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Immunological responses to oxidized ferredoxin and its chemically modified derivatives Gregerson, Dale 1976

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IMMUNOLOGICAL RESPONSES TO OXIDIZED FERREDOXIN AND ITS CHEMICALLY MODIFIED DERIVATIVES  by DALE S. GREGERSON B.A.  L u t h e r C o l l e g e , 1971  A THESIS SUBMITTED IN PARTIAL FULFILLMENT"OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In t h e Department of Microbiology  We accept t h i s t h e s i s as conforming required  t o the  standard  THE UNIVERSITY'OF BRITISH COLUMBIA March, 1976 (c) Dale S. Greecerson  In  presenting  an  advanced degree  the I  Library  further  for  this  shall  agree  thesis  in  at  University  the  make  that  it  freely  permission  s c h o l a r l y p u r p o s e s may  by  his  of  this  representatives.  w r i t ten  thesis  financial  of  University  of  British  F^k. a">  /  <7ic  of  of  Columbia,  British  gain  Ic  Columbia  for  extensive by  the  is understood  , ' c r'O k/o  /M.  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  for  be g r a n t e d  It  fulfilment  available  pe rm i s s i on .  Department The  for  partial  shall  requirements  reference  I  agree  and  Head o f  be a l l o w e d  that  thesis  my D e p a r t m e n t  copying or  for  study.  copying o f ' t h i s  that  not  the  or  publication  without  my  i  ABSTRACT  Guinea p i g lymph node and s p l e e n c e l l s responded c a n a v a l i n A (con A ) , l i p o p o l y s a c c h a r i d e  i n vitro  to con-  (LPS), and a s p e c i f i c a n t i g e n ,  o x i d i z e d f e r r e d o x i n , i n serum f r e e medium, medium w i t h m e r c a p t o e t h a n o l (ME), medium w i t h f o e t a l c a l f serum (FCS), and medium w i t h both FCS and ME.  The  a d d i t i o n o f ME to serum f r e e medium supported a mixed l e u c o c y t e r e a c t i o n as d i d media w i t h FCS or FCS and ME.  Nylon wool f r a c t i o n a t i o n of the c e l l s  e l i m i n a t e d the LPS response and treatment w i t h a n t i - i m m u n o g l o b u l i n complement  ( C ) reduced the LPS response  lymphocyte  mitogen.  ( a l g ) and  i n d i c a t i n g t h a t LPS may be a B  The con A response was enhanced by the a l g and C i  treatment. The s p e c i f i c i t i e s o f the humoral and c e l l u l a r immune responses t o the amino t e r m i n a l h e p t a p e p t i d e a n t i g e n i c determinant o f p e r f o r m i c a c i d ferredoxin  oxidized  (O-Fd) were t e s t e d and compared u s i n g s e v e r a l s y n t h e t i c p e p t i d e s  and analogues  of the determinant  i n leucocyte migration i n h i b i t i o n tests with  guinea p i g s p l e e n c e l l s and i n h i b i t i o n o f complement f i x a t i o n between O-Fd and homologous r a b b i t antiserum.  A t e t r a p e p t i d e c o m p r i s i n g the f o u r amino  a c i d s a t the carboxy t e r m i n a l o f the t e s t determinant was. a b l e to produce significant  i n h i b i t i o n of leucocyte m i g r a t i o n .  M o d i f i c a t i o n s of amino a c i d s  w i t h i n the t e t r a p e p t i d e r e s u l t e d i n a l o s s of m i g r a t i o n i n h i b i t i o n . same p e p t i d e would o n l y i n h i b i t  the complement f i x a t i o n r e a c t i o n i f a  hydrophobic group was a t t a c h e d to i t . produce  This  Otherwise a hexapeptide was r e q u i r e d to  s i g n i f i c a n t i n h i b i t i o n of' complement f i x a t i o n .  w i t h a conjugate of the h e p t a p e p t i d e determinant  Guinea p i g s s e n s i t i z e d  and bovine serum  albumin  gave p o s i t i v e immediate and d e l a y e d s k i n r e a c t i o n s t o c o n j u g a t e s o f the h e p t a p e p t i d e w i t h o t h e r p r o t e i n s i n d i c a t i n g t h a t the h e p t a p e p t i d e f u n c t i o n s as both a hapten and c a r r i e r . was i n h i b i t e d by O-Fd.  A l s o , m i g r a t i o n o f s p l e e n c e l l s from these animals  The  a b i l i t i e s of s e v e r a l c h e m i c a l l y  s t i m u l a t e DNA O-Fd  and  synthesis  i n v i t r o i n spleen  to f i x complement  were t e s t e d .  (C')  O-Fd,  dinitrophenylated  O-Fd  (meth-O-Fd) d i d n o t . f i x e d C' weakly.  native-Fd,  (NEM-vFd) were a b l e  (DNP-O-Fd) f i x e d C' w h i l e methylated  O-Fd  24 hour DNA  s y n t h e t i c response was  mouse immunoglobulin and 120  hour response was  C' and  (TCA-Fd) and  ferredoxin (CM-Fd)  a l l of the  o n l y s e n s i t i v e to t h e ' a n t i - O  and  C'.  C  immunogenic  ferredoxins.,  s e n s i t i v e to treatment w i t h  a n t i - b r a i n a s s o c i a t e d 0 and  (native-Fd)  stimulate  and NEM-Fd were found to be  i n mice when assayed f o r lymphocyte s t i m u l a t i o n by The  to  carboxymethylated f e r r e d o x i n  Only n a t i v e - F d ,  sera  native ferredoxin  acid precipitated ferredoxin  NEM-Fd and  to  c e l l s from mice s e n s i t i z e d to  Of a l l the , f e r r e d o x i n s , only O-Fd,  synthesis.  forms of f e r r e d o x i n  i n the presence of r a b b i t a n t i - O - F d  and N-ethylmaleimide a l k y l a t e d f e r r e d o x i n DNA  modified  1  anti-  while  the  iii  TABLE OF CONTENTS  Page 1  Introduction Part I.  Responses o f Guinea P i g Lymphocytes to M i t o g e n s , and, A n t i g e n and Mixed L e u c o c y t e C u l t u r e i n Media With and Without M e r c a p t o e t h a n o l and F o e t a l C a l f Serum . . . . . . . . . .  3  T i t l e Page  3  Summary  4  Introduction . . . . . .  5  M a t e r i a l s and Methods  7  Results Discussion  10 . . . . . . . .  13  T a b l e s and F i g u r e s  17  References  30  P a r t I I . The Immune Response t o F e r r e d o x i n . I. S p e c i f i c i t y of t h e Response t o the Amino T e r m i n a l Determinant  33  T i t l e Page  33  Summary  34  Introduction  36  M a t e r i a l s and Methods  40  Results  43  Discussion  . . . . . . . . .  46  T a b l e s and F i g u r e s  50  References  59  iv  Page P a r t I I I . The Immune Response t o F e r r e d o x i n . I I . C r o s s R e a c t i v i t y o f C e l l s and A n t i s e r a t o m o d i f i e d f e r r e d o x i n s and t h e Nature of t h e C e l l s Responding I n V i t r o . . . ......... T i t l e Page Summary  Conclusion  67 67  . .  68  Introduction  69  M a t e r i a l s and Methods  71  Results  74  . . . . . .  Discussion  78  T a b l e s and F i g u r e s  82  References  92  96  V  ACKNOWLEDGEMENTS  There a r e two people to whom I would l i k e thanks,  Dr••.  J u l i a Levy and Barbara K e l l y .  to express a v e r y  special  T h e i r a s s i s t a n c e and f r i e n d s h i p  were deeply a p p r e c i a t e d . I would a l s o l i k e to thank t h e f o l l o w i n g p e o p l e who worked w i t h and around me over t h e l a s t  s e v e r a l y e a r s and gave a l o t o f m a t e r i a l s , h e l p ,  i d e a s and d i s c u s s i o n s t h a t a r e n o t obvious i n the f i n a l w r i t e up, but were p a r t o f a good experience':'  Doug H u l l , Fumio T a k e i , Doug Water f i e l d , Rob  McMaster, Bruce A c r e s , Maureen F a i r h u r s t , Barb Pope, Annajane Smith and Dr.'s  Doug K i l b u r n , T e r r y P e a r s o n , Rob Watson, D i c k Whitney and Mary  Lymburner. F i n a l l y , a thank you t o t h e committee members f o r h e l p i n g t o g e t i t a l l finished:  Dr.'s J.J.R. Campbell, P. D e n n i s , A. Sehon, and P. Candido.  1  INTRODUCTION  The  a b i l i t y of the immune system to s p e c i f i c a l l y r e c o g n i z e and  react  w i t h a n t i g e n s has been known f o r some time, but o n l y r e c e n t l y has t h i s been examined i n d e t a i l u s i n g c h e m i c a l l y d e f i n e d , n a t u r a l l y o c c u r r i n g  determinants.  P r e v i o u s work has o f t e n been done without knowledge of the l o c a t i o n o r n a t u r e of the determinants p r e s e n t on the a n t i g e n .  As a r e s u l t , most of these  s t u d i e s , which were done u s i n g a n t i g e n s t h a t were n o n s p e c i f i c a l l y m o d i f i e d by a v a r i e t y of methods, do not r e l a t e the s p e c i f i c i t i e s of the humoral and c e l l u l a r  immune responses to a s i n g l e determinant.  the observed e f f e c t of such m o d i f i c a t i o n s may  To an unknown degree  be due to a l t e r a t i o n s i n the  a c c e s s i b i l i t y of the determinants by changes i n the c o n f o r m a t i o n of the m o l e c u l e r a t h e r than m o d i f i c a t i o n of the determinants themselves. a number of i n t e r e s t i n g r e s u l t s have been found i n s e v e r a l  laboratories  u s i n g u n d e f i n e d a n t i g e n s and c h e m i c a l m o d i f i c a t i o n s of them. a v a r i e t y of t e c h n i q u e s to assay humoral and c e l l u l a r and i n v i t r o  However,  Utilizing  immunity both i n v i v o  to c h e m i c a l l y m o d i f i e d a n t i g e n s , i t has been f r e q u e n t l y  observed t h a t the c e l l u l a r cross r e a c t i v i t y  immune response e x p r e s s e s a g r e a t e r degree  of  than does the humoral response.  There have been s e v e r a l r e p o r t s of work done w i t h d e f i n e d a n t i g e n s , but i n n e a r l y a l l cases the a n t i g e n s used f o r these s t u d i e s were r e c o g n i z e d by e i t h e r thymus dependent lymphocytes comparisons  (T c e l l s ) or a n t i b o d i e s .  Direct  of the s p e c i f i c i t y of T c e l l s and a n t i b o d y to the same determinant  were not p o s s i b l e i n those s t u d i e s .  I t seems c l e a r t h a t t e s t i n g the c r o s s  r e a c t i v i t y of T c e l l s and a n t i b o d y (or thymus independent  B c e l l s ) to a  s i n g l e d e f i n e d , d e t e r m i n a n t i s r e q u i r e d to e s t a b l i s h the r e l a t i v e  specificities  2  of these responses. A p r o m i s i n g approach to the s p e c i f i c i t y problem was a n t i g e n i c n a t u r e of f e r r e d o x i n was polypeptide  isolated  t h a t the p e r f o r m i c major, d e t e c t a b l e  elucidated.  from C l o s t r i d i u m pasteurianum.  a c i d o x i d i z e d form of f e r r e d o x i n  a n t i b o d y , thus p r o v i d i n g  response to f e r r e d o x i n and  In t h i s  f e r r e d o x i n s and  in vitro  t h e s i s the  two  formed.  recognized  for a  direct  s p e c i f i c i t y of the  immune tested  study to  a c o r r e l a t e of  cell  been some doubt r a i s e d r e c e n t l y of immune response.  the parameters of the assay were e x t e n s i v e l y examined u s i n g  For  this  C e r t a i n a s p e c t s of the assay commonly assumed  serum and" long i n c u b a t i o n p e r i o d s were t e s t e d .  proliferative  The  prior  types of c e l l s  later  continued  the l a r g e volume of work a l r e a d y  calf  to l a b e l l i n g w i t h p r e c u r s o r s  (T or B) r e q u i r e d  response were a l s o i n v e s t i g a t e d .  guinea p i g c e l l s and  reason  s e v e r a l means  to be n e c e s s a r y f o r a response, such as the requirement f o r f o e t a l  DNA,  by  serum a n t i b o d i e s .  as a measure of the c e l l u l a r  synthesis.  are  each i s  lymphocyte s t i m u l a t i o n assay used i n t h i s  mediated immunity, a l t h o u g h t h e r e has  of i n d u c i n g DNA  only  s y n t h e t i c analogues of the N7 determinant  examine s p e c i f i c i t y i s g e n e r a l l y b e l i e v e d to be  its validity  (O-Fd) has  the o p p o r t u n i t y  residue  been demonstrated  i t s amino t e r m i n a l d e t e r m i n a n t , N7, was  assays f o r c e l l mediated immunity and The  I t has  a n t i g e n i c determinants to which a n t i b o d i e s  comparison of s p e c i f i c i t i e s .  using modified  i s a 55  Ferredoxin  Both of the determinants have been c h a r a c t e r i z e d and by T c e l l s and  found when the  to  to produce a"  T h i s work was  begun u s i n g  u s i n g mouse c e l l s to take advantage of  done t h a t has  characterized  system i n mice making them more u s e f u l f o r immunological  the  studies.  immune  3  RESPONSES OF GUINEA PIG LYMPHOCYTES TO MITOGENS, AN ANTIGEN, AND MIXED LEUCOCYTE CULTURE IN MEDIA WITH AND WITHOUT MERCAPTOETHANOL AND FOETAL CALF SERUM  D.S.. GREGERSON, BARBARA KELLY AND JULIA G. LEVY  DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, CANADA  4  SUMMARY  The a b i l i t y o f g u i n e a p i g s p l e e n and lymph node c e l l s t o undergo a p r o l i f e r a t i v e response i n v i t r o i n t h e presence o f mitogens  (concanavalin A  and l i p o p o l y s a c c h a r i d e ) , a s p e c i f i c a n t i g e n ( o x i d i z e d f e r r e d o x i n ) , and a l l o g e n e i c c e l l s was.assessed under a v a r i e t y o f c o n d i t i o n s .  Time and  dose dependency o f t h e responses was measured i n RPMI 1640 (1640), 1640 p l u s m e r c a p t o e t h a n o l (ME), 1640 p l u s f o e t a l c a l f serum (FCS), and 1640 w i t h ME and FCS.  M i t o g e n responses were a l s o measured a f t e r t r e a t m e n t o f t h e  c e l l s w i t h sheep a n t i - g u i n e a p i g immunoglobulin  (SaGPIg) and complement  ( C ) o r a f t e r passage t h r o u g h n y l o n wool columns. ?  L i p o p o l y s a c c h a r i d e (LPS)  s t i m u l a t e d t h e c e l l s under a l l media c o n d i t i o n s over a wide range o f c o n c e n t r a t i o n s b u t over a narrow time p e r i o d .  N y l o n wool t r e a t m e n t o f t h e c e l l s  e l i m i n a t e d t h e LPS response w h i l e SaGPIg and C' reduced i t .  Concanavalin A  (con A) s t i m u l a t e d t h e c e l l s under a l l t e s t c o n d i t i o n s and demonstrated a dose-time i n t e r r e l a t i o n s h i p i n terms o f maximum r e s p o n s e .  Pre-treatment  of c e l l s w i t h SaGPIg and C' enhanced t h e response t o con A w h i l e n y l o n wool f r a c t i o n a t i o n d i m i n i s h e d i t somewhat. i n v i t r o to o x i d i z e d f e r r e d o x i n (0-Fd).  Only lymph node c e l l s  responded  I n serum f r e e media t h e O-Fd responses  were maximal a t 48 hours whereas i n media c o n t a i n i n g FCS p r o l i f e r a t i v e responses were s u p p o r t e d f o r a p r o l o n g e d p e r i o d and appeared t o be b i m o d a l . Except f o r an e a r l y response w i t h 1640 and ME, o n l y media c o n t a i n i n g FCS supported s t i m u l a t i o n i n t h e mixed l e u c o c y t e c u l t u r e  (MLC).  5  INTRODUCTION  The I n v i t r o s t i m u l a t i o n o f lymphocytes by m i t o g e n s , a n t i g e n s o r a l l o g e n e i c lymphoid c e l l s has been used as a c o r r e l a t e o f c e l l mediated immunity except i n i n s t a n c e s where B lymphocyte mitogens were used. However, t h e r e i s some q u e s t i o n r e g a r d i n g what p o p u l a t i o n s o f c e l l s a r e r e s p o n d i n g and whether o r n o t macrophages a r e i n v o l v e d (Mugraby, Gery S u l i t z e a n u , 1974).  and  V a r i o u s t y p e s o f r e s p o n s e s o f lymphoid c e l l s t o a  number o f mitogens have been demonstrated i n s e v e r a l a n i m a l s p e c i e s .  In  the mouse i t i s r e c o g n i z e d t h a t d i f f e r e n t lymphoid c e l l p o p u l a t i o n s w i l l respond t o c e r t a i n mitogens.  Con A and LPS have been shown t o be  mitogens f o r T and B l y m p h o c y t e s , r e s p e c t i v e l y  specific  (Anderson, M o l l e r • and  S j b b e r g , 1972b), a l t h o u g h i t has been demonstrated t h a t con A w i l l  stimulate  B lymphocytes i f i t i s a p p r o p r i a t e l y p r e s e n t e d (Anderson, Edelman, M o l l e r and S j b b e r g , 1972a). M u r i n e T lymphocytes may be s e p a r a t e d by passage o f lymphocytes t h r o u g h n y l o n wool columns ( J u l i u s ,  Simpson, and H e r z e n b e r g , 1973), or by t r e a t m e n t  w i t h anti-mouse immunoglobulin p l u s C' ( T a k a h a s h i , O l d , M c l n t i r e . and Boyse, 1971).  C o n v e r s e l y , p o p u l a t i o n s o f c e l l s f r e e o f T lymphocytes may be  p r e p a r e d by t r e a t m e n t w i t h a n t i - 9 and C' ( R a f f , 1969; L a m e l i n , L i s o w s k a B e r n s t e i n , M a t t e r , R y s e r , and V a s s a l l i , not been so w e l l e l u c i d a t e d .  1972).  The g u i n e a p i g system has  I n the present study, treatment of c e l l s  w i t h n y l o n wool o r SaGPIg and C' was examined t o o b s e r v e t h e i r  effects  on t h e con A and LPS r e s p o n s e s . R e c e n t l y , r e p o r t s have appeared d e m o n s t r a t i n g t h a t lymphocytes can  6  be c u l t u r e d f o r r e l a t i v e l y s h o r t p e r i o d s of time w i t h o u t serum 1972; C o u t i n h o , M o l l e r , A n d e r s s o n . and B u l l o c k , 1973).  (Vischer,  In v i t r o  systems  i n w h i c h serum can be e l i m i n a t e d have s e v e r a l advantages; lower back-, ground c o n t r o l s , s i m p l e r c h a r a c t e r i z a t i o n of s u p e r n a t a n t f a c t o r s , e l i m i n a t i o n of v a r i a b i l i t y between b a t c h e s o f serum  s  and r e d u c t i o n of n o n - s p e c i f i c  stimulation! S e v e r a l i n v e s t i g a t o r s have r e p o r t e d t h a t the a d d i t i o n of r e d u c i n g agents such as m e r c a p t o e t h a n o l and c y s t e i n e has a b e n e f i c i a l e f f e c t i n t h e i r c u l t u r e systems.  G r e a t e r v i a b i l i t y and enhanced response t o a n t i -  gens, m i t o g e n s , and MLC have been c l a i m e d (Chen and H i r s c h , 1972;  Bevan,  E p s t e i n , and Cohn, 1974; Broome and J e n g , 1973; F a n g e r , H a r t , W e l l s  and  N i s o n o f f , 1970; Heber-Katz and C l i c k , 1972). T h i s s t u d y was u n d e r t a k e n t o e l u c i d a t e some of the parameters g o v e r n i n g t h e p r o l i f e r a t i v e response of g u i n e a p i g lymph node and s p l e e n c e l l s t o v a r i o u s s t i m u l i when c u l t u r e d i n media w i t h and w i t h o u t FCS and/or ME, t o c o r r e l a t e some of the d a t a p r e s e n t e d here w i t h i n f o r m a t i o n a l r e a d y a v a i l a b l e i n murine  systems.  and  7  MATERIALS AND METHODS  ANIMALS Outbred a l b i n o g u i n e a p i g s o f e i t h e r sex w e i g h i n g a p p r o x i m a t e l y 400 grams were used i n a l l  experiments.  MITOGENS Con A (Sigma, S t . L o u i s , Mo.) saline  was made up i n phosphate b u f f e r e d  (PBS) t o 200 ug/ml, s t e r i l i z e d , and s t o r e d f r o z e n . LPS-W from  S. typhimurium  ( D i f c o , D e t r o i t , Mich.) was d i s s o l v e d i n PBS a t 10 mg/ml,  the pH a d j u s t e d t o 8.0, heated f o r 30 minutes i n a b o i l i n g water b a t h and s t o r e d f r o z e n .  A l l f u r t h e r d i l u t i o n s were made i n serum f r e e medium.  ANTIGEN PREPARATION AND  IMMUNIZATION  F e r r e d o x i n from _C. p a s t e u r i a n u m  (Sigma) was p e r f o r m i c a c i d  oxidized  ( M i t c h e l l , Levyi, and N i t z , 1970) b e f o r e u s e . A n i m a l s were i n j e c t e d i n 5 l o c a t i o n s w i t h 250 ug o f O-Fd i n 50% complete Freund's a d j u v a n t (CFA) u s i n g a t o t a l volume o f 0.5 ml p e r a n i m a l .  The a n i m a l s were b o o s t e d  s i m i l a r l y a f t e r 14 days and s a c r i f i c e d 10 days l a t e r .  O-Fd used i n  c u l t u r e s was d i s s o l v e d i n medium, s t e r i l i z e d , and s t o r e d f r o z e n .  PREPARATION OF CELLS Spleens and lymph nodes were removed a s e p t i c a l l y , t e a s e d i n t o PBS and t h e r e s u l t i n g c e l l s u s p e n s i o n s t r a n s f e r r e d t o p l a s t i c t u b e s . c e l l s were spun down (180 x g f o r 5 min.) and resuspended  The  i n 0.85% NH^Cl  ( i n 0.01 M P O 4 , pH 7.2) f o r 4 t o 5 minutes t o l y s e t h e r e d b l o o d c e l l s . A f t e r 3 washes w i t h PBS they were counted u s i n g t r y p a n b l u e t o a s s e s s viability.  8 MEDIA The b a s i c medium used f o r a l l e x p e r i m e n t s was RPMI 1640 ( G i b c o , Grand I s l a n d , New Y o r k ) supplemented w i t h 100 u n i t s / m l p e n i c i l l i n , 100 ug/ml s t r e p t o m y c i n , and 50 pg/ml f u n g i z o n e .  M e r c a p t o e t h a n o l was made  -4 up a s e p t i c a l l y i n medium a t 2.5 x 10  M and s t o r e d f r o z e n .  Foetal calf  serum (Gibco, #84557) was i n a c t i v a t e d a t 56° " f o r 30 m i n . and made up t o 20% i n medium. CELL CULTURE Spleen and lymph node c e l l s were c u l t u r e d i n m i c r o c u l t u r e p l a t e s . M i t o g e n s o r a n t i g e n i n medium were added i n a volume of 0.05 m l / w e l l . When ME o r FCS was used, 0.05 ml of the p r e v i o u s l y d e s c r i b e d s t o c k s o l u t i o n s was added t o each w e l l .  F i v e t o 10 x 10^ c e l l s i n 0.10 ml of medium  were added t o each w e l l a l o n g w i t h whatever volume of medium was r e q u i r e d t o make a f i n a l volume of 0.25 m l . T h i s p r o c e d u r e r e s u l t e d i n a f i n a l c o n c e n t r a t i o n o f ME a t 5 x 10~ M, FCS a t 4%, and c e l l s between 2-4 x 1 0 / m l . 5  6  The MLC's were s e t up i n a s i m i l a r manner except t h a t 2.5 x 10^ c e l l s i n 0.05 ml from each a n i m a l were used p e r w e l l . 5 x 10^ unmixed c e l l s .  Control wells  contained  Background counts were o b t a i n e d by a v e r a g i n g t h e  c o n t r o l counts o f each a n i m a l .  LABELLING AND HARVESTING T r i t i a t e d thymidine  (Amersham-Searle, A r l i n g t o n H e i g h t s , 111.  s p e c i f i c a c t i v i t y 2.0 Ci/m mole) a t a c o n c e n t r a t i o n of 1.0 u C i i n 0.05 ml was added t o t h e w e l l s 18 hours b e f o r e h a r v e s t i n g .  H a r v e s t i n g was performed  by a s p i r a t i n g t h e c o n t e n t s of each w e l l onto g l a s s f i b e r f i l t e r s u s i n g a  9  m u l t i p l e sample h a r v e s t o r (Hartzman, Bach, Bach, Thurman,% and S e l l , 1972; Thurman, S t r o n g , Ahmed, Green, S e l l , Hartzman  and Bach, 1973).  The  f i l t e r s were d r i e d and counted i n a s c i n t i l l a t i o n c o u n t e r .  NYLON WOOL TREATMENT C e l l s t r e a t e d w i t h NH^Cl and washed as d e s c r i b e d above were suspended i n PBS p l u s 5% FCS and p l a c e d i n p l a s t i c at 37° t o remove adherent c e l l s .  t i s s u e c u l t u r e p l a t e s f o r 1 hour  PBS w i t h 5% FCS was used f o r a l l  subsequent n y l o n wool column m a n i p u l a t i o n s .  The non-adherent c e l l s were  washed i n t o a n y l o n wool column, i n c u b a t e d f o r 1 hour a t 37°y and e l u t e d slowly.  The column c o n s i s t e d o f a 25 ml s y r i n g e b a r r e l f i l l e d t o t h e  20 ml l e v e l w i t h l o o s e l y packed n y l o n wool and s t e r i l i z e d . was adapted from t h a t used by J u l i u s e_t a l . (1973) . was a p p r o x i m a t e l y 15 t o 20% o f t h o s e a p p l i e d . of  The p r o c e d u r e  Recovery o f c e l l s  To o b t a i n s u f f i c i e n t  numbers  c e l l s f o r some e x p e r i m e n t s , lymph node and s p l e e n c e l l s were p o o l e d  before f i l t r a t i o n .  These c e l l s a r e r e f e r r e d t o as p o o l c e l l s .  KILLING WITH ANTI-Ig PLUS COMPLEMENT Ammonium c h l o r i d e t r e a t e d c e l l s were t a k e n up i n s t e r i l e , i n a c t i v a t e d SaGPIg o r g u i n e a p i g C'  (each was d i l u t e d 1:4 i n PBS) o r i n b o t h and i n c u b a t e d  w i t h i n t e r m i t t e n t m i x i n g f o r 1 hour a t t 3 7 ° C a f t e r r w h l c h t h e c e l l s were washed and c o u n t e d . cells.  0.2 ml of t h e d i l u t e d SaGPIg and C' were used per 10^  Where n e c e s s a r y , lymph node and s p l e e n c e l l s were p o o l e d b e f o r e  t r e a t m e n t t o ensure adequate numbers o f c e l l s .  RESULTS  INDUCTION OF DNA  SYNTHESIS BY  LPS  The r e s u l t s of dose response and k i n e t i c s experiments u s i n g s p l e e n c e l l s i n serum f r e e medium a r e p r e s e n t e d i n F i g . 1A. was  found to be 4 ;jg/ml a f t e r 24 hours i n c u l t u r e .  The o p t i m a l dose In cultures with  FCS, c o n c e n t r a t i o n s of 16 and 64 ug/ml s t i m u l a t e d w e l l a f t e r 24 hours ( F i g . S t i m u l a t i o n i n d i c e s (SI) between 2.2 and 3.1 were found w i t h LPS i n d u c e d s p l e e n c e l l s i n medium p l u s ME a t LPS c o n c e n t r a t i o n s from 4-256 ug/ml a f t e r 24 hours i n c u l t u r e . T a b l e 1. out.  LPS s t i m u l a t e d lymph node c e l l s as shown i n  The dose response to LPS was  q u i t e broad i n a l l t e s t s c a r r i e d  P o o l e d c e l l s d i d n o t respond as w e l l as s p l e e n or lymph node c e l l s  a l o n e (Table 1 ) .  INDUCTION OF DNA  SYNTHESIS BY CON  A  S i m i l a r e x p e r i m e n t s were done u u s i n g con A.  The r e s u l t s of s p l e e n  c e l l s t i m u l a t i o n i n serum f r e e medium a r e p r e s e n t e d i n F i g . 2A.  The  maximum r e s p o n s e was found a f t e r 48 hours of c u l t u r e u s i n g 1 ug/ml. doses  Higher  (4 and 8 ug/ml) induced moderate t r a n s f o r m a t i o n a f t e r o n l y 24 h o u r s .  The response w i t h r e s p e c t t o the SI i n medium w i t h FCS was lower than the serum f r e e response  ( F i g . 2B).  W i t h FCS, peak s t i m u l a t i o n s were induced  by 32 ug/ml a t 24 hours and 1 ug/ml a t 48 hours o f c u l t u r e .  I n con A  experiments t h e r e was a t r e n d toward h i g h doses s t i m u l a t i n g e a r l y and lower doses p e a k i n g a t a l a t e r t i m e , u s u a l l y 48 or 72 h o u r s .  Lymph node  c e l l s produced much g r e a t e r a c t i v a t i o n to con A than d i d s p l e e n c e l l s .  T y p i c a l s t i m u l a t i o n s w i t h 2 ug/ml con A over 24, between 40  and 60  fold  i n a l l media t e s t e d .  48,  and  72 hours were  As observed w i t h the LPS  r e s u l t s , the h i g h e s t S i ' s were found u s i n g serum f r e e media.  EFFECT OF ANTI-Ig AND  C' OR NYLON WOOL ON THE RESPONSE TO LPS AND  CON  A  In most cases pretreatment of the c e l l s w i t h SaGPIg and C' enhanced, the  con A response and moderately reduced the response to LPS  (Table  Only treatment of the c e l l s w i t h n y l o n wool c o n s i s t e n t l y e l i m i n a t e d LPS response; however, the con A response was  1).  (Table 20%  Treatment  1). the  a l s o d i m i n i s h e d somewhat  w i t h SaGPIg and C' k i l l e d  23%%of  spleen c e l l s  of lymph node c e l l s compared to c o n t r o l s c o n t a i n i n g normal  and  sheep  serum or C' alone or SaGPIg a l o n e .  ANTIGEN^INDUCED LYMPHOCYTE TRANSFORMATION The responses to O-Fd p l u s ME  ( F i g . 3B)  found i n serum f r e e medium ( F i g . 3A)  were v e r y s i m i l a r .  and medium  In both cases the response peaked  48 hours w i t h 32 ug/ml i n medium o n l y and 16 ug/ml i n medium w i t h By 72 hours the SI had dropped unstimulated c o n t r o l s . medium w i t h FCS and ME  s h a r p l y and by 96  The responses i n medium p l u s FCS ( F i g . 3D)  were bimodal.  responses at 24 hours which d e c l i n e d a t 48 at  96  and 120  hours.  and 120  response were o f t e n lower than those which Spleen c e l l s d i d not respond under  ME.  hours was  below  ( F i g . 3C)  and  Both media supported good  and 72 hours and peaked  The c o n c e n t r a t i o n s of O-Fd  at  which  again  induced the l a t e r  s t i m u l a t e d the 24 hour  any of the c o n d i t i o n s used even  lymph node c e l l s from the same animal were a b l e to respond.  response. though  Lymph node and  s p l e e n c e l l s from c o n t r o l animals immunized w i t h CFA o n l y were not s t i m u l a t e d by  MLC  O-Fd.  RESPONSE OF LYMPH NODE AND Data from the MLC  /-alone was period.  SPLEEN CELLS  t e s t s has been summarized i n T a b l e 2.  Medium  unable to support a response at anytime d u r i n g the c u l t u r e Except i n the case of lymph node c e l l s  s t i m u l a t i o n was  found i n medium w i t h ME.  at 96 hours, v e r y  Media w i t h FCS or FCS and  supported normal r e s p o n s e s , however, the response w i t h FCS b e t t e r than i n medium w i t h FCS o n l y .  little  and ME  ME  was  DISCUSSION  The  r e s u l t s p r e s e n t e d here have shown t h a t g u i n e a p i g lymphocytes  can be r e a d i l y c u l t u r e d and i n serum f r e e medium.  s t i m u l a t e d by mitogens and an a n t i g e n  W i t h the a d d i t i o n of ME  a mixed lymphocyte r e a c t i o n was M e d i a c o m p o s i t i o n had i n the s t i m u l a t i o n s .  also  (medium and FCS),  detected.  Average background counts per minute when c u l t u r e s  and  (medium o n l y ) , 2210  7252 (medium w i t h FCS  counts i n m i t o g e n s t i m u l a t i o n s were h i g h e r FCS,  and ME).  because the u n s t i m u l a t e d  and  The  S i ' s of  of the type of media u s e d , w h i l e supplemented w i t h  ME.  A wide range of LPS DNA  and/or  i n media w i t h o u t  c o n t r o l s were l o w e r .  c u l t u r e s were s i m i l a r r e g a r d l e s s  ME),  Although actual  i n media c o n t a i n i n g ME  the MLC's were c o n s i s t e n t l y b e t t e r when the medium was FCS  (medium p l u s  the s t i m u l a t i o n i n d i c e s were, on the a v e r a g e , h i g h e r  serum and ME O-Fd  t o the medium,  a marked e f f e c t on the c o n t r o l background counts  were t r i t i a t e d at 24 hours were 899 1825  and/or FCS  (O-Fd)  synthesis  and  concentrations  (0.25-256 ug/ml) was  i n the presence of ME  t h e r e was  the response of c e l l s t o 4-256 ug/ml LPS.  The  a b l e to i n d u c e  l i t t l e difference i n  k i n e t i c s of the LPS  was  unusual i n t h a t , regardless  of the media u s e d , s i g n i f i c a n t  was  a c h i e v e d o n l y when the c u l t u r e s were t r i t i a t e d at 24 h o u r s .  48 hours o n l y s l i g h t s t i m u l a t i o n was were below the u n s t i m u l a t e d  controls.  the dose response t o s l i g h t l y h i g h e r  found and by 72 and The LPS  response  stimulation At 0  96 hours the  a d d i t i o n of FCS  or ME  and counts  shifted  concentrations.  Both the dose response and k i n e t i c s of the con A s t i m u l a t i o n s were m o d e r a t e l y a l t e r e d by the use of FCS.  Except f o r con A a t 1 ug/ml, c u l t u r e s  14 c o n t a i n i n g FCS u s u a l l y r e q u i r e d h i g h e r c o n c e n t r a t i o n s of con A and the response was more p r o l o n g e d . of  Other workers have noted t h a t h i g h e r c o n c e n t r a t i o n s  con A a r e r e q u i r e d i n media w i t h FCS and a t t r i b u t e t h i s t o the a b i l i t y of  con A t o b i n d serum p r o t e i n s ( M o l l e r , A n d e r s s o n , P o h l i t ? and S j o b e r g , 1973). ,  Con A-induced DNA  s y n t h e s i s was s u b s t a n t i a l l y h i g h e r w i t h r e s p e c t t o  t o t a l counts and SI w i t h lymph node c e l l s t h a n w i t h s p l e e n c e l l s .  This  o b s e r v a t i o n i s c o n s i s t e n t w i t h the g e n e r a l l y a c c e p t e d v i e w t h a t t h e r e a r e g r e a t e r r e l a t i v e numbers of T lymphocytes i n lymph nodes than i n s p l e e n s . It  has been proposed t h a t con A i s a T lymphocyte s p e c i f i c mitogen i n  c h i c k e n s (Weber, 1973), g u i n e a p i g s and r a b b i t s ( E l f e n b e i n , Harrison.; and Green, 1973),  s  and mice  (Elfenbein et a l . ,  1973; Andersson e t a l . ,  1972b).  The r e s u l t s of our e x p e r i m e n t s u s i n g n y l o n wool and a n t i - I g w i t h C' a r e in  agreement w i t h s t u d i e s i n o t h e r a n i m a l systems which i n d i c a t e t h a t LPS  is  a B lymphocyte mitogen.  Using d i f f e r e n t techniques, Elfenbein et a l .  have suggested t h a t LPS i s B lymphocyte s p e c i f i c i n g u i n e a p i g s .  Using  a n t i - l i g h t c h a i n and C', Gmelig M e y l i n g , K o o y - B l o k ^ and B a l l i e u x  (1974)  (1973)  were a b l e t o k i l l 12% o f p e r i p h e r a l b l o o d lymphocytes and 42% o f lymphocytes from human t o n s i l s .  Takahashi e t .al.  (1971) u s i n g a n t i - l i g h t . c h a i n and  C'  i n mice were a b l e t o k i l l up t o 50% of s p l e e n lymphocytes and 40% of lymph node lymphocytes.  Our r e s u l t s of 23% k i l l i n g of s p l e e n c e l l s  and  20% k i l l i n g of lymph.node c e l l s are c o n s i s t e n t w i t h t h e s e f i n d i n g s s i n c e s p l e e n and lymph node c e l l s r a t h e r than e n r i c h e d p o p u l a t i o n s of lymphocytes were used.  The a b i l i t y of n y l o n wool columns t o remove c e l l s b e a r i n g s u r f a c e  Ig  (presumably B c e l l s ) has been demonstrated by J u l i u s e t . a l .  (1973).  In  our e x p e r i m e n t s , n y l o n wool t r e a t m e n t of g u i n e a p i g c e l l s c o m p l e t e l y  e l i m i n a t e d the response t o LPS, whereas t r e a t m e n t w i t h a n t i - I g and o n l y reduced i t .  U s i n g r a b b i t - a n t i mouse immunoglobulin and C  1  C'  we have  been a b l e t o reduce the LPS observations).  response of mice by more than 80%  That the guinea p i g LPS response i s r e l a t i v e l y  to  SaGPIg and C' treatment c o u l d be due  of  c e l l s w i t h a low d e n s i t y of s u r f a c e immunoglobulin.  a l s o reduced the con A response. adherence,  differential  of  responding to con A are removed.  Nylon wool  treatment  capable  U s u a l l y , the con A response was  enhanced  C'.  induced thymidine uptake  the media t e s t e d .  to an LPS r e s p o n s i v e p o p u l a t i o n  i t i s p o s s i b l e t h a t some of the c e l l s  by treatment w i t h a n t i - I g and  of  insensitive  As n y l o n wool o p e r a t e s on a p r i n c i p l e  of  O-Fd  (unpublished  i n lymph node c e l l s o n l y , but i n a l l  V i s c h e r (1972) u s i n g mice andkKirchner and Oppenheim  (1972) w i t h c h i c k e n s have r e p o r t e d serum f r e e a n t i g e n s t i m u l a t i o n u s i n g KLH of  and  sheep r e d b l o o d c e l l s , r e s p e c t i v e l y , as a n t i g e n s .  the serum f r e e O-Fd  o t h e r and d i s t i n c t l y  different  at  and FCS p l u s ME  72 hours  I t was  from the k i n e t i c s o f medium w i t h FCS unexpected  and  to see the a n t i g e n s t i m u l a t i o n  c o n t a i n i n g media occur as e a r l y as 24 h o u r s ,  decline  (aatime o f t e n c o n s i d e r e d to be o p t i m a l ) and i n c r e a s e a g a i n at  96 and 120 hours.  These r e s u l t s may  i n d i c a t e t h a t two d i f f e r e n t  p o p u l a t i o n s were responding to the a n t i g e n a t d i f f e r e n t response was  kinetics  responses w i t h and w i t h o u t ME were s i m i l a r to each  medium w i t h FCS and ME. i n FCS  The  times.  not observed i n serum f r e e media, p r o b a b l y because  serum, c e l l v i a b i l i t y was  not adequate  cell The  bimodal  without  to support a second r e s p o n s e .  Pre-  l i m i n a r y experiments, not p r e s e n t e d h e r e , t e s t i n g the response of a n t i - I g and G'  or n y l o n wool t r e a t e d c e l l s t o O-Fd  have been i n c o n c l u s i v e but  suggest t h a t SaGPIg and C' t r e a t e d c e l l s may  be a b l e t o respond, whereas  n y l o n wool t r e a t e d c e l l s seem i n c a p a b l e of r e s p o n d i n g .  Mugraby eit al_. (1974)  16  u s i n g mice demonstrated t h a t d e p l e t i o n of T or B lymphocytes reduced the response t o sheep r e d b l o o d c e l l s . The use of ME had i t s most prominent e f f e c t i n the MLC  tests.  The  response i n medium p l u s FCS and ME was g r e a t e r and more p r o l o n g e d than i n medium w i t h FCS o n l y .  No response was found i n serum f r e e medium, but  w i t h the a d d i t i o n of ME,  a d e t e c t a b l e response was o b t a i n e d .  Bevan e t a l .  (1974) u s i n g mouse s p l e e n c e l l s showed t h a t a d d i t i o n of MEEto serum f r e e t e s t s r e s u l t e d i n p o s i t i v e responses. Heber-Katz and C l i c k (1972).  MLC  S i m i l a r r e s u l t s were o b t a i n e d by  A l t h o u g h the mechanism of a c t i o n of ME  and  r e l a t e d compounds i s unknown at t h i s t i m e , i t has been observed t h a t the presence of ME,  cysteine, e t c . , greatly increases  the v i a b i l i t y of c e l l s  i n c u l t u r e (Chenand H i r s c h , 1972; Heber-Katz and C l i c k , 1972).  Chen and  H i r s c h (1972) have used ME as a s u b s t i t u t e f o r macrophages w h i c h a l s o appear t o have a b e n e f i c i a l e f f e c t on v i a b i l i t y .  I t has been proposed t h a t  ME  a c t s as a s u b s t i t u t e f o r a macrophage produced f a c t o r (Broome and J e n g , 1973) . I t would appear t h a t those responses which r e q u i r e p r o l o n g e d c u l t u r i n g of lymphocytes cannot be s u p p o r t e d i n med i a l a c k i n g FCS, as n o t e d i n the l a t e s t i m u l a t i o n by O-Fd,  o r ME,  as seen i n the MLC  tests.  However, r e s p o n s e s  which are maximal w i t h i n the e a r l y days of c u l t u r e are a d e q u a t e l y s u p p o r t e d i n the absence of t h e s e media components.  The d a t a r e p o r t e d h e r e i n d i c a t e  t h a t t h e r e a r e no major d i f f e r e n c e s between the responses of g u i n e a p i g and mouse lymphocytes i n the m i t o g e n , a n t i g e n , and MLC  t e s t systems.  TABLE 1 E f f e c t of N y l o n Wool or SaGPIg and C' Treatment on t h e Con A and LPS , Responses of Guinea P i g C e l l s  t  Medium supplements  FCS  Source of cells  spleen  Hours of culture before labelling  '  Con A ug/ml  Treatment  none  24  n y l o n wool FCS and ME  spleen  FCS and ME  node  FCS and ME  pool  FCS and ME  none  24  l  n  o  n  e  a n t i - I g and C'  pool  24  48  none  1  0.5  256  64  -  -  -  1.68*  -  -  -  0.76  16  4  1  0.25  2.71-  2.64  1.69  1.31  1.25  0.52  0.79  0.80  0.79  0.92  1.10  -  -  -  -  9.10 5.02  1.22  -.  -  -  -  '  9  3 3  70.7  '  2  3  50.8  '  "  9 9  11.9  27.4  3  '  4 2  3  '  3 6  3  '  4 4  -  2.20  2.30  1.94  -  -  2.17  1.86 1.03  nylon wool  14.6  -  -  -  -  0.75  a n t i - I g and C'  31.1  -  -  -  -  1.75  -  -  -  -  -  1.06  0.73  -  -  -  -  -  0.70  0.45  none n y l o n wool  * S t i m u l a t i o n Index  4 2  2  10.5* 3.50  a n t i - I g and C'  24 '  LPS ug/ml  -  TABLE 2 MLC T e s t s o f A l l o g e n e i c Guinea P i g Lymph Node and S p l e e n C e l l s i n FCS and ME Supplemented  Medium Only nodes**  spleen  Medium P l u s ME  Medium P l u s FCS  nodes  spleens  nodes  spleens  Media  Medium p l u s ME and FCS nodes  spleens  Day #  1.38*  1.08  1.33  1.08  1.56  1.22  1.36  1.16  2  0.93  0.92  2.00  0.80  1.34  1.35  2.05  1.45  4  0.89  1.20  1.13  1.22  2.13  2.29  2.54  3.35  6  0.96  1.20  0.76  1.28  1.53  0.82  1.52  3.87  8  1.22  1.34  0.76  1.44  1.28  0.61  0.56  2.09  10  ** Source o f c e l l s * S t i m u l a t i o n Index # The c u l t u r e s w e r e l l a b e l e d f o r 18 hours a f t e r i n c u b a t i o n f o r t h e i n d i c a t e d l e n g t h of time  19  F i g . 1.  The response of g u i n e a p i g s p l e e n  c e l l s t o LPS i n  (A) serum f r e e medium and (B) medium w i t h 4% FCS. The c u l t u r e s were l a b e l l e d w i t h t r i t i a t e d t h y m i d i n e f o r 18 hours a f t e r t h e i n d i c a t e d hours i n c u l t u r e s 0, 0 24, •  • ; 48, A  A ; 72,•  •;  and 96 ,•  •.  0;  20  21  22  F i g . 2.  The Con A responses of guinea p i g s p l e e n c e l l s i n (A) serum f r e e medium and (B) medium w i t h 4% FCS. An 18 hour l a b e l i n g w i t h t r i t i a t e d t h y m i d i n e a f t e r 0, 0 96,•  0; 24, •  •; and 120,A  • ; 48, A  A ;772,«  •; hours i n c u l t u r e .  was done a f t e r •;  25  F i g . 3.  The response of O-Fd immune g u i n e a p i g lymph node c e l l s t o O-Fd a t 1, 0 4, A  A  ug/ml.  ;  8,%  • ;  •; and 32,A  •;  The c u l t u r e s were l a b e l e d f o r 18  hours b e g i n n i n g figures.  •; 16,•  0; 2, •  a t t h e times i n d i c a t e d on t h e  The media used were (A) medium o n l y ,  (B) medium w i t h ME, (C) medium w i t h FCS, and (D) medium w i t h FCS and ME.  26  27  83  29  30  REFERENCES  1.  A n d e r s s o n , J . , Edelman, G.M. , M o l l e r , G., and S j b b e r g , 0.  (1972a).  ' A c t i v a t i o n o f B lymphocytes by l o c a l l y c o n c e n t r a t e d c o n c a n a v a l i n A.' Eur. J . Immunol., 2, 233. 2.  Ande r s s o n , J . , M o l l e r , G., and S j o h e r g , 0. of DNA s y n t h e s i s i n T and B lymphocytes.'  3.  4.  Bevan, M.J., E p s t e i n , R., and Cohn, M.  (1972b).  'Selective induction  C e l l . Immunol., 4, 381.  (1974).  'The e f f e c t o f 2-mercap-  t o e t h a n o l on murine mixed lymphocyte c u l t u r e s . '  J . Exp. Med., 139, 1025.  Broome, J.D., and J e n g , M.W.  (1973).  'Promotion o f r e p l i c a t i o n o f lymphoid  c e l l s by s p e c i f i c t h i o l s and d i s u l f i d e s i n v i t r o .  E f f e c t s on mouse  lymphoma c e l l s i n comparison w i t h s p l e n i c , lymphocytes.'  J . Exp. Med.,  138, 574. 5.  Chen, C. and H i r s c h , J.G.  (1972).  'The e f f e c t s o f m e r c a p t o e t h a n o l and  p e r i t o n e a l macrophages on t h e a n t i b o d y - f o r m i n g c a p a c i t y o f non adherent mouse s p l e e n c e l l s i n v i t r o . ' 6.  J . Exp. Med., 136, 604.  C o u t i n h o , A., M o l l e r , G., A n d e r s s o n , J . , and B u l l o c k , W.W.  (1973).  'In v i t r o a c t i v a t i o n o f mouse lymphocytes i n serum-free medium: of I  Effect  and B c e l l mitogens on p r o l i f e r a t i o n and a n t i b o d y s y n t h e s i s . ' ' E u r . J .  Immunol., 3, 299. 7.  E l f e n b e i n , G.J., H a r r i s o n , M.R., Green, I .  (1973).  'Demonstration o f  p r o l i f e r a t i o n by bone marrow-derived lymphocytes o f g u i n e a p i g s , m i c e , and r a b b i t s i n response t o mitogen s t i m u l a t i o n i n v i t r o . '  J.  Immunol.,  110, 1334. 8.  Fanger, M.W.,  H a r t , D.A., W e l l s , J.V., and N i s o n o f f , A.  (1970).  'Enhancement by r e d u c i n g agents of t h e t r a n s f o r m a t i o n o f human and r a b b i t p e r i p h e r a l lymphocytes.'  J . Immunol., 105, 1043.  9.  Gmelig M e y l i n g , F.H.J., Kooy-Blok, L., and B a l l i e u x , 'Complement-dependent c y t o l y s i s chain antisera.'  10. Hartzman, (1972).  R.E.  (1974).  of human B lymphocytes w i t h a n t i - l i g h t  E u r . J . Immunol., 4, 332.  R . J . , Bach, M.L., 'Precipitation  Bach, F.H., Thurman, G.B. and S e l l ,  of r a d i o a c t i v i t y  automatic multiple-sample harvester.' 11. Heber-Katz, E. and C l i c k , R.E.  l a b e l e d samples:  K.W.  A semi-  C e l l Immunol., 4, 182.  (1972).  'Immune responses i n v i t r o .  V. R o l e o f m e r c a p t o e t h a n o l i n t h e mixed l e u k o c y t e r e a c t i o n . '  Cell.  Immunol., 5, 410. 12. J u l i u s , M.H.,  Simpson, E. and H e r z e n b e r g , L.A.  (1973).  f o r the i s o l a t i o n of f u n c t i o n a l thymus-derived murine  'A r a p i d method  lymphocytes.'  Eur. J . Immunol., 3, 645. 13. K i r c h n e r , H. and Oppenheim, J . J .  (1972).  lymphocytes i n a serum-free medium.'  ' S t i m u l a t i o n of c h i c k e n  C e l l . Immunol., 3, 695. .  14. L a m e l i n , J . P . , L i s o w s k a - B e r n s t e i n , B., M a t t e r , A., R y s e r , J.E. and V a s s a l l i , P.  (1972).  lymphoid c e l l s . '  'Mouse thymus-independent  and thymus-derived  J . Exp. Med., 136, 984.  15. M i t c h e l l , B., L e v y , J.G. and N i t z , R.M.  (1970).  ' S y n t h e s i s of h a p t e n i c  C - t e r m i n a l o c t a p e p t i d e s of two c r o s s - r e a c t i n g f e r r e d o x i n m o l e c u l e s . ' Biochem., 9, 1839. 16. M o l l e r , G., Andersson, J . , P o h l i t , H. arid S j b b e r g , 0. t a t i o n of the number of mitogen m o l e c u l e s a c t i v a t i o n T and B lymphocytes.'  (1973).  'Quanti-  DNA s y n t h e s i s i n  C l i n . Exp. Immunol., 13, 89. •  17. Mugraby, L., Gery, I . and S u l i t z e a n u , D. of thymus-derived and bone marrow-derived  (1974).  'The  lymphocytes o f  participation sensitized  m i c e , i n the p r o l i f e r a t i v e response t o a s p e c i f i c a n t i g e n , i n v i t r o . ' Eur. J . Immunol., 4, 402.  32  18.  R a f f , M.C.  'Theta i s o a n t i g e n as a marker of thymus-derived.  ( 1 9 6 9 ) .  Nature (Lond.), 2 2 4 , 3 7 8 .  lymphocytes i n mice.' 19.  T a k a h a s h i , T., O l d , L . J . , M c l n t i r e , K.R.  and Boyse, E.A.  ( 1 9 7 1 ) .  'Immunoglobulin and other s u r f a c e a n t i g e n s of c e l l s of the immune system.' J . ;Exp. Med., 20.  Thurman, G.B., R.J. of  S t r o n g , D.M., ( 1 9 7 3 ) .  Ahmed, A., Green, S.S.,  S e l l , K.W.,  'Human mixed lymphocyte c u l t u r e s .  Hartzman, Evaluation  a m i c r o c u l t u r e technique u t i l i z i n g the m u l t i p l e automated sample  V i s c h e r , T.L. medium.'  22.  815.  and Bach, F.H.  harvestor.' 21.  .134,  Exp. Immunol., 1 5 , 2 8 9 .  (1972).  ' C u l t u r e of mouse lymphoid c e l l s  i n serum  J . Immunol. Meth., 1 , 1 9 9 .  Weber, W.T. cells  Clin.  ( 1 9 7 3 ) .  ' D i r e c t evidence f o r the response of B and T  to pokeweed mitogen.'  Cell.  Immunol., 9 , 4 8 2 .  free  33  THE IMMUNE RESPONSE TO FERREDOXIN I.  SPECIFICITY OF THE RESPONSE TO THE AMINO TERMINAL DETERMINANT  D. S. GREGERSON, BARBARA KELLY AND JULIA G. LEVY  DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, CANADA  SUMMARY  S e v e r a l s y n t h e t i c p e p t i d e s and analogues of the amino t e r m i n a l a n t i g e n i c determinant ability  to i n h i b i t  of o x i d i z e d f e r r e d o x i n (O-Fd) were t e s t e d f o r t h e i r  the complement f i x a t i o n r e a c t i o n between O-Fd and  homologous antiserum,  and t o i n h i b i t  the m i g r a t i o n of s p l e e n c e l l s  animals  immunized to O-Fd o r t o a conjugate  peptide  (N^) and bovine  of O-Fd. carboxy  of i t s amino t e r m i n a l h e p t a -  serum albumin (N^-BSA).  (H^N-ala-tyr-lys-ile-ala-asp-ser-COOH)  The  heptapeptide  i s known to be an a n t i g e n i c  The r e s u l t s of the m i g r a t i o n i n h i b i t i o n assay t e r m i n a l t r i p e p t i d e of the h e p t a p e p t i d e  suggest  determinant  t h a t the  i s p a r t i a l l y a b l e to  s t i m u l a t e the p r o d u c t i o n of m i g r a t i o n i n h i b i t i o n f a c t o r . and  from  The t e t r a p e p t i d e  l o n g e r p e p t i d e s of the n a t i v e sequence were a l l r e c o g n i z e d t o a much  g r e a t e r degree than the t r i p e p t i d e .  P e p t i d e s m o d i f i e d at the a s p a r t i c  r e s i d u e were p a r t i a l l y a c t i v e w h i l e the s e r i n e m o d i f i e d p e p t i d e was n o t . M o d i f i c a t i o n at the amino end o f the h e p t a p e p t i d e inhibition.  had no e f f e c t on m i g r a t i o n  A c o n t r o l p e p t i d e c o n t a i n i n g the same amino a c i d s i n an  a l t e r e d sequence was not r e c o g n i z e d .  The p e p t i d e s were t e s t e d w i t h  unimmunized s p l e e n c e l l s t o e l i m i n a t e the p o s s i b i l i t y of c y t o t o x i c i t y or n o n s p e c i f i c i n h i b i t i o n of m i g r a t i o n . i t was shown t h a t the  As a d d i t i o n a l s p e c i f i c i t y  p e p t i d e and the N^-BSA conjugate  controls,  inhibited  m i g r a t i o n i n O-Fd immunized animals, w h i l e O-Fd i n h i b i t e d m i g r a t i o n i n N^-BSA immunized  animals.  The hexa, hepta, a s p a r t i c d e l e t e d and s e r i n e m o d i f i e d p e p t i d e s were a b l e to i n h i b i t the complement f i x a t i o n r e a c t i o n w i t h O-Fd and s p e c i f i c r a b b i t antiserum.  I n h i b i t i o n found w i t h the s e r i n e m o d i f i e d p e p t i d e and  the l a c k of i n h i b i t i o n w i t h the amino m o d i f i e d p e p t i d e or the d i , t r i ,  and  tetrapeptides antibodies of MIF  i n d i c a t e s that  the determinant  r e c o g n i z e d by the r a b b i t  i s e i t h e r l a r g e r than the determinant which induces the p r o d u c t i o n  or i s l o c a t e d n e a r e r t o the middle of the h e p t a p e p t i d e .  p e p t i d e d i d not i n h i b i t  complement  fixation.  The  control  INTRODUCTION  The  s p e c i f i c i t y o f a n t i g e n r e c o g n i t i o n by a n t i b o d i e s and thymus  d e r i v e d lymphocytes (T c e l l s ) has been examined i n t h e p a s t . these s t u d i e s have used c h e m i c a l l y m o d i f i e d a n t i g e n s w i t h determinants.  uncharacterized  P a r i s h (1972) u s i n g s e v e r a l h e t e r o l o g o u s . r e d  c h e m i c a l m o d i f i c a t i o n s o f them demonstrated s i g n i f i c a n t  S e v e r a l of  b l o o d c e l l s and  cross r e a c t i v i t y at  the T c e l l l e v e l and an i n v e r s e c o r r e l a t i o n i n t h e a b i l i t i e s o f t h e v a r i o u s m o d i f i e d and u n m o d i f i e d  red blood c e l l s t o s t i m u l a t e delayed  or a n t i b o d y p r o d u c t i o n .  S i m i l a r r e s u l t s were found p r e v i o u s l y u s i n g  n a t i v e and a c e t o a c e t y l a t e d f l a g e l l i n albumins, Schirrmacher  hypersensitivity  ( P a r i s h , 1971 a,b).  Using  modified  and W i g z e l l (1972) and Rubin and W i g z e l l (1973)  a l s o demonstrated a g r e a t e r degree o f c r o s s r e a c t i v i t y i n t h e c e l l mediated. immune response than i n t h e a n t i b o d y r e s p o n s e . using s e v e r a l heterologous  Hoffman and K a p p l e r  (1973)  r e d b l o o d c e l l s showed t h a t c r o s s r e a c t i o n a t  the T c e l l l e v e l was g r e a t e r than t h a t seen w i t h a n t i b o d i e s .  There a r e a  number o f o t h e r r e p o r t s which i m p l y t h a t t h e c e l l u l a r immune response has a w i d e r c r o s s r e a c t i v i t y t h a n t h e a n t i b o d y response (Maron, Webb, Teitelbaum  and A r n o n , 1972; Schirrmacher  and W i g z e l l , 1974; Coon and  H u n t e r , 1973; D a i l e y and H u n t e r , 1974; Champlin and Hunter, 1975). s t u d i e s have been l i m i t e d by t h e l a c k o f d e f i n e d d e t e r m i n a n t s  These s t u  t o which  b o t h T c e l l s and a n t i b o d y a r e d i r e c t e d , making comparisons o f s p e c i f i c i t y questionable. The use o f h a p t e n - c a r r i e r systems (DNP-protein i n most cases produced p r e d o m i n a n t l y  conjugates,  e t c . ) has  a n t i h a p t e n a n t i b o d i e s and a n t i - c a r r i e r  37  c e l l mediated immunity ( K a t z , P a u l , G o i d l and B e n a c e r r a f , 1970; K a t z , G o i d l and  B e n a c e r r a f , 1970;  D a v i e and P a u l , 1971)  a b i l i t y t o compare s p e c i f i c i t i e s .  Paul,  e l i m i n a t i n g the  I n those cases where anti-DNP  cell  mediated immunity has been produced, the s p e c i f i c i t y of the responses has not been compared (Janeway, Cohen, Ben-Sasson and P a u l , 1975;  Goscicka,  1974). The  a n t i g e n i c s p e c i f i c i t y of T c e l l s has  a l s o been t e s t e d  using  s m a l l immunogenic m o l e c u l e s , e s p e c i a l l y a z o b e n z e n e a r s o n a t e - L - t y r o s i n e (ABA-L-tyr).  Data from s e v e r a l l a b o r a t o r i e s has  minor m o d i f i c a t i o n s t o the amino and  indicated that c e r t a i n  carboxy groups of the  tyrosine  m o i e t y or to the a r s o n a t e group do not r e s u l t i n a complete l o s s of r e c o g n i t i o n . As no a n t i b o d y  i s produced to A B A - L - t y r , t h e s e e x p e r i m e n t s do not  a d i r e c t comparison of a n t i b o d y Leskowitz,  1973;  permit  and T c e l l r e c o g n i t i o n (Hanna and  J o k i p i i and J o k i p i i , 1974;  A l k a n , W i l l i a m s , N i t e c k i and Goodman, 1972;  B e c k e r , L e v i n and  Sela,  1973;  Bush, A l k a n , N i t e c k i and.  Goodman, 1972). More d e f i n i t i v e  work has been done on the coat p r o t e i n of tobacco  mosaic v i r u s d e m o n s t r a t i n g t h a t the minimum s i z e of a d e t e r m i n a n t recognized  by a n t i b o d i e s i s 5 amino a c i d r e s i d u e s .  were found t o be nonimmunogenic and  d i d not  These s m a l l  s t i m u l a t e DNA  peptides  synthesis i n v i t r o  w i t h s e n s i t i z e d lymphoid c e l l s , but d i d g i v e p o s i t i v e d e l a y e d s k i n r e a c t i o n s and  s t i m u l a t e d the p r o d u c t i o n  Conservatively modified  inhibition  p e p t i d e s were r e c o g n i z e d  s p e c i f i c i t y of r e c o g n i t i o n was r a b b i t s (Benjamini,  of m i g r a t i o n  factor  (MIF).  and a d i f f e r e n c e i n the  noted between a n t i - s e r a from d i f f e r e n t  S h i m i z u , Young and Leung, 1969'; S p i t l e r ,  Young, K a p l a n and Fudenberg, 1969).  Benjamini,  Specificity  s t u d i e s done on glucagon, a s m a l l p o l y p e p t i d e  of 29  amino a c i d r e s i d u e s , have i n d i c a t e d the presence of two a n t i g e n i c one i n each h a l f of the m o l e c u l e . recognized  by a n t i - g l u c a g o n  The amino t e r m i n a l determinant i s s t r o n g l y  a n t i b o d i e s and a l s o s t i m u l a t e s MIF  The carboxy t e r m i n a l determinant has l e s s antibody but  s t i m u l a t e s DNA  s y n t h e s i s and MIF p r o d u c t i o n  N i t e c k i and Goodman, 1971).  determinants,  production.  directed against i t ,  in vitro  (Senyk, W i l l i a m s ,  Thus, the amino t e r m i n a l determinant has  g r e a t e r "hapten" a c t i v i t y w h i l e  the carboxy t e r m i n a l determinant has  g r e a t e r " c a r r i e r " a c t i v i t y although  n e i t h e r i s e x c l u s i v e l y antibody  or  T c e l l directed. Work i n t h i s l a b o r a t o r y w i t h f e r r e d o x i n i s o l a t e d from C l o s t r i d i u m pasteurianum has r e v e a l e d t h a t t h i s p o l y p e p t i d e has  o n l y two major d e t e c t a b l e determinants,  (Ny) and the carboxy t e r m i n a l p e n t a p e p t i d e and  Christenson,  of 55 amino a c i d r e s i d u e s  the amino t e r m i n a l (C5)  ( N i t z , M i t c h e l l , Gerwing  1969; M i t c h e l l , Levy and N i t z , 1970; M i t c h e l l and Levy,  K e l l y and Levy, 1971).  Both a n t i b o d i e s and T c e l l s r e c o g n i z e  these determinants w i t h N^ b e i n g s t i m u l a t i n g the p r o d u c t i o n  s l i g h t l y more T c e l l d i r e c t e d and C^  of a somewhat g r e a t e r q u a n t i t y of  Levy, H u l l , K e l l y , K i l b u r n and T e a t h e r ,  antibody  A c o n s i d e r a b l e amount of work has been done w i t h  the N 7 determinant  s e v e r a l m o d i f i a b l e amino a c i d s  125 can be l a b e l e d to a h i g h s p e c i f i c a c t i v i t y w i t h  a c e t i c anhydride.  N e i t h e r of these  1972;  1972; Pearson, Levy and K i l b u r n ,  since i t i s r e a d i l y synthesized, contains  and  1970;  each of  ( K e l l y and Levy, 1971; W a t e r f i e l d , Levy, K i l b u r n , and T e a t h e r ,  1975).  heptapeptide  3 I or  H labeled  l a b e l i n g procedures causes a l o s s  of r e c o g n i t i o n ( K e l l y and Levy, 1971; Pearson e t a l , 1975).  In  this  study, the f i n e  specificity  o f the immune response t o the  determinant has been examined by u s i n g s e v e r a l p e p t i d e analogues I n h i b i t i o n o f the complement f i x a t i o n antibody s p e c i f i c i t y .  o f Ny.  r e a c t i o n has been used t o assay  The l e u c o c y t e m i g r a t i o n i n h i b i t i o n  a s s o c i a t e d w i t h delayed h y p e r s e n s i t i v i t y and c e l l u l a r  test,  immunity  classically ( S a l v i n and  Smith, 1960; D a v i d , A l - A s k a r i ; Lawrence and Thomas, 1964; D a v i d , Lawrence and Thomas, 1964; David and Schlossman, specificity. nition  1968) was used t o determine T c e l l  The r e s u l t s d e s c r i b e d here compare the s p e c i f i c i t y  by a n t i b o d i e s and T c e l l s t o a s i n g l e  determinant.  of recog-  MATERIALS AND METHODS  ANIMALS Young a d u l t o u t b r e d all  a l b i n o r a b b i t s and g u i n e a p i g s were used f o r  experiments.  PEPTIDES S e v e r a l p e p t i d e s o f v a r i o u s s t r u c t u r e s were prepared  by s o l i d phase  p e p t i d e s y n t h e s i s as d e s c r i b e d by Hancock, P r e s c o t t , M a r s h a l l and V a g e l o s (1972), a m o d i f i c a t i o n o f t h e M e r r i f i e l d s y n t h e s i s ( M e r r i f i e l d , 1963). The p e p t i d e s and nomenclature a r e shown i n T a b l e 1.  The GEE-Ny p e p t i d e was  made by r e a c t i n g t h e Ny p e p t i d e w i t h 20 e q u i v a l e n t s o f g l y c i n e e t h y l e s t e r and l-ethyl-3-(dimethylaminopropyl) for  carbodiimide hydrochloride  4 hours (Hoare and K o s h l a n d , 1967).  (EDCI) a t pH 4.7  The N ^ - B z l - S e r p e p t i d e was s y n t h e -  s i z e d by s o l i d phase except t h a t i t was c l e a v e d from t h e r e s i n by t r a n s e s t e r i f i c a t i o n w i t h dimethylaminoethanol. removed by h y d r o l y s i s i n DMF-water  (1:2) l e a v i n g t h e 0 - b e n z y l e t h e r b l o c k i n g  group i n t a c t on t h e s e r i n e h y d r o x y l . Savoie and B a r t o n  (1974).  The r e s u l t i n g e s t e r s were  The p r o c e d u r e i s t h a t p u b l i s h e d by  The N-M-Asp p e p t i d e was an e r r o r p e p t i d e  from t h e Ny s y n t h e s i s . . DNP-Ny was a g i f t from D. W a t e r f i e l d .  isolated  The p e p t i d e s  were r o u t i n e l y p u r i f i e d by Sephadex G-15 g e l f i l t r a t i o n i n 0.05 M a c e t i c a c i d and i o n exchange on Dowex 1 x 2 r e s i n u s i n g p y r i d i n e - a c e t i c a c i d - w a t e r buffers.  P e p t i d e p u r i t y was a s s e s s e d  by t h i n l a y e r chromatography on  s i l i c a g e l G u s i n g at. l e a s t 3 s o l v e n t systems.  F u r t h e r t e s t s o f p u r i t y and  q u a n t i t a t i o n were performed on a Beckman 120 amino a c i d a n a l y z e r . r a t i o s from t h e amino a c i d a n a l y s e s a r e shown i n T a b l e 2.  Molar  ANTIGENS F e r r e d o x i n from C_. p a s t e u r i a n u m was  purchased from Sigma ( S t . L o u i s ,  M i s s o u r i ) o r i s o l a t e d from c u l t u r e s as d e s c r i b e d by Mortenson (1964) and Tanaka, Nakashima, Mower and Yasunobu (1964). d i s s o l v i n g the f e r r e d o x i n (20-40mg) i n 1.66 3.33  The  r e a c t i o n was  The N^-BSA c o n j u g a t e  and a n a l y z e d  was  evaporated  made by m i x i n g N^ and BSA  d i a l y z e d , concentrated  degree of c o u p l i n g . (PDG,  N^ was  30%  under vacuum,  f o r amino a c i d c o n t e n t  of EDCI a c c o r d i n g t o Hoare and K o s h l a n d (1967). the s o l u t i o n was  ml of  a l l o w e d to c o n t i n u e f o r 2 hours a t  a f t e r w h i c h the r e a c t i o n m i x t u r e was  d i a l y z e d , concentrated  performed by  ml f o r m i c a c i d f o l l o w e d by  ml of p e r f o r m i c a c i d (3.0 ml of f o r m i c a c i d p l u s 0.33  hydrogen p e r o x i d e ) . -10°,  O x i d a t i o n was  quantity.  (20:1) i n the p r e s e n c e  A f t e r 4 hours r e a c t i o n  and a n a l y z e d  a l s o conjugated  and  to determine the  to p o l y - D - g l u t a m i c  acid  MW-75,000 Sigma) and p o l y - L - l y s i n e (PLL, MW-17,000 Schwarz-Mann)  by the same p r o c e d u r e .  S u b s t i t u t i o n r a t i o s as c a l c u l a t e d from amino a c i d  a n a l y s e s b e f o r e and a f t e r s u b s t i t u t i o n were:; N7-BSA, 6:1;  N-.-PDG, 10.3:1;  N - P L L , 12.6:1. 7  IMMUNIZATIONS R a b b i t s were immunized i n t r a m u s c u l a r l y i n each l e g s e v e r a l times 2 week i n t e r v a l s w i t h 1 mg (FCA)  doses of 0-Fd  to produce the a n t i s e r a .  i n 50% Freund's'complete  Guinea p i g s were immunized t w i c e  m u s c u l a r l y w i t h e i t h e r 250 ug o f O-Fd  or Ny-BSA i n 50%  at  adjuvant intra-  FCA.  SKIN TESTS The  shaved and d e p i l l a t e d f l a n k s of g u i n e a p i g s were i n j e c t e d i n t r a -  d e r m a l l y w i t h 50 ug of a n t i g e n i n 0.1 ml s a l i n e .  C o n t r o l s c o n s i s t e d of  42  t e s t s w i t h 0.1 ml antigens  s a l i n e on immunized animals and  on unimmunized  complete t e s t s w i t h a l l  animals.  LEUCOCYTE MIGRATION INHIBITION TEST Spleen saline  cells  from guinea p i g s were washed twice  (pH 7.2, 0 . 0 1 M PO^)  and  heat-inactivated foetal calf The  The  resuspended i n RPMI 1 6 4 0 medium p l u s  ( F i s h e r ) and  c a p i l l a r y tubes which were s e a l e d  c o n t a i n i n g 1 ml of medium.  placed  cytotoxic effects. at 1 6 ug/ml.  3 7 ° , 5 % C O 2 i n c u b a t o r , the areas c a l i b r a t e d stage of a m i c r o s c o p e .  The  of m i g r a t i o n were measured u s i n g  the r a t i o of areas was  An  this  the  C o n t r o l experiments were performed i n an The  procedure i s s i m i l a r to t h a t  1 9 7 2 ; Levy, et a l , 1 9 7 2 ; W a t e r f i e l d ,  r e s u l t s are expressed  as the r a t i o of  m i g r a t i o n a r e a found w i t h a t e s t a n t i g e n to the m i g r a t i o n c o n t a i n i n g o n l y medium.  at  A f t e r 1 8 hours i n c u b a t i o n i n a  d e s c r i b e d p r e v i o u s l y ( W a t e r f i e l d , et_ al, Kilburn, 1974).  obtained  of  Ny-PDG, N^-PLL and Ny-BSA were  i d e n t i c a l manner on unimmunized animals.  Levy and  i n t o chambers  Test p e p t i d e s were used at a c o n c e n t r a t i o n  0 . 0 5 umoles/ml i n the same medium as good i n h i b i t i o n was  used at 1 0 ug/ml and O-Fd  with  the c e l l s sedimented at 2 0 0 x g f o r 5 m i n u t e s .  tubes were broken at the cell-medium i n t e r f a c e and  c o n c e n t r a t i o n without  5%  serum a t a c o n c e n t r a t i o n of 1 0 % c e l l s / m e d i a .  c e l l s were drawn i n t o 1 x 7 5 mm  p l a s t i c putty  i n phosphate b u f f e r e d  i n h i b i t i o n was  l e s s than 0 . 8 0 and  considered  the  area of c o n t r o l s  significant i f  the t - p r o b a b i l i t y was  l e s s than 0 . 0 5 .  INHIBITION OF COMPLEMENT FIXATION The  complement f i x a t i o n t e s t s were performed as d e s c r i b e d p r e v i o u s l y  (Gerwing and Thompson, 1 9 6 8 ) u s i n g r a b b i t anti-O-Fd been heat i n a c t i v a t e d at 5 6 ° f o r 3 0 minutes.  antiserum  which  had  RESULTS  The a b i l i t y o f preformed a n t i b o d y t o O-Fd t o r e c o g n i z e m o d i f i e d o r partial  p e p t i d e s was assessed by measurement o f t h e i r a b i l i t y t o i n h i b i t  f i x a t i o n o f complement i n t h e presence o f O-Fd and t h e a n t i b o d y a t o p t i m a l proportions.  The r e s u l t s a r e shown i n F i g u r e 1 A and B and r e p r e s e n t t h e  average o f 4 i n d i v i d u a l e x p e r i m e n t s .  P e r c e n t i n h i b i t i o n s were e s t i m a t e d  at t h e range i n w h i c h c o n t r o l tubes c o n t a i n i n g o n l y O-Fd and a n t i s e r u m y i e l d e d 50% h e m o l y s i s  i n t h e presence o f complement.  Of t h e p a r t i a l Ny  p e p t i d e s , o n l y N g was capable of c o n s i s t e n t l y i n h i b i t i n g T h i s suggests  t h a t t h e p o r t i o n of t h e  determinant  the r e a c t i o n .  r e c o g n i z e d by a n t i b o d y  encompasses 6 o f t h e 7 r e s i d u e s o r i s l o c a t e d c e n t r a l l y o r near t h e amino t e r m i n a l end. The p o s s i b i l i t y t h a t t h e s p e c i f i c i t y o f t h e r e c o g n i t i o n may i n v o l v e amino a c i d r e s i d u e s a t t h e amino t e r m i n a l p o r t i o n o f t h e m o l e c u l e i s supported inhibiting  by t h e o b s e r v a t i o n t h a t DNP-N^ ( F i g . IB) was i n c a p a b l e o f  t h e complement f i x a t i o n r e a c t i o n .  U n l i k e o b s e r v a t i o n s made w i t h  the l e u c o c y t e m i g r a t i o n i n h i b i t i o n t e s t (see b e l o w ) , t h e p e p t i d e m o d i f i e d at t h e s e r i n e r e s i d u e ( N ^ - B z l - S e r ) and c o n t a i n i n g o n l y t h e c o n s i s t e n t l y a b l e t o i n h i b i t complement f i x a t i o n a l t h o u g h  amino a c i d s was a l o n e was  u n a b l e t o do s o . I t i s p o s s i b l e t h a t t h e O-Benzyl group may p r o v i d e a hydrophobic  enhancing e f f e c t as noted by o t h e r s ( B e n j a m i n i e_t a l , 1969)  without i n t e r f e r i n g with s p e c i f i c i t y .  Unlike the migration  inhibition  t e s t s , t h e p e p t i d e l a c k i n g a s p a r t i c a c i d (N-M-Asp) was a l s o capable o f inhibiting  complement f i x a t i o n , thus r e i n f o r c i n g t h e p o s s i b i l i t y t h a t t h e  s p e c i f i c i t y f o r antibody r e c o g n i t i o n l i e s c e n t r a l l y w i t h i n the Specificity  determinant.  of t h e r e a c t i o n was e s t a b l i s h e d by n e g a t i v e r e s u l t s o b t a i n e d w i t h  NC^  (the s y n t h e t i c p e p t i d e  a different The  c o n t a i n i n g the same amino a c i d s as Ny,  but i n  sequence).  s p e c i f i c i t y requirements  f o r c e l l u l a r immunity as assessed by  i n h i b i t i o n of l e u c o c y t e m i g r a t i o n were measured u s i n g s p l e e n c e l l s guinea p i g s s e n s i t i z e d to O-Fd.  Spleen  analogues.  determinant  and  T e s t s were a l s o c a r r i e d out w i t h  (a s y n t h e t i c p e p t i d e of 22 amino a c i d s c o n t a i n i n g two l i n k e d by 8 g l y c i n e r e s i d u e s  Ny  The  with N-8-N  O-Fd,  r e s u l t s are summarized i n T a b l e  I t can be seen t h a t , i n terms of s i z e , the N^ p e p t i d e was  observed  Ng p e p t i d e a l s o caused i n h i b i t i o n ; t h i s was  Although  N£ was  i t is  t h a t , o c c a s i o n a l l y , the  observed  w i t h s p l e e n c e l l s which  demonstrated s t r o n g e r than average i n h i b i t i o n i n the presence of N^ Ny.  3.  the s m a l l e s t p a r t '  of the Ny p e p t i d e which c o n s i s t e n t l y i n h i b i t e d m i g r a t i o n . not shown by the averaged d a t a , i t was  of  determinants  (see K e l l y , Levy and H u l l , 1973),  Ny-BSA, Ny-PDG, Ny-PLL and NCy.  from  c e l l s were s e t up w i t h p e p t i d e s  v a r i o u s l e n g t h , each c o n s t i t u t i n g p a r t of the chemically modified  the  c o n s i s t e n t l y unable to i n h i b i t m i g r a t i o n .  The NCy  through  peptide  had  no e f f e c t on the m i g r a t i o n thus e s t a b l i s h i n g the dependence of the response on amino a c i d sequence r a t h e r than on o v e r a l l charge e f f e c t s . on p e p t i d e s  i n which e i t h e r the a s p a r t i c or s e r i n e r e s i d u e s had  showed minimal or no  run  been m o d i f i e d  a b i l i t y to i n h i b i t l e u c o c y t e m i g r a t i o n , i m p l y i n g  importance of the i n t e g r i t y of t h i s end of Ny A l l of the Ny  Tests  conjugates  in cellular recognition.  (Ny-PDG, e t c ) were a b l e to i n h i b i t  emphasizing the l a c k of c a r r i e r e f f e c t . v a r y i n g l e n g t h (^-Ny) and  the  migration,  Both the data on the p e p t i d e s  the data on the s e r i n e and  p e p t i d e s i n d i c a t e t h a t the a c t u a l sequence r e c o g n i z e d  of  a s p a r t i c modified i n both l e n g t h  and  configurational  s p e c i f i c i t y i s c o n t a i n e d w i t h i n the N^ p e p t i d e , s i n c e  l o n g e r p e p t i d e s do not show a g r e a t e r a b i l i t y t o i n h i b i t m i g r a t i o n ,  and  p e p t i d e s m o d i f i e d at the amino t e r m i n a l or l y s i n e r e s i d u e (DNP-Ny) are s t i l l s p e c i f i c a l l y r e c o g n i z e d by s e n s i t i z e d  cells.  I n the e x p e r i m e n t s d e s c r i b e d above, t h e immunogen i n a l l i n s t a n c e s was  O-Fd.  F u r t h e r s t u d i e s were c a r r i e d out to determine the e f f e c t on  c e l l mediated response t o the Ny d e t e r m i n a n t when the Ny p e p t i d e conjugated  w i t h ED^T  to BSA.  tested for skin r e a c t i v i t y carriers.  Guinea p i g s so immunized were  to the Ny d e t e r m i n a n t conjugated  S k i n r e a c t i o n s were measured at 4 and  24 h o u r s .  the  was  subsequently to a v a r i e t y The  of  results  are shown i n T a b l e 4 and demonstrate t h a t b o t h a n t i b o d y and c e l l mediated responses were e l i c i t e d t o the Ny d e t e r m i n a n t .  This observation i s i n  agreement w i t h p r e v i o u s work (Levy e_t_ a l _ , 1972)  which showed t h a t s i m i l a r  conjugates with  e l i c i t e d b o t h types of s k i n r e a c t i o n s i n guinea p i g s immunized  O-Fd. The  s p e c i f i c i t y of the c e l l mediated response i n g u i n e a p i g s immunized  w i t h Ny-BSA was The  assessed  r e s u l t s are presented  u s i n g the l e u c o c y t e m i g r a t i o n i n h i b i t i o n i n T a b l e 5.  I n most r e s p e c t s , the r e s u l t s  analogous t o t h o s e found w i t h c e l l s s e n s i t i z e d t o O-Fd, Ny and O-Fd  caused marked i n h i b i t i o n whereas N-M-Asp was  the a s p a r t i c a c i d m o d i f i e d p e p t i d e  test.  (GEE-Ny) was  weakly  are  i n t h a t N^' t h r o u g h not a c t i v e  and  recognized.  A summary of the r e s u l t s o b t a i n e d w i t h a n t i b o d y or immune c e l l s i s shown i n T a b l e  6.  DISCUSSION  T h e d a t a summary i n T a b l e 6 c l e a r l y d e m o n s t r a t e s t h a t b o t h  antibodies  and T c e l l s f r o m O - F d immune a n i m a l s r e c o g n i z e d e t e r m i n a n t s w i t h i n t h e Ng peptide.  However, the s p e c i f i c i t i e s are not  appears t o be c r i t i c a l (DNF-N^) o r e l i m i n a t i o n DNPpN^ a n d  for of  recognition  it  (N^)  cause a l o s s of  In  contrast,  demonstrates the important N^ p e p t i d e  inhibits  and GEE-Ny) their for  role  role  lack sufficient  l o s s of m i g r a t i o n of  the  migration w e l l .  binding to T c e l l s .  the determinant  (N^)  Conversely, a  at  or  hydrophobicity  inhibition  to  N^-Bzl-Ser  a l l , to  r e c o g n i z e d by a n t i b o d y ,  r e c o g n i z e d by T c e l l s .  et  recognition  bulk  Aspartic acid modified  The N-M-Asp p e p t i d e i s  (Waterfield  for  antibody  peptides  inhibit  since  the  (N-M-Asp  migration  than  implying the a s p a r t i c residue i s necessary  f r o m t h e amino d e t e r m i n a n t  p r e v i o u s work  lysine  serine in T c e l l recognition  a l s o l e s s a b l e to  r e a c t i o n which suggests that  i n the determinant  fragment  the  counterpart  complement f i x a t i o n  the  The a d d i t i o n o f  t h e amino a c i d s c r i t i c a l  are considerably less able, i f  unmodified  residue  t h e s e r i n e r e s i d u e o f N^ ( N ^ - B z l - S e r ) r e s t o r e s  a n d may i n d i c a t e t h a t  binding.  of  recognition.  inhibition.  by a n t i b o d i e s a r e c o n t a i n e d i n N ^ , b u t for  The l y s i n e  a n t i b o d y b i n d i n g as m o d i f i c a t i o n s  give positive migration  h y d r o p h o b i c group t o  identical.  the  the a s p a r t i c residue plays though not  A pentapeptide  was u n a b l e t o  a l , 1974)  inhibit  as important  as  a in  (ala-tyr-lys-ile-ala)  inhibit  migration  emphasizing the importance of  in the  a s p a r t i c and s e r i n e r e s i d u e s . Of t h e p e p t i d e s u s e d i n t h e m i g r a t i o n  inhibition  immune g u i n e a p i g s p l e e n , c e l l s , o n l y N - M - A s p i s n o t migration  inhibition  new d e t e r m i n a n t  t e s t s w i t h N^-BSA  recognized.  found w i t h GEE-N^ i s p r o b a b l y due t o  The  the c r e a t i o n of  o n t h e immunogen N - B S A b y c o u p l i n g t h r o u g h t h e P - c a r b o x y l 7  a of  47  the a s p a r t i c r e s i d u e d u r i n g c o n j u g a t i o n t o BSA. that the s y n t h e t i c  R e c o g n i t i o n of  suggests  on BSA f u n c t i o n s much the same as t h e amino d e t e r m i n a n t  on O-Fd. The minimum s i z e o f an a n t i b o d y b i n d i n g d e t e r m i n a n t appears t o be 4-6 r e s i d u e s i n agreement w i t h s e v e r a l r e p o r t s (Young, B e n j a m i n i , Stewart and Leung, 1967; B e n j a m i n i , Young and Leung, 1968 a and b; reviewed by Goodman, 1969).  I n t h e p r e s e n t work t h e t e t r a p e p t i d e , N^, i s c o n s i s t e n t l y  able to i n h i b i t migration.  S p i t l e r et_ a l (1969) found m i g r a t i o n i n h i b i t i o n  w i t h a p e n t a p e p t i d e from tobacco v i r u s p r o t e i n . The d i f f e r e n c e s observed between a n t i b o d y and T c e l l s p e c i f i c i t y may be due t o two s e p a r a t e but o v e r l a p p i n g d e t e r m i n a n t s w i t h i n N, or t o t h e o  d i f f e r e n t n a t u r e s o f a n t i b o d y and T c e l l r e c e p t o r s .  I f a n t i b o d y and T c e l l  r e c e p t o r s have d i f f e r e n t b i n d i n g c h a r a c t e r i s t i c s , a s i n g l e m o d i f i c a t i o n may not have t h e same e f f e c t on t h e a b i l i t y o f t h e p e p t i d e t o b i n d t o t h e s e two r e c e p t o r s even i f they both r e c o g n i z e i d e n t i c a l d e t e r m i n a n t s on t h e u n m o d i f i e d molecule.  I t i s g e n e r a l l y a c c e p t e d t h a t t h e a n t i g e n r e c e p t o r on thymus  independent lymphocytes  (B c e l l s ) i s a n t i b o d y (reviewed by V i t e t t a and U h r ,  1975) and t h e s p e c i f i c i t y o f B c e l l s i s p r o b a b l y v e r y s i m i l a r o r i d e n t i c a l to  t h e a n t i b o d y they produce (Makela, 1970).  The n a t u r e o f t h e r e c e p t o r on  T c e l l s i s s t i l l u n c e r t a i n (Crone, Kock and Simonsen, 1972; Greaves, 1975; Munro and T a u s s i g , 1975).  E v i d e n c e from experiments where t h e a n t i g e n  s t i m u l a t e d p r o d u c t i o n o f MIF by g u i n e a p i g c e l l s was b l o c k e d by t h e a d d i t i o n of  a n t i - ^ , ^ g o r £ i m p l i e s t h a t the r e c e p t o r i s i m m u n o g l o b u l i n - l i k e o r  c l o s e l y a s s o c i a t e d w i t h i t on t h e c e l l s u r f a c e ( G o s c i c k a , 1974).  Also,  i d i o t y p i c determinants and Lindenmann The  have been found on T c e l l s  ( B i n z , W i g z e l l , Ramseier  (1975)).  a b i l i t y of p e p t i d e s t o s t i m u l a t e MIF  u s e f u l f o r s t u d i e s of T c e l l s p e c i f i c i t y . and used d i r e c t l y w i t h o u t  p r o d u c t i o n makes them v e r y  P e p t i d e s can be  synthesized  c o u p l i n g them t o o t h e r p r o t e i n s or compensating  f o r the presence and e f f e c t of o t h e r d e t e r m i n a n t s . q u a n t i t i e s of p e p t i d e s used to i n h i b i t m i g r a t i o n i s e q u i v a l e n t to 280 ug O-Fd  The  r e l a t i v e l y large  (0.05 umole of  peptide  on the b a s i s of m o l a r i t y ) p r o b a b l y  reflects  the f a c t t h a t the p e p t i d e s have l i t t l e t e r t i a r y s t r u c t u r e t o m a i n t a i n i n the c o n f i g u r a t i o n found i n O-Fd.  them  I t has been shown t h a t o n l y a s m a l l  p e r c e n t a g e of p e p t i d e s i n s o l u t i o n have the same c o n f i g u r a t i o n they would have i n the parent m o l e c u l e (Crumpton and S m a l l , 1967). As found i n o t h e r s t u d i e s w i t h s m a l l p e p t i d e s , the p e p t i d e s here a r e u n a b l e t o s t i m u l a t e DNA from e i t h e r O-Fd  s y n t h e s i s i n v i t r o i n lymph node c e l l s  or N -BSA immunized a n i m a l s . 7  dodecapeptide from g l u c a g o n (Senyk et_ a l , 1971) guinea p i g c e l l s .  The  One  reported exception i s a  which i s a b l e t o  a b i l i t y of s m a l l p e p t i d e s t o g e n e r a t e MIF  s t i m u l a t i n g DNA  s y n t h e s i s i s c o n s i s t e n t w i t h r e p o r t s t h a t MIF  not r e q u i r e DNA  s y n t h e s i s (Bloom, G a f f n e y and Jimenez, 1972;  T h i s d i s s o c i a t i o n between MIF  synthesis.  be g e n e r a t e d w i t h o u t DNA  transform without  p r o d u c t i o n does R o c k l i n , 1973).  p r o d u c t i o n and t r a n s f o r m a t i o n i s  but suggests t h a t two l e v e l s of a c t i v a t i o n can o c c u r i n T w i t h and w i t h o u t DNA  described  unexplained,  cells—activation  The r e p o r t t h a t c y t o t o x i c c e l l s can a l s o  s y n t h e s i s and  c e l l d i v i s i o n supports  this  hypothesis  (MacDonald, S o r d a t , C e r o t t i n i and Brunner, 1975). The p e p t i d e s used i n t h i s study a r e not immunogenic u n l e s s coupled m o l e c u l e such assBSA.  I t i s apparent t h a t N  7  and DNP  to a  ( a l s o a hapten)  do not a c t a l i k e when conjugated t o p r o t e i n s . immunity are d i r e c t e d to In animals  immunized to N^-BSA, both  molecule) and BSA The  w h i l e DNP  Both humoral and  cellular  i s o n l y r e c o g n i z e d by a n t i b o d i e s . (when coupled to a h e t e r o l o g o u s  individually e l i c i t  immediate and d e l a y e d s k i n r e a c t i o n s .  c l a s s i c a l h a p t e n - c a r r i e r phenomena i s not expressed by N^-BSA.  be r e c o g n i z e d when coupled to d i v e r s e c a r r i e r s such as BSA, s u g g e s t i n g t h a t shared determinants  inability  of O-Fd  to be performed  PLL to  response.  to s t i m u l a t e p r o d u c t i o n of a u s e f u l t i t e r of  a n t i b o d y i n guinea p i g s i s u n f o r t u n a t e s i n c e the complement t e s t s had  and  do not c o n t r i b u t e s i g n i f i c a n t l y  e i t h e r the a n t i b o d y or T c e l l mediated The  PDG  can  w i t h r a b b i t serum.  d i f f e r e n c e on the s p e c i f i c i t i e s  i s unknown.  fixation  The e f f e c t of the s p e c i e s  TABLE 1 NAMES AND STRUCTURES OF SYNTHETIC PEPTIDES  N  asp  2  N  4  N  5  N  6  N  7  ser  a l a - asp  3  N  -  l i e - a l a - asp  N-M-Asp  NO;^  DNP-N.  ser -  ser  l y s - l i e - a l a - asp  ser  t y r - l y s - i l e - a l a - asp  ser  a l a - t y r - l y s - i l e - a l a - asp  ser  ala - tyr - l y s - i l e -aala  ser  Q>afa  ser  C0NHCH C00CH CH o  GEE-N.  a l a - t y r - l y s - i l e - a l a - asp  -  o  o  ser - CONHCH COOCH CH 2  2  3  An Ny p e p t i d e d i a m i d a t e d w i t h g l y c i n e e t h y l e s t e r ,  N^-Bzl-Ser  i l e - a l a - asp  An N^ p e p t i d e w i t h i n t a c t NCy Leucine i s s u b s t i t u t e d  O-benzyl  protecting  - ser  group.  s e r - l e u - a l a - t y r - asp - l y s - a l a for isoleucine f o r identification  purposes.  o  TABLE 2  MOLAR RATIOS OF SYNTHETIC PEPTIDES AFTER AMINO ACID ANALYSIS Amino Acid  N. 2 T  X  F  3 +  N,  T  4 F  N.  T  F  5  N  N,  6  r  T  F  F  T  7  F  N  N-M -Asp  7  GEE -N., 7  T  F  T  F  TF  DNP -N, 7 F  T  F  ^4 !? Ser T  NC, 7  F  T  F  ser  1  1.05°  1  1103 1 0.95  1  0.90  1 1.11  1  0.85  1  0.96  1  0.32  1  1.01  1  xx 0.02"  1  1.00  asp  1  0.97  1  0.96  1 1.01  1  1.08  1 1.00  1  1.03  0  0.03  1  0.78  1  0.71  1  1.16  1  1.06  1  0.99  1 0.98  1  0.95  1 0.99  2  2.08  2  1.99  2  2.33  2  1.22  1  1.10  2  1.70  1 0.95  1  1.01  1 1.02  1  0.95  1  0.98  1  1.04  1  1.05  1  0.85  1  1.05  1 0.93  1  0.97  1  1.04  1  1.04  1  0.07  1  1.01  1 0.94  1  1.06  1  1.04  1  0.86  1  1.01  1  0.98  1  0.96  ala ile lys tyr leu gly  2  x - theoretical + - found o - molar r a t i o s xx - d i s a p p e a r a n c e o f amino a c i d i n d i c a t e s m o d i f i c a t i o n * - l o s t during hydrolysis  2.01  X X  x x  A  52  TABLE 3 INHIBITION OF MIGRATION OF SPLEEN CELLS FROM O-Fd IMMUNE AND NON-IMMUNE GUINEA PIGS Antigen  O-Fd Immune  Unimmunized  0.70 I .04*  0.99  0.79 ± .04  1.02  0.73 ± .05  0.92  0.71 ± .05  0.94 + .06  < .005  0.90 ± .05  0.96 + .08  ^ .40  0.91 ±. .07  0.97 + .09  < .40  0.93 ± .06  1.09 + .08  < .10  N-M-Asp  0.88 ± 006  1.14 + .07  <• .005  GEE-N  0.86 ± .09  1.06 + .08  .10  0.92 ± .10  0.90 + .10  > .40  0.67 ±.110  0.87 + .14  < .05  O-Fd 16 ug/ml  0.78 ± .03  1.06 + .06  < .0005  N -BSA 10 ug/ml  0.78 ± .05  1.01 + .11  < .01  N -PDG 10 ug/ml  0.79 ± .05  0.98 + .09  < .025  N -PLL 10 ug/ml  0.73 ± .02  0.92±± .09  < .01  DNP-N  0.69 ± .13  0.92 + .31  < .001  N  7 Q  7  N  6  N  5  N  4  N  3  N  2  NC  7  7  N.-Bzl-Ser 4 N8N  0.025 nmoles/ml  7  7  7  ?  + + +  t-test  .04  <.0005  .08  < .01  .07  < .01  0 _ a l l p e p t i d e s used a t 0.05 umoles/ml * - mean ± s t a n d a r d e r r o r o f t h e mean - p r o b a b i l i t y c a l c u l a t e d from t h e s t u d e n t ' s t - t e s t t h a t t h e r a t i o of m i g r a t i o n i n immune a n i m a l s i s d i f f e r e n t from t h a t i n unimmunized a n i m a l s - from p r e v i o u s l y p u b l i s h e d work u s i n g a n i m a l s immunized t o a s y n t h e t i c analogue o f O-Fd ( W a t e r f i e l d e t a l , 1974).  53  TABLE 4 SKIN REACTIONS OBSERVED ON N -BSA IMMUNIZED AND ?  UNIMMUNIZED GUINEA PIGS SKIN REACTIONS Ny-BSA IMMUNIZED Challenging Antigen** N -BSA ?  Arthus 15  12  D  Arthus  Delayed  7  1  8  7  4  2  N -PDG  12  7  3  2  N -PLL  9  5  3  1  Saline  3  2  3  1  N8N* ?  ?  average o f 4 immunized and 4 unimmunized **-  Delayed  UNIMMUNIZED  animals  a l l a n t i g e n s used a t 50 u g / t e s t  is- two Ny p e p t i d e s b r i d g e d by 8 g l y c i n e r e s i d u e s i n a l i n e a r manner ( K e l l y , Levy and H u l l , 1973) " - diameter o f r e a c t i o n i n mm's  54  TABLE 5  INHIBITION OF MIGRATION OF SPLEEN CELLS FROM N-.-BSA IMMUNE AND NON-IMMUNE GUINEA PIGS  Antigen  N-BSA Immune  Unimmunized  t-test^  N ** ?  0.59 ± .05*  0.99 ± .04  ^.0005  N  5  0.66 ± .07  0.92 ± .07  <.01  0.64 ± .09  0.94 ± .06  <.005  N-M-Asp  0.97 ± .09  1.14 ± .07  <.10  GEE-N  0.80 ± .07  1.06 ± .08  <.025  0-Fd°  0.75 ± .05  1.06 ± .06  < .01  N.  4  ** - P e p t i d e s used a t 0.05 pmole/ml D  - used a t 16 ug/ml  * - Mean ± s t a n d a r d e r r o r of the mean - See T a b l e 3 f o r e x p l a n a t i o n  TABLE 6 SUMMARY OF DATA  ABILITY TO INHIBIT O-Fd Immune Antigen or Peptide  N -BSA Immune 7  Complement F i x a t i o n  Leucocyte M i g r a t i o n  Leucocyte M i g r a t i o n  +  +  +  N^ 6  +  +  ND  N  -  +  +  +  +  N  •  ?  5  N. 4 N  3  -  +-  ND  N  2  -  -  ND  -  -.  ND  NC  ?  N-M-Asp  +  +-.  N.-Bzl-Ser 4  +  -  ND  GEE-N  ?  ND  -  +-  DNP-N  7  —  +  ND  +  +  O-Fd  *  Not determined  56  LEGENDS  F i g . 1 A and B.  I n h i b i t i o n of the complement  fixation  r e a c t i o n between O-Fd and homologous a n t i s e r a by p e p t i d e analogues o f the amino t e r m i n a l determinant of the means.  of O-Fd.  Bars i n d i c a t e the s t a n d a r d e r r o r  N7  N6 N5  Hk 0  N3 N2  •  o lo o| o oP® o1  io  ;o a0 Q  o  9  • 0 o M00 o •I  °o  O  iO Ol  Of %  • d,  0.006  •M  oo o o oo oo o o o o oo o o o9 9 o 9 o o9 9 o • oo 9 o e © • B o o 9 9 a • • > > • .> • 1 c 9 o o 9 9\ • q• • > > • • >  0.012 ^itmoles  g. 1A  o o| o o o| o°o| o°o| 0 o o o 0I0 o O Oj 9  o 999 o o 0 o« 9  o|o  <  •  0 0  •  9 9  i>  o o 9  o ^ o| a  peptide/test  0.025  o d  o a ol °o° 9 D • DIB °o°:9 9 0  0.050  Bl  >  N4-Bzl-Ser  • N-M-Asp a DNP-N7  ko h • NC7  o  30  3 >  20  10  > >  >\  _>  > > > >> > >>  to  >  > >  > >> > > i> > > •  •  > i> >l > >  •  0.003  >  >|  >  t>l  > ,  >  •  •  > • > > > • > > > > > l> M > • • > >  0.006  0.012 ^Umoles  >|  >> > >> > > >  •  > t>  >  > >  >  •  •  •  •  • •  b •a ••a • aa  •  0.025  > > >  • >! •  • • •  • •  > >! • H > • > >! • p > • > >|• • > • > • •a±a[ • •  > > > »la nil 0.050  pepti d e / t e s t 00  Fig.  1B  REFERENCES  A l k a n , S.S., W i l l i a m s , E.B., N i t e c k i , D.E. and Goodman, J.W. ' A n t i g e n r e c o g n i t i o n and t h e immune r e s p o n s e .  (1972).  Humoral and c e l l u l a r  immune responses t o s m a l l mono and b i f u n c t i o n a l a n t i g e n m o l e c u l e s . ' J . Exp. Med., 135, 1228. B e c k e r , M.J., L e v i n , H. and S e l a , M. c e l l u l a r immunity:  (1973).  'The s p e c i f i c i t y o f  Studies i n guinea pigs using defined t e t r a peptides  containing p-azobenzenearsonate-L-tyrosine.'  E u r . J . Immunol., 3, 131.  B e n j a m i n i , E., S h i m i z u , M., Young, J.D. and Leung, C.Y.  (1968a).  'Immuno c h e m i c a l s t u d i e s on t h e tobacco mosaic v i r u s p r o t e i n . VI.  C h a r a c t e r i z a t i o n of antibody p o p u l a t i o n s f o l l o w i n g  w i t h tobacco mosaic v i r u s p r o t e i n . '  immunization  Biochem., 7, 1253.  B e n j a m i n i , E., S h i m i z u , M., Young, J.D. and Leung, C.Y.  (1968b).  'Immunochemical s t u d i e s on tobacco mosaic v i r u s p r o t e i n .  V I I . The  b i n d i n g o f o c t a n o y l a t e d p e p t i d e s o f t h e tobacco mosaic v i r u s p r o t e i n w i t h a n t i b o d i e s t o t h e whole p r o t e i n . '  Biochem., 7, 1261.  B e n j a m i n i , E. , S h i m i z u , M. , Young, J.D. and Leung, C V . 'Immunochemical s t u d i e s on Tobacco M o s a i c V i r u s p r o t e i n .  (1969). IX.  Investi-  g a t i o n s on b i n d i n g and a n t i g e n i c s p e c i f i c i t y o f a n t i b o d i e s t o an a n t i g e n i c a r e a o f Tobacco Mosaic V i r u s p r o t e i n . '  Biochem., 8, 2242.  B i n z , H., B a c h i , T., W i g z e l l , H., Ramseier, H. and Lindenmann, J . (1975).  ' I d i o t y p e p o s i t i v e T c e l l s v i s u a l i z e d by a u t o r a d i o g r a p h y and  e l e c t r o n microscopy.'  P r o c . N a t . Acad. S c i . , U.S.A., 72, 3210.  Bush, M.E., A l k a n , S.S., N i t e c k i , D.E. and Goodman, J.W. 'Antigen r e c o g n i t i o n and t h e immune r e s p o n s e . a n t i g e n m o l e c u l e s . ' , J . Exp. Med., 136, 1478.  (1972).  S e l f - h e l p w i t h symmetrical  60  8.  Bloom, R.,  G a f f n e y , J . and Jimenez, L.  p r o d u c t i o n and c e l l p r o l i f e r a t i o n . ' 9.  Champlin, R. and H u n t e r , R.L.  (1972).  ' D i s s o c i a t i o n of MIF  J . Immunol., 109,  (1975).  1395.  ' S t u d i e s on t h e c o m p o s i t i o n of  a d j u v a n t s which s e l e c t i v e l y enhance d e l a y e d type h y p e r s e n s i t i v i t y t o l i p i d conjugated p r o t e i n antigens.' 10. Coon, J . and Hunter, R.  (1973).  J . Immunol., 114,  76.  ' S e l e c t i v e i n d u c t i o n of d e l a y e d  h y p e r s e n s i t i v i t y by a l i p i d c o n j u g a t e d p r o t e i n a n t i g e n which i s l o c a l i z e d i n thymus dependent lymphoid t i s s u e . ' 11. Crone, M.,  J . Immunol., 110,  Koch, C. and Simonsen, M.  receptor.'  T r a n s p l a n t . R e v l , 10,  12. Crumpton, M.J.  and S m a l l , P.A.  (1972).  'The e l u s i v e T  cell  36.  (1967).  'Conformation of I m m u n o l o g i c a l l y -  a c i t v e fragments of sperm Whale myoglobin B i o l . , 26,  183.  i n aqueous s o l u t i o n . '  J . Mol.  143.  13. D a i l e y , M.O.  and Hunter, R.L.  (1974).  'The r o l e o f l l i p i d i n the  i n d u c t i o n of h a p t e n - s p e c i f i c d e l a y e d h y p e r s e n s i t i v i t y and c o n t a c t sensitivity.' 14. D a v i d , J.R.,  J.- Immunol., 112,  1526.  A l - A s k a r i , S., Lawrence, H.S.  'Delayed h y p e r s e n s i t i v i t y i n v i t r o . c e l l m i g r a t i o n by a n t i g e n s . ' 15. D a v i d , J.R.,  Lawrence, H.S.  sensitivity in vitro.  (1964).  I . The s p e c i f i c i t y of i n h i b i t i o n  J . Immunol., 93, and Thomas, L.  264.  (1964).  'Delayed  and Schlossman,  S.F.  J . Immunol., 93,  (1968).  hyper-  279.  'Immunochemical s t u d i e s on  the s p e c i f i c i t y of c e l l u l a r h y p e r s e n s i t i v i t y .  The i n v i t r o i n h i b i t i o n  p e r i t o n e a l exudate c e l l m i g r a t i o n by c h e m i c a l l y d e f i n e d a n t i g e n s . ' J . Exp. Med.,  128,  of  I I I . The s p e c i f i c i t y o f h a p t e n - p r o t e i n c o n j u g a t e s  i n the i n h i b i t i o n of c e l l m i g r a t i o n . ' 16. D a v i d , J.R.  and Thomas, L.  1451.  of  61  17. D a v i e , J.M. cells.  and P a u l , W.E.  (1971).  'Receptors on immunocompetent  I I . S p e c i f i c i t y and n a t u r e of r e c e p t o r s on  dinitrophenylated  125 guinea p i g albuminJ . Exp. Med.,  134,  I - b i n d i n g lymphocytes of normal g u i n e a p i g s . ' 495.  18. Gerwing, J . and Thompson, K.  (1968).  tobacco mosaic v i r u s p r o t e i n .  'Immunochemical s t u d i e s  V I I . The  on  b i n d i n g of o c t a n o y l a t e d  peptides  of the tobacco mosaic v i r u s p r o t e i n w i t h a n t i b o d i e s '.to the whole protein.'  Biochem., 7,  19. Goodmanj J.W. advances.' 20. G o s c i c k a ,  1261.  (1969).  'Immunochemical s p e c i f i c i t y :  Immunochem., 6, T.  (1974).  'The  lymphocytes i n p r o d u c t i o n 21. Greaves, M.  (1975).  in sight?'  22. Hancock, W.S.,  r o l e of immunoglobulin r e c e p t o r s  of MIF  i n guinea p i g s . '  and L e s k o w i t z ,  i n v i t r o and sitivity.'  M a r s h a l l , G.R.  S.  (1973).  (1972).  c h a r a c t e r i z a t i o n of  J . B i o l . Chem., 247,  6224.  ' S t r u c t u r a l requirements f o r  i n v i v o immunogenicity i n h a p t e n - s p e c i f i c d e l a y e d h y p e r s e n -  and K o s h l a n d , D.E.  189. (1967).  'A method f o r the q u a n t i t a t i v e  m o d i f i c a t i o n and e s t i m a t i o n of c a r b o x y l i c a c i d groups i n p r o t e i n s . ' J . B i o l . Chenu, 242, 25. Hoffman, M. response.  2447. .  and K a p p l e r ,  J.W.  I I . Q u a l i t a t i v e and  (1973).  'Regulation  of the immune  q u a n t i t a t i v e d i f f e r e n c e s between thymus-  and bone marrow-derived lymphocytes i n the r e c o g n i t i o n of a n t i g e n . ' • J . Exp. Med.,  207.  a solution  and V a g e l o s , P.R.  X V I I I . Chemical s y n t h e s i s and  C e l l . Immunol., 7,  24. Hoare, D.G.  C e l l . Immunol., 13,  on T l y m p h o c y t e s :  a protein with acyl carrier protein a c t i v i t y . ' 23. Hanna, N.  on  92.  P r e s c o t t , D.J.,  'Acyl c a r r i e r p r o t e i n .  conceptual  139.  'Antigen r e c e p t o r s  N a t u r e , 256,  recent  137,  721.  62  26. Janeway, C.A. , Cohen, B.E., 'The  Ben-Sasson, S.Z.  and P a u l , W.E.  (1975).  s p e c i f i c i t y of c e l l u l a r immune responses i n guinea p i g s .  s p e c i f i c f o r 2, 4 - d i n i t r o p h e n y l - 0 - t y r o s y l r e s i d u e s . ' 27. J o k i p i i , A.M.M. and J o k i p i i , L i i s a .  (1974).  J . Exp. Med.,  'Progressive  28. K a t z , D.H.,  and  P a u l , W.E.,  G o i d l , E.A.  and  Benacerraf,  B.  42.  lymphocytes.'  (1970).  I . Enhancement of  secondary a n t i - h a p t e n a n t i b o d y responses by  preimmunization.J  141,  241.  ' C a r r i e r f u n c t i o n i n a n t i - h a p t e n immune responses. primary  cells  increase  w i t h time a f t e r s e n s i t i z a t i o n i n the f u n c t i o n a l a f f i n i t y of T C e l l . Immunol., 13,  I. T  J . Exp.  29. K e l l y , B. and Levy, J.G.  Med.,  132,  (1971).  carrier  261.  'Immunological s t u d i e s on the major  h a p t e n i c p e p t i d e s from p e r f o r m i c a c i d o x i d i z e d f e r r e d o x i n from C l o s t r i d i u m pasteurianum.' 301 K e l l y , B.,  Levy, J.G.  Biochem., 9,  and H u l l , D.  responses i n guinea p i g s and peptides.' 31. Levy, J.G., 'The  cellular  pasteurianum.' 32. MacDonald, H.R.,  K e l l y , B.,  humoral  574. K i l b u r n , D.G.  and T e a t h e r ,  R.M.  (1972).  c o n t a i n i n g sequences  i n p e r f o r m i c a c i d - o x i d i z e d f e r r e d o x i n from C l o s t r i d i u m  C e l l . Immunol., 5, Sordat,  B.,  87.  C e r o t t i n i , J . and  Brunner, K.  of c y t o t o x i c T lymphocytes i n v i t r o .  a c t i v a t i o n of memory c e l l s Med.,  ' C e l l u l a r and  immune response to s y n t h e t i c p e p t i d e s  known to be h a p t e n i c  'Generation  (1973).  r a b b i t s to c h e m i c a l l y d e f i n e d s y n t h e t i c  Eur. J . Immunol., 3, H u l l , D.,  2762.  i n the absence of DNA  (1975).  IV. F u n c t i o n a l synthesis.'  J.  Exp.  142, 622.  33. Makela, 0.  (1970).  'Analogies between lymphocyte r e c e p t o r s and  r e s u l t i n g humoral a n t i b o d i e s . '  T r a n s p l a n t . Rev.,  5,  3.  the  63  34. Maron, E. , Webb, C , T e i t e l b a u m , D. and A r n o n , R.  (1972).  'Cell-  mediated v s humoral response i n t h e c r o s s - r e a c t i o n between hen eggw h i t e lysozyme and b o v i n e <* - l a c t a l b u m i n . ' 35. M e r r i f i e l d , R.B.  (1963).  E u r . J . Immunol., 2, 294.  ' S o l i d phase p e p t i d e s y n t h e s i s .  synthesis of a t e t r a peptide.'  I . The  J . Am. Chem. S o c , 85, 2149.  36. M i t c h e l l , B., L e v y , J.G. and N i t z , R.M.  (1970).  'Synthesis of h a p t e n i c  C - t e r m i n a l o c t a p e p t i d e s o f two c r o s s - r e a c t i n g b a c t e r i a l f e r r e d o x i n molecules.'  Biochem., 9, 1839.  37. M i t c h e l l , B. and Levy, J.G.  (1970).  'Immunological s t u d i e s i n p e p t i d e s '  from t h e h a p t e n i c C - t e r m i n a l o c t a p e p t i d e o f C l o s t r i d i u m p a s t e u r i a n u m ferredoxin.'  Biochem., 9, 2762.  38. Mortenson, L.E.  (1964).  ' P u r i f i c a t i o n and a n a l y s i s o f f e r r e d o x i n from  C l o s t r i d i u m pasteurianum.'  Biochim. Biophys. A c t a , , 81, 71;  39. Munro, A . J . and T a u s s i g , M.J.  (1975).  'Two genes i n t h e major  c o m p a t i b i l i t y complex c o n t r o l immune r e s p o n s e . '  histo-  N a t u r e , 256, 103.  40. N i t z , R.M., M i t c h e l l , B., Gerwing, J . and C h r i s t e n s o n , J . ' S t u d i e s on t h e a n t i g e n i c i t y o f b a c t e r i a l f e r r e d o x i n s .  (1969).  I . The e f f e c t o f  m o d i f i c a t i o n o f c y s t e i n y l r e s i d u e s on t h e a n t i g e n i c i t y o f C l o s t r i d i u m pasteurianum f e r r e d o x i n . ' 41.  P a r i s h , C.R. flagellin.  (1971a).  J . Immunol., 103, 319. 'Immune r e s p o n s e t o c h e m i c a l l y m o d i f i e d  I . I n d u c t i o n o f a n t i b o d y t o l e r a n c e t o f l a g e l l i n by a c e t o -  acetylated d e r i v a t i v e s of the protein.' 42. P a r i s h , C.R.  (1971b).  J . Exp. Med., 134, 1.  'Immune response t o c h e m i c a l l y m o d i f i e d f l a g e l l i n .  I I . Evidence f o r a fundamental r e l a t i o n s h i p between humoral and c e l l mediated immunity.'  J . Exp. Med., 134, 21.  43. P a r i s h , C R .  (1972).  ' P r e f e r e n t i a l i n d u c t i o n of c e l l - m e d i a t e d  immunity by c h e m i c a l l y m o d i f i e d  sheep e r y t h r o c y t e s . '  E u r . J . Immunol.,  2, 143. 44. P a u l , W.E., K a t z , D.H., G o i d l , E.A. and B e n a c e r r a f , 'Carrier function i n anti-hapten  immune responses.  p r o p e r t i e s o f c a r r i e r c e l l s capable responses.'  (1970).  II. Specific  o f enhancing a n t i - h a p t e n  antibody  J . Exp. Med., 132, 283.  45. Pearson, T., Levy, J . and K i l b u r n , D. cell  B.  i n a c t i v a t i o n on the c e l l u l a r  (1975).  'The e f f e c t o f s p e c i f i c  immune response t o f e r r e d o x i n p e p t i d e  Eur. J . Immunol., 5, 65. 46. R o c k l i n , R.E.  (1973).  ' P r o d u c t i o n of m i g r a t i o n i n h i b i t o r y f a c t o r by  n o n - d i v i d i n g lymphocytes.' 47. Rubin, B. and W i g z e l l , H.  J . Immunol., 110, 674. (1973).  'The immune response a g a i n s t  hapten-autologous p r o t e i n conjugates c o o p e r a t i n g non-thymus-processed J.  I I I . S p e c i f i c i t y of  (B) and thymus-processed  (T) lymphocyt  Exp. Med., 137, 911.  48. S a l v i n , S.B. and Smith, R.F. reactions. Med.,  (1960).  'The s p e c i f i c i t y o f a l l e r g i c  I . Delayed v e r s u s a r t h u s h y p e r s e n s i t i v i t y . '  J . Exp.  111, 465.  49. Savoie, J.Y. and Barton, M.A.  50.  i n t h e mouse.  (1974).  ' S o l i d phase s y n t h e s i s o f  s e l e c t i v e l y protected peptides.'  Can. J . Chem., 5 2 , 2832.  Schirrmacher,  (1972).  V. and W i g z e l l , H.  n a t i v e and c h e m i c a l l y m o d i f i e d thymus processed methylated  bovine  'Immune responses a g a i n s t  albumins i n mice.  (B) and thymus-processed serum albumin.'  I . A n a l y s i s o f non-  (T) c e l l responses a g a i n s t  J . Exp. Med., 136, 1616.  65  51. S c h i r r m a c h e r , V. and W i g z e l l , H.  (1974).  'Immune responses a g a i n s t  n a t i v e and c h e m i c a l l y m o d i f i e d albumins i n mice.  I I . E f f e c t of e l e c t r i c  charge and c o n f o r m a t i o n on t h e humoral a n t i b o d y response and on h e l p e r T c e l l responses.'  J . Immunol., 113, 1635.  52. Senyk, G., W i l l i a m s , B., N i t e c k i , O.E. and Goodman, J.W. 'The f u n c t i o n a l d i s s e c t i o n o f an a n t i g e n m o l e c u l e : humoral and c e l l u l a r immune responses t o g l u c a g o n . '  (1971).  S p e c i f i c i t y of J . Exp. Med.,  133, 1294. 53. S p i t l e r , L., B e n j a m i n i , E., Young, J.D., K a p l a n , H. and Fudenberg, (1969).  ' S t u d i e s on t h e immune response t o a c h a r a c t e r i z e d  d e t e r m i n a n t o f t h e Tobacco Mosaic V i r u s P r o t e i n . '  H.H.  antigenic  J . Exp. Med.,  131, 133. 54. Tanaka, M., Nakashima, T., Mower, H.F. and Yasunobu, K.T. 'The C- and N- t e r m i n a l amino a c i d sequences ferredoxin.'  (1963).  of C l o s t r i d i u m  pasteurianum  A r c h . Biochem., 105, 570.  55. V i t e t t a , E.S. and U h r , J.W.  (1975).  'Immunoglobulin-receptors  revisited.'  S c i e n c e , 189, 964. 56. W a t e r f i e l d , J.D., L e v y , J.G. and K i l b u r n , D.G.  (1974).  DNP i n a n t i g e n a c t i v a t i o n o f c e l l u l a r immune r e s p o n s e s . ' 8th L e u c o c y t e C u l t u r e Conference  'The r o l e o f Proceedings  (ed. by R. L i n d a h l - K i e s s l i n g and  D. Osoba), p. 229. Academic P r e s s . 57. W a t e r f i e l d , D., L e v y , J.G., K i l b u r n , D.G. and T e a t h e r , R.M. 'The e f f e c t o f h a p t e n i c p e p t i d e s from p e r f o r m i c a c i d o x i d i z e d  (1972). ferredoxin  from C l o s t r i d i u m p a s t e u r i a n u m and p r o t e i n c a r r i e r - h a p t e n c o n j u g a t e s on the immune response o f macrophages and lymphoid c e l l s from a n i m a l s immunized a g a i n s t o x i d i z e d f e r r e d o x i n . '  C e l l . Immunol. 3, 253.  Young, J.D.,  B e n j a m i n i , E., Stewart, J.M.  and Leung, C.Y.  'Immunochemical s t u d i e s on tobacco mosaic v i r u s p r o t e i n . phase s y n t h e s i s  V. The  solid-  of p e p t i d e s of an a n t i g e n i c a l l y a c t i v e d e c a p e p t i d e  tobacco mosaic v i r u s p r o t e i n and antibodies  (1967).  to the whole p r o t e i n . '  of  the r e a c t i o n of these p e p t i d e s w i t h Biochem., 6,  1455.  THE IMMUNE RESPONSE TO FERREDOXIN  II.  CROSS REACTIVITY OF CELLS AND ANTISERA TO  MODIFIED FERREDOXINS AND THE NATURE OF THE CELLS RESPONDING IN VITRO  D.S. GREGERSON, BARBARA KELLY AND JULIA G. LEVY  DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, CANADA  SUMMARY  The  c r o s s r e a c t i v i t y of s e r a from r a b b i t s s e n s i t i z e d to  a c i d o x i d i z e d f e r r e d o x i n (O-Fd) and O-Fd  was  analyzed  Only O-Fd  ferredoxin  Dinitrophenylated-O-Fd  (DNP-O-Fd) and (C)  (CM-Fd) was  a b l e to f i x C'.  acid  precipitated  but d i d not  stimulate  F e r r e d o x i n a l k y l a t e d w i t h N-ethylmaleimide  c e l l t r a n s f o r m a t i o n but  ferredoxin  assay.  forms of f e r r e d o x i n i n the  lymphocyte s t i m u l a t i o n assay.  (TCA-Fd) were able to f i x complement  synthesis i n v i t r o .  induced  the i n v i t r o  f i x e d C' p o o r l y .  unable to s t i m u l a t e DNA  Methylated-O-Fd  s y n t h e s i s and was  (meth-O-Fd) was  Only O-Fd,  not r e c o g n i z e d  marginally i n either  of s e n s i t i z i n g lymphocytes f o r  a p r o l i f e r a t i v e response i n v i t r o to the t e s t a n t i g e n s .  This correlates  w i t h the o b s e r v a t i o n that o n l y these a n t i g e n s were a b l e to induce  The nature  sensitized  i n v i t r o was  c e l l s w i t h r a b b i t anti-mouse immunoglobulin and C'.  s e r a w h i l e the 120  DNA  lymphocytes.  of the c e l l s responding  b r a i n a s s o c i a t e d 0 and  ability  i n the i n v i t r o lymphocyte s t i m u l a t i o n assay.  NEM-Fd and n a t i v e - F d were capable  s y n t h e s i s i n O-Fd  (NEM-Fd)  Carboxymethylated  The v a r i o u s f e r r e d o x i n p r e p a r a t i o n s were t e s t e d f o r t h e i r  to s e n s i t i z e mice f o r use  to both  to  and n a t i v e f e r r e d o x i n ( n a t i v e - F d ) gave p o s i t i v e responses i n  both a s s a y s .  DNA  of s p l e e n c e l l s from mice s e n s i t i z e d  using several chemically modified  complement f i x a t i o n t e s t and  performic  The  the  C' o r r a b b i t anti-mouse  24 hour response was  hour response was  examined by t r e a t i n g  found  to be  sensitive  s e n s i t i v e o n l y to the a n t i - 9 s e r a .  INTRODUCTION  The a b i l i t y of the immune system t o d i r e c t i t s e l f s p e c i f i c a l l y an a n t i g e n i s w e l l e s t a b l i s h e d .  against  A number of i n v e s t i g a t i o n s have been  made to determine the a b i l i t y of the immune response t o d i s c r i m i n a t e between d i f f e r e n t a n t i g e n s .  S i n c e the c l a s s i c work of L a n d s t e i n e r (1946)  t h e s e s t u d i e s have o f t e n been done w i t h p r o t e i n s i n t h e i r n a t i v e and c h e m i c a l l y m o d i f i e d forms.  A l a c k of humoral c r o s s r e a c t i v i t y between  n a t i v e and denatured forms of the same p r o t e i n a n t i g e n has f r e q u e n t l y been found.  A n t i s e r a t o n a t i v e r i b o n u c l e a s e do not r e c o g n i z e p e r f o r m i c a c i d  o x i d i z e d r i b o n u c l e a s e (Brown, 1962; Brown, D e l a n y , L e v i n e and Van V u n a k i s , 1959). S e v e r a l o t h e r r e p o r t s have demonstrated t h a t w h i l e l i t t l e or no humoral c r o s s r e a c t i v i t y was  f o u n d , a h i g h degree of r e c o g n i t i o n  seen at the l e v e l of d e l a y e d h y p e r s e n s i t i v i t y .  was  A n t i b o d i e s to n a t i v e  lysozyme show v e r y l i t t l e r e a c t i o n t o S-carboxymethylated lysozyme and Thompson, 1968), but v i r t u a l l y of  c e l l - m e d i a t e d immunity  1972). the  (Gerwing  complete r e a c t i v i t y was found i n a s s a y s  (Thompson, H a r r i s , B e n j a m i n i , M i t c h e l l and N o b e l ,  The a f f i n i t y of a n t i - f l a g e l l i n  a n t i b o d i e s f o r f l a g e l l i n was l o s t as  degree of a c e t o a c e t y l a t i o n of the f l a g e l l i n i n c r e a s e d , w h i l e even  h i g h l y s u b s t i t u t e d f l a g e l l i n e l i c i t e d delayed h y p e r s e n s i t i v i t y i n animals s e n s i t i z e d to n a t i v e f l a g e l l i n  ( P a r i s h , 1971, a and b ) .  U s i n g v a r i o u s c h e m i c a l l y m o d i f i e d forms of b o v i n e serum a l b u m i n , S c h i r r m a c h e r and W i g z e l l (1972 and 1974) have demonstrated t h a t thymus dependent  lymphocytes (T c e l l s ) r e c o g n i z e a w i d e r range of m o d i f i e d albumins  than thymus independent lymphocytes (B c e l l s ) i n d i c a t i n g d i f f e r e n t  structural  r e q u i r e m e n t s f o r r e c o g n i t i o n by t h e s e two found by P a r i s h  (1972) w i t h m o d i f i e d  c e l l types.  S i m i l a r r e s u l t s were  and h e t e r o l o g o u s e r y t h r o c y t e s .  v e r y s i m i l a r p r o t e i n s , egg w h i t e lysozyme and  b o v i n e <*-lactalbumin, were  h i g h l y c r o s s r e a c t i v e i n c e l l mediated immune r e s p o n s e s w h i l e a complete l a c k of humoral r e a c t i o n s  Two  demonstrating  (Maron, Webb, T e i t e l b a u m and  Arnon,  1972). The  above f i n d i n g s have been t a k e n to suggest t h a t T c e l l s  amino a c i d sequence ( u n a l t e r e d by m o d i f i c a t i o n s ) w h i l e B c e l l s configuration  (changed by m o d i f i c a t i o n s ) .  We  recognize recognize  have examined t h i s phenomena  u s i n g the f e r r e d o x i n m o l e c u l e which has been w e l l c h a r a c t e r i z e d . work has  shown s u b s t a n t i a l a n t i b o d y c r o s s r e a c t i o n s between n a t i v e , a c i d  p r e c i p i t a t e d , S-carboxymethylated and p e r f o r m i c ( N i t z , M i t c h e l l , Gerwing and The  CKristensen,  acid oxidized  ferredoxins  1969).  i n v i t r o t h y m i d i n e uptake assay has been thought to be a c o r r e l a t e  of d e l a y e d h y p e r s e n s i t i v i t y ( M i l l s , 1966; 1967;  Previous  Oppenheim, W o l s t e n c r o f t  M e u v i s s e n , van A l t e n and Good, 1969), ppresumably due  transformation.  However, Mugraby, Gery and  Sulitzeanu  to T  and cell  (1974) have e v i d e n c e  t h a t B c e l l s a r e i n v o l v e d . i n the response- S i n c e t h i s i n v i t r o method been used i n t h i s s t u d y as an assay of c e l l - m e d i a t e d of T and  examined.  The  and  c e l l u l a r and humoral c r o s s r e a c t i v i t i e s of  s e v e r a l forms of f e r r e d o x i n were t e s t e d and relation  has  immunity, the e f f e c t s  B c e l l d e p l e t i o n on the t h y m i d i n e uptake response to a n t i g e n s  mitogens was  Gell,  the r e s u l t s are d i s c u s s e d  to the f i n d i n g s i n the accompanying r e p o r t  in  (Gregerson e t a l , 1976).  71  MATERIALS AND METHODS  ANIMALS  Young a d u l t a l b i n o r a b b i t s were used t o produce a l l t h e a n t i s e r a .  Mice  of t h e s t r a i n s DBA/2J and B6D2/J F l were used f o r t h e i n ' v i t r o s t i m u l a t i o n s .  ANTIGENS  N a t i v e f e r r e d o x i n ( n a t i v e - F d ) and p e r f o r m i c a c i d o x i d i z e d f e r r e d o x i n (O-Fd) were prepared  as d e s c r i b e d p r e v i o u s l y , (Gregerson,  e_t al_, 1976).  Acid  p r e c i p i t a t e d f e r r e d o x i n (TCA-Fd) was made a c c o r d i n g t o Tanaka, Nakashima, Mower and Yasunobu (1964).  D i n i t r o p h e n y l a t e d O-Fd (DNP-O-Fd) was p r e p a r e d  by r e a c t i n g O-Fd w i t h 5% d i n i t r o f l u o r o b e n z e n e i n 10% aqueous sodium b i c a r b o n a t e p l u s 20% e t h a n o l f o r 2 days i n t h e d a r k . e x t r a c t e d s e v e r a l times w i t h e t h e r t o remove u n r e a c t e d benzene and then d i a l y z e d . by F r a e n k e l - C o n r a t  Methylated  dinitrofluoro-  O-Fd (meth-O-Fd) was made as d e s c r i b e d  and O l c o t t (1945) by suspending O-Fd i n anhydrous  methanol made t o 0.1 M w i t h c o n c e n t r a t e d was  The m i x t u r e was  hydrochloric acid.  Esterification  a l l o w e d t o proceed f o r 3 days i n t h e dark f o l l o w e d by d i a l y s i s .  pH o f meth-O-Fd s o l u t i o n s was kept below 6 u n t i l use t o a v o i d h y d r o l y s i s o f t h e e s t e r s (Ram and Maurer, 1959).  The  alkaline  N a t i v e - F d was reduced  w i t h m e r c a p t o e t h a n o l a t pH 8 (1 ul/mg p r o t e i n ) and r e a c t e d w i t h N - e t h y l m a l e i m i d e (2 e q u i v a l e n t s / e q u i v a l e n t of s u l f h y d r y l ) a t pH 6 f o r 4 hours f o l l o w e d by d i a l y s i s .  The r e s u l t i n g a l k y l a t e d product was c a l l e d NEM-Fd.  N a t i v e - F d was S-carboxymethylated w i t h i o d o a c e t a t e as d e s c r i b e d by B a t t e l l , Zarkadas, S m i l l i e and Madsen (1968) and d i a l y z e d t o g i v e carboxymethylated (CM-Fd).  Fd  The v a r i o u s f e r r e d o x i n s were a n a l y z e d and q u a n t i t a t e d on a Beckman 120  amino a c i d a n a l y z e r a f t e r 18 hour h y d r o l y s i s i n 6N HC1 i n evacuated v i a l s . The r e s u l t s a r e p r e s e n t e d i n T a b l e 1.  IMMUNIZATIONS  R a b b i t s were immunized w i t h O-Fd as d e s c r i b e d b e f o r e (Gregerson et_ a l , 1976).  M i c e were immunized  t w i c e a t a 2-week i n t e r v a l w i t h 35 ug of one of  the v a r i o u s f e r r e d o x i n s i n 50% Freund's complete a d j u v a n t .  COMPLEMENT FIXATION TESTS  These were done as d e s c r i b e d by Gerwing and Thompson (1968).  LYMPHOCYTE STIMULATIONS  I n v i t r o m i c r o c u l t u r e s were performed as d e s c r i b e d by G r e g e r s o n , K e l l y and Levy (1975) except t h a t mouse s p l e e n c e l l s were used i n t h e present study.  The non-immune c o n t r o l s f o r t h e DBA mice r e p r e s e n t t h e  p o o l e d and averaged d a t a from 12 s e p a r a t e e x p e r i m e n t s .  Each experiment  c o n s i s t e d of a minimum of n i n e d e t e r m i n a t i o n s and u s u a l l y more than 1 mouse.  CELL KILLING  R a b b i t anti-mouse immunoglobulin (RaMIg) was p r e p a r e d by immunizing a r a b b i t t h r e e times w i t h 1 mg amounts o f mouse immunoglobulin G ( M i l e s Labs.) i n complete Freund's a d j u v a n t a t 2 week i n t e r v a l s . c o l l e c t e d s e v e r a l t i m e s , p o o l e d and heat i n a c t i v a t e d .  The serum was  Rabbit a n t i - b r a i n  73  a s s o c i a t e d 9 (RaBAO) was p r e p a r e d as d e s c r i b e d by K e l l y , Kaye, Y o s h i z a w a , Levy and K i l b u r n  (1974).  Guinea p i g serum was absorbed w i t h s y n g e n e i c  mouse s p l e e n c e l l s and used as a s o u r c e of complement ( C ' ) . A l l s e r a and C' were used a t a 1:4 d i l u t i o n i n phosphate b u f f e r e d s a l i n e (PBS).  Cells  were suspended i n the d i l u t e d s e r a and C' a t a c o n c e n t r a t i o n of 10^ c e l l s / 0 . 1 ml and i n c u b a t e d f o r 1 hour a t 37°.  A f t e r 3 washes i n PBS the c e l l s were used.  C e l l s i n c u b a t e d w i t h heat i n a c t i v a t e d , s p l e e n c e l l absorbed normal serum  (NRS) and C' s e r v e d as c o n t r o l s .  rabbit  RESULTS  PARAMETERS OF LYMPHOCYTE STIMULATION BY ANTIGEN IN VITRO  S e v e r a l experiments were done t o determine the o p t i m a l c o n d i t i o n s f o r the i n v i t r o s t i m u l a t i o n of DNA s e n s i t i z e d mice.  s y n t h e s i s i n s p l e e n c e l l s from  The o p t i m a l dose of O-Fd  was found t o be 16 pg/ml. i n  agreement w i t h e a r l i e r work done u s i n g g u i n e a p i g c e l l s 1975). T a b l e 2.  O-Fd  (Gregerson e_t a l ,  The r e s u l t s of the dose response experiments a r e p r e s e n t e d i n The k i n e t i c s of the response u s i n g O-Fd  i n T a b l e 3.  a t 16 ug/ml are shown  A b i - m o d a l response was found as seen p r e v i o u s l y u s i n g g u i n e a  p i g c e l l s under s i m i l a r c o n d i t i o n s .  A few experiments done at 144 and  hours i n d i c a t e d the response was d e c l i n i n g a t these t i m e s and were not r o u t i n e l y extended beyond 120 h o u r s .  168  experiments  I n a l l cases the r e s u l t s a r e  compared t o t h o s e o b t a i n e d from experiments w i t h u n s e n s i t i z e d c e l l s by c a l c u l a t i n g the t - p r o b a b i l i t i e s from the s t u d e n t ' s t - t e s t .  The  relatively  low s t i m u l a t i o n i n d i c e s observed i n t h i s s t u d y are p r o b a b l y due t o the h i g h background  l e v e l s found i n the medium c o n t r o l s .  THE NATURE OF THE CELLS RESPONDING IN VITRO  I f the i n v i t r o t h y m i d i n e uptake assay as used i n t h i s s t u d y i s a c o r r e l a t e of c e l l mediated immunity and dependent on T c e l l s , p r e t r e a t m e n t of the c e l l s w i t h RaBAO and C' s h o u l d depress the response of O-Fd  sensitized  c e l l s t o O-Fd.  immunoglobulin  The e f f e c t of t r e a t m e n t w i t h RaMIg and C'  p o s i t i v e B c e l l s ) was a l s o t e s t e d . r a b b i t serum (NRS).  (to k i l l  C o n t r o l s were done u s i n g C' and  normal  The r e s u l t s i n T a b l e 4 summarize the e f f e c t s of the  t r e a t m e n t s on c e l l numbers.  S i n c e the C' and NRS were absorbed w i t h s y n g e n e i  s p l e e n c e l l s to e l i m i n a t e n o n - s p e c i f i c c y t o x i c i t y , the c e l l s l o s t t r e a t m e n t w i t h C' and NRS r e p r e s e n t the l o s s due t o the washing and t h e death of v a r i o u s s h o r t l i v e d s p l e n i c l e u c o c y t e s  during  procedure  d u r i n g the  incubation  washing and c o u n t i n g p e r i o d s . . I t has been shown t h a t the responses of mouse s p l e e n c e l l s t o conc a n a v a l i n A (ConA) and l i p o p o l y s a c c h a r i d e  (LPS) a r e dependent on the p r e s e n c e  of T and B c e l l s , r e s p e c t i v e l y (Andersson, M o l l e r and S j b b e r g , 1972). These mitogen r e s p o n s e s were t e s t e d w i t h each experiment and t a k e n as an i n d i c a t i o n of the e f f e c t i v e n e s s of the t r e a t m e n t s w i t h RaBA9, RaMIg and C'.  I t can be seen i n T a b l e 5 t h a t the r e s p o n s e s to'jeSnA and LPS were  g r e a t l y reduced by the r e s p e c t i v e a n t i s e r a . on the response t o O-Fd  The e f f e c t s of t h e s e a n t i s e r a  a r e p r e s e n t e d i n T a b l e 6.  Only t h o s e experiments  i n which the mitogen responses were s u b s t a n t i a l l y reduced by the a n t i s e r a are i n c l u d e d .  The t - p r o b a b i l i t i e s compare the means of t r e a t e d and u n t r e a t e d  (NRS and C') c e l l s .  The 24 hour response was  found t o be s e n s i t i v e t o  b o t h the RaMIg and RaBAG i n d i c a t i n g t h a t B c e l l s c o n t r i b u t e to the r e s p o n s e , but a r e dependent on the presence of T c e l l s t o do so.  The 120 hour  response was not s i g n i f i c a n t l y a f f e c t e d by the RaMIg t r e a t m e n t , but i t was  l a r g e l y reduced by the RaBAG s e r a .  T h i s r e s u l t s u g g e s t s t h a t the 120  hour i n v i t r o response i s T c e l l mediated and not dependent on B c e l l s . The response of the c o n t r o l c e l l s t r e a t e d w i t h C' and NRS  i s somewhat  d e p r e s s e d compared t o the normal c e l l s seen p r e v i o u s l y i n T a b l e 2. cause f o r the decrease i n t h i s response i s unknown.  The  THE CROSS REACTIVITY OF O-Fd AND MODIFIED FERREDOXIN IMMUNIZED SPLEEN CELLS  The a b i l i t y o f O-Fd s e n s i t i z e d s p l e e n c e l l s t o respond t o s e v e r a l c h e m i c a l l y m o d i f i e d forms o f f e r r e d o x i n was examined u s i n g t h e i n v i t r o lymphocyte s t i m u l a t i o n assay. before.  The O-Fd immunized mice were p r e p a r e d as  The d a t a from 5 e x p e r i m e n t s summarized i n T a b l e 7 demonstrates  t h a t o n l y NEM-Fd and n a t i v e - F d s t i m u l a t e d s i g n i f i c a n t from O-Fd s e n s i t i z e d mice.  responses i n c e l l s  These a n t i g e n s d i d n o t e x h i b i t t h e b i m o d a l  response seen p r e v i o u s l y w i t h O-Fd i n T a b l e 3, a l t h o u g h t h e r e s p o n s e t o NEM-Fd was p r e s e n t a t 24 h o u r s . The immunogenicity o f t h e v a r i o u s f e r r e d o x i n s was examined by u s i n g them t o s e n s i t i z e mice i n t h e same manner as O-Fd was used.  4  Spleen c e l l s  from these mice were t e s t e d i n 4 e x p e r i m e n t s a g a i n s t s e v e r a l o f t h e f e r r e d o x i n s in vitro.  Only NEM-Fd and n a t i v e - F d s e n s i t i z e d c e l l s gave a  response i n v i t r o .  proliferative  N a t i v e - F d and NEM-Fd s t i m u l a t e d DNA s y n t h e s i s i n NEM-Fd  s e n s i t i z e d c e l l s w h i l e n a t i v e - F d immunized c e l l s o n l y responded t o n a t i v e - F d as seen i n T a b l e 8.  Meth-Fd, TCA-Fd, CM-Fd and DNP-O-Fd d i d n o t e l i c i t  responses from any o f t h e m o d i f i e d f e r r e d o x i n immune c e l l s and d i d n o t s e n s i t i z e c e l l s f o r a response t o any o f the a n t i g e n s .  These r e s u l t s  suggest t h a t t h e r e may be a c o r r e l a t i o n between a m o l e c u l e b e i n g a b l e t o s t i m u l a t e an i n v i t r o t h y m i d i n e uptake response and t o f u n c t i o n as an immunogen.  T h i s phenomena has been r e p o r t e d p r e v i o u s l y (Levy, H u l l ,  Kelly,  K i l b u r n and T e a t h e r , 1972).  THE CROSS REACTIVITY OF ANTI-O-Fd ANTISERA  U s i n g t h e complement f i x a t i o n  t e s t , the cross r e a c t i v i t y  of r a b b i t  a n t i - O - F d a n t i b o d i e s was examined i n s e v e r a l t e s t s w i t h m o d i f i e d Previous  ferredoxins.  work.using the complement f i x a t i o n t e s t demonstrated p o s i t i v e  f i x a t i o n w i t h O-Fd, n a t i v e - F d , (Nitz, et d ,  1969).  TCA-Fd and iodoacetamide a l k y l a t e d  The r e s u l t s shown i n F i g u r e  1 i n d i c a t e t h a t DNP-O-Fd  a l s o f i x e s complement w e l l w h i l e CM-Fd and NEM-Fd were o n l y a c t i v e i n t h i s assay.  The a c t i v i t y o f meth-O-Fd  ferredoxin  marginally  was q u i t e low.  DISCUSSION The i n v i t r o lymphocyte s t i m u l a t i o n o f mouse s p l e e n c e l l s by t h e a n t i g e n O-Fd, and t h e mitogens Con A and LPS, i n terms o f c u l t u r e  conditions  ( c e l l c o n c e n t r a t i o n and medium), k i n e t i c s and dose r e s p o n s e s , was found to be r e m a r k a b l y s i m i l a r t o t h e responses o f g u i n e a p i g c e l l s as r e p o r t e d p r e v i o u s l y (Gregerson e_t al_, 1975) . Of p a r t i c u l a r i n t e r e s t was t h e b i m o d a l n a t u r e o f t h e i n v i t r o  response  t o O-Fd where two peaks o f DNA s y n t h e s i s were observed when t h e c e l l s were l a b e l e d w i t h t r i t i a t e d t h y m i d i n e a t 24 and 120 hours o f c u l t u r e w h i l e v i r t u a l l y no response was found a t 72 h o u r s .  S i n c e t h e peak response t o  LPS was found when l a b e l i n g was done a t 24 h o u r s , and LPS i s c o n s i d e r e d t o be a B c e l l mitogen f o r mouse c e l l s  (Anderson et_ a l , 1972), i t seemed  p o s s i b l e t h a t B c e l l s were c o n t r i b u t i n g t o t h e 24 hour r e s p o n s e , e i t h e r by DNA s y n t h e s i s and/or by t h e p r o d u c t i o n o f a f a c t o r t h a t c o u l d i n d u c e DNA synthesis i n T c e l l s .  T h i s problem was examined by p r e t r e a t i n g t h e c e l l s  w i t h RaMIg or RaBAO and C' t o k i l l c e l l s w i t h immunoglobulin  (B c e l l s )  ( R a f f , 1970) o r 9 (T c e l l s ) (Golub, 1971) on t h e i r s u r f a c e s , r e s p e c t i v e l y , and o b s e r v i n g t h e e f f e c t s t h e s e t r e a t m e n t s had on t h e O-Fd, LPS and Con A responses.  I t was found t h a t c e l l s i n c u b a t e d w i t h RaMIg and C' l o s t t h e  24 hour s t i m u l a t i o n by O-Fd and L P S , b u t remained n o r m a l l y r e s p o n s i v e t o Con A.  I f t h e c e l l s were p r e i n c u b a t e d w i t h RaBAO and C' t h e r e s p o n s e s t o  O-Fd and Con A were l o s t ' w h i l e t h e i n d u c t i o n o f DNA s y n t h e s i s by LPS was s l i g h t l y s t r o n g e r than i n u n t r e a t e d c e l l s . I.  These r e s u l t s suggest t h a t :  t h e a n t i s e r a were w o r k i n g p r o p e r l y as seen by t h e a n t i - I g  sensitivity  of t h e LPS s t i m u l a t i o n and t h e a n t i - B A 0 s e n s i t i v i t y o f t h e Con A r e s p o n s e , ii.  b o t h T and B c e l l s a r e r e q u i r e d i n s i g n i f i c a n t numbers ( i t i s assumed  t h a t some c e l l s escape k i l l i n g by the a n t i s e r a but o b v i o u s l y . n o t enough t o maintain r e a c t i v i t y ) the  DNA  i i i . e i t h e r o r b o t h c e l l types may  s y n t h e s i z e d and d e t e c t e d by the a s s a y , but b o t h r e q u i r e the p r e s e n c e  of the o t h e r c e l l type f o r i n d u c t i o n of DNA the  c o n t r i b u t e to  need f o r a c o o p e r a t i v e r e s p o n s e .  proliferative  response by NRS  s y n t h e s i s , w h i c h may  indicate  The r e d u c t i o n o f • t h e 24 hour  and C', as compared t o u n t r e a t e d c e l l s ,  may  be due t o the l o s s of an adherent a c c e s s o r y c e l l , p o s s i b l y macrophages, s i n c e the e x t r a i n c u b a t i o n and. washings do reduce the.numbers o f adherent cells.  Macrophages a r e r e q u i r e d f o r s t i m u l a t i o n i n some i n v i t r o  (Seeger and Oppenheim, 1970; Waldron, Horn and R o s e n t h a l , 1973).  systems This  problem c o u l d be r e s o l v e d by an i n v e s t i g a t i o n of the r o l e of macrophages i n the assay as performed i n t h i s s t u d y . The response t o O-Fd RaMIg or NRS  a t 120 hours was n o t a f f e c t e d by p r e i n c u b a t i o n w i t h  and C', but was v i r t u a l l y e l i m i n a t e d by the RaBAO and  C'  t r e a t m e n t s u g g e s t i n g t h a t the 120 hour response i s , as c l a s s i c a l l y t a k e n , dependent  on the presence of T c e l l s .  can i n d u c e DNA  I t i s p o s s i b l e t h a t the T c e l l  response  s y n t h e s i s i n B c e l l s by the p r o d u c t i o n of m i t o g e n i c f a c t o r s ,  but the s t i m u l a t i o n a t 120 hours depends on the presence of adequate numbers of T c e l l s . sera.  The 72 hour response was too low t o show any a f f e c t by the  Mugraby, et_ a l , (1974), u s i n g e r y t h r o c y t e s as the a n t i g e n have shown  t h a t b o t h T and B c e l l s a r e r e q u i r e d f o r an i n v i t r o response.  t h y m i d i n e uptake  These r e s u l t s are c o n s i s t e n t w i t h the assumption t h a t the  lymphocyte s t i m u l a t i o n assay i s a c o r r e l a t e of c e l l mediated  immunity,  but the c o n t r i b u t i o n of B c e l l s t o the response i s a f a c t o r t h a t s h o u l d be c o n s i d e r e d . The experiments done t o t e s t the c r o s s r e a c t i v i t y  of the c e l l - m e d i a t e d  response I n v i t r o r e v e a l e d  t h a t o n l y t h r e e of the f e r r e d o x i n s were a c t i v e ,  e i t h e r as immunogens or t e s t a n t i g e n s  i n culture.  Only O-Fd  f o r i t s a b i l i t y to s t i m u l a t e a n t i b o d y  production.  I t was  the v a r i o u s f e r r e d o x i n s , o n l y O-Fd, s y n t h e s i s i n v i t r o i n O-Fd  NEM-Fd and n a t i v e - F d  sensitized cells.  was  examined  found t h a t of induced  DNA  When t e s t e d f o r i m m u n o g e n i c i t y ,  NEM-Fd immune c e l l s were s t i m u l a t e d by NEM-Fd and native-Fd--and l e s s s t r o n g l y by O-Fd.  Native-Fd  immune c e l l s responded w e l l to  but not to the o t h e r f e r r e d o x i n s . of O-Fd, O-Fd  P r e v i o u s work w i t h a s y n t h e t i c analogue  N-10-C (which c o n t a i n s o n l y the two  b r i d g e d by 10 g l y c i n e r e s i d u e s ) , has  c r o s s r e a c t i v i t y between N-10-C and O-Fd  t e r m i n a l d e t e r m i n a n t s of  shown t h a t a h i g h degree of can be demonstrated i n the  lymphocyte s t i m u l a t i o n a s s a y , i n immediate and i n the m i g r a t i o n N-10-C or O-Fd  native-Fd,  i n h i b i t i o n t e s t and a n t i b o d y  delayed s k i n r e a c t i o n s , production  with either  f u n c t i o n i n g as the immunogen (Levy, H u l l , K e l l y , K i l b u r n  and T e a t h e r , 1972;  K e l l y , Levy and H u l l , 1973).  I n the p r e s e n t s t u d y ,  f e r r e d o x i n s w i t h m o d i f i c a t i o n s w i t h i n the d e t e r m i n a n t s of O-Fd and DNP-O-Fd) d i d not s t i m u l a t e DNA  synthesis.  (meth-O-Fd  Taken t o g e t h e r ,  these  r e s u l t s i n d i c a t e t h a t m o d i f i c a t i o n s which l e a v e the two d e t e r m i n a n t s of O-Fd  u n o b s t r u c t e d may  sensitized cells.  The  not s e r i o u s l y a f f e c t t h e i r r e c o g n i t i o n by  O-Fd  l a c k of r e a c t i v i t y of those f e r r e d o x i n s whose  m o d i f i c a t i o n s were not i n e i t h e r of the d e t e r m i n a n t s (TCA-Fd and CM-Fd) was  p r o b a b l y due  to the c e l l s .  t o s t r u c t u r a l changes which made the d e t e r m i n a n t s i n a c e s s i b l e  The  i n a b i l i t y of some of the f e r r e d o x i n s to immunize even  when t e s t e d w i t h the homologous a n t i g e n i n c u l t u r e i s u n e x p l a i n a b l e t h i s time. N-ethylmalyl  I t i s i n t e r e s t i n g t h a t NEM-Fd, w h i c h c o n t a i n s  two  groups, f u n c t i o n e d q u i t e w e l l as an immunogen and  at  hydrophobic elicited  a  stronger  i n v i t r o c e l l u l a r response than the more h i g h l y charged  but d i d n o t r e a c t w e l l w i t h a n t i - O - F d s e r a .  Somewhat s i m i l a r  were o b s e r v e d by P a r i s h (1971 a and b) u s i n g a c e t o a c e t y l a t e d He found t h a t the a c e t o a c e t y l a t i o n  O-Fd,  results flagellin.  (which i n c r e a s e s h y d r o p h o b i c i t y  reduces charge) o f f l a g e l l i n produced a m o l e c u l e which  and  preferentially  s t i m u l a t e d and e l i c i t e d a c e l l u l a r immune response b u t d i d not c r o s s react w i t h antibodies to the n a t i v e molecule. The c r o s s r e a c t i v i t y  o f t h e r a b b i t a n t i - O - F d a n t i b o d i e s was  than was observed w i t h the c e l l mediated response i n v i t r o . was v i r t u a l l y u n a b l e t o f i x ' complement.  greater  Only meth-O-Fd  Since t h i s m o d i f i c a t i o n a f f e c t s  c a r b o x y l groups, w h i c h a r e p r e s e n t i n b o t h t h e d e t e r m i n a n t s o f O-Fd and known t o be i m p o r t a n t i n the r e c o g n i t i o n o f O-Fd,  (Gregerson e t a l ,  1976 and u n p u b l i s h e d o b s e r v a t i o n s ) , t h i s may account f o r i t s l a c k o f r e a c t i v i t y . S t r o n g c r o s s r e a c t i v i t y was found w i t h TCA-Fd and DNP-O-Fd, u n l i k e t h e lymphocyte s t i m u l a t i o n assay i n w h i c h they were n o t r e c o g n i z e d . recognized  well i n culture, fixed  C' p o o r l y .  A number of r e p o r t s have  suggested t h a t r e c o g n i t i o n by T c e l l s i s l e s s s p e c i f i c by a n t i b o d i e s .  NEM-Fd,  than r e c o g n i t i o n  T h i s has not been seen i n t h i s s t u d y where the a n t i b o d i e s  seemed.to be l e s s demanding.  A l s o , i n the previous  s e c t i o n (Gregerson,  et a l , 1976), t h e m i g r a t i o n i n h i b i t i o n assay demonstrated a h i g h of s p e c i f i c i t y .  level  The n a t u r e and a c c e s s i b i l i t y o f t h e r e c e p t o r s , whether  c e l l s u r f a c e bound o r f r e e i n s o l u t i o n , may have an e f f e c t on s p e c i f i c i t y . The p o s s i b i l i t y t h a t the r e s u l t s p r e s e n t e d h e r e and i n o t h e r s t u d i e s simply r e f l e c t  t h e s e n s i t i v i t i e s of t h e assays must be c o n s i d e r e d .  may Because  of t h e d i f f i c u l t y o f o b t a i n i n g a n t i - O - F d s e r a i n m i c e , r a b b i t a n t i - s e r a was used.  The e f f e c t o f t h i s s p e c i e s d i f f e r e n c e i s u n c e r t a i n .  F u t u r e work  on s p e c i f i c i t y w i l l i n v o l v e an i n v e s t i g a t i o n of the s p e c i f i c i t y o f B c e l l a c t i v a t i o n and T-T c e l l  cooperation.  i.H.JJJjEi  X  AMINO ACID ANALYSES OF THE FERREDOXIN PREPARATIONS  Amino Acid  Theoretical  Native-Fd  O-Fd  Meth-O-Fd  NEM-Fd  TCA-Fd  9.6  8.8  8.9  8.0  10.0  9.7  7.6  DNP-O-Fd  CM-Fd  asp  8  thr  1  1.6  1.4  1.4  1.1  0.9  1.5  1.2  ser  5  3.8  4.6  4.7  4.0  4.6  4.5  5.2  glu  4  5.4  4.9  4.9  4.0  4.1  5.7  5.0  pro  3  3.7  4.6  3.0  3.8  3.6  3.0  2.8  cys  8  6.5  0+  0  3.6+  2.6  0  0  giy  4  4.8  4.6  4.4  4.0  3.0  5.0  4.4  ala  8  6.0  7.2  6.4  6.9  6.0  5.5"^  6.6  val  6  5.9  6.2  5.0  5.8  5.7  5.2  4.6  ile  5  3.6  4.6  3.5  3.8  4.8  3.9  3.4  tyr  1  1.3  1.2  -  -  1.3  1.2  1.2  phe  1  1.3  1.2  0.9  -  1.3  1.3  1.2  lys  1  1.7  1.5  1.1  1.1  1.6  0.09"^  1.0  8.2  7.8  3.8  6.2  X  cys tele acid  Y  carboxymethyl 1cys  3.4  +  S-syccinyl xi;  cys  1.9  C  molar r a t i o s  00 .produced by the o x i d a t i o n +  a  "derivative of cysteine derivative  of cysteine  ~^~low v a l u e s i n d i c a t e  of c y s t e i n e  and c y s t i n e  produced by a l k y l a t i o n w i t h  iodoacetate  by a l k y l a t i o n w i t h N - e t h y l m a l e i m i d e and subsequent a c i d  modification  hydrolysis oo N3  TABLE 2 DOSE RESPONSE OF O-Fd SENSITIZED AND NON-IMMUNE DBA SPLEEN CELLS TO O-Fd IN VITRO  O-Fd  ug/ml  Immune S t a t u s 4_  8_  16_  32_  hours i n c u l t u r e ^  Immune Non-Immune  1.13  ± . 0 3 ^ 1.20 ± .09 0.96 ± 009 c .05*  Immune Non-Immune  1.30 ± .06  1.34 ± .10  1.36 ± .12 0.96 ± .08 <.01  1.08 ± .12 0.77 ± .19  72 72  >.10  1.91 ± .10  11.98 ± .21  120  1.21 ± .15 1.10 * .12  0.95 + .09  120  ?>.10  C.005  -C.01  r a t i o o f s t i m u l a t e d cpm t o u n s t i m u l a t e d cpm and t h e standard e r r o r of t h e means ( s t i m u l a t i o n i n d e x ± SEM) a  hours i n c u l t u r e p r i o r t o t h e 18 hour l a b e l i n g t-feprobability c a l c u l a t e d from t h e s t u d e n t s t - t e s t t h a t t h e response o f t h e immune c e l l s i s d i f f e r e n t than t h a t o f t h e non immune c e l l s  ** r e p r e s e n t s 6 e x p e r i m e n t s , per experiment  each done w i t h a minimum of 2 mice and w i t h 9 d e t e r m i n a t i o n s  oo  TABLE 3  KINETICS OF THE I N VITRO RESPONSE OF DBA SPLEEN CELLS TO O-Fd AT 16 ug/ML  Hours i n C u l t u r e  Immune  1.60  ± .14  Non-immune  1.01  ± .04  1.12  ± .07  hours b e f o r e a d d i t i o n  1.35  ± .03  1.81  ± .18  0.96  ± .08  1.10  ± .12  < .01  < .005  120  72  48  24  Immune S t a t u s  < .01  of l a b e l  * t - p r o b a b i l i t y - see T a b l e 2 f o r e x p l a n a t i o n  sstimulation  i n d e x ± SEM-H  ** r e p r e s e n t s t h e d a t a from 4 e x p e r i m e n t s  and 9 d e t e r m i n a t i o n s  pereexperiment  oo  85  TABLE 4 PERCENT LOSS OF DBA SPLEEN CELLS AFTER TREATMENT WITH ANTISERA AND C'  C' + NRS 29%"^~  RaMIg + C  RaBAO + C'  56%  55%  NRS - normal r a b b i t serum a  :t incubated simultaneously l o s s of v i a b l e c e l l s d e t e c t e d by t r y p a n b l u e  exclusion  RaMIG + RaBAO + C' 72%  TABLE 5  EFFECT OF TREATMENT WITH RaBAG AND RaMIg PLUS C  ON THE  RESPONSES OF DBA SPLEEN CELLS TO CohA AND LPS ConA 2 ug/ml. NRS and  Hours 24 448  89,913 ± 9571*  RaBAG and C'  89,225 ± 18,873  4525 ± 2479  114,568 ± 24,754  72  23,388 ± 3295  96  3243 ± 5308 -387 ± 1534^"  120 144  RaMIg and C'  C  8388 ±  3723  14,633 ± 9247  5864 ±  2699  6890 ± 3549  11276 ± 3283 LPS 16 ug/ml NRS and  Hours  C  24  57,995 ± 8443"  48  38,946 ± 10,665  72  13,929 ± 3218  96  -1667 ± 3374  120  511 ± 2318  144  -1783 ± 1608  '  RaMIg and C'  RaBAG and C'  8888 ± 1523  62,352 ± 8096  -630 ± 1283  29,979 ± 2677  474 ± 1004  7647 ± 2801  ^ hours i n c u l t u r e b e f o r e l l a b e l i n g *stimulated  cpm minus u n s t i m u l a t e d  controls  r e s p o n s e s below the u n s t i m u l a t e d background are expressed as n e g a t i v e s * * r e p r e s e n t s the p o o l e d r e s u l t s o f 6 experiments, each done on a minimum o f 4 mice w i t h a t l e a s t 9 d e t e r m i n a t i o n s per experiment  87  TABLE 6 EFFECT OF TREATMENT WITH RaBAO AND RaMIg PLUS C  ON THE  RESPONSE OF O-FD IMMUNE DBA SPLEEN CELLS TO O-Fd  O-Fd 16 ug/ml Hours °  C' and NRS  RaMIg and C'  24  1.36 ± . 0 8 ^  1.08 ± .06  <.01*  72  0.99 ± .09  0.95 ± .08  120  1.70 ± .17  1.70 ± .29  RaBAO and C' -1.07 ± .08  <.05*  >.25  0.96 ± .05  >.25  >.25  1.15 ± .11  <.01  * t - p r o b a b i l i t y t h a t t h e response i s d i f f e r e n t than t h e response of c e l l s t r e a t e d w i t h NRS and C'  a  hour i n c u l t u r e p r i o r t o l a b e l i n g  ** r e p r e s e n t s t h e p o o l e d r e s u l t s o f 6 e x p e r i m e n t s , each done on a minimum o f 4 mice w i t h a t l e a s t 9 d e t e r m i n a t i o n s p e r experiment  stimulation  i n d e x ± SEM  TABLE 7 RESPONSES OF O-Fd SENSITIZED AND UNSENSITIZED SPLEEN CELLS FROM DBA MICE TO MODIFIED FERREDOXIN ANTIGENS AT 16 ug/ml Hours •  NEM-Fd  Meth-O-Fd  CM-Fd  TCA-Fd  Native-Fd  DNP-O-Fd  Immune S t a t u s  24  1.79 ± .32  1.00 ± .03  0.84 ± .08  1.18 ± .17  1.13 ± .13  1.05 ± .04  immune  24  1.08 ± . 23  1.00 ± .05  0.83 ± .15  1.10 ± .08  1.12 ± .04  0.93 ± .09  non-immune  0.74 ± .09  0.95 ± .26  0.99 ± .14  1.32 ± .47  0.83 ± .26  immune  1.01 ± .07  0.72 ± .08  0.63 ± .15  non-immune  <• .05 „ 72 772  2.19  ± .55  1.01 ± .27  0.79 ± .12  0.72 ± .29  <.10  <.05 120  2.99  ± .68  120  0.86 ± 1*17  0.97 ± .11  0.90 ± .18  1.16 ± .13  1.94 ± .31  1.04 ±..28  immune  0.98 ± .166  1.03 ± .12  1.18 ± .18  1.04 ± .07  0.73 ± .09  non-immune  <.025  <.005  hours i n c u l t u r e p r i o r t o l a b e l i n g * t-probabilities ~$~sstimulation  i n d e x ± SEM  ** r e p r e s e n t s t h e p o o l e d r e s u l t s o f 5 experiments  and 9 d e t e r m i n a t i o n s p e r experiment  oo oo  89  TABLE 8 IN VITRO RESPONSES OF B6D2/J F l MICE IMMUNIZED TO MODIFIED FERREDOXINS  Hours"  NEM-Fd*  O-Fd  24  1.26 ± . 1 8  24  1.16 ± .23  X  Native-Fd  Immune  status  1.25 ± .25  1.11 ± .11  NEM-Fd immune  0.98 ± .04  1.07 ± .04  Non-immune  <.10^ 72  1.16 ± .18  1.08 ± .14  1.07 ± .18  NEM-Fd immune  72  0.80 * .22  0.97 ± .06  0.70 ± .07  Non-immune  120  2.13 ± .54  1.50 ± .45  2.44 ± .64  NEM-Fd immune  120  0.75 ± .15  1.08 ± .09  0.96 ± .10  Non-immune  ^.01  <.10  <.05  24  1.19 ± .14  1.04 ± .06  1.24 ± .14  Native-Fd-immune  24  1.16 ± .23  0.98 ± .04  1.07 ± .04  Non-immune  72  0.67 ± .07  0.98 ± .16  1.04 ± .11  Native-Fd-immune  72  0.80 ± .22  0.97 ± .06  0.70 ± .07  Non-immune  120  1.32 ± .27  1.14 ± .19  1.87 ± .55  Native-Fd-immune  120  0.75 ± .15  1.08 ± .09  0.96 ± .10  Non-immune  < .05 ** r e p r e s e n t s the r e s u l t s from 4 experiments and n i n e d e t e r m i n a t i o n s p e r experiment ^ t-probability a  hours i n c u l t u r e x stimulation  p r i o r to 18 hour  labeling  index ± SEM  * a l l ferredoxin  a n t i g e n s used i n c u l t u r e  a t 16 ug/ml  90  Fig.  1.  Complement f i x a t i o n r e a c t i o n s of pooled a g a i n s t O-Fd a t a 1:40 d i l u t i o n w i t h preparations • •  of f e r r e d o x i n : •  A, DNP-0-Fd;A • , CM-Fd ;Q  antisera  several  •, TCA-Fd;  A,NEM-Fd ;• O , me th-O-Fd.  •,0-Fd;  UO I j\n I I p 4U3W9 [dwoo  92 REFERENCES  1.  Andersson, J . , M b l l e r , G. and Sj'oberg, 0.  (1972).  of  o f DNA s y n t h e s i s i n T and B lymphocytes.'  C e l l . Immunol., 4, 381.  2.  B a t t e l l , M.L., Zarkadas, C.G., S m i l l i e , L.B. and Madsen, N.B. 'The s u l f h y d r y l groups of muscle  phosphorylase.  of c y s t e i n y l p e p t i d e s r e l a t e d t o f u n c t i o n . ' 3.  Brown, R.K. nuclease.  (1962).  'Selective  induction  (1968).  III. Identification  J . B i o l . Chem., 243, 6202.  'Studies on the a n t i g e n i c s t r u c t u r e o f r i b o -  I I I . I n h i b i t i o n by p e p t i d e s of a n t i b o d y t o p e r f o r m i c  a c i d - o x i d i z e d r i b o n u c l e a s e . ' J . B i o l . Chem., 237, 1162. 4.  Brown, R.K., Delany, R., L e v i n e , L. and Van V u n a k i s , H. 'Studies on the a n t i g e n i c s t r u c t u r e o f r i b o n u c l e a s e . r o l e of hydrogen  5.  and d i s u l f i d e bonds.'  F r a e n k e l - C o n r a t , H. and O l c o t t , H.S.  Gerwing,  J . and Thompson, K.  p r o p e r t i e s o f egg-white  (1945).  (1968).  lysozyme.  I. General  J . B i o l . Chem., 234, 2043. ' E s t e r i f i c a t i o n of  p r o t e i n s w i t h a l c o h o l s o f low m o l e c u l a r weight.' 6.  J . B i o l . Chem., 161, 259.  'Studies on the a n t i g e n i c  I . I s o l a t i o n and c h a r a c t e r i z a t i o n  of a t r y p t i c p e p t i d e from reduced and a l k y l a t e d lysozyme haptenic a c t i v i t y . ' 7.  Golub, E.S.  8.  exhibiting  Biochem., 7, 3888.  (1971).  r a b b i t anti-mouse  (1959).  'Brain-associated 0 antigen:  b r a i n w i t h mouse lymphoid  Gregerson, D.S., K e l l y , B. and Levy, J.G. g u i n e a - p i g lymphocytes  t o mitogens,  cells.' (1975).  r e a c t i v i t y of C e l l . Immunol., 2, 353. 'Responses o f  an a n t i g e n , and mixed l e u c o c y t e  c u l t u r e i n media w i t h and without mercaptoethanol and f o e t a l serum.' 9.  calf  Immunol., 29, 237.  Gregerson, D.S., K e l l y , B., and Levy, J.G. to o x i d i z e d f e r r e d o x i n . t e r m i n a l determinant.'  (1976).  'The immune response  I . S p e c i f i c i t y of the response t o the amino Immunol.  93  10. K e l l y , B., Levy, J.G. and H u l l , D.  (1973).  ' C e l l u l a r and humoral  immune responses i n g u i n e a p i g s and r a b b i t s t o c h e m i c a l l y d e f i n e d synthetic peptides.'  E u r . J . Immunol., 3, 574.  11. K e l l y , B., Kaye, B., Yoshizawa, (1974).  W.,  Levy, J.G. and K i l b u r n ,  D.G.  ' S e l e c t i v e b i n d i n g of c h e m i c a l l y d e f i n e d a n t i g e n i c p e p t i d e s  to mouse lymphocytes.' 12. L a n d s t e i n e r , K.  E u r . J . Immunol., 4, 356.  (1946).  'The s p e c i f i c i t y o f s e r o l o g i c a l  reactions.'  Harvard U n i v e r s i t y P r e s s . 13. Levy, J.G., H u l l , D., K e l l y , B., K i l b u r n , D.G. and T e a t h e r , (1972).  'The c e l l u l a r  sequences  R.M.  immune response to s y n t h e t i c p e p t i d e s c o n t a i n i n g  known to be h a p t e n i c i n p e r f o r m i c a c i d - o x i d i z e d  from C l o s t r i d i u m pasteurianum.'  ferredoxin  C e l l . Immunol., 5, 87.  14. Maron, E., Webb, C., T e i t e l b a u m , D. and Arnon, R.  (1972).  'Cell-  mediated vs humoral response i n the c r o s s r e a c t i o n between hen eggw h i t e lysozyme  and b o v i n e < * - l a c t a l b u m i n . '  E u r . J . Immunol., 2, 294.  15. Meuvissen, N.J., van A l t e n , P.J. and Good, R.A.  (1969).  'Antigen-  induced c e l l p r o l i f e r a t i o n i n c u l t u r e s from antibody d e f i c i e n t c h i c k e n s . ' J . Immunol., 102, 1079. 16. M i l l s , J.A. lymphocyte  (1966).  'The immunologic  transformation i n v i t r o . '  s i g n i f i c a n c e of a n t i g e n induced J . Immunol., 97, 239.  17. Mugraby, L., Gery, I . and S u l i t z e a n u , D.  (1974).  o f thymus-derived and o f bone marrow-derived  'The p a r t i c i p a t i o n  lymphocytes  of s e n s i t i z e d  mmice, i n the p r o l i f e r a t i v e response t o s p e c i f i c a n t i g e n , i n v i t r o . ' Eur. J . Immunol., 4, 402. 18. N i t z , R.M.,  M i t c h e l l , B., Gerwing,  J . and C h r i s t e n s e n , J .  'Studies on t h e a n t i g e n i c i t y o f b a c t e r i a l f e r r e d o x i n s .  (1969).  I . The e f f e c t of  m o d i f i c a t i o n of c y s t e i n y l r e s i d u e s on the a n t i g e n i c i t y o f C l o s t r i d i u m  94  p<>  pasteurianum f e r r e d o x i n . '  J . Immunol., 103, 319.  19. Oppenheim, J . J . , W o l s t e n c r o f t , R.A. and G e l l , P.G.H. h y p e r s e n s i t i v i t y i n the guinea-pig  to a protein-hapten  (1967).  'Delayed  conjugate  and i t s  r e l a t i o n s h i p t o i n v i t r o t r a n s f o r m a t i o n o f lymph node, s p l e e n , thymus and p e r i p h e r a l b l o o d lymphocytes.' 20. P a r i s h , C.R.  (1971a).  Immunol., 12, 89.  'Immune response t o c h e m i c a l l y m o d i f i e d  flagellin.  1. I n d u c t i o n o f a n t i b o d y t o l e r a n c e t o f l a g e l l i n by a c e t o a c e t y l a t e d d e r i v a t i v e s of the p r o t e i n . ' 21. P a r i s h , C.R. flagellin.  (1971b).  J . Exp. Med., 134, 1.  'Immune response t o c h e m i c a l l y m o d i f i e d  I I . E v i d e n c e f o r a fundamental r e l a t i o n s h i p between  humoral and c e l l - m e d i a t e d immunity.' 22. P a r i s h , C.R.  (1972).  J . Exp. Med., 134, 21.  ' P r e f e r e n t i a l - induction of cell-mediated  immunity by c h e m i c a l l y m o d i f i e d sheep e r y t h r o c y t e s . '  E u r . J . Immunol.,  2, 143. 23. R a f f , M.C.  (1970).  'Two d i s t i n c t p o p u l a t i o n s o f p e r i p h e r a l lymphocytes  i n mice d i s t i n g u i s h a b l e by immunofluorescence.' 24. Ram, J . S . and Maurer, P.H.  (1959).  ' M o d i f i e d b o v i n e serum a l b u m i n .  V I I I . E s t i m a t i o n and some p h y s i c o c h e m i c a l derivative.' 25. S c h i r r m a c h e r ,  A r c h . Biochem. B i o p h y s . , V. and W i g z e l l , H.  s t u d i e s of the methylated  85, 512.  (1972).  'Immune responses a g a i n s t  n a t i v e and c h e m i c a l l y m o d i f i e d albumins i n m i c e . thymus p r o c e s s e d methylated  I . A n a l y s i s o f non-  (B) and thymus-processed (T) c e l l responses a g a i n s t  b o v i n e serum a l b u m i n . '  26. S c h i r r m a c h e r ,  Immunol., 19, 637.  J . Exp. Med., 136, 1616.  V. and W i g z e l l , H.  (1974).  'Immune responses a g a i n s t  n a t i v e and c h e m i c a l l y m o d i f i e d albumins i n mice. of e l e c t r i c charge and c o n f o r m a t i o n and on h e l p e r T c e l l responses.'  I I . E f f e c t of a l t e r a t i o n  on t h e humoral a n t i b o d y  J . Immunol., 113, 1635.  response  95  27. Seeger, R. and Oppenheim, J . J . macrophages and lymphocytes lymphocytes.' 28. Tanaka, M.,  J . Exp. Med.,  (1970)..  ' S y n e r g i s t i c i n t e r a c t i o n of  i n antigen-induced transformation i n 132,  44.  Nakashima, T., Mower, H.F.  and Yasunobu, K.T.  (1963).  'The C- and N- t e r m i n a l amino a c i d sequences o f C l o s t r i d i u m ferredoxin.'  A r c h . Biochem., 105,  29. Thompson, K., H a r r i s , M., (1972).  570.  B e n j a m i n i , E., M i t c h e l l , G. and N o b l e ,  ' C e l l u l a r and humoral immunity:  recognition.'  Nature  (New  30. Waldron, J.A., Horn, R.G.  pasteurianum  B i o l . ) , 238,  M.  a distinction i n antigenic 20.  and R o s e n t h a l , A.S.  i n d u c e d p r o l i f e r a t i o n of g u i n e a p i g lymphocytes  (1973).  'Antigen-  in vitro:  Obligatory  r o l e of macrophages i n the r e c o g n i t i o n of a n t i g e n by immune T-lymphocytes.'  J . Immunol., I l l ,  58.  CONCLUSION  E x a m i n a t i o n o f t h e r e s u l t s o f t h e i n v i t r o lymphocyte  stimulation  assay r e v e a l e d t h a t t h e responses o f g u i n e a p i g and mouse c e l l s t o t h e mitogens Con A and LPS and t h e a n t i g e n O-Fd a r e v e r y s i m i l a r i n terms o f dose response and k i n e t i c s .  The LPS r e s p o n s e s o f b o t h s p e c i e s were  s e n s i t i v e t o t r e a t m e n t w i t h a n t i - i m m u n o g l o b u l i n and complement a l t h o u g h the s t i m u l a t i o n o f mouse s p l e e n c e l l s by LPS was reduced t o a much g r e a t e r degree than was t h e response o f g u i n e a p i g c e l l s . Of p a r t i c u l a r i n t e r e s t was t h e s p e c i f i c response i n v i t r o t o t h e a n t i g e n , O-Fd.  The a b i l i t y o f O-Fd t o s t i m u l a t e a p r o l i f e r a t i v e r e s p o n s e  i n serum f r e e medium w i t h and w i t h o u t m e r c a p t o e t h a n o l was t e s t e d u s i n g g u i n e a p i g lymph node c e l l s .  Both o f t h e s e media s u p p o r t e d an e a r l y  response w h i c h peaked a t 48 hours and r a p i d l y d e c l i n e d .  The a d d i t i o n o f  f o e t a l c a l f serum (FCS) t o e i t h e r o f t h e s e media r e s u l t e d i n a b i m o d a l response t o O-Fd w i t h peak s t i m u l a t i o n s o c c u r i n g a t 24 and 96 t o 120 h o u r s . O-Fd s e n s i t i z e d mouse s p l e e n c e l l s a l s o gave a b i m o d a l response t o O-Fd i n medium w i t h FCS.  Peak r e s p o n s e s were found a t 24 and 120 hours as seen  with the guinea p i g c e l l s .  .By p r e t r e a t i n g t h e mouse s p l e e n c e l l s w i t h  a n t i - i m m u n o g l o b u l i n and complement or a n t i - b r a i n a s s o c i a t e d 9 and complement, i t was found t h a t t h e 24 hour response r e q u i r e d t h e presence o f b o t h T and B c e l l s i n l a r g e numbers w h i l e t h e 120 hour response r e q u i r e d o n l y T c e l l s . These r e s u l t s c o r r o b o r a t e t h e T c e l l dependency o f t h e i n v i t r o  lymphocyte  s t i m u l a t i o n assay and t h e v a l i d i t y o f i t s use as a c o r r e l a t e of c e l l mediated  immunity.  The s p e c i f i c i t y o f t h e c e l l u l a r and humoral immune responses t o s e v e r a l forms o f f e r r e d o x i n were t e s t e d u s i n g t h e lymphocyte  stimulation  assay to measure c e l l mediated immunity and d e t e c t antibody  reactivity.  e s t e r i f i e d form  (meth^-O-Fd) was  l a c k of r e c o g n i t i o n was  the complement f i x a t i o n t e s t  Only one m o d i f i e d not r e c o g n i z e d  probably  both of the,determinants p r e s e n t  f e r r e d o x i n , the methyl by the anti-O-Fd  by the anti-O-Fd  sera.  i n e i t h e r determinant  (O-Fd, n a t i v e - F d  and NEM-Fd) were a b l e to  synthesis i n v i t r o  i n O-Fd  Only f e r r e d o x i n s not  s e n s i t i z e d mouse s p l e e n c e l l s .  This in  Other f e r r e d o x i n s were at  moderately r e c o g n i z e d  DNA  sera.  a r e s u l t of meth-O-Fd being m o d i f i e d on O-Fd.  to  least  modified  stimulate These r e s u l t s  i n d i c a t e t h a t i n the f e r r e d o x i n system, a n t i b o d i e s a r e more c r o s s r e a c t i v e than i s the c e l l u l a r immune response. work i n other a n t i g e n  systems and may  which c o n t a i n s o n l y two I t was  of i n t e r e s t  determinants,  to n o t e , t h a t  T h i s i s i n c o n t r a s t to most p u b l i s h e d reflect one  the a n t i g e n i c nature  of  O-Fd  on each terminus of the m o l e c u l e .  o n l y f e r r e d o x i n s which s t i m u l a t e d  DNA  s y n t h e s i s i n v i t r o were immunogenic. The  s p e c i f i c i t y of the a n t i - O - F d  response was  also studied i n greater  d e t a i l u s i n g the amino t e r m i n a l determinant of O-Fd. i n d i c a t e d t h a t both a n t i b o d i e s and i n t h e i r s p e c i f i c i t y requirements.  These s t u d i e s  T c e l l s are h i g h l y s p e c i f i c , but There are s e v e r a l p o s s i b l e  of the r e s u l t s found u s i n g the p e p t i d e e i t h e r i . ) the r e c e p t o r s of T c e l l s and  differ  explanations  analogues of the amino d e t e r m i n a n t : the a n t i b o d i e s have d i f f e r e n t  c h a r a c t e r i s t i c s but r e a c t to the same determinant, or i i . ) r e c e p t o r s and  a n t i b o d i e s are s i m i l a r but are r e a c t i n g to  determinants,  or i i i . )  both p o s s i b i l i t i e s  the T  cell  overlapping  occur.  The more d e f i n i t i v e r e s u l t s o b t a i n a b l e u s i n g s m a l l monovalent  peptide  determinants to assay immune s p e c i f i c i t y i n d i c a t e t h a t s p e c i f i c i t y  studies  done by m o d i f i c a t i o n of an e n t i r e antigen.may be m i s l e a d i n g ,  as  such  8  modifications  g r e a t l y a f f e c t m o l e c u l a r c o n f o r m a t i o n and may simply  the a c c e s s i b i l i t y o f some determinants r a t h e r to t h e determinants themselves. using modified  of t h e p e p t i d e  i n d i c a t e that antibodies  a r e more  cross  However, t h i s i s not c o n s i s t e n t w i t h the r e s u l t s  s t u d i e s which show t h a t both r e s p o n s e s a r e s p e c i f i c .  supports the c o n t e n t i o n may not p r o v i d e  than e f f e c t i n g a change  The r e s u l t s of s t u d i e s i n t h i s t h e s i s  f e r r e d o x i n antigens  r e a c t i v e than T c e l l s .  decrease  This  t h a t the i n d i s c r i m i n a t e m o d i f i c a t i o n o f p r o t e i n s  u s e f u l antigens  for specificity  studies.  The  peptide  s t u d i e s p r e s e n t e d h e r e suggest t h a t T c e l l s and a n t i b o d i e s have s i m i l a r s p e c i f i c i t i e s i n that neither  seems more c r o s s . r e a c t i v e than t h e o t h e r ,  however, t h e i r manner o f r e c o g n i t i o n may be d i f f e r e n t .  

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