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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 U n i v e r s i t y o f Washington, 1975  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE  FACULTY OF GRADUATE STUDIES  Department o f M i c r o b i o l o g y  We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA October 1981 ©  Tom Maier  In p r e s e n t i n g  this  thesis  in partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e of B r i t i s h Columbia, I agree that it  freely  the L i b r a r y s h a l l  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 .  agree t h a t p e r m i s s i o n for  University  f o r extensive  s c h o l a r l y p u r p o s e s may  for  financial  shall  I C ^ 0 ia T c?  of  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5 Date  DE-6  (2/79)  b*(  .  V-  ,  ^  y  Columbia  my  It is thesis  n o t be a l l o w e d w i t h o u t my  permission.  Department  thesis  be g r a n t e d by t h e h e a d o f  copying or p u b l i c a t i o n of this  gain  further  copying of t h i s  d e p a r t m e n t 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 . understood that  I  make  written  ii  ABSTRACT The work r e p o r t e d here i n v o l v e s s t u d i e s of suppressor T c e l l s ( T C ) and t h e i r g  suppressor f a c t o r  (SF) which s p e c i f i c a l l y  suppress  the i n v i t r o 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 f o r a syngeneic tumor, P815, i n DBA/2 mice.  T h i s work can be d i v i d e d  immunogenetic p r o p e r t i e s and requirements  i n t o t h r e e s e c t i o n s : a) the of t h i s T C and SF, b) the g  L y t phenotype o f the T C as w e l l as t h a t of the c e l l s g  c y t o t o x i c response  t o the syngeneic tumor, c )  i n v o l v e d i n the  the p r o p e r t i e s o f  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 t o the P815 s p e c i f i c SF. a)  P815-antigen  s p e c i f i c T C and s u p p r e s s i v e e x t r a c t s g  . '  o b t a i n e d from the thymuses of DBA/2 mice b e a r i n g s m a l l syngeneic P815 tumors, were compared f o r t h e i r ments.  immunogenetic p r o p e r t i e s and r e q u i r e -  I t was shown t h a t pretreatment  of T C p o p u l a t i o n s w i t h g  a n t i - l a ^ a n t i s e r u m p l u s r a b b i t complement removed the s u p p r e s s i v e activity.  S i m i l a r l y , a b s o r p t i o n o f the SF w i t h a n t i - l a ^  removed the s u p p r e s s i v e p r o p e r t i e s of the m a t e r i a l . the TjC and SF were c a p a b l e of s p e c i f i c a l l y response  H-2  I t was found  that  s u p p r e s s i n g the anti-P815  of B6D2F^ r a d i a t i o n chimeras p o s s e s s i n g lymphoid  b  antiserum  c e l l s of the  12 or H-2  response  h a p l o t y p e e q u a l l y as w e l l as they c o u l d suppress the  of H-2^ b e a r i n g c e l l s .  T h i s i n d i c a t e s t h a t the T C and SF g  are n o t H-2 r e s t r i c t e d w i t h r e s p e c t t o K or D markers on responder in this b)  system. T C were a l s o i d e n t i f i e d i n the s p l e e n s of DBA/2 mice s  i n t r a p e r i t o n e a l l y w i t h membrane e x t r a c t s of the P815 tumor.  cells  injected  The L y t  phenotypes o f v a r i o u s e f f e c t o r c e l l s was d e t e r m i n e d .  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 t h e syngeneic e f f e c t o r +  +  c e l l s were found t o be p r e d o m i n a n t l y L y t - 1 2 . +  The s u p p r e s s o r 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 t o suppress t h e i n v i t r o c y t o t o x i c a n t i - P 8 1 5 response a f t e r t r e a t m e n t w i t h a n t i - L y t - 1 serum p l u s complement b u t n o t a f t e r t r e a t m e n t w i t h a n t i - L y t - 2 serum, i n d i c a t i n g t h a t an L y t - l 2 +  cell  i s e s s e n t i a l i n t h i s suppression. c)  P815 t u 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  s u p p r e s s i v e s p l e e n 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 t h a t these a n t i s e r a , b u t n o t  t h e i r c o n t r o l s were c a p a b l e o f a b s o r b i n g o u t t h e SF.  The a n t i s e r a were  a l s o c a p a b l e , i n the p r e s e n c e o f complement, o f e l i m i n a t i n g T C from g  s u p p r e s s i v e s p l e e n 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 n o t c a p a b l e o f 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 g e n e r a t e d  killer  c e l l s , i n d i c a t i n g t h a t the r e c e p t o r m o l e c u l e s on s u p p r e s s o r and e f f e c t o r c e l l s i n t h i s system a r e d i s t i n c t from each o t h e r .  Only t h e a n t i s e r a  "raised i n syngeneic DBA/2 mice had any o b s e r v a b l e e f f e c t on P815 tumor growth i n v i v o .  iv  TABLE OF CONTENTS  ABSTRACT  Page i i  LIST OF FIGURES  v  LIST OF TABLES  v  ACKNOWLEDGEMENTS  i  v  CHAPTER I - INTRODUCTION  8  A.  Introduction  B.  M a t e r i a l s and Methods  10  C.  Results  1  8  D.  Discussion  3  3  3  3  A.  Introduction  B.  M a t e r i a l s and Methods  C.  Results  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 A.  Introduction  B.  M a t e r i a l s and Methods  C.  Results  D.  Discussion  l  2  CHAPTER I I - CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIRMENTS OF P815 TUMOR-SPECIFIC SUPPRESSOR T CELLS AND THEIR SOLUBLE FACTOR  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  l  8  43 ^  5  5  ^ 6  8  oo  CHAPTER V - SUMMARY DISCUSSION Literature Cited  94 100  i  l  V  LIST OF FIGURES  Figure 1  2 3  4  5  6.  7  8 9  10  11  Title  Page  The a b i l i t y o f P815 s u p p r e s s o r c e l l s t o suppress the i n v i t r o p r i m a r y c y t o t o x i c response o f normal DBA/2 s p l e n o c y t e s t o s y n g e n e i c m i t o m y c i n C - t r e a t e d P815 tumor c e l l s  19  The a b i l i t y o f a n t i - l a ^ a l l o a n t i s e r u m to e l i m i n a t e P815 s u p p r e s s o r c e l l s  20  plus rabbit C  The a b i l i t y of DBA/2 P815 s u p p r e s s o r c e l l s t o suppress t h e c y t o t o x i c response o f s p l e n o c y t e s of H-2 r a d i a t i o n chimeras t o P815  22  The a b i l i t y o f DBA/2 P815 s u p p r e s s o r c e l l s t o suppress t h e c y t o t o x i c response o f s p l e n o c y t e s from H-2 (A.TH) r a d i a t i o n chimeras t o P815.  23  The a b i l i t y o f t h e s u p p r e s s o r f a c t o r i s o l a t e d from P815 s u p p r e s s o r thymocyte p o p u l a t i o n s t o suppress the i n v i t r o p r i m a r y c y t o t o x i c response of normal DBA/2 s p l e n o c y t e s t o syngeneic mitomycin C - t r e a t e d tumor c e l l s .  24  The e f f e c t o f a n t i - l a a n t i s e r u m a d s o r p t i o n on t h e 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 s u p p r e s s o r thymocyte p o p u l a t i o n s . . .  26  The a b i l i t y o f P815 s u p p r e s s o r f a c t o r t o suppress t h e c y t o t o x i c response o f s p l e n o c y t e s from H~2 r a d i a t i o n chimeras t o P815  27  Flow diagram i l l u s t r a t i n g t h e p r e p a r a t i o n o f P815 tumor-specific suppressor c e l l s . .  40  The a b i l i t y o f s p l e n i c P815 s u p p r e s s o r c e l l s t o suppress the i n v i t r o p r i m a r y c y t o t o x i c response o f normal DBA/2 s p l e n o c y t e s t o syngeneic mitomycin-C t r e a t e d P815 tumor cells  44  The a b i l i t y o f n y l o n wool-passed s p l e n i c P815 s u p p r e s s o r c e l l s t o suppress t h e i r i v i t r o p r i m a r y c y t o t o x i c response of normal DBA/2 s p l e n o c y t e s t o syngeneic mitomycin-C t r e a t e d P815 tumor c e l l s  45  Time c o u r s e o f appearance and d i s a p p e a r a n c e o f a n t i g e n 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 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  vi LIST OF FIGURES Continued Figure 12  13  14  15  16  17  Title  Page  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 DBA/2 a l l o g e n e i c e f f e c t o r c e l l s r a i s e d a g a i n s t C57BL/6, on a EL4 t a r g e t  48  The e f f e c t o f 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 DBA/2 e f f e c t o r c e l l s primed a g a i n s t the syngeneic P815 mastocytoma  49  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  50  Flow diagram i l l u s t r a t i n g t u m o r - s p e c i f i c suppressor  60  the p r e p a r a t i o n o f P815 f a c t o r (P815-SF)  The a b i l i t y of P 8 1 5 - s p e c i f i c suppressor f a c t o r t o suppress the i n v i t r o primary c y t o t o x i c response of normal DBA/2J s p l e n o c y t e s to. syngeneic mitomycin C t r e a t e d P815 tumor c e l l s  69  T i t r a t i o n of immunoadsorbent p u r i f i e d suppressor f a c t o r  70  P815-specific  18  C h a r a c t e r i z a t i o n of P815-SF by ELISA assay  19  The a b i l i t y o f 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  20  21  22  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 P815 tumor growth i n syngeneic DBA/2 mice. E f f e c t o f anti-P815*-SF a n t i s e r a i n v i v o on L1210 tumor growth i n syngeneic DBA/2 mice The i n a b i l i t y of anti-P815-SF a n t i s e r a , i n the presence of complement, t o l y s e C r l a b e l e d P815 tumor c e l l s . ... 5 1  23  Does a n t i - S F a n t i s e r a have any a c t i v i t y d i r e c t e d towards P815 tumor membrane determinants?  7  5  77  .  80  8  3  85  vii  LIST OF TABLES  Table I II  III  IV  V VI  Title 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 Anti-P815 a n t i s e r a  Page 37  i s a b l e t o r e a c t and b i n d to P815-  s p e c i f i c suppressor f a c t o r  73  R e s u l t s o f S t u d e n t s ' t - t e s t r u n on 3 s e p a r a t e 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 b l e e d s o f mice immunized w i t h P815-SF o r c o n t r o l B a l e n t l - " S F "  74  A n t i - P 8 1 5 - S F a n t i s e r a p l u s complement ( C ) treatment has no e f f e c t on t h e a b i l i t y o f 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 lyse the appropriate target  79  E f f e c t o f a n t i - P 8 1 5 - S F a n t i s e r a j m v i v o on s u r v i v a l time o f DBA/2 mice a f t e r P815 tumor i n j e c t i o n  81  E f f e c t o f 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 o f 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 t o Dr. Levy f o r her v a l u a b l e i n s t r u c t i o n s ,  guidance, and enthusiasm throughout the course of my I thank Dr. Doug K i l b u r n f o r h i s very  research.  u s e f u l d i s c u s s i o n s of  t h i s work. I would l i k e  to thank Primrose G o n t i e r  f o r h e l p i n g f i n i s h the  t y p i n g of t h i s t h e s i s . 1 would a l s o l i k e lab  t o extend a s p e c i a l thanks t o everyone i n the  f o r h e l p i n g t o make my stay  i n Vancouver so very  enjoyable.  iX  FOR PE8BI  1  CHAPTER I INTRODUCTION  2  CHAPTER I Introduction It  i s now apparent t h a t the immune response i s the r e s u l t of a  complex s e r i e s o f ongoing i n t e r c o n n e c t i n g events which i s o n l y t i a t e d by a n t i g e n i c s t i m u l a t i o n  (1).  Initially,  ini-  the immune response  was thought o f as a p h y s i o l o g i c a l response o f the body t o a n t i g e n , i n which t h e s o l e end product was a n t i b o d y . of  L a t e r , t h e two main limbs  the immune response were r e c o g n i z e d (humoral and c e l l - m e d i a t e d ) ,  and w i t h i n the past two decades,  the two lymphocyte  m e d i a t i n g these responses were d e f i n e d .  subpopulations  Thus i t was r e a l i z e d  that at.',  l e a s t two types o f i n t e r a c t i o n s o c c u r r e d between a n t i g e n and the two responding c e l l  t y p e s , the T and B lymphocytes.  T h i s was f o l l o w e d  by the r e a l i z a t i o n t h a t the immune response i s r e g u l a t e d v i a T c e l l - B cell  i n t r e a c t i o n s , and a l s o T cell-macrophage, and T c e l l - T c e l l  actions.  I t i s now r e c o g n i z e d t h a t the T lymphocyte  made up o f d i s t i n c t  inter-  population i s  subsets which i n t e r a c t w i t h each o t h e r as w e l l  as B c e l l s and macrophages, to both a m p l i f y 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  e f f e c t s on t h e immune response by way o f h e l p e r T c e l l s  enhancing ( T ) (2-10). u  rl  T c e l l s have a l s o been found t o i n h i b i t suppressor T c e l l s demonstrated  ( T ) (11-44). g  the immune response by way o f  Although some o f these s t u d i e s have  the e x i s t a n c e of n o 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 , the  antigen s p e c i f i c regulatory T c e l l s are expecially i n t r i g u i n g they appear response.  because  t o possess the s p e c i f i c i t y t h a t u s u a l l y marks t h e immune  3  One o f t h e f i r s t o b s e r v a t i o n s o f a n t i g e n - s p e c i f i c T regarding  a n t i b o d y f o r m a t i o n i n r a t s (45-47).  were made  T h i s was q u i c k l y  expanded t o 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 48-58).  g  (il-17,22-44,  From t h i s and o t h e r work some g e n e r a l c h a r a c t e r i s t i c s o f  antigen-specific T  g  or i d i o t y p e b i n d i n g l a determinants.  b e g i n t o emerge. potential.  F i r s t they show s p e c i f i c a n t i g e n  Antigen s p e c i f i c T  T h i s i s u s u a l l y I - J coded.  a l s o express  g  The i n h i b i t o r y a c t i v i t y  of t h e s e c e l l s may or may n o t by g e n e t i c a l l y r e s t r i c t e d t o 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 e x p e r i m e n t a l systems have a  s u r f a c e phenotype o f L y t - 1 of T  having the L y t - l 2 +  g  2"*". However, t h e r e a r e a l s o some r e p o r t s phenotype o r even L y t - l 2 . +  +  I n many o f t h e i n v e s t i g a t i o n s c o n c e r n i n g a n t i g e n - s p e c i f i c  reg-  u l a t o r y T c e l l s , i t has been shown t h a t s o l u b l e f a c t o r s d e r i v e d t h e s e c e l l s can a p p a r e n t l y d u p l i c a t e t h e i r f u n c t i o n (59-61).  from  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 i n d e p e n d a n t l y o f the T c e l l s w h i c h produced them.  T h i s would have t h e e f f e c t o f  i n c r e a s i n g the e f f e c t i v e radius of the r e g u l a t o r y  T c e l l , much i n  the same way t h a t a n t i b o d y f u n c t i o n s w i t h r e s p e c t t o t h e B c e l l . These . f a c t o r s 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 t o induce t h e a c t i v a t i o n o f o t h e r l y m p h o c y t e s , which e v e n t u a l l y to t h e f i n a l n e t e f f e c t on t h e immune r e s p o n s e . studied  leads  T h i s has been b e s t  i n t h e a n t i g e n - s p e c i f i c s u p p r e s s i o n systems ( 6 0 ) .  As i n t h e 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  discovery  of a n t i g e n - s p e c i f i c s u p p r e s s o r f a c t o r (SF) was made by Tada and c o l l e a g u e s (62-64).  Again, t h i s observation  was q u i c k l y broadened t o  many o t h e r e x p e r i m e n t a l systems (47,59-61,65-80).  The a n t i g e n - s p e c i f i c  4  s u p p r e s s o r 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 i s t i c s i n common. T . g  They a r e produced o r e x t r a c t e d  from a n t i g e n primed  They a r e p r o t e i n s w i t h s p e c i f i c a n t i g e n ( o r i d i o t y p e )  potential.  character-  binding  T h e i r m o l e c u l a r w e i g h t i s u s u a l l y between 35-75,000.  l a c k constant region  immunoglobulin d e t e r m i n a n t s b u t may c a r r y  ( i d i o t y p i c ) d e t e r m i n a n t s o f t h e immunoglobulin heavy c h a i n . d e t e r m i n a n t s w h i c h a r e encoded by t h e I r r e g i o n the a n t i g e n - s p e c i f i c T  g  They  variable  They have  ( u s u a l l y I - J ) , and l i k e  they may o r may n o t 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 t o a c t i n g on c e l l s o f t h e same H-2 t y p e .  Finally,  s i n c e T c e l l s would be u n l i k e l y t o e x p r e s s a n t i g e n - s p e c i f i c m o l e c u l e s u n l e s s t h e y were r e 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 o b v i o u s c a n d i d a t e f o r t h e a n t i g e n r e c e p t o r have been p u b l i s h e d one  g  Several  reviews  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  i s d i r e c t e d t h e r e f o r a more comprehensive e x a m i n a t i o n o f a n t i g e n -  s p e c i f i c suppressor f a c t o r s The  (59-61).  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 o u t by T a k e i and c o l l e a g u e s i n  this laboratory  p r o v i d e d p r e l i m i n a r y d a t a on w h i c h t h e work i n t h i s  t h e s i s i s based. P815  of T .  They found t h a t DBA/2 mice i n j e c t e d w i t h  syngeneic  mastocytoma c e l l s 8 days p r e v i o u s l y , c o n t a i n e d i n t h e i r thymus  P815-specific  T  of P 8 1 5 - s p e c i f i c  g  (P815-T ), which could g  i n h i b i t the i n v i t r o  c y t o t o x i c T c e l l s (P815-T ) (11,12). c  d e s i g n f o r d e m o n s t r a t i n g t h i s was as f o l l o w s . cytotoxic T c e l l s involved  generation  The e x p e r i m e n t a l  The i n v i t r o assay f o r  t h e c u l t u r i n g o f "immune" DBA/2 s p l e n o c y t e s  ( t a k e n 12 days f o l l o w i n g subcutaneous i n j e c t i o n o f a s m a l l number o f P815  c e l l s i n t o t h e DBA/2 mice) w i t h mitomycin-C t r e a t e d P815 c e l l s f o r  4 days p r i o r t o 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 i n this system, the generation of cytotoxic T c e l l s was greatly reduced as assessed by the reduction i n ^"*"Cr release mediated by these cultures i n comparison to appropriate.controls.  This suppressive effect was shown to be  antigen-specific. It was l a t e r shown that a P815-specific  suppressor factor  could be isolated from sonicated P815-T "extract (65). replace the P815-T  s  i n the i n v i t r o functional assay.  (P815-SF)  This SF could The P815-SF  was capable of suppressing the i n v i t r o generation of syngeneic c e l l s cytotoxic  for P815 tumor c e 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 i n the range of  40-60,000, with an i s o e l e c t r i c point i n 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 c e l l s and the suppressor factor extracted from them.  Both  the c e l l and factor are P815 s p e c i f i c and obtained from DBA/2 mice primed with P815 tumor c e l l s or P815 membrane fragments.  The function-  a l assay involves the a b i l i t y of either c e l l s or their extracts to s p e c i f i c a l l y i n h i b i t a primary i n v i t r o cytotoxic response of normal DBA/2 splenocytes to P815 tumor c e l l s .  The work i n this report has  been divided into three self-contained chapters for ease of reading.  6  Chapter  I I examines some of the immunogenetic p r o p e r t i e s and r e q u i r e -  ments of the P815-T  .  T h i s work was c a r r i e d out between 6/77 and  s 9/78.  Chapter I I I examines the L y t phenotype of the P815-T  as t h a t of the c e l l s i n v o l v e d P815 tumor. 2/79.  g  as w e l l  i n the c y t o t o x i c response to the syngeneic  T h i s 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  The work r e p o r t e d  i n Chapter IV examines the i n v i t r o  and i n  v i v o e f f e c t s of syngeneic DBA/2 and a l l o g e n e i c C57BL/6 a n t i s e r a r a i s e d to P815-SF.  T h i s work was done between 10/78 and 5/81.  a summary d i s c u s s i o n of the s t u d i e s r e p o r t e d chapters.  Chapter V i s  i n the p r e c e e d i n g  three  7  CHAPTER I I CHARACTERIZATION OF THE H-2 PROPERTIES AND REQUIREMENTS OF P815 TUMOR-SPECIFIC  SUPPRESSOR  T CELLS AND THEIR SOLUBLE FACTOR  8  CHAPTER I I Introduction There have been a number o f r e p o r t s showing t h a t the lymphoid organs  o f tumor-bearing mice c o n t a i n T c e l l s t h a t a r e capable o f  specifically  s u p p r e s s i n g the response o f normal c e l l s t o t h a t tumor  (12,15,36,83-85).  I t has a l s o been shown t h a t , i n some c a s e s , i t i s  possible to i s o l a t e  from such p o p u l a t i o n s of suppressor c e l l s ,  f a c t o r ( s ) a l s o capable o f i n h i b i t i n g of normal lymphoid  cells  the s p e c i f i c  anti-tumor  response  (65,76,86,87).  S t u d i e s on t h e c h a r a c t e r i z a t i o n o f s o l u b l e s p e c i f i c f a c t o r s i n tumor-bearing  soluble  suppressor  (65,76,86) animals have shown t h a t they do  not appear t o d i f f e r  significantly  f a c t o r s demonstrated  i n animals immunized w i t h a v a r i e t y of a n t i g e n i c  stimuli  (59-61,77,81,82,88).  been found  t o have m.w.  from a n t i g e n - s p e c i f i c s u p p r e s s i v e  G e n e r a l l y , s u p p r e s s i v e f a c t o r s have  i n the range o f 30-75,000.  have been shown t o e x h i b i t a n t i g e n s p e c i f i c i t y  Moreover, they  (65,75-78,87) and not  to share a n t i g e n i c s i m i l a r i t i e s w i t h t h e c o n s t a n t r e g i o n s o f immunoglobulin  (65,77,78,87).  S t u d i e s w i t h these f a c t o r s have a l s o shown  t h a t they can be removed by a b s o r p t i o n w i t h a n t i s e r a r a i s e d of  to products  t h e major h i s t o c o m p a t i b i l i t y complex (MHC) e x c l u d i n g the K and the  D regions ( a n t i - l a  serum).  F u r t h e r a n a l y s e s of these a b s o r p t i o n s  have shown t h a t the s u b r e g i o n o f the l a t h a t i s expressed on the s u p p r e s s o r f a c t o r i s encoded by the I - J r e g i o n (77,81,82,86). r e c e n t o b s e r v a t i o n t h a t treatment amounts o f a n t i - I - J  A  o f tumor b e a r i n g animals w i t h s m a l l  a n t i s e r u m caused  a significant  s l o w i n g i n the  growth o f syngeneic tumor (89) i n d i c a t e t h a t e l i m i n a t i o n o f I - J -  9  bearing c e l l s  i n v i v o may  The p r e s e n t study was cells elicited  be of a c l i n i c a l undertaken  i n tumor-bearing  significance.  t o determine whether suppressor  animals were capable of s u p p r e s s i n g  the primary i n v i t r o c y t o t o x i c response o f normal  c e l l s to tumor  a n t i g e n s , and to compare the suppressor c e l l s , thus generated, w i t h the e q u i v a l e n t s o l u b l e f a c t o r s i n terms of l a - b e a r i n g  characteristics  and g e n e t i c r e s t r i c t i o n s r e g a r d i n g t h e i r s u p p r e s s i v e q u a l i t i e s .  M a t e r i a l s and Methods M i c e and  tumors.  Female DBA/2, B 6 D 2 F  1>  B10.D2, BIO and C57BL/6 mice (6 t o  10 weeks o l d ) were o b t a i n e d from the J a c k s o n L a b o r a t o r y (Bar Harbor, Maine).  A.TH  a n i m a l s were o b t a i n e d from the B a n t i n g  and B e s t I n s t i t u t e from Dr. T. D e l o v i t c h and were b r e d i n our own a n i m a l u n i t .  P815 mastocytoma and L1210 l e u k e m i a were  o b t a i n e d from Dr. Bruce Smith ( I n s t i t u t e f o r Cancer R e s e a r c h , P h i l a d e l p h i a , P e n n s y l v a n i a ) and m a i n t a i n e d as an a s c i t e s tumor as d e s c r i b e d p r e v i o u s l y (11,12). C u l t u r e system f o r g e n e r a t i o n of c y t o t o x i c e f f e c t o r  cells.  S i n g l e - c e l l s u s p e n s i o n s were prepared from s p l e e n s o f 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 gauge mesh i n RPMI 1640 I s l a n d , N.Y.)  (Grand I s l a n d B i o l o g i c a l Company, Grand  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  serum (FCS), 10 mM HEPES and 5 x 1 0 ~  5  calf  mM of 2-mercaptoethanol.  Gentamycin was a l s o added t o a f i n a l c o n c e n t r a t i o n of 50 Suspended  60-  c e l l s were washed t h r o u g h 3.0 ml o f FCS,  g/ml.  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 t r y p a n b l u e . Tumor c e l l s were washed t w i c e i n medium b e f o r e r e s u s p e n s i o n i n complete medium f o r c o u n t i n g .  Specific 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^ m i t o m y c i n C - t r e a t e d tumor c e l l s i n L i n b r o 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) a t 37°C i n a h u m i d i f i e d i n c u b a t o r w i t h 5% 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.  C0  2  After  11  5 days, c e l l s were h a r v e s t e d by c e 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 t r y p a n for  b l u e e x c l u s i o n and t e s t e d  cytotoxicity.  Assay f o r suppressor c e l l s and suppressor f a c t o r s . When t h e assay was used f o r measuring suppressor a c t i v i t y , 5 x 10  cell  normal s p l e n i c lymphocytes were incubated w i t h  6 5 x 10 suppressor thymocytes; a c o n t r o l c o n t a i n e d  6 5 x 10 normal  s p l e n i c lymphocytes and 5 x 10^ normal thymocytes.  On a l l o c c a -  s i o n s , experiments were r u n a t l e a s t i n d u p l i c a t e w i t h lymphocytes from i n d i v i d u a l p o o l s o f c e l l s i n o r d e r t o m i n i m i z e e r r o r s i n c u r r e d by i n a c c u r a t e involved  incubation  centrations  counts.  of 10  Assay f o r thymic suppressor f a c t o r  s p l e n i c lymphocytes w i t h v a r i o u s  7  o f t h e f a c t o r under the same c o n d i t i o n s  con-  as s t a t e d  above. When t h e degree o f s u p p r e s s i o n was q u a n t i t a t e d , was t e s t e d a t v a r i o u s  e f f e c t o r to t a r g e t c e l l r a t i o s and the  d e c r e a s e i n t o t a l l y t i c u n i t s was a s s e s s e d . which was d e f i n e d  cytotoxicity  One l y t i c  unit,  as t h e number o f 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  4 50%  o f t h e 10  target c e l l s  by l i n e a r r e g r e s s i o n logarithm  i n 18 hr i n c u b a t i o n ,  was e s t i m a t e d  a n a l y s i s o f percentage o f c y t o t o x i c i t y v s  of e f f e c t o r to target c e l l r a t i o .  were c a l c u l a t e d from c e l l  Total l y t i c  r e c o v e r y i n c u l t u r e s and percentage  of s u p p r e s s i o n was estimated from the d e c r e a s e i n t o t a l units.  units  lytic  No s i g n i f i c a n t d i f f e r e n c e s i n c e l l r e c o v e r y between t e s t  c u l t u r e s and c o n t r o l s were n o r m a l l y observed.  12  When thymocytes on t h e i r own, a t a c o n c e n t r a t i o n  o f 1 0 per 7  w e l l were c u l t u r e d w i t h m i c o m y c i n - t r e a t e d P815, no d e t e c t a b l e c e l l s were generated, i n d i c a t i n g t h a t i n t h e syngeneic  killer  killing  system, thymocytes do n o t c o n t a i n s i g n i f i c a n t l e v e l s o f p r e c u r sors.  T h i s was a l s o found to be t h e case when 5 x 10  thymocytes  were c u l t u r e d w i t h 5 x 10, m i t o m y c i n - t r e a t e d DBA/2 s p l e e n or C57BL/6 c h i m e r i c  cells  cells.  C y t o t o x i c i t y assay. Target  tumor c e l l s were l a b e l e d w i t h "'"'"Cr-sodium chromate  (New England N u c l e a r , Boston, Mass.) as d e s c r i b e d  p r e v i o u s l y (11).  4 51 Various  numbers o f lymphocytes and 10  Cr-labeled  target  cells  i n RPMI 1640 c u l t u r e medium were d i s p e n s e d i n the w e l l s o f m u l t i dish microculture  plates  ( L i n b r o Chemical, 1S-FB-96-TC, New  Haven, Conn.) and the f i n a l volume was a d j u s t e d p l a t e s were incubated incubator  a t 37°C.  t o 0.20 ml. The  f o r 18 hr i n a CO2 e n r i c h e d ,  humidified  The c e l l s were then sedimented by c e n t r i f u g i n g  the p l a t e s a t 200 x G f o r 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 o f s p e c i f i c c y t o t o x i c i t y was c a l -  c u l a t e d as f o l l o w s : S p e c i f i c c y t o t o x i c i t y (%) t e s t r e l e a s e (CPM) - spontaneous r e l e a s e (CPM) maximum.release (CPM) - spontaneous r e l e a s e (CPM) Spontaneous r e l e a s e was measured by i n c u b a t i n g alone,  4 10 t a r g e t  4 and maximum r e l e a s e was t e s t e d by l y s i n g 10 t a r g e t  x  cells cells  13  w i t h 5% TRITON X 100 (Sigma Chemical Company, S t . L o u i s , Mo.). Spontaneous r e l e a s e o f P815 c e l l s was 20 t o 25% and t h a t o f L1210 c e l l s was 15 t o 20% o f maximum r e l e a s e .  Generation  of suppressor  cells.  3 DBA/2 mice were i n j e c t e d subcutaneously w i t h 2 x 10 cells  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  t h e i r thymocytes used as. a source o f suppressor t o c o l had been shown p r e v i o u s l y for the generation  P815  cells.  This  pro-  (65) to.be r e l i a b l e and r e p r o d u c i b l e  of T lymphocytes s p e c i f i c a l l y s u p p r e s s i v e i n  the assay system. Preparation  of suppressor  factor.  T h i s method has been d e s c r i b e d suppressive sonication  i n d e t a i l p r e v i o u s l y <(65). B r i e f l y ,  thymocytes prepared as s t a t e d above, were d i s r u p t e d by ( B i o s o n i c 80) a t 0°C f o r t h r e e 1-min b u r s t s and t h e  s o l u b l e m a t e r i a l was s u b j e c t e d  to preparative  isoelectric  a f t e r u l t r a c e n t r i f u g a t i o n a t 60,000 x G f o r 60 min.  focusing  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 b e a r e r ext 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  m a t e r i a l was s t a b l e a t 4°C and a c t i v e up to d i l u t i o n s e q u i v a l e n t to 1/125 o f a s i n g l e thymus.  Assays f o r t h i s m a t e r i a l were  always r u n a g a i n s t c o n t r o l s c o n t a i n i n g equal the e q u i v a l e n t Isoelectric  c o n c e n t r a t i o n s of  f r a c t i o n s from normal thymocytes.  focusing.  Preparative  isoelectirc  f o c u s i n g was r u n on a 110 ml LKB-  f o c u s i n g column through a 5 t o 50% l i n e a r sucrose  g r a d i e n t and a  14  t o t a l o f 1.6% LKB ampholines (1.2%, pH 4 t o 6, and 0.4%, pH 5 t o 7).  Crude thymic e x t r a c t s i n volumes o f 2.5 t o 3.0 ml w i t h 280  nm absorbance o f about 10.0/ml were used.  The e l e c t r o d e  c o n s i s t e d o f 7.0 ml H 0 and 3.0 ml 1 M NaOH ( l i g h t 2  and  15 g s u c r o s e ,  13 m l ^ 0 and 3 m l 1 M H^PO^  solutions  electrode)  (dense e l e c t r o d e ) .  F o c u s i n g was c a r r i e d out a t 4°C f o r 24 h r a t 950 v o l t s and 0.8 mA. A f t e r t h e f o c u s i n g was c o m p l e t e , d i s t i l l e d water was pumped s l o w l y i n t o t h e column and 1.0 ml f r a c t i o n s o f t h e focused m a t e r i a l s were collected.  These f r a c t i o n s were m o n i t o r e d f o r pH and absorbance  a t 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 t h a t t h e s u p p r e s s i v e  m a t e r i a l focused a t between pH 4.6 and 4.7.  Therefore,  when  f r a c t i o n s were b e i n g pooled f o r subsequent t e s t i n g , b o t h pH and absorbance a t 280 nm were used as i n d i c e s f o r c u t t i n g t h e f r a c t i o n s . P o o l e d samples were d i a l y z e d a g a i n s t p h y s i o l o g i c s a l i n e t o e l i m i n a t e t h e s u c r o s e and ampholines.  They were then  concentrated  down t o 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 a UM10 membrane.  with  F r a c t i o n s were then d i a l y z e d a g a i n s t RPMI 1640  a f t e r w h i c h t h e i r absorbance a t 280 nm was r e c o r d e d .  They were  t h e n 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 s t o r e d a t 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 .  Preparation of a l l o a n t i s e r a . The a n t i - l a ^ a n t i s e r u m used h e r e was prepared by immunizing B10 mice r e p e a t e d l y w i t h B10-D2 s p l e n o c y t e s . i n j e c t e d i . p . e v e r y week w i t h 1 0  7  A n i m a l s were  B10.D2 s p l e n o c y t e s  f o r 8 weeks.  S u b s e q u e n t l y , t h e y were b l e d from t h e r e t r o o r b i t a l s i n u s 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 p e r i o d o f 2 months.  The a n t i - H - 2 ^ a n t i s e r u m  t h u s prepared  was 100% c y t o -  t o x i c t o P815 c e l l s i n t h e presence o f r a b b i t complement (C) up t o a f i n a l d i l u t i o n o f 1/1200.  I n o r d e r t o render  the a n t i -  serum s p e c i f i c f o r t h e l a r e g i o n o f t h e H-2^ MHC i t was absorbed e x h a u s t i v e l y a g a i n s t P815 c e l l s u n t i l i t was no l o n g e r c y t o t o x i c i n t h e presence o f r a b b i t C f o r these t a r g e t s . antiserum  The absorbed  ( a n t i - l a ' * ) , when t e s t e d f o r d i r e c t k i l l i n g o f DBA/2  s p l e n i c lymphocytes k i l l e d 60 t o 65% o f these c e l l s up t o a f i n a l d i l u t i o n o f 1/80 b u t had no a p p r e c i a b l e c y t o t o x i c i t y f o r thymocytes a t a d i l u t i o n o f 1/4.  When t e s t e d by ^ C r r e l e a s e  a s s a y w i t h LPS b l a s t s , i t caused 75% "^Cr r e l e a s e up t o a 1/100 dilution.  I n t e s t s r e p o r t e d h e r e t h e a n t i - l a ' * a n t i s e r a was  used a t a f i n a l c o n c e n t r a t i o n o f 1/40 i n t h e presence o f a 1/15 d i l u t i o n o f r a b b i t C (Lowtox Cedar Lane L a b s , 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 o b t a i n e d  Dr. T. D e l o v i t c h ( B a n t i n g and B e s t I n s t i t u t e ) . H-2^ h a p l o t y p e was prepared  from  Antiserum t o  by immunizing B10.D2 mice w i t h B10  s p l e n o c y t e s as d e s c r i b e d 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 r e c o n s t i t u t e d w i t h 1 0 bone marrow c e l l s from e i t h e r DBA/2, C57BL/6 7  o r 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. was o b t a i n e d  The a n t i s e r u m  from M. L e t a r t e ( O n t a r i o Cancer I n s t i t u t e ) and  16  was used a t a d i l u t i o n of 1/200 w i t h a 1/15 d i l u t i o n of r a b b i t C.  Animals were kept f o r 8 weeks b e f o r e they were used  experiments.  i n any  When these animals were s a c r i f i c e d , t h e i r s p l e e n  c e l l s were t e s t e d w i t h the a p p r o p r i a t e a l l o a n t i s e r a p l u s C t o a s c e r t a i n t h a t 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 In experiments  t e s t i n g f o r the presence of l a markers on  suppressor c e l l s , 4 x 1 0 were suspended  cells.  7  tumor b e a r e r or normal  thymocytes  i n 1.0 ml of a 1/40 d i l u t i o n of a n t i s e r u m i n  complete medium and i n c u b a t e d f o r 45 min a t 37°C a f t e r which 0.5 ml of a 1/5 d i l u t i o n of r a b b i t C i n the same medium was added and  i n c u b a t i o n was c o n t i n u e d f o r another 45 min.  Appropriate  c o n t r o l s of C and medium o n l y were r u n c o n c u r r e n t l y . washed, resuspended  i n medium, and counted  no i n s t a n c e were v i a b l e counts i n c e l l  C e l l s were  for v i a b i l i t y .  In  suspensions c o n t a i n i n g  the a n t i - l a ^ a n t i s e r u m s i g n i f i c a n t l y d i f f e r e n t  from those o f  the c o n t r o l s and i n no i n s t a n c e were counts lower than 90% of the number of c e l l s b e f o r e treatment. s e q u e n t l y used  i n suppressor c e l l  These c e l l s were  sub-  assays.  Immunoadsorbent a s s a y s . The  a b i l i t y of a n t i - l a ^ to remove the s u p p r e s s i v e f a c t o r  from thymic e x t r a c t s was t e s t e d by u s i n g an immunoadsorbent assay described previously isoelectric  ( 6 5 ) . " B r i e f l y , both normal.and suppressor  focused f r a c t i o n s were mixed w i t h e i t h e r the a n t i -  17  la  a n t i s e r u m o r a c o n t r o l o f anti-H-2  o v e r n i g h t a t 4°C. adsorbent  Sepharose  a n t i s e r u m , and i n c u b a t e d  These m a t e r i a l s were then passed over 4B columns t o which goat anti-mouse  been a t t a c h e d by cyanogen bromide.  immunoI g had  The c a p a c i t y o f the column  was s u f f i c i e n t t o remove a t l e a s t t w i c e t h e amount o f the mouse Ig added t o t h e f r a c t i o n s . was t i t r a t e d  The m a t e r i a l e l u t i n g from the columns  f o r s u p p r e s s i v e a c t i v i t y over d i l u t i o n s r a n g i n g  from 1/5 t o 1/125 (these d i l u t i o n s o f e l u t e d m a t e r i a l s r e p r e s e n t between 0.2 and 0.04 of a thymus e q u i v a l e n t ) .  18 Results The  a b i l i t y of suppressor  c e l l s generated as p r e v i o u s l y d e s c r i b e d  (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 g e n e r a t i o n DBA/2 c e l l s c y t o t o x i c f o r the syngeneic F i g u r e 1. P815  P815  A primary c y t o t o x i c response was  or L1210  mitomycin-treated  normal or suppressor response to P815  thymocytes.  of  mastocytoma i s shown i n generated a g a i n s t e i t h e r  c e l l s i n the presence of e i t h e r As can be seen i n F i g u r e l a , the  i n the presence of suppressor  thymocytes was  i c a n t l y suppressed whereas the response to L1210  was  not.  signif-  It  was  thus shown t h a t these suppressor  c e l l s t h a t had been found p r e v i o u s l y  (12)  of P815  to suppress the g e n e r a t i o n  of s p l e n o c y t e s  cytotoxic c e l l s in  primed i n v i v o by exposure to P815,  the primary response i n an analogous manner.  c o u l d a l s o suppress  These r e s u l t s  e n t i r e l y r e p r o d u c i b l e , the o n l y d i f f e r e n c e b e i n g i n the numbers of l y t i c u n i t s between c u l t u r e s .  populations  The  are  relative  d a t a shown i n F i g u r e 1  are r e p r e s e n t a t i v e i n t h a t c o n t r o l c u l t u r e s c o n t a i n r o u g h l y number of l y t i c u n i t s as do c u l t u r e s c o n t a i n i n g suppressor  twice cells.  I t has been r e p o r t e d by o t h e r s t h a t s p e c i f i c T suppressor bear d e t e c t a b l e l a a n t i g e n s  on t h e i r  surface  (81,90-94).  the  cells  Experiments  were run to determine whether 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 c o u l d e l i m i n a t e suppressor  c e l l s i n t h i s system.  The  s e n t a t i v e experiment are shown i n F i g u r e 2. showed t h a t suppressor  c e l l s were p r e s e n t  r e s u l t s of a r e p r e -  Although the C c o n t r o l  i n the t r e a t e d  population  of thymocytes ( F i g . 2b), the a n t i - l a treatment removed  suppressive  a c t i v i t y so t h a t the c y t o t o x i c i t y generated i n the t e s t  population  was  not  significantly different  from the c o n t r o l p o p u l a t i o n  contain-  19  F i g u r e 1.  The a b i l i t y o f P815 s u p p r e s s o r c e l l s t o suppress t h e i n v i t r o primary c y t o t o x i c response o f normal DBA/2 s p l e n o c y t e s t o syngeneic mitomycin C - t r e a t e d P815 tumor c e l l s . Anti-P815: the c y t o t o x i c response o f 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 e d P815 c e l l s i n an 18 h r - a s s a y . • • , primary c u l t u r e o f normal DBA/2 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 33.3); o o, primary c u l t u r e of normal DBA/2 s p l e n o c y t e s and P815 s u p p r e s s o r thymocytes ( l y t i c u n i t s were 15.4). Anti-L1210: t h e cytotoxjLj response o f 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 e d L1210 c e l l s i n an 18 h r assay. • 1 , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and normal DBA/2 thymocytes; 0 0 , primary c u l t u r e of normal DBA/2 s p l e n o c y t e s and P815 s u p p r e s s o r thymocytes. L y t i c u n i t s f o r both c u l t u r e s were 11.1.  20  F i g u r e 2.  The a b i l i t y o f 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 t o e l i m i n a t e P815 s u p p r e s s o r c e l l s . Anti-la: t h e c y t o t o x i c response of c e l l s primed in_ v i t r o w i t h P815 a f t e r treatment o f thymocytes w i t h a n t i - l a antiserum p l u s C. 0 0 , p r i m a r y c u l t u r e o f normal DBA/2 s p l e n o c y t e s p l u s a n t i - l a - t r e a t e d normal thymocytes; • — - j * , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s p l u s a n t i - l a t r e a t e d s u p p r e s s o r thymocytes. L y t i c u n i t s i n b o t h c u l t u r e s were 19.2. C control: C c o n t r o l of the a n t i - l a experiment 0 0, primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and C - t r e a t e d normal thymocytes ( l y t i c u n i t s were 24.4); • • , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and C - t r e a t e d P815 s u p p r e s s o r thymocytes. L y t i c u n i t s f o r s u p p r e s s o r c e l l p o p u l a t i o n s i n t h i s i n s t a n c e c o u l d not be c a l c u l a t e d because 50% l y s i s o f c e l l s was n o t a c h i e v e d i n t h e assay.  21  ing anti-la-treated normal thymocytes (Fig. 2a). The potential of suppressor cells to operate across a histocompatibility 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 haplotypes rendered tolerant to H-2^ 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 suppressor cells with either H-2  d or H-2  splenocytes. A l l 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 c e l l 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  Figure 3.  The a b i l i t y of DBA/2 P815 s u p p r e s s o r 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. 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 t u t e d ) r a d i a t i o n chimera s p l e n o c y t e s ^ p r i m e d i_n v i t r o w i t h P815. 0 0 , p r i m a r y 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 p r i m a r y c u l t u r e of H-2 chimera s p l e n o c y t e s and P815 supp 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 a s s a y f o r s u p p r e s s o r c e l l s . 0 0, primary 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 , p r i m a r y c u l t u r e o f H-2 chimera s p l e n o c y t e s and P815 s u p p r e s s o r thymocytes ( l y t i c u n i t s were 2 7 . 4 ) . b  :  23  ol  I 12-5:1  1 25:1  • 50:1  —  i  —  "  100:1 12-5:1  EFFECTOR : T A R G E T  1  25:1  1  50:1  •  ' 100:1  RATIO  J  Figure 4.  The a b i l i t y o f DBA/2 P815 s u p p r e s s o r c e l l s t o s u p p r e s s the c y t o t o x i c response o f s p l e n o c y t e s from H-2 (A.TH) radiajj^on chimeras t o P815. ^2 H-2 : t h e c y t o t o x i c response o f 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 , primary 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 1 0 . 9 ) ; * primary c u l t u r e o f H-2 chimera s p l e n o c y t e s and P815 s u p p r e s s o r thymocytes ( l y t i c u n i t s c o u l d n o t be c a l c u l a t e d because of low^ l e v e l s o f c y t o t o x i c i t y ) . H-2 : c o n t r o l a s s a y ^ f o r s u p p r e s s o r c e l l s . 0 0, p r i m a r y 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; • • , p r i m a r y c u l t u r e o f H-2 chimera s p l e n o c y t e s and P815 s u p p r e s s o r thymocytes (Lytic units c o u l d n o t 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  50.1  1 0 0 : i 12.5M  E f f e c t o r : Target  Figure  5.  25:1  50M  100:1  Ratio  The a b i l i t y o f t h e s u p p r e s s o r f a c t o r i s o l a t e d from P815 s u p p r e s s o r thymocyte p o p u l a t i o n s t o suppress t h e i n v i t r o primary c y t o t o x i c response o f normal DBA/2 s p l e n o c y t e s t o syngeneic mitomycin C - t r e a t e d tumor c e l l s . The d i l u t i o n s used i n t h i s assay r e p r e s e n t a p p r o x i m a t e l y 1/25 o f a s i n g l e thymus. Anti-P815: t h e c ^ J o t o x i c response o f 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 e d P815 c e l l s i n an 18 h r - a s s a y . • • , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and . . normal DBA/2 thymocyte e x t r a c t ( l y t i c u n i t s were 59.1); 0 0 , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and P815 s u p p r e s s o r thymocyte e x t r a c t ( l y t i c u n i t s were 31.8). Anti-Ll210: t h e c y t o t o x j L j response o f 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 e d L1210 c e l l s i n an 18 h r - a s s a y . • • , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and normal DBA/2 thymocyte e x t r a c t ( l y t i c u n i t s were 45.6) 0 0 , primary c u l t u r e o f normal DBA/2 s p l e n o c y t e s and P815 s u p p r e s s o r thymocyte e x t r a c t ( l y t i c u n i t s 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 . E x p e r i m e n t s were r u n t o d e t e r m i n e 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 s u p p r e s s o r 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 s u b j e c t e d t o t r e a t m e n t 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 a n t i - m o u s e i m m u n o g l o b u l i n . 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 . F i g u r e 6.  The e l u t e d m a t e r i a l s  The r e s u l t s a r e shown i n  W h i l e s u p p r e s s i v e a c t i v i t y was p r e s e n t i n the a n t i - H - 2  suppressor f a c t o r e l u a t e s ,  the suppressive 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 e x p r e s s e s d e t e r m i n a n t s encoded by the l a r e g i o n of t h e MHC. The a b i l i t y of t h e f a c t o r , when added t o B6D2F^ C57BL/6 c h i m e r i c s p l e n o c y t e s , t o suppress t h e g e n e r a t i o n o f c e l l s c y t o t o x i c t o P815, was a l s o t e s t e d . cells,  The r e s u l t s ( F i g . 7) show, as w i t h t h e s u p p r e s s o r  t h a t t h e 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 t h e DBA/2 c e l l s t e s t e d a t the same time.  26  F i g u r e 6.  The e f f e c t o f a n t i - l a a n t i s e r u m a d s o r p t i o n on t h e s u p p r e s s i v e a c t i v i t y o f t h e s u p p r e s s o r f a c t o r i s o l a t e d from P.815 s u p p r e s s o r -thymocyte p o p u l a t i o n s . The d i l u t i o n s used i n t h i s assav r e p r e s e n t a p p r o x i m a t e l y 1/25 o f 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 a n t i s e r u m , o—-^o, p r i m a r y c u l t u r e o f n o r m a l DBA/2 s p l e n o c y t e s and. a n t i - l a adsorbed n o r m a l thymocyte e x t r a c t s ; • — p r i m a r y c u l t u r e o f normal DBA/2 s p l e n o c y t e s and a n t i - l a adsorbed s u p p r e s s o r thymocyte e x t r a c t s . ^ L y t i c u n i t s i n b o t h c a s e s were 57.1. Anti-H-2 : t h e e f f e c t o f a d s o r p t i o n o f t h e s u p p r e s s i v e factor 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 s p l e n o c y t e s 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, p r i m a r y c u l t u r e o f n o r m a l DBA/2 s p l e n o c y t e s and a n t i - l a adsorbed s u p p r e s s o r thymocyte e x t r a c t ( l y t i c u n i t s were 38.5).  27  pi • 12.5n  i  i  1—i  '  251  5CM  1OO0 1250  251  :  ;  1  5CM  1000  EFFECTOR :TARGET RATIO  F i g u r e 7.  The a b i l i t y o f P815 suppressor f a c t o r t g suppress t h e c y t o t o x i c response o f s p l e n o c y t e s from H-2 r a d i a t i o n chimeras to P815. The amount of f a c t o r used i n these experiments was a p p r o x i m a t e l y 1/25 thymus e q u i v a l e n t . H-2 : t h e c y t o t o x i c response o f 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, primary 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 thymocyte e x t r a c t ( l y t i c u n i t s were 62.5); • •, primary c u l t u r e of H-2 chimera s p l e n o c y t e s and s u p p r e s s o r f a c t o r ( l y t i c u n i t s ^ w e r e 16.1). H-2 : c o n t r o l a s s a y ^ f o r s u p p r e s s o r f a c t o r , o o, primary 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 thymocyte e x t r a c t ( l y t i c u n i t s were 48.8); • • , primary c u l t u r e o f H-2 chimera s p l e n o c y t e s and s u p p r e s s o r f a c t o r ( l y t i c u n i t s were 33.9).  28  Discussion In p r e v i o u s s t u d i e s on s u p p r e s s o r c e l l s and the s p e c i f i c p o s s i b l y r e l e a s e d from them, i t was demonstrated t h a t such  factors  suppressive  elements were c a p a b l e of s p e c i f i c a l l y s u p p r e s s i n g the g e n e r a t i o n of c y t o t o x i c c e l l s i n v i t r o by s p l e e n c e l l s from P815-primed (11,12,65).  I n t h i s c h a p t e r , i t was  p r e s s o r c e l l s and P815  specific  animals  shown t h a t P 8 1 5 - s p e c i f i c sup-  suppressor  f a c t o r are a l s o capable  s u p p r e s s i n g a p r i m a r y i n v i t r o c y t o t o x i c response  t o P815  of  ( F i g . 1,5).  Thus, the s t a t e (primed o r v i r g i n ) o f t h e c e l l p o p u l a t i o n on w h i c h i t a c t s appears t o be u n i m p o r t a n t  f o r the P 8 1 5 - s p e c i f i c suppressor  or P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r t o e x p r e s s t h e i r  cell  activity.  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 t o the l a r e g i o n ( t h e I - J s u b r e g i o n i n most s t u d i e s ) t o k i l l by o t h e r s (81,90-93).  s u p p r e s s o r T c e l l s has been r e p o r t e d  These s t u d i e s i n v o l v e d m a i n l y the c h a r a c t e r -  i z a t i o n of T s u p p r e s s o r c e l l s p a r t i c i p a t i n g i n t h e r e g u l a t i o n of the a n t i b o d y response  to s o l u b l e antigens.  The  suppressor c e l l s studied  here are those i n v o l v e d i n r e g u l a t i n g the g e n e r a t i o n o f c y t o t o x i c cells.  However, t h e y a l s o appear t o possess  s u r f a c e markers r e c o g n i z e d  by a n t i - h a p l o t y p e s e r a t o the l a r e g i o n , so i n t h i s r e s p e c t t h e y do n o t d i f f e r from o t h e r p o p u l a t i o n s of a n t i g e n - s p e c i f i c T cells. specific  suppressor  S i n c e a t t h i s time i t i s not p o s s i b l e t o d e v e l o p an f o r the I - J s u b r e g i o n of t h e I a ^ h a p l o t y p e , i t was  antiserum not p o s s i b l e  t o 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 a l t h o u g h many o t h e r s t u d i e s have shown t h a t t h i s s u b r e g i o n of t h e l a i s e x p r e s s e d on suppressor T c e l l s .  antigen-specific  29  I n t h i s c h a p t e r i t was u n d e r t a k e n t o determine whether t h e popul a t i o n o f T s u p p r e s s o r c e l l s under study was H-2 r e s t r i c t e d . the  immune  response  Because  being assessed f o r suppression i n v o l v e d the  development o f c e l l s c y t o t o x i c f o r a s y n g e n e i c tumor c e l l l i n e , i t was n e c e s s a r y t o use s p l e e n c e l l s from r a d i a t i o n chimeras t h a t were t o l e r a n t t o normal H-2  d  alloantigens.  b Thus B6D2F^ a n i m a l s (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 o r DBA/2) o r w i t h A.TH c e l l s  o n l y t h e D l o c u s w i t h H-2^.  (H-2' ' ) t h a t shares t  2  Suppressor c e l l s were r a i s e d i n DBA/2  tumor b e a r e r s and then c u l t u r e d i n v i t r o w i t h mitomycin C - t r e a t e d P815 c e l l s and c h i m e r i c s p l e e n c e l l s o f e i t h e r DBA/2, C57BL/6, o r A.TH o r i g i n .  The e x p e r i m e n t s , which were r e p e a t e d t h r e e t i m e s showed  u n e q u i v o c a l l y t h a t t h e s u p p r e s s o r c e l l s were c a p a b l e o f s u p p r e s s i n g the a n t i - P 8 1 5 c y t o t o x i c response o f s p l e e n c e l l s d i f f e r i n g  a t the K  (A.TH) o r b o t h t h e K and D l o c u s (C57BL/6) and thus d i d n o t e x h i b i t the c l a s s i c a l possibility  form o f H-2 r e s t r i c t i o n .  T h i s does n o t e x c l u d e t h e  t h a t some form o f l a r e c o g n i t i o n was r e q u i r e d s i n c e t h e s e  c h i m e r i c c e l l s had been i n f l u e n c e d by t h e thymus o f t h e i r r a d i a t e d B6D2 r e c i p i e n t s , b u t does show t h a t K o r 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 o f i n t e r a c t i o n . The s u p p r e s s o r f a c t o r ( s ) i s o l a t e d from t h e s u p p r e s s o r thymocyte p o p u l a t i o n and p a r t i a l l y p u r i f i e d f o c u s i n g appeared  isoelectric  t o share t h e same p r o p e r t i e s as t h e s u p p r e s s o r  c e l l s i n that i t could s p e c i f i c a l l y  suppress t h e p r i m a r y i n v i t r o  c y t o t o x i c response o f DBA/2 s p l e e n c e l l s specifically  by p r e p a r a t i v e  cell  ( F i g . 5) and c o u l d be  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 this  30  r e s p e c t t h i s f a c t o r i s not d i f f e r e n t (77,81,82,86,89).  from f a c t o r s d e s c r i b e d by o t h e r s  As mentioned above, i t was  the s u b r e g i o n s p e c i f i c i t y w i t h the H-2^  not p o s s i b l e t o t e s t  system.  The d a t a r e p o r t e d so f a r on the H-2  r e s t r i c t i o n s observed i n  s u p p r e s s i v e f a c t o r s a r e somewhat e q u i v o c a l .  In a s e r i e s of r e p o r t s by  Tada and h i s a s s o c i a t e s (78,95,96) i n v o l v i n g s u p p r e s s i v e f a c t o r ( s ) c o n t r o l the a n t i b o d y response, a syngeneic t a r g e t c e l l  i t was  (presumably  found  that t h i s f a c t o r r e q u i r e d  a helper T c e l l ) before i t could  e f f e c t i v e l y suppress the a n t i b o d y response. a specific  Moorhead, who  produced  suppressor f a c t o r i n v i t r o t h a t c o u l d suppress the d e v e l -  opment of d e l a y e d - t y p e h y p e r s e n s i t i v i t y to DNFB i n mice, found t h i s m a t e r i a l was  H-2  or D end of the MHC mice (79,80,97). syngeneic MHC  restricted  was  s u f f i c i e n t t o permit s u p p r e s s i o n i n r e c i p i e n t  A I^E c l a s s s p e c i f i c s u p p r e s s o r f a c t o r a l s o shows  r e s t r i c t i o n o f a c t i o n (66).  the a n t i b o d y response (GT)  that  and t h a t i d e n t i t y at e i t h e r the K  A l t e r n a t e l y , Waltenbaugh  and h i s c o l l e a g u e s (98) i n s t u d y i n g a s o l u b l e f a c t o r t h a t  tyrosine"^  that  suppresses  t o the s y n t h e t i c copolymer L - g l u t a m y l ^ ^ - L - .  showed t h a t a s u p p r e s s i v e 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 o f GT a l o n e , c o u l d e f f e c t i v e l y suppress the response GT coupled to c a r r i e r p r o t e i n a c r o s s a l l o g e n e i c b a r r i e r s . 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).  Similar  to MHC  A SRBC s p e c i f i c  s u p p r e s s o r f a c t o r of a T - h y b r i d l i n e has a l s o r e c e n t l y been shown to act across allogeneic b a r r i e r s observed no H-2  (100).  K o n t i a i n e n and c o l l e g u e s a l s o  r e s t r i c t i o n of t h e i r suppressor f a c t o r s  (101).  The r e s u l t s here show t h a t the suppressor c e l l s under  study  (and the presumed e q u i v a l e n t f a c t o r ) , a r e capable of s u p p r e s s i n g the  31  generation  o f c y t o t o x i c i t y t o P815 by h i s t o i n c o m p a t i b l e  cells.  The  r e s u l t s show c l e a r l y t h a t t h e K or D gene p r o d u c t s do n o t have t o be shared by t h e s u p p r e s s o r c e l l  ( o r 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 c h i m e r a s , i n which t h e r e c i p i e n t s were B6D2F^ a n i m a l s , were used f o r t e s t i n g r e s t r i c t i o n , t h e c o n c l u s i o n s  can o n l y  extend t o a l a c k o f r e s t r i c t i o n a t t h e K and D l o c i of the MHC thymic i n f l u e n c e by the r e c i p i e n t may have i n f l u e n c e d mechanisms on t h e p a r t o f t h e c h i m e r i c  cells.  since  recognition  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 Introduction A n t i g e n i c determinants  found on lymphocyte c e l l s u r f a c e  molecules  can be used as markers f o r s t u d y i n g the r e l a t i o n s h i p of the e x p r e s s i o n of these d e t e r m i n a n t s  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  y e a r s , many s t u d i e s have examined c e l l s u r f a c e a n t i g e n i c d e t e r m i n a n t s w h i c h c o u l d be used to s t u d y and c h a r a c t e r i z e d i s t i n c t s u b s e t s of T cells.  One  o f the most w i d e l y s t u d i e d a n t i g e n i c systems o f T c e l l s  has been the one determined  by the Ly-1 and Ly-2  S i n c e these a n t i g e n s appear t o be expressed  l o c i i n the mouse (102).  s e l e c t i v e l y on v a r i o u s sub-  s e t s 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 t h a t they be c a l l e d L y t a n t i g e n (103).  suggested  The gene c o n t r o l l i n g the e x p r e s s -  ion  of L y t - 1 a n t i g e n i s found on chromosome 19, whereas the gene c o n t r o l l -  ing  L y t - 2 e x p r e s s i o n i s found on chromosome 6 (104).  Lyt  3  Two  allelic  and L y t * , o f the L y t - 1 and L y t - 2 a n t i g e n s have been found and 5  d e s i g n a t e d L y t - 1 . 1 , -2.1  and L y t - 1 . 2 , -2.2  respectively  forms, are  (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 b e a r i n g the Lyt* a l l e l e . 3  been made.  I n such animals a number of b a s i c o b s e r v a t i o n s have  K i l l e r c e l l s raised against allogeneic c e l l s (allogeneic  k i l l e r c e l l s ) have been d e s i g n a t e d as L y t - 1 2  (105-107).  +  Recently, i t  has been r e p o r t e d t h a t 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 + be o f the L y t - 1 2 accounted  may  + phenotype (108-110).  T h i s apparent  d i s c r e p a n c y may  be  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 g a i n s t syngeneic  tumor c e l l s ( s y n g e n e i c k i l l e r c e l l s )  have a l s o been r e p o r t e d to express the L y t - l 2 +  +  phenotype (107,111,112).  Helper c e l l s have been described by a number of investigators as expressing the L y t - l 2  phenotype (114-116). Antigen-specific suppress-  +  or c e l l s isolated from antigen-primed mice have been i d e n t i f i e d as Lyt-1  2  (115).  +  Suppressor c e l l s generated i n v i t r o by concanavalin A  were also found to be Lyt-1 2 , while the helper c e l l s thus generated +  were L y t - l 2 ~ (114,117). +  There have been fewer studies carried out on mice bearing the Lyt allele.  In these studies allogeneic k i l l e r c e l l s have been i d e n t i f i e d  as L y t - l 2 +  +  (108,118). K i l l e r s of syngeneic tumor c e l l s i n C3H mice  were found to be predominantly Lyt-1 2 a population of Lyt-1 2  +  although i t was indicated that  c e l l s might also be involved (119).  In the  case of suppressor c e l l s , i t was found that two d i s t i n c t populations of c e l l s were present, those which suppressed the delayed type hypersensit i v i t y reaction and were L y t - l 2 +  response and were Lyt-1 2  +  and those which suppressed a humoral  (120).  I t has also been reported that i n vitro-induced suppressor c e l l s i n CBA mice are Lyt-1 2  +  (118), while the helper c e l l s are L y t - l 2 +  +  (118,121).  The present study was carried out i n DBA/2 mice (Lyt-1.1, -2.1) and involved the response of their c e l l s to the syngeneic mastocytoma P815.  The Lyt phenotypes of i n vitro-generated allogeneic k i l l e r s ,  syngeneic k i l l e r c e l l s , and i n vivo-generated T suppressor c e l l s were characterized using anti-Lyt-1.1 and anti-Lyt-2.1 serum.  M a t e r i a l s and Methods E x p e r i m e n t a l A n i m a l s and Tumors Female DBA/2J mice ( H - 2 , L y t - 1 . 1 , -2.1) d  (H-2* , L y t - 1 . 2 , -2.2) 3  and C57BL/6 mice  (The J a c k s o n L a b o r a t o r i e s , Bar H a r b o r ,  Maine) between the ages o f 2 and 5 months of age were used exclusively. and L1210  The tumor l i n e s used were the P815 mastocytoma  l e u k e m i a , b o t h s y n g e n e i c f o r DBA/2J mice, and b o t h  m a i n t a i n e d and t r a n s p l a n t e d 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 m a i n t a i n e d a t -70°C i n l i q u i d  nitrogen.  The methods o f tumor maintenance have been d e s c r i b e d elsewhere (11).  EL-4 lymphoma, s y n g e n e i c f o r C57BL/6 mice was m a i n t a i n e d  similarly. Cells Single-cell  s u s p e n s i o n s were p r e p a r e d from s p l e e n s 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 mesh i n RPMT 1640 N.Y.)  (Grand I s l a n d B i o l o g i c a l  60-gauge  Company, Grand  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  Island,  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 t o a f i n a l c o n c e n t r a t i o n o f 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 u s i n g t r y p a n blue.  Tumor c e l l s were washed twice-: i n medium b e f o r e r e s u s p e n -  s i o n i n complete medium f o r c o u n t i n g . C u l t u r e system f o r the g e n e r a t i o n of c y t o t o x i c e f f e c t o r  cells  Allogeneic effectors The method f o r the jLn v i t r o g e n e r a t i o n of a l l o g e n e i c k i l l e r c e l l s have been d e s c r i b e d i n d e t a i l elsewhere ( 1 2 2 ) .  36 B r i e f l y , 5 x 10  s p l e e n c e l l s from DBA/2J mice were c u l t u r e d ,  f o r 4 days i n t h e presence o f . 5 x 10  mitomycin-treated spleen  c e l l s from C57BL/6 mice i n 2 4 - w e l l 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 - m e r c a p t o e t h a n o l , and 50 mycin.  g/ml g e n t a -  C e l l s were h a r v e s t e d a t 4 d a y s , counted by t r y p a n  b l u e e x c l u s i o n , and assayed f o r t h e i r l a b e l e d EL4 c e l l s i n a s t a n d a r d  a b i l i t y to k i l l "^Cr-  4 hr assay, using t a r g e t :  e f f e c t o r c e l l s from 30:1 t o 3.75:1. Syngeneic e f f e c t o r s The method by which a p r i m a r y i n v i t r o c y t o t o x i c response to s y n g e n e i c tumor c e l l s can be generated has been d e s c r i b e d where (113).The method i s b a s i c a l l y  else-  t h a t d e s c r i b e d above  w i t h t h e e x c e p t i o n t h a t 5 x 10^ DBA/2 s p l e e n 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"* m i t o m y c i n - t r e a t e d P815 cells.  H a r v e s t e d c e l l s were r u n i n t h e s t a n d a r d "'"'"Cr r e l e a s e  assay f o r 18 h r 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 : . t a r g e t ratios  o f between 100:1 and 12.5:1.  P r e v i o u s work i n t h i s  l a b o r a t o r y has demonstrated t h a t t h e k i l l e r c e l l s generated i n t h i s way a r e s p e c i f i c this  i n s t a n c e ) (123,124).  Cytotoxicity  f o r the s t i m u l a t o r c e l l s  (P815 i n  ( A l s o see T a b l e I ) .  test  T h i s has been d e s c r i b e d i n d e t a i l  i n Chapter I I .  Generation of suppressor c e l l s I n most o f t h e p r e v i o u s work c a r r i e d out i n t h i s  laboratory,  a n t i g e n - s p e c i f i c T s u p p r e s s o r c e l l s i n v o l v e d i n r e g u l a t i o n o f the c y t o t o x i c response of s y n g e n e i c mice t o 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  In v i t r o c u l t u r e Responding C e l l s  generated i n v i t r o  ,.  Cytotoxocity  Stimulating c e l l s  Target  cells  Assay  % cytotoxicity  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  a  51 Cr r e l e a s e a f t e r 18 h r i n c u b a t i o n ^ e f f e c t o r : target r a t i o of 100:1 f o r P815 s t i m u l a t e d c e l l s . Cr r e l e a s e a f t e r 4 h r i n c u b a t i o n , effector: t a r g e t r a t i o of 715:1 f o r EL-4 s t i m u l a t e d c e l l s .  38  have been o b t a i n e d from the thymuses o f animals i n t o which 3 x  3 10 ly  tumor c e l l s had been i n j e c t e d (65).  subcutaneously 8-9 days p r e v i o u s -  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 s o l u b l e membrane e x t r a c t s of P815 c e l l s .  P815 c e l l s ,  from mice b e a r i n g the tumor as  a s c i t e s , were drawn from t h e p e r i t o n e a l c a v i t y , washed i n PBS, and l y s e d by f r e e z i n g and thawing Large membrane fragments  t h r e e times i n d i s t i l l e d  and o t h e r c e l l d e b r i s were removed by  c e n t r i f u g a t i o n a t 12,000g f o r 30 min.  Membrane components were  then p e l l e t e d by c e n t r i f u a t i o n a t 105,000g f o r 120 min. membrane p e l l e t was resuspended 1-min  water.  The  i n PBS and s u b j e c t e d t o t h r e e  b u r s t s o f u l t r a s o u n d ( B i o s o n i c probe, 30 s e t t i n g ) a t 0°C.  The m a t e r i a l was a g a i n c e n t r i f u g e d and the supernatant  presumably  c o n t a i n i n g s o l u b i l i z e d membrane components and s m a l l membrane fragments was used. determined  The p r o t e i n content o f t h i s m a t e r i a l was  by the standard Lowry t e s t .  Suppressor c e l l s i n the  s p l e e n s 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 o f between 80 and 200 y g p r o t e i n p e r a n i m a l .  Animals  were s a c r i f i c e d 4 t o 5 days a f t e r a n t i g e n i n e c t i o n , and t h e i r s p l e e n s used as a source o f suppressor c e l l s .  T h i s method i s an  a d a p t a t i o n of t h a t d e s c r i b e d r e c e n t l y f o r the g e n e r a t i o n of suppressor T c e l l s t o m e t h y l c h o l a n t h r e n e - i n d u c e d  sarcomas 0-25).  Because the s p l e e n s o f these animals c o n t a i n e d both a n t i g e n 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  (see below),  n e c e s s a r y t o s e p a r a t e these two p o p u l a t i o n s .  i t was  Spleen c e l l s were  passed through n y l o n wool columns a c c o r d i n g t o the method o f  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 h e r e were t h a t c e l l s ,  a f t e r a p p l i c a t i o n t o t h e column, were i n c u b a t e d f o r 30 min a t 37°C and then e l u t e d a t 37°C.  The columns were then i n c u b a t e d f o r a  f u r t h e r 30 min a t 4°C b e f o r e a second e l u t i o n i n c o l d medium.  The  c e l l s from b o t h e l u t i o n s were p o o l e d and used as a s o u r c e o f a n t i g e n s p e c i f i c suppressor c e l l s .  T h i s procedure  u t i l i z e d columns w h i c h  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 o f n y l o n w o o l through w h i c h 20 ml o f supplemented medium a t 37°C had been passed 8 b e f o r e u s e . A t o t a l o f 2 x 10  s p l e e n c e l l s were a p p l i e d t o columns  of t h i s s i z e and a p p r o x i m a t e l y 20% of t h e c e l l s were recovered, by elution.  The p r o p e r t i e s o f t h e 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 p o p u l a t i o n s i n t h a t they were 95% s e n s i t i v e t o a n t i - t h y - 1 serum p l u s complement and c o n t a i n e d v e r y few p h a g o c y t i c cells  (<1.0%).  The g e n e r a t i o n o f s u p p r e s s o r c e l l s i s i l l u s t r a t e d i n  F i g u r e 8. Assay f o r s u p p r e s s o r  cells  The method used t o assay f o r s u p p r e s s o r c e l l s has been d e s c r i b e d 6 i n d e t a i l elsewhere.(123).  B r i e f l y , 5 x 10  normal s p l e e n c e l l s were  c o c u l t u r e d w i t h 5 x 10^ ( o r fewer i n some cases) s u p p r e s s o r c e l l popul a t i o n s i n the presence  o f 5 x 10^ m i t o m y c i n - t r e a t e d P815 c e l l s i n  2 4 - w e l l L i n b r o t r a y s i n 2.5 ml o f medium.  C e l l s were c u l t u r e d f o r  5 days b e f o r e assay f o r g e n e r a t i o n o f c e l l s c y t o t o x i c f o r P815. C o n t r o l s i n these s t u d i e s c o n s t i t u t e d t h e use o f 5 x 10 c e l l s p l u s 5 x 10  normal s p l e e n  normal s p l e e n o r s p l e e n - d e r i v e d c e l l s w h i c h had been  t r e a t e d i n a manner analogous t o t h e s u p p r e s s o r p o p u l a t i o n . When t h e degree o f s u p p r e s s i o n 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 P r e p a r a t i o n of P815 Tumor-Specific Suppressor C e l l s (P815-T )  I n j e c t i o n of 80-200 vg of S o l u b l e P815 Tumor Membrane E x t r a c t  IP  DBA/2J Mice  4 Days  S p l e n o c y t e s - C o n t a i n i n g both S p e c i f i c Suppressor C e l l s ( T - C e l l s ) And N o n - S p e c i f i c Suppressor C e l l s (Probably M0)  Passed Through Nylon Wool Column (30' at  37°)  E l u t e d w i t h 37° media  Further  30' a t  4°  E l u t e d w i t h 4° media  Eluted Enriched T - c e l l Population C o n t a i n i n g P815~Specif i c T ..  F i g u r e 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 t u m o r - s p e c i f i c suppressor c e l l s .  P815  :  41  was  t e s t e d a t v a r i o u s e f f e c t o r to t a r g e t r a t i o s and the d e c r e a s e  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  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 t o l y s e  was 50%  4 of  the 10  t a r g e t c e l l s i n 18 hr i n c u b a t i o n , was  e s t i m a t e d by  l i n e a r r e g r e s s i o n a n a l y s i s of percentage of c y t o t o x i c i t y vs l o g a r i t h m of e f f e c t o r to t a r g e t c e l l r a t i o .  Total l y t i c  were c a l u c l a t e d from c e l l r e c o v e r y i n c u l t u r e s , and of  s u p p r e s s i o n was  units.  .  units  percentage  e s t i m a t e d from the decrease i n t o t a l  lytic  In a l l experiments, no s i g n i f i c a n t d i f f e r e n c e s i n c e l l  r e c o v e r y between t e s t c u l t u r e s and c o n t r o l s were observed.  In  all  s u p p r e s s i o n experiments r e p o r t e d here, except the time c o u r s e  one  (Fig- H ) ,  a  minimum of 37% s u p p r e s s i o n was  seen.  A n t i - L y t treatment of c e l l s The c e l l s t e s t e d f o r t h e i r L y t phenotype allogeneic k i l l e r  cells,  syngeneic k i l l e r  tumor s p e c i f i e d suppressor c e l l s . 1.1  and Lyt-2.1 phenotype.  kind g i f t  of Dr. R. Nowinski  c e l l s , and  syngeneic  DBA/2J mice a r e of the L y t -  The a n t i s e r a used here were the (The Fred Hutchinson Cancer  C e n t e r , S e a t t l e , Washington) who the  i n t h i s study were:  had r a i s e d and  Research  characterized  a n t i s e r a a c c o r d i n g the p r e v i o u s l y d e s c r i b e d procedures  The a n t i - L y t - 1 . 1 serum t i t e r e d  a t 1:640  t i t e r e d a t 1:160  i n the presence of r a b b i t  when t i t r a t e d  (127).  and the a n t i - L y t - 2 . 1 comple-  ment on B 6 ^ L y t - l . l and B 6 L y t - 2 . 1 , - 3 . 1 thymocytes,  respectively.  The a n t i - L y t - 1 . 1 serum had no e f f e c t on B6Lyt-2.1,  -3.1  thymocytes,  l i k e w i s e , the a n t i - L y t - 2 . 1 serum had no e f f e c t on B 6 . L y t - l . l thymocytes.  In the experiments run h e r e , h i g h e r l e v e l s of the  42  a n t i s e r a were found to be n e c e s s a r y i n order t o k i l l cells effectively. cells,  the a p p r o p r i a t e  For both the a l l o g e n e i c and syngeneic  the procedure was i d e n t i c a l .  counted, and d i s t r i b u t e d  E f f e c t o r c e l l s were h a r v e s t e d ,  to s m a l l p l a s t i c  c o n c e n t r a t i o n of 3.0 x 1 0  7  killer  t e s t tubes a t a t o t a l  c e l l s per tube.  c e n t r i f u g e d and the supernatant removed.  These c e l l s were Three hundred  microliters  of medium was added t o each tube and 50 y l o f e i t h e r a n t i - L y t - 1 . 1 , L y t - 2 . 1 , o r medium was added t o the a p p r o p r i a t e tube. were mixed and i n c u b a t e d a t room temperature  The c e l l s  f o r 45 min f o l l o w i n g  which 50 y l of neat r a b b i t complement (Low-tox, Cedar  Lane,  O n t a r i o , Canada) was added to each tube except the c o n t r o l , to which 50 y l of medium was added. another 45 min.  I n c u b a t i o n was c o n t i n u e d f o r  C e l l s were washed t h r e e times i n supplemented  medium, counted by t r y p a n b l u e e x c l u s i o n , and subsequently t e s t e d for of and  their cytotoxic activity.  One p o p u l a t i o n of a 1:1 m i x t u r e  a n t i - L y t - 1 and a n t i - L y t - 2 - t r e a t e d c e l l s were a l s o  reconstituted  tested f o r c y t o t o x i c i t y . In  the assay of suppressor c e l l s , l a r g e r c e l l numbers had to  be t r e a t e d because  t h e c e l l s were subsequently g o i n g i n t o t h e  l a r g e c u l t u r e s f o r 5 days.  For t h i s r e a s o n , 6 x 10^ c e l l s were  t r e a t e d , and double the amounts of a l l reagents were used. p r o t o c o l as d e s c r i b e d above was used, except t h a t a f t e r  A  treatment,  washing, and c o u n t i n g , the c e l l s were put i n t o c u l t u r e a t a c o n c e n t r a t i o n of 5 x 1 0  6  mitomycin-C t r e a t e d P815  with 5 x 10 cells.  6  normal  spleen c e l l s plus  43  Results The  s p l e e n c e l l s o f DBA/2J m i c e , 4 days f o l l o w i n g  intraperitoneal  i n j e c t i o n o f P815 membrane e x t r a c t s , were v e r y s u p p r e s s i v e when they were c o c u l t u r e d w i t h 5 x 10^ DBA/2 s p l e e n c e l l s i n t h e presence o f mitomycin-treated  P815 c e l l s .  The g e n e r a t i o n o f 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 t h a t seen i n c o n t r o l c u l t u r e s ( F i g . 9 a ) . When these s u p p r e s s o r c e l l s were assayed  i n c u l t u r e w i t h normal s p l e e n  c e l l s p l u s m i t o m y c i n - t r e a t e d L1210 c e l l s , t h e g e n e r a t i o n o f c e l l s c y t o t o x i c f o r t h i s tumor were a l s o markedly suppressed  ( F i g . 9b).  S i n c e t h e s e two systems do n o t c r o s s r e a c t a t t h i s l e v e l , i t was concluded  ( s e e Fig.T) ,(12)  t h a t i n t r a p e r i t o n e a l i n j e c t i o n o f membrane e x t r a c t s  of P815 c e l l s c o u l d generate b o t h s p e c i f i c and n o n s p e c i f i c s u p p r e s s o r c e l l s . S i n c e t h e most common n o n s p e c i f i c s u p p r e s s o r c e l l i s an adherent  cell.,- p r o -  b a b l y o f t h e 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 p a s s i n g t h e s p l e e n c e l l s from a n t i g e n i n j e c t e d a n i m a l s through n y l o n wool columns was attempted.  The c e l l s  e l u t e d from these columns a t e i t h e r 37 o r 4°C were t e s t e d f o r t h e i r suppressive activity'.  S i n c e b o t h p o p u l a t i o n s were found t o c o n t a i n  s p e c i f i c s u p p r e s s o r c e l l s , subsequent s t u d i e s i n v o l v e d a p o o l o f t h e two p o p u l a t i o n s ( F i g . 1 0 ) . The time c o u r s e o f appearance and d i s a p p e a r a n c e s p e c i f i c s u p p r e s s o r c e l l s was examined.  of the antigen-  As can be seen i n F i g u r e l i ,  DBA/2 s p l e n o c y t e s show a n t i g e n - s p e c i f i c s u p p r e s s i o n 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 o f P815 membrane e x t r a c t s .  This  s u p p r e s s i o n peaks a t 4-5 d a y s , then f a l l s o f f r a p i d l y t o c o n t r o l l e v e l s by 7-9 days post  injection.  44  SOI  100=1 125=1  EFFECTOR ^TARGET RATIO  F i g u r e 9.  C y t o t o x i c i t y t i t r a t i o n s o f DBA/2 s p l e e n c e l l s primed a g a i n s t e i t h e r P815 s t i m u l a t o r ^a) or L1210 s t i m u l a t o r s (b). (•) C o n t r o l c o n t a i n i n g 10 normal DBA/2 splenocyjzes i n t h e p r i m i n g c u l t u r e ; (o) c u l t u r e g c o 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 and 5 x 10 s p l e n o c y t e s from mice i n o c u l a t e d 4 days p r e v i o u s l y w i t h P815 membrane e x t r a c t s (80 yg per mouse).  45  125:1  50:1  l O O ' l 125:1  EFFECTOR TARGET RATIO ;  F i g u r e 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 s p l e e n c e l l s primed a g a i n s t e i t h e r P815 (a) or L1210 £b) s t i m u l a t o r s . (•) C o n t r o l c u l t u r e g o 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 and 5 x 10 normal DBA/2 n y l o n wool-pgssed s p l e n i c lymphocytes; (o) c u l t u r e s c o 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 and 5 x 10 P815 primed DBA/2 n y l o n woolpassed s p l e n i c lymphocytes ( c e l l s e l u t e d a t 37 and 4 C were p o o l e d ) .  i  i  2  Days  F i g u r e 11.  aftar  Time course o f  \  4 Lp.  injection  of  \ 6  8  P815  appearance  specific  suppressor  cells  injected  intraperitoneally  membrane  \ 10  extract  and d i s a p p e a r a n c e i n the spleen with  on day 0. C o n t r o l c u l t u r e g DBA s p l e n o c y t e s a n d 5 x 10  P815  of  of antigenDBA/2 m i c e  membrang  extracts  5 x 10 normal DBA/2 n y l o n w o o l -  contained normal  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 p l o t t e d 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 passedgSplenic  spleen  lymphocytes.  as compared  to  controls.  47  The L y t phenotypes o f a v a r i e t y o f e f f e c t o r c e l l s i n DBA/2 mice was  investigated.  allogeneic k i l l e r s considered  Since previous  f i n d i n g s by o t h e r s had c h a r a c t e r i z e d  i n mice o f t h e L y t  important  a  + + a l l e l e as L y t - 1 2 , i t was  t o t e s t t h e a n t i s e r a by d e t e r m i n i n g  on a l l o g e n e i c k i l l e r c e l l s .  i t s effect  The r e s u l t s a r e shown i n F i g u r e 12, i n  w h i c h i t can be seen t h a t b o t h a n t i - L y t - 1 . 1 and a n t i - L y t - 2 . 1 p l u s complement e f f e c t i v e l y abrogated t h e a l l o g e n e i c k i l l e r c e l l s .  Mixing  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 t o t h e c y t o t o x i c i t y a s s a y d i d n o t r e c o n s t i t u t e t h e r e s p o n s e ( d a t a n o t shown).  These  r e s u l t s , t h e r e f o r e , a r e i n agreement w i t h t h e o b s e r v a t i o n s  of others  and  i n d i c a t e t h a t t h e a l l o g e n e i c k i l l e r c e l l i n t h e DBA/2J a n i m a l  + + expresses the Lyt-1 2  phenotype.(108,118).  An experiment was r u n c o n c u r r e n t l y t o d e t e r m i n e t h e L y t phenotype of t h e c y t o t o x i c e f f e c t o r t o syngeneic tumor c e l l s . a r e shown i n F i g u r e 13.  I n t h i s case complement p l u s  The r e s u l t s anti-Lyt-2.1  but n o t a n t i - L y t - 1 . 1 reduced t h e k i l l i n g e f f i e n c y o f t h i s  population,  i n d i c a t i n g t h a t t h e L y t phenotype o f t h i s c e l l i s L y t - 1 2 . +  Mixing  of t h e two t r e a t e d p o p u l a t i o n s d i d n o t r e s t o r e t h e response. F i n a l l y , t h e L y t phenotype o f t h e DBA/2 a n t i g e n - s p e c i f i c s u p p r e s s o r 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 a r e shown i n F i g u r e 14.  instance, the a n t i - L y t - 1 antiserum  In this  e l i m i n a t e d suppressor c e l l s , or  t h e i r development, whereas t h e a n t i - L y t - 2 serum had no e f f e c t . t h i s case a l s o , m i x t u r e s  In  o f 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 n o t  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,  t h a t .the s u p p r e s s o r c e l l , i t s p r o g e n i t o r , o r a c e l l v i t a l development e x p r e s s e s t h e L y t - 1 2  phenotype.  in its  48 i  3-75:1  I  75:1 15:1 30:1 EFFECTOR : TARGET RATIO  ! I-  F i g u r e 12.  The e f f e c t o f 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 DBA/2 a l l o g e n e i c e f f e c t o r . c e l l s r a i s e d a g a i n s t C57BL/6, on a EL4 t a r g e t . (o) C o n t r o l c u l t u r e ; (t) a n t i - L y t - 1 . 1 - t r e a t e d 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  CYTOTOXIC  All  60 ' a n t i Ly-1-1 < anti Ly-2-7 40  ^? ON  20 -  •  1X5:1  I  25:1  50:1  100:1  EFFECTOR: TARGET RATIO  F i g u r e 13.  The e f f e c t o f a r i 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 DBA/2 e f f e c t o r c e l l s primed a g a i n s t t h e syngeneic P815 mastocytoma. (o) C o n t r o l ; (•) a n t i - L y t 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 - t r e a t e d effector c e l l s .  50  1001 •  80  60r-  O  I-  A Control  40  0 Suppressor Cell  >• u  Control  QN  • Anti-Lyl-1 A Anti-Ly 2~1  20  •  12.5 1  i  •  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 w o o l - p a s s e d 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 w o o l - p a s s e d 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 s p l e n o c y t e s p l u s 5 x 10 n y l o n w o o l passedgP815 s u p p r e s s o r s 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 anti-Lyt-1.1treated^ P815 s u p p r e s s o r 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 s u p p r e s s o r 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 n o t shown. Cell r e c o v e r i e s of antiserum-treated suppressor T c e l l populati 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 , 7 0 . 3 % . . These d a t a a r e based on "estimates from t h e complem e n t - t r e a t e d c o n t r o l c e l l s w h i c h were r e g a r d e d as 100% recovery. L y t i c u n i t s were: (A) 8 4 . 3 , (o) 4 6 . 1 , (•) 78.6, (A) 4 1 . 7 .  51 Discussion In  t h i s c h a p t e r , a method f o r t h e i n v i v o i n d u c t i o n o f s p e c i f i c  s u p p r e s s o r c e l l s w h i c h i n h i b i t t h e hi v i t r o g e n e r a t i o n o f c e l l s c y t o t o x i c f o r a syngeneic to  tumor has been d e s c r i b e d .  The method i s s i m i l a r  the method d e s c r i b e d by F u j i m o t o f o r t h e i n d u c t i o n of t h e same type  of s u p p r e s s o r c e l l s w i t h a d i f f e r e n t tumor (125).  However, i n t h e  work shown h e r e , the s u p p r e s s i o n was n o t s p e c i f i c when whole s p l e e n c e l l p o p u l a t i o n s o f 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 t o suppress  the j m v i t r o c y t o t o x i c response  leukemia c e l l s as w e l l as P815 mastocytoma c e l l s .  t o L1210  Since i t i s w e l l  documented t h a t a n t i g e n - s t i m u l a t e d s p l e e n c e l l s may c o n t a i n a p o p u l a t ion  o f n o n s p e c i f i c s u p p r e s s o r c e l l s w h i c h 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 t o p l a s t i c or n y l o n wool and p h a g o c y t i c a c t i v i t y  (18,128,  129), i t was f e l t t h a t t h e passage o f the s t i m u l a t e d s p l e e n c e l l s through n y l o n wool might remove the n o n s p e c i f i c s u p p r e s s i v e T h i s proved  t o be the c a s e .  activity.  That the a n t i g e n - s p e c i f i c s u p p r e s s o r  cell  was o f 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 t h a t t h e n y l o n wool nonadherent p o p u l a t i o n (which i s 9 5 % >  thy-1 p o s i t i v e ) c o n t a i n e d t h e a n t i g e n - s p e c i f i c s u p p r e s s o r The  cell.  s t u d i e s on t h e L y t phenotypes o f the v a r i o u s e f f e c t o r  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 .  cells  The f i n d i n g t h a t t h e 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 mouse was L y t - l 2 " * " was n o t s u r p r i s i n g , +  s i n c e i t c o n f i r m s the o b s e r v a t i o n s o f o t h e r s t h a t , i n mice b e a r i n g t h e L y t - 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 (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 r e g a r d i n g  k i l l e r and s u p p r e s s o r c e l l s i n mice e x p r e s s i n g t h i s  allele.  Lyt-l 2  syngenic  +  +  52  The  f i n d i n g t h a t t h e syngeneic k i l l e r d i f f e r s  killer  c e l l i n e x p r e s s i n g the L y t - 1 2  +  from the a l l o g e n e i c  phenotype i s somewhat s u r p r i s i n g .  However, o t h e r i n v e s t i g a t o r s have r e p o r t e d that anti-tumor killer  c e l l s i n C3H mice were predominantly  of the Lyt-1 2  These workers a l s o noted t h e p r o b a b l e involvement i n the syngeneic  syngeneic phenotype.  +  of L y t - l 2 +  +  cells  system.  In a r e c e n t r e p o r t v e r y a p p l i c a b l e t o t h i s  study, M i l l s and  c o l l e a g u e s found t h a t i n DBA/2 mice, syngeneic tumor k i l l e r s d i r e c t e d towards P815 tumor c e l l s expressed'Lyt-1 a n t i g e n . ( 1 3 0 ) . not l o o k a t L y t - 2 a n t i g e n e x p r e s s i o n on these c e l l s .  They however d i d  T h e r e f o r e , as  f a r as e x p r e s s i o n of L y t - 1 on syngeneic k i l l e r s goes, t h a t study and the one r e p o r t e d here seem t o be i n c o n f l i c t . may be the d i f f e r e n t Syngeneic  One reason f o r t h i s  methods o f g e n e r a t i o n of c y t o t o x i c c e l l s t o P815.  c y t o t o x i c c e l l s i n the M i l l s  ( i n t h e presence o f IL-2)  study were generated  i n vitro  from P815 tumor b e a r i n g DBA/2 mice.  they were p r o b a b l y g e n e r a t i n g secondary to primary c y t o t o x i c c e l l s i n t h i s  Therefore,  c y t o t o x i c k i l l e r s as opposed  investigation.  Thus, t h e r e may  be a d i f f e r e n c e i n t h e L y t - 1 a n t i g e n e x p r e s s i o n on primary v s secondary syngeneic k i l l e r s i n the DBA/2-P815 system.  T h i s of course leads to  the r e c e n t f i n d i n g s t h a t a l l T c e l l s p r o b a b l y express L y t - 1 and L y t - 2 , a l t h o u g h i n v a r y i n g amounts (131). T h e r e f o r e , i t i s i m p o s s i b l e t o conclude a t t h i s  time t h a t t h e  syngeneic k i l l e r c e l l i s L y t - 1 2 , s i n c e i t may o n l y be q u a n t i t a t i v e +  d i f f e r e n c e s between i t and t h e a l l o g e n e i c k i l l e r i n e x p r e s s i o n o f t h e L y t - 1 a l l o a n t i g e n which appear t o render them as d i s t i n c t  populations.  The o b s e r v a t i o n made here abrogated  suppressor  that a n t i - L y t - 1 antiserum  effectively  f u n c t i o n , i n the in v i t r o system used, i s  cell  e x p l i c a b l e i n a t l e a s t two p o s s i b l e ways.  First,  i t has been r e p o r t e d  p r e v i o u s l y t h a t suppressor c e l l s which lower DTH r e a c t i o n s i n mice of a + the L y t a l l e l e are o f the L y t - 1 2 phenotype  (120).  Second, the c e l l  t h a t was i s o l a t e d from immune spleens may n o t be the e f f e c t o r c e l l f o r s u p p r e s s i o n b u t may induce  the s u p p r e s s o r s  mechanism such as t h i s has been p r e s e n t e d mediated responses Benacaraf  (42-44, 132,133).  and c o l l e a g u e s  i n culture.  Evidence  for a  f o r b o t h humoral and c e l l -  In the model p r e s e n t e d by Germain,  (22,23,44,60,134-136), the f i r s t c e l l  i n immune  s u p p r e s s i o n i s a Lyt l"*", I - J , a n t i g e n - b i n d i n g " i n d u c e r " of s u p p r e s s i o n . -  +  T h i s c e l l may "induce", actively  suppress  i n a c a s c a d e - l i k e mechanism, o t h e r T c e l l s t o  i n the same system.  The 5-day c u l t u r e p e r i o d o f the  assay here might a l l o w the i n d u c t i o n o f suppressors influence of t h i s L y t - l 2 +  cell.  t h a t i n a p o p u l a t i o n o f suppressor Lyt-l 2 +  in  cell  in s i t u under the  A t p r e s e n t , i t can o n l y be c o n c l u d e d cells,  or t h e i r p r e c u r s o r s , an  i s e s s e n t i a l f o r the s u p p r e s s i v e e f f e c t t o the observed  i n v i t r o c y t o t o x i c assays.  CHAPTER IV IN VITRO AND IN VIVO EFFECTS OF SYNGENEIC AND ALLOGENEIC ANTISERA RAISED TO TUMORSPECIFIC SUPPRESSOR FACTOR FROM DBA/2 MICE  55  CHAPTER IV Introduction I t i s now  r e c o g n i z e d t h a t T lymphocytes  are made up of d i s t i n c t  s e t s which i n t e r a c t w i t h each o t h e r as w e l l as B lymphocytes phages t o b o t h a m p l i f y and  by way hibit  of T h e l p e r c e l l s  and macro-  i n h i b i t a v a r i e t y of immunological  Thus T c e l l s have demonstrated  enhancing  (2-10).  the immune response by way  responses.  e f f e c t s on the immune  response  T c e l l s have a l s o been found of T suppressor c e l l s  to i n -  (11-17,19~44,65).  In many of the i n v e s t i g a t i o n s c o n c e r n i n g a n t i g e n - s p e c i f i c latory T c e l l s ,  sub-  regu-  i t has been shown t h a t s o l u b l e f a c t o r s d e r i v e d from  these  c e l l s can a p p a r e n t l y d u p l i c a t e t h e i r f u n c t i o n s (14,59-61,67-74,76,77,82, 137-139).  Little  i s known of these f a c t o r s b u t , as i n d i c a t e d  i n Chap-  t e r 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 , c h e m i c a l a n t i g e n i c p r o p e r t i e s i n common.  They are p r o t e i n s w i t h s p e c i f i c  b i n d i n g p o t e n t i a l , have m o l e c u l a r weights minants which are coded by  heavy c h a i n .  carry variable  A l l this  t h a t these " f a c t o r s " may  antigen  50,000, have d e t e r -  the I r r e g i o n , l a c k c o n s t a n t r e g i o n immuno-  g l o b u l i n d e t e r m i n a n t s , but may of the I  of about  and  (idiotypic)  l e a d s to the i n t r i g u i n g  determinants possibility  i n f a c t be the a n t i g e n r e c e p t o r s of these  specific regulatory T c e l l s . Yamauchi e_t al_ (125) have r e c e n t l y shown t h a t i n j e c t i o n of S1509a tumor e x t r a c t s i n t o syngeneic A/J mice a c t i v a t e s , t u m o r - s p e c i f i c T suppressor c e l l s . to the syngeneic P815  tumor system  i n the s p l e e n  T h i s has r e c e n t l y been i n DBA/2J mice  soluble  (Chapther  extended I I I , 13).  t h i s c h a p t e r , i t i s shown t h a t by p a s s i n g e x t r a c t s of t h i s s u p p r e s s i v e  In  p o p u l a t i o n over P815  membrane - Sepharose columns, P 8 1 5 - s p e c i f i c  suppressor f a c t o r can be  eluted.  T h i s P 8 1 5 - s p e c i f i c suppressor f a c t o r was syngeneic and a l l o g e n e i c groups of mice. the syngeneic mice the- u m i q u e found  then i n j e c t e d  into  The hope b e i n g t h a t i n  determinants  (idiotypic?),  i n or near the a n t i g e n b i n d i n g s i t e of the P 8 1 5 - s p e c i f i c sup-  p r e s s o r f a c t o r , would e l i c i t of the a n i m a l s .  an a n t i b o d y response  While both these and  the  i n a t l e a s t some  ©onstant  determinants  on the s u p p r e s s o r f a c t o r might induce an a n t i b o d y response a l l o g e n e i c mice.  Both groups of mice produced  i n the  a n t i s e r a which  was  capable of r e a c t i n g i n v i t r o s p e c i f i c a l l y w i t h the P 8 1 5 - s p e c i f i c suppressor f a c t o r , and w i t h complement was  a b l e t o abrogate  f u n c t i o n of P 8 1 5 - s p e c i f i c T suppressor c e l l s , but was effect  unable  the to  the a c t i o n of P 8 1 5 - s p e c i f i c T c y t o t o x i c e f f e c t o r c e l l s .  a n t i s e r a produced  i n syngeneic mice was  a l s o shown to be  effective  i n v i v o i n s l o w i n g P815-tumor growth and p r o l o n g i n g s u r v i v a l of DBA/2 mice i n j e c t e d w i t h t h i s tumor.  The  times  57  M a t e r i a l s and Methods M i c e and Tumors. Female DBA/2J and C57BL/6 mice were o b t a i n e d Jackson Laboratory  (Bar H a r b o r , M a i n e ) .  r a t i o n of a n t i - s u p p r e s s o r  Except f o r the p r e p a -  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. used were the P815 f o r DBA/2J mice.  from the  mastocytoma and L1210  The  tumor l i n e s  leukemia,  They have b o t h been m a i n t a i n e d  both  and  syngeneic  trans-  p l a n t e d as a s c i t e s tumors i n DBA/2J mice ( 1 1 ) , o r as f r o z e n 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 y e a r s .  B a l e n t l tumor, syngeneic maintained  t o B a l b / c m i c e , was  a l s o used  and  as above.  Cells. T h i s has been d e s c r i b e d 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 g e n e r a t i o n 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 p r i m a r y t o syngeneic  tumor c e l l s can be generated has been d e s c r i b e d  v i o u s l y • (123):. P815  or L1210  i n v i t r o c y t o t o x i c response  was  pre-  " B r i e f l y , specif i c . c y t o t o x i c i t y .against e i t h e r generated i n v i t r o by i n c u b a t i n g 5 x  10  s p l e e n c e l l s w i t h 5 x 10^ m i t o m y c i n t r e a t e d tumor c e l l s i n L i n b r o 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 , a t 37°C i n a h u m i d i f i e d i n c u b a t o r w i t h 5% CO2 t o t a l volume of each c u l t u r e w e l l was complete 1640  medium.  f o r 5 days.  The  made up 2.5 ml w i t h  A f t e r i n c u b a t i o n , c e l l s were h a r v e s t e d ,  c o u n t e d , - r e s u s p e n d e d ' i n complete: 1640 medium'at 'a c o n c e n t r a t i o n  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 a t 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 s t a n d a r d 18 h ^ C r r e l e a s e assay which has been d e s c r i b e d i n p r e v i o u s chapters  ( I I and I I I ) .  syngeneic  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  tumor system have p r e v i o u s l y been shown t o be T  lymphocytes  (11,13).  G e n e r a t i o n of suppressor  cells.  The method, f o r g e n e r a t i o n of t u m o r - s p c i f i c T suppressor cells,  i n v o l v e d i n the r e g u l a t i o n of c y t o t o x i c response  of DBA/2J  mice to the syngeneic P815 tumor, has been d e s c r i b e d p r e v i o u s l y (Chapter  III).  were generated  B r i e f l y , suppressor c e l l s used  study  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 s o l u b l e  membrane e x t r a c t s of P815 c e l l s . induced  i n this  Suppressor  c e l l s were r e a d i l y  i n the s p l e e n s 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 p r o t e i n per animal 4-5 days b e f o r e  sacrifice.  Because the s p l e e n s of these animals c o n t a i n e d both a n t i g e n 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 to s e p a r a t e the two p o p u l a t i o n s .  (13) i t was  Spleen c e l l s were  necessary  passed  through n y l o n wool columns by a m o d i f i e d method of J u l i u s e t a l (126). The method i s b a s i c a l l y t h a t d e s c r i b e d b e f o r e (Chapter I I I , 13) w i t h a few m o d i f i c a t i o n s o u t l i n e d below.  In s h o r t , the c e l l s ,  a f t e r a p p l i c a t i o n to the n y l o n wool column, were i n c u b a t e d f o r 60 min a t room temperature 37°C complete 1640 medium.  (20°C) and then e l u t e d w i t h warmed Approximately  r e c o v e r e d by t h i s e l u t i o n p r o c e s s .  15% of the c e l l s were  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 p o p u l a t i o n s i n t h a t  59  they were >95% s e n s i t i v e t o a n t i - T h y 1 serum p l u s complement and c o n t a i n e d v e r y few p h a g o c y t i c c e l l s (<1.0%).  T h i s 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 s u p p r e s s i v e a c t i v i t y , and has been found r e p e a t e d l y t o c o n t a i n c e l l s , w h i c h under t h e c o n d i t i o n s used h e r e , were c a p a b l e o f s u p p r e s s i n g t h e i n v i t r o p r i m a r y c y t o t o x i c .response P r e p a r a t i o n of suppressor  30-70%. factor.  A n t i g e n - s p e c i f i c T s u p p r e s s o r c e l l s , o b t a i n e d from s p l e e n s 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 t h e p r e p a r a t i o n of P815 a n t i g e n - s p e c i f i c s u p p r e s s o r  f a c t o r (P815-SF).  Control  f a c t o r p r e p a r a t i o n s were made from normal DBA/2 s p l e n o c y t e s , 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 , s u p p r e s s i v e or o t h e r w i s e ) .  The s p l e n o -  c y t e s were s o n i c a t e d f o r 3 one minute b u r s t s ( B i o s o n i c 80, 60 watt s e t t i n g ) a t 0°C. T h i s  m a t e r i a l was  f o r 30 min a t 15,000 x G a t 0°C.  then  centrifuged  The s u p e r n a t a n t was then  t a k e n and mixed w i t h normal DBA/2J membrane fragments l i n k e d t o Sepharose 4B.  T h i s s t e p was t a k e n t o ensure t h a t any m a t e r i a l  w h i c h n o n - s p e c i f i c a l l y bound t o DBA/2J membrane would be removed here.  A f t e r 60 min a t 0°C any non-bound m a t e r i a l was e l u t e d  w i t h i c e c o l d phosphate b u f f e r e d s a l i n e (PBS).  This material  was then mixed w i t h r a b b i t anti-mouse immunoglobulin Sepharose 4B.  linked to  T h i s was t o remove any a n t i - b o d y m o l e c u l e s  which  60 P r e p a r a t i o n of P815 Tumor-Specific Suppressor F a c t o r (P815-SF)  IP I n j e c t i o n of 100-200 ug of S o l u b l e P815 Membrane E x t r a c t i n t o DBA/2J Mice  4 Days  Splenocytes  (Contain P 8 1 5 - s p e c i f i c T suppressor  cells)  Sonication + Centrifugation  Supernate  (Crude E x t r a c t )  v Normal DBA/2 Membrane: Sepharose Column (60',  Elute with  Rabbit  PBS  Anti-MIg: Sepharose Column (60',  Elute with  P815  E l u t e Absorbed M a t e r i a l w i t h  0°)  PBS  Membrane: Sepharose Column (60',  Wash w i t h  0°)  0°)  PBS  2M  NaCl  si/  P815-Specific  F i g u r e 15.  Flow diagram i l l u s t r a t i n g t u m o r - s p e c i f i c suppressor  SF  the p r e p a r a t i o n of f a c t o r (P815-SF)  P815  61  might b i n d  i n the f o l l o w i n g s t e p .  The c a p a c i t y o f the a n t i -  mouse immunoglobulin column was s u f f i c i e n t  t o remove a t l e a s t  t w i c e t h e amount o f I g found i n the e x t r a c t s . 0°C  A f t e r 60 min a t  any non-bound m a t e r i a l , was e l u t e d w i t h i c e c o l d PBS.  This  m a t e r i a l was then mixed w i t h P815 membranes l i n k e d t o Sepharose 4B. ice  A f t e r 60 min a t 0°C the beads were washed t h o r o u g h l y w i t h c o l d PBS.  Bound m a t e r i a l was e l u t e d under m i d l y d i s s o c i a t i n g  c o n d i t i o n s w i t h 2M NaCl.  The absorbance a t 280 nm of t h i s  m a t e r i a l was i n a l l cases O.01; t h e r e f o r e , to q u a n t i f y i t other filter  sterilized  i t has been  than by i t s b i o l o g i c a l p r o p e r t i e s .  and s t o r e d a t -70°C.  Concentration  equivalents corrected  f o r suppressive  when mentioned i s quoted as s p l e e n  based on the volume o f o r i g i n a l s p l e e n homogenates  f o r the change i n volume f o l l o w i n g passage through  the columns.  V a l u e s f o r the e l u t e d f r a c t i o n s a r e based on the  assumption t h a t a l l the a c t i v i t y was r e c o v e r e d . mentioned columns were prepared as d e s c r i b e d by  I t was  T h i s m a t e r i a l which was  e l u t e d from the P815 column was then t i t r a t e d activity.  difficult  the cyanogen bromide method (141).  or f a c t o r i s i l l u s t r a t e d  A l l the above-  previously  The p r e p a r a t i o n  (65,140) of suppress-  i n F i g u r e 15.  Assay f o r suppressor c e l l s and suppressor f a c t o r s . The described  method used t o assay f o r s u p p r e s s o r c e l l s has been 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 c o c u l t u r e d w i t h 5 x 10^ (or fewer i n some cases) suppressor c e l l populations  i n the presence o f 5 x 10^ mitomycin  62  C t r e a t e d tumor c e l l s ,  i n 24 w e l l L i n b r o  complete 1640  C e l l s were c u l t u r e d f o r 5 days  being  medium.  assayed f o r c e l l s c y t o t o x i c to the tumor.  these s t u d i e s c o n s t i t u t e d the use c e l l s p l u s 5 x 10 had  t r a y s i n 2.5  of 5 x 10  normal s p l e e n c e l l s  ml  before  Controls  normal  or s p l e e n c e l l s which  Assay f o r the s p l e n i c suppressor  i n c u b a t i o n of 5 x 10^  in  spleen  been t r e a t e d i n a manner analogous to the suppressor  lation.  of  popu-  factor activity  normal s p l e e n c e l l s w i t h v a r i o u s  involved  concen-  t r a t i o n s of the f a c t o r p r e p a r a t i o n , under the same c o n d i t i o n s as o u t l i n e d above.  A l l experiments r e p o r t e d h e r e i n , u s i n g  assays were repeated ( t h e r e f o r e separate  at l e a s t  t h r e e times on separate  preparations  of both suppressor  these  occasions  cells  and  factor).  Preparation The  of a n t i s e r a a g a i n s t P 8 1 5 - s p e c i f i c  a n t i - P 8 1 5 - s p e c i f i c suppressor  factor antisera  P815-SF a n t i s e r a ) used i n t h i s study was syngeneic DBA/2J mice and with P815-specific (see above). parallel  suppressor  prepared by  a l l o g e n e i c C57BL/6 mice  ( B a l e n t l - " S F " ) was  from DBA/2J mice i n j e c t e d 4-5  B a l e n t l tumor membrane e x t r a c t s .  (antiimmunizing  repeatedly  immunoadsorbent e l u t e d suppressor  Control material  factor.  factor  prepared i n  days p r e v i o u s l y  T h i s m a t e r i a l was  with  injected  i n t o c o n t r o l groups of mice from the same b a t c h .  Animals were  injected  (0.036  subcutaneously every 2 weeks w i t h  equivalent)  of e i t h e r P 8 1 5 - s p e c i f i c  Balentl-"suppressor  0.1  suppressor  ml  spleen  f a c t o r or c o n t r o l  f a c t o r " i n complete Freund's adjuvant.  After  63  4 months, t h e mice were b l e d from t h e r e t r o o r b i t a l s i n u s once a month (one week a f t e r t h e l a s t immunization) w h i l e 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  a t 56°C f o r 30 m i n , then t e s t e d f o r i t s a b i l i t y t o a b r o g a t e i n t h e presence o f complement t h e f u n c t i o n o f P815 s p e c i f i c T suppressor c e l l s .  I n a l l experiments r e p o r t e d here t h e a n t i s e r a  from t h e mice w i t h i n each group were pooled b e f o r e t e s t i n g .  Each  of t h e f o u r groups c o n t a i n e d a t 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 t e s t e d f o r t h e i r e x p r e s s i o n o f P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r d e t e r m i n a n t s i n t h i s s t u d y were: P815  syngeneic  t u m o r - s p e c i f i c s u p p r e s s o r T c e l l s and s y n g e n e i c P815 tumor-  cytotoxic T cells.  I n p r e l i m i n a r y t e s t s t h e two a n t i - P 8 1 5 - S F  a n t i s e r a p o o l s were found t o be e f f e c t i v e , i n c o n j u n c t i o n w i t h r a b b i t complement ( R a b b i t Low Tox Complement, Cedar Lane L a b s , Hornby, O n t a r i o ) , i n e l i m i n a t i n g s u p p r e s s o r c e l l f u n c t i o n , a t d i l u t i o n s o f up t o 1:40.  I n experiments r u n h e r e a l l t h e a n t i s e r a  was used a t a f i n a l c o n c e n t r a t i o n o f 1:10, i n t h e presence o f a 1:9 f i n a l c o n c e n t r a t i o n o f complement.  At t h i s concentration  no a n t i - n o r m a l DBA/2J o r a n t i - t u m o r a c t i v i t y was d e t e c t e d i n t h e anti-P815-SF  antisera.  I n experiments t e s t i n g f o r t h e presence o f P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r d e t e r m i n a n t s on s u p p r e s s o r c e l l s , 2 x 1 0  7  s p l e n i c T s u p p r e s s o r 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^ s p l e n o c y t e s from  normal  64  DBA/2 c o n t r o l s , were suspended i n 0.40 ml of a 1:10 d i l u t i o n of a n t i s e r a and incubated f o r 45 min at room temperature.  Then  50 y l of neat r a b b i t complement was added and incubation was continued f o r 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 v i a b l e counts i n  c e l l suspensions containing the anti-P815-SF a n t i s e r a 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 c o n t r o l s , 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 t e s t i n g f o r the presence of P815-specific suppressor f a c t o r determinants on c y t o t o x i c 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 w i t h mitomycin C treated tumor c e l l s , were treated and subsequently tested i n the c y t o t o x i c i t y assay.  Since  t h i s experiment e s s e n t i a l l y resulted i n negative r e s u l t s , cytot o x i c c e l l s were also treated w i t h monoclonal anti-Thy 1 a n t i s e r a ( k i n d l y 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 a n t i s e r a absorption of suppressor f a c t o r a c t i v i t y . The a b i l i t y of anti-P815-SF a n t i s e r a to remove the suppressive a c t i v i t y from P815-specific suppressor factor preparations was tested by an immunoadsorbent assay s i m i l a r to the one described previously (Chapter 11,65). - I n short, P815-specific suppressor  65  f a c t o r p r e p a r a t i o n s or normal c o n t r o l m a t e r i a l prepared  in  p a r a l l e l were mixed w i t h e i t h e r the anti-P815-SF a n t i s e r a or the c o n t r o l a n t i - B a l e n t l - " S F " a n t i s e r a , and min  a t 0°C.  adsorbent  incubated  These m a t e r i a l s were then passed  through  for  120  immuno-  Sepharose 4B columns to which r a b b i t anti-mouse immuno-  g l o b u l i n had been a t t a c h e d by cyanogen bromide treatment. c a p a c i t y of the column was  s u f f i c i e n t to remove a t l e a s t  the amount of mouse immunoglobulin added to the factor preparations.  The twice  suppressor  The m a t e r i a l e l u t e d from the columns  was  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 over d i l u t i o n s r a n g i n g from 1:25  to 1:300  (these d i l u t i o n s of e l u t e d m a t e r i a l r e p r e s e n t  between 0.036 and  ELISA  0.0033 s p l e e n e q u i v a l e n t s ) .  assay. T h i s t e s t was  0.2  ml of a n t i g e n (e.g. P815-SF i n F i g 18)  b u f f e r was  i n pH 9.6  Briefly,  carbonate  a t t a c h e d to s u b s t r a t e m i c r o t i t e r p l a t e s (Cooke  E n g i n e e r i n g Co., 4°C.  c a r r i e d out as d e s c r i b e d b e f o r e . ( 1 4 2 ) .  A l e x a n d i a , Va., No.  1-220-295) f o r 18 h r a t  A f t e r washing w i t h PBS-tween b u f f e r , a n t i s e r a to be t e s t e d  f o r t h e i r a c t i v i t y a g a i n s t the coated a n t i g e n s were added to the w e l l s i n 0.2  ml a l i q u o t s .  a t room temperature and  subsequent washing, the  a l k a l i n e phosphatase-linked goat a n t i - r a b b i t  F o l l o w i n g i n c u b a t i o n f o r 2 hr developing  r a b b i t anti-mouse Ig (RaMI ) or  (GoRI ) , a t a d i l u t i o n of 1:600  r e s p e c t i v e l y , was 2 h i n c u b a t i o n and  added i n 0.2  ml a l i q u o t .  1:400  After a further  f i n a l washing w i t h b u f f e r , 0.2  enzyme s u b s t r a t e s o l u t i o n  or  ml of the  (Sigma-104-105, p - n i t r o p h e n y l phosphate  66  disodium) was added t o each w e l l and t h e enzyme s u b s t r a t e  reaction  was  allowed t o c o n t i n u e w h i l e c o l o r developed i n t h e w e l l s .  The  c o l o r change was f o l l o w e d  Laboratories).  on a T i t e r t e k M u l i s k a n  (Flow  A l l t e s t s were done a t l e a s t i n t r i p l i c a t e .  G o n t r o l s o f normal mouse serum (NMS), normal r a b b i t serum (NRS) or a n t i g e n alone w i t h no added serum were always r u n and t h e appropriate  c o n t r o l response was s u b t r a c t e d  t o get the s p e c i f i c  response.  Antisera P815 viously  p l u s complement k i l l i n g of P815 tumor c e l l s . tumor c e l l s were l a b e l e d w i t h " ^ C r as d e s c r i b e d (Chapter II) .  The "'"'"Cr l a b e l e d  P815 tumor c e l l s were  resuspended a t 2 x 10^/ml i n complete medium. of t h e s e c e l l s were d i s p e n s e d i n t o t h e w e l l s microculture  plates.  pre-  100 u l (2 x 10^) of m u l t i - d i s h  50 u l o f a n t i s e r a p l u s 50 u l o f 1:2 d i l u t i o n  of r a b b i t complement was then added t o each w e l l .  Appropriate  c o n t r o l s o f complement and medium o n l y were r u n c o n c u r r e n t l y . They were then incubated f o r 90 minutes a t room temperature. ml  0.10  o f t h e 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 v i v o e f f e c t s o f anti-P815-SF a n t i s e r a . The out  i n vivo  experiments o f anti-P815-SF a n t i s e r a were c a r r i e d  t o 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 t o i n j e c t i o n o f P815 tumor c e l l s 50 u l o f a n t i P815-SF a n t i s e r a , a n t i - B a l e n t l - " S F " jected  i . v . i n t o DBA/2 mice.  a n t i s e r a , or PBS were i n -  3  Two-days l a t e r 2 .x 10. P815 tumor  67  c e l l s were i n j e c t e d  s.c. i n t o these mice.  Tumor growth was f o l l o w e d  by two d i m e n s i o n a l measurements w i t h c a l i p e r s . groups,  and these experiments  occasions.  There were 7 mice/  were c a r r i e d out on t h r e e s e p a r a t e  T h e r e f o r e , a t o t a l o f 21 mice i n each o f the f o u r  groups.  The combined r e s u l t s a r e r e p o r t e d here. A n a l y s i s o f Data. On a l l o c c a s i o n s , experiments times.  W i t h i n each experiment,  were repeated a minimum o f t h r e e  a l l treatment  groups were t e s t e d f o r  c y t o t o x i c i t y i n q u a d r u p l i c a t e over an e f f e c t o r : t a r g e t r a t i o range o f 100:1 to 12.5:1 i n the ^ C r r e l e a s e 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 u n i t s was c a l c u l a t e d as d e s c r i b e d p r e v i o u s l y (Chapters I I and I I I ) and t o t a l l y t i c u n i t s from r e c o v e r e d c e l l s in vitro cal  c u l t u r e i s r e c o r d e d i n the f i g u r e o r t a b l e legends.  a n a l y s i s o f d a t a from repeated  t-test.  experiments  after  Statisti-  was done by S t u d e n t s '  These r e s u l t s are shown i n t a b l e s where a p p r o p r i a t e .  68  Results S i n c e i t had been found (Chapter 111,13,125) t h a t i n t r a p e r i t o n e a l i n j e c t i o n o f s o l u b i l i z e d tumor membrane a n t i g e n s i n d u c e , i n t h e s p l e e n , T s u p p r e s s o r c e l l s s p e c i f i c f o r t h a t tumor, an attempt was made t o p r e p a r e from t h e s e c e l l s r e l a t i v e l y pure s p e c i f i c s u p p r e s s o r 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 o f t h e P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r was a c h i e v e d by p a s s i n g crude s p l e e n 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 s u b s e q u e n t l y e l u t i n g from t h e column t h e P815s p e c i f i c s u p p r e s s o r f a c t o r w i t h 2M N a C l . F i g u r e 16a shows,, t h a t when added t o i n v i t r o c u l t u r e s , t h e 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 t o P815 tumor c e l l s when.compared control cultures.  to untreated  The s p e c i f i c i t y o f t h e P 8 1 5 - s p e c i f i c s u p p r e s s o r  f a c t o r i s shown by i t s i n a b i l i t y t o a f f e c t t h e g e n e r a t i o n o f c y t o t o x i c c e l l s t o L1210, another tumor syngeneic t o DBA/2J mice ( F i g . 16b) . The P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r was found t o be r e l a t i v e l y s t a b l e when s t o r e d a t -70°C, and was a c t i v e a t v e r y h i g h d i l u t i o n s . F i g u r e 17 shows a t y p i c a l t i t r a t i o n c u r v e f o r t h i s s u p p r e s s o r f a c t o r . W h i l e t h e s u p p r e s s o r f a c t o r was v e r y i n h i b i t o r y a t h i g h d i l u t i o n s , at h i g h c o n c e n t r a t i o n s i t was n o t , i n f a c t sometimes i t was somewhat s t i m u l a t o r y .  The r e a s o n f o r t h i s i s n o t u n d e r s t o o d , but t h i s  was a r e p r o d u c i b l e o b s e r v a t i o n .  The t i t r a t i o n c u r v e ( F i g u r e 17)  may p o s s i b l y be accounted f o r by the presence o f P 8 1 5 - s p e c i f i c h e l p e r  69  F i g u r e 16.  The a b i l i t y of P 8 1 5 - s p e e i f i c s u p p r e s s o r f a c t o r t o suppress the i n v i t r o p r i m a r y c y t o t o x i c response o f normal DBA/2J s p l e n o c y t e s t o s y n g e n e i c m i t o m y c i n C t r e a t e d P815 tumor cells. ,(a) A n t i - P 8 1 5 : 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) A n t i - L 1 2 1 0 : t h e 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. • •, p r i m a r y c u l t u r e of normal DBA/2J s p l e n o c y t e s and 1/150 d i l u t i o n (0.0071. s p l e e n e q u i v a l e n t ) 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. • • , p r i m a r y c u l t u r e of normal DBA/2J s p l e n o c y t e s , no s u p p r e s s o r 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  -40  \-  1:150  1:600  1:1200  DILUTION  F i g u r e 17.  T i t r a t i o n o f immunoadsorbent p u r i f i e d P 8 1 5 - s p e c i f i c supp r e s s o r f a c t o r . C y t o t o x i c v a l u e s were c a l c u l a t e d a t an e f f e c t o r : t a r g e t r a t i o o f 50:1 and converted to % supp r e s s i o n by comparison w i t h e q u i v a l e n t c u l t u r e s incubated i n the presence o f a c o n t r o l suppressor f a c t o r i s o l a t e d i n an analogous f a s h i o n from L1210 primed mice.  71  f a c t o r which c o u l d c o - p u r i f y w i t h the suppressor m a t e r i a l .  However,  o t h e r i n v e s t i g a t o r s , working w i t h suppressor f a c t o r p r o d u c i n g h y b r i domas have a l s o found t h a t h i g h c o n c e n t r a t i o n s of hybridoma  super-  n a t a n t s a r e sometimes much l e s s s u p p r e s s i v e than are h i g h e r d i l u t i o n s (143), so t h i s may  not be the e x p l a n a t i o n .  While the methods used f o r the  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 s u p p r e s s o r f a c t o r were c a r r i e d to maximize the p u r i t y o f the f a c t o r , i t i s q u i t e p r o b a b l e t h a t m a t e r i a l e l u t e d from P815  columns c o n t a i n e d a number of  (<0.01 absorbance  to e s t a b l i s h i t s degree However, i t appears  a t 280 nm),  the  contaminants.  Because of the e x c e e d i n g l y s m a l l amounts of p r o t e i n i n our preparations  out  factor  a t t h i s time i t i s d i f f i c u l t  of p u r i t y by c o n v e n t i o n a l b i o c h e m i c a l means.  t h a t t h i s m a t e r i a l has p r o p e r t i e s analogous  to  those d e s c r i b e d p r e v i o u s l y (Chapter 11,65,123) f o r a s u p p r e s s o r i s o l a t e d by s i m i l a r methods from thymocytes of P815-bearing  factor  mice  (Chapter 11,65). Taken t o g e t h e r , these r e s u l t s show t h a t a v e r y i n h i b i t o r y highly specific  suppressor f a c t o r can be prepared  DBA/2J mice p r e v i o u s l y i n j e c t e d  and  from s p l e e n s of  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 l e tumor  membrane e x t r a c t s . S i n c e 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  s e t up t o immunize groups of both syngeneic DBA/2J and  a l l o g e n e i c C57BL/6 mice w i t h P 8 1 5 - s p e c i f i c suppressor f a c t o r . was  t h a t i n the syngeneic mice the u n i q u e  (idiotypic?),  found  i n or around  determinants  the a n t i g e n b i n d i n g s i t e of the  s p e c i f i c suppressor f a c t o r , would e l i c i t l e a s t some o f the animals.  The hope  an a n t i b o d y response  While both these and  the  P815-  i n at  constant  72  determinants on the suppressor f a c t o r might i n the a l l o g e n e i c mice.  induce an a n t i b o d y response  C o n t r o l groups of mice were i n j e c t e d w i t h  m a t e r i a l ( B a l e n t l - " S F " ) prepared i n an i d e n t i c a l f a s h i o n ( i . e . d i l u t i o n o f f o f P815 membrane columns) from DBA/2J mice, i n t r a p e r i t o n e a l l y w i t h B a l e n t l tumor e x t r a c t s .  injected  A l l the r e s u l t s  r e p o r t e d here a r e from a n t i s e r a pooled w i t h i n each of the f o u r The  final  groups.  f a c t t h a t the DBA/2J and C57BL/6 a n t i s e r a thus prepared  c o n t a i n e d 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 P 8 1 5 - s p e c i f i c suppressor m a t e r i a l . A r e p r e s e n t a t i v e s e t of d a t a i s shown i n T a b l e I I . P 8 1 5 - s p e c i f i c s u p p r e s s o r f a c t o r was mixed w i t h e i t h e r the anti-P815-SF a n t i - B a l e n t l - " S F " a n t i s e r a , and subsequently absorbed adsorbent columns c o n t a i n i n g anti-mouse  a n t i s e r a or  on immuno-  immunoglobulin.  The unattached  e l u t e d m a t e r i a l was then t e s 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 . s u p p r e s s i v e a c t i v i t y was s t i l l  p r e s e n t i n the c o n t r o l  While  anti-Balentl-  "SF" a n t i s e r a t r e a t e d 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 anti-P815-SF  antisera lost  DBA/2J anti-P815-SF recognize  i t s a c t i v i t y , thus showing t h a t  a n t i s e r a and C57BL/56 anti-P815-SF  both  antisera  and can b i n d determinants expressed on the P 8 1 5 - s p e c i f i c  suppressor f a c t o r .  T h i s experiment was r u n on t h r e e s e p a r a t e o c c a s i o n s  and, t h e d i f f 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  a n t i - B a l e n t l - " S F " a n t i s e r a to b i n d t o determinants expressed on P815-SF i s shown u s i n g a d i f f e r e n t method i n F i g u r e 18.  The ELISA  assay was used to show t h a t both.the DBA/2 and the C57BL/6  anti-  P815-SF a n t i s e r a r e a c t e d p o s i t i v e l y w i t h P815-SF, w h i l e n e i t h e r of  Table I I .  P815-Specific SF added  l  A n t i - P 8 1 5 a n t i s e r a i s a b l e t o r e a c t and b i n d 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 r e p a r a t i o n s 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 anti-mouse Ig.  P815-Specific SF Absorbed W i t h  a  b  . . % Cytotoxicity , 100:1 50:1  Total L y t i c Units  6  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  23.1  <.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 "SF"  4 9 . 1 ± 1.9  20.9 ± 1 . 1  25.8  <.005  <.005  +  -  41.1±3.9  17.5 ±1.4  21.7  —  —  56.9 ± 0 . 9  36.7 ± 1 . 3  39.6  SF added a t t h e s t a r t of 5 day i n v i t r o c u l t u r e s f o r the g e n e r a t i o n 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 n e x t column p r i o r t o a d d i t i o n . A 1/150 d i l u t i o n ( 0 . 0 0 7 1 s p l e e n e q u i v a l e n t ) of SF was u s e d .  'SF was mixed w i t h the a n t i s e r a then passed through an a n t i - m o u s e I g column. M a t e r i a l w h i c h passed t h r o u g h was t e s t e d f o r s u p p r e s s i v e a b i l i t y . C o n t r o l SF p r e p a r a t i o n s were o n l y passed through a n t i - m o u s e I g column t h e n t e s t e d . Cytotoxicity  was t e s t e d by 18 h ~^Cr r e l e a s e a s s a y .  Effector:Target  Numbers a r e % s p e c i f i c r e l e a s e ± S . E . M .  ratio.  4 One l y t i c u n i t was d e f i n e d as t h e 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 T o t a l l y t i c u n i t s were t h e number of l y t i c u n i t s found i n t h e r e c o v e r e d c e l l p o p u l a t i o n . D a t a were a n a l y s e d by S t u d e n t s ' t - t e s t . significant.  N . S . - not s i g n i f i c a n t .  cells.  P v a l u e s of <.05 were c o n s i d e r e d  Table I I I .  R e s u l t s o f Students' t - t e s t r u n on 3 s e p a r a t e experiments t e s t i n g 3 i n d i v i d u a l b l e e d s of mice immunized w i t h P815-SF or c o n t r o l B a l e n t l "SF'^ D i f f e r e n c e s between groups were c a l c u l a t e d from d a t a o b t a i n e d in Cr r e l e a s e assay a t e f f e c t o r - t a r g e t r a t i o s o f 50:1 ( l e v e l s a t which k i l l i n g i s i n the l i n e a r p a r t o f the c u r v e ) . A l l groups were compared t o the k i l l i n g i n c u l t u r e s c o n t a i n i n g suppressor c e l l s which had been t r e a t e d o n l y w i t h C.  Treatment  of Suppressor  Cells Expt. 1  P Expt. 2  Expt. 3  anti-P815-SF  <.025  <.005  <.005  DBA a n t i - B a l e n t l - " S F "  N.S.  N.S.  N.S.  DBA  C57BL/6  anti-P815-SF  C57BL/6 a n t i - B a l e n t l - " S F "  P v a l u e s o f <.05 a r e c o n s i d e r e d s i g n i f i c a n t .  a  <.05 ' <.025  <.05 N.S.  <.005 N.S.  75  0.8  o  J3 0 M  0.6  0.4  < 0.2  '/10  V40 Antisera  F i g u r e 18.  Vo40  Dilution  C h a r a c t e r i z a t i o n o f P815-SF by ELISA a s s a y . P815-SF (0.0023 s p l e e n e q u i v a l e n t ) was used as a n t i g e n i n t h e ELISA. V a r i o u s a n t i s e r a were added t o d e t e r m i n e what c h a r a c t e r i s t i c s were e x p r e s s e d on t h e 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-P815SF, • •, C57BL/6 a n t i - B a l e n t l - " S F " , k 4, a n t i - H - 2 ; A A, a n t i - l a ; x x, anti-MIg. I n c u b a t i o n o f p l a t e a f t e r a d d i t i o n o f enzyme s u b s t r a t e was f o r 45 m i n u t e s .  76  the a n t i - B a l e n t l - " S F " a n t i s e r a r e a c t e d These r e s u l t s c o n f i r m (Table I I ) .  the r e s u l t s of the a b s o r b t i o n  levels.  experiments  Another important f e a t u r e to note i s which of the  antisera preparations and  above background  r e a c t s w i t h the P815-SF.  other  While both a n t i - H Z ^  a n t i - l a * * a n t i s e r a r e a c t w i t h P815-SF, anti-mouse Ig a n t i s e r a  does n o t .  T h i s shows t h a t P815-SF has  r e g i o n of the H-2  determinants coded by  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.  are the same as those d e s c r i b e d  previously for P815-specific  pressor II,  f a c t o r i s o l a t e d from thymocytes of P815  the l a  These p r o p e r t i e s sup-  b e a r i n g mice  (Chapter  65,21). The  p o s s i b i l i t y t h a t the a c t i v i t y of the P 8 1 5 - s p e c i f i c  T  suppressor c e l l s c o u l d be abrogated by anti-P815-SF a n t i s e r a p l u s complement was spleen  investigated.  P r i o r to a d d i t i o n to normal DBA/2J  c e l l s p l u s mitomycin C t r e a t e d P815  c e l l s a t the s t a r t of i n  v i t r o c u l t u r e , the s p e c i f i c T s u p p r e s s o r c e l l  population  was  treated  w i t h complement p l u s e i t h e r anti-P815-SF a n t i s e r a or a n t i - B a l e n t l "SF"  antisera.  syngeneic and  Both of the anti-P815-SF a n t i s e r a produced i n the a l l o g e n e i c mice were e f f e c t i v e i n e l i m i n a t i n g the  p r e s s i v e e f f e c t n o r m a l l y produced by these c e l l s as w i t h the a d s o r p t i o n  of the P 8 1 5 - s p e c i f i c  ( F i g . 19).  sup-  Again,  suppressor f a c t o r , n e i t h e r  of the a n t i - B a l e n t l - " S F " a n t i s e r a c o n t r o l s had  an e f f e c t .  the b l e e d s t e s t e d the DBA/2J anti-P815-SF a n t i s e r a and  With a l l  the C57BL/6  anti-P815-SF a n t i s e r a were a p p r o x i m a t e l y e q u a l l y e f f e c t i v e (Table I I I ) . The it  d a t a p r e s e n t e d i n F i g u r e 19  i s from experiment 2.  appears t h a t i n t h i s system the suppressor c e l l ,  Therefore,  i t s progenator,  77  1? 5 1  •Figure 19.  25:1  50-1 EFFECTOR:TARGET  12.5:1 RATIO  251  50:1  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 t o 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 p r i m e d i n v i t r o w i t h P815 a f t e r t r e a t m e n t of the s u p p r e s s i v e s p l e n o c y t e s 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 t r e a t m e n t of the s u p p r e s s i v e s p l e n o c y t e s w i t h C57BL/6 a n t i - S F a n t i s e r a p l u s complement . • ^ , p r i m a r y c u l t u r e of normal DBA/2J s p l e n o c y t e s 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 splenocytes. T o t a l l y t i c u n i t s (a) 101.02 (b) 90.13 • • , p r i m a r y c u l t u r e of normal DBA/2J s p l e n o c y t e s 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 splenocytes. T o t a l l y t i c u n i t s (a) 6 3 . 1 9 (b) 52.25 0 0 , p r i m a r y c u l t u r e of normal DBA/2J s p l e n o c y t e s p l u s complement o n l y t r e a t e d P 8 1 5 - s p c i f i c s u p p r e s s o r s p l e n o cytes. T o t a l l y t i c u n i t s (a) and (b) 5 7 . 5 4 .  78  or a c e l l v i t a l  f o r i t s development, expresses  on i t s s u r f a c e ,  sup-  p r e s s o r f a c t o r , or a t l e a s t determinants common t o i t . The p o s s i b i l i t y t h a t i n v i t r o generated syngeneic c e l l s c y t o t o x i c f o r P815 c o u l d a l s o be k i l l e d by these a n t i s e r a was a l s o i n vestigated. P815  I n v i t r o generated c y t o t o x i c T c e l l s s p e c i f i c  o r L1210 were t r e a t e d w i t h complement p l u s e i t h e r  a n t i s e r a or anti-Balentl-"SF" antisera. n e i t h e r t h e syngeneic DBA/2J anti-P815-SF C57BL/6 anti-P815-SF  P815  anti-P815-SF  As can be seen i n T a b l e IV, a n t i s e r a n o r the a l l o g e n e i c  a n t i s e r a had any e f f e c t on t h e k i l l i n g  out by t h e c y t o t o x i c T c e l l s .  for either  carried  T h e r e f o r e , showing t h a t i n t h e syngeneic  tumor system o f DBA/2J mice t h e s p e c i f i c c y t o t o x i c T c e l l s  lack  determinants i n common w i t h those found on e i t h e r t h e s p e c i f i c T s u p p r e s s o r c e l l s or t h e s p e c i f i c suppressor f a c t o r as d e t e c t e d by the a n t i - S F a n t i s e r a .  I n r e p e a t s o f 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 o c c a s i o n s . F i n a l l y t h e i n v i v o e f f e c t o f t h e anti-P815-SF tumor growth i n DBA/2 mice was a s s e s s e d .  a n t i s e r a on P815  Two days p r i o r t o i n j e c t i o n  w i t h P815 tumor c e l l s , DBA/2 mice were i n j e c t e d i n t r a v e n o u s l y w i t h 50 y l o f one o f t h e f o u r a n t i s e r a p r e p a r a t i o n s on PBS.  F i g u r e 20  shows t h e growth o f t h e tumor a f t e r i n j e c t i o n subcutaneously. can be seen t h e DBA/2 anti-P815-SF growth o f t h e P815 tumor. e f f e c t on tumor growth.  As  a n t i s e r u m g r e a t l y slowed t h e  The o t h e r a n t i s e r a p r e p a r a t i o n s had no These r e s u l t s a r e a l s o born-out  improved  s u r v i v a l time o f animals i n j e c t e d w i t h DBA/2  antisera  (Table V).  i n the  anti-P815-SF  The mice i n j e c t e d w i t h DBA/2 anti-P815-SF  antisera  had a 48% i n c r e a s e d s u r v i v a l time compared t o those i n j e c t e d w i t h PBS  79  Table IV.  E f f e c t o r s Generated Against  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 lyse the appropriate target.  C e l l s Treated W i t h Antisera  P815  % Cytotoxicity  b  C  100:l  d  +  6.6 ± 3.3  2.5 ± 1.4  DBA anti-P815-SF  +  40.5 ± 2.5  28.5 ± 4.7  N.S.  DBA a n t i - B a l e n t l - " S F "  +  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 a n t i - B a l e n t l - " S F "  +  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  25:l  d  12.5:l  <.005  d  Anti-Thy 1  +  9.8 ± 2.7  1.2 ± 0.8  DBA anti-P815-SF  +  40.5 + 3.9  24.1 ± 1.6  N.S.  DBA a n t i - B a l e n t l - " S F "  +  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 a n t i - B a l e n t l - " S F "  +  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  50:l  e  Anti-Thy 1  L1210  d  p  0  <.005  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 f o r 5 days as described i n the M a t e r i a l s and Methods.  ^The e f f e c t o r c e l l s were treated with a n t i s e r a plus complement then washed and tested f o r t h e i r a b i l i t y to lyse the appropriate ^ l c r l a b e l l e d targets as described i n M a t e r i a l s and Methods. Control e f f e c t o r populations were treated with only complement or media alone. c  C y t o t o x i c i t y was tested by 18 h  51  Cr r e l e a s e assay.  . Numbers are % s p e c i f i c release ± S.E.M.  d  Effector:Target  ratio.  e  D a t a were a n a l y s e d b y Students, t - t e s t . N.S. - not s i g n i f i c a n t . In three 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 any of the anti-SF  groups.  80  255  SIZE OF  195  TUMOR  135  75  -2  / a n t i s e r a iv  F i g u r e 20.  0  11  15  19  21  \ 2M0 P815 sc 3  DAYS  E f f e c t o f anti-P815-SF a n t i s e r a i n v i v o on P815 tumor growth^in syngeneic DBA/2 mice. Mice were i n j e c t e d w i t h 2 x .10 P815 subcutaneously on day 0. Two days p r i o r 50 y l o f a n t i s e r a had been i n j e c t e d i n t r a v e n e o u s l y . Tumor s i z e was measured i n two dimensions with c a l i p e r s . This f i g u r e shows t h e combined r e s u l t s o f t h r e e s e p a r a t e experiments w i t h a t o t a l o f 21 mice/group. A A, DBA/2 anti-P815-SF; • •, C57BL/6 anti-P815-SF; A A, DBA/2 anti-Balentl-"SF"; • • , C57BL/6 a n t i - B a l e n t l - " S F " ; • • , PBS.  81  T a b l e V.  E f f e c t o f anti-P815-SF a n t i s e r a i n v i v o on s u r v i v a l time o f DBA/2 mice a f t e r P815 ..... a tumor i n j e c t i o n .  DBA/2 mice i n j e c t e d i . v . with DBA/2 anti-P815-SF  anitsera  DBA/2 a n t i - B a l e n t l - " S F " C57BL/6 anti-P815-SF  antisera  antisera  C57BL/6 a n t i - B a l e n t l - " S F " PBS  mean s u r v i v a l time 10 20 30  antisera  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  (days)  c  40  *Three s e p a r a t e experiments were r u n and t h e r e s u l t s were combined f o r this table. "T)BA/2 mice were i n j e c t e d i . v . wij^h 50 u l o f a n t i s e r a two days p r i o r to P815 tumor c h a l l e n g e . 2 x 10 P815 tumor c e l l s were i n j e c t e d s.c. on day z e r o . "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) a f t e r i n j e c t i o n o f 2 x 10 P815 tumor c e l l s s . c .  o f mice  82  alone.  T h e r e f o r e , even though both DBA anti-P815-SF a n t i s e r a and  C57BL/6 anti-P815-SF a n t i s e r a were r e a c t i v e i n v i t r o o n l y t h e syngeneic DBA/2 p r e p a r a t i o n shows any e f f e c t  i n vivo.  Experiments were a l s o c a r r i e d out i n v i v o w i t h t h e anfc.i-P815-SF a n t i s e r a t o a s s e s s 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 F i g u r e 21 and T a b l e VI show t h a t n e i t h e r t h e syngeneic DBA/2 anti-P815-SF n o r t h e 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 v i v o . S i n c e t h e p r e p a r a t i o n o f t h e P815-SF used t o induce t h e a r i t i P815-SF a n 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 s t e p o f f a P815 membrane: Sepharose column, t h e r e i s a p o s s i b i l i t y t h a t t h e P815-SF m a t e r i a l c o u l d a l s o c o n t a i n s m a l l amounts o f P815 a n t i g e n which was shed o f f t h e column a l o n g w i t h t h e bound s u p p r e s s o r f a c t o r .  I t i s there-  f o r e p o s s i b l e t h a t t h e a n t i s e r a may c o n t a i n anti-P815 a n t i g e n a c t i v i t y , which c o u l d i n t e r f e r e w i t h t h e i n t e r p r e t a t i o n of some o f t h e r e s u l t s r e p o r t e d here. this  Two d i f f e r e n t experiments were s e t up t o e x p l o r e  possibility. The f i r s t  i s shown i n F i g u r e 22.  Here t h e two anti-P815-SF  a n t i s e r a were t e s t e d f o r t h e i r a b i l i t y t o l y s e "^Cr l a b e l e d P815 tumor c e l l s i n t h e presence o f complement.  As can be seen n e i t h e r  the DBA/2 anti-P815-SF a n t i s e r a nor t h e C57BL/6 anti-P815-SF had any anti-P815 l y t i c  antisera  ability.  The second experiment, t o determine i f t h e two anti-P815-SF a n t i s e r a c o n t a i n any anti-P815 a c t i v i t y , was t o r e a c t these a n t i s e r a w i t h P815 membrane e x t r a c t s i n an ELISA assay.  I t can be seen i n  F i g u r e 23 t h a t both DBA/2 and C57BL/6 anti-P815-SF a n t i s e r u m c o n t a i n  83  -2  antisera iv  0  8  2x10 P815sc 3  9  10  DAYS  11  12  13  j , ! i  Figure  21. E f f e c t o f a n t i - P 8 1 5 - S F a n t i s e r a i n v i v o on L1210 tumor g r o w t h ^ i n s y n g e n e i c DBA/2 mice. M i c e were i n j e c t e d w i t h 2 x 10 L1210 s u b c u t a n e o u s l y on day 0. Two days p r i o r 50 y l o f a n t i s e r a had been i n j e c t e d i n t r a v e n e o u s l y . Tumor s i z e was measured i n two dimensions w i t h c a l i p e r s . T h i s f i g u r e shows t h e combined r e s u l t s o f two s e p a r a t e experiments w i t h a t o t a l o f 18 mice/group. A A, DBA/2 a n t i - P 8 1 5 - S F ; •- •, C57BL/6 a n t i - P 8 1 5 - S F ; • -A, DBA/2 anti-Balentl-"SF"; • • , 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 o f anti-P815-SF a n t i s e r a i n v i v o om' s u r v i v a l time o f DBA/2 mice a f t e r L1210 tumor i n j e c t i o n .  *• b DBA/2 mice i n j e c t e d i . v . with DBA/2 anti-P815-SF  antisera  DBA/2 a n t i - B a l e n t l - " S F " C57BL/6 anti-P815-SF  antisera  antisera  C57BL/6 a n t i - B a l e n t l - " S F " PBS  mean s u r v i v a l time (days) 5 10 15 20  antisera  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 s e p a r a t e experiments were r u n and t h e r e s u l t s were combined f o r this table. 'DBA/2 mice were i n j e c t e d i . v . witb^ 50 y l o f a n t i s e r a two days p r i o r to L1210 tumor c h a l l e n g e . 2 x 10 L1210 tumor c e l l s were i n j e c t e d s.c. on day z e r o . 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) a f t e r i n j e c t i o n o f 2 x 10 L1210 tumor c e l l s s . c .  of mice  85  80  60  40  20  Vt  J/64  Dilution  F i g u r e 22.  of  '/256  Antisera  The i n a b i l i t y of anti-P^15-SF a n t i s e r a , i n the presence g£ complement, to l y s e Cr l a b e l e d P815 tumor c e l l s . Cr l a b e l e d P815 tumor c e l l s were i n c u b a t e d w i t h v a r i o u s d i l u t i o n s o f a n t i s e r a p l u s complement DBA/2 anti-P815-SF; o o, C57BL/6 anti-P815-SF; Hi, a n t i H-2 ; • •, a n t i - l a d  86  '4  '/20  '/80  '/320  '^280  I I  Dilution  F i g u r e 23.  of  Antisera  Does a n t i - S F a n t i s e r a have any a c t i v i t y d i r e c t e d towards P815 tumor membrane d e t e r m i n a n t s ? Crude P815 membrane e x t r a c t was used as a n t i g e n i n the ELISA assay to determine i f a n t i - S F a n t i s e r a has any a c t i v i t y d i r e c t e d a g a s i n t P815. • • , DBA/2 anti-P815-SF, 0 o, C57BL/6 a n t i P815-SF, • •, r a b b i t anti-P815; P — r a b b i t anti-DBA/2. I n c u b a t i o n o f p l a t e s a f t e r a d d i t i o n of enzyme s u b s t r a t e was f o r 90 minutes.  o n l y a v e r y s m a l l amount of anti-P815  reactivity.  I t should be noted  t h a t the ELISA assay i s a v e r y s e n s i t i v e method of d e t e c t i n g a n t i b o d y r e a c t i v i t y , w i t h o n l y v e r y s m a l l amounts specific  antibody necessary  s h o r t time  (142).  i n the anti-P815-SF of  ment .  as 50 ng/ml) o f  t o show a p o s i t i v e r e a c t i o n i n a very  T h e r e f o r e the amount of anti-P815  reactivity  a n t i s e r a p r e p a r a t i o n s i s a very s m a l l  the t o t a l r e a c t i v i t y  experiments  (as l i t t l e  found  percentage  and p r o b a b l y p l a y s no r o l e i n any of the  r e p o r t e d here, e s p e c i a l l y the ones mediated through  comple-  88  Discussion The r e s u l t s presented p r e v i o u s l y (Chapter I I ) show t h a t t o n e a l i n j e c t i o n o f s o l u b i l i z e d P815  intraperi-  tumor membrane a n t i g e n i n t o  syn-  g e n e i c DBA/2J mice i n d u c e s , i n the s p l e e n , T suppressor c e l l s  specific  f o r P815.  and  I t was  shown i n t h i s chapter t h a t a v e r y i n h i b i t o r y  relatively purified  P 8 1 5 - s p e c i f i c suppressor f a c t o r c o u l d be  from these s u p p r e s s i v e s p l e e n c e l l s . f a c t o r was  (b)  T h i s P 8 1 5 - s p e c i f i c suppressor  i n j e c t e d i n t o syngeneic DBA/2J and a l l o g e n e i c C57BL/6  mice and the r e s u l t i n g a n t i s e r a was specific  shown to (a)  interact with  P815-  suppressor f a c t o r by a b s o r p t i o n s t u d i e s and ELISA a s s a y s ,  i n t e r a c t w i t h P 8 1 5 - s p e c i f i c T suppressor c e l l s by  mediated  prepared  k i l l i n g s t u d i e s , and  (c)  not t o i n t e r a c t w i t h P 8 1 5 - s p e c i f i c  T c y t o t o x i c c e l l s by complement mediated o n l y the syngeneic a n t i s e r a was  complement  k i l l i n g studies, while  (d)  shown to be e f f e c t i v e i n v i v o .  The r e s u l t s shown i n F i g u r e 16 i n d i c a t e t h a t a s u p p r e s s o r e x t r a c t e d from a s u p p r e s s i v e c e l l p o p u l a t i o n can d u p l i c a t e the o g i c a l f u n c t i o n of these c e l l s .  factor biol-  In t h i s case both the T suppressor  c e l l and the suppressor f a c t o r presumed d e r i v e d from i t are a b l e to s p e c i f i c a l l y suppress the primary i n v i t r o g e n e r a t i o n of c y t o t o x i c T c e l l s t o P815  (8, Chapter  II).  These r e s u l t s p l u s those i n F i g u r e  18 show t h a t t h i s suppressor f a c t o r has p r o p e r t i e s analogous d e s c r i b e d p r e v i o u s l y f o r a suppressor f a c t o r i s o l a t e d by methods from thymocytes of P815-bearing  to those  similar  DBA/2 mice (9,21,65,123,  Chapter I I ) . I t was anti-P815-SF  then p o s s i b l e to use the P815-suppressor antisera.  T h i s was  f a c t o r to prepare  done by i n j e c t i n g both  syngeneic  89  DBA/2 and a l l o g e n e i c C57BL/6 mice w i t h P815-SF.  As T a b l e  I I and  F i g u r e s 18 and 19 show the r e s u l t i n g anti-P815-SF a n t i s e r a c o u l d r e a c t w i t h determinants expressed suppressor  cell  on the suppressor  i t i s presumed d e r i v e d from.  T a b l e IV i n d i c a t e t h a t determinants expressed suppressor pressed  f a c t o r and the  The r e s u l t s shown i n on P815  f a c t o r and P 8 1 5 - s p e c i f i c T suppressor  specific  c e l l s a r e not ex-  on P 8 1 5 - s p e c i f i c T c y t o t o x i c c e l l s .  S i n c e the p r e p a r a t i o n of the P815-SF used t o induce  the a n t i -  P815-SF a n 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 of a P815 membrane column, t h e r e i s a p o s s i b i l i t y t h a t the P815-SF m a t e r i a l c o u l d a l s o c o n t a i n s m a l l amounts of P815 a n t i g e n which was shed o f f the column along with  the bound suppressor  factor.  t h a t the a n t i s e r a p o s s i b l y c o n t a i n e d could i n t e r f e r e with r e p o r t e d here.  I t i s therefore possible  anti-P815 a n t i g e n a c t i v i t y , which  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  The r e s u l t s shown i n F i g u r e 22 and 23 show t h a t the  amount of anti-P815 r e a c t i v i t y found i n the anti-P815-SF a n t i s e r a i s a t most a v e r y s m a l l percentage of the t o t a l r e a c t i v i t y and p l a y s no r o l e i n any of the experiments r e p o r t e d here, ones mediated through complement. "SF"  was prepared  And o f c o u r s e ,  probably  e s p e c i a l l y the  s i n c e the B a l e n t l -  i n an i d e n t i c a l f a s h i o n 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 a n t i s e r a prepared  to i t  s e r v e s as an e x c e l l e n t i n t e r n a l c o n t r o l f o r these experiments.  It  should be r e s t a t e d here t h a t a t the c o n c e n t r a t i o n used i n t h i s work t h e r e was no d e t e c t a b l e a n t i - n o r m a l the a n t i s e r a p r e p a r a t i o n s . f a c t o r was prepared  DBA/2J a c t i v i t y found i n any of  A l s o i t should be noted t h a t the  suppressor  from DBA/2J c e l l s primed i n v i v o , and d u r i n g  90  p r e p a r a t i o n and p r i o r t o immunization and a l l o g e n e i c mice, was serum.  i n t o the a p p r o p r i a t e syngeneic  o n l y suspended i n PBS without f e t a l  calf  T h e r e f o r e , i t i s r e a s o n a b l e to assume t h a t any a n t i b o d i e s  b e i n g formed i n immunized animals would be d i r e c t e d e x c l u s i v e l y t o a n t i g e n s of DBA/2J o r i g i n .  Thus, i t seems p r o b a b l e , t h a t the  anti-  P815-SF a n t i s e r a from the syngeneic DBA/2J mice i s d i r e c t e d t o unique determinants  ( i d i o t y p i c ? ) expressed a t or near the a n t i g e n b i n d i n g  s i t e of the suppressor f a c t o r .  The anti-P815-SF  a n t i s e r a from the  a l l o g e n e i c C57BL/6 mice would p r o b a b l y be d i r e c t e d to both r e c e p t o r s i t e d e t e r m i n a n t s as w e l l as the c o n s t a n t d e t e r m i n a n t s .  Similar  c o n c l u s i o n s were drawn by o t h e r s u s i n g v e r y s i m i l a r p r o t o c o l s  (26,  144-146). The o b s e r v a t i o n t h a t these a n t i s e r a do not k i l l t o x i c T c e l l s but do k i l l t h a t t h e s e two in  anti-P815 c y t o -  P 8 1 5 - s p e c i f i c suppressor T c e l l s ,  c e l l types bear u n r e l a t e d d e t e r m i n a n t s .  As mentioned  the i n t r o d u c t i o n , i t i s v e r y p o s s i b l e t h a t the s p e c i f i c  f a c t o r s may  be the r e c e p t o r s of s p e c i f i c  implies  suppressor  suppressor T c e l l s .  f o r e , s i n c e the a n t i s e r a used c o n t a i n e d a n t i - S F a c t i v i t y  There-  (Table I I  and F i g u r e 18), i t i s p o s s i b l e t h a t the determinants r e c o g n i z e d on the s u p p r e s s o r T c e l l s a r e a s s o c i a t e d w i t h the r e c e p t o r m o l e c u l e s of  these c e l l s .  P815  The  f a c t t h a t these a n t i s e r a d i d not k i l l  anti-  c y t o t o x i c T c e l l s i m p l i e s the p o s s i b i l i t y t h a t these two  s p e c i f i c T c e l l types may  P815-  bear u n r e l a t e d r e c e p t o r determinants a t  l e a s t a t the a n t i g e n b i n d i n g 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 c y t o t o x i c c e l l s to the s e r a p l u s complement treatment may  anti-  r e f l e c t a lower d e n s i t y or d i f -  91  f e r e n t arrangement o f r e c e p t o r s on t h e i r s u r f a c e when compared t o T suppressor  cells.  I t i s becoming v e r y apparent t h a t d i f f e r e n t i a l a c t i v a t i o n o f t h e v a r i o u s T c e l l subsets immunization  can be accomplished by v a r y i n g t h e mode of  (30,125,147-152).  I t a l s o seems t h e r e may be d e t e r -  minants which s e l e c t i v e l y r e a c t w i t h T h e l p e r c e l l s and other minants which r e a c t w i t h T s u p p r e s s o r • c e l l s . most d e f i n i t i v e l y w i t h  deter-  T h i s can be demonstrated  s m a l l c h e m i c a l l y d e f i n e d m o l e c u l e s i n which  v a r i o u s fragments o f t h e whole m o l e c u l e have been shown t o s e l e c t i v e l y a c t i v a t e e i t h e r T suppressor  c e l l s or T helper c e l l s  s t r a i n s of mice (20,153,157).  i n certain  Furthermore, Yamauchi e t a l . , i n  experiments i n v o l v i n g v a r y i n g immunization p r o t o c o l s and b l o c k i n g s t u d i e s , have v e r y r e c e n t l y shown t h a t i n one tumor system T supp r e s s o r c e l l s and T c y t o t o x i c c e l l s r e c o g n i z e d i f f e r e n t determinants (125).  In t h i s chapter,  these  f i n d i n g s a r e extended  by showng t h a t a n t i s e r a , from both syngeneic  DBA/2J and a l l o g e n e i c  C57BL/6 mice, d i r e c t e d a g a i n s t P 8 1 5 - s p e c i f i c suppressor w i t h P 8 1 5 - s p e c i f i c T suppressor  antigeneic  factor reacts  c e l l s but n o t s p e c i f i c T c y t o t o x i c  c e l l s t o t h i s same tumor. F i n a l l y t h e in. v i v o e f f e c t o f t h e anti-P815-SF a n t i s e r a on P815 tumor growth i n DBA/2 mice was a s s e s s e d .  As shown i n F i g u r e 20 and  T a b l e V o n l y t h e DBA/2 a n t i s e r a had any o b s e r v a b l e  effect  i n vivo.  T h i s i s i n c o n t r a s t t o the i n v i t r o work which showed t h a t b o t h the syngeneic  DBA/2 anti-P815-SF and t h e a l l o g e n e i c C57BL/6 a n t i -  P815-SF a n t i s e r a r e a c t e d w i t h The  reason.for  suppressor  t h i s i s unknown.  factor/cell  determinants.  A l t h o u g h i t may be r e l a t e d t o t h e  92  fact  t h a t the a l l o g e n e i c C57BL/6 anti-P815-SF  a n t i s e r a would  r e a c t w i t h many more determinants p r e s e n t on P815-SF and suppressor T c e l l s , antisera.  than would the syngeneic DBA/2  T h e r e f o r e , the a l l o g e n e i c anti-P815-SF  likely  P815-specific  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 v i v o than the syngeneic a n t i s e r a . example, the a l l o g e n e i c anti-P815-SF P815-specific in vivo.  a n t i s e r a could  possibly  For  stimulate  suppressor T c e l l i n d u c t i o n as w e l l as i n h i b i t i n g i t  Thus, e f f e c t i v e l y " c a n c e l l i n g o u t " i t s e f f e c t  i n vivo.  CHAPTER V SUMMARY DISCUSSION  94  CHAPTER V Summary D i s c u s s i o n The work r e p o r t e d i n the p r e c e e d i n g t h r e e c h a p t e r s i n v o l v e d s t u d i e s o f DBA/2 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 a n t i g e n s p e c i f i c suppressor f a c t o r d e r i v e d from them.  Both t h e s u p p r e s s o r  c e l l s and t h e suppressor f a c t o r s s p e c i f i c a l l y i n h i b i t t h e i n v i t r o g e n e r a t i o n of DBA/2 c y t o t o x i c T c e l l s f o r t h e syngeneic tumor, P815. In Chapter I I P815-T  and P815-SF were o b t a i n e d from the thymuses  g  of DBA/2 mice primed p r e v i o u s l y w i t h P815 tumor c e l l s The experiments  subcutaneously.  r e p o r t e d i n t h a t c h a p t e r showed t h a t t h e P815-T  g  and -SF both expressed I a ^ determinants and were n o t 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 t o e f f e c t i v e l y suppress t h e i n v i t r o response t o P815 by r a d i a t i o n chimeras o f a d i f f e r e n t H-2 h a p l o t y p e . T  g  used i n experiments  The P815-  r e p o r t e d i n Chapter I I I were prepared  from  the s p l e e n s o f 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 ^ w i t h membrane  fragments  R e s u l t s i n t h a t c h a p t e r showed  o f t h e P815 tumor c e l l .  among o t h e r t h i n g s t h a t t h e DBA/2 P815-T  g  expressed t h e c e l l s u r f a c e  + -  phenotype o f L y t - 1 2 , whereas t h e DBA/2 P815-T Chapter IV used P815-T  e  -+  G  was L y t - 1 2 .  prepared as i n Chapter I I I .  prepared from t h e s e s u p p r e s s o r T c e l l s .  P815-SF was then  Syngeneic DBA/2 and a l l o -  geneic C57BL/6 a n t i s e r a was prepared a g a i n s t t h e P815-SF. the anti-P815-SF  a n t i s e r a r e a c t e d i n v i t r o w i t h determinants  on t h e P815-SF and the P815-T  generated i n v i t r o . observable e f f e c t  Both o f expressed  , but d i d not r e a c t w i t h t h e P815-T s' c  Only t h e DBA/2 anti-P815-SF  a n t i s e r a had any  i n v i v o on P815 tumor growth i n DBA/2 mice.  95  The s t u d i e s o f some of the g e n e t i c p r o p e r t i e s of the P815-T i n Chapter I I i n d i c a t e t h a t t h i s s u p p r e s s o r c e l l on i t s s u r f a c e . out  I t was  s  express l a a n t i g e n  a l s o shown t h a t the P815-SF c o u l d be  absorbed  w i t h a n t i - l a * * a n t i s e r a , and thus a l s o expressed l a coded d e t e r -  minants.  T h i s l a s t r e s u l t was  a l s o shown i n Chapter IV u s i n g a  d i f f e r e n t method of p r e p a r i n g P815-SF.  There the ELISA assay  was  used to show t h a t P815-SF prepared from DBA/2 s p l e n o c y t e s expressed H-2** and more s p e c i f i c a l l y la** coded d e t e r m i n a n t s . t e s t e d t o date the a n t i g e n - s p e c i f i c T produced  In e v e r y system  and the a n t i g e n - s p e c i f i c  g  from i t have been shown to express l a determinants  T h e r e f o r e , these r e s u l t s i n the DBA/2-P815 system a r e not and f i t i n n i c e l y w i t h the assumption  SF  (59-61).  surprising  that a l l a n t i g e n - s p e c i f i c  s u p p r e s s o r T c e l l s and t h e i r s u p p r e s s i v e f a c t o r s p r o b a b l y express determinants encoded  w i t h i n the I - J to IE/C r e g i o n  (59-61).  Some of the immunogenetic requirements f o r the e x p r e s s i o n of P 8 1 5 - s p e c i f i c s u p p r e s s i o n were l o o k e d a t i n Chapter I I . showed t h a t the P815-T  The  results  and the P815-SF d e r i v e d from them, were s  capable of s u p p r e s s i n g the i n v i t r o g e n e r a t i o n 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 a b l e c e l l s .  genes w i t h i n the MHC (or  do not have to be shared by the s u p p r e s s o r  i t s f a c t o r ) and i t s t a r g e t .  systems  i n which H-2  These r e s u l t s showed t h a t the  In the a n t i g e n - s p e c i f i c s u p p r e s s o r  r e s t r i c t i o n has been addressed, the p u b l i s h e d  r e s u l t s of o t h e r s are somewhat e q u i v o c a l .  In most systems  c e l l - m e d i a t e d responses t h e r e has been no H-2 l e a s t f o r the f i r s t p a r t of the systems In  cell  involving  r e s t r i c t i o n found, a t  activity  (see below)  (59-61).  one system s t u d i e d by Moorhead of a a n t i g e n - s p e c i f i c SF which  96  suppresses c o n t a c t s e n s i t i v i t y t o DNP, i t was found t h a t the SF .. r e q u i r e d homology a t the K and/or D l o c i o f the H-2 w i t h the t a r g e t c e l l s f o r a c t i v i t y t o be observed  (79,80,97).  The P815-SF c l e a r l y  does n o t need t h i s homology w i t h i t s t a r g e t t o show s u p p r e s s i v e activity. The work i n Chapter I I I p r e s e n t s evidence o f t h e L y t phenotype of  t h e c y t o t o x i c 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 t h a t the primary i n v i t r o  generated DBA/2 P815-T  were L y t - 1 2 . +  c  As was p o i n t e d out i n the  d i s c u s s i o n i n Chapter I I I , t h e r e may be a d i f f e r e n c e i n the L y t - 1 a n t i g e n e x p r e s s i o n on primary v s . secondary syngeneic tumor-T i n c  the DBA/2-P815 system  (13,130), and a l s o between syngeneic-T  a l l o g e n e i c - ^ i n DBA/2 mice (13).  and  Of course these r e s u l t s p r o b a b l y  r e f l e c t a p u r e l y q u a n t i t a t i v e n o t 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 p r o b a b l y express L y t - 1 a n t i g e n s , a l t h o u g h i n v a r y i n g amounts (131). Even though most a n t i g e n s p e c i f i c - T  g  i n other systems have been  shown t o express t h e s u r f a c e phenotype o f L y t - 1 2  (114-118,120),  +  it l 2 +  of  i s n o t s u r p r i s i n g t h a t t h e DBA/2 P815-T .  T h i s i s because  systems  132,133).  g  were shown t o be L y t -  t h e r e have been s e v e r a l r e p o r t s i n a v a r i e t y  of a n t i g e n - s p e c i f i c T  which a r e L y t - l 2 +  g  (42-44, 120,  As was d i s c u s s e d i n Chapter I I I , these c e l l s a r e u s u a l l y  found t o n o t be the a c t u a l e f f e c t o r c e l l f o r s u p p r e s s i o n but may " i n d u c e " other T c e l l s t o a c t i v e l y suppress Thus, t h e L y t - l 2 +  (22,23,44,60,134-136).  , I a , a n t i g e n - b i n d i n g , H-2 u n r e s t r i c t e d T ^ -inducer +  g  i s thought t o produce a I a , a n t i g e n - b i n d i n g , H-2 u n r e s t r i c t e d SF, +  97  which induces a second Ia  suppressor T c e l l ,  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 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 +  T cell, T  g 3  . T  g 3  i s Lyt-l~2t  which may suppress  This T  i nactivity.  i s L y t - 1 2 ", -1  g 2  I t produces an  S F , which induces a t h i r d 2  Ia ,. idiotype , " f i n a l " +  +  effector  suppressor cell,  i n a n o n - s p e c i f i c f a s h i o n , p o s s i b l y v i a an i n t e r -  a c t i o n w i t h macrophage. Thus, i t can be seen t h a t both t h e P815-T  and P815-SF from s  the thymus or s p l e e n o f DBA/2 mice would f i t n i c e l y i n t o t h e above mentioned model of s u p p r e s s i o n a t the T x and SF^ stage. s  at  first  seem s t r a n g e s i n c e t h e P 8 1 5 - T  s>  which appears  T h i s may  after  intra-  p e r i t o n e a l 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 i n j e c t e d  subcutaneously.  One e x p l a n a t i o n f o r t h i s may be t h a t i t takes s e v e r a l days f o r t h e a n t i g e n l o a d produced  by t h e subcutaneously  i n j e c t e d l i v e P815 t o  e q u a l t h e amount i n j e c t e d d i r e c t l y v i a t h e i n t r a p e r i t o n e a l method.  Thus t h e two P 8 1 5 - s p e c i f i c suppressor T c e l l s may be i d e n t i c a l  and e q u i v a l e n t t o t h e T ^- i n other systems.  I t may be worth n o t i n g  t h a t 4-6 days a f t e r maximum s u p p r e s s i o n appears p r e s s i o n appears mice (148).  i n the thymus, sup-  i n t h e s p l e e n and lymph nodes o f tumor b e a r i n g  T h i s may be an i n d i c a t i o n o f t h e time i t takes f o r t h e  thymus P815-T vivo.  injection  g  t o " i n d u c e " the f u l l suppressor e f f e c t o r f u n c t i o n i n  The 5 day c u l t u r e p e r i o d o f t h e i n v i t r o assay may a l s o a l l o w  the " i n d u c t i o n " o f s u p p r e s s i o n through t h e i n f l u e n c e o f t h e L y t l 2" +  P815-T . s In t h e e a r l i e r s t u d i e s o f P815-T  suppressor c e l l  and suppressor  s  and P815-SF i n DBA/2 mice t h e  f a c t o r were o b t a i n e d from t h e thymuses  98  of DBA/2 mice which had been primed p r e v i o u s l y w i t h P815 tumor c e l l s (123,124, Chapter I I ) . was  The s u p p r e s s i v e a c t i v i t y o f these m a t e r i a l s  assayed by t h e i r a b i l i t y t o s p e c i f i c a l l y suppress t h e secondary  i n v i t r o c y t o t o x i c response o f primed DBA/2 s p l e n o c y t e s t o mitomycinC t r e a t e d P815 c e l l s .  I n t h e work r e p o r t e d i n t h i s t h e s i s P815-T s  and P815-SF, e i t h e r prepared as above from thymocytes  or from s p l e n o -  c y t e s of DBA/2 mice which had been primed p r e v i o u s l y w i t h P815 tumor membrane e x t r a c t , c o u l d s p e c i f i c a l l y i n h i b i t t h e primary i n v i t r o c y t o t o x i c response o f normal DBA/2 s p l e n o c y t e s t o mitomycin-C P815.  Therefore, the s t a t e  on which  P815  (primed o r v i r g i n ) o f t h e c e l l p o p u l a t i o n  i t a c t s appears t o be unimportant  SF a c t i v i t y .  treated  S i n c e t h e number o f P815-T  c  f o r t h e P815-T  or P815-  g  i s probably higher i n the  primed DBA/2 s p l e e n p o p u l a t i o n than the normal DBA/2 s p l e e n  population  (130), i t would seem t h a t t h e P815-T  a c t on t h e h e l p e r T c e l l p o p u l a t i o n .  s  and P815-SF may  Thus, i n h i b i t i n g t h e T^ p r o v i d e d  2nd s i g n a l , which i s p r o b a b l y n e c e s s a r y f o r t h e P815-T e n t i a t e and become a c t i v e e f f e c t o r s o f c y t o t o x i c i t y . p r e s e n t s no evidence s u p p o r t i n g t h i s p o s s i b i l i t y ,  c  to d i f f e r -  While t h i s  study  t h e r e has been good  r e c e n t e x p e r i m e n t a l evidence f o r SF e f f e c t i n g T^ a c t i v i t y  directly  (158). The r e s u l t s p r e s e n t e d i n Chapter IV show t h a t anti-P815-SF r a i s e d both i n syngeneic DBA/2 and a l l o g e n e i c C57BL/6 mice, absorb out t h e P815-SF.  b u t n o t P815-T s  As mentioned  could  These a n t i s e r a were a l s o c a p a b l e , i n t h e  presence o f complement, o f e l i m i n a t i n g P815-T  DBA/2 mice.  antisera,  i n Chapters I and IV, i t i s l i k e l y  from s t h a t the  a h f i g e n - s p e c x f c SF may be the r e c e p t o r molecule of a n t i g e n - s p e c i f i c T .  Therefore,  s i n c e the a n t i s e r a used c o n t a i n e d  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 a s s o c i a t e d w i t h the r e c e p t o r m o l e c u l e s .  on the P815-T a r e s  Thus, the f a c t t h a t the  a n t i - P 8 1 5 - S F a n t i s e r a d i d n o t 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  t h a t t h e s e 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 determinants,  a t l e a s t a t the a n t i g e n b i n d i n g  site.  receptor  100  LITERATURE CITED  1.  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Properties o f s y n g e n e i c 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 t o tumors p e c i f i c s u p p r e s s o r f a c t o r from DBA/2 mice. Cancer Immunol. Immunotherapy ( I n 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 o f 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 •  -*y • A«l-',-  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, (  Wit  ;  o-£ v^o~> oclow<\ \  a^tL  

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