l s to t h i s same tumor. F i n a l l y the in. vivo e f f e c t of the anti-P815-SF antisera on P815 tumor growth i n DBA/2 mice was assessed. As shown i n Figure 20 and Table V only the DBA/2 antisera had any observable e f f e c t i n vivo. This i s i n contrast to the i n v i t r o work which showed that both the syngeneic DBA/2 anti-P815-SF and the allogeneic C57BL/6 a n t i - P815-SF an t i s e r a reacted with suppressor f a c t o r / c e l l determinants. The reason.for t h i s i s unknown. Although i t may be rela t e d to the 92 fact that the allogeneic C57BL/6 anti-P815-SF antisera would l i k e l y react with many more determinants present on P815-SF and P815-specific suppressor T c e l l s , than would the syngeneic DBA/2 anti-P815-SF ant i s e r a . Therefore, the allogeneic anti-P815-SF a n t i s e r a may have much more complex a c t i v i t y i n vivo than the syngeneic antisera. For example, the allogeneic anti-P815-SF antisera could possibly stimulate P815-specific suppressor T c e l l induction as well as i n h i b i t i n g i t i n vivo. Thus, e f f e c t i v e l y " c a n c e l l i n g out" i t s e f f e c t i n vivo. CHAPTER V SUMMARY DISCUSSION 94 CHAPTER V Summary Discussion The work reported i n the preceeding three chapters involved studies of DBA/2 antig e n - s p e c i f i c suppressor T c e l l s and the antigen- s p e c i f i c suppressor factor derived from them. Both the suppressor c e l l s and the suppressor factors s p e c i f i c a l l y i n h i b i t the i n v i t r o generation of DBA/2 cytotoxic T c e l l s for the syngeneic tumor, P815. In Chapter II P815-T g and P815-SF were obtained from the thymuses of DBA/2 mice primed previously with P815 tumor c e l l s subcutaneously. The experiments reported i n that chapter showed that the P815-T g and -SF both expressed Ia^ determinants and were not H-2 r e s t r i c t e d i n t h e i r a b i l i t y to e f f e c t i v e l y suppress the i n v i t r o response to P815 by r a d i a t i o n chimeras of a d i f f e r e n t H-2 haplotype. The P815- T g used i n experiments reported i n Chapter I I I were prepared from the spleens of DBA/2 mice injected i n t r a p e r i t o n e a l ^ with membrane fragments of the P815 tumor c e l l . Results i n that chapter showed among other things that the DBA/2 P815-T g expressed the c e l l surface + - - + phenotype of Lyt-1 2 , whereas the DBA/2 P815-T G was Lyt-1 2 . Chapter IV used P815-T e prepared as i n Chapter I I I . P815-SF was then prepared from these suppressor T c e l l s . Syngeneic DBA/2 and a l l o - geneic C57BL/6 antisera was prepared against the P815-SF. Both of the anti-P815-SF antisera reacted i n v i t r o with determinants expressed on the P815-SF and the P815-T , but did not react with the P815-T s' c generated i n v i t r o . Only the DBA/2 anti-P815-SF a n t i s e r a had any observable e f f e c t i n vivo on P815 tumor growth i n DBA/2 mice. 95 The studies of some of the genetic properties of the P815-T s in Chapter II indi c a t e that t h i s suppressor c e l l express l a antigen on i t s surface. I t was also shown that the P815-SF could be absorbed out with anti-la** antisera, and thus also expressed l a coded deter- minants. This l a s t r e s u l t was also shown i n Chapter IV using a d i f f e r e n t method of preparing P815-SF. There the ELISA assay was used to show that P815-SF prepared from DBA/2 splenocytes expressed H-2** and more s p e c i f i c a l l y la** coded determinants. In every system tested to date the antigen-specific T g and the a n t i g e n - s p e c i f i c SF produced from i t have been shown to express l a determinants (59-61). Therefore, these r e s u l t s i n the DBA/2-P815 system are not s u r p r i s i n g and f i t i n n i c e l y with the assumption that a l l a n t i g e n - s p e c i f i c suppressor T c e l l s and t h e i r suppressive factors probably express determinants encoded within the I-J to IE/C region (59-61). Some of the immunogenetic requirements for the expression of P815-specific suppression were looked at i n Chapter I I . The r e s u l t s showed that the P815-T and the P815-SF derived from them, were s capable of suppressing the i n v i t r o generation of c y t o t o x i c i t y to P815 by histoincompatable c e l l s . These r e s u l t s showed that the genes within the MHC do not have to be shared by the suppressor c e l l (or i t s factor) and i t s target. In the a n t i g e n - s p e c i f i c suppressor systems i n which H-2 r e s t r i c t i o n has been addressed, the published r e s u l t s of others are somewhat equivocal. In most systems involving cell-mediated responses there has been no H-2 r e s t r i c t i o n found, at l e a s t for the f i r s t part of the systems a c t i v i t y (see below) (59-61). In one system studied by Moorhead of a a n t i g e n - s p e c i f i c SF which 96 suppresses contact s e n s i t i v i t y to DNP, i t was found that the SF .. required homology at the K and/or D l o c i of the H-2 with the target c e l l s for a c t i v i t y to be observed (79,80,97). The P815-SF c l e a r l y does not need t h i s homology with i t s target to show suppressive a c t i v i t y . The work i n Chapter I I I presents evidence of the Lyt phenotype of the cytotoxic T c e l l and the suppressor T c e l l i n the P815 tumor system i n DBA/2 mice. I t was found that the primary i n v i t r o generated DBA/2 P815-T c were Lyt-1 2 +. As was pointed out i n the discussion i n Chapter I I I , there may be a dif f e r e n c e i n the Lyt-1 antigen expression on primary vs. secondary syngeneic tumor-T c i n the DBA/2-P815 system (13,130), and also between syngeneic-T and a l l o g e n e i c - ^ i n DBA/2 mice (13). Of course these r e s u l t s probably r e f l e c t a purely quantitative not q u a l i t a t i v e d i f f e r e n c e , as a l l T c e l l s probably express Lyt-1 antigens, although i n varying amounts (131). Even though most antigen s p e c i f i c - T g i n other systems have been shown to express the surface phenotype of Lyt-1 2+ (114-118,120), i t i s not s u r p r i s i n g that the DBA/2 P815-T g were shown to be Ly t - l + 2 . This i s because there have been several reports i n a v a r i e t y of systems of ant i g e n - s p e c i f i c T g which are L y t - l + 2 (42-44, 120, 132,133). As was discussed i n Chapter I I I , these c e l l s are usu a l l y found to not be the actual e f f e c t o r c e l l for suppression but may "induce" other T c e l l s to a c t i v e l y suppress (22,23,44,60,134-136). Thus, the L y t - l + 2 , I a + , antigen-binding, H-2 unr e s t r i c t e d T g^ -inducer i s thought to produce a I a + , antigen-binding, H-2 unr e s t r i c t e d SF, 97 which induces a second suppressor T c e l l , This T g 2 i s Lyt-1 2-1", I a + a n t i - i d i o t y p i c , and H-2 r e s t r i c t e d i n a c t i v i t y . I t produces an I a + , a n t i - i d i o t y p i c , H-2 r e s t r i c t e d SF 2, which induces a t h i r d suppressor T c e l l , T g 3 . T g 3 i s Lyt-l~ 2 t Ia +,. i d i o t y p e + , " f i n a l " e f f e c t o r c e l l , which may suppress i n a non-specific fashion, possibly v i a an i n t e r - a ction with macrophage. Thus, i t can be seen that both the P815-T and P815-SF from s the thymus or spleen of DBA/2 mice would f i t n i c e l y into the above mentioned model of suppression at the T s x and SF^ stage. This may at f i r s t seem strange since the P815-T s > which appears a f t e r i n t r a - p e r itoneal i n j e c t i o n of P815 membrane fragments, does so about 4 days sooner than .the P815-T s induced by P815 c e l l s injected subcutaneously. One explanation for t h i s may be that i t takes several days for the antigen load produced by the subcutaneously injected l i v e P815 to equal the amount injected d i r e c t l y v i a the i n t r a p e r i t o n e a l i n j e c t i o n method. Thus the two P815-specific suppressor T c e l l s may be i d e n t i c a l and equivalent to the T -̂ i n other systems. It may be worth noting that 4-6 days a f t e r maximum suppression appears i n the thymus, sup- pression appears i n the spleen and lymph nodes of tumor bearing mice (148). This may be an i n d i c a t i o n of the time i t takes for the thymus P815-T g to "induce" the f u l l suppressor e f f e c t o r function i n vivo. The 5 day culture period of the i n v i t r o assay may also allow the "induction" of suppression through the influence of the Ly t - l + 2 " P815-T . s In the e a r l i e r studies of P815-T and P815-SF i n DBA/2 mice the s suppressor c e l l and suppressor factor were obtained from the thymuses 98 of DBA/2 mice which had been primed previously with P815 tumor c e l l s (123,124, Chapter I I ) . The suppressive a c t i v i t y of these materials was assayed by t h e i r a b i l i t y to s p e c i f i c a l l y suppress the secondary i n v i t r o cytotoxic response of primed DBA/2 splenocytes to mitomycin- C treated P815 c e l l s . In the work reported i n t h i s thesis P815-T s and P815-SF, either prepared as above from thymocytes or from spleno- cytes of DBA/2 mice which had been primed previously with P815 tumor membrane extract, could s p e c i f i c a l l y i n h i b i t the primary i n v i t r o cytotoxic response of normal DBA/2 splenocytes to mitomycin-C treated P815. Therefore, the state (primed or v i r g i n ) of the c e l l population on which i t acts appears to be unimportant for the P815-T g or P815- SF a c t i v i t y . Since the number of P815-T c i s probably higher i n the P815 primed DBA/2 spleen population than the normal DBA/2 spleen population (130), i t would seem that the P815-T s and P815-SF may act on the helper T c e l l population. Thus, i n h i b i t i n g the T^ provided 2nd s i g n a l , which i s probably necessary for the P815-T c to d i f f e r - e ntiate and become active e f f e c t o r s of c y t o t o x i c i t y . While t h i s study presents no evidence supporting t h i s p o s s i b i l i t y , there has been good recent experimental evidence for SF e f f e c t i n g T^ a c t i v i t y d i r e c t l y (158). The r e s u l t s presented i n Chapter IV show that anti-P815-SF an t i s e r a , raised both i n syngeneic DBA/2 and allogeneic C57BL/6 mice, could absorb out the P815-SF. These antisera were also capable, i n the presence of complement, of eliminating P815-T but not P815-T from s s DBA/2 mice. As mentioned i n Chapters I and IV, i t i s l i k e l y that the ahfigen-specxfc SF may be the receptor molecule of antig e n - s p e c i f i c T . Therefore, since the a n t i s e r a used contained anti~SF a c t i v i t y , i t very p o s s i b l e that the determinants recognized on the P815-T s are as s o c i a t e d w i t h the receptor molecules. Thus, the f a c t that the anti-P815-SF a n t i s e r a d i d not k i l l P815-T c i m p l i e s the p o s s i b i l i t y that these two P 8 1 5 - s p e c i f i c T c e l l s may bear d i f f e r e n t receptor determinants, at l e a s t at the antigen b i n d i n g s i t e . 100 LITERATURE CITED 1. Jerne, N.K. (J974) Toward a network theory of the immune system. Ann. Immunol. ( P a r i s ) , 125C:373. 2. Cantor, H. and E.A. Boyse. 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C e l l . Immunol., 56:392 (1980). Maier, T., D.G. K i l b u r n , and J .G. Levy. P r o p e r t i e s of syngeneic and a l l o g e n e i c a n t i s e r a r a i s e d to tumor- s p e c i f i c suppressor f a c t o r from DBA/2 mice. J . . Supramol. S t r u c t . _5:52 (1981). Maier, T., D.G. K i l b u r n , and J .G. Levy. P r o p e r t i e s of syngeneic and a l l o g e n e i c a n t i s e r a r a i s e d to tumor- s p e c i f i c suppressor f a c t o r from DBA/2 mice. Cancer Immunol. Immunotherapy (In P r e s s ) . -M^i-e-Tv-I—ajrd-J-rGv---L-evy-;—-ffi—v-i-vo a c t i v i t y of syn^eneijc ^ i ^ t ^ 4 ^ e f i € 4 c - ^ t - + s - e r u r a i s e d to-UmtQ-p-'Spocifie -s^ppe^m'-^a-ekQ-r—f-mtr-B-BA/2-- mice-;—(^u-bm44ie4— HoJtr, T • L<-*y • A«l-',- K>*M>r t-C-Ctc-W Uc\- t V o ,b-,~T. I^V.e^ ( T . 6-. U.uy ., K>; 6-. H, Ta^zrs, a^tL W i t o-£ v̂ o~> oclow<\ \

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