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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. Luther College, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT"OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY'OF BRITISH COLUMBIA March, 1976 (c) Dale S. Greecerson In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f ' t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t ten pe rm i ss i on . Department o f /M. , ' c r'O k/o Ic The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e F^k. a"> / <7ic i ABSTRACT Guinea pig lymph node and spleen c e l l s responded i n v i t r o to con-canavalin A (con A), lipopolysaccharide (LPS), and a s p e c i f i c antigen, oxidized ferredoxin, i n serum free medium, medium with mercaptoethanol (ME), medium with f o e t a l c a l f serum (FCS), and medium with both FCS and ME. The addition of ME to serum free medium supported a mixed leucocyte reaction as did media with 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 eliminated the LPS response and treatment with anti-immunoglobulin (alg) and complement ( C ) reduced the LPS response i n d i c a t i n g that LPS may be a B lymphocyte mitogen. The con A response was enhanced by the alg and Ci treatment. 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 the amino terminal heptapeptide antigenic determinant of performic acid oxidized ferredoxin (O-Fd) were tested and compared using several synthetic peptides and analogues of the determinant i n leucocyte migration i n h i b i t i o n tests with guinea pig spleen c e l l s and i n h i b i t i o n of complement f i x a t i o n between O-Fd and homologous rabbit antiserum. A tetrapeptide comprising the four amino acids at the carboxy terminal of the test determinant was. able to produce s i g n i f i c a n t i n h i b i t i o n of leucocyte migration. Modifications of amino acids within the tetrapeptide r e s u l t e d i n a loss of migration i n h i b i t i o n . This same peptide would only i n h i b i t the complement f i x a t i o n reaction i f a hydrophobic group was attached to i t . Otherwise a hexapeptide was required to produce 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 . Guinea pigs s e n s i t i z e d with a conjugate of the heptapeptide determinant and bovine serum albumin gave p o s i t i v e immediate and delayed skin reactions to conjugates of the hepta-peptide with other proteins i n d i c a t i n g that the heptapeptide functions as both a hapten and c a r r i e r . Also, migration of spleen c e l l s from these animals was i n h i b i t e d by O-Fd. The a b i l i t i e s of several chemically modified forms of ferredoxin to stimulate DNA synthesis i n v i t r o i n spleen c e l l s from mice s e n s i t i z e d to O-Fd and to f i x complement (C') i n the presence of rabbit anti-O-Fd sera were tested. Of a l l the ,ferredoxins, only O-Fd, native ferredoxin (native-Fd) and N-ethylmaleimide alkylated ferredoxin (NEM-vFd) were able to stimulate DNA synthesis. O-Fd, native-Fd, acid p r e c i p i t a t e d ferredoxin (TCA-Fd) and dinitrophenylated O-Fd (DNP-O-Fd) fi x e d C' while methylated ferredoxin (meth-O-Fd) did not. NEM-Fd and carboxymethylated ferredoxin (CM-Fd) fi x e d C' weakly. Only native-Fd, O-Fd and NEM-Fd were found to be immunogenic i n mice when assayed for lymphocyte stimulation by a l l of the ferredoxins., The 24 hour DNA synthetic response was s e n s i t i v e to treatment with a n t i -mouse immunoglobulin and C' and a n t i - b r a i n associated 0 and C 1 while the 120 hour response was only s e n s i t i v e to the'anti-O and C'. i i i TABLE OF CONTENTS Page Introduction 1 Part I. Responses of Guinea Pig Lymphocytes to Mitogens, and, Antigen and Mixed Leucocyte Culture i n Media With and Without Mercaptoethanol and Foetal Calf Serum . . . . . . . . . . 3 T i t l e Page 3 Summary 4 I n t r o d u c t i o n . . . . . . 5 Materials and Methods 7 Results 10 Discussion . . . . . . . . 13 Tables and Figures 17 References 30 Part I I . The Immune Response to Ferredoxin. I. S p e c i f i c i t y of the Response to the Amino Terminal Determinant 33 T i t l e Page 33 Summary 34 Introduction 36 Materials and Methods 40 Results 43 Discussion . . . . . . . . . 46 Tables and Figures 50 References 59 i v Page Par t I I I . The Immune Response to Ferredoxin. I I . Cross R e a c t i v i t y of C e l l s and A n t i s e r a to modified f e r r e d o x i n s and the Nature of the C e l l s Responding I n V i t r o . . . ......... 67 T i t l e Page 67 Summary . . 68 I n t r o d u c t i o n 69 M a t e r i a l s and Methods 71 Results . . . . . . 74 Di s c u s s i o n 78 Tables and Figures 82 References 92 Conclusion 96 V ACKNOWLEDGEMENTS There are two people to whom I would l i k e to express a very s p e c i a l thanks, Dr••. J u l i a Levy and Barbara K e l l y . Their assistance and friendship were deeply appreciated. I would also l i k e to thank the following people who worked with and around me over the l a s t several years and gave a l o t of materials, help, ideas and discussions that are not obvious i n the f i n a l write up, but were part of a good experience':' Doug H u l l , Fumio Takei, Doug Water f i e l d , Rob McMaster, Bruce Acres, Maureen F a i r h u r s t , Barb Pope, Annajane Smith and Dr.'s Doug Kilburn, Terry Pearson, Rob Watson, Dick Whitney and Mary Lymburner. F i n a l l y , a thank you to the committee members for helping to get i t a l l f i n i s h e d : Dr.'s J.J.R. Campbell, P. Dennis, 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 recognize and react with antigens has been known for some time, but only recently has t h i s been examined i n d e t a i l using chemically defined, n a t u r a l l y occurring determinants. Previous work has often been done without knowledge of the l o c a t i o n or nature of the determinants present on the antigen. As a r e s u l t , most of these studies, which were done using antigens that were n o n s p e c i f i c a l l y modified 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 si n g l e determinant. To an unknown degree the observed e f f e c t of such modifications may 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 conformation of the molecule rather than modification of the determinants themselves. However, 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 several laboratories using undefined antigens and chemical modifications of them. U t i l i z i n g a v a r i e t y of techniques to assay humoral and c e l l u l a r immunity both i n vivo and i n v i t r o to chemically modified antigens, i t has been frequently observed that the c e l l u l a r immune response expresses a greater degree of cross r e a c t i v i t y than does the humoral response. There have been several reports of work done with defined antigens, but i n nearly a l l cases the antigens used for these studies were recognized by either thymus dependent lymphocytes (T c e l l s ) or antibodies. D i r e c t comparisons of the s p e c i f i c i t y of T c e l l s and antibody to the same determinant were not possible i n those studies. I t seems clear that t e s t i n g the cross r e a c t i v i t y of T c e l l s and antibody (or thymus independent B c e l l s ) to a sin g l e defined,determinant i s required to e s t a b l i s h the r e l a t i v e s p e c i f i c i t i e s 2 of these responses. A promising approach to the s p e c i f i c i t y problem was found when the antigenic nature of ferredoxin was elucidated. Ferredoxin i s a 55 residue polypeptide i s o l a t e d from Clostridium pasteurianum. It has been demonstrated that the performic acid oxidized form of ferredoxin (O-Fd) has only two major, detectable antigenic determinants to which antibodies are formed. Both of the determinants have been characterized and each i s recognized by T c e l l s and antibody, thus providing the opportunity for a d i r e c t comparison of s p e c i f i c i t i e s . In t h i s thesis the s p e c i f i c i t y of the immune response to ferredoxin and i t s amino terminal determinant, N7, was tested using modified ferredoxins and synthetic analogues of the N7 determinant by assays for c e l l mediated immunity and serum antibodies. The i n v i t r o lymphocyte stimulation assay used i n t h i s study to examine s p e c i f i c i t y i s generally believed to be a correlate of c e l l mediated immunity, although there has been some doubt ra i s e d recently of i t s v a l i d i t y as a measure of the c e l l u l a r immune response. For t h i s reason the parameters of the assay were extensively examined using several means of inducing DNA synthesis. Certain aspects of the assay commonly assumed to be necessary for a response, such as the requirement for f o e t a l c a l f serum and" long incubation periods p r i o r to l a b e l l i n g with precursors to DNA, were tested. The types of c e l l s (T or B) required to produce a" p r o l i f e r a t i v e response were also investigated. This work was begun using guinea pig c e l l s and l a t e r continued using mouse c e l l s to take advantage of the large volume of work already done that has characterized the immune system i n mice making them more useful for immunological studies. 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 of guinea p i g spleen and lymph node c e l l s to 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 the presence of 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 antigen ( 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 of c o n d i t i o n s . Time and dose dependency of the responses was measured i n RPMI 1640 (1640), 1640 plus mercaptoethanol (ME), 1640 plus f o e t a l c a l f serum (FCS), and 1640 wi t h ME and FCS. Mitogen responses were a l s o measured a f t e r treatment of the c e l l s w i t h sheep anti - g u i n e a p i g immunoglobulin (SaGPIg) and complement (C ?) or a f t e r passage through nylon wool columns. Lipopolysaccharide (LPS) stim u l a t e d the c e l l s under a l l media c o n d i t i o n s over a wide range of concent-r a t i o n s but over a narrow time p e r i o d . Nylon wool treatment of the c e l l s e l i m i n a t e d the LPS response while SaGPIg and C' reduced i t . Concanavalin A (con A) sti m u l a t e d the 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 of maximum response. Pre-treatment of c e l l s w i t h SaGPIg and C' enhanced the response to con A while nylon wool f r a c t i o n a t i o n diminished i t somewhat. Only lymph node c e l l s responded 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). In serum f r e e media the O-Fd responses were maximal at 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 supported f o r a prolonged p e r i o d and appeared to be bimodal. Except f o r an e a r l y response w i t h 1640 and ME, only 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 the mixed leucocyte c u l t u r e (MLC). 5 INTRODUCTION The In v i t r o s t i m u l a t i o n of lymphocytes by mitogens, antigens or 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 of c e l l mediated immunity except i n instances where B lymphocyte mitogens were used. However, there i s some question regarding what populations of c e l l s are responding and whether or not macrophages are in v o l v e d (Mugraby, Gery and S u l i t z e a n u , 1974). Various types of responses of lymphoid c e l l s to a number of mitogens have been demonstrated i n s e v e r a l animal s p e c i e s . In the mouse i t i s recognized that d i f f e r e n t lymphoid c e l l p opulations w i l l respond to c e r t a i n mitogens. Con A and LPS have been shown to be s p e c i f i c mitogens f o r T and B lymphocytes, r e s p e c t i v e l y (Anderson, M o l l e r • and Sjbberg, 1972b), although i t has been demonstrated that con A w i l l s t i m u l a t e B lymphocytes i f i t i s a p p r o p r i a t e l y presented (Anderson, Edelman, M o l l e r and Sjbberg, 1972a). Murine T lymphocytes may be separated by passage of lymphocytes through nylon wool columns ( J u l i u s , Simpson, and Herzenberg, 1973), or by treatment w i t h anti-mouse immunoglobulin plus C' (Takahashi, Old, M c l n t i r e . and Boyse, 1971). Conversely, populations of c e l l s f r e e of T lymphocytes may be prepared by treatment w i t h a n t i - 9 and C' (Raff, 1969; Lamelin, Lisowska-B e r n s t e i n , Matter, Ryser, and V a s s a l l i , 1972). The guinea p i g system has not been so w e l l e l u c i d a t e d . In the present study, treatment of c e l l s w i t h nylon wool or SaGPIg and C' was examined to observe t h e i r e f f e c t s on the con A and LPS responses. Recently, r e p o r t s have appeared demonstrating that 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 short periods of time without serum (V i s c h e r , 1972; Coutinho, M o l l e r , Andersson. and B u l l o c k , 1973). In v i t r o systems i n which 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 , simpler c h a r a c t e r i z a t i o n of supernatant 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 batches of serum s and r e d u c t i o n of n o n - s p e c i f i c s t i m u l a t i o n ! Several i n v e s t i g a t o r s have reported that the a d d i t i o n of reducing agents such as mercaptoethanol 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. Greater v i a b i l i t y and enhanced response to a n t i -gens, mitogens, and MLC have been claimed (Chen and H i r s c h , 1972; Bevan, E p s t e i n , and Cohn, 1974; Broome and Jeng, 1973; Fanger, Hart, Wells and N i s o n o f f , 1970; Heber-Katz and C l i c k , 1972). This study was undertaken to e l u c i d a t e some of the parameters governing the p r o l i f e r a t i v e response of guinea p i g lymph node and spleen c e l l s to 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 without FCS and/or ME, and to c o r r e l a t e some of the data presented here w i t h i n f o r m a t i o n already a v a i l a b l e i n murine systems. 7 MATERIALS AND METHODS ANIMALS Outbred a l b i n o guinea pigs of e i t h e r sex weighing approximately 400 grams were used i n a l l experiments. MITOGENS Con A (Sigma, St. L o u i s , Mo.) was made up i n phosphate buffered s a l i n e (PBS) to 200 ug/ml, s t e r i l i z e d , and stored 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 at 10 mg/ml, the pH adjusted to 8.0, heated f o r 30 minutes i n a b o i l i n g water bath and stored 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 Ferredoxin from _C. pasteurianum (Sigma) was performic a c i d o x i d i z e d ( M i t c h e l l , Levyi, and N i t z , 1970) before use. Animals 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 of O-Fd i n 50% complete Freund's adjuvant (CFA) using a t o t a l volume of 0.5 ml per animal. The animals were boosted 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 stored 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 , teased i n t o PBS and the r e s u l t i n g c e l l suspensions t r a n s f e r r e d to p l a s t i c tubes. The c e l l s were spun down (180 x g f o r 5 min.) and resuspended i n 0.85% NH^Cl ( i n 0.01 M P O 4 , pH 7.2) f o r 4 to 5 minutes to l y s e the red blood c e l l s . A f t e r 3 washes w i t h PBS they were counted using trypan blue to assess v i a b i l i t y . 8 MEDIA The b a s i c medium used f o r a l l experiments was RPMI 1640 (Gibco, Grand I s l a n d , New York) 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 streptomycin, and 50 pg/ml fungizone. Mercaptoethanol was made -4 up a s e p t i c a l l y i n medium at 2.5 x 10 M and stored f r o z e n . F o e t a l c a l f serum (Gibco, #84557) was i n a c t i v a t e d at 56° " f o r 30 min. and made up to 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 . Mitogens or antigen i n medium were added i n a volume of 0.05 m l / w e l l . When ME or FCS was used, 0.05 ml of the p r e v i o u s l y described stock s o l u t i o n s was added to each w e l l . F i v e to 10 x 10^ c e l l s i n 0.10 ml of medium were added to each w e l l along w i t h whatever volume of medium was re q u i r e d to make a f i n a l volume of 0.25 ml. This procedure r e s u l t e d i n a f i n a l c o n centration of ME at 5 x 10~ 5M, FCS at 4%, and c e l l s between 2-4 x 10 6/ml. The MLC's were set up i n a s i m i l a r manner except that 2.5 x 10^ c e l l s i n 0.05 ml from each animal were used per w e l l . C o n t r o l w e l l s contained 5 x 10^ unmixed c e l l s . Background counts were obtained by averaging the c o n t r o l counts of each animal. LABELLING AND HARVESTING T r i t i a t e d thymidine (Amersham-Searle, A r l i n g t o n Heights, 111. s p e c i f i c a c t i v i t y 2.0 Ci/m mole) at a concentration of 1.0 u C i i n 0.05 ml was added to the w e l l s 18 hours before h a r v e s t i n g . Harvesting was performed by a s p i r a t i n g the contents of each w e l l onto g l a s s f i b e r f i l t e r s using a 9 m u l t i p l e sample harvestor (Hartzman, Bach, Bach, Thurman,% and S e l l , 1972; Thurman, Strong, 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 counter. NYLON WOOL TREATMENT C e l l s t r e a t e d w i t h NH^Cl and washed as described above were suspended i n PBS plus 5% FCS and placed i n p l a s t i c t i s s u e c u l t u r e p l a t e s f o r 1 hour at 37° to remove adherent c e l l s . PBS w i t h 5% FCS was used f o r a l l subsequent nylon wool column manipulations. The non-adherent c e l l s were washed i n t o a nylon wool column, incubated f o r 1 hour at 37°y and e l u t e d s l o w l y . The column c o n s i s t e d of a 25 ml syringe b a r r e l f i l l e d to the 20 ml l e v e l w i t h l o o s e l y packed nylon wool and s t e r i l i z e d . The procedure was adapted from that used by J u l i u s e_t a l . (1973) . Recovery of c e l l s was approximately 15 to 20% of those a p p l i e d . To o b t a i n s u f f i c i e n t numbers of c e l l s f o r some experiments, lymph node and spleen c e l l s were pooled before f i l t r a t i o n . These c e l l s are r e f e r r e d to as pool 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 taken up i n s t e r i l e , i n a c t i v a t e d SaGPIg or guinea p i g C' (each was d i l u t e d 1:4 i n PBS) or i n both and incubated w i t h i n t e r m i t t e n t mixing f o r 1 hour att37°C a f t e r r w h l c h the c e l l s were washed and counted. 0.2 ml of the d i l u t e d SaGPIg and C' were used per 10^ c e l l s . Where necessary, lymph node and spleen c e l l s were pooled before treatment to ensure adequate numbers of 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 using spleen c e l l s i n serum f r e e medium are presented i n F i g . 1A. The optimal dose was found to be 4 ;jg/ml a f t e r 24 hours i n c u l t u r e . In c u l t u r e s w i t h FCS, concentrations of 16 and 64 ug/ml sti 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 induced spleen c e l l s i n medium plus ME at LPS concentrations from 4-256 ug/ml a f t e r 24 hours i n c u l t u r e . LPS s t i m u l a t e d lymph node c e l l s as shown i n Table 1. 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 out. Pooled c e l l s d i d not respond as w e l l as spleen or lymph node c e l l s alone (Table 1). INDUCTION OF DNA SYNTHESIS BY CON A S i m i l a r experiments were done uusing con A. The r e s u l t s of spleen c e l l s t i m u l a t i o n i n serum f r e e medium are presented i n F i g . 2A. The maximum response was found a f t e r 48 hours of c u l t u r e using 1 ug/ml. Higher doses (4 and 8 ug/ml) induced moderate transformation a f t e r only 24 hours. The response w i t h respect to the SI i n medium w i t h FCS was lower than the serum f r e e response ( F i g . 2B). With FCS, peak s t i m u l a t i o n s were induced by 32 ug/ml at 24 hours and 1 ug/ml at 48 hours of c u l t u r e . In con A experiments there was a trend toward high doses s t i m u l a t i n g e a r l y and lower doses peaking at a l a t e r time, u s u a l l y 48 or 72 hours. Lymph node c e l l s produced much greater a c t i v a t i o n to con A than d i d spleen c e l l s . T y p i c a l stimulations with 2 ug/ml con A over 24, 48, and 72 hours were between 40 and 60 f o l d i n a l l media tested. As observed with the LPS r e s u l t s , the highest Si's were found using serum free 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 with SaGPIg and C' enhanced, the con A response and moderately reduced the response to LPS (Table 1). Only treatment of the c e l l s with nylon wool consistently eliminated the LPS response; however, the con A response was also diminished somewhat (Table 1). Treatment with SaGPIg and C' k i l l e d 23%%of spleen c e l l s and 20% of lymph node c e l l s compared to controls containing normal sheep serum or C' alone or SaGPIg alone. ANTIGEN^INDUCED LYMPHOCYTE TRANSFORMATION The responses to O-Fd found i n serum free medium (Fig. 3A) and medium plus ME (Fig. 3B) were very s i m i l a r . In both cases the response peaked at 48 hours with 32 ug/ml i n medium only and 16 ug/ml i n medium with ME. By 72 hours the SI had dropped sharply and by 96 and 120 hours was below unstimulated controls. The responses i n medium plus FCS (Fig. 3C) and medium with FCS and ME (Fig. 3D) were bimodal. Both media supported good responses at 24 hours which declined at 48 and 72 hours and peaked again at 96 and 120 hours. The concentrations of O-Fd which induced the l a t e r response were often lower than those which stimulated the 24 hour response. Spleen c e l l s did not respond under any of the conditions used even though lymph node c e l l s from the same animal were able to respond. Lymph node and spleen c e l l s from co n t r o l animals immunized with CFA only were not stimulated by O-Fd. MLC RESPONSE OF LYMPH NODE AND SPLEEN CELLS Data from the MLC tests has been summarized i n Table 2. Medium /-alone was unable to support a response at anytime during the culture period. Except i n the case of lymph node c e l l s at 96 hours, very l i t t l e s t imulation was found i n medium with ME. Media with FCS or FCS and ME supported normal responses, however, the response with FCS and ME was better than i n medium with FCS only. DISCUSSION The r e s u l t s presented here have shown that guinea p i g lymphocytes can be r e a d i l y c u l t u r e d and s t i m u l a t e d by mitogens and an antigen (O-Fd) i n serum f r e e medium. With the a d d i t i o n of ME and/or FCS to the medium, a mixed lymphocyte r e a c t i o n was a l s o detected. Media composition had a marked e f f e c t on the c o n t r o l background counts i n the s t i m u l a t i o n s . Average background counts per minute when c u l t u r e s were t r i t i a t e d at 24 hours were 899 (medium o n l y ) , 2210 (medium p l u s ME), 1825 (medium and FCS), and 7252 (medium w i t h FCS and ME). Although a c t u a l counts i n mitogen s t i m u l a t i o n s were higher i n media c o n t a i n i n g ME and/or FCS, the s t i m u l a t i o n i n d i c e s were, on the average, higher i n media without serum and ME because the unstimulated c o n t r o l s were lower. The S i ' s of O-Fd 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 of the type of media used, w h i l e 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 supplemented w i t h FCS and ME. A wide range of LPS concentrations (0.25-256 ug/ml) was able to induce DNA s y n t h e s i s and i n the presence of ME there was l i t t l e d i f f e r e n c e i n the response of c e l l s to 4-256 ug/ml LPS. The k i n e t i c s of the LPS response was unusual i n t h a t , regardless of the media used, s i g n i f i c a n t s t i m u l a t i o n was achieved only when the c u l t u r e s were t r i t i a t e d at 24 hours. At 0 and 48 hours only s l i g h t s t i m u l a t i o n was found and by 72 and 96 hours the counts were below the unstimulated c o n t r o l s . The a d d i t i o n of FCS or ME s h i f t e d the dose response to s l i g h t l y higher LPS 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 moderately a l t e r e d by the use of FCS. Except f o r con A at 1 ug/ml, c u l t u r e s 14 co n t a i n i n g FCS u s u a l l y r equired higher concentrations of con A and the response was more prolonged. Other workers have noted that higher concentrations of con A are required i n media w i t h FCS and a t t r i b u t e t h i s to the a b i l i t y of con A to bind serum p r o t e i n s ( M o l l e r , Andersson, P o h l i t ? and Sjoberg, 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 higher w i t h respect to t o t a l counts and SI w i t h lymph node c e l l s than w i t h spleen c e l l s . This observation 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 accepted view that there are greater r e l a t i v e numbers of T lymphocytes i n lymph nodes than i n spleens. I t has been proposed that con A i s a T lymphocyte s p e c i f i c mitogen i n chickens (Weber, 1973), guinea pigs and r a b b i t s ( E l f e n b e i n , Harrison.; and Green, 1973),s and mice ( E l f e n b e i n et a l . , 1973; Andersson et a l . , 1972b). The r e s u l t s of our experiments using nylon wool and a n t i - I g w i t h C' are i n agreement w i t h s t u d i e s i n other animal systems which i n d i c a t e that LPS i s a B lymphocyte mitogen. Using d i f f e r e n t techniques, E l f e n b e i n et a l . (1973) have suggested that LPS i s B lymphocyte s p e c i f i c i n guinea p i g s . Using a n t i - l i g h t chain and C', Gmelig Meyling, Kooy-Blok^ and B a l l i e u x (1974) were able to k i l l 12% of p e r i p h e r a l blood lymphocytes and 42% of lymphocytes from human t o n s i l s . Takahashi et . a l . (1971) using a n t i - l i g h t . c h a i n and C' i n mice were able to k i l l up to 50% of spleen 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 spleen 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 these f i n d i n g s s i n c e spleen and lymph node c e l l s r a t h e r than enriched populations of lymphocytes were used. The a b i l i t y of nylon wool columns to remove c e l l s bearing surface 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 experiments, nylon wool treatment of guinea p i g c e l l s completely e l i m i n a t e d the response to LPS, whereas treatment w i t h a n t i - I g and C' only reduced i t . Using r a b b i t - a n t i mouse immunoglobulin and C 1 we have been able to reduce the LPS response of mice by more than 80% (unpublished observations). That the guinea pig LPS response i s r e l a t i v e l y i n s e n s i t i v e to SaGPIg and C' treatment could be due to an LPS responsive population of c e l l s with a low density of surface immunoglobulin. Nylon wool treatment also reduced the con A response. As nylon wool operates on a p r i n c i p l e of d i f f e r e n t i a l adherence, i t i s possible that some of the c e l l s capable of responding to con A are removed. Usually, the con A response was enhanced by treatment with anti - I g and C'. O-Fd induced thymidine uptake i n lymph node c e l l s only, but i n a l l of the media tested. Vischer (1972) using mice andkKirchner and Oppenheim (1972) with chickens have reported serum free antigen stimulation using KLH and sheep red blood c e l l s , r e s p e c t i v e l y , as antigens. The k i n e t i c s of the serum free O-Fd responses with and without ME were s i m i l a r to each other and d i s t i n c t l y d i f f e r e n t from the k i n e t i c s of medium with FCS and medium with FCS and ME. It was unexpected to see the antigen stimulation i n FCS and FCS plus ME containing media occur as early as 24 hours, decline at 72 hours (aatime often considered to be optimal) and increase again at 96 and 120 hours. These r e s u l t s may ind i c a t e that two d i f f e r e n t c e l l populations were responding to the antigen at d i f f e r e n t times. The bimodal response was not observed i n serum free media, probably because without serum, c e l l v i a b i l i t y was not adequate to support a second response. Pre-liminary experiments, not presented here, t e s t i n g the response of a n t i - I g and G' or nylon wool treated c e l l s to O-Fd have been inconclusive but suggest that SaGPIg and C' treated c e l l s may be able to respond, whereas nylon wool treated c e l l s seem incapable of responding. Mugraby eit al_. (1974) 16 using mice demonstrated that d e p l e t i o n of T or B lymphocytes reduced the response to sheep red blood 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 t e s t s . The response i n medium plus FCS and ME was greater and more prolonged than i n medium w i t h FCS only. 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 detectable response was obtained. Bevan et a l . (1974) using mouse spleen c e l l s showed that a d d i t i o n of MEEto serum f r e e MLC t e s t s r e s u l t e d i n p o s i t i v e responses. S i m i l a r r e s u l t s were obtained by Heber-Katz and C l i c k (1972). Although 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 time, i t has been observed that the presence of ME, c y s t e i n e , e t c . , g r e a t l y 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 which a l s o appear to 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 that ME acts 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 Jeng, 1973) . I t would appear that those responses which r e q u i r e prolonged c u l t u r i n g of lymphocytes cannot be supported i n med i a l a c k i n g FCS, as noted i n the l a t e s t i m u l a t i o n by O-Fd, or ME, as seen i n the MLC t e s t s . However, responses 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 adequately supported i n the absence of these media components. The data reported here i n d i c a t e that there are no major d i f f e r e n c e s between the responses of guinea p i g and mouse lymphocytes i n the mitogen, antigen, and MLC t e s t systems. TABLE 1 E f f e c t of Nylon Wool or SaGPIg and C' Treatment on the Con A and LPS t, Responses of Guinea P i g C e l l s Medium Source Hours of ' Con A ug/ml LPS ug/ml  supple- of c u l t u r e ments c e l l s before l a b e l l i n g Treatment 2 1 0.5 256 64 16 4 1 0.25 FCS spleen 24 FCS and spleen 24 ME ME none - - - 1.68* 2.71- 2.64 1.69 1.31 1.25 nylon wool - - - 0.76 0.52 0.79 0.80 0.79 0.92 none 10.5* 3.50 1.10 - - - -a n t i - I g and C' 9.10 5.02 1.22 -. - - - -FCS and node 24 l' n o n e 4 2 ' 9 3 3 ' 2 3 ' 9 9 " 3 ' 4 2 3 ' 3 6 3 ' 4 4 a n t i - I g and C' 70.7 50.8 11.9 - 2.20 2.30 1.94 FCS and pool 24 ME none 27.4 - - 2.17 1.86 nylon wool 14.6 - - - - 0.75 1.03 a n t i - I g and C' 31.1 - - - - 1.75 FCS and ME pool 48 none - - - - - 1.06 0.73 nylon wool - - - - - 0.70 0.45 * S t i m u l a t i o n Index TABLE 2 MLC Tests of A l l o g e n e i c Guinea P i g Lymph Node and Spleen C e l l s i n FCS and ME Supplemented Media Medium Only Medium Plu s ME Medium Plus FCS Medium plu s ME and FCS nodes** spleen nodes spleens nodes spleens 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 of 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 the i n d i c a t e d length of time 19 F i g . 1. The response of guinea p i g spleen c e l l s to 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 thymidine f o r 18 hours a f t e r the i n d i c a t e d hours i n c u l t u r e s 0, 0 0; 24, • • ; 48, A A ; 72,• • ; and 96 ,• •. 20 21 22 F i g . 2. The Con A responses of guinea p i g spleen 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 thymidine was done a f t e r a f t e r 0, 0 0; 24, • • ; 48, A A ;772,« •; 96,• •; and 120,A •; hours i n c u l t u r e . 25 F i g . 3. The response of O-Fd immune guinea p i g lymph node c e l l s to O-Fd at 1, 0 0; 2, • • ; 4, A A ; 8,% •; 16,• •; and 32,A • ; ug/ml. The c u l t u r e s were l a b e l e d f o r 18 hours beginning at the times i n d i c a t e d on the f i g u r e s . The media used were (A) medium onl 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. Andersson, J . , Edelman, G.M. , M o l l e r , G., and Sjbberg, 0. (1972a). ' A c t i v a t i o n of B lymphocytes by l o c a l l y concentrated concanavalin A.' Eur. J . Immunol., 2, 233. 2. Ande rsson, J . , M o l l e r , G., and Sjoherg, 0. (1972b). ' S e l e c t i v e i n d u c t i o n of DNA synth e s i s i n T and B lymphocytes.' C e l l . Immunol., 4, 381. 3. Bevan, M.J., E p s t e i n , R., and Cohn, M. (1974). 'The e f f e c t of 2-mercap-toethanol on murine mixed lymphocyte c u l t u r e s . ' J . Exp. Med., 139, 1025. 4. Broome, J.D., and Jeng, M.W. (1973). 'Promotion of r e p l i c a t i o n of 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. 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Weber, W.T. ( 1 9 7 3 ) . 'Direct evidence f or the response of B and T c e l l s to pokeweed mitogen.' C e l l . Immunol., 9 , 4 8 2 . 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 Several synthetic peptides and analogues of the amino terminal antigenic determinant of oxidized ferredoxin (O-Fd) were tested f o r t h e i r a b i l i t y to i n h i b i t the complement f i x a t i o n reaction between O-Fd and homologous antiserum, and to i n h i b i t the migration of spleen c e l l s from animals immunized to O-Fd or to a conjugate of i t s amino terminal hepta-peptide (N^) and bovine serum albumin (N^-BSA). The heptapeptide (H^N-ala-tyr-lys-ile-ala-asp-ser-COOH) i s known to be an antigenic determinant of O-Fd. The r e s u l t s of the migration i n h i b i t i o n assay suggest that the carboxy terminal t r i p e p t i d e of the heptapeptide i s p a r t i a l l y able to stimulate the production of migration i n h i b i t i o n f a c t o r . The tetrapeptide and longer peptides of the native sequence were a l l recognized to a much greater degree than the t r i p e p t i d e . Peptides modified at the asp a r t i c residue were p a r t i a l l y active while the serine modified peptide was not. Modif i c a t i o n at the amino end of the heptapeptide had no e f f e c t on migration i n h i b i t i o n . A control peptide containing the same amino acids i n an altered sequence was not recognized. The peptides were tested with unimmunized spleen c e l l s to eliminate 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 nonspecific i n h i b i t i o n of migration. As a d d i t i o n a l s p e c i f i c i t y c o ntrols, i t was shown that the peptide and the N^-BSA conjugate i n h i b i t e d migration i n O-Fd immunized animals, while O-Fd i n h i b i t e d migration i n N^-BSA immunized animals. The hexa, hepta, aspartic deleted and serine modified peptides were able to i n h i b i t the complement f i x a t i o n reaction with O-Fd and s p e c i f i c rabbit antiserum. I n h i b i t i o n found with the serine modified peptide and the lack of i n h i b i t i o n with the amino modified peptide or the d i , t r i , and tetrapeptides indicates that the determinant recognized by the rabbit antibodies i s e i t h e r larger than the determinant which induces the production of MIF or i s located nearer to the middle of the heptapeptide. The co n t r o l peptide did not i n h i b i t complement f i x a t i o n . INTRODUCTION The s p e c i f i c i t y of antigen r e c o g n i t i o n by an t i b o d i e s and thymus derived lymphocytes (T c e l l s ) has been examined i n the past. Several of these s t u d i e s have used chemically modified antigens w i t h uncharacterized determinants. P a r i s h (1972) using s e v e r a l heterologous.red blood c e l l s and chemical m o d i f i c a t i o n s of them demonstrated s i g n i f i c a n t 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 inverse c o r r e l a t i o n i n the a b i l i t i e s of the v a r i o u s modified and unmodified red blood c e l l s to s t i m u l a t e delayed h y p e r s e n s i t i v i t y or antibody production. 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 using n a t i v e and acet o a c e t y l a t e d f l a g e l l i n ( P a r i s h , 1971 a,b). Using modified albumins, Schirrmacher and W i g z e l l (1972) and Rubin and W i g z e l l (1973) als o demonstrated a greater degree of cross r e a c t i v i t y i n the c e l l mediated. immune response than i n the antibody response. Hoffman and Kappler (1973) using s e v e r a l heterologous red blood c e l l s showed that cross r e a c t i o n at the T c e l l l e v e l was greater than that seen w i t h a n t i b o d i e s . There are a number of other reports which imply that the c e l l u l a r immune response has a wider cross r e a c t i v i t y than the antibody response (Maron, Webb, Teitelbaum and Arnon, 1972; Schirrmacher and W i g z e l l , 1974; Coon and Hunter, 1973; D a i l e y and Hunter, 1974; Champlin and Hunter, 1975). These s t u st u d i e s have been l i m i t e d by the l a c k of defined determinants to which both T c e l l s and antibody are d i r e c t e d , making comparisons of s p e c i f i c i t y questionable. The use of ha p t e n - c a r r i e r systems (DNP-protein conjugates, etc.) has i n most cases produced predominantly antihapten 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 (Katz, P a u l , G o i d l and Benacerraf, 1970; P a u l , Katz, G o i d l and Benacerraf, 1970; Davie and P a u l , 1971) e l i m i n a t i n g the a b i l i t y to compare s p e c i f i c i t i e s . In those cases where anti-DNP c e l l 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 small immunogenic molecules, e s p e c i a l l y azobenzenearsonate-L-tyrosine (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 i n d i c a t e d that c e r t a i n minor m o d i f i c a t i o n s to the amino and carboxy groups of the t y r o s i n e moiety or to the arsonate 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 antibody i s produced to ABA-L-tyr, these experiments do not permit a d i r e c t comparison of antibody and T c e l l r e c o g n i t i o n (Hanna and Leskowitz, 1973; J o k i p i i and J o k i p i i , 1974; Becker, L e v i n and S e l a , 1973; Alkan, W i l l i a m s , N i t e c k i and Goodman, 1972; Bush, Al 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 demonstrating that the minimum s i z e of a determinant 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 . These small peptides were found to be nonimmunogenic and d i d not s t i m u l a t e DNA s y n t h e s i s 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 give p o s i t i v e delayed s k i n r e a c t i o n s and stimulated the production 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 (MIF). C o n s e r v a t i v e l y modified peptides were recognized and a d i f f e r e n c e i n the s p e c i f i c i t y of r e c o g n i t i o n was noted between a n t i - s e r a from d i f f e r e n t r a b b i t s (Benjamini, Shimizu, Young and Leung, 1969'; S p i t l e r , Benjamini, Young, Kaplan and Fudenberg, 1969). S p e c i f i c i t y studies done on glucagon, a small polypeptide of 29 amino acid residues, have indicated the presence of two antigenic determinants, one i n each h a l f of the molecule. The amino terminal determinant i s strongly recognized by anti-glucagon antibodies and also stimulates MIF production. The carboxy terminal determinant has less antibody directed against i t , but stimulates DNA synthesis and MIF production i n v i t r o (Senyk, Williams, N i t e c k i and Goodman, 1971). Thus, the amino terminal determinant has greater "hapten" a c t i v i t y while the carboxy terminal determinant has greater " c a r r i e r " a c t i v i t y although neither 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 laboratory with ferredoxin i s o l a t e d from Clostridium pasteurianum has revealed that t h i s polypeptide of 55 amino acid residues has only two major detectable determinants, the amino terminal heptapeptide (Ny) and the carboxy terminal pentapeptide (C5) (Nitz, M i t c h e l l , Gerwing and Christenson, 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, 1970; K e l l y and Levy, 1971). Both antibodies and T c e l l s recognize each of these determinants with N^ being s l i g h t l y more T c e l l directed and C^ stimulating the production of a somewhat greater quantity of antibody (Kelly and Levy, 1971; Waterfield, Levy, Kilburn, and Teather, 1972; Levy, H u l l , K e l l y , Kilburn and Teather, 1972; Pearson, Levy and Kilburn, 1975). A considerable amount of work has been done with the N 7 determinant since i t i s r e a d i l y synthesized, contains several modifiable amino acids 125 3 and can be labeled to a high s p e c i f i c a c t i v i t y with I or H labeled a c e t i c anhydride. Neither of these l a b e l i n g procedures causes a loss of recognition (Kelly and Levy, 1971; Pearson et a l , 1975). In t h i s study, the f i n e s p e c i f i c i t y of the immune response to the determinant has been examined by using several peptide analogues of Ny. I n h i b i t i o n of the complement f i x a t i o n r eaction has been used to assay antibody s p e c i f i c i t y . The leucocyte migration i n h i b i t i o n t e s t , c l a s s i c a l l y associated with 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 immunity (Salvin and Smith, 1960; David, A l - A s k a r i ; Lawrence and Thomas, 1964; David, Lawrence and Thomas, 1964; David and Schlossman, 1968) was used to determine T c e l l s p e c i f i c i t y . The r e s u l t s described here compare the s p e c i f i c i t y of recog-n i t i o n by antibodies and T c e l l s to a sing l e determinant. MATERIALS AND METHODS ANIMALS Young adult outbred a l b i n o r a b b i t s and guinea pigs were used f o r a l l experiments. PEPTIDES Several peptides of va r i o u s s t r u c t u r e s were prepared by s o l i d phase peptide synthesis as described by Hancock, P r e s c o t t , M a r s h a l l and Vagelos (1972), a m o d i f i c a t i o n of the 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 peptides and nomenclature are shown i n Table 1. The GEE-Ny peptide was made by r e a c t i n g the Ny peptide w i t h 20 equivalents of g l y c i n e e t h y l e s t e r and l-ethyl-3-(dimethylaminopropyl) carbodiimide h y d r o c h l o r i d e (EDCI) at pH 4.7 fo r 4 hours (Hoare and Koshland, 1967). The N^-Bzl-Ser peptide was synthe-s i z e d by s o l i d phase except that i t was cleaved from the 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. The r e s u l t i n g e s t e r s were 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 the 0-benzyl ether b l o c k i n g group i n t a c t on the se r i n e h y d r o x y l . The procedure i s that published by Savoie and Barton (1974). The N-M-Asp peptide was an e r r o r peptide i s o l a t e d from the 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 . The peptides 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 using p y r i d i n e - a c e t i c acid-water b u f f e r s . Peptide p u r i t y was assessed by t h i n l a y e r chromatography on s i l i c a g e l G using at. l e a s t 3 solvent systems. Further t e s t s of 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 analyzer. Molar r a t i o s from the amino a c i d analyses are shown i n Table 2. ANTIGENS Ferredoxin from C_. pasteurianum was purchased from Sigma (St. L o u i s , M i s s o u r i ) or i s o l a t e d from c u l t u r e s as described by Mortenson (1964) and Tanaka, Nakashima, Mower and Yasunobu (1964). O x i d a t i o n was performed by 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 ml formic a c i d f o l l o w e d by 3.33 ml of performic a c i d (3.0 ml of formic a c i d plus 0.33 ml of 30% hydrogen p e r o x i d e ) . The r e a c t i o n was allowed to continue f o r 2 hours at -10°, a f t e r which the r e a c t i o n mixture was evaporated under vacuum, d i a l y z e d , concentrated and analyzed f o r amino a c i d content and q u a n t i t y . The N^-BSA conjugate was made by mixing N^ and BSA (20:1) i n the presence of EDCI according to Hoare and Koshland (1967). A f t e r 4 hours r e a c t i o n the s o l u t i o n was d i a l y z e d , concentrated and analyzed to determine the degree of coupling. N^ was a l s o conjugated to poly-D-glutamic a c i d (PDG, 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 procedure. 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 analyses before 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 7-PLL, 12.6:1. IMMUNIZATIONS Rabbits 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 at 2 week i n t e r v a l s w i t h 1 mg doses of 0-Fd i n 50% Freund's'complete adjuvant (FCA) to produce the a n t i s e r a . Guinea pigs were immunized twice i n t r a -muscularly w i t h e i t h e r 250 ug of O-Fd or Ny-BSA i n 50% FCA. SKIN TESTS The shaved and d e p i l l a t e d f l a n k s of guinea pigs were i n j e c t e d i n t r a -dermally w i t h 50 ug of antigen i n 0.1 ml s a l i n e . Controls c o n s i s t e d of 42 tests with 0.1 ml s a l i n e on immunized animals and complete t e s t s with a l l antigens on unimmunized animals. LEUCOCYTE MIGRATION INHIBITION TEST Spleen c e l l s from guinea pigs were washed twice i n phosphate buffered s a l i n e (pH 7.2, 0.01 M PO^) and resuspended i n RPMI 1640 medium plus 5% heat-inactivated f o e t a l c a l f serum at a concentration of 1 0 % cells/media. The c e l l s were drawn into 1 x 75 mm c a p i l l a r y tubes which were sealed with p l a s t i c putty (Fisher) and the c e l l s sedimented at 200 x g for 5 minutes. The tubes were broken at the cell-medium i n t e r f a c e and placed into chambers containing 1 ml of medium. Test peptides were used at a concentration of 0.05 umoles/ml i n the same medium as good i n h i b i t i o n was obtained at t h i s concentration without cytotoxic e f f e c t s . Ny-PDG, N^-PLL and Ny-BSA were used at 10 ug/ml and O-Fd at 16 ug/ml. A f t e r 18 hours incubation i n a 3 7 ° , 5% C O 2 incubator, the areas of migration were measured using the c a l i b r a t e d stage of a microscope. Control experiments were performed i n an i d e n t i c a l manner on unimmunized animals. The procedure i s s i m i l a r to that described previously (Waterfield, et_ al, 1972; Levy, et a l , 1972; Waterfield, Levy and Kilburn, 1 9 7 4 ) . The r e s u l t s are expressed as the r a t i o of the migration area found with a test antigen to the migration area of controls containing only medium. An i n h i b i t i o n was considered s i g n i f i c a n t i f the r a t i o of areas was less than 0.80 and the t - p r o b a b i l i t y was l e s s than 0.05. INHIBITION OF COMPLEMENT FIXATION The complement f i x a t i o n t e s t s were performed as described previously (Gerwing and Thompson, 1968) using rabbit anti-O-Fd antiserum which had been heat inactivated at 5 6 ° for 30 minutes. RESULTS The a b i l i t y of preformed antibody to O-Fd to recognize modified or p a r t i a l peptides was assessed by measurement of t h e i r a b i l i t y to i n h i b i t f i x a t i o n of complement i n the presence of O-Fd and the antibody at optimal p r o p o r t i o n s . The r e s u l t s are shown i n Figure 1 A and B and represent the average of 4 i n d i v i d u a l experiments. Percent i n h i b i t i o n s were estimated at the range i n which c o n t r o l tubes c o n t a i n i n g only O-Fd and antiserum y i e l d e d 50% hemolysis i n the presence of complement. Of the p a r t i a l Ny peptides, 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 the r e a c t i o n . This suggests that the p o r t i o n of the determinant recognized by antibody encompasses 6 of the 7 residues or i s l o c a t e d c e n t r a l l y or near the amino te r m i n a l end. The p o s s i b i l i t y that the s p e c i f i c i t y of the r e c o g n i t i o n may i n v o l v e amino a c i d residues at the amino t e r m i n a l p o r t i o n of the molecule i s supported by the observation that DNP-N^ ( F i g . IB) was incapable of i n h i b i t i n g the complement f i x a t i o n r e a c t i o n . U n l i k e observations made w i t h the leucocyte m i g r a t i o n i n h i b i t i o n t e s t (see below), the peptide modified at the ser i n e residue (N^-Bzl-Ser) and con t a i n i n g only the amino acids was c o n s i s t e n t l y able to i n h i b i t complement f i x a t i o n although alone was unable to do so. I t i s p o s s i b l e that the O-Benzyl group may provide a hydrophobic enhancing e f f e c t as noted by others (Benjamini e_t a l , 1969) without i n t e r f e r i n g w i t h s p e c i f i c i t y . U n l i k e the m i g r a t i o n i n h i b i t i o n t e s t s , the peptide 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 of i n h i b i t i n g complement f i x a t i o n , thus r e i n f o r c i n g the p o s s i b i l i t y that the 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 determinant. S p e c i f i c i t y of the r e a c t i o n was e s t a b l i s h e d by negative r e s u l t s obtained w i t h NC^ (the synthetic peptide containing the same amino acids as Ny, but i n a d i f f e r e n t sequence). The s p e c i f i c i t y requirements for c e l l u l a r immunity as assessed by the i n h i b i t i o n of leucocyte migration were measured using spleen c e l l s from guinea pigs s e n s i t i z e d to O-Fd. Spleen c e l l s were set up with peptides of various length, each c o n s t i t u t i n g part of the determinant and with chemically modified analogues. Tests were also c a r r i e d out with N-8-N (a synthetic peptide of 22 amino acids containing two Ny determinants linked by 8 glycine residues (see K e l l y , Levy and H u l l , 1973), O-Fd, Ny-BSA, Ny-PDG, Ny-PLL and NCy. The r e s u l t s are summarized i n Table 3. It can be seen that, i n terms of s i z e , the N^ peptide was the smallest part' of the Ny peptide which consistently i n h i b i t e d migration. Although i t i s not shown by the averaged data, i t was observed that, occasionally, the Ng peptide also caused i n h i b i t i o n ; t h i s was observed with spleen c e l l s which demonstrated stronger than average i n h i b i t i o n i n the presence of N^ through Ny. N£ was consistently unable to i n h i b i t migration. The NCy peptide had no e f f e c t on the migration thus e s t a b l i s h i n g the dependence of the response on amino acid sequence rather than on o v e r a l l charge e f f e c t s . Tests run on peptides i n which either the a s p a r t i c or serine residues had been modified showed minimal or no a b i l i t y to i n h i b i t leucocyte migration, implying the importance of the i n t e g r i t y of t h i s end of Ny i n c e l l u l a r recognition. A l l of the Ny conjugates (Ny-PDG, etc) were able to i n h i b i t migration, emphasizing the lack of c a r r i e r e f f e c t . Both the data on the peptides of varying length (^-Ny) and the data on the serine and aspartic modified peptides indicate that the actual sequence recognized i n both length and c o n f i g u r a t i o n a l s p e c i f i c i t y i s contained w i t h i n the N^ peptide, s i n c e longer peptides do not show a greater a b i l i t y to i n h i b i t m i g r a t i o n , and peptides modified at the amino t e r m i n a l or l y s i n e residue (DNP-Ny) are s t i l l s p e c i f i c a l l y recognized by s e n s i t i z e d c e l l s . I n the experiments described above, the immunogen i n a l l i n s t a n c e s was O-Fd. Further 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 the c e l l mediated response to the Ny determinant when the Ny peptide was conjugated w i t h ED^T to BSA. Guinea pigs so immunized were subsequently t e s t e d f o r s k i n r e a c t i v i t y to the Ny determinant conjugated to a v a r i e t y of c a r r i e r s . Skin r e a c t i o n s were measured at 4 and 24 hours. The r e s u l t s are shown i n Table 4 and demonstrate that both antibody and c e l l mediated responses were e l i c i t e d to the Ny determinant. This observation i s i n agreement with previous work (Levy e_t_ al_, 1972) which showed that s i m i l a r conjugates e l i c i t e d both types of s k i n r e a c t i o n s i n guinea pigs immunized w i t h O-Fd. The s p e c i f i c i t y of the c e l l mediated response i n guinea p i g s immunized w i t h Ny-BSA was assessed using the leucocyte m i g r a t i o n i n h i b i t i o n t e s t . The r e s u l t s are presented i n Table 5. In most r e s p e c t s , the r e s u l t s are analogous to those found w i t h c e l l s s e n s i t i z e d to O-Fd, i n that N^ ' through Ny and O-Fd caused marked i n h i b i t i o n whereas N-M-Asp was not a c t i v e and the a s p a r t i c a c i d modified peptide (GEE-Ny) was weakly recognized. A summary of the r e s u l t s obtained w i t h antibody or immune c e l l s i s shown i n Table 6. DISCUSSION The 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 a n t i b o d i e s and T c e l l s f r o m O-Fd 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 p e p t i d e . H o w e v e r , t h e s p e c i f i c i t i e s a r e n o t i d e n t i c a l . The l y s i n e r e s i d u e a p p e a r s t o be c r i t i c a l f o r 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 o f t h e l y s i n e (DNF-N^) o r e l i m i n a t i o n o f i t (N^) c a u s e a l o s s o f r e c o g n i t i o n . C o n v e r s e l y , DNPpN^ and g i v e p o s i t i v e m i g r a t i o n i n h i b i t i o n . The a d d i t i o n o f a h y d r o p h o b i c g roup t o 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 t i b o d y r e c o g n i t i o n and may i n d i c a t e t h a t t h e amino a c i d s c r i t i c a l f o r r e c o g n i t i o n 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 l a c k s u f f i c i e n t b u l k o r h y d r o p h o b i c i t y f o r b i n d i n g . I n c o n t r a s t , t h e l o s s o f m i g r a t i o n i n h i b i t i o n t o N ^ - B z l - S e r d e m o n s t r a t e s t h e i m p o r t a n t r o l e o f t h e s e r i n e i n T c e l l r e c o g n i t i o n s i n c e t h e N^ p e p t i d e i n h i b i t s m i g r a t i o n w e l l . 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 s ( N - M - A s p and G E E - N y ) a r e c o n s i d e r a b l y l e s s a b l e , i f a t a l l , t o i n h i b i t m i g r a t i o n t h a n t h e i r u n m o d i f i e d c o u n t e r p a r t (N^) i m p l y i n g t h e a s p a r t i c r e s i d u e i s n e c e s s a r y f o r b i n d i n g t o T c e l l s . The N -M-Asp p e p t i d e i s a l s o l e s s a b l e t o i n h i b i t t h e complement f i x a t i o n r e a c t i o n w h i c h s u g g e s t s t h a t t h e a s p a r t i c r e s i d u e p l a y s a r o l e i n t h e d e t e r m i n a n t r e c o g n i z e d by a n t i b o d y , t h o u g h n o t as i m p o r t a n t as i n t h e d e t e r m i n a n t r e c o g n i z e d by T c e l l s . A p e n t a p e p t i d e ( a l a - t y r - l y s - i l e - a l a ) f r a g m e n t f r o m t h e amino d e t e r m i n a n t was u n a b l e t o i n h i b i t m i g r a t i o n i n p r e v i o u s wo rk ( W a t e r f i e l d e t a l , 1974) e m p h a s i z i n g t h e i m p o r t a n c e o f t h e 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 i n h i b i t i o n t e s t s w i t h N ^ - B S A 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-Asp i s n o t r e c o g n i z e d . The m i g r a t i o n i n h i b i t i o n f o u n d w i t h G E E - N ^ i s p r o b a b l y due t o t h e c r e a t i o n o f a new d e t e r m i n a n t on t h e immunogen N 7 - B S A by 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 o f 47 the a s p a r t i c residue during conjugation to BSA. Recognition of suggests that the s y n t h e t i c on BSA fu n c t i o n s much the same as the amino determinant on O-Fd. The minimum s i z e of an antibody b i n d i n g determinant appears to be 4-6 residues i n agreement w i t h s e v e r a l r e p o rts (Young, Benjamini, Stewart and Leung, 1967; Benjamini, Young and Leung, 1968 a and b; reviewed by Goodman, 1969). In the present work the 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 m i g r a t i o n . 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 pentapeptide 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 antibody and T c e l l s p e c i f i c i t y may be due to two separate but overlapping determinants w i t h i n N, or to the o d i f f e r e n t natures of antibody and T c e l l r e c e p t o r s . I f antibody and T c e l l r eceptors 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 the same e f f e c t on the a b i l i t y of the peptide to bind to these two receptors even i f they both recognize i d e n t i c a l determinants on the unmodified molecule. I t i s g e n e r a l l y accepted that the antigen receptor on thymus independent lymphocytes (B c e l l s ) i s antibody (reviewed by V i t e t t a and Uhr, 1975) and the s p e c i f i c i t y of B c e l l s i s probably very s i m i l a r or i d e n t i c a l to the antibody they produce (Makela, 1970). The nature of the receptor 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 Taussig, 1975). Evidence from experiments where the antigen stimulated production of MIF by guinea p i g c e l l s was blocked by the a d d i t i o n of a n t i - ^ , ^ g or £ i m p l i e s that the receptor i s immunoglobulin-like or c l o s e l y a s s o c i a t e d w i t h i t on the c e l l surface (Goscicka, 1974). A l s o , i d i o t y p i c determinants have been found on T c e l l s ( B i n z , W i g z e l l , Ramseier and Lindenmann (1975)). The a b i l i t y of peptides to s t i m u l a t e MIF production makes them very 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 . Peptides can be synthesized and used d i r e c t l y without coupling them to other p r o t e i n s or compensating f o r the presence and e f f e c t of other determinants. The r e l a t i v e l y l a r g e q u a n t i t i e s of peptides used to i n h i b i t m i g r a t i o n (0.05 umole of peptide i s equivalent to 280 ug O-Fd on the b a s i s of m o l a r i t y ) probably r e f l e c t s the f a c t that the peptides have l i t t l e t e r t i a r y s t r u c t u r e to maintain them i n the c o n f i g u r a t i o n found i n O-Fd. I t has been shown that only a small percentage of peptides 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 molecule (Crumpton and Small, 1967). As found i n other s t u d i e s w i t h small peptides, the peptides described here are unable to s t i m u l a t e DNA 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 from e i t h e r O-Fd or N7-BSA immunized animals. One reported exception i s a dodecapeptide from glucagon (Senyk et_ a l , 1971) which i s able to transform guinea p i g c e l l s . The a b i l i t y of small peptides to generate MIF without s t i m u l a t i n g DNA synthesis i s c o n s i s t e n t w i t h r e p o r t s that MIF production does not r e q u i r e DNA s y n t h e s i s (Bloom, Gaffney and Jimenez, 1972; R o c k l i n , 1973). This d i s s o c i a t i o n between MIF production and transformation i s unexplained, but suggests that two l e v e l s of a c t i v a t i o n can occur i n T c e l l s — a c t i v a t i o n w i t h and without DNA s y n t h e s i s . The report that c y t o t o x i c c e l l s can a l s o be generated without DNA s y n t h e s i s and c e l l d i v i s i o n supports t h i s hypothesis (MacDonald, Sordat, C e r o t t i n i and Brunner, 1975). The peptides used i n t h i s study are not immunogenic unless coupled to a molecule such assBSA. I t i s apparent that N 7 and DNP (also a hapten) do not act a l i k e when conjugated to proteins. Both humoral and c e l l u l a r immunity are directed to while DNP i s only recognized by antibodies. In animals immunized to N^-BSA, both (when coupled to a heterologous molecule) and BSA i n d i v i d u a l l y e l i c i t immediate and delayed skin r e a c t i o n s . The c l a s s i c a l hapten-carrier phenomena i s not expressed by N^-BSA. can be recognized when coupled to diverse c a r r i e r s such as BSA, PDG and PLL suggesting that shared determinants do not contribute s i g n i f i c a n t l y to either the antibody or T c e l l mediated response. The i n a b i l i t y of O-Fd to stimulate production of a u s e f u l t i t e r of antibody i n guinea pigs i s unfortunate since the complement f i x a t i o n tests had to be performed with rab b i t serum. The e f f e c t of the species difference on the s p e c i f i c i t i e s i s unknown. TABLE 1 NAMES AND STRUCTURES OF SYNTHETIC PEPTIDES N2 asp - ser N 3 a l a - asp ser N4 l i e - a l a - asp - ser N 5 lys - l i e - a l a - asp ser N 6 tyr - l y s - i l e - a l a - asp ser N7 al a - ty r - l y s - i l e - a l a - asp ser N-M-Asp ala - tyr - l y s - i l e -aala ser DNP-N. GEE-N. NO; ^Q > a f a ser C0NHCHoC00CHoCHo a l a - t y r - l y s - i l e - a l a - asp - ser - CONHCH2COOCH2CH3 An Ny peptide diamidated with glycine e t h y l ester, N^-Bzl-Ser i l e - a l a - asp - ser An N^ peptide with i n t a c t O-benzyl protecting group. NCy ser - leu - a l a - t y r - asp - ly s - ala Leucine i s substituted for is o l e u c i n e f o r i d e n t i f i c a t i o n purposes. o TABLE 2 MOLAR RATIOS OF SYNTHETIC PEPTIDES AFTER AMINO ACID ANALYSIS N-M GEE DNP ^4 Amino N. N, N. N r N, N 7 -Asp -N., -N, !? NC, 2 3 4 5 6 7 7 7 Ser 7 Aci d T X F + T F T F T F F T F T F T F TF F T F T 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 0.02" A 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 a l a 1 0.99 1 0.98 1 0.95 1 0.99 2 2.08 2 1.99 2 2.33 2 1.22 X X 1 1.10 2 1.70 i l e 1 0.95 1 1.01 1 1.02 1 0.95 1 0.98 1 1.04 1 1.05 1 0.85 l y s 1 1.05 1 0.93 1 0.97 1 1.04 1 1.04 1 0.07 x x 1 1.01 t y r 1 0.94 1 1.06 1 1.04 1 0.86 1 1.01 1 0.98 xx l e u g l y 2 2.01 x - t h e o r e t i c a l + - found o - molar r a t i o s xx - disappearance of 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 h y d r o l y s i s 1 0.96 52 TABLE 3 INHIBITION OF MIGRATION OF SPLEEN CELLS FROM O-Fd IMMUNE AND NON-IMMUNE GUINEA PIGS Antigen O-Fd Immune Unimmunized t - t e s t N 7 Q 7 0.70 I .04* 0.99 + .04 <.0005 N 6 0.79 ± .04 1.02 + .08 < .01 N 5 0.73 ± .05 0.92 + .07 < .01 N 4 0.71 ± .05 0.94 + .06 < .005 N 3 0.90 ± .05 0.96 + .08 ^ .40 N 2 0.91 ±. .07 0.97 + .09 < .40 NC 7 0.93 ± .06 1.09 + .08 < .10 N-M-Asp 0.88 ± 006 1.14 + .07 <• .005 GEE-N7 0.86 ± .09 1.06 + .08 .10 N.-Bzl-Ser 4 0.92 ± .10 0.90 + .10 > .40 N8N 0.025 nmoles/ml 0.67 ±.110 0.87 + .14 < .05 O-Fd 16 ug/ml 0.78 ± .03 1.06 + .06 < .0005 N 7-BSA 10 ug/ml 0.78 ± .05 1.01 + .11 < .01 N7-PDG 10 ug/ml 0.79 ± .05 0.98 + .09 < .025 N 7-PLL 10 ug/ml 0.73 ± .02 0.92±± .09 < .01 DNP-N? 0.69 ± .13 0.92 + .31 < .001 0 _ a l l peptides used at 0.05 umoles/ml * - mean ± standard e r r o r of the mean - p r o b a b i l i t y c a l c u l a t e d from the student's t - t e s t that the r a t i o of m i g r a t i o n i n immune animals i s d i f f e r e n t from that i n unimmunized animals - from p r e v i o u s l y published work using animals immunized to a s y n t h e t i c analogue of O-Fd (W a t e r f i e l d et a l , 1974). 53 TABLE 4 SKIN REACTIONS OBSERVED ON N?-BSA IMMUNIZED AND UNIMMUNIZED GUINEA PIGS SKIN REACTIONS Ny-BSA IMMUNIZED UNIMMUNIZED Challenging Antigen** Arthus Delayed Arthus Delayed N?-BSA 15 D 12 7 1 N8N* 8 7 4 2 N?-PDG 12 7 3 2 N ?-PLL 9 5 3 1 S a l i n e 3 2 3 1 average of 4 immunized and 4 unimmunized animals **- a l l antigens used at 50 ug/test is- two Ny peptides bridged by 8 g l y c i n e residues i n a l i n e a r manner ( K e l l y , Levy and H u l l , 1973) " - diameter of 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 N. 0.64 ± .09 0.94 ± .06 <.005 4 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 ** - Peptides used at 0.05 pmole/ml D - used at 16 ug/ml * - Mean ± standard error of the mean - See Table 3 for explanation TABLE 6 SUMMARY OF DATA ABILITY TO INHIBIT O-Fd Immune N7-BSA Immune Antigen or Peptide Complement F i x a t i o n Leucocyte Migration Leucocyte Migration N ? • + + + N^ + + ND 6 N 5 - + + N. + + 4 N 3 - +- ND N 2 - - ND NC ? - -. ND N-M-Asp + +-. N.-Bzl-Ser + - ND 4 GEE-N? ND - +-DNP-N7 — + 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 f i x a t i o n reaction between O-Fd and homologous antisera by peptide analogues of the amino terminal determinant of O-Fd. Bars indicate the standard error of the means. 0 • N7 N6 N5 Hk N3 N2 P® o o 1 lo o| o i oo ;oQa o o °o iO Ol M 0 0 9 o • 0 0 •I % O Of • d, • M 0.006 g. 1A o o o o o o o o o o o o o o o o o o o o o o o o o o c > o o 9 9 9 9 9 9 e © 9 9 9 9 9\ a • • • q - • • B • • • > > > > • . 1 • > • o o| o o o| o°o| o°o| o o o o O Oj o o o < o o o ^ ao| o« 0 0I0 9 9 9 9 0 9 0 0 9 9 9 • • i> o | o o 0 d °o° °o°: o 9 9 9 a ol • I D D B Bl 0.012 0.025 0.050 ^ i t m o l e s p e p t i d e / t e s t o ko h 30 20 10 > N 4 - B z l - S e r • N-M-Asp a DNP-N7 • NC7 3 > >\ _> > > > > > > > > > > > > > i> > > • • > > to > > > i> >l > > >| > > t>l > , > • • > > > > > > > > > > > > • • • l> M • • > >| > > > > > > > > > t> > > > • • • • • • • b • • • • a • a aa > > > >! > > >! > > >! > > >| > > > > • • • • • • • • H • • p • • • • • • • a±a[ • > »la nil 0.003 0.006 0.012 0.025 0.050 F i g . 1B ^Umoles p e p t i d e / t e s t 00 REFERENCES Alkan, 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. 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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. Parish, CR. (1972). ' P r e f e r e n t i a l induction of cell-mediated immunity by chemically modified sheep erythrocytes.' Eur. J . Immunol., 2, 143. 44. Paul, W.E., Katz, D.H., Goidl, E.A. and Benacerraf, B. (1970). 'Carrier function i n anti-hapten immune responses. I I . S p e c i f i c properties of c a r r i e r c e l l s capable of enhancing anti-hapten antibody responses.' J . Exp. Med., 132, 283. 45. Pearson, T., Levy, J . and Kilburn, D. (1975). 'The e f f e c t of s p e c i f i c c e l l i n a c t i v a t i o n on the c e l l u l a r immune response to ferredoxin peptide Eur. J . Immunol., 5, 65. 46. Rocklin, R.E. (1973). 'Production of migration i n h i b i t o r y factor by non-dividing lymphocytes.' J. Immunol., 110, 674. 47. Rubin, B. and Wigzell, H. (1973). 'The immune response against hapten-autologous protein conjugates i n the mouse. I I I . S p e c i f i c i t y of cooperating non-thymus-processed (B) and thymus-processed (T) lymphocyt J . Exp. Med., 137, 911. 48. S a l v i n , S.B. and Smith, R.F. (1960). 'The s p e c i f i c i t y of a l l e r g i c reactions. I. Delayed versus arthus h y p e r s e n s i t i v i t y . ' J . Exp. Med., 111, 465. 49. Savoie, J.Y. and Barton, M.A. (1974). 'Solid phase synthesis of s e l e c t i v e l y protected peptides.' Can. J . Chem., 52, 2832. 50. Schirrmacher, V. and Wigzell, H. (1972). 'Immune responses against native and chemically modified albumins i n mice. I. Analysis of non-thymus processed (B) and thymus-processed (T) c e l l responses against methylated bovine serum albumin.' J . Exp. Med., 136, 1616. 65 51. Schirrmacher, V. and W i g z e l l , H. (1974). 'Immune responses against n a t i v e and chemically modified albumins i n mice. I I . E f f e c t of e l e c t r i c charge and conformation on the humoral antibody response and on helper 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. (1971). 'The f u n c t i o n a l d i s s e c t i o n of an antigen molecule: S p e c i f i c i t y of humoral and c e l l u l a r immune responses to glucagon.' J . Exp. Med., 133, 1294. 53. S p i t l e r , L., Benjamini, E., Young, J.D., Kaplan, H. and Fudenberg, H.H. (1969). 'Studies on the immune response to a c h a r a c t e r i z e d a n t i g e n i c determinant of the Tobacco Mosaic V i r u s P r o t e i n . ' J . Exp. Med., 131, 133. 54. Tanaka, M., 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 of C l o s t r i d i u m pasteurianum f e r r e d o x i n . ' Arch. Biochem., 105, 570. 55. V i t e t t a , E.S. and Uhr, J.W. (1975). 'Immunoglobulin-receptors r e v i s i t e d . ' Science, 189, 964. 56. W a t e r f i e l d , J.D., Levy, J.G. and K i l b u r n , D.G. (1974). 'The r o l e of DNP i n antigen a c t i v a t i o n of c e l l u l a r immune responses.' Proceedings 8th Leucocyte C u l t u r e Conference (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 Press. 57. W a t e r f i e l d , D., Levy, J.G., K i l b u r n , D.G. and Teather, R.M. (1972). 'The e f f e c t of haptenic peptides from performic 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 and p r o t e i n c a r r i e r - h a p t e n conjugates on the immune response of macrophages and lymphoid c e l l s from animals immunized against 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., Benjamini, E., Stewart, J.M. and Leung, C.Y. (1967). 'Immunochemical studies on tobacco mosaic v i r u s p r o t e i n . V. The s o l i d -phase synthesis of peptides of an a n t i g e n i c a l l y a c t i v e decapeptide of tobacco mosaic v i r u s protein and the re a c t i o n of these peptides with antibodies to the whole protein.' Biochem., 6, 1455. THE IMMUNE RESPONSE TO FERREDOXIN I I . 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 cross r e a c t i v i t y of sera from rabbits s e n s i t i z e d to performic a c i d oxidized ferredoxin (O-Fd) and of spleen c e l l s from mice s e n s i t i z e d to O-Fd was analyzed using several chemically modified forms of ferredoxin i n the complement f i x a t i o n test and the i n v i t r o lymphocyte stimulation assay. Only O-Fd and native ferredoxin (native-Fd) gave p o s i t i v e responses i n both assays. Dinitrophenylated-O-Fd (DNP-O-Fd) and acid p r e c i p i t a t e d ferredoxin (TCA-Fd) were able to f i x complement ( C ) but did not stimulate DNA synthesis i n v i t r o . Ferredoxin a l k y l a t e d with N-ethylmaleimide (NEM-Fd) induced c e l l transformation but f i x e d C' poorly. Carboxymethylated ferredoxin (CM-Fd) was unable to stimulate DNA synthesis and was marginally able to f i x C'. Methylated-O-Fd (meth-O-Fd) was not recognized i n e i t h e r assay. The various ferredoxin preparations were tested for t h e i r a b i l i t y to s e n s i t i z e mice for use i n the i n v i t r o lymphocyte stimulation assay. Only O-Fd, NEM-Fd and native-Fd were capable of s e n s i t i z i n g lymphocytes for a p r o l i f e r a t i v e response i n v i t r o to the test antigens. This correlates with the observation that only these antigens were able to induce DNA synthesis i n O-Fd s e n s i t i z e d lymphocytes. The nature of the c e l l s responding i n v i t r o was examined by t r e a t i n g the c e l l s with rabbit anti-mouse immunoglobulin and C' or rabbit anti-mouse brain associated 0 and C'. The 24 hour response was found to be s e n s i t i v e to both sera while the 120 hour response was s e n s i t i v e only to the anti-9 sera. INTRODUCTION The a b i l i t y of the immune system to d i r e c t i t s e l f s p e c i f i c a l l y against an antigen i s w e l l e s t a b l i s h e d . 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 to d i s c r i m i n a t e between d i f f e r e n t antigens. Since the c l a s s i c work of Landsteiner (1946) these 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 chemically modified forms. A l a c k of humoral cross 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 antigen has f r e q u e n t l y been found. A n t i s e r a to n a t i v e ribonuclease do not recognize performic a c i d o x i d i z e d ribonuclease (Brown, 1962; Brown, Delany, Levine and Van Vunakis, 1959). Several other r e p o r t s have demonstrated that w hile l i t t l e or no humoral cross r e a c t i v i t y was found, a h i g h degree of r e c o g n i t i o n was seen at the l e v e l of delayed h y p e r s e n s i t i v i t y . A n t i b o d i e s to n a t i v e lysozyme show very l i t t l e r e a c t i o n to S-carboxymethylated lysozyme (Gerwing and Thompson, 1968), but v i r t u a l l y complete r e a c t i v i t y was found i n assays of cell-mediated immunity (Thompson, H a r r i s , Benjamini, M i t c h e l l and Nobel, 1972). 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 the 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 ncreased, while 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 ) . Using v a r i o u s chemically modified forms of bovine serum albumin, Schirrmacher and W i g z e l l (1972 and 1974) have demonstrated that thymus dependent lymphocytes (T c e l l s ) recognize a wider range of modified 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 s t r u c t u r a l requirements f o r r e c o g n i t i o n by these two c e l l types. S i m i l a r r e s u l t s were found by P a r i s h (1972) w i t h modified and heterologous e r y t h r o c y t e s . Two very s i m i l a r p r o t e i n s , egg white lysozyme and bovine <*-lactalbumin, were h i g h l y cross r e a c t i v e i n c e l l mediated immune responses w h i l e demonstrating a complete l a c k of humoral r e a c t i o n s (Maron, Webb, Teitelbaum and Arnon, 1972). The above f i n d i n g s have been taken to suggest that T c e l l s recognize amino a c i d sequence (unaltered by m o d i f i c a t i o n s ) while B c e l l s recognize c o n f i g u r a t i o n (changed by m o d i f i c a t i o n s ) . We have examined t h i s phenomena using the f e r r e d o x i n molecule which has been w e l l c h a r a c t e r i z e d . Previous work has shown s u b s t a n t i a l antibody cross 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 performic a c i d o x i d i z e d f e r r e d o x i n s ( N i t z , M i t c h e l l , Gerwing and CKristensen, 1969). The i n v i t r o thymidine uptake assay has been thought to be a c o r r e l a t e of delayed h y p e r s e n s i t i v i t y ( M i l l s , 1966; Oppenheim, Wolstencroft and G e l l , 1967; Meuvissen, van A l t e n and Good, 1969), ppresumably due to T c e l l t r a n s f o r m a t i o n . However, Mugraby, Gery and S u l i t z e a n u (1974) have evidence that B c e l l s are i n v o l v e d . i n the response- Since t h i s i n v i t r o method has been used i n t h i s study as an assay of cell-mediated immunity, the e f f e c t s of T and B c e l l d e p l e t i o n on the thymidine uptake response to antigens and mitogens was examined. The c e l l u l a r and humoral cross 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 test e d and the r e s u l t s are discussed i n r e l a t i o n to the f i n d i n g s i n the accompanying report (Gregerson et a l , 1976). 71 MATERIALS AND METHODS ANIMALS Young adult a l b i n o r a b b i t s were used to produce a l l the a n t i s e r a . Mice of the s t r a i n s DBA/2J and B6D2/J F l were used f o r the i n ' v i t r o s t i m u l a t i o n s . ANTIGENS Native f e r r e d o x i n (native-Fd) and performic 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 described previously, (Gregerson, e_t al_, 1976). A c i d 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 according to 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 prepared 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 bicarbonate plus 20% ethanol f o r 2 days i n the dark. The mixture was ex t r a c t e d s e v e r a l times w i t h ether to remove unreacted d i n i t r o f l u o r o -benzene and then d i a l y z e d . Methylated O-Fd (meth-O-Fd) was made as described by Fraenkel-Conrat and O l c o t t (1945) by suspending O-Fd i n anhydrous methanol made to 0.1 M w i t h concentrated h y d r o c h l o r i c a c i d . E s t e r i f i c a t i o n was allowed to proceed f o r 3 days i n the dark followed by d i a l y s i s . The pH of meth-O-Fd s o l u t i o n s was kept below 6 u n t i l use to avoid a l k a l i n e h y d r o l y s i s of the e s t e r s (Ram and Maurer, 1959). Native-Fd was reduced w i t h mercaptoethanol at pH 8 (1 ul/mg p r o t e i n ) and reacted w i t h N - e t h y l -maleimide (2 equ 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 ) at pH 6 f o r 4 hours followed 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. Native-Fd was S-carboxymethylated w i t h iodoacetate as described 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 to give carboxymethylated Fd (CM-Fd). The various f e r r e d o x i n s were analyzed and qu a n t i t a t e d on a Beckman 120 amino a c i d analyzer 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 are presented i n Table 1. IMMUNIZATIONS Rabbits were immunized w i t h O-Fd as described before (Gregerson et_ a l , 1976). Mice were immunized twice at 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 adjuvant. COMPLEMENT FIXATION TESTS These were done as described by Gerwing and Thompson (1968). LYMPHOCYTE STIMULATIONS In v i t r o m i c r o c u l t u r e s were performed as described by Gregerson, K e l l y and Levy (1975) except that mouse spleen c e l l s were used i n the present study. The non-immune c o n t r o l s f o r the DBA mice represent the pooled and averaged data from 12 separate experiments. Each experiment consisted of a minimum of nine determinations and u s u a l l y more than 1 mouse. CELL KILLING Rabbit anti-mouse immunoglobulin (RaMIg) was prepared by immunizing a rabbit three times w i t h 1 mg amounts of mouse immunoglobulin G (Miles Labs.) i n complete Freund's adjuvant at 2 week i n t e r v a l s . The serum was c o l l e c t e d s e v e r a l times, pooled and heat i n a c t i v a t e d . Rabbit a n t i - b r a i n 73 as s o c i a t e d 9 (RaBAO) was prepared as described by K e l l y , Kaye, Yoshizawa, Levy and K i l b u r n (1974). Guinea p i g serum was absorbed w i t h syngeneic mouse spleen c e l l s and used as a source of complement (C'). A l l sera and C' were used at a 1:4 d i l u t i o n i n phosphate bu f f e r e d s a l i n e (PBS). C e l l s were suspended i n the d i l u t e d sera and C' at a concentration of 10^ c e l l s / 0 . 1 ml and incubated f o r 1 hour at 37°. A f t e r 3 washes i n PBS the c e l l s were used. C e l l s incubated w i t h heat i n a c t i v a t e d , spleen c e l l absorbed normal r a b b i t serum (NRS) and C' served as c o n t r o l s . RESULTS PARAMETERS OF LYMPHOCYTE STIMULATION BY ANTIGEN IN VITRO Several experiments were done to determine the optimal 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 syn t h e s i s i n spleen c e l l s from O-Fd s e n s i t i z e d mice. The optimal dose of O-Fd was found to be 16 pg/ml. i n agreement w i t h e a r l i e r work done using guinea p i g c e l l s (Gregerson e_t a l , 1975). The r e s u l t s of the dose response experiments are presented i n Table 2. The k i n e t i c s of the response using O-Fd at 16 ug/ml are shown i n Table 3. A bi-modal response was found as seen p r e v i o u s l y using guinea 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 168 hours i n d i c a t e d the response was d e c l i n i n g at these times and experiments were not r o u t i n e l y extended beyond 120 hours. In a l l cases the r e s u l t s are compared to those obtained 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 student's t - t e s t . The r e l a t i v e l y 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 study are probably due to the high 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 thymidine uptake assay as used i n t h i s study 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 , pretreatment of the c e l l s w i t h RaBAO and C' should depress the response of O-Fd s e n s i t i z e d c e l l s to O-Fd. The e f f e c t of treatment w i t h RaMIg and C' (to k i l l immunoglobulin 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 . Controls were done using C' and normal r a b b i t serum (NRS). The r e s u l t s i n Table 4 summarize the e f f e c t s of the treatments on c e l l numbers. Since the C' and NRS were absorbed w i t h syngenei spleen 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 during treatment w i t h C' and NRS represent the l o s s due to the washing procedure and the death of v a r i o u s short l i v e d s p l e n i c leucocytes during the i n c u b a t i o n washing and counting periods.. I t has been shown that the responses of mouse spleen c e l l s to con-ca 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) are dependent on the presence 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 Sjbberg, 1972). These mitogen responses were t e s t e d w i t h each experiment and taken 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 treatments w i t h RaBA9, RaMIg and C'. I t can be seen i n Table 5 that the responses 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 . The e f f e c t s of these a n t i s e r a on the response to O-Fd are presented i n Table 6. Only those 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 untreated (NRS and C') c e l l s . The 24 hour response was found to be s e n s i t i v e to both the RaMIg and RaBAG i n d i c a t i n g that B c e l l s c o n t r i b u t e to the response, but are dependent on the presence of T c e l l s to 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 treatment, but i t was l a r g e l y reduced by the RaBAG sera. This r e s u l t suggests that 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 depressed compared to the normal c e l l s seen p r e v i o u s l y i n Table 2. The cause f o r the decrease i n t h i s response i s unknown. THE CROSS REACTIVITY OF O-Fd AND MODIFIED FERREDOXIN IMMUNIZED SPLEEN CELLS The a b i l i t y of O-Fd s e n s i t i z e d spleen c e l l s to respond to s e v e r a l chemically modified forms of f e r r e d o x i n was examined using the i n v i t r o lymphocyte s t i m u l a t i o n assay. The O-Fd immunized mice were prepared as before. The data from 5 experiments summarized i n Table 7 demonstrates that only NEM-Fd and native-Fd s t i m u l a t e d s i g n i f i c a n t responses i n c e l l s from O-Fd s e n s i t i z e d mice. These antigens d i d not e x h i b i t the bimodal response seen p r e v i o u s l y w i t h O-Fd i n Table 3, although the response to NEM-Fd was present at 24 hours. The immunogenicity of the va r i o u s f e r r e d o x i n s was examined by using 4 them to s e n s i t i z e mice i n the same manner as O-Fd was used. Spleen c e l l s from these mice were t e s t e d i n 4 experiments against s e v e r a l of the f e r r e d o x i n s i n v i t r o . Only NEM-Fd and native-Fd s e n s i t i z e d c e l l s gave a p r o l i f e r a t i v e response i n v i t r o . Native-Fd and NEM-Fd sti m u l a t e d DNA sy 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 native-Fd immunized c e l l s only responded to native-Fd as seen i n Table 8. Meth-Fd, TCA-Fd, CM-Fd and DNP-O-Fd d i d not e l i c i t responses from any of the modified f e r r e d o x i n immune c e l l s and d i d not s e n s i t i z e c e l l s f o r a response to any of the antigens. These r e s u l t s suggest that there may be a c o r r e l a t i o n between a molecule being able to stim u l a t e an i n v i t r o thymidine uptake response and to f u n c t i o n as an immunogen. This phenomena has been reported p r e v i o u s l y (Levy, H u l l , K e l l y , K i l b u r n and Teather, 1972). THE CROSS REACTIVITY OF ANTI-O-Fd ANTISERA Using the 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 anti-O-Fd antibodies was examined i n several tests with modified ferredoxins. Previous work.using the complement f i x a t i o n test demonstrated p o s i t i v e f i x a t i o n with O-Fd, native-Fd, TCA-Fd and iodoacetamide alkylated ferredoxin (Nitz, et d , 1969). The r e s u l t s shown i n Figure 1 indicate that DNP-O-Fd also f i x e s complement well while CM-Fd and NEM-Fd were only marginally a c t i v e i n t h i s assay. The a c t i v i t y of meth-O-Fd was quite low. DISCUSSION The i n v i t r o lymphocyte s t i m u l a t i o n of mouse spleen c e l l s by the antigen O-Fd, and the mitogens Con A and LPS, i n terms of c u l t u r e c o n d i t i o n s ( 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 responses, was found to be remarkably s i m i l a r to the responses of guinea p i g c e l l s as reported 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 the bimodal nature of the i n v i t r o response to O-Fd where two peaks of DNA s y n t h e s i s were observed when the 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 thymidine at 24 and 120 hours of 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 at 72 hours. Since the peak response to LPS was found when l a b e l i n g was done at 24 hours, and LPS i s considered to 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 that B c e l l s were c o n t r i b u t i n g to the 24 hour response, e i t h e r by DNA synth e s i s and/or by the production of a f a c t o r that could induce DNA syn t h e s i s i n T c e l l s . This problem was examined by p r e t r e a t i n g the c e l l s w i t h RaMIg or RaBAO and C' to k i l l c e l l s w i t h immunoglobulin (B c e l l s ) (Raff, 1970) or 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 observing the e f f e c t s these treatments had on the O-Fd, LPS and Con A responses. I t was found that c e l l s incubated w i t h RaMIg and C' l o s t the 24 hour s t i m u l a t i o n by O-Fd and LPS, but remained normally responsive to Con A. I f the c e l l s were preincubated w i t h RaBAO and C' the responses to O-Fd and Con A were l o s t ' w h i l e the i n d u c t i o n of DNA synthesis by LPS was s l i g h t l y stronger than i n untreated c e l l s . These r e s u l t s suggest t h a t : I . the a n t i s e r a were working p r o p e r l y as seen by the a n t i - I g s e n s i t i v i t y of the LPS s t i m u l a t i o n and the anti-BA0 s e n s i t i v i t y of the Con A response, i i . both T and B c e l l s are re 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 that 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 obviously.not enough to maintain r e a c t i v i t y ) i i i . e i t h e r or both c e l l types may c o n t r i b u t e to the DNA synthesized and detected by the assay, but both r e q u i r e the presence of the other c e l l type f o r i n d u c t i o n of DNA s y n t h e s i s , which may i n d i c a t e the need f o r a cooperative response. The r e d u c t i o n of•the 24 hour p r o l i f e r a t i v e response by NRS and C', as compared to untreated c e l l s , may be due to the l o s s of an adherent accessory c e l l , p o s s i b l y macrophages, sin c e the e x t r a i n c u b a t i o n and. washings do reduce the.numbers of adherent c e l l s . Macrophages are 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 systems (Seeger and Oppenheim, 1970; Waldron, Horn and Rosenthal, 1973). This problem could be resolved 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 study. The response to O-Fd at 120 hours was not 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 RaMIg or NRS 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' treatment suggesting that the 120 hour response i s , as c l a s s i c a l l y taken, dependent on the presence of T c e l l s . I t i s p o s s i b l e that the T c e l l response can induce DNA synthesis i n B c e l l s by the production of mitogenic f a c t o r s , but the s t i m u l a t i o n at 120 hours depends on the presence of adequate numbers of T c e l l s . The 72 hour response was too low to show any a f f e c t by the sera. Mugraby, et_ a l , (1974), using e r y t h r o c y t e s as the antigen have shown that both T and B c e l l s are r e q u i r e d f o r an i n v i t r o thymidine uptake response. 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 that 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 to the response i s a f a c t o r that should be considered. The experiments done to t e s t the cross 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 In v i t r o revealed that only three 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 antigens i n c u l t u r e . Only O-Fd was examined f o r i t s a b i l i t y to s t i m u l a t e antibody production. I t was found that of the v a r i o u s f e r r e d o x i n s , only O-Fd, NEM-Fd and native-Fd induced DNA s y n t h e s i s i n v i t r o i n O-Fd s e n s i t i z e d c e l l s . When tested f o r immunogenicity, 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 n a t i v e - F d , but not to the other f e r r e d o x i n s . Previous work w i t h a s y n t h e t i c analogue of O-Fd, N-10-C (which contains only the two t e r m i n a l determinants of O-Fd bridged by 10 g l y c i n e r e s i d u e s ) , has shown that a high degree of cross r e a c t i v i t y between N-10-C and O-Fd can be demonstrated i n the lymphocyte s t i m u l a t i o n assay, i n immediate and delayed s k i n r e a c t i o n s , i n the m i g r a t i o n i n h i b i t i o n t e s t and antibody production w i t h e i t h e r N-10-C or O-Fd 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 Teather, 1972; K e l l y , Levy and H u l l , 1973). In the present study, 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 determinants of O-Fd (meth-O-Fd and DNP-O-Fd) d i d not s t i m u l a t e DNA s y n t h e s i s . Taken together, these r e s u l t s i n d i c a t e that m o d i f i c a t i o n s which leave the two determinants of O-Fd unobstructed may 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 s e n s i t i z e d c e l l s . The 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 determinants (TCA-Fd and CM-Fd) was probably due to s t r u c t u r a l changes which made the determinants i n a c e s s i b l e to the c e l l s . 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 teste d w i t h the homologous antigen i n c u l t u r e i s unexplainable at t h i s time. I t i s i n t e r e s t i n g that NEM-Fd, which contains two hydrophobic N-ethylmalyl groups, functioned q u i t e w e l l as an immunogen and e l i c i t e d 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 O-Fd, but d i d not react w e l l w i t h anti-O-Fd sera. Somewhat s i m i l a r r e s u l t s were observed by P a r i s h (1971 a and b) using acetoacetylated f l a g e l l i n . He found that the a c e t o a c e t y l a t i o n (which increases hydrophobicity and reduces charge) of f l a g e l l i n produced a molecule which p r e f e r e n t i a l l y 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 but d i d not cross r e a c t w i t h a n t i b o d i e s to the n a t i v e molecule. The cross r e a c t i v i t y of the r a b b i t anti-O-Fd a n t i b o d i e s was greater than was observed w i t h the c e l l mediated response i n v i t r o . Only meth-O-Fd was v i r t u a l l y unable to f i x ' complement. Since t h i s m o d i f i c a t i o n a f f e c t s carboxyl groups, which are present i n both the determinants of O-Fd and known to be important i n the r e c o g n i t i o n of O-Fd, (Gregerson et a l , 1976 and unpublished 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 of r e a c t i v i t y . Strong cross 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 the lymphocyte s t i m u l a t i o n assay i n which they were not recognized. NEM-Fd, recognized w e l l i n c u l t u r e , f i x e d C' p o o r l y . A number of r e p o r t s have suggested that 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 than r e c o g n i t i o n by a n t i b o d i e s . This has not been seen i n t h i s study 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), the m i g r a t i o n i n h i b i t i o n assay demonstrated a high l e v e l of s p e c i f i c i t y . The nature and a c c e s s i b i l i t y of the r e c e p t o r s , whether c e l l surface bound or 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 that the r e s u l t s presented here and i n other s t u d i e s may simply r e f l e c t the s e n s i t i v i t i e s of the assays must be considered. Because of the d i f f i c u l t y of o b t a i n i n g anti-O-Fd sera i n mice, r a b b i t a n t i - s e r a was used. The e f f e c t of t h i s species d i f f e r e n c e i s u n c e r t a i n . Future 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 of 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 . J J J j E i X AMINO ACID ANALYSES OF THE FERREDOXIN PREPARATIONS Amino Acid T h e o r e t i c a l Native-Fd O-Fd Meth-O-Fd NEM-Fd TCA-Fd DNP-O-Fd CM-Fd asp 8 X 9.6 8.8 8.9 8.0 10.0 9.7 7.6 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 Y 3.6+ 2.6 0 0 giy 4 4.8 4.6 4.4 4.0 3.0 5.0 4.4 a l a 8 6.0 7.2 6.4 6.9 6.0 5.5"^ 6.6 v a l 6 5.9 6.2 5.0 5.8 5.7 5.2 4.6 i l e 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 cys tele acid 8.2 7.8 3.8 6.2 carboxymethyl 1-cy s + S-syccinyl cys C xi; molar r a t i o s 1.9 00 .produced by the oxidation of cysteine and cystine + "derivative of cysteine produced by a l k y l a t i o n with iodoacetate a d e r i v a t i v e of cysteine by a l k y l a t i o n with N-ethylmaleimide and subsequent acid hydrolysis ~^~low values ind i c a t e modification 3.4 oo N3 Immune Status Immune TABLE 2 DOSE RESPONSE OF O-Fd SENSITIZED AND NON-IMMUNE DBA SPLEEN CELLS TO O-Fd IN VITRO O-Fd ug/ml 4_ 8_ 16_ 32_ hours i n c u l t u r e ^  1.13 ± .03^ 1.20 ± .09 1.36 ± .12 1.08 ± .12 72 Non-Immune 0.96 ± 009 0.96 ± .08 0.77 ± .19 72 c .05* <.01 >.10 Immune 1.30 ± .06 1.34 ± .10 1.91 ± .10 11.98 ± .21 120 Non-Immune 1.21 ± .15 1.10 * .12 0.95 + .09 120 ?>.10 C.005 -C.01 r a t i o of stimulated cpm to unstimulated cpm and the standard e r r o r of the means ( s t i m u l a t i o n index ± SEM) a hours i n c u l t u r e p r i o r to the 18 hour l a b e l i n g t-feprobability c a l c u l a t e d from the student s t - t e s t that the response of the immune c e l l s i s d i f f e r e n t than that of the non immune c e l l s ** represents 6 experiments, each done w i t h a minimum of 2 mice and with 9 determinations per experiment oo TABLE 3 KINETICS OF THE IN VITRO RESPONSE OF DBA SPLEEN CELLS TO O-Fd AT 16 ug/ML Immune Status 24 Hours i n Culture 48 72 120 Immune Non-immune 1.60 ± .14 1.01 ± .04 < .005 1.12 ± .07 1.35 ± .03 0.96 ± .08 < .01 1.81 ± .18 1.10 ± .12 < .01 hours before a d d i t i o n of l a b e l * t - p r o b a b i l i t y - see Table 2 f o r e x p l a n a t i o n s s t i m u l a t i o n index ± SEM-H ** represents the data from 4 experiments and 9 determinations pereexperiment oo 85 TABLE 4 PERCENT LOSS OF DBA SPLEEN CELLS AFTER TREATMENT WITH ANTISERA AND C' C' + NRS RaMIg + C RaBAO + C' RaMIG + RaBAO + C' 29%"^~ 56% 55% 72% 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 detected by trypan blue e x c l u s i o n TABLE 5 EFFECT OF TREATMENT WITH RaBAG AND RaMIg PLUS C ON THE RESPONSES OF DBA SPLEEN CELLS TO CohA AND LPS  Hours 24 448 72 96 120 144 Hours 24 48 72 96 120 144 NRS and C 89,913 ± 9571* 114,568 ± 24,754 23,388 ± 3295 3243 ± 5308 -387 ± 1534^" 11276 ± 3283 NRS and C 57,995 ± 8443" 38,946 ± 10,665 13,929 ± 3218 -1667 ± 3374 511 ± 2318 -1783 ± 1608 ConA 2 ug/ml.  RaMIg and C' 89,225 ± 18,873 8388 ± 3723 5864 ± 2699 LPS 16 ug/ml  RaMIg and C' 8888 ± 1523 -630 ± 1283 ' 474 ± 1004 RaBAG and C' 4525 ± 2479 14,633 ± 9247 6890 ± 3549 RaBAG and C' 62,352 ± 8096 29,979 ± 2677 7647 ± 2801 ^ hours i n culture b e f o r e l l a b e l i n g *stimulated cpm minus unstimulated controls responses below the unstimulated background are expressed as negatives **represents the pooled r e s u l t s of 6 experiments, each done on a minimum of 4 mice with at lea s t 9 determinations 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' RaBAO and C' 24 1.36 ± . 0 8 ^ 1.08 ± .06 <.01* -1.07 ± .08 <.05* 72 0.99 ± .09 0.95 ± .08 >.25 0.96 ± .05 >.25 120 1.70 ± .17 1.70 ± .29 >.25 1.15 ± .11 <.01 * t - p r o b a b i l i t y that the response i s d i f f e r e n t than the 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 to l a b e l i n g ** represents the pooled r e s u l t s of 6 experiments, each done on a minimum of 4 mice w i t h at l e a s t 9 determinations per experiment s t i m u l a t i o n index ± SEM TABLE 7 RESPONSES OF O-Fd SENSITIZED AND UNSENSITIZED SPLEEN CELLS FROM DBA MICE TO MODIFIED FERREDOXIN ANTIGENS AT 16 ug/ml Hours 24 24 72 772 120 120 • NEM-Fd 23 1.79 ± .32 1.08 ± . <• .05 „ 2.19 ± .55 1.01 ± .27 <.05 2.99 ± .68 0.86 ± 1*17 <.025 Meth-O-Fd 1.00 ± .03 1.00 ± .05 0.74 ± .09 0.79 ± .12 0.97 ± .11 0.98 ± .166 CM-Fd 0.84 ± .08 0.83 ± .15 0.95 ± .26 0.72 ± .29 0.90 ± 1.03 ± .18 .12 TCA-Fd 1.18 ± .17 1.10 ± .08 0.99 ± .14 1.01 ± .07 1.16 ± .13 1.18 ± .18 Native-Fd 1.13 ± .13 1.12 ± .04 1.32 ± .47 0.72 ± .08 <.10 1.94 ± .31 1.04 ± .07 <.005 DNP-O-Fd 1.05 ± .04 0.93 ± .09 0.83 ± .26 0.63 ± .15 1.04 ±..28 0.73 ± .09 Immune Status immune non-immune immune non-immune immune non-immune hours i n c u l t u r e p r i o r to l a b e l i n g * t - p r o b a b i l i t i e s ~$~sstimulation index ± SEM ** represents the pooled r e s u l t s of 5 experiments and 9 determinations per experiment oo oo 89 TABLE 8 IN VITRO RESPONSES OF B6D2/J F l MICE IMMUNIZED TO MODIFIED FERREDOXINS Hours" NEM-Fd* O-Fd Native-Fd Immune status 24 1.26 ± .18 X 1.25 ± .25 1.11 ± .11 NEM-Fd immune 24 1.16 ± .23 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 ** represents the r e s u l t s from 4 experiments and nine determinations per experiment ^ t - p r o b a b i l i t y a hours i n culture p r i o r to 18 hour l a b e l i n g x stimulation index ± SEM * a l l ferredoxin antigens used i n culture at 16 ug/ml 90 F i g . 1. 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'Studies on the a n t i g e n i c i t y of b a c t e r i a l ferredoxins. I. The e f f e c t of modification of c y s t e i n y l residues on the a n t i g e n i c i t y of Clostridium 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. (1967). 'Delayed 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 conjugate and i t s r e l a t i o n s h i p to i n v i t r o t ransformation of lymph node, spleen, thymus and p e r i p h e r a l blood lymphocytes.' Immunol., 12, 89. 20. P a r i s h , C.R. (1971a). 'Immune response to chemically modified f l a g e l l i n . 1. Induction of antibody t o l e r a n c e to f l a g e l l i n by ac 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 . ' J . Exp. Med., 134, 1. 21. P a r i s h , C.R. (1971b). 'Immune response to chemic a l l y modified 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 cell-mediated immunity.' J . Exp. Med., 134, 21. 22. 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 cel l - m e d i a t e d immunity by chemically modified sheep e r y t h r o c y t e s . ' Eur. J . Immunol., 2, 143. 23. R a f f , M.C. (1970). 'Two d i s t i n c t populations of 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.' Immunol., 19, 637. 24. Ram, J.S. and Maurer, P.H. (1959). 'Modified bovine serum albumin. V I I I . E s t i m a t i o n and some physicochemical s t u d i e s of the methylated d e r i v a t i v e . ' Arch. Biochem. Biophys., 85, 512. 25. Schirrmacher, V. and W i g z e l l , H. (1972). 'Immune responses against n a t i v e and chemically modified albumins i n mice. I . A n a l y s i s of non-thymus processed (B) and thymus-processed (T) c e l l responses against methylated bovine serum albumin.' J . Exp. Med., 136, 1616. 26. Schirrmacher, V. and W i g z e l l , H. (1974). 'Immune responses against n a t i v e and chemically modified albumins i n mice. I I . E f f e c t of a l t e r a t i o n of e l e c t r i c charge and conformation on the humoral antibody response and on helper T c e l l responses.' J . Immunol., 113, 1635. 95 27. Seeger, R. and Oppenheim, J . J . (1970).. ' S y n e r g i s t i c i n t e r a c t i o n of macrophages and lymphocytes i n antigen-induced transformation i n lymphocytes.' J . Exp. Med., 132, 44. 28. Tanaka, M., 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 of C l o s t r i d i u m pasteurianum f e r r e d o x i n . ' Arch. Biochem., 105, 570. 29. Thompson, K., H a r r i s , M., Benjamini, E., M i t c h e l l , G. and Noble, M. (1972). ' C e l l u l a r and humoral immunity: a d i s t i n c t i o n i n a n t i g e n i c r e c o g n i t i o n . ' Nature (New B i o l . ) , 238, 20. 30. Waldron, J.A., Horn, R.G. and Rosenthal, A.S. (1973). 'Antigen-induced p r o l i f e r a t i o n of guinea p i g lymphocytes i n v i t r o : O b l i g a t o r y r o l e of macrophages i n the r e c o g n i t i o n of antigen by immune T-lympho-cytes.' J . Immunol., I l l , 58. CONCLUSION Examination of the r e s u l t s of the i n v i t r o lymphocyte s t i m u l a t i o n assay revealed that the responses of guinea p i g and mouse c e l l s to the mitogens Con A and LPS and the antigen O-Fd are very s i m i l a r i n terms of dose response and k i n e t i c s . The LPS responses of both species were s e n s i t i v e to treatment w i t h anti-immunoglobulin and complement although the s t i m u l a t i o n of mouse spleen c e l l s by LPS was reduced to a much greater degree than was the response of guinea 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 the s p e c i f i c response i n v i t r o to the antigen, O-Fd. The a b i l i t y of O-Fd to s t i m u l a t e a p r o l i f e r a t i v e response i n serum f r e e medium w i t h and without mercaptoethanol was teste d using guinea p i g lymph node c e l l s . Both of these media supported an e a r l y response which peaked at 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 of f o e t a l c a l f serum (FCS) to e i t h e r of these media r e s u l t e d i n a bimodal response to O-Fd w i t h peak s t i m u l a t i o n s occuring at 24 and 96 to 120 hours. O-Fd s e n s i t i z e d mouse spleen c e l l s a l s o gave a bimodal response to O-Fd i n medium w i t h FCS. Peak responses were found at 24 and 120 hours as seen w i t h the guinea p i g c e l l s . .By p r e t r e a t i n g the mouse spleen c e l l s w i t h anti-immunoglobulin 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 that the 24 hour response r e q u i r e d the presence of both T and B c e l l s i n l a r g e numbers w h i l e the 120 hour response r e q u i r e d only T c e l l s . These r e s u l t s corroborate the T c e l l dependency of the i n v i t r o lymphocyte s t i m u l a t i o n assay and the v a l i d i t y of 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 of the c e l l u l a r and humoral immune responses to s e v e r a l forms of f e r r e d o x i n were test e d using the lymphocyte s t i m u l a t i o n assay to measure c e l l mediated immunity and the complement f i x a t i o n test to detect antibody r e a c t i v i t y . Only one modified ferredoxin, the methyl e s t e r i f i e d form (meth^-O-Fd) was not recognized by the anti-O-Fd sera. This lack of recognition was probably a r e s u l t of meth-O-Fd being modified i n both of the,determinants present on O-Fd. Other ferredoxins were at least moderately recognized by the anti-O-Fd sera. Only ferredoxins not modified i n either determinant (O-Fd, native-Fd and NEM-Fd) were able to stimulate DNA synthesis i n v i t r o i n O-Fd s e n s i t i z e d mouse spleen c e l l s . These r e s u l t s indicate that i n the ferredoxin system, antibodies are more cross reactive than i s the c e l l u l a r immune response. This i s i n contrast to most published work i n other antigen systems and may r e f l e c t the antigenic nature of O-Fd which contains only two determinants, one on each terminus of the molecule. 8 It was of i n t e r e s t to note,that only ferredoxins which stimulated DNA synthesis i n v i t r o were immunogenic. The s p e c i f i c i t y of the anti-O-Fd response was also studied i n greater d e t a i l using the amino terminal determinant of O-Fd. These studies indicated that both antibodies and T c e l l s are highly s p e c i f i c , but d i f f e r i n t h e i r s p e c i f i c i t y requirements. There are several possible explanations of the r e s u l t s found using the peptide analogues of the amino determinant: either i . ) the receptors of T c e l l s and the antibodies 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 react to the same determinant, or i i . ) the T c e l l receptors and antibodies are s i m i l a r but are reacting to overlapping determinants, or i i i . ) both p o s s i b i l i t i e s occur. The more d e f i n i t i v e r e s u l t s obtainable using small monovalent peptide determinants to assay immune s p e c i f i c i t y indicate that s p e c i f i c i t y studies done by modification of an e n t i r e antigen.may be misleading, as such modifications greatly a f f e c t molecular conformation and may simply decrease the a c c e s s i b i l i t y of some determinants rather than e f f e c t i n g a change to the determinants themselves. The r e s u l t s of studies i n t h i s t h e s i s using modified ferredoxin antigens indicate that antibodies are more cross reactive than T c e l l s . However, t h i s i s not consistent with the r e s u l t s of the peptide studies which show that both responses are s p e c i f i c . This supports the contention that the indiscriminate modification of proteins may not provide useful antigens for s p e c i f i c i t y studies. The peptide studies presented here suggest that T c e l l s and antibodies 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 cross.reactive than the other, however, t h e i r manner of recognition may be d i f f e r e n t . 

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