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Characterization of a T lymphocyte-derived, antigen-binding molecule with suppressive activity Chu, Nelson Randall 1989

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CHARACTERIZATION OF A T LYMPHOCYTE-DERIVED ANTIGEN-BINDING MOLECULE WITH SUPPRESSIVE ACTIVITY By NELSON RANDALL CHU B.Sc, Queen's University, 1980 M.Sc, Queen's University, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH NOVEMBER 198 9 ® Nelson Randall Chu, COLUMBIA 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT R e g u l a t i o n o f t h e immune r e s p o n s e i s m e d i a t e d , i n p a r t , b y t h e a c t i o n o f s u p p r e s s o r T c e l l s ( T s ) . One i n t r i g u i n g a s p e c t o f t h e s e c e l l s i s t h e d e s c r i p t i o n o f T c e l l s u p p r e s s o r f a c t o r ( T s F ) : a s o l u b l e a n a l o g o f t h e c e l l t h a t s h a r e s many o f i t s p r o p e r t i e s , s u c h a s t h e a b i l i t y t o b i n d f r e e a n t i g e n (Ag) a n d s u p p r e s s an A g -s p e c i f i c immune r e s p o n s e . The e x a c t m o l e c u l a r n a t u r e o f T s F a n d t h e r e l a t i o n s h i p o f T s F t o Ts a r e unknown. The immune r e s p o n s e t o t h e s m a l l , b a c t e r i a l p r o t e i n , f e r r e d o x i n ( F d ) , was u s e d a s a m o d e l s y s t e m t o s t u d y T s F . A F d - s p e c i f i c s u p p r e s s o r c e l l n e t w o r k h a s b e e n d e s c r i b e d i n m i c e t h a t a r e g e n e t i c a l l y n o n r e s p o n s i v e t o t h i s Ag. P r e v i o u s l y , a s o l u b l e m e d i a t o r , known a s F d l l F , was f o u n d i n t h e c u l t u r e s u p e r n a t a n t (SN) o f t h e Ts h y b r i d o m a , F d l l . F d l l F p o s s e s s e d b o t h A g - b i n d i n g a c t i v i t y a n d t h e a b i l i t y t o s u p p r e s s t h e a n t i - F d Ab r e s p o n s e i n m i c e . The T s F - s p e c i f i c m o n o c l o n a l a n t i b o d y , B16G, was u s e d f o r b o t h t h e r e c o v e r y o f F d l l F - e n r i c h e d m a t e r i a l f r o m SN a n d i t s d e t e c t i o n b y t h e enzyme-l i n k e d i m m u n o s o r b e n t a s s a y . ' F u r t h e r i m m u n o c h e m i c a l , b i o l o g i c a l , a n d b i o c h e m i c a l c h a r a c t e r i z a t i o n o f F d l l F was done w i t h e m p h a s i s on d e s c r i b i n g t h e A g - b i n d i n g p r o p e r t i e s o f F d l l F . I t was f o u n d t h a t F d l l F b o u n d t o s o l i d - a n d l i q u i d - p h a s e F d , a n d d e m o n s t r a t e d p r e f e r e n t i a l b i n d i n g t o t h e c a r r i e r d e t e r m i n a n t o f t h e Ag. A s p l e e n c e l l c u l t u r e a s s a y was d e v i s e d w h i c h showed t h a t F d l l F s u p p r e s s e d Ab p r o d u c t i o n i n a c o n c e n t r a t i o n - d e p e n d e n t manner. A d d i t i o n a l e x p e r i m e n t s s u g g e s t e d t h a t t h e s u p p r e s s i v e e f f e c t was A g - s p e c i f i c . The i d e n t i f i c a t i o n o f t h e A g - b i n d i n g m o l e c u l e was a t t e m p t e d by t h e f r a c t i o n a t i o n o f F d l l F - e n r i c h e d m a t e r i a l u s i n g h i g h performance g e l f i l t r a t i o n o r p r e p a r a t i v e SDS-PAGE (run under n o n - r e d u c i n g c o n d i t i o n s ) . U s i n g SDS-PAGE, a u n i q u e , s i n g l e p o l y p e p t i d e o f about 30k r e l a t i v e m o l e c u l a r mass (M r) was i d e n t i f i e d as t h e A g - b i n d i n g m o i e t y o f F d l l F . The p o s s i b l e r e l a t i o n s h i p o f t h i s m o i e t y t o o t h e r i d e n t i f i e d m a t e r i a l s i s d i s c u s s e d . i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v L I S T OF TABLES v i i L I S T OF FIGURES v i i i ACKNOWLEDGEMENTS x ABBREVIATIONS x i INTRODUCTION S u p p r e s s i o n a n d t h e Immune R e s p o n s e 1 P r o p e r t i e s o f T c e l l S u p p r e s s o r F a c t o r s 2 Summary 11 The Immune R e s p o n s e t o F e r r e d o x i n as a M o d e l f o r t h e S t u d y o f M e c h a n i s m s o f I m m u n o r e g u l a t i o n 13 The c h o i c e o f F d a s a m o d e l a n t i g e n 14 E p i t o p e m a p p i n g a n d H-2 l i n k e d r e s p o n s i v e n e s s t o F d . . 15 Summary 18 I d i o t y p e - a n t i - i d i o t y p e i n t e r a c t i o n s 19 Summary 22 B16G a n d t h e D e v e l o p m e n t o f t h e F d l l h y b r i d o m a 23 Summary 27 P u r p o s e o f t h e I n v e s t i g a t i o n 28 MATERIALS AND METHODS B u f f e r s a n d S o l u t i o n s 29 E x p e r i m e n t a l A n i m a l s 2 9 C e l l L i n e s 29 C o n v e n t i o n a l a n d M o n o c l o n a l A n t i b o d i e s 30 A n t i g e n s 32 V E n z y m e - l i n k e d I m m u n o s o r b e n t A s s a y 33 I m m u n i z a t i o n o f A n i m a l s 35 E n r i c h m e n t o f F d l l F u s i n g I m m u n o a f f i n i t y C h r o m a t o g r a p h y . 35 I n v i t r o M e a s u r e m e n t o f F d l l F S u p p r e s s i v e A c t i v i t y . . . . 37 S u p p r e s s i o n o f t h e Serum A n t i b o d y R e s p o n s e u s i n g F d l l F . . 39 S o d i u m D o d e c y l S u l p h a t e P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 40 H i g h P e r f o r m a n c e G e l F i l t r a t i o n 41 P r e p a r a t i v e S o d i u m D o d e c y l S u l p h a t e P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 42 T w o - d i m e n s i o n a l G e l E l e c t r o p h o r e s i s ( I E F x SDS-PAGE) . . . 43 RESULTS R e c o v e r y o f F d l l F f r o m C u l t u r e S u p e r n a t a n t s 45 I m m u n o c h e m i c a l P r o p e r t i e s o f F d l l F 45 B i o l o g i c a l P r o p e r t i e s o f F d l l F 57 D e v e l o p m e n t o f an a n t i b o d y c u l t u r e a s s a y f o r t h e measu r e m e n t o f s u p p r e s s i o n 57 C o r r e l a t i o n b e t w e e n b i o a c t i v i t y a n d a n t i g e n - b i n d i n g a c t i v i t y o f F d l l F 61 S u p p r e s s i o n o f a n t i - F d a n t i b o d y l e v e l s i n v i v o . . . . 69 B i o c h e m i c a l A n a l y s i s o f F d l l F 74 I n t r o d u c t i o n 74 A n a l y s i s o f F d l l F u s i n g h i g h p e r f o r m a n c e g e l f i l t r a t i o n : c o r r e l a t i o n b e t w e e n a n t i g e n - b i n d i n g a n d s u p p r e s s i v e a c t i v i t y 75 I d e n t i f i c a t i o n o f t h e a n t i g e n - b i n d i n g m o l e c u l e u s i n g SDS-PAGE 81 DISCUSSION B16G a s a P r o b e f o r T S u p p r e s s o r F a c t o r 91 I m m u n o c h e m i c a l P r o p e r t i e s o f F d l l F . 93 B i o l o g i c P r o p e r t i e s o f F d l l F 96 v i Biochemical Analysis of F d l l F 101 Summary 106 LITERATURE CITED 108 APPENDIX THE COMPOSITIONS OF BUFFERS AND SOLUTIONS . . . 117 v i i L I S T OF TABLES I . The b i n d i n g o f F d l l F t o F d 52 I I . C o m p a r i s o n o f t h e s u p p r e s s i v e c a p a b i l i t y o f F d l l F a n d A10F i n two a s s a y s o f c e l l u l a r f u n c t i o n . 68 I I I . A b i l i t y o f v a r i o u s f r a c t i o n s e l u t i n g f r o m F P L C S u p e r o s e 12 c o l u m n t o i n h i b i t t h e i n v i t r o Ab r e s p o n s e o f Fd-immune s p l e e n c e l l s 82 v i i i L I S T OF FIGURES 1. B i n d i n g o f B16G t o F d l l F a d s o r b e d t o p o l y s t y r e n e w e l l s 46 2. B i n d i n g o f F d l l F t o F d a d s o r b e d t o p o l y s t y r e n e w e l l s a n d i t s d e t e c t i o n w i t h B16G 49 3. The r e a c t i v i t y o f B16G a n d t h e i r r e l e v a n t mAb, CAMAL-1, w i t h F d l l F 50 4. The b i n d i n g o f s o l u b l e F d t o F d l l F i n f l u i d - p h a s e . . 53 5. A n t i g e n i c a n a l y s i s o f F d a n d i t s a n a l o g s u s i n g d e t e r m i n a n t -s p e c i f i c mAb 54 6. I n h i b i t i o n o f F d l l F b i n d i n g b y F d a n d i t s a n a l o g s . . 56 7. D e t e c t i o n o f a n t i - F d Ab i n t h e SN o f s p l e e n c e l l c u l t u r e s p r e p a r e d f r o m B10.D2 m i c e t h a t were i m m u n i z e d w i t h C - m o l e c u l e a n d F d t w i c e 58 8. R e l a t i o n s h i p b e t w e e n A b s o r b a n c e a t 405 nm v s . c o n c e n t r a t i o n o f a n t i - F d Ab 60 9A. S p e c i f i c i t y o f a n t i - F d Ab f r o m Fd-immune s p l e e n c e l l c u l t u r e s f o r F d a n a l o g s ' 63 9B. S p e c i f i c i t y o f se r u m a n t i - F d Ab f r o m Fd-immune B10.D2 m i c e f o r F d a n a l o g s 63 10. The r e l a t i o n s h i p o f s u p p r e s s i v e a c t i v i t y o f F d l l F t o i t s A g -b i n d i n g a c t i v i t y 64 11. S p e c i f i c i t y o f t h e s u p p r e s s i v e a c t i v i t y o f F d l l F . . 67 12. S u p p r e s s i v e a c t i v i t y o f a f f i n i t y - e n r i c h e d F d l l F c o m p a r e d t o BW5147 c o n t r o l m a t e r i a l u s i n g t h e " r e v i s e d " p r o t o c o l . . . 70 13. X-Y p l o t s h o w i n g a n t i - F d Ab l e v e l s o f a n i m a l s b e f o r e arid a f t e r t r e a t m e n t w i t h F d l l F a n d Ag 72 14. X-Y p l o t s h o w i n g a n t i - K L H Ab l e v e l s o f a n i m a l s b e f o r e a n d a f t e r t r e a t m e n t w i t h F d l l F a n d Ag 73 15. SDS-PAGE o f e l u a t e s f r o m B16G a f f i n i t y c o l u m n s . . . 76 16. P r o t e i n p r o f i l e s (280 nm) o f B16G e l u a t e s d e r i v e d f r o m medium ( A ) , BW5147 ( B ) , a n d F d l l F (C) SN f r a c t i o n a t e d on a S u p e r o s e 12 c o l u m n u s i n g F PLC 78 17. A g - b i n d i n g a c t i v i t y o f FPLC f r a c t i o n s o b t a i n e d f r o m F d l l F IX (•), BW5147 (a), and medium (O) e l u t i o n p r o f i l e s o f F i g . 16 79 18. SDS-PAGE a n a l y s i s of FPLC f r a c t i o n s (shown i n F i g . 17) 16 t o 19 i n c l u s i v e 80 19. Ag-binding a c t i v i t y of DC10 and F d l l F m a t e r i a l e l u t e d from s l i c e s of a 12% p r e p a r a t i v e SDS-PAGE g e l run under non-reducing c o n d i t i o n s 84 20. PAGE of m a t e r i a l from s e l e c t e d f r a c t i o n s o f F i g . 19 us i n g a 10% SDS g e l run under r e d u c i n g c o n d i t i o n s 85 21. Two-dimensional g e l e l e c t r o p h o r e s i s of the 80k M r p r o t e i n s of medium and F d l l F 89 X ACKNOWLEDGEMENT S I w o u l d l i k e t o t h a n k A n t h e a T e n c h Stammers, f o r b o t h p e r f o r m i n g t h e e x p e r i m e n t s t h a t c o m p l e m e n t e d t h o s e p r e s e n t e d h e r e a n d h e r h e l p f u l comments a n d s u g g e s t i o n s . S p e c i a l t h a n k s s h o u l d a l s o go t o A g n e s Chan a n d J o a n S h e l l a r d f o r t h e i r d i s c u s s i o n s a n d s u g g e s t i o n s a b o u t some o f t h e t e c h n i c a l a s p e c t s o f t h e work. I w o u l d a l s o l i k e t o a c k n o w l e d g e J a n N o r t h , M a rk C u r r y , a n d M a r y M a c D o n a l d f o r t h e i r u s e f u l d i s c u s s i o n s a b o u t t h e p r o j e c t . I h a v e b e e n f o r t u n a t e t o r e c e i v e enormous s u p p o r t f r o m b o t h my f a m i l i e s d u r i n g t h i s t i m e . The k n o w l e d g e , e n c o u r a g e m e n t a n d g u i d a n c e r e c e i v e d f r o m my s u p e r v i s o r , J u l i a L e v y w i l l a l w a y s be remembered. L a s t l y , I t h a n k my w i f e , E s t h e r , f o r h e r u n d e r s t a n d i n g a n d b e l i e f i n me. x i ABBREVIATIONS Ag a n t i g e n Ab a n t i b o d y BSA b o v i n e s e r u m a l b u m i n CS c o n t a c t s e n s i t i v i t y CTL c y t o t o x i c T c e l l DNP 2 , 4 - d i n i t r o p h e n y l E L I S A e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y FACS f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r FCS f e t a l c a l f s e r u m F P L C h i g h p e r f o r m a n c e g e l f i l t r a t i o n F d f e r r e d o x i n GAT 1 - g l u t a m i c a c i d ^ - l - a l a n i n e ^ - l - t y r o s i n e HPLC h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y i . v . i n t r a v e n o u s * . . . I E F i s o e l e c t r i c f o c u s i n g KLH k e y h o l e l i m p e t h e m o c y a n i n MHC m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x mAb m o n o c l o n a l a n t i b o d y M r r e l a t i v e m o l e c u l a r mass NP 4 - h y d r o x y - 3 - n i t r o p h e n y l a c e t y l OFd p e r o x y f o r m i c a c i d - o x i d i z e d f e r r e d o x i n PBS p h o s p h a t e b u f f e r e d s a l i n e RaMIg-AP a c o n j u g a t e o f r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n a n d a l k a l i n e p h o s p h a t a s e x i i s . c . s u b c u t a n e o u s SDS-PAGE s o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s SN s u p e r n a t a n t TCR T c e l l r e c e p t o r Th T h e l p e r c e l l Ts T s u p p r e s s o r c e l l T s F T c e l l s u p p r e s s o r f a c t o r M o n o c l o n a l a n t i b o d y B16G CAMAL-1 16a 6-90 S p e c i f i c i t y i n v a r i a n t r e g i o n o f T c e l l s u p p r e s s o r f a c t o r l e u k e m i a - a s s o c i a t e d a n t i g e n c a r b o x y l - t e r m i n a l d e t e r m i n a n t o f f e r r e d o x i n a m i n o - t e r m i n a l d e t e r m i n a n t o f f e r r e d o x i n A b b r e v i a t i o n s m a r k e d w i t h an a s t e r i s k w e r e c o n s i d e r e d as s t a n d a r d f o r m s a n d w e r e n o t d e f i n e d i n t h e t e x t . The f o r m s o f a b b r e v i a t i o n s were t a k e n f r o m t h e s t a n d a r d l i s t p u b l i s h e d b y t h e J o u r n a l o f Immunology ( J . Immunol. 1988. 140 ( 4 ) . ) 1 INTRODUCTION S u p p r e s s i o n a n d t h e Immune R e s p o n s e The manner i n w h i c h t h e immune s y s t e m r e g u l a t e s i t s e l f f o l l o w i n g s t i m u l a t i o n b y a n t i g e n (Ag) i s n o t w e l l u n d e r s t o o d . I t i s known t h a t t h e r e s p o n s e i s m o d u l a t e d b y b o t h T h e l p e r c e l l s (Th) a n d T s u p p r e s s o r c e l l s ( T s ) . The s t u d y o f Th h a s b e e n a i d e d b y t h e e l u c i d a t i o n o f t h e s t r u c t u r e o f i t s c l o n a l l y d i s t r i b u t e d r e c e p t o r , t h e a/R T c e l l r e c e p t o r (TCR), a n d t h e a b i l i t y t o c l o n e t h e s e c e l l s d i r e c t l y . I n c o n t r a s t , t h e s t u d y o f s u p p r e s s i o n h a s b e e n h a m p e r e d b y t h e r e l a t i v e d i f f i c u l t y i n c l o n i n g T c e l l s w i t h s u p p r e s s i v e c a p a b i l i t y . F u r t h e r m o r e t h e i n a b i l i t y t o i d e n t i f y £> c h a i n r e a r r a n g e m e n t s f r o m Ts h y b r i d o m a s o r c e l l l i n e s h a s l e d t o t h e s u g g e s t i o n t h a t s u p p r e s s o r c e l l s may p o s s e s s an a l t e r n a t e t y p e o f TCR ( H e d r i c k e t a l . 1 9 8 5 ) . I n s p i t e o f t h i s , p r o g r e s s h a s b e e n made i n t h e c h a r a c t e r i z a t i o n o f t h e Ts s u b s e t . One i n t r i g u i n g a s p e c t t h a t h a s r e c e i v e d much a t t e n t i o n , i s t h e d e s c r i p t i o n o f A g - s p e c i f i c T c e l l s u p p r e s s o r f a c t o r s ( T s F ) : s o l u b l e a n a l o g s o f t h e Ts t h a t a p p e a r t o s h a r e many o f i t s p r o p e r t i e s , s u c h a s Ag s p e c i f i c i t y a n d t h e c a p a c i t y t o s u p p r e s s t h e immune r e s p o n s e . T s F h a v e b e e n d e s c r i b e d a t b o t h t h e f u n c t i o n a l a n d b i o c h e m i c a l l e v e l s ; h o w e v e r , a c o n s e n s u s h a s n o t b e e n r e a c h e d c o n c e r n i n g t h e i r p r e c i s e m o l e c u l a r n a t u r e a n d a c t u a l r e l a t i o n s h i p t o t h e T s . 2 P r o p e r t i e s o f T c e l l S u p p r e s s o r F a c t o r s The e x i s t e n c e o f s o l u b l e T - c e l l d e r i v e d m a t e r i a l c a p a b l e o f b o t h A g - b i n d i n g a n d s u p p r e s s i v e a c t i v i t y i s s u b s t a n t i a l l y d o c u m e n t e d . I n i t i a l l y , T s F was p r e p a r e d a s c r u d e t h y m u s o r s p l e e n e x t r a c t s o b t a i n e d f r o m m i c e t h a t w e re p r i m e d w i t h c a r r i e r A g w i t h o u t a d j u v a n t . T h i s m e t h o d o f i m m u n i z a t i o n f a v o r e d t h e i n d u c t i o n o f T s . F o r e x a m p l e , T a k e m o r i a n d T a d a (1975) showed t h a t s u p p r e s s i o n o f t h e a n t i - h a p t e n a n t i b o d y (Ab) r e s p o n s e was p o s s i b l e when m i c e were i m m u n i z e d w i t h t h e h a p t e n - c a r r i e r c o n j u g a t e ( u s i n g a d j u v a n t ) a n d g i v e n t h e s p l e e n e x t r a c t a t t h e same t i m e . T h i s d e m o n s t r a t e d t h a t t h e e x t r a c t c o n t a i n e d s u p p r e s s i v e m a t e r i a l t h a t was d i r e c t e d t o t h e c a r r i e r , i n t h i s c a s e , k e y h o l e l i m p e t h e m o c y a n i n (KLH) a n d n o t t h e h a p t e n ( 2 , 4 -d i n i t r o p h e n y l , DNP) p o r t i o n o f t h e immunogen. F u r t h e r m o r e , t h e r e s p o n s e was c a r r i e r - s p e c i f i c s i n c e an e x t r a c t p r e p a r e d f r o m KLH-p r i m e d m i c e w o u l d n o t s u p p r e s s t h e a n t i - D N P Ab r e s p o n s e o f m i c e made immune t o DNP c o n j u g a t e d t o a d i f f e r e n t c a r r i e r , b o v i n e gamma g l o b u l i n (BGG). O n l y an e x t r a c t o b t a i n e d f r o m B G G - p r i m e d m i c e w o u l d now e f f e c t t h e s u p p r e s s i o n o f t h e a n t i - D N P Ab r e s p o n s e . The s u p p r e s s i v e a c t i v i t y o f K L H - s p e c i f i c T s F was a l s o g e n e t i c a l l y r e s t r i c t e d : i e . g e n e t i c i d e n t i t y b e t w e e n t h e T s F d o n o r a n d t h e T s F r e c i p i e n t was r e q u i r e d , a t a c e r t a i n gene l o c u s , known as I - J ( s e e b e l o w ) , f o r s u p p r e s s i o n t o o c c u r . I m m u n o c h e m i c a l a n a l y s i s i n d i c a t e d t h a t t h e T s F c o u l d be b o u n d by A g a n d b y an a n t i s e r u m r a i s e d t o t h e M a j o r H i s t o c o m p a t i b i l i t y 3 Complex (MHC) o f t h e TsF donor. L a t e r work showed t h a t A g - s p e c i f i c TsF e x p r e s s e d d e t e r m i n a n t s t h a t were encoded by t h e I - J s u b r e g i o n , a g e n e t i c l o c u s t h a t mapped s u p p o s e d l y t o w i t h i n t h e I r e g i o n o f t h e murine MHC (Murphy e t a l . 1976; Tada, T a n i g u c h i , and D a v i d 1976). The MHC o r H-2 complex, i s l o c a t e d on chromosome 17 o f t h e mouse. The gene p r o d u c t s from t h e I s u b r e g i o n s , I-A and I-E, p l a y a fundamental r o l e i n g o v e r n i n g immune r e s p o n s i v e n e s s because Ag i s r e c o g n i z e d by t h e Th o n l y i n a s s o c i a t i o n w i t h t h e s e c e l l - s u r f a c e p r o t e i n s , a l s o known as c l a s s I I a n t i g e n s . P a r a d o x i c a l l y , m o l e c u l a r g e n e t i c a n a l y s i s had demonstrated t h a t I - J encoded gene p r o d u c t s c o u l d not have a c t u a l l y o r i g i n a t e d from w i t h i n t h e I r e g i o n (Kronenberg e t a l . 1983). At p r e s e n t , t h e o r i g i n o f t h e I - J p r o d u c t ( s ) remains unknown but I - J d e t e r m i n a n t s , as r e c o g n i z e d by a n t i s e r a o r monoclonal a n t i b o d i e s (mAb), a r e s t i l l u n d e r s t o o d as markers f o r Ts and TsF (Murphy 1987; D o r f and B e n a c e r r a f 1985) . The H-2 l i n k e d nonresponder s t a t u s o f c e r t a i n murine s t r a i n s t o s p e c i f i c Ag has f r e q u e n t l y been a s s o c i a t e d w i t h t h e p r o d u c t i o n o f b o t h Ts and TsF. F o r example, H-2^ a n i m a l s do not mount an Ab 6 0 r e s p o n s e t o t h e s y n t h e t i c t e r p o l y m e r , 1 - g l u t a m i c a c i d - 1 -a l a n i n e ^ ^ - l - t y r o s i n e ' ' " ^ (GAT) . T h i s n o n r e s p o n s i v e n e s s was found t o be m e d i a t e d by Ts (Kapp e t a l . 1974) . However, H-2^ mice became r e s p o n s i v e t o GAT when t h e y were immunized w i t h GAT t h a t was c o v a l e n t l y l i n k e d t o m e t h y l a t e d BSA (GAT-MBSA). Kapp e t a l . (1976) d e m o n s t r a t e d t h a t thymus and s p l e e n e x t r a c t s from GAT-4 p r i m e d m i c e s u p p r e s s e d t h e Ab r e s p o n s e o f GAT-MBSA-immunized m i c e i f t h e e x t r a c t was a d m i n i s t e r e d a t t h e same t i m e a s A g . I n v i t r o s u p p r e s s i o n o f a n t i - G A T Ab p r o d u c t i o n b y T s F was shown b y t h e a d d i t i o n o f e x t r a c t t o a p r i m a r y c u l t u r e o f s p l e e n c e l l s t h a t was s t i m u l a t e d w i t h GAT-MBSA. S p e c i f i c i t y o f s u p p r e s s i o n was shown by t h e i n a b i l i t y o f GAT-TsF t o s u p p r e s s t h e i n v i t r o r e s p o n s e t o a n i r r e l e v a n t A g . L i k e KLH-TsF, G A T - s p e c i f i c T s F e x p r e s s e d I - J e n c o d e d d e t e r m i n a n t s a n d c o u l d be b o u n d b y i m m o b i l i z e d A g . O t h e r s t u d i e s h a v e shown t h a t T c e l l - m e d i a t e d s u p p r e s s i o n was n o t a c h i e v e d b y j u s t one t y p e o f T s . I n g e n e r a l , s u p p r e s s i o n was a c c o m p l i s h e d t h r o u g h s e v e r a l p h e n o t y p i c a l l y d i f f e r e n t T c e l l s u b s e t s , e a c h p r o d u c i n g i t s own t y p e o f T s F o r m e d i a t o r . C a n t o r a n d G e r s h o n (1979) p r o p o s e d t h a t T c e l l s u b s e t s c o u l d be d i s t i n g u i s h e d b y b o t h t h e e x p r e s s i o n o f c e l l s u r f a c e g l y c o p r o t e i n s ( c a l l e d L y m a r k e r s ) a n d t h e i r f u n c t i o n a l p r o p e r t i e s . They d e s c r i b e d L y l + 2 ~ , I - J + i n d u c e r c e l l s , t h a t i n d u c e d r e s t i n g nonimmune T c e l l s t o e x e r t s u p p r e s s i v e a c t i v i t y , L y l ~ 2 + e f f e c t o r c y t o t o x i c / s u p p r e s s o r c e l l s , t h a t c o u l d be f u r t h e r c l a s s i f i e d a c c o r d i n g t o t h e i r I - J d e t e r m i n a n t e x p r e s s i o n ( c y t o t o x i c c e l l s b e i n g I - J , s u p p r e s s o r c e l l s b e i n g I - J + ) a n d L y l , 2 + r e g u l a t o r y c e l l s , t h a t were t h e t a r g e t s o f t h e i n d u c e r c e l l s . Y a m a u c h i e t a l . (1981a,b) r e p o r t e d t h a t L y l + 2 ~ c e l l s s e c r e t e d an A g - s p e c i f i c T s F t h a t was t a r g e t e d f o r L y 2 + c e l l s . L y l - 2 + c e l l s , i n t u r n , p r o d u c e d a s e c o n d f a c t o r ( e f f e c t o r TsF) t h a t c a u s e d s u p p r e s s i o n . T a n i g u c h i a n d T o k u h i s a (1979) a l s o r e p o r t e d t h a t a t l e a s t one o t h e r t y p e o f T c e l l was r e q u i r e d , i n 5 a d d i t i o n t o t h e one p r o d u c i n g A g - s p e c i f i c T s F , t o c a u s e s u p p r e s s i o n o f t h e a n t i - h a p t e n Ab r e s p o n s e . T h i s o t h e r c e l l ( e f f e c t o r Ts) was t h e t a r g e t o f t h e A g - s p e c i f i c T s F a n d r e q u i r e d t h a t t h e c e l l s be p r i m e d w i t h Ag. However, o n c e t h e s e e f f e c t o r Ts w e r e c o m b i n e d w i t h T s F , i n t h e p r e s e n c e o f Ag, t h e y w e r e c a p a b l e o f s u p p r e s s i n g t h e Ab r e s p o n s e t o o t h e r Ag a s w e l l . A c c o r d i n g t o Hausman, S h e r r , a n d D o r f (1986) f o r t h e 4-h y d r o x y - 3 - n i t r o p h e n y l a c e t y l (NP) h a p t e n s y s t e m , s u p p r e s s i o n was a c h i e v e d t h r o u g h a c a s c a d e o f a t l e a s t t h r e e p h e n o t y p i c a l l y d i f f e r e n t T c e l l s u b s e t s t h a t e a c h s e c r e t e d i t s own t y p e o f T s F . T h u s , i m m u n i z a t i o n o f m i c e w i t h N P - c o u p l e d s p l e e n c e l l s i n d u c e d a p r i m a r y l e v e l o f Ts known as f i r s t o r d e r Ts (Ts-^) . T h e s e c e l l s s e c r e t e d T s F ^ t h a t b o u n d NP, e x p r e s s e d I - J d e t e r m i n a n t s a n d a l s o e x p r e s s e d i d i o t y p i c d e t e r m i n a n t s a s s o c i a t e d w i t h t h e N P - b i n d i n g s t r u c t u r e o f t h e T s F . J u s t as Ab c a n p o s s e s s a n t i g e n i c d e t e r m i n a n t s l o c a t e d w i t h i n t h e A g - c o m b i n i n g s i t e o f t h e Ab m o l e c u l e ( i d i o t y p e ) , T c e l l r e c e p t o r s t r u c t u r e s c a n h a v e i d i o t y p i c d e t e r m i n a n t s . TsF-^ a c t i v i t y was d e m o n s t r a t e d b y i t s a b i l i t y t o s u p p r e s s t h e c o n t a c t s e n s i t i v i t y (CS) r e s p o n s e t o NP i n m i c e t h a t w e re e i t h e r s y n g e n e i c o r a l l o g e n e i c w i t h t h e T s F d o n o r . TsF-^ w o u l d o n l y s u p p r e s s i f a d m i n i s t e r e d d u r i n g t h e i n i t i a l p r i m i n g o f t h e a n i m a l w i t h A g . T s F ^ a l s o i n d u c e d s e c o n d l e v e l o r s e c o n d o r d e r Ts (TS2) i n a n i m a l s . I n t h i s way, TsF-^ i s t h e i n d u c e r T s F d e s c r i b e d a b o v e . T h i s d i d n o t r e q u i r e t h e p r e s e n c e o f e x o g e n o u s Ag. TS2 s e c r e t e d TSF2 t h a t d i f f e r e d f r o m T s F ^ i n s e v e r a l a s p e c t s : TSF2 was a n t i - i d i o t y p i c a n d b o u n d t o 6 anti-NP Ab i e . the i d i o t y p i c determinants of the Ab. T s F 2 expressed I-J encoded determinants and could suppress the CS response of previously primed animals. Furthermore, i t was gen e t i c a l l y r e s t r i c t e d for suppressive a c t i v i t y . Lastly, t h i r d order Ts (TS3), were primed at the same time as Th during Ag exposure, but were only activated by TsF 2- T s F 3 bound to NP, expressed I-J encoded and i d i o t y p i c determinants of the NP-binding moiety. The f i n a l target of the TsF^ was the T c e l l responsible for mediating CS. Once NP-specific TsF^ interacted with NP, the suppression of other CS responses i n the immediate v i c i n i t y occurred (bystander suppression). It i s not known i f actual physical contact between the c e l l types i s required for the expression of suppressive a c t i v i t y . However the i d i o t y p i c determinants of Ts^ and Ts-^  and the a n t i - i d i o t y p i c s p e c i f i c i t y of Ts 2 (TSF2) could be viewed as a possible way of bridging a l l three suppressor components together. Other suppressor pathways have been proposed and are reviewed by Tada (1984). A more det a i l e d description of the functional properties and molecular composition of TsF became possible with the development of T c e l l hybridomas and clones. Factor-secreting T c e l l hybridomas and clones served as homogeneous and continuous sources for the biochemical study of TsF. It has been estimated that TsF i n |ig quantities can be recovered from the hybridoma supernatant (SN) instead of i n ng quantities from mice (Krupen et a l . 1982). Ts hybridomas were created v i a the fusion of the BW5147 7 thymoma w i t h s p l e e n c e l l s e n r i c h e d f o r Ts a c t i v i t y a n d ( o r ) t h e e x p r e s s i o n o f Ts m a r k e r s ( e g . T a n i g u c h i , S a i t o , a n d Tada 1979; Kapp, A r a n e o , a n d C l e v i n g e r 1 9 8 0 ) . Ts c l o n e s w e re d e r i v e d f r o m s p l e e n c e l l s e n r i c h e d f o r Ts a c t i v i t y a n d ( o r ) t h e e x p r e s s i o n o f Ts m a r k e r s , a n d t h e n p r o p a g a t e d u s i n g i n t e r l e u k i n - 2 , ( I L - 2 ) ( e g . F r e s n o e t a l . 1 9 8 1 ; N a k a u c h i e t a l . 1 9 8 4 ) . T s F , s p e c i f i c f o r KLH, p r o d u c e d f r o m a Ts h y b r i d o m a t h a t e x p r e s s e d I - J e n c o d e d d e t e r m i n a n t s a n d b o u n d t o A g - c o a t e d p l a t e s , d e m o n s t r a t e d t h e same p r o p e r t i e s a s t h e s u p p r e s s i v e m a t e r i a l e x t r a c t e d f r o m s p l e e n c e l l s , i e . , A g - b i n d i n g , g e n e t i c a l l y r e s t r i c t e d i n a c t i v i t y , a n d I - J + . G e l f i l t r a t i o n s howed t h a t TsF h a d a r e l a t i v e m o l e c u l a r mass (M r) o f 4 2 k - 6 8 k . A c o m p a r i s o n o f t h e T s F m a t e r i a l p r e p a r e d f r o m SN ( s e c r e t e d form) o r c e l l u l a r e x t r a c t s ( c y t o p l a s m i c form) d e m o n s t r a t e d t h a t t h i s p a r t i c u l a r T s F was c o m p o s e d o f two c h a i n s : an A g - b i n d i n g one a n d an I - J d e t e r m i n a n t - b e a r i n g one ( T a n i g u c h i , S a i t o , a n d T a d a , 1979; T a n i g u c h i , T a k e i , a n d Tada 1 9 8 0 ) . An e f f e c t o r T s F , so c a l l e d f o r i t s a b i l i t y t o a c t d i r e c t l y on L y l + Th t o s u p p r e s s t h e a n t i - K L H Ab r e s p o n s e , was p r e p a r e d f r o m a c l o n e d Ts l i n e ( K i t a m u r a e t a l . 1984; N a k a u c h i e t a l . 1 9 8 4 ) . T h i s K L H - s p e c i f i c T s F was p h e n o t y p i c a l l y d i f f e r e n t f r o m t h e p r e v i o u s l y m e n t i o n e d m o n o c l o n a l KLH-TsF i n t h a t i t d i d n o t d e m o n s t r a t e MHC r e s t r i c t i o n f o r i t s e x p r e s s i o n o f a c t i v i t y a n d i t d i d n o t e x p r e s s I - J c o d e d d e t e r m i n a n t s . U n l i k e t h e o t h e r e f f e c t o r T s F m e n t i o n e d ( T s F ^ , Hausman, S h e r r , a n d D o r f 1986) i t was A g - s p e c i f i c i n a c t i o n . I t was a l s o c l e a r l y shown t h a t t h e 8 s i m u l t a n e o u s p r e s e n c e o f Ag a n d T s F were r e q u i r e d f o r s u p p r e s s i o n t o o c c u r . A G A T - s p e c i f i c T s F f r o m a Ts h y b r i d o m a h a s a l s o b e e n r e p o r t e d (Kapp, A r a n e o , a n d C l e v i n g e r 1 9 8 0 ) . T h i s T s F d e m o n s t r a t e d t h e same p r o p e r t i e s a s t h e m a t e r i a l e x t r a c t e d f r o m s p l e e n c e l l s (Kapp e t a l . , 1 9 7 6 ) ; i e . , b i n d s Ag, e x p r e s s e s I - J e n c o d e d d e t e r m i n a n t s , a n d s u p p r e s s e s t h e p r i m a r y r e s p o n s e t o GAT-MBSA-primed c e l l s . I n a d d i t i o n , t h e T c e l l p r o l i f e r a t i v e r e s p o n s e o f m i c e p r i m e d w i t h GAT-MBSA was i n h i b i t e d when TsF was a d d e d t o c u l t u r e s c o n t a i n i n g GAT. The e f f e c t o r KLH-TsF o f K i t a m u r a e t a l . (1984) was a l s o a b l e t o s u p p r e s s t h e T c e l l p r o l i f e r a t i v e r e s p o n s e o f A g - p r i m e d c e l l s . I n t h i s c a s e , t h e t a r g e t 'of t h e T s F was shown d i r e c t l y t o be L y l c e l l s s i n c e i n c u b a t i o n o f A g - p r i m e d L y l c e l l s w i t h Ag a n d T s F r e s u l t e d i n t h e i n h i b i t i o n o f T c e l l p r o l i f e r a t i o n . Ts h y b r i d o m a s h a v e f a c i l i t a t e d t h e b i o c h e m i c a l s t u d y o f T s F . I n p r a c t i c a l l y a l l c a s e s , h y b r i d o m a s w e r e s e l e c t e d f o r t h e a b i l i t y t o c o n s t i t u t i v e l y s e c r e t e a c t i v e m a t e r i a l i n t o t h e SN. K r u p e n e t a l . (1982) p u r i f i e d t h e G A T - s p e c i f i c T s F o f Kapp, A r a n e o , a n d C l e v i n g e r (1980, s e e above) t o h o m o g e n e i t y . U s i n g a f f i n i t y c h r o m a t o g r a p h y , two r o u n d s o f r e v e r s e - p h a s e HPLC a n d i o n - e x c h a n g e HPLC, t h e y r e c o v e r e d 1.8 ug o f b i o a c t i v e m a t e r i a l f r o m 6 1 o f c r u d e SN ( c o n t a i n i n g 26 g o f p r o t e i n ) . When t h e p u r i f i e d m a t e r i a l was a n a l y z e d u s i n g SDS-PAGE u n d e r r e d u c i n g c o n d i t i o n s , one b a n d a t 24k M r was o b s e r v e d . A f i n a l p u r i f i c a t i o n f a c t o r , b a s e d on b i o a c t i v i t y a n d t o t a l p r o t e i n 7 c o n t e n t , was c a l c u l a t e d as 5.2 x 10 . 9 U s i n g t h e same G A T - s p e c i f i c Ts h y b r i d o m a , W i e d e r e t a l . (1982) i s o l a t e d mRNA f r o m i t a n d a c h i e v e d c e l l - f r e e t r a n s l a t i o n o f b i o a c t i v e GAT-TsF. The t r a n s l a t e d p r o d u c t s u p p r e s s e d b o t h GAT-s p e c i f i c T c e l l p r o l i f e r a t i o n a n d t h e a n t i - G A T Ab r e s p o n s e . I t e x p r e s s e d I - J d e t e r m i n a n t s , a n d when p u r i f i e d t o h o m o g e n e i t y u s i n g a f f i n i t y c h r o m a t o g r a p h y , r e v e r s e - p h a s e HPLC a n d SDS-PAGE, t h e T s F r e s o l v e d as a 19k M r p r o t e i n . They e x p l a i n e d t h e d i s c r e p a n c y i n M r w i t h t h e s e c r e t e d m a t e r i a l (24k M ) a s a r e s u l t o f a l a c k o f c a r b o h y d r a t e m o d i f i c a t i o n o f t h e f a c t o r , s i n c e t h e t r a n s l a t i n g s y s t e m u s e d ( r a b b i t r e t i c u l o c y t e ) d i d n o t p o s s e s s g l y c o s y l a t i n g a c t i v i t y . M o l e c u l a r c o m p o s i t i o n o f an e f f e c t o r T s F s p e c i f i c f o r GAT h a s a l s o b e e n d e t e r m i n e d ( T u r c k , Kapp, a n d Webb 1 9 8 6 ) . T h i s T s F d i f f e r e d i n m o l e c u l a r s t r u c t u r e f r o m t h e p r e v i o u s l y d e s c r i b e d GAT-TsF. E f f e c t o r GAT-TsF was a d i s u l p h i d e - l i n k e d h e t e r o d i m e r . When a n a l y z e d b y t w o - d i m e n s i o n a l n o n - r e d u c i n g / r e d u c i n g SDS-PAGE, one s p o t , l y i n g o f f t h e d i a g o n a l o f t h e g e l ( i n d i c a t i n g t h a t i t c o n t a i n e d a d i s u l p h i d e l i n k a g e ) , was r e c o v e r e d . When t h e m a t e r i a l was a p p l i e d t o a r e v e r s e - p h a s e HPLC c o l u m n , two p r o t e i n s w e r e r e s o l v e d . S u p p r e s s i v e a c t i v i t y was r e s t o r e d when t h e y were m i x e d t o g e t h e r a n d a d d e d t o c u l t u r e . F o l l o w i n g HPLC s e p a r a t i o n , one c h a i n was shown t o b i n d A g w h i l e t h e o t h e r c h a i n d i s p l a y e d I -J d e t e r m i n a n t s . U s i n g SDS-PAGE, b o t h c h a i n s w e r e r e s o l v e d a t a b o u t 30k M . L i k e t h e s i n g l e c h a i n GAT-TsF, b o t h c o m p o n e n t s o f e f f e c t o r T s F were g l y c o s y l a t e d . However, i n c o n t r a s t t o t h e s i n g l e c h a i n f a c t o r , b i o a c t i v i t y was l o s t f o l l o w i n g n e u r a m i n i d a s e 10 t r e a t m e n t . Work w i t h KLH-TsF h a s p r o g r e s s e d i n a s i m i l a r manner t o t h a t o f GAT-TsF. T a n i g u c h i e t a l . ( 1 9 8 2 ) , u s i n g a Ts h y b r i d o m a ( d e s c r i b e d b y T a n i g u c h i , T a k e i , a n d Tada 1979, s e e above) s p e c i f i c f o r KLH, o b t a i n e d s e v e r a l mRNA s p e c i e s t h a t , when t r a n s l a t e d b y t h e f r o g o o c y t e s y s t e m y i e l d e d an A g - b i n d i n g c h a i n a n d a c h a i n e x p r e s s i n g I - J d e t e r m i n a n t s . S u p p r e s s i v e a c t i v i t y was r e c o v e r e d when t h e two c h a i n s w e re c o m b i n e d a n d t e s t e d i n c u l t u r e . S a i t o a n d T a n i g u c h i (1984) b i o c h e m i c a l l y c h a r a c t e r i z e d t h e t w o - c h a i n K L H - s p e c i f i c T s F . The n o n - c o v a l e n t l y a s s o c i a t e d c h a i n s b o u n d A g a n d e x p r e s s e d I - J d e t e r m i n a n t s . The 45k M r c h a i n b o u n d t o b o t h KLH a n d t o a mAb s p e c i f i c f o r a c o n s t a n t r e g i o n o f T s F . The 27k M r c h a i n b o u n d t o b o t h p o l y c l o n a l a n d m o n o c l o n a l a n t i - I -J r e a g e n t s . S u p p r e s s i o n o f t h e a n t i - h a p t e n Ab r e s p o n s e was d e m o n s t r a t e d b y T s F r e c o v e r e d f r o m i m m u n o a d s o r b e n t s made w i t h t h e m o n o c l o n a l r e a g e n t s . A l t h o u g h e x t e n s i v e work h a s b e e n done w i t h t h e GAT a n d KLH s y s t e m s , o t h e r T s F w i t h d i f f e r e n t s p e c i f i c i t i e s h a v e b e e n p u r i f i e d t o h o m o g e n e i t y . Webb e t a l . (1989) d e s c r i b e d a T s F s p e c i f i c f o r 1 - t y r - p - a z o p h e n y l t r i - m e t h y l - a m m o n i u m (TMA). I t was d e s c r i b e d a s an i n d u c e r T s F t h a t b o u n d Ag, e x p r e s s e d b o t h I - J d e t e r m i n a n t s a n d t h e c r o s s - r e a c t i v e i d i o t y p e a s s o c i a t e d w i t h a n t i - T M A Ab. E x t r a c t e d m a t e r i a l f r o m t h e Ts h y b r i d o m a was s u b j e c t e d t o ( N H ^ ^ S O ^ p r e c i p i t a t i o n , I E F , r e v e r s e - p h a s e HPLC, a n d h i g h p e r f o r m a n c e g e l f i l t r a t i o n t o y i e l d a m a j o r b i o a c t i v e 11 peak at 62k M . Using SDS-PAGE, a major peak was observed at 2 6k M . They noted that the biochemical c h a r a c t e r i s t i c s of TMA-TsF were very s i m i l a r to that of single-chain GAT-TsF. I n fact, they demonstrated that a mAb raised to GAT-TsF bound to TMA-TsF. The authors suggested that both factors belonged to the same family of suppressor inducer proteins. A TsF i s o l a t e d from a Ts clone (as opposed to a hybridoma) has been reported. Fresno et a l . (1981) recovered a 70k M , s i n g l e -chain, Ag-binding factor from a Ly23 + T c e l l clone. It did not express I-J determinants. It was p u r i f i e d using gel f i l t r a t i o n , ion-exchange chromatography and IEF. The target of t h i s TsF was the Th and required the presence of Ag for the expression of suppressive a c t i v i t y . Summary A variety of properties have been attributed to TsF. I n i t i a l l y , such features as the s p e c i f i c suppression of the Ab response, the c a p a b i l i t y to bind Ag, the expression of I - J encoded determinants and the requirement for g e n e t i c a l l y r e s t r i c t e d c e l l u l a r interactions were discovered using c e l l u l a r extracts. These findings were v e r i f i e d and extended using Ts hybridomas and the TsF produced by them. Indeed, the l i n e a r cascade model for the suppression of the NP response (Hausman, Sherr, and Dorf 1986, see above) was based on data obtained using hybridomas and t h e i r respective factors made from the three o r d e r s o f T s : T s ^ , TS2 a n d TSg. O t h e r work s u g g e s t e d t h a t s u p p r e s s i o n was a c h i e v e d t h r o u g h i n t e r a c t i n g c e l l p a t h w a y s o r c i r c u i t s w i t h e a c h p h e n o t y p i c a l l y d i s t i n c t Ts s u b s e t e l a b o r a t i n g i t s own t y p e o f T s F ( e g . i n d u c e r T s F , e f f e c t o r T s F ) . The t a r g e t o f t h e A g - s p e c i f i c T s F was e i t h e r t h e n e x t Ts i n t h e p a t h w a y o r t h e t e r m i n a l c e l l o f t h e p a t h , t h e e f f e c t o r T c e l l , w h i c h m e d i a t e d t h e f u n c t i o n t h a t was u n d e r s u p p r e s s i v e c o n t r o l ( e g . T h ) . S i n c e T s F a n d Ts c a n e x p r e s s i d i o t y p i c d e t e r m i n a n t s , one p o s s i b l e way o f c e l l t o c e l l r e c o g n i t i o n was t h r o u g h t h e i d i o t y p e - a n t i - i d i o t y p e n e t w o r k . G e n e t i c r e s t r i c t i o n c o u l d a l s o h e l p t o i n s u r e t h a t t h e a p p r o p r i a t e c e l l s a n d f a c t o r s w o u l d i n t e r a c t . A l t h o u g h s u p p r e s s i o n o f t h e e f f e c t o r f u n c t i o n was A g -d e p e n d e n t a n d s p e c i f i c , o t h e r T c e l l r e s p o n s e s i n t h e i m m e d i a t e v i c i n i t y w o u l d be s u p p r e s s e d ( b y s t a n d e r s u p p r e s s i o n ) . T h i s e x p l a i n s how a m o n o c l o n a l Ts F c o u l d s u p p r e s s a p o l y c l o n a l T c e l l r e s p o n s e . F a c t o r - s e c r e t i n g Ts h y b r i d o m a s h a v e a l s o made i t p o s s i b l e t o s t u d y T s F a t t h e m o l e c u l a r l e v e l . S e v e r a l T s F h a v e b e e n p u r i f i e d u s i n g d i f f e r e n t s e p a r a t i o n t e c h n i q u e s i n c l u d i n g , a f f i n i t y a n d i o n - e x c h a n g e c h r o m a t o g r a p h y , I E F a n d r e v e r s e - p h a s e HPLC, i n v a r i o u s c o m b i n a t i o n s , t o y i e l d homogeneous a n d b i o a c t i v e m a t e r i a l . T s F h a s b e e n i s o l a t e d a s a one c h a i n o r two c h a i n m o l e c u l e . The one c h a i n T s F was b i o a c t i v e , b o u n d Ag a n d e x p r e s s e d I - J e n c o d e d d e t e r m i n a n t s . The two c h a i n s t r u c t u r e c o n s i s t e d o f an A g - b i n d i n g c h a i n a n d a n o n - A g - b i n d i n g c h a i n t h a t d i s p l a y e d I - J d e t e r m i n a n t s . B o t h o f t h e c h a i n s w e re r e q u i r e d f o r b i o a c t i v i t y a n d h a v e b e e n r e p o r t e d as e i t h e r n o n - c o v a l e n t l y 13 a s s o c i a t e d o r d i s u l p h i d e - l i n k e d . M e s s e n g e r RNA h a s b e e n p r e p a r e d f r o m a t l e a s t two d i f f e r e n t Ts h y b r i d o m a s a n d u s e d t o a c h i e v e t r a n s l a t i o n o f b i o a c t i v e T s F . B o t h t h e one c h a i n a n d two c h a i n t y p e s o f T s F h a v e b e e n s y n t h e s i z e d u s i n g i n v i t r o t r a n s l a t i o n t e c h n i q u e s . A p r e c i s e d e s c r i p t i o n o f t h e T s F m o l e c u l e h a s b e e n d i f f i c u l t b e c a u s e o f t h e v a r i e t y i n b o t h i t s r e p o r t e d m o l e c u l a r a n d f u n c t i o n a l p r o p e r t i e s . W h e t h e r t h e v a r i e t y o f r e s u l t s i s a r e f l e c t i o n o f t h e d i f f e r e n t A g s y s t e m s u n d e r s t u d y , t h e l a b i l e n a t u r e o f t h e T s F m o l e c u l e , t h e c o m p l e x i t y o f t h e s u p p r e s s o r m e c h a n i s m s a t work o r a c o m b i n a t i o n o f a l l t h r e e , i s unknown. D e s p i t e t h e e f f o r t b e i n g d e v o t e d t o t h e s t u d y o f T s F , a c o n s e n s u s on i t s s t r u c t u r e a n d f u n c t i o n w i l l o n l y be r e a c h e d o n l y b y s t r i n g e n t t e s t i n g e g . m o l e c u l a r c l o n i n g o f a g e n e ( s ) e n c o d i n g an A g - s p e c i f i c T s F . The Immune R e s p o n s e t o F e r r e d o x i n as a M o d e l f o r t h e S t u d y o f  M e c h a n i s m s o f I m m u n o r e g u l a t i o n The immune r e s p o n s e t o f e r r e d o x i n (Fd) h a s b e e n s t u d i e d e x t e n s i v e l y i n t h i s l a b o r a t o r y . C o l l e c t i v e l y , t h e f i n d i n g s h a v e s e r v e d a s a d e f i n e d m o d e l f o r t h e immune r e s p o n s e t o a n a t u r a l p r o t e i n A g . 14 The c h o i c e o f F d as a m o d e l a n t i g e n F d i s a s m a l l (5500 M ) m o l e c u l e p r o d u c e d b y C l o s t r i d i u m  p a s t e u r i a n u m w h i c h f u n c t i o n s a s an e l e c t r o n - a c c e p t o r p r o t e i n i n t h e e l e c t r o n t r a n s p o r t p a t h w a y o f t h a t o r g a n i s m . I t h a s b e e n w e l l - c h a r a c t e r i z e d b i o c h e m i c a l l y a n d p u r i f i e d t o h o m o g e n e i t y ( R a b i n o w i t z 1 9 7 2 ) . The a m i n o - a c i d s e q u e n c e o f F d was c o n f i r m e d u s i n g gene c l o n i n g t e c h n i q u e s ( G r a v e s , M u l l e n b a c h , a n d R a b i n o w i t z 1985) a n d i t s t h r e e - d i m e n s i o n a l s t r u c t u r e h a s b e e n d e t e r m i n e d u s i n g X - r a y c r y s t a l l o g r a p h y ( J e n s e n 1 9 7 4 ) . T h i s 55 a m i n o - a c i d p r o t e i n i s d i s t i n g u i s h e d b y t h e p r e s e n c e o f e i g h t c y s t e i n e r e s i d u e s , t h e s u l f u r atoms o f w h i c h , f o r m c o o r d i n a t i o n b o n d s w i t h two i r o n - s u l f u r c l u s t e r s , t h a t make up t h e c e n t r a l c o r e o f t h e p r o t e i n . I t h a s b e e n h y p o t h e s i z e d t h a t F d was c r e a t e d t h r o u g h a gene d u p l i c a t i o n e v e n t a s t h e p r o t e i n h a s a t w o - f o l d a x i s o f sy m m e t r y a n d 13 i d e n t i c a l p a i r s o f a m i n o - a c i d r e s i d u e s t h a t a r e p r e s e n t i n b o t h h a l v e s o f t h e m o l e c u l e . I n a d d i t i o n t o h a v i n g k n o w l e d g e o f i t s b i o c h e m i s t r y , t h e r e was a n o t h e r r e a s o n f o r c h o o s i n g F d a s a m o d e l Ag f o r t h e s t u d y o f i m m u n o r e g u l a t o r y m e c h a n i s m s . S t a t e d s i m p l y , F d i s a n a t u r a l p r o t e i n A g o f d e f i n e d s t r u c t u r e . T h i s t y p e o f m o l e c u l e h a s two d i s t i n c t a d v a n t a g e s o v e r some o f t h e o t h e r e x p e r i m e n t a l A g . F i r s t l y , n a t u r a l p r o t e i n s a r e what i s e n c o u n t e r e d b y t h e immune s y s t e m u n d e r p h y s i o l o g i c a l c o n d i t i o n s a n d t h e r e f o r e any m e c h a n i s m s e l u c i d a t e d b y i t s s t u d y w o u l d p r e s u m a b l y h a v e b r o a d b a s e d i m p l i c a t i o n s . S u c h c o n c l u s i o n s c a n n o t n e c e s s a r i l y be d r a w n 15 f r o m s y n t h e t i c h a p t e n s o r A g . S e c o n d l y , n a t u r a l p r o t e i n s a r e o f d e f i n e d p r i m a r y s t r u c t u r e , as o p p o s e d t o s y n t h e t i c Ag l i k e GAT, o r d e r i v a t i z e d a n t i g e n s l i k e DNP-KLH. K n o w l e d g e o f t h e p r e c i s e a n t i g e n i c s t r u c t u r e i s v i t a l f o r t h e s t u d y o f immunodominance: t h e phenomenon i n w h i c h , o f t h e p o t e n t i a l l y l a r g e number o f e p i t o p e s e x p r e s s e d by t h e Ag, o n l y a l i m i t e d number a r e s e l e c t e d t o i n t e r a c t w i t h l y m p h o c y t e c l o n e s . H a v i n g a d e f i n e d s t r u c t u r e a l l o w s t h e i d e n t i f i c a t i o n o f t h e s e immunodominant e p i t o p e s . As w i l l be d e s c r i b e d b e l o w , F d h a s b e e n u s e f u l as a p r o t o t y p e f o r t h e s t u d y o f t h e immune r e s p o n s e t o T c e l l - d e p e n d e n t n a t u r a l p r o t e i n A g . Some o f t h e work w i t h F d h a s i n v o l v e d t h e u s e o f d e r i v a t i z e d o r s y n t h e t i c f o r m s o f t h e Ag. However t h e d a t a h a v e a l w a y s b e e n r e l a t e d t o o r i n t e r p r e t e d i n t e r m s o f t h e r e s p o n s e t o t h e n a t i v e Ag. E p i t o p e m a p p i n g a n d H-2 l i n k e d r e s p o n s i v e n e s s t o F d I n i t i a l s t u d i e s w e r e a i m e d a t m a p p i n g t h e m a j o r a n t i g e n i c d e t e r m i n a n t s o f F d u s i n g b o t h r a b b i t a n t i s e r u m a n d A g - p r i m e d T c e l l s f r o m g u i n e a p i g s . I t was d e m o n s t r a t e d t h a t F d c o n t a i n e d a l i m i t e d number o f d e t e r m i n a n t s s i n c e p e r o x y f o r m i c a c i d m o d i f i c a t i o n o f t h e c y s t e i n e r e s i d u e s o f t h e n a t i v e m o l e c u l e d i d n o t a l t e r t h e b i n d i n g o f a n t i s e r u m r a i s e d t o t h e u n m o d i f i e d p r o t e i n ( N i t z e t a l . 1 9 6 9 ) . F u r t h e r work, by K e l l y a n d L e v y ( 1 9 7 1 ) , u s i n g r a b b i t a n t i s e r u m r a i s e d t o p e r o x y f o r m i c a c i d -t r e a t e d F d (OFd) a n d s y n t h e t i c p e p t i d e s r e p r e s e n t i n g v a r i o u s 16 s e q u e n c e s i n t h e F d m o l e c u l e showed t h a t e s s e n t i a l l y a l l o f t h e Ab a c t i v i t y was d i r e c t e d t o e i t h e r t h e a m i n o - t e r m i n a l h e p t a p e p t i d e ( N H ^ - a l a - t y r - l y s - i l e - v a l - a s p - s e r - C O O H , known as N-d e t e r m i n a n t ) o r t h e c a r b o x y l - t e r m i n a l p e n t a p e p t i d e ( N H ^ - a l a - p r o -v a l - g l n - g l u - C O O H , known as C - d e t e r m i n a n t ) . The a b i l i t y o f b o t h t h e a m i n o - t e r m i n a l a n d c a r b o x y l - t e r m i n a l p e p t i d e s t o a c t a s T c e l l d e t e r m i n a n t s was shown u s i n g s y n t h e t i c p e p t i d e s c o n s i s t i n g o f t h e immunodominant s e q u e n c e s l i n k e d t o g e t h e r v i a a r e p e a t i n g g l y c i n e b r i d g e . The s y n t h e t i c p e p t i d e s p r o v e d t o be i m m u n o g e n i c i n g u i n e a p i g s s i n c e t h e T c e l l s o b t a i n e d f r o m t h e s e p r i m e d a n i m a l s w o u l d p r o l i f e r a t e i n r e s p o n s e t o t h e r e s p e c t i v e p e p t i d e s a s w e l l a s t o t h e i n t a c t A g ( L e v y e t a l . 1972; K e l l y , L e v y , a n d H u l l 1 9 7 3 ) . E v i d e n c e t h a t t h e N - d e t e r m i n a n t a n d C - d e t e r m i n a n t -p r i m e d c e l l s s y n e r g i z e d f o r t h e g e n e r a t i o n o f a Th c e l l r e s p o n s e was p r o v i d e d when i t was d e m o n s t r a t e d t h a t o n l y a m i x t u r e o f t h e two s e p a r a t e l y p r i m e d c e l l p o p u l a t i o n s w o u l d r e c o g n i z e t h e h a p t e n - c a r r i e r c o m p l e x , DNP-OFd i n an a n t i - h a p t e n (DNP) Ab r e s p o n s e ( F e l d m a n n , K i l b u r n , a n d L e v y 1 9 7 5 ) . S i k o r a a n d L e v y (1980) c a r r i e d o u t a g e n e t i c a n a l y s i s u s i n g i n b r e d s t r a i n s o f m i c e a n d d e t e r m i n e d t h a t t h e Ab r e s p o n s e t o F d was H-2 l i n k e d . U s i n g c o n g e n i c s t r a i n s o f m i c e , t h e r e s p o n s e was v mapped t o t h e I-A s u b r e g i o n . H-2 a n i m a l s w e re i d e n t i f i e d a s h i g h r e s p o n d e r s , H-2 ' m i c e as i n t e r m e d i a t e r e s p o n d e r s a n d H-2 a n i m a l s w e r e d e s i g n a t e d as n o n r e s p o n d e r s . An i m p o r t a n t s t u d y b y S i k o r a , Weaver, a n d L e v y (1982) c o n f i r m e d a n d e x t e n d e d t h e p r e v i o u s work t h a t d e s c r i b e d t h e d e t e r m i n a n t m a p p i n g o f F d . They showed t h a t F d c o n s i s t e d o f two a n t i g e n i c d e t e r m i n a n t s , t h r o u g h t h e u s e o f s t r u c t u r a l a n a l o g s w h i c h w e re g e n e r a t e d b y e n z y m a t i c d i g e s t i o n . T r y p s i n c l e a v a g e o f F d d e s t r o y s t h e N - d e t e r m i n a n t b y r e m o v i n g t h e t e r m i n a l t r i p e p t i d e , l e a v i n g t h e r e m a i n d e r o f t h e m o l e c u l e , i n c l u d i n g t h e C - d e t e r m i n a n t , i n t a c t . C a r b o x y p e p t i d a s e A p a r t i a l l y d i g e s t s t h e C - d e t e r m i n a n t b y r e m o v i n g t h e t e r m i n a l 3 amino a c i d s , l e a v i n g t h e r e s t o f t h e m o l e c u l e , i n c l u d i n g t h e N - d e t e r m i n a n t , i n t a c t . A d o u b l y d i g e s t e d m o l e c u l e d i d n o t r e a c t w i t h a n t i - F d a n t i s e r u m p r e p a r e d f r o m h i g h r e s p o n d e r m i c e , c o n f i r m i n g t h e e a r l i e r o b s e r v a t i o n t h a t Ab a c t i v i t y was d i r e c t e d e x c l u s i v e l y t o t h e two d e t e r m i n a n t s . M o n o c l o n a l a n t i b o d i e s w i t h s p e c i f i c i t y f o r e i t h e r t h e N - d e t e r m i n a n t o r C - d e t e r m i n a n t w e re p r o d u c e d a n d shown t o b i n d t o t h e r e s p e c t i v e e n z y m e - d i g e s t e d m o l e c u l e b e a r i n g t h e a p p r o p r i a t e d e t e r m i n a n t . I t was a l s o o b s e r v e d t h a t t h e sum o f t h e N - d e t e r m i n a n t a n d C - d e t e r m i n a n t r e a c t i v i t i e s o f an a n t i s e r u m was e q u a l t o i t s t o t a l a n t i - F d r e a c t i v i t y . T h i s f i n d i n g was u s e d a s b a s i s o f c o m p a r i s o n b e t w e e n s t r a i n s o f m i c e t h a t w e re k r e s p o n s i v e t o F d . The h i g h r e s p o n d i n g s t r a i n s ( I - A ) p r o d u c e d Ab, o f w h i c h , 60 t o 80% was d i r e c t e d t o t h e C - d e t e r m i n a n t . Of t h e i n t e r m e d i a t e r e s p o n d e r s ( e x p r e s s i n g t h e b a n d s a l l e l e s a t t h e I-A s u b r e g i o n ) , o n l y 25 t o 5 0 % o f t h e t o t a l a n t i - F d Ab was s p e c i f i c f o r t h e C - d e t e r m i n a n t . I t was f o u n d t h a t i n j e c t i o n o f n o n r e s p o n d e r m i c e (H-2 ) w i t h t r y p s i n - d i g e s t e d F d ( a l s o r e f e r r e d t o a s C - m o l e c u l e ) made them r e s p o n s i v e t o s u b s e q u e n t c h a l l e n g e s o f F d ( S i k o r a a n d L e v y 1 9 8 4 ) . 18 Furthermore, i f the mice were primed with carboxypeptidase A-treated Fd (also referred to as N-molecule) and then challenged with C-molecule, t h i s resulted i n an accelerated anti-Fd Ab response as compared to control animals that received C-molecule immunization only. It was postulated that a second overlapping determinant resided within the N-determinant heptapeptide region that was "masked" by the a l a - t y r - l y s t r i p e p t i d e , but was exposed in the case of C-molecule. Interestingly, for the C-molecule-primed mice, less than 10% of the anti-Fd Ab was s p e c i f i c for the C-determinant (see F i g . 9B) . Summary Fd was chosen as a model Ag because i t i s a small, natural protein with defined structure. I n i t i a l studies i d e n t i f i e d the amino-terminal heptapeptide and the carboxyl-terminal pentapeptide as the only antigenic determinants or epitopes of the molecule. These determinants were reactive with both Ab and T c e l l s . This suggested that either epitope had the p o t e n t i a l for being assigned a hapten or c a r r i e r function. Monoclonal antibodies were raised to either determinant and through the use of analogs helped to v e r i f y the o r i g i n a l epitope mapping of Fd. The immune response to Fd was observed to be H-2 linked and determinant s e l e c t i v i t y of the anti-Fd Ab response was also related to the haplotype (genotype) of the responding mouse. H-2 animals were i d e n t i f i e d as high responders and 60 to 80% of t h e Ab r e s p o n s e was d i r e c t e d t o t h e C - d e t e r m i n a n t . H-2 ' a n i m a l s w e r e d e s i g n a t e d as i n t e r m e d i a t e r e s p o n d e r s a n d 25 t o 50% o f t h e Ab was s p e c i f i c f o r t h e C - d e t e r m i n a n t . L a s t l y , t o l e r a n c e t o F d , e x p r e s s e d b y t h e n o n r e s p o n d e r H-2 m i c e , c o u l d be b r o k e n b y p r i m i n g t h e a n i m a l s w i t h C - m o l e c u l e , t h e t r y p s i n - d i g e s t e d a n a l o g o f F d . L e s s t h a n 10% o f t h e a n t i - F d Ab o f t h e s e a n i m a l s was d i r e c t e d t o t h e C - d e t e r m i n a n t . I d i o t y p e - a n t i - i d i o t y p e i n t e r a c t i o n s The s t u d y o f t h e immune r e s p o n s e t o F d h a s a l s o b e e n p u r s u e d by d e f i n i n g a n d e x a m i n i n g i d i o t y p i c - a n t i - i d i o t y p i c i n t e r a c t i o n s a s s o c i a t e d w i t h t h e a n t i - F d r e s p o n s e . The c o n c e p t o f t h e immune s y s t e m a s an a u t o r e g u l a t o r y n e t w o r k w i t h c o m p o n e n t s t h a t r e c o g n i z e e a c h o t h e r v i a s e t s o f a n t i g e n i c d e t e r m i n a n t s e x p r e s s e d b y t h e V r e g i o n s o f Ab o r TCR ( i d i o t y p e ) , i s now g e n e r a l l y a c c e p t e d . N e t w o r k i n t e r a c t i o n s t h a t i n f l u e n c e d t h e immune r e s p o n s e t o F d were f i r s t r e v e a l e d t h r o u g h t h e u s e o f an a n t i - i d i o t y p i c r e a g e n t ( r a b b i t a n t i s e r u m ) s p e c i f i c f o r t h e i d i o t y p e e x p r e s s e d b y t h e a n t i - F d mAb, F d - 1 . T h i s mAb i s s p e c i f i c f o r t h e N - d e t e r m i n a n t o f F d . The i d i o t y p e i d e n t i f i e d b y t h e a n t i - F d - 1 r e a g e n t r e p r e s e n t e d k a m a j o r i d i o t y p e e x p r e s s e d i n a n t i - F d Ab o b t a i n e d f r o m b o t h H-2 h i g h r e s p o n d e r s a n d H-2 S i n t e r m e d i a t e r e s p o n d e r s (Weaver, S i k o r a , a n d L e v y 1 9 8 2 ) . Weaver e t a l . (1983) d e s c r i b e d T c e l l s , w h i c h e x p r e s s e d t h e 20 Fd- 1 i d i o t y p e , t h a t h e l p e d t o r e g u l a t e t h e a n t i - F d Ab r e s p o n s e o f B10.BR ( H - 2 k ) a n i m a l s . T h i s was done b y t r e a t i n g B10.BR T c e l l s w i t h a n t i - F d - 1 p l u s c o m p l e m e n t a n d t h e n a d o p t i v e l y t r a n s f e r r i n g t h e m t o s u b l e t h a l l y i r r a d i a t e d B10.BR r e c i p i e n t s . The r e c i p i e n t s w e r e i m m u n i z e d w i t h F d a n d m o n i t o r e d f o r Ab p r o d u c t i o n . T h e r e was a s i g n i f i c a n t i n c r e a s e i n a n t i - C - d e t e r m i n a n t - s p e c i f i c Ab i n t h e a n i m a l s t h a t r e c e i v e d T c e l l s t r e a t e d w i t h t h e a n t i - F d - 1 r e a g e n t a s c o m p a r e d t o c o n t r o l a n i m a l s . T h i s s u g g e s t e d t h a t a p o p u l a t i o n o f Fd-1 i d i o t y p e - b e a r i n g T c e l l s was b e i n g i n f l u e n c e d b y t h e a n t i - i d i o t y p i c r e a g e n t . I n t e r e s t i n g l y , t h e l e v e l o f a n t i -N - d e t e r m i n a n t Ab o r t h e p e r c e n t a g e o f e x p r e s s i o n o f t h e Fd-1 i d i o t y p e i n t h e a n t i s e r u m d i d n o t c h a n g e w i t h t h e a n t i - i d i o t y p e t r e a t m e n t . When F d - 1 , t h e mAb d i s p l a y i n g t h e Fd-1 i d i o t y p e , was a d m i n i s t e r e d t o nonimmune B10.BR m i c e s e v e n d a y s p r i o r t o F d i m m u n i z a t i o n , t h e r e was a s i g n i f i c a n t i n c r e a s e i n a n t i - F d Ab p r o d u c t i o n i n t h o s e a n i m a l s t h a t h a d r e c e i v e d t h e mAb i n j e c t i o n . T h i s s u g g e s t e d t o t h e a u t h o r s t h a t an a n t i - i d i o t y p i c p o p u l a t i o n o f T c e l l s a l s o e x i s t e d a n d t h a t t h e i n t e r a c t i o n b e t w e e n t h e i d i o t y p e - b e a r i n g a n d t h e a n t i - i d i o t y p e b e a r i n g T c e l l s was i m p o r t a n t i n t h e r e g u l a t i o n o f t h e a n t i - F d Ab r e s p o n s e i n B10.BR a n i m a l s . S i n g h a i e t a l . (1984) c h a r a c t e r i z e d a n o t h e r a n t i - F d mAb, F d -2, t h a t was s p e c i f i c f o r t h e C - d e t e r m i n a n t o f F d a n d p r e p a r e d an a n t i - i d i o t y p i c r e a g e n t t o i t . When t h i s a n t i - F d - 2 i d i o t y p e r e a g e n t was a d m i n i s t e r e d t o B10.BR m i c e , t h a t w e r e s u b s e q u e n t l y i m m u n i z e d w i t h F d , t h e a n t i - F d Ab r e s p o n s e was e n h a n c e d i n t r e a t e d a n i m a l s a s c o m p a r e d t o c o n t r o l g r o u p s . The l e v e l o f a n t i - C - d e t e r m i n a n t Ab h a d i n c r e a s e d , as was o b s e r v e d b y Weaver e t a l . (1983). U n l i k e t h e Fd-1 i d i o t y p e , t h e Fd-2 i d i o t y p e was n o t d e t e c t e d i n BIO.BR immune s e r a . More i n t e r e s t i n g l y , t h e a d m i n i s t r a t i o n o f a n t i - F d - 2 t o n o n r e s p o n d e r B10.D2 m i c e , t h a t w e r e t h e n i m m u n i z e d t o F d , r e s u l t e d i n t h e p r o d u c t i o n o f a n t i - F d Ab i n t h e s e a n i m a l s . T h i s a b r o g a t i o n o f n o n r e s p o n s i v e n e s s was i n v e s t i g a t e d f u r t h e r b y S i n g h a i , H o f f m a n n , a n d L e v y (1985). They t r e a t e d B10.D2 T c e l l s w i t h e i t h e r Fd-2 a n d co m p l e m e n t o r a n t i -Fd-2 a n d com p l e m e n t a n d a d o p t i v e l y t r a n s f e r r e d t h e r e m a i n i n g c e l l s i n t o s y n g e n e i c i r r a d i a t e d B10.D2 r e c i p i e n t s . The r e c i p i e n t s w e r e i m m u n i z e d w i t h F d a n d m o n i t o r e d f o r t h e p r o d u c t i o n o f a n t i - F d Ab. B o t h g r o u p s o f a n i m a l s r e s p o n d e d t o t h e F d c h a l l e n g e . R e c o n s t i t u t i o n o f n o n r e s p o n s i v e n e s s was d e m o n s t r a t e d b y a d o p t i v e l y t r a n s f e r r i n g a m i x t u r e o f T c e l l s t h a t was c o m p o s e d o f t h e F d - 2 - d e p l e t e d s u s p e n s i o n a n d t h e a n t i - F d - 2 -d e p l e t e d s u s p e n s i o n i n t o r e c i p i e n t s , t h a t w e re t h e n c h a l l e n g e d w i t h F d . The a n t i - F d Ab l e v e l s o f t h e s e a n i m a l s w e re n e g l i g i b l e as c o m p a r e d t o t h e o t h e r i m m u n i z e d r e c i p i e n t s who r e c e i v e d e i t h e r t h e F d - 2 - d e p l e t e d o r t h e a n t i - F d - 2 - d e p l e t e d T c e l l s . T h i s s u g g e s t e d t h a t b o t h p o p u l a t i o n s o f T c e l l s e x p r e s s i n g t h e Fd-2 i d i o t y p e a n d t h e a n t i - F d - 2 i d i o t y p e w e r e r e q u i r e d f o r t h e m a i n t e n a n c e o f t h e n o n r e s p o n d e r s t a t u s i n H-2 a n i m a l s . 22 Summary I d i o t y p e was d e f i n e d a s t h e s e t o f a n t i g e n i c d e t e r m i n a n t s e x p r e s s e d b y t h e V r e g i o n s o f Ab o r TCR. I d i o t y p i c - a n t i -i d i o t y p i c i n t e r a c t i o n s w e r e i n v e s t i g a t e d u s i n g a n t i - F d mAb as t h e i d i o t y p e - e x p r e s s i n g r e a g e n t s . A f f i n i t y - p u r i f i e d a n t i - i d i o t y p i c a n t i s e r a a g a i n s t t h e s e i d i o t y p e s w e r e r a i s e d i n r a b b i t s . The F d -1 i d i o t y p e was e x p r e s s e d i n t h e m a j o r i t y o f s e r a t e s t e d f r o m h i g h a n d i n t e r m e d i a t e r e s p o n d e r a n i m a l s . T h i s i d i o t y p e was a l s o e x p r e s s e d b y a p o p u l a t i o n o f B10.BR T c e l l s t h a t a p p e a r e d t o r e g u l a t e t h e a n t i - F d Ab r e s p o n s e . The o b s e r v a t i o n t h a t an en h a n c e m e n t i n a n t i - F d Ab i n B10.BR a n i m a l s was c a u s e d b y t h e a d m i n i s t r a t i o n o f Fd-1 i d i o t y p e i t s e l f s u g g e s t e d t h a t a n t i - F d - 1 -i d i o t y p e - b e a r i n g c e l l s e x i s t e d as w e l l . I t was s p e c u l a t e d t h a t t h e i d i o t y p e - b e a r i n g a n d a n t i - i d i o t y p e - b e a r i n g c e l l p o p u l a t i o n s w o r k e d i n c o n c e r t t o h e l p r e g u l a t e t h e Ab r e s p o n s e i n B10.BR a n i m a l s . R e s u l t s f r o m t h e i n v e s t i g a t i o n o f a s e c o n d immune n e t w o r k i n t e r a c t i o n ( d e s i g n a t e d Fd-2 i d i o t y p e ) i n B10.D2 m i c e showed t h a t two p o p u l a t i o n s o f i d i o t y p e - b e a r i n g a n d a n t i - i d i o t y p e b e a r i n g T c e l l s g o v e r n e d r e s p o n s i v e n e s s t o F d . When e i t h e r p o p u l a t i o n was d e p l e t e d , r e s p o n s i v e n e s s e n s u e d f o l l o w i n g i m m u n i z a t i o n . When t h e two p o p u l a t i o n s w e r e c o m b i n e d , a n d r e t u r n e d t o t h e a n i m a l a l m o s t c o m p l e t e r e s t o r a t i o n o f n o n r e s p o n s i v e n e s s r e s u l t e d . 23 B16G a n d t h e D e v e l o p m e n t o f t h e F d l l h y b r i d o m a A n o t h e r a r e a o f i n t e r e s t i n t h i s l a b o r a t o r y h a s b e e n t h e d e v e l o p m e n t a n d u s e o f B16G, a mAb h a v i n g a p p a r e n t s p e c i f i c i t y f o r a n i n v a r i a n t e p i t o p e on T s F . B16G h a s b e e n u s e d f o r b o t h t h e s c r e e n i n g o f Ts h y b r i d o m a s a n d t h e i s o l a t i o n o f s u p p r e s s i v e m a t e r i a l u s i n g i m m u n o a f f i n i t y c o l u m n s . I n o r d e r t o e x t e n d o u r s t u d i e s o f t h e immune r e s p o n s e t o F d , a T c e l l h y b r i d o m a , F d l l , was r a i s e d a g a i n s t t h a t Ag a n d s c r e e n e d u s i n g B16G. B16G i t s e l f was d e v e l o p e d as a r e s u l t o f s t u d i e s on t h e P815 t u m o r s y s t e m . The m u r i n e m a s t o c y t o m a P815 i s a w e l l c h a r a c t e r i z e d t u m o r m o d e l w h i c h i s l e t h a l t o DBA/2 m i c e when i n j e c t e d a t r e l a t i v e l y l o w d o s e s . I t was shown t h a t s o o n a f t e r t u m o r i n o c u l a t i o n ( t h r e e t o f i v e d a y s ) a P 8 1 5 - s p e c i f i c CTL r e s p o n s e was e v i d e n t . However t h i s p h a s e was q u i c k l y o v e r w h e l m e d b y t h e e m e r g e n c e o f T s . The a p p e a r a n c e o f Ts c o r r e l a t e d w i t h an a c c e l e r a t i o n i n t u m o r g r o w t h ( T a k e i , L e v y , a n d K i l b u r n 1 9 7 6 ) . O t h e r w o r k e r s h a v e a l s o d e s c r i b e d t h e i n v o l v e m e n t o f Ts i n t h e immune r e s p o n s e t o t h i s a n d o t h e r t u m o r s ( G r a n s t e i n e t a l . 1984; F i s h e r a n d K r i p k e 1982; M i l l s a n d N o r t h 1 9 8 5 ) . F u r t h e r i n v e s t i g a t i o n l e d t o t h e i d e n t i f i c a t i o n o f a s o l u b l e f a c t o r , e x t r a c t a b l e f r o m t h e t h y m o c y t e s o f P 8 1 5 - b e a r i n g m i c e , t h a t was c a p a b l e o f s u p p r e s s i n g t h e s y n g e n e i c i n v i t r o c y t o l y t i c T c e l l (CTL) r e s p o n s e t o P815 ( T a k e i , L e v y , a n d K i l b u r n 1 9 7 8 ) . The f a c t o r a p p e a r e d t o h a v e a d e g r e e o f t u m o r s p e c i f i c i t y . The s u p p r e s s i v e a c t i v i t y c o u l d be a b s o r b e d o u t o f s o l u t i o n b y p a s s a g e 24 o v e r a P 8 1 5 - S e p h a r o s e c o l u m n b u t n o t by a c o n t r o l c o l u m n . A t t e m p t s a t p u r i f i c a t i o n o f t h i s m a t e r i a l w e r e n o t s u c c e s s f u l . I n o r d e r t o s t u d y t u m o r - s p e c i f i c f a c t o r s more d i r e c t l y , M a i e r , Stammers, a n d L e v y (1983) d e v e l o p e d a n d c h a r a c t e r i z e d t h e T s F -s p e c i f i c mAb, B16G. A f f i n i t y - e n r i c h e d t h y m o c y t e - d e r i v e d T s F was u s e d t o i m m u n i z e m i c e f r o m w h i c h B16G was d e r i v e d . Once i t was i s o l a t e d a n d c l o n e d , B16G was t e s t e d b o t h i n v i v o a n d i n v i t r o . P r e t r e a t m e n t o f DBA/2 m i c e w i t h B16G n o t o n l y i n h i b i t e d t h e g r o w t h o f P815 t u m o r b u t t h e s y n g e n e i c t u m o r , M-1 as w e l l . T h a t B16G was n o n s p e c i f i c a l l y i m m u n o e n h a n c i n g was a l s o s u b s t a n t i a t e d b y n o t i n g t h a t DBA/2 s p l e e n c e l l s f r o m B 1 6 G - t r e a t e d a n i m a l s r e s p o n d e d b e t t e r t o i r r a d i a t e d h i s t o i n c o m p a t i b l e c e l l s b y p r o l i f e r a t i o n t h a n d i d u n t r e a t e d c o n t r o l s . T h i s e n h a n c i n g e f f e c t on T c e l l p r o l i f e r a t i o n s u g g e s t e d t h a t B16G was r e s p o n s i b l e f o r t h e i n a c t i v a t i o n o f a r e g u l a t o r y ( s u p p r e s s o r ? ) T c e l l . P a n n i n g e x p e r i m e n t s , w h e r e T c e l l s were i n c u b a t e d on o r " p a n n e d o v e r " B l 6 G - c o a t e d p l a t e s , d e m o n s t r a t e d t h a t B16G was r e s p o n s i b l e f o r t h e r e m o v a l o f a r e g u l a t o r y T c e l l f r o m t h e s p l e e n p o p u l a t i o n . W i t h t h e a v a i l a b i l i t y o f a " T s F " - s p e c i f i c mAb f o r s e l e c t i o n p u r p o s e s , a T c e l l h y b r i d o m a , A10, was c r e a t e d f r o m t h e m u r i n e thymoma l i n e , BW5147 a n d T c e l l s s t i m u l a t e d i n v i v o w i t h P815 membrane e x t r a c t s ( S t e e l e , Stammers, a n d L e v y 1 9 8 5 ) . A10 f a c t o r ( A 1 0 F ) , was r e c o v e r e d f r o m SN u s i n g e i t h e r B16G- o r P 8 1 5 -S e p h a r o s e c o l u m n s . When A10F was a d m i n i s t e r e d i n t r a v e n o u s l y ( i . v . ) t o DBA/2 m i c e , i t c a u s e d t h e e n h a n c e m e n t o f P815 t u m o r g r o w t h , b u t n o t t h a t o f L1210 o r M-1 t u m o r . I n a d d i t i o n , A10F 25 did not enhance or suppress the T c e l l p r o l i f e r a t i v e allogeneic response of DBA/2 spleen c e l l s . The i n a b i l i t y of AlOF to affe c t the growth of two unrelated tumors or the T c e l l response of DBA/2 c e l l s to histoincompatible c e l l s indicated that i t might be tumor-specific. Molecular characterization revealed that a f f i n i t y - e n r i c h e d A10F resolved as 140k, 80k and 45k M r moieties when analyzed by SDS-PAGE under reducing conditions (Steele, Stammers, and Levy 1985; Chan 1988; Chan et a l . 1988). The 80k M r species had the propensity for "aggregation" to the larger M r species or degradation to a smaller size of approximately 30k M . SDS-PAGE under non-reducing conditions of A10F showed that suppressive a c t i v i t y was associated with the 140k and 80k M r forms while the 45k and 32k M r forms showed marginal b i o a c t i v i t y . Biosynthetic 35 l a b e l i n g of A10 c e l l s with S-methionine and cysteine revealed that the 80k Mr form was actually secreted into the SN and formally ruled out the p o s s i b i l i t y that the 80k M r A10F moiety was a medium or FCS a r t i f a c t . Amino-acid sequencing of the amino-terminal of the 140k and 80k Mr forms indicated that they shared the same sequence while the 30k M r polypeptide had a d i f f e r e n t amino-terminal sequence. Because of the p o s s i b i l i t y that sequence data could have been derived from co-migrating impurities, assignment of these sequences to the TsF molecules could not be done. Much information has been gathered about the immune response to Fd. Of p a r t i c u l a r interest i s the existence of an idiotype-26 a n t i - i d i o t y p e i n t e r a c t i o n , a t t h e T c e l l l e v e l , w h i c h f u n c t i o n s a s p a r t o f t h e s u p p r e s s o r n e t w o r k t o c o n t r o l t h e immune r e s p o n s e t o F d i n n o n r e s p o n d e r B10.D2 H-2 m i c e . W i t h t h e k n o w l e d g e t h a t F d - s p e c i f i c Ts e x i s t e d , a T c e l l h y b r i d o m a was made u s i n g BW5147 a n d T c e l l s o b t a i n e d f r o m F d -p r i m e d B10.D2 a n i m a l s ( S t e e l e , Chu, Chan, N o r t h , a n d L e v y e t a l . 1 9 8 7 ) . B e c a u s e t h e n o m i n a l Ag o f t h e F d l l h y b r i d o m a was known, u n l i k e t h a t o f t h e A10 h y b r i d o m a , t h e h y b r i d o m a c e l l s w e r e s c r e e n e d f o r t h e a b i l i t y t o s e c r e t e m o l e c u l e s c a p a b l e o f b i n d i n g t o F d a n d B16G. The s e c r e t e d m a t e r i a l , known a s F d l l F , c o u l d be r e c o v e r e d b y a f f i n i t y c h r o m a t o g r a p h y f r o m B 1 6 G - S e p h a r o s e o r F d -S e p h a r o s e c o l u m n s . F d l l F b o u n d t o F d - c o a t e d E L I S A p l a t e s w h i l e c o n t r o l m a t e r i a l f r o m an i r r e l e v a n t h y b r i d o m a d i d n o t . When a n a l y z e d b y SDS-PAGE, u n d e r r e d u c i n g c o n d i t i o n s , F d l l F - e n r i c h e d m a t e r i a l a p p e a r e d a s two m a j o r u n i q u e b a n d s , one m i g r a t i n g a t a b o u t 80k M r a n d a n o t h e r a t a b o u t 30k M r a s w e l l a s a number o f o t h e r m i n o r b a n d s . T h i s was t h e c a s e w h e t h e r t h e m a t e r i a l was r e c o v e r e d f r o m e i t h e r Ag o r B16G c o l u m n s . When F d l l F a n d F d were a d m i n i s t e r e d t o Fd-immune DBA/2 m i c e c o n c o m i t a n t l y , t h e Ab r e s p o n s e was s u p p r e s s e d a s c o m p a r e d t o t h e c o n t r o l g r o u p s r e c e i v i n g PBS o r A10F. E v i d e n c e t h a t F d l l c a n a c t u a l l y r e c o g n i z e F d , p r e s u m a b l y b y a c e l l - a s s o c i a t e d TCR, was p r o v i d e d b y c a l c i u m f l u x s t u d i e s . The i n t r a c e l l u l a r f l u x o f c a l c i u m i o n s i s one o f t h e e v e n t s a s s o c i a t e d w i t h a c e l l s u r f a c e r e c e p t o r - l i g a n d r e a c t i o n . F d l l c e l l s w e r e l o a d e d w i t h t h e c a l c i u m - s e n s i t i v e d y e , Q u i n - 2 . S e c o n d s a f t e r t h e e x p o s u r e o f F d l l c e l l s t o F d , Q u i n - 2 27 f l u o r e s c e n c e was d e t e c t e d , i n d i c a t i n g an i n c r e a s e i n t h e l e v e l o f i n t r a c e l l u l a r c a l c i u m a n d t h e p r e s u m e d b i n d i n g o f F d by F d l l F e x p r e s s e d on t h e s u r f a c e o f t h e c e l l . Summary The s t u d y o f t h e immune r e s p o n s e t o a s y n g e n e i c t u m o r p r o v i d e d t h e i m p e t u s f o r t h e d e v e l o p m e n t o f t h e mAb, B16G. I n t h e r e s p o n s e t o t h e P815 t u m o r , Ts p l a y t h e p r e d o m i n a n t r o l e a n d a l l o w t h e g r o w t h o f t h e t u m o r . S o l u b l e e x t r a c t s w i t h s u p p r e s s i v e a c t i v i t y s p e c i f i c f o r P815 were i n i t i a l l y p r e p a r e d f r o m t h y m o c y t e s o f P 8 1 5 - b e a r i n g m i c e a n d t h e n l a t e r f r o m t h e A10 h y b r i d o m a . I n t e r e s t i n g l y , B16G was n o t A g - s p e c i f i c i t s e l f b u t a l l o w e d t h e s e l e c t i o n o f Ts h y b r i d o m a s a n d t h e r e c o v e r y o f a p p a r e n t l y A g - s p e c i f i c T s F f r o m t h e s e c e l l s . When B l 6 G - r e a c t i v e m a t e r i a l was a n a l y z e d u s i n g SDS-PAGE, u n d e r r e d u c i n g c o n d i t i o n s , p r o t e i n s o f s i m i l a r M r were r e c o v e r e d f r o m e i t h e r t h e A10 o r F d l l h y b r i d o m a s . A s a c o r o l l a r y o f t h e d u a l b i n d i n g o f F d l l F t o F d a n d B16G, t h e SDS-PAGE a p p e a r a n c e o f F d l l F was i d e n t i c a l w h e t h e r i t was e l u t e d f r o m t h e F d - o r B l 6 G - S e p h a r o s e c o l u m n . The m a j o r a d v a n t a g e t h a t t h e F d l l h y b r i d o m a o f f e r e d o v e r t h e A10 h y b r i d o m a was t h e a v a i l a b i l i t y o f t h e n o m i n a l A g o f F d l l . I n d e e d , t h e s t u d y o f t h e A g - b i n d i n g p r o p e r t i e s o f F d l l F i s a m a j o r s u b j e c t o f t h i s t h e s i s . 28 P u r p o s e o f t h e I n v e s t i g a t i o n T h a t F d l l F was s u p p r e s s i v e i n v i v o a n d a b l e t o b i n d A g s u g g e s t e d i t m i g h t be c l a s s i f i e d a l o n g w i t h t h e p r e v i o u s l y m e n t i o n e d g r o u p o f p r o t e i n s known as T s F . T h i s p r o m p t e d us t o s t u d y F d l l F f u r t h e r w i t h t h e hope o f p r o v i d i n g a d d i t i o n a l i n s i g h t i n t o t h e n a t u r e o f T c e l l - d e r i v e d , A g - b i n d i n g m o l e c u l e s . F u r t h e r m o r e , an i n v e s t i g a t i o n i n t o t h e p r o p e r t i e s o f F d l l F was l i k e l y t o c o n t r i b u t e t o t h e u n d e r s t a n d i n g o f t h e a n t i - F d r e s p o n s e . The p r e s e n t work c o m p r i s e s a d e t a i l e d e x a m i n a t i o n o f F d l l F a t t h e i m m u n o c h e m i c a l , b i o l o g i c a l , a n d b i o c h e m i c a l l e v e l s w i t h e m p h a s i s on d e s c r i b i n g t h e A g - b i n d i n g p r o p e r t i e s o f F d l l F . 2 9 MATERIALS AND METHODS B u f f e r s a n d S o l u t i o n s The c o m p o s i t i o n s o f t h e b u f f e r s a n d s o l u t i o n s u s e d a r e l i s t e d i n t h e A p p e n d i x , THE COMPOSITIONS OF BUFFERS AND SOLUTIONS. E x p e r i m e n t a l A n i m a l s B10.BR, DBA/2, B10.D2 a n d ( B a l b / c x DBA/2)F 1 m i c e w ere p u r c h a s e d f r o m t h e A n i m a l C a r e U n i t o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . They were u s e d b e t w e e n 6 a n d 12 weeks o f a g e . C e l l L i n e s The d e v e l o p m e n t o f t h e F d l l h y b r i d o m a a n d a p r e l i m i n a r y d e s c r i p t i o n o f t h e i m m u n o l o g i c a l p r o p e r t i e s o f i t s s e c r e t e d p r o d u c t , F d l l F h a v e b e e n d e s c r i b e d p r e v i o u s l y ( S t e e l e , Chu, Stammers, N o r t h a n d L e v y 1 9 8 7 ) . The F d l l h y b r i d o m a was o r i g i n a l l y made i n 1985 a n d h a s r e m a i n e d s t a b l e w i t h r e s p e c t t o i t s p r o d u c t i o n o f F d l l F f o r a t l e a s t f o u r y e a r s . A s a s s e s s e d by f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r (FACS) a n a l y s i s , - t h e F d l l h y b r i d o m a was f o u n d t o be Thy 1 + a n d F 2 3 . 1 - " The h y b r i d o m a d k e x p r e s s e s b o t h H-2 a n d H-2 A g . The DC10 h y b r i d o m a was d e r i v e d b y f u s i o n o f t h e BW5147 c e l l l i n e w i t h T c e l l s o b t a i n e d f r o m 30 DBA/2 m i c e p r i m e d t o a l e u k e m i a - a s s o c i a t e d Ag a n d was u s e d a s a c o n t r o l i n t h e s e s t u d i e s . The h y p o x a n t h i n e g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e n e g a t i v e BW5147 c e l l l i n e was o b t a i n e d f r o m t h e A m e r i c a n Type C u l t u r e C o l l e c t i o n ( R o c k v i l l e , MD). Two c e l l l i n e s p r o d u c i n g t h e a n t i - F d mAb, 16a a n d 6-90, r e s p e c t i v e l y , w e r e c r e a t e d i n t h i s l a b o r a t o r y a n d h a v e b e e n p r e v i o u s l y d e s c r i b e d (Weaver, S i k o r a , a n d L e v y 1982; S i k o r a , Weaver, a n d L e v y 1 9 8 2 ) . The c e l l l i n e p r o d u c i n g t h e T s F - s p e c i f i c mAb, B16G was a l s o p r o d u c e d i n t h i s l a b o r a t o r y ( M a i e r , Stammers, a n d L e v y 1 9 8 5 ) . The BW5147, DC10 a n d F d l l c e l l l i n e s w e r e t e s t e d b y t h e t i s s u e c u l t u r e s e r v i c e o f t h e A m e r i c a n Type C u l t u r e C o l l e c t i o n a n d shown t o be m y c o p l a s m a - f r e e . A l l c e l l s w e r e m a i n t a i n e d i n D u l b e c c o ' s M o d i f i e d E a g l e ' s medium ( G i b c o , G r a n d I s l a n d , NY) c o n t a i n i n g 10% f e t a l c a l f s e r u m (FCS, CPSR-3, S i g m a , S t . L o u i s , MO), a t 37°C, i n a 10% C 0 2 i n c u b a t o r . C o n v e n t i o n a l a n d M o n o c l o n a l A n t i b o d i e s The mAb, 16a, 6-90 a n d B16G w e r e r a i s e d as a s c i t e s b y i n j e c t i n g a p p r o x i m a t e l y 5 X 10^ c e l l s i n t r a p e r i t o n e a l l y i n t o ( B a l b / c X D B A / 2 ) F ^ a n i m a l s . T h r e e t o f i v e d a y s p r i o r t o r e c e i v i n g t h e i n j e c t i o n o f c e l l s , t h e m i c e were p r e t r e a t e d w i t h an i n t r a p e r i t o n e a l i n j e c t i o n o f 0.5 ml o f 2 ,6,10,14-T e t r a m e t h y l p e n t a d e c a n e ( P r i s t a n e , A l d r i c h , M i l w a u k e e , W I ) . I m m e d i a t e l y b e f o r e i n o c u l a t i o n w i t h c e l l s , t h e m i c e r e c e i v e d 250 r a d s o f gamma r a d i a t i o n ( G a m m a - c e l l , A t o m i c E n e r g y o f C a n a d a , O t t a w a , O n t . ) . T a p p i n g o f t h e a s c i t i c f l u i d was i n i t i a t e d a b o u t 10 d a y s a f t e r t h e i n t r o d u c t i o n o f c e l l s . I m m e d i a t e l y a f t e r t h e c o l l e c t i o n o f f l u i d , t h e a s c i t i c c e l l s a n d d e b r i s w e r e p e l l e t e d a t 400 g f o r 10 m i n . The c e l l s w e r e e i t h e r r e s u s p e n d e d i n PBS a n d u s e d t o i n o c u l a t e a d d i t i o n a l m i c e , o r a d d e d t o d i m e t h y l s u l f o x i d e a n d FCS a n d f r o z e n i n l i q u i d n i t r o g e n . The s u p e r n a t a n t (SN) was p o o l e d a n d s t o r e d a t 4°C. When a p p r o x i m a t e l y 25 m l o f SN was c o l l e c t e d , an e q u a l v o l u m e o f PBS was a d d e d t o i t . To p r e c i p i t a t e t h e gamma g l o b u l i n f r a c t i o n , s a t u r a t e d ( N H ^ ^ S O ^ was a d d e d d r o p - w i s e , w i t h c o n s t a n t s t i r r i n g , t o t h e mAb s o l u t i o n t o a c h i e v e 5 0 % s a t u r a t i o n a t room t e m p e r a t u r e . The s o l u t i o n was s t i r r e d a t 4°C o v e r n i g h t t o c o m p l e t e t h e p r e c i p i t a t i o n o f mAb. The p r e c i p i t a t e was s p u n a t 9000 g f o r 30 m i n a t 4°C. F o l l o w i n g c e n t r i f u g a t i o n , t h e p e l l e t was g e n t l y r e s u s p e n d e d i n PBS t o a v o l u m e e q u a l t o t h e o r i g i n a l s u p e r n a t a n t a n d d i a l y z e d a g a i n s t PBS ( 2 x 2 1 c h a n g e s ) , a t 4°C u s i n g 14,000 M r c u t - o f f d i a l y s i s t u b i n g ( S p e c t r u m , L o s A n g e l e s , C A ) . F o l l o w i n g d i a l y s i s , t h e mAb s o l u t i o n was d i v i d e d i n t o 50 | i l , 1 a n d 5 ml a l i q u o t s a n d f r o z e n a t -70'C. M o n o c l o n a l Ab p r e p a r a t i o n s were t h a w e d i m m e d i a t e l y b e f o r e u s e . A f f i n i t y - p u r i f i e d CAMAL-1, a mAb s p e c i f i c f o r a l e u k e m i a -a s s o c i a t e d A g , was u s e d a s a c o n t r o l p r e p a r a t i o n , a n d was s u p p l i e d b y J o a n S h e l l a r d . A f f i n i t y - p u r i f i e d B10.BR a n t i - F d Ab was u s e d a s a s t a n d a r d f o r t h e measu r e m e n t o f Ab. A n t i s e r u m was p r e p a r e d a n d p a s s e d o v e r a F d - S e p h a r o s e c o l u m n . A f t e r w a s h i n g t h e c o l u m n , Ab was 32 e l u t e d u s i n g 0.1 M HC1, n e u t r a l i z e d , d i a l y z e d a g a i n s t PBS a n d t h e n made i n t o a l i q u o t s w h i c h were f r o z e n a t -20'C. The p r o t e i n c o n c e n t r a t i o n o f t h e Ab s o l u t i o n was m e a s u r e d a c c o r d i n g t o a m e t h o d m o d i f i e d f r o m L o w r y e t a l . (1951) ( s e e b e l o w f o r d e t a i l s ) . A n t i g e n s F d i s o l a t e d f r o m C. p a s t e u r i a n u m ( t y p e V, F 7 6 2 9 , S i g m a ) , KLH ( 3 7 4 8 0 5 , C a l b i o c h e m , L a J o l l a , C A ) , b o v i n e s e r u m a l b u m i n (BSA, F r a c t i o n V, S i g m a ) , l y s o z y m e ( L y s f 8D8, W o r t h i n g t o n , F r e e h o l d , NJ) a n d c y t o c h r o m e C (Type I I I , C-2506, S i g m a ) , w e re r e c o n s t i t u t e d w i t h d i s t i l l e d w a t e r t o a c o n c e n t r a t i o n o f 1 mg/ml, a l i q u o t e d , a n d s t o r e d a t -20°C. OFd, N-, a n d C - m o l e c u l e w e re p r e p a r e d f r o m n a t i v e F d t h a t h a d b e e n p r e v i o u s l y d e n a t u r e d w i t h t r i c h l o r o a c e t i c a c i d ( T C A - F d ) . TCA-Fd was p r o d u c e d by a d d i n g an e q u a l v o l u m e o f 2 0 % TCA t o a F d s o l u t i o n a t 1 mg/ml. P r e c i p i t a t i o n was a l l o w e d t o c o n t i n u e f o r 1 h r on i c e . I n s o l u b l e p r o t e i n was p e l l e t e d a t 11,600 g f o r 10 m i n . The p e l l e t was w a s h e d o n c e w i t h i c e - c o l d 1 0 0 % e t h a n o l . A f t e r c e n t r i f u g a t i o n , t h e p e l l e t was d r i e d u n d e r vacuum. C - m o l e c u l e was p r e p a r e d by d i s s o l v i n g 750 ug o f TCA-Fd i n 720 p i o f 46 mM T r i s - H C l b u f f e r , pH 8.1, s u p p l e m e n t e d w i t h 11.5 mM C a C l 2 . T r y p s i n (TRTPCK, W o r t h i n g t o n ) , a t a 1:40 enyzme t o p r o t e i n r a t i o (w/w; 18.75 u.g i n 30 p i o f 1 mM HC1) was a d d e d a n d t h e d i g e s t i o n was c o n t i n u e d o v e r n i g h t a t room t e m p e r a t u r e w i t h g e n t l e s t i r r i n g i n a s e a l e d v i a l . N - m o l e c u l e was p r e p a r e d by 33 resuspending 750 ug of TCA-Fd i n 720 ul of 25 mM Tris-HCl buffer, pH 7.5, supplemented with 0.5 M NaCl. Carboxypeptidase A (COAPMS, Worthington) was added at a 1:25 enzyme to protein r a t i o (w/w; 30 ug i n 40 ul of 10% LiCl) and digestion was continued overnight at room temperature with gentle s t i r r i n g i n a sealed v i a l . Following overnight incubation, both digests were aliquoted and stored at -20"C. OFd was prepared as described by Tanaka et a l . (1964) by resuspending 750 ug of TCA-Fd i n 33 ul of formic acid and 67 ul of performic acid. Performic acid was prepared by mixing nine parts of formic acid with one part of 30% ^2^2 f o r ^ h r a t r o o m temperature. The Fd solution was reacted for 2.5 hr at -10"C, maintained at t h i s temperature with a 5 M NaCl-dry ice bath. The reaction was stopped by l y o p h i l i z i n g the protein. Enzyme-linked Immunosorbent Assay The amount of anti-Fd Ab present i n the spleen c e l l culture SN was measured using ELISA. Polystyrene ELISA wells (Immulon I, Dynatech, Alexandria, VA) were coated with 100 ul of Fd solution prepared at 1 ug/ml i n bicarbonate buffer, overnight, at 37'C, under humid conditions. Between each incubation step, the plates were washed three times using PBS-Tween. After washing, 100 ul of SN were added to the wells and allowed to react for 2 hr at 37*C under humid conditions. The plates were washed again and 100 ul of rabbit anti-mouse immunoglobulin-alkaline phosphatase 34 (RaMIg-AP, J a c k s o n , West G r o v e , PA) , u s e d a t 2 u g / m l , d i l u t e d i n PBS-Tween, were a d d e d a n d a l l o w e d t o r e a c t f o r 1 h r u n d e r t h e same c o n d i t i o n s a s a b o v e . A f t e r w a s h i n g , 100 u l o f p -n i t r o p h e n y l p h o s p h a t e s u b s t r a t e (one t a b l e t / 5 m l b u f f e r , 104-105, Sigma) i n d i e t h a n o l a m i n e b u f f e r were a d d e d . F d l l F was a l s o m e a s u r e d u s i n g s o l i d - p h a s e E L I S A . F d - c o a t e d p l a t e s w e r e p r e p a r e d a s a b o v e . F d l l F o r c o n t r o l m a t e r i a l a t v a r i o u s c o n c e n t r a t i o n s d i l u t e d i n PBS, o r f r a c t i o n a t e d m a t e r i a l c o n t a i n i n g F d l l F w e r e a d d e d i n 100 u l v o l u m e s t o F d - c o a t e d w e l l s f o r 2 h r a t 37'C u n d e r h u m i d c o n d i t i o n s . A f t e r w a s h i n g , 100 u l o f t h e T s F - s p e c i f i c mAb, B16G, d i l u t e d i n PBS-Tween, was a d d e d a t 18 u g / m l , a n d i n c u b a t e d f o r 1 h r a t t h e c o n d i t i o n s d e s c r i b e d a b o v e . RaMIg-AP a n d s u b s t r a t e were u s e d as m e n t i o n e d p r e v i o u s l y . The i n h i b i t i o n o f F d l l F b i n d i n g t o F d - c o a t e d w e l l s b y f l u i d -p h a s e F d was m e a s u r e d u s i n g E L I S A . V a r i o u s c o n c e n t r a t i o n s o f F d , i t s a n a l o g s , o r t h e c o n t r o l , c y t o c h r o m e C d i l u t e d i n PBS, were a d d e d t o an e q u a l v o l u m e o f F d l l F s o l u t i o n , u s e d a t a c o n c e n t r a t i o n o f 16 u g / m l . The m i x t u r e was g e n t l y s h a k e n a n d i n c u b a t e d a t 4°C f o r 2 h r . The amount o f f r e e F d l l F r e m a i n i n g i n s o l u t i o n was t h e n d e t e r m i n e d u s i n g t h e E L I S A d e s c r i b e d a b o v e . F o r a l l t h r e e i m m u n o a s s a y s , t h e s u b s t r a t e was d e v e l o p e d f o r 1 t o 3 h r a t 37'C u n d e r h u m i d c o n d i t i o n s . The p l a t e s w e r e r e a d u s i n g a n a u t o m a t e d E L I S A r e a d e r ( M u l t i s k a n , F l o w , M i s s i s s a u g a , Ont.) s e t a t 405 nm. E a c h E L I S A d e t e r m i n a t i o n was p e r f o r m e d i n t r i p l i c a t e a n d t h e a v e r a g e v a l u e was u s e d f o r d a t a c a l c u l a t i o n s . 35 I m m u n i z a t i o n o f A n i m a l s B10.D2 a n i m a l s were i m m u n i z e d t o F d u s i n g C - m o l e c u l e f o l l o w e d b y a b o o s t w i t h F d . The m i c e w ere p r i m e d w i t h 50 ug o f C-m o l e c u l e i n PBS p r e p a r e d as a 1:1 ( v / v ) e m u l s i o n w i t h c o m p l e t e F r e u n d ' s a d j u v a n t (H37 Ra, D i f c o , D e t r o i t , MI) i n a t o t a l v o l u m e o f 0.1 m l . The i m m u n i z a t i o n was a d m i n i s t e r e d s u b c u t a n e o u s l y ( s . c . ) i n t h e f l a n k on t h e v e n t r a l s i d e . A f t e r 21 d a y s , t h e s e c o n d a r y i m m u n i z a t i o n was g i v e n i n an i d e n t i c a l manner w i t h t h e e x c e p t i o n t h a t 50 ug o f F d w e r e . p r e p a r e d u s i n g i n c o m p l e t e F r e u n d ' s a d j u v a n t ( D i f c o ) a n d t h e s i t e o f i n j e c t i o n was i n t h e o p p o s i t e f l a n k t o t h a t o f t h e f i r s t i n j e c t i o n . Hyperimmune B10.BR a n i m a l s w e re p r e p a r e d i n a s i m i l a r manner w i t h t h e e x c e p t i o n t h a t b o t h t h e p r i m a r y a n d s e c o n d a r y i m m u n i z a t i o n s were w i t h F d . A n i m a l s w e re i m m u n i z e d w i t h 50 ug o f l y s o z y m e f o l l o w e d by a b o o s t 21 d a y s l a t e r . A n t i g e n e m u l s i o n a n d r o u t e o f a d m i n i s t r a t i o n w e r e a s f o r F d . C o - i m m u n i z a t i o n o f DBA/2 a n i m a l s w i t h F d a n d KLH was a c c o m p l i s h e d by g i v i n g t h e Ag s e p a r a t e l y t o o p p o s i t e s i d e s o f t h e m i c e . KLH was a d m i n i s t e r e d a t 50 ug/mouse. E n r i c h m e n t o f F d l l F u s i n g I m m u n o a f f i n i t y C h r o m a t o g r a p h y F d l l F - e n r i c h e d m a t e r i a l was r e c o v e r e d f r o m t h e SN u s i n g i m m u n o a f f i n i t y c h r o m a t o g r a p h y . C u l t u r e s o f F d l l h y b r i d o m a c e l l s were grown to a density of about 5 X 10 to 1 X 10 /ml. Typically, 2 1 of c e l l s were grown for each l o t of F d l l F . Supernatant was recovered by centrifugation using a Sorvall GS-3 rotor (Dupont, Newtown CT) spun at 4,300 g for 20 min. Supernatant was pumped onto a column (2.5 x 23 cm, Econo, Bio-Rad, Richmond, CA) at a flow rate of 60 ml/hr, containing 5 ml of B16G-Sepharose CL-4B beads. B16G was coupled to Sepharose CL-4B (Pharmacia, Montreal, P.Q.) using CNBr (F964-5, Baker, P h i l l i p s b u r g , NJ) as described by Cuatrecasas (1970). However, just before SN was pumped onto the column, the beads were preconditioned with 5 ml of 0.1 M HC1 followed by washing with 200 ml of PBS. Once the SN had been loaded, the beads were washed with PBS at a rate of 100 ml/hr u n t i l the absorbance (280 nm) of the effluent was less the 0.005. Protein was eluted using 10 ml of 0.01 M HC1. The entire 10 ml of eluate was c o l l e c t e d and then neutralized by adding 1.5 M Tris-HCl, pH 7.5, 100 ul at a time. This was continued u n t i l the pH of the solution reached 7.5. Following ne u t r a l i z a t i o n , the solution was immediately dialyzed against PBS and then concentrated using microconcentrators (30k Mr cut-off, Centricon-30, Amicon, Danvers, MA). After concentration, aliquots of FdllF-enriched material were prepared using polypropylene tubes and then frozen at -70"C. If the sample was to be used the next day for preparative, non-reducing SDS-PAGE or high performance gel f i l t r a t i o n analysis, then i t was concentrated to 500 ul or 200 ul, respectively, and stored at 4°C u n t i l use. Samples used for 37 b i o l o g i c a l t e s t i n g i n v i t r o w e r e d i a l y z e d a g a i n s t RPMI 1640 i n s t e a d o f PBS. C o n t r o l m a t e r i a l was p r e p a r e d c o n c u r r e n t l y w i t h F d l l F u s i n g a d u p l i c a t e s e t o f e q u i p m e n t . A l l p r o c e d u r e s , i n c l u d i n g a f f i n i t y c h r o m a t o g r a p h y , were c o n d u c t e d a t 4°C. The p r o t e i n c o n c e n t r a t i o n o f t h e B 1 6 G - e l u t e d m a t e r i a l was d e t e r m i n e d b y a m o d i f i c a t i o n o f t h e m e t h o d o f L o w r y e t a l . ( 1 9 5 1 ) . The p r o t e i n s a m p l e was d i s s o l v e d i n 200 u l o f d i s t i l l e d w a t e r . T h i s s o l u t i o n was t h e n m i x e d w i t h 200 u l o f 1 N NaOH. Two s e p a r a t e s t o c k s o l u t i o n s o f 2% Na2CO-3 i n d i s t i l l e d w a t e r a n d 0.5% C u S O ^.S^O i n 1% s o d i u m c i t r a t e were p r e p a r e d a n d p o r t i o n s o f e a c h w e re m i x e d i n a r a t i o o f 49 p a r t s (Na2C03 s o l u t i o n ) t o 1 p a r t (CuSO^ s o l u t i o n ) , v / v . Two m i l l i t r e s o f t h i s s o l u t i o n were a d d e d t o t h e p r o t e i n m i x t u r e , s h a k e n i m m e d i a t e l y a n d a f t e r e x a c t l y 5 m i n , 200 u l o f 1 N P h e n o l r e a g e n t ( F i s h e r , F a i r l a w n , NJ) w e r e a d d e d . A g a i n , t h e t u b e was s h a k e n i m m e d i a t e l y . The r e a c t i o n was t h e n d e v e l o p e d f o r 30 m i n . A t t h e e n d o f t h i s t i m e , t h e c o l o u r o f t h e s o l u t i o n was r e a d a t 750 nm. A s e r i e s o f BSA s o l u t i o n s , c o n t a i n i n g f r o m 2.5 t o 50 ug o f p r o t e i n , was u s e d f o r t h e s t a n d a r d c u r v e . I n v i t r o M e a s u r e m e n t o f F d l l F S u p p r e s s i v e A c t i v i t y Fd-immune B10-D2 m i c e were k i l l e d 13 t o 15 d a y s f o l l o w i n g t h e b o o s t e r i m m u n i z a t i o n w i t h F d . S p l e e n s w e re r e m o v e d a s e p t i c a l l y a n d made i n t o s i n g l e c e l l s u s p e n s i o n s b y p a s s a g e t h r o u g h a w i r e s c r e e n . The c e l l s w e r e w a s h e d t h r e e t i m e s u s i n g RPMI 1640 ( 1 0 -38 601-24, F l o w , M c l e a n , VA) s u p p l e m e n t e d w i t h 10% FCS a n d p e n i c i l l i n a n d s t r e p t o m y c i n (100 u n i t s / m l a n d 100 u g / m l , r e s p e c t i v e l y , G i b c o ) a n d s p u n a t 400 g. a) " O r i g i n a l " p r o t o c o l : A f t e r w a s h i n g , t h e c e l l s w e r e p u l s e d w i t h F d w h i c h h a d b e e n p r e v i o u s l y s t e r i l i z e d u s i n g a 0.22 um f i l t e r ( 4 1 92, Gelman, Ann A r b o r , M I ) . P r i o r t o p u l s i n g , a b o u t 1 7 X 10 c e l l s w e r e p l a c e d i n t o a s e p a r a t e t u b e a n d d i d n o t r e c e i v e F d . P u l s i n g c o n d i t i o n s w e re 250 ug F d / 1 . 5 ml m e d i u m / s p l e e n . The p u l s i n g medium was s u p p l e m e n t e d w i t h FCS t o a f i n a l c o n c e n t r a t i o n o f 2 0 % . The p u l s e d a n d u n p u l s e d c e l l s were c u l t u r e d a t 37'C i n a 10% CO2 a t m o s p h e r e f o r 2 h r u s i n g p o l y p r o p y l e n e t u b e s . The c e l l s w e r e r e s u s p e n d e d g e n t l y t w i c e d u r i n g t h e 2 h r p e r i o d . A f t e r w a s h i n g , t h e c e l l s w e r e a d j u s t e d t o a c o n c e n t r a t i o n o f 2.5 X 10 / m l , s u p p l e m e n t e d w i t h 2 - m e r c a p t o e t h a n o l (M 6250, Sigma) t o a f i n a l c o n c e n t r a t i o n o f 50 uM, a n d p l a t e d o u t as 2 ml c u l t u r e s . F d l l F o r c o n t r o l m a t e r i a l was a d d e d a t t h i s t i m e . The c e l l s were c u l t u r e d f o r s e v e n d a y s a t 37°C i n a 10% C 0 2 a t m o s p h e r e . C u l t u r e SN w e r e c a r e f u l l y r e m o v e d on d a y 7 a n d u n d i l u t e d s a m p l e s w e r e m e a s u r e d f o r Ab c o n t e n t u s i n g E L I S A . b) " R e v i s e d " p r o t o c o l : C e l l s w e r e p u l s e d i n i t i a l l y w i t h F d l l F o r c o n t r o l m a t e r i a l , i n s t e a d o f F d . A t t h e e n d o f 2 h r , F d was a d d e d a t a c o n c e n t r a t i o n a s d e s c r i b e d a b o v e . P u l s i n g was c o n t i n u e d f o r a n o t h e r 2 h r . A f t e r w a s h i n g , t h e c e l l s w e r e t r e a t e d a s a b o v e b u t n o t c o - c u l t u r e d w i t h F d l l F . P e r c e n t s u p p r e s s i o n f o r b o t h p r o t o c o l s was c a l c u l a t e d u s i n g t h e f o r m u l a : 39 ( (Ab l e v e l o f F d - p u l s e d c u l t u r e s - Ab l e v e l o f F d l l F c u l t u r e s ) H-Ab l e v e l o f F d - p u l s e d c u l t u r e s ) x 100% The Ab l e v e l o f t h e u n p u l s e d c u l t u r e was u s e d a s t h e b a c k g r o u n d a n d s u b t r a c t e d f r o m t h e l e v e l s o f t h e F d - p u l s e d a n d F d l l F c u l t u r e s p r i o r t o t h e c a l c u l a t i o n o f p e r c e n t s u p p r e s s i o n . S u p p r e s s i o n o f t h e Serum A n t i b o d y R e s p o n s e u s i n g F d l l F DBA/2 m i c e were p r i m e d w i t h C - m o l e c u l e a n d t h e n c o - i m m u n i z e d w i t h F d a n d KLH 21 d a y s l a t e r . The a n i m a l s w e r e t h e n b l e d f r o m t h e t a i l 21 d a y s l a t e r a n d r a n k e d , u s i n g E L I S A , a s " h i g h " r e s p o n d e r s (>0.5) o r " l o w " r e s p o n d e r s (<0.5) t o F d . They were d i v i d e d i n t o F d l l F , A 10F, a n d PBS " t r e a t m e n t " g r o u p s w i t h e a c h g r o u p c o n t a i n i n g an e q u a l number o f " h i g h " a n d " l o w " r e s p o n d e r s . M i c e w e re t h e n g i v e n e i t h e r 10 ug F d l l F , 10 ug A l O F o r PBS i n t r a v e n o u s l y ( i . v . ) , a l l o w e d t o r e c o v e r f o r s e v e r a l h o u r s , a n d b o o s t e d w i t h F d a n d KLH. They were b l e d s e v e n d a y s l a t e r a n d t h e a n t i - F d a n d a n t i - K L H Ab r e s p o n s e s w e re a s s e s s e d as p a i r e d s a m p l e s f o r e a c h i n d i v i d u a l a n i m a l . P r i o r t o t h e i r a d d i t i o n t o t h e E L I S A p l a t e s , s e r u m s a m p l e s were d i l u t e d a t 1/100 a n d 1/200 u s i n g PBS-Tween. The d a t a a t 1/100 w e re r e c o r d e d a s an " X - Y " p l o t ( s e e R E S U L T S ) . S i m i l a r v a l u e s were o b t a i n e d a t b o t h d i l u t i o n s . 40 S o d i u m D o d e c y l S u l p h a t e P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s B 1 6 G - e l u t e d s a m p l e s a n d i n d i v i d u a l f r a c t i o n s p r e p a r e d f r o m F P L C a n d p r e p a r a t i v e SDS-PAGE were a n a l y z e d u s i n g 10% (% T) p o l y a c r y l a m i d e g e l s b a s e d on t h e m e t h o d o f L a e m m l i (1970) . The m i n i - P r o t e a n I I s l a b g e l a p p a r a t u s ( B i o - R a d ) was u s e d a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . S a m p l e s w e r e c o n c e n t r a t e d by a c e t o n e p r e c i p i t a t i o n a n d t h e n s o l u b i l i z e d i n SDS s a m p l e b u f f e r c o n s i s t i n g o f 10% g l y c e r o l ( w / v ) , 2% SDS (w/v; B 44244, BDH, T o r o n t o , O n t . ) , 62.5 mM T r i s - H C l b u f f e r , b r o m o p h e n o l b l u e a n d 10 mM d i t h i o t h r e i t o l (DTT, B i o - R a d ) . P r i o r t o l o a d i n g , t h e s a m p l e s w e r e h e a t e d t o 100'C f o r 4 m i n . B e t w e e n 1 t o 2 ug o f p r o t e i n w e r e l o a d e d p e r l a n e . When a p r o t e i n d e t e r m i n a t i o n was n o t made, b e t w e e n 5 t o 10% o f t h e t o t a l v o l u m e o f t h e u n c o n c e n t r a t e d s a m p l e was u s e d f o r t h e a n a l y s i s . See APPENDIX f o r f u r t h e r d e t a i l s a b o u t g e l b u f f e r s a n d s o l u t i o n s . P r o t e i n b a n d s were v i s u a l i z e d u s i n g s i l v e r s t a i n i n g b a s e d on t h e m e t h o d o f Wray e t a l . ( 1 9 8 1 ) . The g e l was f i x e d i n 100 m l o f 5 0 % m e t h a n o l : 1 0 % g l a c i a l a c e t i c a c i d f o r a minimum o f 30 m i n . A f t e r t h e r e m o v a l o f f i x a t i v e , t h e g e l was r e h y d r a t e d i n 10% g l a c i a l a c e t i c a c i d : 1 0 % m e t h a n o l . The p r o c e s s was a s s i s t e d b y h e a t i n g t h e g e l i n a m i c r o w a v e o v e n f o r 1 m i n . R e h y d r a t i o n was c o n t i n u e d b y t h e r e m o v a l o f t h e m e t h a n o l - a c e t i c a c i d m i x t u r e and r e p l a c e m e n t w i t h d i s t i l l e d w a t e r . The g e l was r e h y d r a t e d f o r a n o t h e r 5 m i n a t room t e m p e r a t u r e . A f t e r t h e w a t e r was r e m o v e d , t h e g e l was e q u i l i b r a t e d i n 100 m l o f d i s t i l l e d w a t e r c o n t a i n i n g 33 u l o f 100 mM DTT. A g a i n , t h e g e l was h e a t e d i n a microwave oven f o r 1 min. The s o l u t i o n was r e p l a c e d w i t h 100 ml o f d i s t i l l e d w a ter c o n t a i n i n g 1 ml o f 10% AgNOj (S 6506, Sigma). T h i s was c o n t i n u e d f o r 15 min a t room t e m p e r a t u r e . D u r i n g t h i s t i m e , d e v e l o p i n g r e a g e n t , c o n s i s t i n g o f 40 ml 15% ^ 2 0 0 ^ and 200 u l o f 37% formaldehyde (F-79B, F i s h e r ) i n 200 ml o f d i s t i l l e d w a t e r , was p r e p a r e d . The AgNO^ s o l u t i o n was p o u r e d o f f and t h e g e l was r i n s e d w i t h about 20 ml o f d i s t i l l e d w a t e r . Immediately f o l l o w i n g t h e removal of t h e water, t h e g e l was r i n s e d w i t h about 20 ml o f d e v e l o p i n g r e a g e n t . T h i s was r e p l a c e d w i t h t h e remainder of t h e d e v e l o p i n g r e a g e n t . The r e a c t i o n was c o n t i n u e d u n t i l t h e bands began t o appear. The r e a c t i o n was s t o p p e d w i t h 20 ml o f 2.3 M c i t r i c a c i d . L a s t l y , g e l s were t r a n s f e r r e d t o d i s t i l l e d water f o r s t o r a g e u n t i l d r i e d u s i n g a s l a b g e l d r y e r ( B i o - R a d ) . H i g h Performance G e l F i l t r a t i o n B 1 6 G - e l u t e d F d l l F was f r a c t i o n a t e d by h i g h performance g e l f i l t r a t i o n u s i n g t h e Pharmacia chromatography system (FPLC). Samples were c o n c e n t r a t e d t o a volume of 200 u l , f i l t e r e d u s i n g a 0.22 um f i l t e r (SLGVL04S, M i l l i p o r e , B e d f o r d , MA) and l o a d e d onto a Superose 12 column (HR 10/30, Pharmacia) a t a f l o w r a t e o f 0.25 ml/min. Between 150 t o 200 ug o f t o t a l p r o t e i n were l o a d e d p e r r u n . Absorbance at 280 nm was r e c o r d e d u s i n g t h e FPLC i n t e g r a t o r u n i t . M a t e r i a l was e l u t e d w i t h PBS and 48 0.5 ml f r a c t i o n s , 42 e q u i v a l e n t t o one c o l u m n v o l u m e , were c o l l e c t e d . The c o l u m n was c a l i b r a t e d u s i n g t h e f o l l o w i n g M r m a r k e r s : t h y r o g l o b u l i n , 669k M ; I g G , 150k M r; BSA, 66k M r; c y t o c h r o m e C, 12.4k M r F r a c t i o n s w e r e h e l d a t 4°C u n t i l a s s a y e d f o r A g - b i n d i n g a c t i v i t y . F r a c t i o n s a s s a y e d f o r b i o l o g i c a c t i v i t y w e r e s t e r i l i z e d b y f i l t r a t i o n u s i n g a 0.22 um f i l t e r ( M i l l i p o r e ) . P r e p a r a t i v e S o d i u m D o d e c y l S u l p h a t e P o l y a c r y l a m i d e G e l  E l e c t r o p h o r e s i s B 1 6 G - e l u t e d F d l l F was a l s o f r a c t i o n a t e d u s i n g p r e p a r a t i v e SDS-PAGE. The P r o t e a n I I s l a b g e l a p p a r a t u s ( B i o - R a d ) was u s e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n s t r u c t i o n s . Two h u n d r e d m i c r o g r a m s o f F d l l F a n d DC10 m a t e r i a l w e r e s o l u b i l i z e d i n SDS s a m p l e b u f f e r c o n s i s t i n g o f 10% g l y c e r o l ( w / v ) , 2% SDS ( w / v ) , 62.5 mM T r i s - H C l b u f f e r a n d b r o m o p h e n o l b l u e . The s a m p l e s were r e s o l v e d i n a d j a c e n t l a n e s u s i n g a 12% (% T) p o l y a c r y l a m i d e g e l . See APPENDIX f o r f u r t h e r d e t a i l s a b o u t g e l b u f f e r s a n d s o l u t i o n s . The g e l s w e r e r u n a t c o n s t a n t c u r r e n t (35 itiA) a n d c o o l e d w i t h c i r c u l a t i n g p o l y e t h y l e n e g l y c o l . E l e c t r o p h o r e s i s was c o n t i n u e d u n t i l t h e d y e f r o n t r e a c h e d t o w i t h i n 1 cm o f t h e e n d o f t h e g e l . A f t e r e l e c t r o p h o r e s i s , t h e g e l l a n e s w e r e c u t i n t o 5 mm s l i c e s a n d e a c h p i e c e was m i n c e d a n d p l a c e d i n t o p o l y p r o p y l e n e t u b e s c o n t a i n i n g 0.75 ml o f 0.05 M NH^HCC^. A f t e r e l u t i o n f o r 18 h a t 4°C, t h e m a t e r i a l was t r a n s f e r r e d t o a m i c r o d i a l y s i s u n i t (1200 MD, BRL, G a i t h e r s b u r g , MD) a n d d i a l y z e d a g a i n s t RPMI 1640 f o r 18 43 h. A s e c o n d r o u n d o f e l u t i o n a n d d i a l y s i s was c a r r i e d o u t a n d t h e e l u a t e s w e r e c o m b i n e d f o r b o t h r e d u c i n g SDS-PAGE a n a l y s i s a n d A g - b i n d i n g E L I S A d e t e r m i n a t i o n s . T w o - d i m e n s i o n a l G e l E l e c t r o p h o r e s i s ( I E F x SDS-PAGE) I n s t r u c t i o n s p r o v i d e d w i t h t h e m i n i - P r o t e a n I I I E F a n d s l a b g e l a p p a r a t u s ( B i o - R a d ) w e re f o l l o w e d . M o d i f i c a t i o n s a r e l i s t e d b e l o w . G l a s s t u b e s f o r t h e f i r s t d i m e n s i o n w e re w a s h e d i n d e t e r g e n t (RBS-35, P i e r c e , R o c k f o r d , I L ) a n d r i n s e d w i t h t a p w a t e r f o l l o w e d b y d i s t i l l e d w a t e r . The t u b e s w e r e t h e n b o i l e d i n 0.IM HC1 f o r 30 m i n a n d r i n s e d w i t h t a p w a t e r , f o l l o w e d b y d i s t i l l e d w a t e r a n d d r i e d . The f i r s t d i m e n s i o n g e l s o l u t i o n was p r e p a r e d b y a d d i n g u r e a (B10290, BDH), H P L C - q u a l i t y w a t e r , a c r y l a m i d e s o l u t i o n , 3, [ ( 3 -c h o l a m i d o p r o p y l ) - d i m e t h y l a m m o n i o ] - 1 - p r o p a n e s u l f o n a t e (CHAPS), a n d a m p h o l i n e s (pH 3.5-10, LKB, P h a r m a c i a ) t o g e t h e r a n d t h e n h e a t i n g t h e m i x t u r e i n a 37'C w a t e r b a t h t o d i s s o l v e t h e u r e a . Any p a r t i c u l a t e m a t t e r was r e m o v e d b y f i l t e r i n g (0.45 urn, 4184, G e l m a n ) . The s o l u t i o n was d e g a s s e d f o r 10 t o 15 m i n u s i n g w a t e r s u c t i o n . I m m e d i a t e l y f o l l o w i n g t h e a d d i t i o n o f ammonium p e r s u l p h a t e (APS; B i o - R a d ) a n d N , N , N ' , N ' - T e t r a m e t h y l -e t h y l e n e d i a m i n e (TEMED; B i o - R a d ) t o t h e g e l s o l u t i o n , t h e t u b e g e l s w e r e c a s t e d a n d a l l o w e d t o p o l y m e r i z e f o r 2 t o 3 h r . P r i o r t o s a m p l e l o a d i n g , d e g a s s e d s o l u t i o n s o f a n o l y t e a n d c a t h o l y t e w e r e p r e p a r e d . E x c e s s u n p o l y m e r i z e d g e l was r e m o v e d f r o m t h e t o p o f t h e s o l i d i f i e d g e l s . Any t u b e s w i t h t r a p p e d a i r b u b b l e s w e r e d i s c a r d e d . Sample r e s e r v o i r s w e r e c o n n e c t e d t o e a c h o f t h e t u b e g e l s a n d f i l l e d w i t h c a t h o l y t e . S a mple ( a b o u t 10 ug i n 20 u l o f s a m p l e b u f f e r ) was l o a d e d u s i n g a m i c r o s y r i n g e b y u n d e r l a y i n g t h e c a t h o l y t e . The s a m p l e was p r o t e c t e d b y t h e a p p l i c a t i o n o f 30 u l o f o v e r l a y b u f f e r . The s a m p l e g e l s w e re f o c u s s e d a l o n g w i t h a t u b e g e l c o n t a i n i n g 6 p i o f M e t h y l -Red m a r k e r ( l m g / m l , M 5768, S i g m a ) , a n d two o t h e r b l a n k g e l s ( u s e d f o r t h e m e a s u r e m e n t o f t h e pH g r a d i e n t ) . F o c u s i n g was c a r r i e d o u t a t 500 V f o r 10 m i n a n d 1500 V f o r 90 m i n . D u r i n g t h e I E F s t a g e , 12% SDS g e l s w e re c a s t e d . T h i r t y m i n u t e s b e f o r e t h e c o n c l u s i o n o f I E F , s t a c k i n g g e l s (5 mm i n l e n g t h ) w e re p o u r e d . F o l l o w i n g p o l y m e r i z a t i o n , t h e w e l l s w e re w a s h e d o u t w i t h d i s t i l l e d w a t e r a n d M r s t a n d a r d s w e re p r e p a r e d f o r l o a d i n g . A t t h e c o m p l e t i o n o f I E F , e a c h t u b e g e l was e x t r u d e d l e n g t h - w i s e i n t o t h e w e l l o f t h e s e c o n d d i m e n s i o n SDS g e l a n d e q u i l i b r a t e d f o r 3 m i n i n 2 x SDS s a m p l e b u f f e r w i t h DTT. M o l e c u l a r w e i g h t s t a n d a r d s w e re l o a d e d a n d e l e c t r o p h o r e s i s was p e r f o r m e d as d e s c r i b e d . See APPENDIX f o r f u r t h e r d e t a i l s a b o u t g e l b u f f e r s a n d s o l u t i o n s . The t h r e e t u b e g e l s w i t h o u t s a m p l e s w ere e x t r u d e d a n d c u t i n t o 1 cm s e c t i o n s a n d e a c h g r o u p o f 3 x 1 cm g e l p i e c e s was p l a c e d i n t o a t u b e c o n t a i n i n g 250 u l o f H P L C - q u a l i t y , d e g a s s e d d i s t i l l e d w a t e r . The a m p h o l i n e s w ere e l u t e d f o r 2 h r a t room t e m p e r a t u r e . pH e s t i m a t e s w e re made by i m m e r s i n g pH t e s t s t r i p s (pH 5 - 1 0 , BDH) i n t o e a c h o f t h e t u b e s . 45 RESULTS R e c o v e r y o f F d l l F f r o m C u l t u r e S u p e r n a t a n t s The SN o f o v e r g r o w n c u l t u r e s o f F d l l h y b r i d o m a c o n t a i n i n g F d l l F , w e r e p a s s e d o v e r a c o l u m n o f S e p h a r o s e CL-4B b e a d s t o w h i c h B16G was c o v a l e n t l y l i n k e d . A f t e r e x t e n s i v e w a s h i n g o f t h e c o l u m n w i t h p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , F d l l F was e l u t e d u s i n g 0.1 N HC1 a n d i m m e d i a t e l y n e u t r a l i z e d w i t h T r i s - H C l . I t was t h e n d i a l y z e d a g a i n s t PBS a n d c o n c e n t r a t e d , d i v i d e d i n t o a l i q u o t s a n d f r o z e n a t -70"C. S a m p l e s were t a k e n f r o m e a c h b a t c h f o r p r o t e i n d e t e r m i n a t i o n , SDS-PAGE a n a l y s i s a n d meas u r e m e n t o f i m m u n o r e a c t i v i t y . C o m p l e t e t i s s u e c u l t u r e medium i n c l u d i n g 10% FCS, o r SN f r o m t h e f u s i o n p a r e n t BW5147 o r DC10 h y b r i d o m a , a n o t h e r BW5147 f u s i o n p r o d u c t o f unknown s p e c i f i c i t y w e r e u s e d t o p r e p a r e c o n t r o l m a t e r i a l w i t h a d u p l i c a t e B 1 6 G - S e p h a r o s e c o l u m n . I m m u n o c h e m i c a l P r o p e r t i e s o f F d l l F The r e c o v e r y o f F d l l F f r o m t h e i m m u n o a d s o r b e n t c o l u m n s c o u l d be m o n i t o r e d u s i n g a s o l i d - p h a s e E L I S A . V a r i o u s c o n c e n t r a t i o n s o f F d l l F a n d DC10 c o n t r o l m a t e r i a l were a d s o r b e d t o E L I S A w e l l s o v e r n i g h t . A f t e r w a s h i n g , B16G a n d an i r r e l e v a n t mAb o f t h e same s u b c l a s s (IgG2a) w ere a d d e d a n d d e v e l o p e d w i t h r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n - a l k a l i n e p h o s p h a t a s e ( R a M I g - A P ) . F i g . 1 shows t h a t when s o l i d - p h a s e m a t e r i a l was a d s o r b e d a t a 46 F i g . 1. B i n d i n g o f B16G t o F d l l F a d s o r b e d t o p o l y s t y r e n e w e l l s . F d l l F a n d DC10 m a t e r i a l w e re a d s o r b e d t o E L I S A w e l l s a n d d e t e c t e d w i t h B16G, u s e d a t 18 u g / m l . The E L I S A was d e v e l o p e d w i t h R a M I g -AP, u s e d a t 0.5 u g / m l . The i r r e l e v a n t mAb, CAMAL-1, u s e d a t t h e same c o n c e n t r a t i o n a s B16G, b o u n d t o n e i t h e r F d l l F o r DC10 m a t e r i a l . (•) F d l l F + B16G; (-) F d l l F + CAMAL-1; (O) DC10 + B16G; (•) DC10 + CAMAL-1. 47 c o n c e n t r a t i o n o f 6.6 u g / m l , B16G r e a c t e d s t r o n g l y w i t h s o l i d -p h a s e F d l l F b u t n o t w i t h DC10 m a t e r i a l . The i r r e l e v a n t mAb, CAMAL-1, d i d n o t b i n d t o e i t h e r t h e F d l l F o r DC10 l a y e r . R e p r e s e n t a t i v e d a t a o f t h r e e e x p e r i m e n t s a r e shown. The E L I S A was a l s o u s e d t o t e s t t h e b i n d i n g o f F d l l F t o s o l i d -p h a s e F d . I n t h i s a s s a y s y s t e m , v a r i o u s c o n c e n t r a t i o n s o f F d l l F w e r e a d d e d t o F d - c o a t e d p l a t e s . The b o u n d F d l l F was t h e n d e t e c t e d w i t h B16G a n d RaMIg-AP. F i g . 2A shows a t y p i c a l c u r v e w h ere v a r i o u s c o n c e n t r a t i o n s o f F d l l F w e r e a d d e d t o F d - c o a t e d p l a t e s . E v i d e n t l y , B16G c o u l d s t i l l r e a c t w i t h F d l l F i n s p i t e o f i t s b i n d i n g t o F d . The n e g a t i v e c o n t r o l o f F i g . 2A i s t h e m e d i u m - d e r i v e d B16G e l u a t e p r e p a r e d c o n c u r r e n t l y w i t h F d l l F . E s s e n t i a l l y no r e a c t i v i t y f r o m t h e c o n t r o l p r e p a r a t i o n was o b s e r v e d . F i g . 2B shows t h a t e l u t e d c o n t r o l m a t e r i a l f r o m c e l l u l a r SN (DC10 SN) a l s o d i d n o t show any r e a c t i v i t y i n t h e a s s a y . Thus f o r any g i v e n F d l l F p r e p a r a t i o n , i t s r e a c t i v i t y i n t h e " s a n d w i c h " E L I S A s u g g e s t e d t h a t b o t h i t s A g - b i n d i n g s i t e a n d B 1 6 G - b i n d i n g s i t e w e r e i s o l a t e d i n t a c t . F i g . 3 i s a c o n t r o l e x p e r i m e n t w here an i r r e l e v a n t mAb was c o m p a r e d t o B16G. B16G a n d CAMAL-1 were u s e d a t e q u i v a l e n t c o n c e n t r a t i o n s o f 18 ug/ml t o d e t e c t F d l l F . I t c a n be s e e n t h a t n o n s p e c i f i c b i n d i n g o f CAMAL-1 f o r F d l l F b e g a n o n l y when h i g h c o n c e n t r a t i o n s o f m a t e r i a l w e re e m p l o y e d ( i e . > 4 u g / m l ) . H owever, e v e n a t t h e s e c o n c e n t r a t i o n s , t h e n o n s p e c i f i c s i g n a l was e x c e e d e d by t h e r e a c t i o n o f B16G a n d F d l l F . R e p r e s e n t a t i v e r e s u l t s f r o m t h r e e d i f f e r e n t e x p e r i m e n t s a r e shown. 48 F i g . 2. B i n d i n g o f F d l l F t o F d a d s o r b e d t o p o l y s t y r e n e w e l l s a n d i t s d e t e c t i o n w i t h B16G. F d was a d s o r b e d t o E L I S A w e l l s a t 1 u g / m l . M a t e r i a l s e l u t e d f r o m B16G a f f i n i t y c o l u m n s w e r e a d d e d t o s o l i d - p h a s e F d a t v a r i o u s c o n c e n t r a t i o n s a n d t h e n d e t e c t e d w i t h B16G a n d RaMIg-AP, u s e d a t t h e same c o n c e n t r a t i o n s a s f o r F i g . 1. A. F d l l F (•) c o m p a r e d t o medium c o n t r o l ( • ) . R e p r e s e n t a t i v e r e s u l t s f r o m s e v e n d i f f e r e n t p r e p a r a t i o n s a r e shown. B. F d l l F (•) c o m p a r e d t o t h e c e l l u l a r SN c o n t r o l DC10 ( • ) . R e s u l t s (mean + SEM) f r o m 3 s e p a r a t e p r e p a r a t i o n s a r e shown. 49 0.00 0.25 0.50 1.00 2.00 4.00 8.00 16.0 ug/ml Factor I : o-i 1 1 — , 1 0.00 0.05 1.00 2.00 4.00 ug/ml Factor 50 OH ¥ 1 ¥ 1 0.00 1.00 2.00 4.00 8.00 ug/ml Factor F i g . 3. The r e a c t i v i t y o f B16G a n d t h e i r r e l e v a n t mAb, CAMAL-1, w i t h F d l l F . F d l l F a n d DC10 m a t e r i a l w e r e a d d e d t o F d - c o a t e d w e l l s a t v a r i o u s c o n c e n t r a t i o n s . They were d e t e c t e d w i t h e i t h e r B16G o r CAMAL-1, u s e d a t 18 u g / m l , a n d RaMIg-AP. (-) B16G + DC10; (•) B16G + F d l l F ; (O) CAMAL-1 + DC10; (•) CAMAL-1 + F d l l F . 51 F d l l F s howed s e l e c t i v i t y f o r F d i n b i n d i n g when c o m p a r e d t o o t h e r A g . The d a t a p r e s e n t e d i n T a b l e I d e m o n s t r a t e t h a t when F d , s e r u m a l b u m i n , a n d a n o t h e r i r o n - c o n t a i n i n g p r o t e i n , c y t o c h r o m e C were a d s o r b e d t o s e p a r a t e w e l l s , F d l l F b o u n d t o F d o n l y . None o f t h e o t h e r B16G e l u a t e s showed a n y b i n d i n g p r e f e r e n c e f o r t h e Ag t h a t were u s e d . The b i n d i n g o f F d l l F t o F d i n s o l u t i o n was a l s o shown. V a r i o u s c o n c e n t r a t i o n s o f F d were i n c u b a t e d w i t h a c o n s t a n t amount o f F d l l F . The m i x t u r e was t h e n a d d e d t o F d - c o a t e d w e l l s a n d t h e amount o f f r e e F d l l F was m e a s u r e d as b e f o r e i n t h e " s a n d w i c h " E L I S A . F i g . 4 shows t h a t i n c r e a s i n g c o n c e n t r a t i o n s o f F d c o u l d i n h i b i t t h e amount o f F d l l F b i n d i n g t o F d - c o a t e d p l a t e s . C y t o c h r o m e C, u s e d a s t h e c o n t r o l p r o t e i n , d i d n o t b i n d t o F d l l F , a n d c o n s e q u e n t l y d i d n o t i n h i b i t F d l l F b i n d i n g . R e p r e s e n t a t i v e d a t a o f t h r e e e x p e r i m e n t s a r e shown. The f i n e s p e c i f i c i t y o f F d l l F b i n d i n g was e x a m i n e d u s i n g t h i s i n h i b i t i o n a s s a y a n d a n a l o g s t o F d . A n a l o g s , e x p r e s s i n g e i t h e r t h e N - d e t e r m i n a n t o r t h e C - d e t e r m i n a n t , a n d a t h i r d a n a l o g , OFd, were m i x e d a t v a r i o u s c o n c e n t r a t i o n s w i t h a c o n s t a n t amount o f F d l l F . The amount o f u n b o u n d F d l l F was t h e n be m e a s u r e d u s i n g t h e " s a n d w i c h " E L I S A . P r i o r t o p e r f o r m i n g t h i s a s s a y , t h e a n t i g e n i c i t y o f t h e a n a l o g s was c o n f i r m e d u s i n g F d d e t e r m i n a n t - s p e c i f i c mAb. The a n a l o g s w e re a d s o r b e d t o E L I S A w e l l s a n d t h e n i n c u b a t e d w i t h t h e mAb s p e c i f i c f o r t h e a m i n o - t e r m i n a l a n d c a r b o x y l - t e r m i n a l d e t e r m i n a n t s . F i g . 5 shows t h a t t h e a m i n o - t e r m i n a l d e t e r m i n a n t 52 Table I. The binding of F d l l F to Fd. Fd or other antigens were coated onto ELISA wells at 1 ug/ml. F d l l F or control materials were added at 4 ug/ml and detected with B16G and RaMIg-AP. Representative data of three d i f f e r e n t experiments are shown. HSA: human serum albumin Source of B16G eluate Adsorbed antigen Fd HSA cytochrome C F d l l .567 .090 . 103 medium .060 . 015 . 087 DC10 . 050 . 000 BW5147 . 013 . 035 53 701 60-• 60- • c o a 40-•C C • / rcent 30-• Q. 20-• ^ • 10- • • ' • • • 0 -.312 .625 1.25 2.5 ug/ml Inhibitor 1 5 10 F i g . 4 . The binding of soluble Fd to F d l l F i n fluid-phase. F d l l F was mixed with various concentrations of Fd (•) or cytochrome C (•) p r i o r i t s addition to Fd-coated wells. F d l l F was detected as for Fi g . 2. 54 F i g . 5. A n t i g e n i c a n a l y s i s o f F d a n d i t s a n a l o g s u s i n g d e t e r m i n a n t - s p e c i f i c mAb. N - m o l e c u l e ( N ) , C - m o l e c u l e ( C ) , OFd, F d , KLH a n d BSA w e re c o a t e d o n t o E L I S A w e l l s a n d t h e n i n c u b a t e d w i t h t h e F d - s p e c i f i c mAb, 16a a n d 6-90, b o t h u s e d a t 2 u g / m l . 16a i s s p e c i f i c f o r C-d e t e r m i n a n t , t h e c a r b o x y l - t e r m i n a l e p i t o p e o f F d a n d 6-90 i s s p e c i f i c f o r N - d e t e r m i n a n t , t h e a m i n o - t e r m i n a l e p i t o p e o f F d . M o n o c l o n a l a n t i b o d i e s w e re d e v e l o p e d w i t h RaMIg-AP. 55 s p e c i f i c mAb, 6-90, bound to both the carboxypeptidase A-treated analog (N-molecule) and Fd. It did not bind to the t r y p s i n -treated analog, known as C-molecule or the i r r e l e v a n t Ag, K L H or bovine serum albumin (BSA). In contrast, the carboxyl-terminal determinant-specific mAb, 16a bound to C-molecule and Fd; there was a s l i g h t degree of binding to N-molecule (approximately 10% of the C-molecule s i g n a l ) . This probably indicated that carboxypeptidase A digestion was not complete and a small percentage of the N-molecule preparation was contaminated with the undigested Ag. Incomplete digestion of N-molecule has been noted previously i n t h i s laboratory. 16a did not bind to either K L H or BSA. OFd was reactive with both 6-90 and 16a. Typical r e s u l t s of seven d i f f e r e n t experiments are shown. The fine s p e c i f i c i t y of F d l l F binding was subsequently determined using the analogs. F i g . 6 shows that C-molecule was the most e f f e c t i v e i n h i b i t o r of F d l l F binding causing 50% i n h i b i t i o n at .312 ug/ml. Fd was the next best i n h i b i t o r followed by OFd. More than 15 times the amount of N-molecule was required to achieve the l e v e l of i n h i b i t i o n observed with C-molecule and Fd. In fact, the amount of N-molecule that was required suggested that i n h i b i t i o n was most l i k e l y due to the contamination of the preparation with undigested Fd. The data indicate that F d l l F i s s p e c i f i c for the C-determinant of Fd. Representative data from three d i f f e r e n t experiments are shown. In summary, F d l l F was shown to bind to both Fd and the mAb, B16G which allowed i t s qu a n t i f i c a t i o n , at least at the 56 0 J O Q 1—Q 1 A 1— .312 .625 1.25 2.50 5.00 10.0 ug/ml Inhibitor F i g . 6. I n h i b i t i o n o f F d l l F b i n d i n g b y F d a n d i t s a n a l o g s . F d l l F was m i x e d w i t h v a r i o u s c o n c e n t r a t i o n s o f N - m o l e c u l e (o), C-m o l e c u l e (•), OFd (•), F d (•), o r c y t o c h r o m e C (A) p r i o r t o i t s a d d i t i o n t o F d - c o a t e d w e l l s . F d l l F was d e t e c t e d a s i n F i g . 2. 57 i m m u n o c h e m i c a l l e v e l . F d l l F was shown t o b i n d p r e f e r e n t i a l l y t o F d a n d t h r o u g h t h e u s e o f a n a l o g s i t was d e t e r m i n e d t o be s p e c i f i c f o r t h e c a r b o x y l - t e r m i n a l r e g i o n o f F d . The n e x t s e t o f s t u d i e s e x a m i n e d some o f t h e b i o l o g i c a l p r o p e r t i e s o f F d l l F . B i o l o g i c a l P r o p e r t i e s o f F d l l F D e v e l o p m e n t o f an a n t i b o d y c u l t u r e a s s a y f o r t h e meas u r e m e n t o f s u p p r e s s i o n H a v i n g e s t a b l i s h e d t h e i m m u n o c h e m i c a l p r o p e r t i e s o f F d l l F , i t was o f i n t e r e s t t o e x a m i n e some o f i t s b i o l o g i c p r o p e r t i e s . To s t u d y t h e e f f e c t o f F d l l F on t h e Ab r e s p o n s e , an i n v i t r o Ab p r o d u c t i o n a s s a y was d e v e l o p e d . S e v e r a l d i f f e r e n t p r o t o c o l s w e r e t r i e d b e f o r e one was c h o s e n . I n i t i a l l y , t h e C - m o l e c u l e - p r i m e d a n i m a l s w e re i m m u n i z e d t w i c e w i t h F d b e f o r e t h e h a r v e s t i n g o f s p l e e n s t o i n s u r e t h a t t h e c e l l s w o u l d s y n t h e s i z e e n o u g h Ab i n v i t r o . I n a d d i t i o n , i t was f o u n d t h a t t h e c e l l s h a d t o be p u l s e d w i t h A g j u s t p r i o r t o c u l t u r i n g i n o r d e r t o p r o v i d e e n o u g h s t i m u l u s f o r Ab p r o d u c t i o n i n v i t r o . D u r i n g t h e p u l s i n g p e r i o d , a s u f f i c i e n t amount o f F d i s p i n o c y t o s e d a n d " p r o c e s s e d " b y s p l e n i c a n t i g e n - p r e s e n t i n g c e l l s t o d r i v e t h e Ab r e s p o n s e d e s p i t e t h e l a c k o f s o l u b l e A g i n c u l t u r e . T y p i c a l r e s u l t s ( o f s i x d i f f e r e n t c u l t u r e e x p e r i m e n t s ) o f t h i s i m m u n i z a t i o n a n d p u l s i n g p r o t o c o l a r e s e e n i n F i g . 7. I t c a n be s e e n t h a t Ab was e a s i l y d e t e c t e d i n t h e SN f r o m day 5 58 0 .9-, CD impulsed ^ fd pulsed 5 6 7 Day F i g . 7. D e t e c t i o n o f a n t i - F d Ab i n t h e SN o f s p l e e n c e l l c u l t u r e s p r e p a r e d f r o m B10.D2 m i c e t h a t w e re i m m u n i z e d w i t h C-m o l e c u l e a n d F d t w i c e . S p l e e n c e l l s w e r e i n c u b a t e d w i t h e i t h e r F d o r medium f o r 2 h b e f o r e c u l t u r i n g . SN were c o l l e c t e d f i v e , s i x , a n d s e v e n d a y s a f t e r i n i t i a t i o n o f c u l t u r e . Ab c o n t e n t o f SN was m e a s u r e d u s i n E L I S A . 59 c u l t u r e s u s i n g E L I S A . I n f a c t , Ab p r o d u c t i o n c o n t i n u e d t h r o u g h t o d a y 6 a n d d a y 7 . A c u l t u r e c o n t a i n i n g medium p u l s e d c e l l s s e r v e d a s b a c k g r o u n d c o n t r o l f o r t h e E L I S A m e a s u r e m e n t . Once i t was e s t a b l i s h e d t h a t Ab c o u l d be p r o d u c e d i n v i t r o , e x p e r i m e n t s w e r e done t o m a x i m i z e t h e amount o f s u p p r e s s i o n . I n one m o d i f i c a t i o n , h y p e r i m m u n e a n i m a l s w e re t r e a t e d w i t h F d l l F i n v i v o b e f o r e t h e i r s p l e e n s w e re r e m o v e d f o r c u l t u r i n g . T h i s was t h o u g h t t o make t h e c e l l s more " s u s c e p t i b l e " t o t h e i n v i t r o e f f e c t s o f F d l l F . P r e l i m i n a r y e x p e r i m e n t s w e re a l s o t r i e d where t h e s p l e e n c e l l s w e r e p u l s e d c o n c u r r e n t l y w i t h F d a n d F d l l F . E v e n t u a l l y , one m e t h o d was s e l e c t e d . S p l e e n c e l l s w e r e p r e p a r e d f r o m Fd-immune B10.D2 m i c e t h a t w e r e p r e v i o u s l y i m m u n i z e d w i t h C - m o l e c u l e ( t o e s t a b l i s h i m m u n i t y ) a n d b o o s t e d w i t h F d o n c e . The c e l l s were p u l s e d w i t h F d , w a s h e d f r e e o f s o l u b l e A g , a n d c u l t u r e d w i t h e i t h e r c o n t r o l m a t e r i a l , F d l l F -c o n t a i n i n g m a t e r i a l o r medium f o r s e v e n d a y s . SN w e r e c o l l e c t e d a n d t h e a n t i - F d Ab l e v e l s w e r e m e a s u r e d u s i n g E L I S A . A c o m p a r i s o n o f t h e l e v e l s s e r v e d as a m e a s u r e o f s u p p r e s s i o n . S u b s t r a t e d e v e l o p m e n t was a l l o w e d u n t i l t h e m a x i m a l a b s o r b a n c e on t h e p l a t e r e a c h e d a p p r o x i m a t e l y one. A c u r v e p l o t t i n g t h e r e l a t i o n s h i p b e t w e e n a b s o r b a n c e p r o d u c e d a n d t h e c o n c e n t r a t i o n o f Ab t h a t b o u n d t o F d - c o a t e d w e l l s i s shown i n F i g . 8. Mouse Ab e l u t e d f r o m a F d - S e p h a r o s e c o l u m n was u s e d as a s o u r c e o f a f f i n i t y - p u r i f i e d m a t e r i a l . The c u r v e was a p p r o x i m a t e l y l i n e a r f r o m 0.1 t o 1.0 a b s o r b a n c e u n i t s . T h e r e f o r e i t c o u l d be a s s u m e d t h a t a b s o r b a n c e w o u l d be d i r e c t l y 60 F i g . 8. R e l a t i o n s h i p b e t w e e n A b s o r b a n c e a t 4 05 nm v s . c o n c e n t r a t i o n o f a n t i - F d Ab. A f f i n i t y - p u r i f i e d a n t i - F d Ab was i n c u b a t e d on F d - a n d K L H - c o a t e d p l a t e s . Ab was d e t e c t e d u s i n g RaMIg-AP. R e a c t i o n was d e v e l o p e d f o r 2 h. F d : (•) KLH: (O) 61 related to the concentration of Ab that was act u a l l y present i n the SN. The s p e c i f i c i t y of the a f f i n i t y - p u r i f i e d Ab for Fd was shown by i t s lack of r e a c t i v i t y with KLH. Representative data of three experiments are shown. The s p e c i f i c i t y of the anti-Fd Ab secreted into the SN for the Fd analogs and unrelated Ag was then determined. In F i g . 9A i t can be seen that e s s e n t i a l l y a l l of the Ab was s p e c i f i c for the amino-terminal determinant. The r e l a t i v e lack of Ab directed to the C-molecule was consistent with the finding that C-molecule immunization of B 1 0 . D 2 animals induces l i t t l e Ab directed to that determinant (data not shown). The SN did not react with the irr e l e v a n t Ag, KLH and BSA. For comparison, the serum Ab a c t i v i t y of Fd-immune B 1 0 . D 2 animals i s shown i n F i g . 9B. The serum a c t i v i t y i s quite s i m i l a r to that of the SN. The apparent increase of Ab reactive with N-molecule over Fd was probably due to differences i n binding of Fd or N-molecule to ELISA plates or some nonspecific binding of serum Ab. Indeed, the nonspecific binding a c t i v i t y of the serum, as shown by i t s r e a c t i v i t y with KLH, i s greater than that of the SN ( c f . F i g . 9 A ) . Correlation between b i o a c t i v i t y and antigen-binding a c t i v i t y of F d l l F F i g . 1 0 i s the "dose-response" curve of F d l l F c o r r e l a t i n g suppressive a c t i v i t y i n v i t r o (ie. the response) with Ag-binding 62 F i g . 9A. S p e c i f i c i t y o f a n t i - F d Ab f r o m Fd-immune s p l e e n c e l l c u l t u r e s f o r F d a n a l o g s . SN f r o m s e v e n d a y c u l t u r e s w e r e h a r v e s t e d a n d Ab l e v e l d e t e r m i n e d u s i n g E L I S A . T y p i c a l r e s u l t s o f s i x d i f f e r e n t c u l t u r e s a r e shown. N: N - m o l e c u l e C: C - m o l e c u l e F i g . 9B. S p e c i f i c i t y o f s e r u m a n t i - F d Ab f r o m Fd-immune B10.D2 m i c e f o r F d a n a l o g s . Ab l e v e l a t a 1/400 d i l u t i o n o f s e r u m was d e t e r m i n e d u s i n g E L I S A . R e s u l t s (mean + SEM) f r o m t h r e e s e p a r a t e s e r u m s a m p l e s a r e shown. N: N - m o l e c u l e C: C - m o l e c u l e Antigen 64 F i g . 10. The r e l a t i o n s h i p o f s u p p r e s s i v e a c t i v i t y o f F d l l F t o i t s A g - b i n d i n g a c t i v i t y . S u p p r e s s i v e a c t i v i t y i s e x p r e s s e d a s p e r c e n t s u p p r e s s i o n . P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g Ab l e v e l s o f c u l t u r e s c o n t a i n i n g F d l l F t o t h e l e v e l s m e a s u r e d i n t h e c o n t r o l c u l t u r e . A g - b i n d i n g a c t i v i t y i s e x p r e s s e d a s t h e s a n d w i c h E L I S A v a l u e o f t h e F d l l F l o t t e s t e d . The p o o l e d d a t a f r o m s e v e n l o t s a r e shown. L o t s 1 t o 5 w e re a s s a y e d a t t w o o r t h r e e c o n c e n t r a t i o n s w h i l e l o t s 6 a n d 7 w e r e a s s a y e d a t o n l y one c o n c e n t r a t i o n . F o l l o w i n g i t s e l u t i o n f r o m B16G a f f i n i t y c o l u m n s , e a c h l o t was m e a s u r e d f o r p r o t e i n c o n t e n t , A g - b i n d i n g a c t i v i t y , a n d b i o a c t i v i t y . A n e g a t i v e c o n t r o l s a m p l e was p r e p a r e d c o n c u r r e n t l y w i t h e a c h F d l l F l o t . T h i s s a m p l e was u s e d as t h e r e s p e c t i v e n e g a t i v e c o n t r o l f o r t h e d e t e r m i n a t i o n o f n e t A g -b i n d i n g a c t i v i t y a n d b i o a c t i v i t y . 65 a c t i v i t y as measured by "sandwich" ELISA ( i e . t h e d o s e ) . Response v a l u e s were e x p r e s s e d as p e r c e n t s u p p r e s s i o n ( c a l c u l a t e d u s i n g t h e Ab l e v e l o f t h e c u l t u r e c o n t a i n i n g F d l l F as compared t o t h e u n t r e a t e d c u l t u r e ) . Dose v a l u e s were e x p r e s s e d as A g - b i n d i n g a c t i v i t y , r a t h e r t h a n t o t a l p r o t e i n c o n c e n t r a t i o n t o a l l o w c o m parison o f t h e d a t a from d i f f e r e n t b a t c h e s o f F d l l F . The c u r v e i s a c o m p i l a t i o n o f r e s u l t s from seven d i f f e r e n t l o t s o f F d l l F . The c u r v i l i n e a r r e l a t i o n s h i p between b i o a c t i v i t y and A g - b i n d i n g a c t i v i t y suggest t h a t t h e two a c t i v i t i e s a r e r e l a t e d and can be a s c r i b e d t o F d l l F . The d a t a r e p r e s e n t i n g l o t s 4 and 7 d i d not conform w i t h t h e m a j o r i t y o f o b s e r v a t i o n s . The r e a s o n (s) f o r t h i s i s unknown. One p o s s i b i l i t y i s t h a t t h e t o t a l Ab l e v e l s p r o d u c e d by t h e u n t r e a t e d , F d - p u l s e d c u l t u r e s i n t h e s e e x p e r i m e n t s were c o m p a r a t i v e l y low. T h i s r e s u l t e d i n a d i s p r o p o r t i o n a l l y h i g h p e r c e n t a g e o f s u p p r e s s i o n . I t was a l s o o b s e r v e d t h a t t h e s u p p r e s s i o n p r o d u c e d by F d l l F d i d not exceed 60%. Almost complete s u p p r e s s i o n was e v e n t u a l l y a c h i e v e d f o l l o w i n g a r e v i s i o n t o t h e c u l t u r i n g p r o t o c o l (see below, F i g . 12) . None o f t h e c u l t u r e s c o n t a i n i n g F d l l F showed an i n c r e a s e i n Ab p r o d u c t i o n as compared t o u n t r e a t e d c o n t r o l s . B16G e l u a t e s made w i t h n e g a t i v e c o n t r o l SN were p r e p a r e d i n p a r a l l e l w i t h each l o t o f F d l l F . Of t h e n e g a t i v e c o n t r o l p r e p a r a t i o n s , o n l y one l o t ( l o t 4) o f t h e seven demonstrated any s u p p r e s s i v e a c t i v i t y . F o r t h e s e c u l t u r e s , t h e d e c r e a s e was c o r r e c t e d f o r t h e e f f e c t o f t h e n e g a t i v e c o n t r o l m a t e r i a l . 66 F d l l F - m e d i a t e d s u p p r e s s i o n was a l s o shown t o be s p e c i f i c f o r t h e a n t i - F d Ab r e s p o n s e . F i g . 11 shows t h a t when F d l l F , a t t h r e e d i f f e r e n t c o n c e n t r a t i o n s , was a d d e d t o l y s o z y m e - i m m u n e c e l l s , c o n c e n t r a t i o n - d e p e n d e n t s u p p r e s s i o n was n o t o b s e r v e d . However, t h i s same p r e p a r a t i o n o f F d l l F d i d s u p p r e s s t h e a n t i - F d Ab r e s p o n s e . BW5147 c o n t r o l m a t e r i a l , w h i c h d i d n o t p o s s e s s A g -b i n d i n g a c t i v i t y , i s shown f o r c o m p a r i s o n . I t a l s o d i d n o t d e m o n s t r a t e s u p p r e s s i v e a c t i v i t y a n d was r e p r e s e n t a t i v e o f t h e r e s p o n s e g e n e r a t e d b y t h e o t h e r c o n t r o l m a t e r i a l s ( i e . medium and DC10 S N ) . R e p r e s e n t a t i v e d a t a o f t h r e e e x p e r i m e n t s a r e shown. A d d i t i o n a l w o r k, a d d r e s s i n g t h e i n v i t r o " s p e c i f i c i t y " o f F d l l F b i o a c t i v i t y was done. I n b r i e f , a n o t h e r p r o d u c t d e r i v e d f r o m t h e T c e l l h y b r i d o m a , A10, i s s u p p r e s s i v e f o r t h e CTL r e s p o n s e t o t h e m u r i n e m a s t o c y t o m a , P815 ( S t e e l e , Stammers, a n d L e v y 1 9 8 5 ) . The m a t e r i a l , A10F, i s r e c o v e r e d f r o m t h e c u l t u r e SN by a f f i n i t y c h r o m a t o g r a p h y u s i n g B 1 6 G - S e p h a r o s e . When A l O F was a d d e d t o CTL c u l t u r e s p r e p a r e d f r o m P 8 1 5 - p r i m e d m i c e , t h e r e was a d e c r e a s e i n s p e c i f i c c e l l l y s i s a s c o m p a r e d t o c u l t u r e s t h a t c o n t a i n e d F d l l F . By c o n t r a s t , when F d l l F was a d d e d t o c u l t u r e s o f Fd-immune s p l e e n c e l l s , t h e r e was a d e c r e a s e i n Ab l e v e l s o f t h e c u l t u r e s a s c o m p a r e d t o t h e c u l t u r e s t h a t c o n t a i n e d A 1 0F. T h a t i s , F d l l F was o n l y s u p p r e s s i v e when u s e d i n t h e Ab c u l t u r e a s s a y a n d A10F was o n l y a c t i v e when u s e d w i t h t h e CTL a s s a y . The r e s u l t s f r o m two o f t h e s e " c r i s s - c r o s s " e x p e r i m e n t s a r e shown i n T a b l e I I . 67 so-i to I I 1 1 0.00 .147(1) .486(2) 1.10(4) Net antigen-binding activity & (ug/ml) F i g . 11. S p e c i f i c i t y of the s u p p r e s s i v e a c t i v i t y of F d l l F . A s i n g l e p r e p a r a t i o n of F d l l F , used at t h r e e d i f f e r e n t c o n c e n t r a t i o n s , was.added t o c u l t u r e s of lysozyme-immune c e l l s and Fd-immune c e l l s . A nti-lysozyme Ab l e v e l s of c u l t u r e s c o n t a i n i n g F d l l F (•) were compared with the "untreated c o n t r o l c u l t u r e . The percent s u p p r e s s i o n i s shown. A l s o i n c l u d e d i s the response o b t a i n e d w i t h BW5147- c o n t r o l m a t e r i a l (O) when added to the lysozyme-immune c e l l s . I t d i d not possess any A g - b i n d i n g a c t i v i t y and was added to the c u l t u r e s at the p r o t e i n c o n c e n t r a t i o n s t h a t are i n d i c a t e d . To demonstrate t h a t the same p r e p a r a t i o n of F d l l F was a c t i v e , i t was added to Fd-immune c e l l s . The percent s u p p r e s s i o n of these c u l t u r e s , at three c o n c e n t r a t i o n s , are shown (•). 68 T a b l e I I . C o m p a r i s o n o f t h e s u p p r e s s i v e c a p a b i l i t y o f F d l l F a n d A10F i n two a s s a y s o f c e l l u l a r f u n c t i o n . F d l l F a n d A10F w e r e e a c h a d d e d t o c u l t u r e s c o n t a i n i n g e i t h e r P 8 1 5 - s p e c i f i c CTL o r Fd-immune s p l e e n c e l l s . F o r t h e P815 CTL a s s a y , d a t a a r e e x p r e s s e d a s p e r c e n t l y s i s a n d p e r c e n t s u p p r e s s i o n f o r t h e c u l t u r e . P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g p e r c e n t l y s i s o f t h e c u l t u r e s c o n t a i n i n g a f f i n i t y -e n r i c h e d m a t e r i a l t o t h e u n t r e a t e d c o n t r o l c u l t u r e . F o r t h e a n t i - F d Ab a s s a y , d a t a a r e e x p r e s s e d a s Ab l e v e l o f t h e c u l t u r e ( A b s o r b a n c e 405 nm) a n d p e r c e n t s u p p r e s s i o n . P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g t h e Ab l e v e l o f c u l t u r e s c o n t a i n i n g a f f i n i t y - e n r i c h e d m a t e r i a l t o t h e u n t r e a t e d c o n t r o l c u l t u r e . The r e s u l t s f r o m two s e p a r a t e e x p e r i m e n t s a r e shown. Ab c u l t u r e m e a s u r e m e n t s w e r e o b t a i n e d u s i n g t h e " r e v i s e d " p r o t o c o l ( s e e MATERIALS AND METHODS). A: m a t e r i a l a d d e d a t 1 ug/ml B: m a t e r i a l a d d e d a t 1.5 ug/ml M a t e r i a l A s s a y P815 CTL A n t i - F d Ab % l y s i s % s u p p r e s s i o n Ab l e v e l % s u p p r e s s i o n E x p t . A no f a c t o r 24.5 - .197 A l OF 0 100 .183 7.1 F d l l F 22 10.7 .143 27.4 E x p t . B no f a c t o r 15 - .301 A10F 5.6 62.4 .280 7 F d l l F 14.5 3.3 .100 67 69 The " d o s e - r e s p o n s e " c u r v e o f F i g . 10 i n d i c a t e d t h a t t h e amount o f s u p p r e s s i o n a c h i e v e d was d e p e n d e n t on t h e A g - b i n d i n g a c t i v i t y o f t h e s a m p l e . However, t h e m a x i m a l l e v e l o f s u p p r e s s i o n t h a t was e v e r a t t a i n e d w i t h t h e a s s a y d i d n o t e x c e e d 60%. W h e t h e r t h i s was a l i m i t a t i o n o f F d l l F o r o f t h e a s s a y i t s e l f was unknown. T h i s o r i g i n a l a s s a y u s e d Fd-immune s p l e e n c e l l s t h a t w e r e p u l s e d f o r 2 h w i t h F d t o a l l o w f o r Ag p r o c e s s i n g . The c e l l s w e r e t h e n w a s h e d t o remove s o l u b l e F d a n d t h e n c o - c u l t u r e d w i t h F d l l F ( o r c o n t r o l m a t e r i a l ) f o r s e v e n d a y s . A t t h e e n d o f t h e i n c u b a t i o n p e r i o d , t h e c u l t u r e SN were a s s e s s e d f o r a n t i - F d Ab u s i n g E L I S A . F u r t h e r d e v e l o p m e n t work showed t h a t m o d i f i c a t i o n s t o t h e Ab c u l t u r e p r o t o c o l y i e l d e d s u p p r e s s i v e e f f e c t s b y F d l l F , w h i c h a p p r o a c h e d 100%. The c h a n g e s t o t h e p r o t o c o l i n c l u d e d p u l s i n g t h e Fd-immune c e l l s f i r s t w i t h F d l l F a n d t h e n i n t r o d u c i n g F d 2 h l a t e r . A f t e r a n o t h e r 2 h, t h e c e l l s were washed, p l a t e d a n d i n c u b a t e d f o r s e v e n d a y s w i t h o u t r e c e i v i n g any a d d i t i o n s o f f a c t o r . The d a y 7 SN were a s s e s s e d as t h e i n t h e o r i g i n a l p r o t o c o l . R e p r e s e n t a t i v e d a t a ( o f t h r e e d i f f e r e n t e x p e r i m e n t s ) i n F i g . 12 show t h a t t h e s u p p r e s s i v e e f f e c t was c o n c e n t r a t i o n -d e p e n d e n t a n d r e a c h e d a maximum o f 85%. BW5147 c o n t r o l m a t e r i a l was n o t s u p p r e s s i v e a t any c o n c e n t r a t i o n . S u p p r e s s i o n o f a n t i - F d a n t i b o d y l e v e l s i n v i v o The f o l l o w i n g e x p e r i m e n t was d e s i g n e d t o t e s t t h e e f f e c t o f 70 F i g . 12. S u p p r e s s i v e a c t i v i t y o f a f f i n i t y - e n r i c h e d F d l l F c o m p a r e d t o BW5147 c o n t r o l m a t e r i a l u s i n g t h e " r e v i s e d " p r o t o c o l . V a r i o u s c o n c e n t r a t i o n s o f e i t h e r BW5147 o r F d l l F m a t e r i a l w e r e f i r s t a d d e d t o Fd-immune s p l e e n c e l l s f o l l o w e d b y t h e i n v i t r o p u l s i n g w i t h F d . P e r c e n t s u p p r e s s i o n was d e t e r m i n e d a s f o r F i g . 10. (•), Fd-immune c e l l s p u l s e d w i t h F d l l F ; (•), Fd-immune c e l l s p u l s e d w i t h BW5147 c o n t r o l m a t e r i a l . 71 F d l l F on t h e a n t i - F d Ab r e s p o n s e i n v i v o , u s i n g a n o t h e r s t r a i n o f H - 2 d a n i m a l s , DBA/2 m i c e . DBA/2 m i c e were p r i m e d w i t h C - m o l e c u l e f o l l o w e d b y an i m m u n i z a t i o n w i t h F d . The m i c e were a l s o i m m u n i z e d w i t h t h e c o n t r o l Ag, KLH a t t h i s t i m e . A l l i m m u n i z a t i o n s w e re made s u b c u t a n e o u s l y ( s . c . ) u s i n g c o m p l e t e F r e u n d ' s a d j u v a n t . The a n i m a l s w e re b l e d 21 d a y s l a t e r a n d r a n k e d a c c o r d i n g t o t h e m a g n i t u d e o f t h e i r r e s p o n s e t o F d as m e a s u r e d u s i n g E L I S A . They were d e s i g n a t e d as l o w o r h i g h r e s p o n d e r s a n d t h e n d i v i d e d i n t o t h r e e g r o u p s s u c h t h a t e a c h one h a d an a p p r o x i m a t e l y e q u a l number o f l o w a n d h i g h r e s p o n d e r s . The a n i m a l s w i t h i n e a c h g r o u p r e c e i v e d e i t h e r 10 ug F d l l F , 10 ug A l O F o r PBS i . v . , w e re a l l o w e d t o r e c o v e r f o r s e v e r a l h o u r s a n d t h e n b o o s t e d w i t h F d a n d KLH, s . c . The a n i m a l s w e re b l e d s e v e n d a y s l a t e r a n d t h e a n t i - F d a n d a n t i - K L H Ab r e s p o n s e s b e f o r e a n d a f t e r t r e a t m e n t w e r e a s s e s s e d a s p a i r e d s a m p l e s f o r e a c h i n d i v i d u a l a n i m a l . The d a t a a r e p r e s e n t e d a s an X-Y p l o t w h ere t h e p r e t r e a t m e n t . s e r u m Ab l e v e l i s p l o t t e d on t h e x a x i s a n d t h e p o s t t r e a t m e n t s e r u m Ab l e v e l i s i n d i c a t e d on t h e y - a x i s . A d e c r e a s e i n Ab r e s p o n s e f o l l o w i n g t r e a t m e n t a p p e a r s as d a t a b e l o w t h e "no c h a n g e " l i n e . E a c h d a t a p o i n t r e p r e s e n t s one a n i m a l . F i g . 13 shows t h a t f i v e o u t o f s i x a n i m a l s t h a t r e c e i v e d F d l l F a t t h e t i m e o f Ag b o o s t h a d a d e c r e a s e i n c i r c u l a t i n g a n t i - F d Ab s e v e n d a y s p o s t t r e a t m e n t . I n c o n t r a s t , a s shown i n F i g . 14, t h e m a j o r i t y o f a n i m a l s f r o m b o t h g r o u p s o f a n i m a l s r e c e i v i n g A10F o r PBS showed an i n c r e a s e d Ab r e s p o n s e . The X-Y p l o t shows t h e 72 0.82-1 0 . 4 8 -0.44 « 0 . 4 0 -O a © C 0 . 36 cd A o 01 n 0 . 3 2 < 0.28 0.24 0 .20 0 . 20 0 .24 0.28 0 .32 0 . 3 6 0 .40 Absorbance (pre) 0.44 0.48 0 .62 F i g . 13. X-Y p l o t s h o w i n g a n t i - F d Ab l e v e l s o f a n i m a l s b e f o r e a n d a f t e r t r e a t m e n t w i t h F d l l F a n d Ag. A n i m a l s w e r e i m m u n i z e d w i t h C - m o l e c u l e a n d t h e n c o - i m m u n i z e d w i t h F d a n d KLH. Once i m m u n i t y was e s t a b l i s h e d , t h e y w e r e b o o s t e d w i t h F d a n d KLH a n d g i v e n F d l l F , PBS o r c o n t r o l m a t e r a l , i . v . . M a t c h e d p a i r s o f s e r a w e re o b t a i n e d f r o m t h e i n d i v i d u a l a n i m a l s t h a t w e r e b l e d b e f o r e a n d a f t e r t r e a t m e n t . S y m b o l s r e p r e s e n t t h e x - y c o o r d i n a t e s o f p r e t r e a t m e n t a n d p o s t t r e a t m e n t s e r u m Ab l e v e l s f o r i n d i v i d u a l a n i m a l s . A d e c r e a s e i n Ab l e v e l i s p l o t t e d as a s y m b o l b e l o w t h e "no c h a n g e l i n e " . A n i m a l s w e re t r e a t e d w i t h e i t h e r F d l l F (•), A10F (•) o r PBS ( • ) . 73 Absorbance (pre) F i g . 14. X-Y p l o t showing anti-KLH Ab l e v e l s of animals b e f o r e and a f t e r treatment with F d l l F and Ag. See F i g . 13 f o r e x p l a n a t i o n . Animals were t r e a t e d with e i t h e r F d l l F (•), A10F (•) or PBS ( • ) . r e s u l t s f r o m t h e same a n i m a l s , o n l y t h i s t i m e t h e a n t i - K L H Ab r e s p o n s e i s p l o t t e d . The l a r g e m a j o r i t y o f a n i m a l s showed an i n c r e a s e i n a n t i - K L H Ab w h e t h e r t r e a t e d w i t h A l O F o r F d l l F . T a k e n t o g e t h e r , t h e d a t a r e v e a l t h a t F d l l F a p p e a r s t o h a v e s p e c i f i c i t y f o r t h e a n t i - F d Ab r e s p o n s e when c o m p a r e d t o B 1 6 G - e l u t e d c o n t r o l m a t e r i a l a n d a n o t h e r Ab r e s p o n s e t o an i r r e l e v a n t A g . (B16G-r e a c t i v e m a t e r i a l was t e s t e d i n v i v o , f o u r t i m e s . R e p r e s e n t a t i v e r e s u l t s a r e s h o w n ) . To s u m m a r i z e , t h e b i o l o g i c a c t i v i t y o f F d l l F was d e m o n s t r a t e d b o t h i n v i t r o a n d i n v i v o . A s p l e e n c e l l a s s a y was d e v e l o p e d a n d u s e d t o show t h a t F d l l F s u p p r e s s e d t h e Ab r e s p o n s e i n a c o n c e n t r a t i o n - d e p e n d e n t manner. D a t a f r o m o t h e r e x p e r i m e n t s s u g g e s t e d t h a t t h e s u p p r e s s i v e e f f e c t was A g - s p e c i f i c . I n v i v o , F d l l F t r e a t m e n t s u p p r e s s e d t h e s e r u m Ab r e s p o n s e o f Fd-immune H-2 m i c e f o l l o w i n g A g c h a l l e n g e . A s c o n t r o l s , A l O F a n d PBS h a d no e f f e c t on t h e d e v e l o p m e n t o f t h e s e c o n d a r y r e s p o n s e . The a n t i -KLH Ab r e s p o n s e was n o t a f f e c t e d b y any o f t h e t r e a t m e n t s . W i t h t h e a v a i l a b i l i t y o f b o t h an i m m u n o c h e m i c a l a n d b i o l o g i c a l means o f m e a s u r i n g F d l l F , b i o c h e m i c a l a n a l y s i s o f t h e F d l l F A g -b i n d i n g m o l e c u l e ( s ) was now p o s s i b l e . B i o c h e m i c a l A n a l y s i s o f F d l l F I n t r o d u c t i o n When t h e e l u a t e s f r o m B16G c o l u m n s were a n a l y z e d u s i n g SDS-PAGE, run under reducing conditions, a c h a r a c t e r i s t i c and reproducible banding pattern was observed with s i l v e r staining. F i g . 15 shows the res u l t from a representative F d l l F recovery experiment. Equal amounts of protein (1 to 2 ug) were loaded into each well. A f f i n i t y chromatography yielded only p a r t i a l l y p u r i f i e d preparations of F d l l F since bands resembling BSA and probably other serum components co-eluted with F d l l F . Two other predominant bands always appeared. Of primary i n t e r e s t was a unique band at approximately 30k M . In addition, a band at approximately 80k M r was routinely recovered. Lesser amounts of 80k M r material also appeared i n the lane containing the sample derived from non-Fdll SN, whether i t was prepared from medium, BW5147 or DC10 SN. (Note: Subsequent references i n the text to the "80k" or "30k" M r proteins refer to the protein at approximately 80k M r and the protein at approximately 30k M r of F i g . 15, respectively.) From t h i s s t a r t i n g point, e f f o r t s were directed at the biochemical i d e n t i f i c a t i o n of the Ag-binding moiety of F d l l F . The aim was to further resolve the FdllF-enriched material from the B16G-Sepharose column and i d e n t i f y the molecule(s) by both t h e i r Ag-binding and b i o l o g i c a c t i v i t i e s . Analysis of F d l l F using high performance gel f i l t r a t i o n : c o r r e l a t i o n between antigen-binding and suppressive a c t i v i t y High performance gel f i l t r a t i o n (FPLC, Pharmacia, Fig. 15. SDS-PAGE of eluates from B16G a f f i n i t y columns. Eluates derived from F d l l SN (A) or medium containing 10% FCS (B) were analyzed using a 10% SDS gel run under reducing conditions. Position of M markers are indicated. Actual M r equals number shown m u l t i p l i e d by 1000. The predominant bands at 80k and 30k Mr were consistently obtained with F d l l SN. Bands were developed by s i l v e r s taining. Typical results of s ix d i f f e r e n t preparations of F d l l F (and control material) are shown. 77 S u p e r o s e 12 c o l u m n ) was t h e f i r s t s e p a r a t i o n m e t h o d c h o s e n s i n c e i t a l l o w e d f a s t r e c o v e r y o f t h e n a t i v e m o l e c u l e s w i t h o u t t h e u s e o f d e n a t u r i n g c o n d i t i o n s o r b u f f e r s w h i c h w o u l d c a u s e d i s s o c i a t i o n o f n o n - c o v a l e n t l y a s s o c i a t e d m a t e r i a l s . The s t a r t i n g m a t e r i a l s w e re t h e e l u a t e s f r o m t h e B16G-S e p h a r o s e c o l u m n s . C o n t r o l m a t e r i a l was c h r o m a t o g r a p h e d f i r s t t o a v o i d c r o s s - c o n t a m i n a t i o n o f p r o t e i n s . A f t e r t h e c o l u m n was w a s h e d w i t h e l u e n t , F d l l F m a t e r i a l was l o a d e d . E a c h f r a c t i o n was a s s a y e d f o r A g - b i n d i n g a c t i v i t y u s i n g t h e " s a n d w i c h " E L I S A a n d a n a l y z e d by SDS-PAGE r u n u n d e r r e d u c i n g c o n d i t i o n s . F i g . 16 shows t h e F PLC p r o t e i n p r o f i l e s o f medium, BW5147, a n d F d l l F m a t e r i a l . E q u a l amounts o f p r o t e i n w e r e l o a d e d f o r e a c h s a m p l e . A l a r g e p r o t e i n p e a k b e g i n n i n g a t t h e v o i d v o l u m e was r e c o v e r e d f r o m t h e F d l l F s a m p l e (The v o i d v o l u m e o f t h e c o l u m n was c a l c u l a t e d b a s e d on e x p e r i m e n t a l d a t a a n d t h e p e r f o r m a n c e s p e c i f i c a t i o n s p r o v i d e d b y P h a r m a c i a ) . T h i s l a r g e amount was s p r e a d o v e r s e v e r a l f r a c t i o n s ( f r a c t i o n s 16 t o 22) a s s u g g e s t e d b y t h e A g - b i n d i n g a c t i v i t y p r o f i l e , shown i n F i g . 17. ( R e p r e s e n t a t i v e r e s u l t s o f t h r e e d e t e r m i n a t i o n s a r e shown.) The p e a k o f i m m u n o r e a c t i v i t y ( f r a c t i o n 17) c o i n c i d e d e x a c t l y w i t h t h e p r e s e n c e o f a 80k a n d 30k M r b a n d i d e n t i f i e d i n t h e f r a c t i o n as r e v e a l e d b y SDS-PAGE ( F i g . 1 8 C ) . F r a c t i o n s 16 t o 19 a r e shown. I n a d d i t i o n , two 45k M r b a n d s u n i q u e t o t h e F d l l F s a m p l e were p r e s e n t i n f r a c t i o n 16. They d i d n o t a p p e a r t o c o n t r i b u t e t o A g -b i n d i n g a c t i v i t y . L e s s e r q u a n t i t i e s o f t h e 80k M r m a t e r i a l a p p e a r e d i n b o t h c o n t r o l l a n e s , c o n s i s t e n t w i t h 78 TG IgG BSA cytochrome C F i g . 16. P r o t e i n p r o f i l e s (280 nm) o f B16G e l u a t e s d e r i v e d f r o m medium ( A ) , BW5147 ( B ) , a n d F d l l F (C) SN f r a c t i o n a t e d on a S u p e r o s e 12 c o l u m n u s i n g F P L C . E n h a n c e d r e c o v e r y o f m a t e r i a l a t t h e v o i d v o l u m e d e r i v e d f r o m F d l l F SN i s shown. E q u i v a l e n t amounts o f p r o t e i n (188 ug) were l o a d e d a n d c h r o m a t o g r a p h e d a c c o r d i n g t o t h e c o n d i t i o n s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . R e l a t i v e m o l e c u l a r mass m a r k e r s a r e i n d i c a t e d . M a r k e r s i n c l u d e d t h y r o g l o b u l i n (TG) , 669k M r; IgG, 150k M r; BSA, 66k M r; a n d c y t o c h r o m e C, 12.4k M r. I n t o t a l , 48 f r a c t i o n s (0.5 ml) were c o l l e c t e d . P r o t e i n t r a c e s a r e f o r f r a c t i o n s 15 t o 47. T y p i c a l d a t a o f f i v e d i f f e r e n t f r a c t i o n a t i o n s a r e shown. 79 13 15 17 19 21 23 28 27 29 31 33 Fraotlon no. F i g . 17. Ag-binding a c t i v i t y of FPLC f r a c t i o n s o b t a i n e d from F d l l F (•), BW5147 (•), and medium (O) e l u t i o n p r o f i l e s of F i g . 16. A g - b i n d i n g a c t i v i t y was measured as f o r F i g . 2. One hundred m i c r o l i t r e s of each f r a c t i o n were added t o Fd-coated p l a t e s and developed with B16G. ELISA val u e s are the mean of d u p l i c a t e d e t e r m i n a t i o n s . A p r e l i m i n a r y FPLC experiment had shown t h a t Ag-b i n d i n g a c t i v i t y was not d e t e c t a b l e i n the r e g i o n of f r a c t i o n s 35 to 48. R e p r e s e n t a t i v e r e s u l t s of t h r e e d i f f e r e n t d e t e r m i n a t i o n s are shown. 80 16 17 18 19 16 17 18 19 16 17 18 19 A B C F i g . 18. SDS-PAGE a n a l y s i s o f FPLC f r a c t i o n s (shown i n F i g . 17) 16 t o 19 i n c l u s i v e . Ten p e r c e n t SDS g e l s c o n t a i n i n g r e d u c i n g a g e n t w e r e d e v e l o p e d by s i l v e r s t a i n i n g . T w e n t y - f i v e m i c r o l i t r e s f r o m e a c h f r a c t i o n were c o n c e n t r a t e d a n d e l e c t r o p h o r e s e d . P o s i t i o n o f M m a r k e r s a r e i n d i c a t e d . A c t u a l M r e q u a l s number shown m u l t i p l i e d b y 1000. R e p r e s e n t a t i v e d a t a o f t h r e e a n a l y s e s a r e shown. A: medium c o n t r o l B: BW5147 m a t e r i a l C: F d l l F 81 p r e v i o u s r e s u l t s . T h e r e were no o t h e r v i s i b l e b a n d s u n i q u e t o F d l l F ( o r t o t h e c o n t r o l s a m p l e s ) i n t h e r e m a i n d e r o f t h e f r a c t i o n s t h a t w e re a n a l y z e d . A s e c o n d F P L C e x p e r i m e n t was c o n d u c t e d u s i n g a f r e s h s e t o f BW5147 a n d F d l l F e l u a t e s w i t h t h e i n t e n t o f c o l l e c t i n g f r a c t i o n s f o r b i o a s s a y . F r a c t i o n s 15 t o 20 f r o m e a c h s a m p l e w e r e s e l e c t e d f o r t e s t i n g . T a b l e I I I shows t h e p e r c e n t s u p p r e s s i o n o f Ab p r o d u c t i o n b y s p l e e n c e l l s t h a t w e re c o - c u l t u r e d w i t h t h e f r a c t i o n a t e d m a t e r i a l . I t c a n be s e e n t h a t m a x i m a l s u p p r e s s i o n o c c u r r e d w i t h f r a c t i o n 18 f r o m t h e F d l l F s a m p l e . T h e r e f o r e m a t e r i a l f r o m t h i s r e g i o n o f t h e f r a c t i o n a t i o n p r o f i l e d e m o n s t r a t e d s u p p r e s s i v e a c t i v i t y . T h u s , A g - b i n d i n g ( F i g . 17) a n d s u p p r e s s i v e ( T a b l e I I I ) m a t e r i a l w e r e r e c o v e r e d f r o m t h e v o i d v o l u m e o f t h e g e l f i l t r a t i o n c o l u m n . However, when t h e f r a c t i o n s w e r e a n a l y z e d by p o l y a c r y l a m i d e - S D S g e l s r u n w i t h r e d u c i n g a g e n t , a u n i q u e b a n d o f 30k M r was r e c o v e r e d a l o n g w i t h an 80k M r p r o t e i n . A d d i t i o n a l b a n d s a t 45k M , n o t d i r e c t l y r e l a t e d t o A g - b i n d i n g , w e r e a l s o s e e n . T o g e t h e r , t h e s e d a t a s u g g e s t t h a t t h e A g - b i n d i n g , s u p p r e s s i v e m a t e r i a l e l u t i n g f r o m B16G i s c o m p o s e d o f a h i g h M r m o i e t y made up o f a number o f i d e n t i f i a b l e p e p t i d e s w i t h M r b e t w e e n 30k a n d 80k. I d e n t i f i c a t i o n o f t h e a n t i g e n - b i n d i n g m o l e c u l e u s i n g SDS-PAGE A l t h o u g h F PLC f r a c t i o n a t i o n p r o v i d e d more e v i d e n c e r e l a t i n g 8 2 T a b l e I I I . A b i l i t y o f v a r i o u s f r a c t i o n s e l u t i n g f r o m FPLC S u p e r o s e 12 c o l u m n t o i n h i b i t t h e i n v i t r o Ab r e s p o n s e o f F d -immune s p l e e n c e l l s . P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g l e v e l s o f Ab f o r m a t i o n i n c u l t u r e s i n c u b a t e d w i t h e l u t e d m a t e r i a l s t o l e v e l s d e t e c t e d i n u n t r e a t e d c o n t r o l s . A t o t a l o f 475 u l f r o m e a c h f r a c t i o n w e r e a d d e d t o t h e c u l t u r e s . C u l t u r e s c o n t a i n i n g t h e f r a c t i o n a t e d m a t e r i a l w e re i n c u b a t e d f o r s e v e n d a y s . Ab c u l t u r e m e a s u r e m e n t s were o b t a i n e d u s i n g t h e " o r i g i n a l " p r o t o c o l ( s e e MATERIALS AND METHODS). R e p r e s e n t a t i v e r e s u l t s o f t h r e e d i f f e r e n t c u l t u r i n g e x p e r i m e n t s a r e shown. F r a c t i o n No. P e r c e n t s u p p r e s s i o n BW5147 F d l l F 15 -8 0 16 6 9 17 2 -5 18 6 53 19 13 -7 20 -10 13 83 A g - b i n d i n g a c t i v i t y a n d b i o a c t i v i t y w i t h F d l l F , i t d i d n o t i n d i c a t e w h i c h m o l e c u l e ( s ) was a c t u a l l y r e s p o n s i b l e f o r A g -b i n d i n g . A n o t h e r a t t e m p t t o i d e n t i f y t h e A g - b i n d i n g m o i e t y was made u s i n g p r e p a r a t i v e SDS-PAGE ( r u n w i t h o u t r e d u c i n g r e a g e n t ) . B16G e l u a t e s o f F d l l F a n d DC10 SN were e l e c t r o p h o r e s e d on a 12% s l a b g e l . The g e l was c u t i n t o e q u a l - s i z e s t r i p s a n d t h e m a t e r i a l e l u t e d f r o m t h e i n d i v i d u a l s l i c e s was a n a l y z e d b y SDS-PAGE r u n u n d e r r e d u c i n g c o n d i t i o n s a n d a s s a y e d f o r A g - b i n d i n g a c t i v i t y . The A g - b i n d i n g p r o f i l e i s shown i n F i g . 19. M a j o r r e g i o n s o f a c t i v i t y w e r e l o c a t e d a t s l i c e s 12 t o 14 a n d a t t h e e n d o f t h e g e l ( s l i c e s 20 t o 2 2 ) . DC10 e l u t e d m a t e r i a l d i d n o t h a v e a n y A g - b i n d i n g a c t i v i t y . The a c t i v i t y o f s l i c e s 12 t o 14 c o i n c i d e d e x a c t l y w i t h t h e p r e s e n c e o f b a n d s i n t h e 30k M r r e g i o n t h a t w e r e u n i q u e t o t h e F d l l F s a m p l e a s a s s e s s e d b y r e d u c i n g SDS-PAGE. The g e l s a c t u a l l y r e s o l v e d a d o u b l e t i n t h i s r e g i o n . T h i s i s shown i n F i g . 20. No A g - b i n d i n g a c t i v i t y was d e t e c t e d w i t h t h e 80k M r b a n d s t h a t r e s o l v e d a t s l i c e 4 o f F d l l F m a t e r i a l ( s e e F i g . 2 0 ) . I n f a c t , no o t h e r d i s t i n c t b a n d s u n i q u e t o F d l l F were d e t e c t e d . Two f a i n t b a n d s a t 68k M r were v i s i b l e i n l a n e s 13 and 14, f o r b o t h " F " a n d "D" o f F i g . 20. W h e t h e r t h e y w e re p r o t e i n o r a c h e m i c a l a r t i f a c t was unknown. T h e i r a p p e a r a n c e i n a c o n t r o l l a n e n o t l o a d e d w i t h a p r o t e i n s a m p l e ("C") s u g g e s t e d t h a t t h e y w e r e s i l v e r s t a i n i n g a r t i f a c t s . The p r o t e i n b a n d s e l u t e d f r o m s l i c e s 20 t o 22 o f t h e DC10 o r F d l l F s a m p l e s , i f a c t u a l l y p r e s e n t , w e r e n o t v i s i b l e ; m ost l i k e l y 84 • M - 1 Fraction no. F i g . 19. A g - b i n d i n g a c t i v i t y o f DC10 a n d F d l l F m a t e r i a l e l u t e d f r o m s l i c e s o f a 12% p r e p a r a t i v e SDS-PAGE g e l r u n u n d e r n o n -r e d u c i n g c o n d i t i o n s . Two h u n d r e d m i c r o g r a m s e a c h o f a f f i n i t y - e n r i c h e d F d l l F (•) o r DC10 (•) m a t e r i a l were f r a c t i o n a t e d u s i n g p r e p a r a t i v e SDS-PAGE u n d e r n o n - r e d u c i n g c o n d i t i o n s . R e c o v e r e d m a t e r i a l f r o m i n d i v i d u a l s l i c e s was t e s t e d f o r A g - b i n d i n g a c t i v i t y as i n F i g . 85 4 12 13 14 C F D F D F D F D F i g . 20. PAGE o f m a t e r i a l f r o m s e l e c t e d f r a c t i o n s o f F i g . 19 u s i n g a 10% SDS g e l r u n u n d e r r e d u c i n g c o n d i t i o n s . F r a c t i o n 4 shows t h e 80k M r p r o t e i n r e c o v e r e d f r o m b o t h F d l l F a n d DC10 c o n t r o l m a t e r i a l . F r a c t i o n s 12 t o 14, w h i c h e x h i b i t e d A g -b i n d i n g a c t i v i t y ( F i g . 19), show b a n d s u n i q u e t o F d l l F f r o m t h e 30k M r r e g i o n . F: F d l l F D: DC10 m a t e r i a l C: c o n t r o l l a n e s h o w i n g s i l v e r s t a i n i n g " a r t i f a c t " 86 due t o t h e i n a b i l i t y o f t h e g e l t o r e s o l v e l o w M r p r o t e i n s ( i e . < 14k M r ) . W h e t h e r t h e m a t e r i a l r e s p o n s i b l e f o r t h e E L I S A a c t i v i t y was a b r e a k d o w n p r o d u c t o f t h e 30k M r s p e c i e s o r r e p r e s e n t s a d i f f e r e n t p r o t e i n t h a t b i n d s t o b o t h F d a n d B16G i s unknown. F u r t h e r w ork w o u l d be r e q u i r e d t o c l a r i f y i t s r e l a t i o n s h i p , i f any, t o t h e 30k M r p r o t e i n . I t s h o u l d be n o t e d t h a t t h r e e s e p a r a t e p r e p a r a t i v e SDS-PAGE f r a c t i o n a t i o n e x p e r i m e n t s w e re p e r f o r m e d w i t h t h e a i m o f r e c o v e r i n g b i o a c t i v i t y f r o m g e l s l i c e s , w i t h o u t s u c c e s s . I t was p o s s i b l e t h a t t h e r e s i d u a l amounts o f SDS i n t h e e l u t e d f r a c t i o n m a t e r i a l h a d a d e t r i m e n t a l e f f e c t on t h e g r o w t h o f t h e Ab c u l t u r e s . An a l t e r n a t i v e e x p l a n a t i o n was t h a t SDS-PAGE f r a c t i o n a t i o n h a d d e s t r o y e d b i o a c t i v i t y b y t h e s e p a r a t i o n o f two o r more n o n - c o v a l e n t l y a s s o c i a t e d m o l e c u l e s , one o f w h i c h was A g -b i n d i n g . On o c c a s i o n , o t h e r b a n d s u n i q u e t o t h e F d l l F s a m p l e ( e g . F i g . 18C) h a v e b e e n r e c o v e r e d a l o n g w i t h t h e 30k M r m o l e c u l e . The a p p e a r a n c e o f t h e 30k M r p r o t e i n i n b o t h t h e FPLC f r a c t i o n s a n d t h e m a t e r i a l e l u t e d f r o m t h e n o n - r e d u c i n g p r e p a r a t i v e SDS-PAGE f r a c t i o n s s t r o n g l y s u g g e s t e d t h a t i t was t h e m o i e t y r e s p o n s i b l e f o r t h e A g - b i n d i n g a c t i v i t y . H owever, t o d e t e r m i n e i f t h e 80k M r m o l e c u l e was a c t u a l l y u n i q u e t o F d l l F , i t was s u b j e c t e d t o t w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . The r e m a i n d e r o f t h e s e l e c t e d f r a c t i o n s f r o m t h e FPLC e x p e r i m e n t o f F i g . 18 w e r e u s e d . M a t e r i a l f r o m f r a c t i o n 17 o f t h e F d l l F s a m p l e a n d p o o l e d m a t e r i a l f r o m f r a c t i o n s 17 t o 19 o f t h e medium s a m p l e 87 were a n a l y z e d i n p a r a l l e l . F i g . 21 shows t h a t , w i t h i n t h e l i m i t s o f t h e t e c h n i q u e , t h e 80k M r p r o t e i n s f r o m medium a n d F d l l F s a m p l e s w ere n o t o n l y o f t h e same M , b u t h a d t h e same i s o e l e c t r i c p o i n t as w e l l . I t i s g e n e r a l l y a s s u m e d t h a t two d i f f e r e n t p r o t e i n s w o u l d n o t h a v e t h e i d e n t i c a l p i a n d M . T h e r e f o r e i n s p i t e o f t h e p r e p o n d e r a n c e o f t h e 80k M r p r o t e i n o f F d l l F o v e r c o n t r o l m a t e r i a l , t h e two p r o t e i n s w e r e p r o b a b l y i d e n t i c a l a n d were d e r i v e d n o t f r o m t h e F d l l h y b r i d o m a , b u t t h e FCS o f t h e c o m p l e t e medium m i x t u r e . 88 F i g . 2 1 . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f t h e 80k M r p r o t e i n s o f medium a n d F d l l F . E q u a l amounts o f 8 0k M r m a t e r i a l f r o m b o t h medium ( p a n e l A) a n d F d l l F ( p a n e l B) were s e p a r a t e d b y I E F u s i n g pH 3-10 a m p h o l y t e s a n d r e d u c i n g SDS-PAGE. A f t e r f o c u s s i n g , t h e t u b e g e l s w e r e l a y e r e d on t o p o f 12% SDS-PAGE g e l s a n d i n c u b a t e d i n s a m p l e b u f f e r c o n t a i n i n g r e d u c i n g a g e n t f o r 5 m i n . F o l l o w i n g i n c u b a t i o n , e l e c t r o p h o r e s i s i n t h e s e c o n d d i m e n s i o n was b e g u n c o n c u r r e n t l y f o r b o t h s a m p l e s . G e l s w e re f i x e d a n d s i l v e r -s t a i n e d . I s o e l e c t r i c p o i n t a n d M c a l i b r a t i o n s a r e i n d i c a t e d . 89 90 DISCUSSION I t has been r e p o r t e d t h a t F d l l F , p roduced by t h e Ts hybridoma, F d l l , was r e c o v e r e d u s i n g t h e T s F - s p e c i f i c mAb, B16G. F d l l F p o s s e s s e d b o t h A g - b i n d i n g a c t i v i t y and t h e a b i l i t y t o s u p p r e s s t h e a n t i - F d Ab response i n v i v o ( S t e e l e , Chu, Chan, N o r t h , and Levy 1987). I n t h i s t h e s i s , F d l l F was c h a r a c t e r i z e d f u r t h e r a t t h e immunochemical, b i o l o g i c a l , and b i o c h e m i c a l l e v e l s . I t was shown t h a t F d l l F bound t o and d e m o n s t r a t e d s e l e c t i v i t y f o r s o l i d -phase Fd. The f a c t o r r e a c t e d a l s o w i t h f l u i d - p h a s e Ag. T h i s p r o p e r t y was used t o d e t e r m i n e t h e f i n e s p e c i f i c i t y o f F d l l F b i n d i n g . The b i o l o g i c a l e f f e c t o f F d l l F on t h e a n t i - F d Ab response was measured u s i n g a s p l e e n c e l l c u l t u r e system. W i t h t h i s a s s a y , F d l l F was shown t o s u p p r e s s Ab p r o d u c t i o n i n a c o n c e n t r a t i o n - d e p e n d e n t manner. F u r t h e r m o r e , a d d i t i o n a l e x p e r i m e n t s s u g g e s t e d t h a t t h e s u p p r e s s i v e e f f e c t was Ag-s p e c i f i c . B i o c h e m i c a l c h a r a c t e r i z a t i o n was aimed a t i s o l a t i n g t h e A g - b i n d i n g m o i e t y o f F d l l F . By u s i n g g e l f i l t r a t i o n , b o t h A g - b i n d i n g and b i o l o g i c a c t i v i t y were r e c o v e r e d from a h i g h M r peak i e . > 500,000. When n o n - r e d u c i n g p r e p a r a t i v e SDS-PAGE was used, a s i n g l e F d l l - a s s o c i a t e d , 30k M r p o l y p e p t i d e , w h i c h appeared t o b i n d Ag, was i s o l a t e d . These f i n d i n g s a r e d i s c u s s e d below. 9 1 B16G a s a P r o b e f o r T S u p p r e s s o r F a c t o r I s o l a t i o n o f F d l l F was p o s s i b l e b e c a u s e o f t h e T s F - s p e c i f i c mAb, B16G. As m e n t i o n e d i n t h e INTRODUCTION, B16G was i n i t i a l l y d e v e l o p e d t o s t u d y t h e P815 t u m o r s y s t e m . The Ab i t s e l f was shown t o be a c t i v e , b o t h i n v i t r o a n d i n v i v o . I t was t h e n u s e d t o i s o l a t e d A 10F, a T s F w h i c h s u p p r e s s e d t h e CTL r e s p o n s e t o P 8 1 5 . B16G h a s b e e n e m p l o y e d a s a t o o l t o s t u d y s u p p r e s s i o n i n o t h e r A g s y s t e m s . S t e e l e e t a l . (1987) showed t h a t B16G r e c o g n i z e d T s F 1 , b u t n o t T s F 2 o r T s F ^ o f t h e NP s y s t e m . T h i s i n d i c a t e d t h a t B16G was n o t o n l y u s e f u l o u t s i d e t h e s y s t e m s i n w h i c h i t was d e v e l o p e d , b u t t h a t B16G was " c l a s s - s p e c i f i c " w i t h r e s p e c t t o r e c o g n i z i n g d i f f e r e n t t y p e s o f T s F . I n a c o m p l e t e l y d i f f e r e n t e x p e r i m e n t a l s y s t e m , B16G was u s e d t o s t u d y UV r a d i a t i o n - i n d u c e d s u p p r e s s i o n . I t i s known t h a t e x p o s u r e o f m i c e t o UV r a d i a t i o n c a n l e a d t o an i m p a i r m e n t i n immune f u n c t i o n . A d o p t i v e l y t r a n s f e r r e d l y m p h o i d c e l l s f r o m U V - t r e a t e d a n i m a l s f a i l t o r e j e c t a U V - i n d u c e d s y n g e n e i c t u m o r a s c o m p a r e d t o u n t r e a t e d c o n t r o l s . F a i l u r e o f r e j e c t i o n was a t t r i b u t e d t o t h e i n d u c t i o n o f t u m o r - s p e c i f i c Ts ( F i s h e r a n d K r i p k e 1 9 7 7 ) . An a s s o c i a t i o n b e t w e e n d e f e c t i v e Ag p r e s e n t a t i o n a n d s y s t e m i c s u p p r e s s i o n o f t h e CS r e s p o n s e was n o t e d i n m i c e f o l l o w i n g e x p o s u r e t o UV r a d i a t i o n (Noonan e t a l . 1 9 8 1 ) . B16G was u s e d t o show t h a t T s F as w e l l , was i m p l i c a t e d i n t h e U V - i n d u c e d s y s t e m i c s u p p r e s s i o n o f t h e CS r e s p o n s e (Yee e t a l . 1 9 8 9 ) . I n v i v o , B16G a b r o g a t e d t h e e f f e c t o f UV r a d i a t i o n on t h e CS r e s p o n s e . The 92 i n v e s t i g a t o r s a l s o p r e p a r e d B16G i m m u n o a d s o r b e n t s a n d r e c o v e r e d s u p p r e s s i v e m a t e r i a l f r o m Ts c u l t u r e SN. L a s t l y , S ehon a n d c o l l e a g u e s h a v e u s e d B16G f o r t h e i s o l a t i o n o f s u p p r e s s i v e m a t e r i a l i n v o l v e d i n t h e r e g u l a t i o n o f t h e I g E r e s p o n s e (Sehon e t a l . 1 9 8 9 ) . T h u s , B16G h a s b e e n e f f e c t i v e i n t h e s t u d y o f s u p p r e s s i o n i n s e v e r a l l a b o r a t o r i e s . I t h a s a m o d u l a t i n g e f f e c t on t h e immune r e s p o n s e when i t i s a d m i n i s t e r e d i n v i v o a n d c a n be u s e d f o r t h e r e c o v e r y o f s u p p r e s s i v e m o l e c u l e s i n d i f f e r e n t Ag s y s t e m s . The u s e o f Ab r e a g e n t s s p e c i f i c f o r a c o n s t a n t r e g i o n o f t h e T s F m o l e c u l e i s n o t e x c l u s i v e t o B16G. I n an a t t e m p t t o i d e n t i f y p u t a t i v e TCR i s o t y p e s , T o k u h i s a a n d T a n i g u c h i (1982) r a i s e d an a l l o a n t i s e r u m s p e c i f i c f o r t h e m u r i n e i m m u n o g l o b u l i n h e a v y c h a i n . The a n t i s e r u m was f o u n d t o b i n d T s F . U s i n g t h e r e a g e n t , t h e y went on t o i d e n t i f y a l l o t y p i c d e t e r m i n a n t s on t h e c o n s t a n t r e g i o n o f t h e A g - b i n d i n g c h a i n o f a K LH-TsF. S o r e n s e n a n d P i e r c e (1985) p r e p a r e d mAb t h a t w e r e e i t h e r s p e c i f i c f o r i n d u c e r o r e f f e c t o r T s F . A l t h o u g h t h e y p r o d u c e d a b a t t e r y o f mAb, none o f t h e m were c l o n o t y p i c i e . none were r e a c t i v e w i t h t h e A g - b i n d i n g , v a r i a b l e r e g i o n s o f any o f t h e T s F , w h i c h w e r e u s e d as immunogens. S i m i l a r l y , I v e r s o n a n d c o l l e a g u e s i s o l a t e d mAb t h a t w e re s p e c i f i c f o r t h e A g - b i n d i n g c h a i n o f i n d u c e r a n d e f f e c t o r T s F , r e s p e c t i v e l y ( F e r g u s o n , Beaman, a n d I v e r s o n 1 9 8 5 ; F e r g u s o n a n d I v e r s o n 1986) . B o t h g r o u p s u s e d t h e mAb i n t h e r e c o v e r y o f T s F f o r f u n c t i o n a l a n d b i o c h e m i c a l a n a l y s i s . 93 I m m u n o c h e m i c a l P r o p e r t i e s o f F d l l F D i r e c t b i n d i n g o f F d l l F t o F d was shown u s i n g E L I S A . I n i t i a l l y , h o w e v e r , t h e d a t a o f F i g . 1 showed d i r e c t a n d s p e c i f i c b i n d i n g o f B16G t o F d l l F - c o a t e d p l a t e s . W i t h a v a r i a t i o n i n t h e i m m u n o a s s a y , A g - b i n d i n g by F d l l F was d e m o n s t r a t e d a s w e l l . T h a t i s , F d l l F , b u t n o t m a t e r i a l p r e p a r e d f r o m medium c o n t a i n i n g FCS o r f r o m t h e i r r e l e v a n t h y b r i d o m a , DC10, b o u n d t o F d - c o a t e d p l a t e s . The d a t a o f T a b l e I , w h i c h showed b i n d i n g o f F d l l F t o F d , b u t n o t t o two o t h e r u n r e l a t e d A g , i n d i c a t e d t h a t F d l l F r e a c t i v i t y was n o t e x p l a i n e d by n o n s p e c i f i c a d s o r p t i o n t o a p r o t e i n - c o a t e d s u r f a c e . The p o s s i b i l i t y t h a t t h e b i n d i n g was due t o a p a r t i c u l a r , s o l i d - p h a s e c o n f o r m a t i o n o f F d was t e s t e d by d e t e r m i n i n g w h e t h e r F d l l F c o u l d r e a c t w i t h F d i n s o l u t i o n . I t was f o u n d t h a t v a r i o u s c o n c e n t r a t i o n s o f A g c o u l d i n h i b i t t h e b i n d i n g o f F d l l F t o s o l i d -p h a s e F d . A s shown i n F i g . 6, t h e l e v e l o f i n h i b i t i o n a p p r o a c h e d 100% w i t h F d a n d C - m o l e c u l e . T h i s v e r i f i e d t h a t F d l l F b i n d i n g was i d e n t i c a l w h e t h e r F d was i n f l u i d - o r s o l i d - p h a s e a n d was i n d e p e n d e n t o f any " f i x e d " c o n f o r m a t i o n o f F d . The i n h i b i t i o n E L I S A a l l o w e d t h e b i n d i n g o f F d l l F t o be q u a n t i f i e d a s w e l l . I t was shown i n F i g . 6 t h a t F d l l F was s p e c i f i c f o r t h e C - d e t e r m i n a n t o f F d . The o b s e r v a t i o n t h a t C - m o l e c u l e a n d F d w e r e b e t t e r i n h i b i t o r s t h a n OFd (0.312 ug/ml v s . 1.25 ug/ml f o r 5 0 % i n h i b i t i o n ) s u g g e s t e d t h a t F d l l F b i n d i n g t o F d ( o r i t s a n a l o g s ) was n o t s i m p l y due t o c h a r g e e f f e c t s . S i n c e F d i s an a c i d i c protein ( i s o e l e c t r i c point of Fd i s 3.7), i t was conceivable that binding was due to the net negative charge of Fd while i n solution. If so, then OFd would be expected to be a better i n h i b i t o r of F d l l F binding as peroxyformic acid oxidation imparted an even greater negative charge to the protein. Furthermore, the intermediate i n h i b i t o r y a c t i v i t y of OFd correlated with i t s expressed l e v e l of C-determinant r e a c t i v i t y , as measured with the C-determinant-specific mAb, 16a. This was seen i n F i g . 5 where the l e v e l of 16a binding to OFd was intermediate between C-molecule and N-molecule. This lends additional support to the suggestion that F d l l F i s s p e c i f i c for the C-determinant of Fd. That F d l l F should bind to either N- or C-determinant, as opposed to other s i t e s on the molecule, was consistent with the pattern of Fd a n t i g e n i c i t y . It i s known that a l l of the B and T c e l l interactions with Fd reported so far, are focussed at the N-and C-determinants. The si g n i f i c a n c e of the C-determinant s p e c i f i c i t y of F d l l F , with respect to regulatory mechanisms, cannot be assessed at t h i s time. However, i t i s of i n t e r e s t to compare F d l l F binding with the determinant s p e c i f i c i t y of the Ab secreted by the c e l l s used i n the suppression assay. As shown in F i g . 9A, a l l of the anti-Fd Ab produced by the c e l l s obtained from the C-molecule and Fd-immunized mice was directed to N-molecule. The lack of r e a c t i v i t y with C-molecule was not explained by the "hidden" expression of C-determinant due to i t s binding to solid-phase, as the anti-Fd mAb, 16a ( s p e c i f i c for the 9 5 C - m o l e c u l e ) , was a b l e t o b i n d ( F i g . 5 ) . The p a t t e r n o f Ab a c t i v i t y o f t h e c u l t u r e d c e l l s was c o n f i r m e d b y t h e s i m i l a r i t y i n b i n d i n g d i s t r i b u t i o n o f s e r u m Ab o f t h e m i c e ( F i g . 9 B ) . The r e s u l t s i m p l i e d t h a t , i n v i t r o , C - d e t e r m i n a n t - s p e c i f i c F d l l F s u p p r e s s e d t h e a n t i - N - d e t e r m i n a n t Ab r e s p o n s e . The d e s i g n a t i o n o f C - d e t e r m i n a n t a s t h e c a r r i e r a n d N - d e t e r m i n a n t a s t h e h a p t e n o f t h e F d m o l e c u l e w o u l d be c o n s i s t e n t w i t h t h i s o b s e r v a t i o n . C a r r i e r s p e c i f i c i t y was d e m o n s t r a t e d f o r KLH-TsF ( T a k e m o r i a n d T a d a 1975) a n d Ts i n t h e immune r e s p o n s e t o i n s u l i n ( J e n s e n , P i e r c e , a n d Kapp 1 9 8 4 ) . The h a p t e n - c a r r i e r m o d e l o f F d was a l s o u s e d t o e x p l a i n t h e e f f e c t s o f a n t i - i d i o t y p i c Ab on t h e immune r e s p o n s e i n H-2 a n i m a l s (Weaver e t a l 1983) . M o s t o f t h e r e p o r t e d A g - s p e c i f i c T s F h a v e e x h i b i t e d A g -b i n d i n g a c t i v i t y b y t h e i r a b i l i t y t o b i n d t o i m m u n o a d s o r b e n t c o l u m n s . R e l a t i v e l y few e x a m p l e s h a v e shown i m m u n o c h e m i c a l p r o p e r t i e s s i m i l a r t o F d l l F , s u c h as i t s d i r e c t d e t e c t i o n u s i n g E L I S A o r t h e d e t e r m i n a t i o n o f f i n e s p e c i f i c i t y o f b i n d i n g u s i n g Ag a n d s t r u c t u r a l a n a l o g s . However, a T c e l l - d e r i v e d , A g - b i n d i n g m o l e c u l e d e s c r i b e d b y Cone e t a l . (1987) d o e s s h a r e some f e a t u r e s w i t h F d l l F . U s i n g a r a b b i t a n t i s e r u m , t h e y d e t e c t e d t h e m o l e c u l e i n t h e s e r u m o f d e s e n s i t i z e d m i c e . I t d i s p l a y e d s i m i l a r i m m u n o c h e m i c a l p r o p e r t i e s t o F d l l F a n d h a d a M r o f a b o u t 37k as a n a l y z e d b y SDS-PAGE, w i t h r e d u c i n g a g e n t . The f i n e A g s p e c i f i c i t y shown b y F d l l F was a l s o s e e n w i t h two o t h e r T s F r a i s e d t o n a t u r a l p r o t e i n A g : l y s o z y m e ( A d o r i n i e t a l . 1984) a n d i n s u l i n ( J e n s e n a n d Kapp 1 9 8 6 ) . T h e s e i n v e s t i g a t o r s u s e d 96 immunoadsorbent columns made w i t h s t r u c t u r a l a n a l o g s o f t h e r e s p e c t i v e Ag t o demonstrate s p e c i f i c i t y o f TsF b i n d i n g . A more d i r e c t r e l a t i o n s h i p between a d i f f e r e n t Ag system and F d l l F was shown by u s i n g an a n t i s e r u m d e v e l o p e d by N o r t h e t a l . (1988). A r a b b i t a n t i s e r u m (known as a n t i - p 3 0 ) was r a i s e d t o t h e 30k M r p r o t e i n o f F d l l F and shown, u s i n g FACS a n a l y s i s , t o b i n d t o not o n l y F d l l and t h e A10 hybridoma, but t o a p r o p o r t i o n o f normal murine T c e l l s as w e l l . A n t i - p 3 0 r e a c t e d w i t h t h e CD4 and CD8 d i f f e r e n t i a t e d T c e l l s u b s e t s i n a d d i t i o n t o thymocytes t h a t d i d not e x p r e s s e i t h e r marker. R e l a t e d t o t h i s work, but i n an independent l i n e o f s t u d y , Askenase and c o l l e a g u e s d e s c r i b e d T c e l l - d e r i v e d , A g - b i n d i n g m o l e c u l e s t h a t were found t o i n i t i a t e t h e CS r e s p o n s e (Ptak e t a l . 1986a,b). I n an e f f o r t t o c h a r a c t e r i z e t h e o r i g i n o f t h e f a c t o r - p r o d u c i n g c e l l , t h e y found t h a t T c e l l s from nude mice were c a p a b l e o f p r o d u c i n g t h i s Ag-b i n d i n g f a c t o r (Herzog e t a l . 1989). S u b s e q u e n t l y , t h e y d e r i v e d a c l o n e d c e l l l i n e from nude mice t h a t r e a c t e d s p e c i f i c a l l y w i t h a n t i - p 3 0 (P. Askenase, u n p u b l i s h e d ) . Whether t h e a n t i s e r u m i s r e a c t i v e w i t h a n o v e l t y p e o f TCR, p o s s i b l y e x p r e s s e d on an u n d i f f e r e n t i a t e d " p r i m i t i v e " T c e l l s u b s e t , remains t o be seen. -B i o l o g i c P r o p e r t i e s o f F d l l F B i o l o g i c a c t i v i t y o f F d l l F was shown i n two ways. F i r s t l y , t h e a d d i t i o n o f F d l l F t o Ag-primed s p l e e n c e l l s s u p p r e s s e d t h e p r o d u c t i o n o f a n t i - F d Ab. T h i s was i n comparison t o c u l t u r e s 9 7 c o n t a i n i n g c o n t r o l m a t e r i a l o r medium a l o n e . P a s t e x p e r i e n c e i n t h i s l a b o r a t o r y h a d shown t h a t i t was n o t t e c h n i c a l l y f e a s i b l e t o c o u p l e F d t o s h e e p r e d b l o o d c e l l s . T h e r e f o r e a d i f f e r e n t s p l e e n c e l l a s s a y was d e v i s e d , t o r e p l a c e t h e c o n v e n t i o n a l p l a q u e -f o r m i n g c e l l a s s a y , f o r t h e measurement o f F d l l F - m e d i a t e d s u p p r e s s i o n . P r e l i m i n a r y e x p e r i m e n t s h a d a l s o shown t h a t p r i m a r y c u l t u r e s o f s p l e e n c e l l s p r o d u c e d an i n s u f f i c i e n t amount o f Ab f o r r e l i a b l e m e asurement u s i n g E L I S A . T h e r e f o r e , A g - p r i m e d c e l l s w o u l d be u s e d f o r t h e a s s a y . F u r t h e r m o r e , t h e s p l e e n c e l l s were p u l s e d ( i e . c u l t u r e d b r i e f l y w i t h a h i g h d o s e o f Ag) t o s t i m u l a t e t h e p r o d u c t i o n o f Ab i n v i t r o . B10.D2 (H-2 ) m i c e w e r e u s e d as t h e s o u r c e o f s p l e e n c e l l s i n o r d e r t o t e s t t h e e f f e c t o f F d l l F i n a s y n g e n e i c s y s t e m . T h e r e f o r e , a s p a r t o f t h e i m m u n i z a t i o n p r o t o c o l , t h e B10.D2 a n i m a l s w e re made immune t o F d b y p r i m i n g t h e m w i t h C - m o l e c u l e . The d a t a o f F i g . 7 showed t h a t t h e amount o f s e c r e t e d Ab r e p r e s e n t e d a s t r o n g " s i g n a l " a b o v e b a c k g r o u n d , t o m e a s u r e s u p p r e s s i o n w i t h . A s t h e m e c h a n i s m o f a c t i o n o f F d l l F was unknown, s e v e r a l v a r i a t i o n s i n t h e a s s a y h a d t o be t r i e d b e f o r e t h e m a x i m a l l e v e l o f s u p p r e s s i o n w i t h F d l l F was d e t e r m i n e d . The r e s u l t s f r o m two p r o t o c o l s w e r e shown i n t h i s t h e s i s . The m a j o r i t y o f i n v i t r o t e s t i n g w i t h F d l l F was done w i t h A g - p r i m e d c e l l s t h a t w e re p u l s e d w i t h F d a n d t h e n c o n t i n u o u s l y c o - c u l t u r e d w i t h F d l l F f o r t h e e n t i r e i n c u b a t i o n p e r i o d ( F i g . 10). T h i s p r o t o c o l was l a t e r m o d i f i e d s o t h a t t h e s p l e e n c e l l s w e r e f i r s t i n c u b a t e d w i t h F d l l F a n d t h e n e x p o s e d t o F d , w i t h o u t w a s h i n g b e t w e e n a d d i t i o n s . 98 F o l l o w i n g a 2 h e x p o s u r e t o F d , t h e c e l l s w e r e w a s h e d a n d c u l t u r e d w i t h o u t r e c e i v i n g any f u r t h e r a d d i t i o n s . S u p p r e s s i o n o f t h e a n t i - F d Ab r e s p o n s e b y F d l l F was a c h i e v e d i n a c o n c e n t r a t i o n - d e p e n d e n t manner u s i n g e i t h e r p r o t o c o l . A maximum o f 60% s u p p r e s s i o n was a t t a i n e d u s i n g t h e o r i g i n a l a s s a y o f A g p u l s i n g f o l l o w e d b y c o - c u l t u r i n g w i t h F d l l F . I t s h o u l d be e m p h a s i z e d t h a t t h e d a t a o f F i g . 10 was c o m p i l e d u s i n g t h e r e s u l t s f r o m d i f f e r e n t b a t c h e s o f F d l l F . The o b s e r v e d c u r v i l i n e a r r e l a t i o n s h i p b e t w e e n p e r c e n t s u p p r e s s i o n a n d A g -b i n d i n g a c t i v i t y s u g g e s t e d t h a t b o t h t h e a s s a y a n d t h e F d l l F p r e p a r a t i o n t e c h n i q u e were r e p r o d u c i b l e . By r e v i s i n g t h e p r o t o c o l , s u p p r e s s i o n a p p r o a c h i n g 100% was n o t e d ( F i g . 1 2 ) . O t h e r i n v e s t i g a t o r s h a v e a c h i e v e d n e a r l y 100% s u p p r e s s i o n when n a i v e o r A g - p r i m e d s p l e e n c e l l s w e r e c o - c u l t u r e d w i t h b o t h s o l u b l e A g a n d T s F a t t h e same t i m e ( T a n i g u c h i , S a i t o , a n d Tada 1979; Kapp e t a l . 1 9 7 6 ) . F o r t h e p r e s e n t work, s u p p r e s s i o n was o b s e r v e d when t h e s p l e e n c e l l s w e r e p u l s e d w i t h f a c t o r f o l l o w e d b y A g . The e f f e c t i v e n e s s o f p u l s i n g s p l e e n c e l l s t o "arm th e m " w i t h f a c t o r f o r s u p p r e s s i o n h a s b e e n r e p o r t e d b y M o s e r , K a u f f m a n , a n d A b b a s ( 1 9 8 5 ) . An a l t e r n a t e e x p l a n a t i o n , f o r t h e e f f e c t i v e n e s s o f F d l l F p u l s i n g , i s p r o v i d e d b y t h e " p l u s - m i n u s " n e t w o r k t h e o r y , p r o p o s e d b y H o f f m a n n ( 1 9 8 0 ) . I t was p o s t u l a t e d t h a t d u r i n g t h e s u p p r e s s e d s t a t e , m o n o v a l e n t A g - b i n d i n g f a c t o r s w o u l d b i n d t o T c e l l s o f c o m p l e m e n t a r y s p e c i f i c i t y a n d i n h i b i t t h e i r m u t u a l s t i m u l a t i o n , p r e v e n t i n g t h e i n d u c t i o n o f an Ab r e s p o n s e . When t h e s p l e e n c e l l s w e r e f i r s t e x p o s e d t o A g 99 f o l l o w e d b y c o - c u l t u r i n g w i t h F d l l F , t h e p e r c e n t a g e s u p p r e s s i o n r e a c h e d a maximum a n d a p p e a r e d t o p l a t e a u ( F i g . 1 0 ) . I t i s p o s s i b l e t h a t t h e Ag p u l s i n g p e r i o d h a d g i v e n t h e Th a " h e a d s t a r t " t o w a r d s Ab p r o d u c t i o n b e f o r e t h e s u p p r e s s i v e e f f e c t s o f F d l l F b e g a n . D e s p i t e t h e d i f f e r e n c e i n o b s e r v e d l e v e l s o f s u p p r e s s i o n , a d d i t i o n a l e x p e r i m e n t s u s i n g t h e " o r i g i n a l " ( F i g s . 10 a n d 11) o r " r e v i s e d " p r o t o c o l ( T a b l e I I ) s u p p o r t e d t h e i d e a t h a t t h e s u p p r e s s i v e a c t i v i t y o f F d l l F was A g - s p e c i f i c . A s s e e n i n F i g . 11, F d l l F d i d n o t s u p p r e s s t h e Ab r e s p o n s e t o an u n r e l a t e d A g . T a b l e I I showed t h a t i n r e c i p r o c a l c r i s s - c r o s s e x p e r i m e n t s , F d l l F was s u p p r e s s i v e i n t h e Ab c u l t u r e a s s a y , w h e r e a s A10F was n o t . C o n v e r s e l y , A10F s u p p r e s s e d t h e CTL r e s p o n s e t o P 8 1 5 , w h i l e F d l l F d i d n o t . I t c o u l d be a r g u e d t h a t a l t h o u g h t h e f a c t o r s w e r e t e s t e d i n d i f f e r e n t c e l l u l a r a s s a y s , t h e m o l e c u l e s t h e m s e l v e s w e r e s i m i l a r a s t h e y w e re b o t h i s o l a t e d u s i n g t h e same mAb. I n a d d i t i o n , A10F d o e s s u p p r e s s t h e s y n g e n e i c CTL r e s p o n s e t o P815 i n a c o n c e n t r a t i o n - d e p e n d e n t manner a n d d o e s n o t a l t e r t h e m a g n i t u d e o f a m i x e d l y m p h o c y t e r e a c t i o n w i t h DBA/2 s p l e e n c e l l s on h i s t o i n c o m p a t i b l e t a r g e t s ( S t e e l e , Chan, Stammers, S i n g h a i , a n d L e v y 1987; Chan e t a l . 1 9 8 8 ) . C o l l e c t i v e l y , t h e d a t a a r e c o n s i s t e n t w i t h F d l l F b e i n g A g - s p e c i f i c . B i o l o g i c a l a c t i v i t y o f F d l l F was a l s o shown i n v i v o . The s e r u m Ab r e s p o n s e s o f Fd-immune, DBA/2 H-2 m i c e w ere s u p p r e s s e d , a s c o m p a r e d t o c o n t r o l a n i m a l s , when c h a l l e n g e d w i t h F d a n d F d l l F . Due t o t h e w i d e r a n g e i n Ab r e s p o n s e s o f t h e m i c e 100 f o l l o w i n g t h e i r i n i t i a l i m m u n i z a t i o n , t h e a n i m a l s w e r e r a n k e d a c c o r d i n g t o t h e i r s e r u m Ab l e v e l s a n d p l a c e d i n t o F d l l F , A10F, o r PBS " t r e a t m e n t " g r o u p s p r i o r t o r e c e i v i n g f a c t o r . I n t h i s way, a p p r o x i m a t e l y e q u a l numbers o f a n i m a l s h a v i n g s i m i l a r p r e t r e a t m e n t Ab l e v e l s w o u l d r e c e i v e e i t h e r F d l l F , A 10F, o r PBS. A g a i n , b e c a u s e o f t h e w i d e r a n g e o f r e s p o n s e s , t h e d a t a was r e c o r d e d a s i n d i v i d u a l c h a n g e s i n Ab l e v e l s b e f o r e a n d a f t e r t r e a t m e n t . I t was c l e a r f r o m t h e d a t a o f F i g . 13 t h a t t h e Ab l e v e l s o f t h e F d l l F - t r e a t e d a n i m a l s h a d d e c r e a s e d f o l l o w i n g f a c t o r a d m i n i s t r a t i o n a n d A g c h a l l e n g e . I n c o n t r a s t , t h e a n t i - F d Ab l e v e l s o f t h e PBS o r A10F g r o u p s h a d b y a n d l a r g e i n c r e a s e d . The r e l a t i v e l y p o o r i m m u n o g e n i c i t y o f F d c a n p r o b a b l y a c c o u n t f o r t h e Ab l e v e l s o f most o f t h e c o n t r o l g r o u p a n i m a l s t h a t r e m a i n e d c l o s e t o t h e "no c h a n g e " l i n e . E a r l i e r w ork h a d shown t h a t a s e c o n d a r y r e s p o n s e t o F d d e v e l o p s more s l o w l y a s c o m p a r e d t o t h e r e s p o n s e t o a more c o m p l e x Ag s u c h KLH. I n d e e d , t h e d a t a o f F i g . 14, s h o w i n g t h e Ab r e s p o n s e o f t h e i d e n t i c a l a n i m a l s t o KLH, i n d i c a t e d t h a t t h e y were c a p a b l e o f r e s p o n d i n g r e a d i l y t o t h i s A g . I t was n o t e d t h a t t h e t i m i n g o f f a c t o r a d m i n i s t r a t i o n a n d A g c h a l l e n g e a p p e a r e d t o be c r i t i c a l f o r t h e s u p p r e s s i v e e f f e c t . I n t h e e x p e r i m e n t o f F i g s . 13 a n d 14, Ag was g i v e n w i t h i n s e v e r a l h o u r s o f F d l l F t r e a t m e n t . The i n a b i l i t y t o g i v e A g s o o n a f t e r f a c t o r r e s u l t e d i n a f a i l u r e t o s u p p r e s s t h e r e s p o n s e ( d a t a n o t s h o w n ) . I f an immune r e s p o n s e i s d e t e r m i n e d b y a b a l a n c e b e t w e e n 101 h e l p a n d s u p p r e s s i o n , t h e n i t i s p o s s i b l e t h a t t h e t i m e o f d e l i v e r y o f a " h e l p e r " o r " s u p p r e s s o r " s i g n a l c a n i n f l u e n c e t h e o u t c o m e . T h i s i s a l s o s u p p o r t e d b y t h e d i f f i c u l t i e s i n d e v e l o p i n g t h e i n v i t r o a s s a y f o r t h e m e a s u r e m e n t o f s u p p r e s s i o n . I n t h i s i n s t a n c e , t h e o r d e r o f e x p o s u r e o f t h e c e l l s t o F d l l F a nd A g h e l p e d t o d e t e r m i n e t h e o b s e r v e d l e v e l o f s u p p r e s s i o n . B i o c h e m i c a l A n a l y s i s o f F d l l F The b i o c h e m i c a l i d e n t i f i c a t i o n o f t h e A g - b i n d i n g m o l e c u l e o f F d l l F was made p o s s i b l e b e c a u s e B16G p e r m i t t e d t h e i m m u n o a f f i n i t y e n r i c h m e n t o f t h e a c t i v e m a t e r i a l f r o m F d l l s p e n t c u l t u r e SN. The e l u t e d m a t e r i a l , a s a n a l y z e d b y r e d u c i n g SDS-PAGE, showed a l i m i t e d number o f b a n d s t h a t a p p e a r e d t o be u n i q u e t o t h e F d l l c u l t u r e SN i n c o m p a r i s o n t o c o n t r o l m a t e r i a l . A p r e d o m i n a n t b a n d a t 30k M r was a l w a y s p r e s e n t i n t h e e l u t e d m a t e r i a l ( F i g . 1 5 ) . I n a d d i t i o n , a p r o t e i n a t 80k M r was r e c o v e r e d . A l t h o u g h t h e b a n d was n o t u n i q u e t o F d l l SN, i t was p r e s e n t a t g r e a t e r c o n c e n t r a t i o n s i n c o m p a r i s o n t o c o n t r o l s . F o r t h i s r e a s o n , i t was i n i t i a l l y c o n s i d e r e d a s a n o t h e r p o s s i b l e F d l l F c o m p o n e n t . T h e r e w e r e o t h e r , m i n o r c o m p o n e n t s a l s o p r e s e n t i n t h e r a n g e o f 45 t o 50k M r b u t b e c a u s e t h e y w e re p r e s e n t a t l o w c o n c e n t r a t i o n s , t h e y w e r e i n i t i a l l y n o t t h o u g h t t o h a v e s i g n i f i c a n c e . I n an a t t e m p t t o i s o l a t e F d l l F i n i t s n a t i v e f o r m , B16G-e l u t e d m a t e r i a l was f r a c t i o n a t e d u s i n g h i g h p e r f o r m a n c e g e l f i l t r a t i o n ( F P L C ) . The F d l l F m a t e r i a l was r e s o l v e d a s a s i n g l e 102 p e a k , b u t u n e x p e c t e d l y , a t a M r g r e a t e r t h a n 200k. The n a t i v e M r o f p r e v i o u s l y r e p o r t e d T c e l l - d e r i v e d A g - b i n d i n g m o l e c u l e s h a v e n o t e x c e e d e d 145k (Beaman e t a l . 1 9 8 4 ) . One e x p l a n a t i o n was t h a t F d l l F h a d a g g r e g a t e d u n d e r t h e c o n d i t i o n s u s e d f o r e i t h e r a f f i n i t y e n r i c h m e n t o r g e l f i l t r a t i o n . A g g r e g a t i o n o f T s F h a s b e e n p r e v i o u s l y r e p o r t e d (Webb, Kapp, a n d P i e r c e 1983). B e s i d e s t h e i n t r i n s i c h y d r o p h o b i c n a t u r e o f T s F i n a q u e o u s s o l u t i o n , t h e r e a r e o t h e r e x p l a n a t i o n s f o r a g g r e g a t i o n . The i m m u n o a f f i n i t y c o l u m n p r o t o c o l may h a v e c a u s e d F d l l F m a t e r i a l t o d e n a t u r e a n d a g g r e g a t e u p o n r e c o v e r y u n d e r v e r y a c i d i c c o n d i t i o n s . T h i s c o u l d h a v e b e e n compounded by f u r t h e r c o n c e n t r a t i o n o f t h e s a m p l e , w h i c h was r e q u i r e d f o r h i g h p e r f o r m a n c e g e l f i l t r a t i o n . The p e a k was c o m p r i s e d o f f r a c t i o n s t h a t p o s s e s s e d A g - b i n d i n g a c t i v i t y . I n a s e p a r a t e e x p e r i m e n t , s u p p r e s s i v e a c t i v i t y was a l s o i d e n t i f i e d i n t h e v i c i n i t y o f t h i s h i g h M r p e a k . T h i s a c t i v e m a t e r i a l f r o m i n d i v i d u a l f r a c t i o n s , when a n a l y z e d by r e d u c i n g SDS-PAGE, c o n t a i n e d l a r g e amounts o f b o t h t h e 80k a n d 30k M r p r o t e i n s d e s c r i b e d a b o v e . One o f t h e p e a k f r a c t i o n s a l s o c o n t a i n e d a p a i r o f b a n d s a t a b o u t 45k M . However, t h e r o l e o f t h e s e p r o t e i n s was t h o u g h t t o h a v e l i t t l e s i g n i f i c a n c e f o r A g -b i n d i n g a c t i v i t y b e c a u s e o f t h e l o w c o n c e n t r a t i o n s o b s e r v e d , i n c o m p a r i s o n t o t h e o t h e r p r o t e i n b a n d s . D e s p i t e t h e f a i l u r e t o d e t e r m i n e t h e n a t i v e M r o f F d l l F b y g e l f i l t r a t i o n , t h e c o r r e l a t i o n o f t h e 80k a n d 30k M r p r o t e i n s w i t h A g - b i n d i n g a n d s u p p r e s s i v e a c t i v i t y was c o n f i r m e d w i t h t h e s e e x p e r i m e n t s . 103 The FdllF-enriched material was also fractionated using preparative SDS-PAGE, run under non-reducing conditions. This same technique was used successfully for both A10F (Chan et a l . 1988) and GAT-TsF (Sorensen, Pierce, and Webb 1983). As shown in Fig . 20, heavy bands at 80k M r were recovered from both samples in f r a c t i o n 4. No Ag-binding a c t i v i t y was associated with these or any fra c t i o n s i n the v i c i n i t y ; i e . f r a c t i o n s 1 to 11 ( f i g . 19) from either F d l l - or DClO-derived material. To determine i f the 80k M r materials were d i f f e r e n t by some other physical parameter, besides Mr, they were analyzed by two-dimensional gel electrophoresis and observed to be i d e n t i c a l , within l i m i t s of the technique. However, 30k M r bands, unique to F d l l material, were detected in f r a c t i o n s 12 to 14. This was coincident with the f i r s t Ag-binding a c t i v i t y peak of the F d l l - d e r i v e d material. A single band was observed i n f r a c t i o n 12, whereas two or three bands were seen i n fractio n s 13 and 14. When preparative SDS-PAGE was repeated, using 10 times the amount of s t a r t i n g sample, the unique 30k Mr material from the fractions was recovered and analyzed by two-dimensional gel electrophoresis. In spite of the numerous corresponding spots shared between the control and F d l l F material, there were some unique polypeptides (and one major one in p a r t i c u l a r ) i n the F d l l F sample at around the 30k M r p o s i t i o n . However, these two-dimensional gel results also demonstrated how heterogeneous the recovered 30k Mr material was (data not shown). It was therefore shown that an unique 30k Mr protein with 104 a s s o c i a t e d A g - b i n d i n g a c t i v i t y c o u l d be r e c o v e r e d f r o m t h e F d l l -d e r i v e d SN. T h i s m o i e t y i t s e l f may h a v e u n d e r g o n e some p r o t e o l y s i s y i e l d i n g s m a l l e r p e p t i d e f r a g m e n t s ( l e s s t h a n 14k M ) w i t h A g - b i n d i n g a c t i v i t y . An a l t e r n a t i v e p o s s i b i l i t y was t h a t t h e 30k M r m o l e c u l e was p a r t o f a l a r g e r p r o t e i n t h a t was c a p a b l e o f b o t h A g - b i n d i n g a n d s u p p r e s s i v e a c t i v i t y . S i n c e no s u p p r e s s i v e a c t i v i t y was e v e r d e m o n s t r a t e d i n t h e r e c o v e r e d 30k M r m a t e r i a l one c o u l d c o n c l u d e t h a t e i t h e r i t was p a r t i a l l y d e n a t u r e d o r c o n s t i t u t e d o n l y a p a r t o f t h e T s F m o l e c u l e . The A g - b i n d i n g a c t i v i t y r e c o v e r e d f r o m t h e b o t t o m o f t h e n o n - r e d u c i n g SDS g e l ( F i g . 19) may be an a d d i t i o n a l b r e a k d o w n p r o d u c t . The T s F r e p o r t e d by F r e s n o , M c V a y - B o u d r e a u , a n d C a n t o r (1982) was s u s c e p t i b l e t o p r o t e o l y s i s . I t d e g r a d e d i n t o a 24k M r A g -b i n d i n g m o l e c u l e a n d a 45k M r n o n s p e c i f i c s u p p r e s s o r m o l e c u l e . W h e t h e r F d l l F i s c o mposed o f more t h a n one p r o t e i n c omponent i s unknown. I t i s p o s s i b l e t h a t t h e 30k M r p r o t e i n a s s o c i a t e s w i t h e i t h e r t h e 80k o r 45k ( f i g . 18) M r p o l y p e p t i d e . I n d e e d , t h e " i d e n t i c a l " s p o t t i n g p a t t e r n s e e n w i t h t h e 80k M r m a t e r i a l , t h a t was a n a l y z e d b y t w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s ( f i g . 21) d o e s n o t f o r m a l l y r u l e o u t t h a t i t i s a component p a r t o f F d l l F . C o n c e i v a b l y , t h e e l o n g a t e d s p o t s may a c t u a l l y c o n s i s t o f more t h a n one m o l e c u l a r s p e c i e s a n d t h a t t h e " u n i q u e " F d l l F m a t e r i a l i s h i d d e n . A n o t h e r p o s s i b i l i t y i s t h a t B16G i s s p e c i f i c f o r a " c o n s t a n t r e g i o n " o f t h e T s F , a n d t h a t u n i q u e b a n d i n g a t t h e 80k Mr p o s i t i o n b e t w e e n t h e c o n t r o l a n d F d l l m a t e r i a l w o u l d n o t be e x p e c t e d . 105 Two b a n d s a t 45k M r were i d e n t i f i e d as u n i q u e F d l l p r o t e i n s i n f i g . 18. An u n i q u e 45k M r component i s f r e q u e n t l y r e c o v e r e d f r o m t h e B16G e l u a t e s , b u t i t i s n o t a l w a y s c l e a r l y p r e s e n t due t o t h e l o w e r amount o f i t s r e c o v e r y a s c o m p a r e d w i t h t h e 30k M r p r o t e i n ( d a t a n o t s h o w n ) . I f F d l l F was a c t u a l l y c o mposed o f a 30k a n d 45k M r c h a i n , f o r m i n g a h e t e r o d i m e r , t h e n t h e two c o m p o n e n t s w o u l d be e x p e c t e d t o be o b s e r v e d i n e q u i m o l a r q u a n t i t i e s , a s s u m i n g e q u i v a l e n t r e c o v e r y o f t h e p r o t e i n s f r o m t h e i m u n o a f f i n i t y c o l u m n . T h e r e i s c l e a r e v i d e n c e ( f r o m t w o - d i m e n s i o n a l g e l s ) t h a t t h e r e c o v e r e d 30k M r p r o t e i n was h e a v i l y c o n t a m i n a t e d w i t h o t h e r p r o t e i n s . A d d i t i o n a l l y , t h e n o n - r e d u c i n g p r e p a r a t i v e SDS-PAGE f r a c t i o n a t i o n y i e l d e d A g - b i n d i n g f r a c t i o n s t h a t c o n t a i n e d s e v e r a l p o l y p e p t i d e b a n d s ( f i g . 20, f r a c t i o n s 13 a n d 1 4 ) . T h e r e f o r e e q u i m o l a r q u a n t i t i e s o f b o t h t h e 30k a n d 45k M r c o m p o n e n t s may a c t u a l l y be p r e s e n t i n t h e e l u t e d m a t e r i a l , o b s c u r e d b y o t h e r c o n t a m i n a t i n g p r o t e i n s . The i d e a o f a h e t e r o d i m e r i c f o r m o f F d l l F i s i n k e e p i n g w i t h t h e known s t r u c t u r e s o f r e p o r t e d T s F ( e g . S a i t o a n d T a n i g u c h i 1984) a n d t h e TCR t h a t h a v e b e e n c h a r a c t e r i z e d b y gene c l o n i n g a n d mAb. A t p r e s e n t , i t i s unknown i f F d l l F i s r e l a t e d t o any o f t h e h e t e r o d i m e r i c TCR m o l e c u l e s : a/15 o r 876 ( B r e n n e r e t a l . 1986; S a i t o e t a l . 1987; L o h e t a l . 1 9 8 7 ) . R e c e n t r e p o r t s h a v e s u g g e s t e d s i m i l a r i t i e s b e t w e e n h y b r i d o m a - d e r i v e d T s F a n d t h e a/15 TCR. Z h e n g e t a l . (1988) d e s c r i b e d an A g - b i n d i n g m o l e c u l e s e c r e t e d b y a Th h y b r i d o m a t h a t c o m b i n e d w i t h t h e I - J + c h a i n o f 106 an i n d u c e r T s F t o g i v e s u p p r e s s i v e a c t i v i t y . F a i r c h i l d , Kubo, a n d M o o r h e a d (1988) c h a r a c t e r i z e d a D N P - s p e c i f i c T s F t h a t r e a c t e d w i t h two mAb s p e c i f i c f o r t h e p r o d u c t s o f V j j 8 , a f a m i l y o f m u r i n e TCR fi c h a i n g e n e s . K u c h r o o e t a l . (1988) showed t h a t s e l e c t i o n o f C D 3 - e x p r e s s i n g T s ^ h y b r i d o m a s c o r r e l a t e d w i t h t h e a b i l i t y t o b i n d A g a n d p r o d u c e T s F . I n a d d i t i o n , a mAb s p e c i f i c f o r t h e TCR a c h a i n , i m m u n o p r e c i p i t a t e d a p r o t e i n o f t h e same M r (45k) as t h e a c h a i n , f r o m t h e h y b r i d o m a c e l l s . (The CD3 m o l e c u l e , i s c o -e x p r e s s e d w i t h t h e a / f i TCR on t h e T c e l l s u r f a c e ( C l e v e r s e t a l . 1 9 8 8 ) ) . On t h e o t h e r h a n d , i t i s known t h a t Ts ( a n d t h e i r s o l u b l e f a c t o r s ) d i f f e r f r o m t h e a / f i T C R - b e a r i n g c e l l b y b e i n g a b l e t o b i n d A g i n t h e a b s e n c e o f MHC (Webb e t a l . 1 9 8 9 ) . T h i s s u g g e s t s t h a t a l t h o u g h t h e Ts r e c e p t o r may be s i m i l a r , i t i s n o t i d e n t i c a l t o t h e a / f i TCR. Summary The T s F - s p e c i f i c mAb, B16G, h a s b e e n u s e d t o i s o l a t e F d l l F , a s o l u b l e m e d i a t o r s e c r e t e d b y t h e Ts h y b r i d o m a , F d l l . A l t h o u g h t h e Ab was o r i g i n a l l y r a i s e d f o r t h e c h a r a c t e r i z a t i o n o f a P 8 1 5 -s p e c i f i c T s F , i t h a s b e e n u s e f u l f o r t h e s t u d y a n d i s o l a t i o n o f TsF i n o t h e r A g s y s t e m s . B16G was a c r i t i c a l t o o l i n t h e d e v e l o p m e n t o f t h e F d l l h y b r i d o m a . I n s e v e r a l r e s p e c t s , t h i s c e l l l i n e was a g o o d c h o i c e a s a c o n t i n u o u s s o u r c e f o r F d l l F . F i r s t l y , F d l l r e m a i n e d s t a b l e f o r a t l e a s t f o u r y e a r s w i t h r e s p e c t t o t h e p r o d u c t i o n o f F d l l F . 107 S e c o n d l y , t h e e x p o s u r e o f F d l l t o F d i n d u c e s t h e i n t r a c e l l u l a r 2+ . i n f l u x o f Ca i o n s , a s m e a s u r e d u s i n g a c a l c i u m - s e n s i t i v e f l u o r e s c e n t d y e ( S t e e l e , Chu, Chan, N o r t h , a n d L e v y 1 9 8 7 ) . T h i s e v e n t h a s b e e n a s s o c i a t e d w i t h t h e i n t e r a c t i o n o f l i g a n d w i t h i t s c e l l s u r f a c e r e c e p t o r (Imboden e t a l . 1 9 8 6 ) . W h e t h e r F d l l F i s a s e c r e t e d f o r m o f a r e c e p t o r i s unknown. However, t h e c a l c i u m f l u x s t u d i e s do s u g g e s t t h e p r e s e n c e o f a m e m b r a n e - a s s o c i a t e d , A g - s p e c i f i c m o l e c u l e . L a s t l y , u s i n g FACS a n a l y s i s , t h e r a b b i t a n t i s e r u m , a n t i - p 3 0 , was shown t o b i n d t o t h e F d l l h y b r i d o m a ( N o r t h e t a l . 1 9 8 8 ) . T h e s e d a t a a l s o s u g g e s t t h e p r e s e n c e o f a m e m b r a n e - a s s o c i a t e d m o i e t y , p o s s i b l y F d l l F , e x p r e s s e d b y F d l l h y b r i d o m a . The p r o p e r t i e s o f F d l l F were c o n s i s t e n t w i t h t h o s e shown by o t h e r r e p o r t e d T s F . I t b o u n d Ag, h a d s u p p r e s s i v e a c t i v i t y b o t h i n v i t r o a n d i n v i v o , a n d h a s as a p a r t o f i t s m o l e c u l a r c o m p o s i t i o n , a 30k M r A g - b i n d i n g c h a i n . The m a i n t h r u s t o f t h e p r e s e n t work was t h e c h a r a c t e r i z a t i o n o f t h e A g - b i n d i n g m o l e c u l e o f F d l l F . I t i s a c k n o w l e d g e d t h a t s e v e r a l i m p o r t a n t a r e a s , s u c h a s e x p r e s s i o n o f s u p p r e s s o r d e t e r m i n a n t s ( e g . I - J ) a n d g e n e t i c r e s t r i c t i o n s f o r a c t i v i t y were n o t a d d r e s s e d . However, i t i s c o n c l u d e d t h a t F d l l F i s s u f f i c i e n t l y c h a r a c t e r i z e d t o w a r r a n t f u r t h e r i n v e s t i g a t i o n . Of i n t e r e s t i s t h e p o s s i b i l i t y t h a t F d l l F i s a c t u a l l y made up o f more t h a n one c h a i n . 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S i l v e r s t a i n i n g o f p r o t e i n s i n p o l y a c r y l a m i d e g e l s . A n a l . B i o c h e m . 1 1 8 : 1 9 7 - 2 0 3 . Y a m a u c h i , K., D. Murphy, H. C a n t o r , a n d R. K. G e r s h o n . 1 9 8 1 a . A n a l y s i s o f + a n t i g e n - s p e c i f i c , I g - r e s t r i c t e d c e l l - f r e e m a t e r i a l made b y I - J L y - 1 c e l l s ( L y - 1 T s i F ) t h a t i n d u c e s L y - 2 c e l l s t o e x p r e s s s u p p r e s s i v e a c t i v i t y . E u r . J . Immunol. 1 1 : 9 0 5 - 1 2 . Y a m a u c h i , K., D. Murphy, H. C a n t o r , a n d R. K. G e r s h o n . 1981b. A n a l y s i s o f an a n t i g e n - s j j e c i f i c , H - 2 - r e s t r i c t e d c e l l - f r e e p r o d u c t ( s ) made b y " I - J L y - 2 c e l l s ( L y - 2 TsF) t h a t s u p p r e s s e s L y - 2 c e l l - d e p l e t e d s p l e e n c e l l a c t i v i t y . E u r . J . Immunol. 1 1 : 9 1 3 - 1 8 . Yee, G. K., J . G. L e v y , M. L. K r i p k e , a n d S. E. U l l r i c h . 1989. The r o l e o f s u p p r e s s o r f a c t o r s i n t h e r e g u l a t i o n o f immune r e s p o n s e s b y u l t r a v i o l e t r a d i a t i o n - i n d u c e d s u p p r e s s o r T-l y m p h o c y t e s . I I I . I s o l a t i o n o f a s u p p r e s s o r f a c t o r w i t h t h e B16G m o n o c l o n a l a n t i b o d y . C e l l . Immunol, ( i n p r e s s ) . Z heng, H., M. B o y e r , A. F o t e d a r , B. S i n g h , a n d D. R. G r e e n . 1988. An a n t i g e n - s p e c i f i c h e l p e r T c e l l h y b r i d o m a p r o d u c e s an a n t i g e n - s p e c i f i c s u p p r e s s o r i n d u c e r m o l e c u l e w i t h i d e n t i c a l a n t i g e n i c f i n e s p e c i f i c i t y . J . Immunol. 1 4 0 : 1 3 5 1 - 5 8 . 117 APPENDIX THE COMPOSITIONS OF BUFFERS AND SOLUTIONS B i c a r b o n a t e b u f f e r 1.59 g N a 2 C 0 3 2.93 g N a H C 0 3 0.2 g N a N 3 i n 1 1 o f d i s t i l l e d w a t e r , a d j u s t t o pH 9.6 w i t h HC1; s t o r e a t 4°C ( V o l l e r , B i d w e l l , a n d B a r t l e t t 1 9 7 6 ) . D i e t h a n o l a m i n e b u f f e r 97 m l d i e t h a n o l a m i n e 0.1 g M g C l 2 . 6 H 2 0 0.2 g N a N 3 i n 800 m l o f d i s t i l l e d w a t e r , a d j u s t t o pH 9.8 w i t h HC1; a d d d i s t i l l e d w a t e r t o 1 1; s t o r e a t 4°C ( V o l l e r , B i d w e l l , a n d B a r t l e t t 1976) . P h o s p h a t e b u f f e r e d s a l i n e (PBS) 0.137 M N a C l 27.0 mM KC1 71.9 mM N a 2 H P 0 4 . 1 2 H 2 0 14.7 mM K H 2 P 0 4 P h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g Tween 20 (PBS-Tween) PBS 0.05% Tween 20 ( v / v ) ( V o l l e r , B i d w e l l , a n d B a r t l e t t 1 9 7 6 ) . s a t u r a t e d ( N H 4 ) 2 S Q 4 900 g ( N H 4 ) 2 S 0 4 a r e a d d e d t o 1 1 o f d i s t i l l e d w a t e r ; t h i s i s h e a t e d u n t i l ( N H 4 ) 2 S 0 4 i s d i s s o l v e d c o m p l e t e l y ; t h e h o t s o l u t i o n i s t h e n q u i c k l y f i l t e r e d u s i n g Whatman No.1 p a p e r ; t h e s o l u t i o n i s c o o l e d a t room t e m p e r a t u r e t o a l l o w f o r c r y s t a l f o r m a t i o n ; a t t h i s t i m e t h e s o l u t i o n i s a d j u s t e d t o pH 7.4 u s i n g NH 4OH ( M i s h e l l a n d S h i i g i 1 9 8 0 ) . SDS-PAGE S o l u t i o n s 1) S t o c k s o l u t i o n s 118 a) 10% APS 0.05 g APS 500 u l d i s t i l l e d w a t e r , p r e p a r e d a i l y b) 30% a c r y l a m i d e (30% T, 3.3% C, s e e b e l o w ) 29 g a c r y l a m i d e ( B i o - R a d ) 1 g b i s - a c r y l a m i d e ( B i o - R a d ) i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n i s f i l t e r e d (0.45 um) s t o r e d a t 4°C, a n d p r o t e c t e d f r o m l i g h t . N o t e : % T= ( (g a c r y l a m i d e + g b i s - a c r y l a m i d e ) -r t o t a l v o l u m e ) x 100% % C= (g b i s - a c r y l a m i d e -f (g a c r y l a m i d e + g b i s - a c r y l a m i d e ) ) x 100% c) 2 5 % a c r y l a m i d e ( 2 5 % T, 2.5% C) 25 g a c r y l a m i d e 0.65 g b i s - a c r y l a m i d e i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n i s f i l t e r e d (0.45 um) s t o r e d a t 4°C, a n d p r o t e c t e d f r o m l i g h t . d) s t a c k i n g ( u p p e r ) g e l b u f f e r (5 x) 0.625 M T r i s ( T r i z m a , b a s e , T-1503, Sigma) 0.5% SDS a d j u s t t o pH 6.8 u s i n g HC1; s t o r e a t 4°C. e) s e p a r a t i n g ( l o w e r ) g e l b u f f e r (5 x) 1.87 M T r i s 0.5% SDS a d j u s t t o pH 8.8 u s i n g HC1; s t o r e a t 4°C. f ) r u n n i n g b u f f e r (10 x) 0.25 M T r i s 1.92 M g l y c i n e (G 7126, Sigma) 1.0% SDS 2) f o r 15 m l o f a 10% (% T) s e p a r a t i n g g e l s o l u t i o n : 6 m l 2 5 % a c r y l a m i d e 3 m l l o w e r g e l b u f f e r 6 m l H P L C - p u r e w a t e r 119 m i x w i t h o u t i n t r o d u c i n g a i r ; t o p o l y m e r i z e , a d d : 70 u l 10% APS 7 u l TEMED 3) f o r 7.5 m l o f a 4% (% T) s t a c k i n g g e l s o l u t i o n : 1 m l 3 0 % a c r y l a m i d e 1.5 m l u p p e r g e l b u f f e r 5 m l H P L C - p u r e w a t e r m i x w i t h o u t i n t r o d u c i n g a i r ; t o p o l y m e r i z e , a d d : 100 u l 10% APS 10 u l TEMED I E F s o l u t i o n s 1) S t o c k s o l u t i o n s a) 10% CHAPS 0.5 g CHAPS i n 5 ml d i s t i l l e d w a t e r . b) s a m p l e b u f f e r 9.8 M u r e a 3% CHAPS 2% a m p h o l i n e s 100 mM DTT c) o v e r l a y b u f f e r 8 M u r e a 3% CHAPS 2% a m p h o l i n e s 100 mM DTT s a m p l e a n d o v e r l a y b u f f e r s a r e a l i q u o t e d a n d s t o r e d a t -70'C. d) 3 0 % a c r y l a m i d e s o l u t i o n ( 3 0 % T, 2.7% C) 2 9 g a c r y l a m i d e 0.8 g b i s - a c r y l a m i d e i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n i s f i l t e r e d (0.45 urn) s t o r e d a t 4°C, a n d p r o t e c t e d f r o m l i g h t . e) a n o l y t e 120 20 mM NaOH, prepare fresh, degas before use. f) catholyte 10 mM HoP0 4, prepare fresh, degas before use. 2) for 5 ml of IEF gel solution: 2.75 g urea 0.65 ml 30% acrylamide 1 ml 10% CHAPS 0.85 ml HPLC-pure water 0.36 ml pH 3-10 ampholines to polymerize, add: 5 ul 10% APS 3.5 ul TEMED IEF solutions were adapted from the method of Bravo (1984) with the exception of the CHAPS and 30% acrylamide solution. The use of CHAPS i n place of Triton X-100 was o r i g i n a l l y described by Perdew, Schaup, and Selivonchick (1983). The composition of the 30% acrylamide solution was described i n the Bio-Rad technical b u l l e t i n 1144. 

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