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Allograft enhancement in a rat heart transplant model : effect of T and B cell immunization 1985

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ALLOGRAFT ENHANCEMENT IN A RAT HEART TRANSPLANT MODEL EFFECT OF T AND B CELL IMMUNIZATION BY CHARLES HENRY SCUDAMORE B . S c , 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 1972 M . D . , 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 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF SURGERY a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a JULY 1985 ® C h a r l e s Henry Scudamore 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 Surgery The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e October 9, 1985 DE-6(3/81) i i i ABSTRACT A c u t e and c h r o n i c r e j e c t i o n c o n t i n u e t o be t h e most i m p o r t a n t p rob lems i n m a i n t a i n i n g a f u n c t i o n i n g o r g a n a l l o g r a f t . D e s p i t e advances i n i m m u n o s u p p r e s s i o n , o r g a n s a re s t i l l damaged o r d e s t r o y e d by t h e r e c i p i e n t ' s immune s y s t e m . I n o r d e r t o p r o t e c t t h e t r a n s p l a n t e d o rgan i t i s n e c e s s a r y t o o v e r w h e l m t h e h o s t ' s immune s y s t e m and t h u s expose t h e hos t t o t h e c o m p l i c a t i o n s o f i n v a s i o n f r o m f u n g i , b a c t e r i a , p r o t a z o a , r e d u c t i o n i n o n c o l o g i c s u r v e i l l a n c e , and r e d u c t i o n o f s tem c e l l p r o d u c t i o n . Donor s p e c i f i c i m m u n o s u p p r e s s i o n w o u l d p r o v i d e g r a f t p r o t e c t i o n and a l l o w m a i n t e n a n c e o f t h e h o s t ' s immuno log i c compe tence . G r a f t enhancement has been d e s c r i b e d f o r many y e a r s . C u r r e n t p r a c t i c e uses t h i s p r i n c i p l e by p r e t r a n s f u s i n g p r o s p e c t i v e k i d n e y t r a n s p l a n t r e c i p i e n t s w i t h t y p e s p e c i f i c b l o o d . P r e v i o u s work has s u p p o r t e d t h e c o n c e p t t h a t t h i s c l i n i c a l e f f o r t can be p roduced by c e r t a i n c e l l s i n t h e b l o o d , s p e c i f i c a l l y , l y m p h o c y t e s . To s t u d y t h e e f f e c t s o f p r e i m m u n i z a t i o n w i t h T o r B l y m p h o c y t e s and p l a t e l e t s i n a r a t h e t e r o t o p i c h e a r t t r a n s p l a n t m o d e l , , t h e f o l l o w i n g e x p e r i m e n t s were p e r f o r m e d . E x p e r i m e n t 1 : The e f f e c t o f p r e t r a n s p l a n t i m m u n i z a t i o n w i t h l x l O 7 donor s p e c i f i c T c e l l s o r B c e l l s showed t h a t T c e l l s have l i t t l e a f f e c t on r e j e c t i o n o f h e t e r o t o p i c h e a r t a l l o g r a f t and B c e l l s caused i v p r o l o n g a t i o n o f g r a f t f u n c t i o n . T h i s e f f e c t i s s p e c i e s s p e c i f i c and n o t due t o a pu re a n t i - i d i o t y p e phenomenon. E x p e r i m e n t 2 : The e f f e c t o f h e a t i n g t h e p u r i f i e d T and B c e l l s a t 56°C f o r 30 m i n u t e s i s known t o d e n a t u r e t h e p r e s e n t i n g p r o t e i n a n t i g e n s on t h e c e l l membranes w i t h o u t d e s t r o y i n g t h e c e l l membrane. A f t e r p r e t r a n s p l a n t i m m u n i z a t i o n , seven days p r i o r t o h e t e r o t o p i c h e a r t t r a n s p l a n t i n t h e r a t m o d e l , t h e p r e v i o u s l y o b s e r v e d p r o l o n g a t i o n o f g r a f t s u r v i v a l a f t e r n o n h e a t e d B c e l l i m m u n i z a t i o n was s t i l l p r e s e n t b u t no t as m a r k e d . E x p e r i m e n t 4: The e f f e c t o f p r e t r a n s p l a n t i m m u n i z a t i o n o f donor s p e c i f i c T and B c e l l s t r e a t e d by h e a t i n g t o 85°C f o r 10 m i n u t e s f o l l o w e d by h e t e r o t o p i c h e a r t g r a f t showed t h a t t h e r e was a s i g n i f i c a n t p r o l o n g a t i o n o f t h e e n g r a f t e d h e a r t f o l l o w i n g i m m u n i z a t i o n w i t h t h e d e n a t u r e d B c e l l s . C e l l u l a r p r o t e i n s a r e d e n a t u r e d by t h i s p r e t r e a t m e n t bu t p o l y s a c c h a r i d e s a r e n o t . E x p e r i m e n t 5: The e f f e c t o f p r e t r a n s p l a n t i m m u n i z a t i o n w i t h p u r i f i e d donor s p e c i f i c p l a t e l e t s f o l l o w e d by h e t e r o t o p i c h e a r t r a t t r a n s p l a n t showed no p r o l o n g a t i o n o r s h o r t e n i n g o f g r a f t s u r v i v a l . I t i s c o n c l u d e d t h a t , i n t h e h e t e r o t o p i c r a t h e a r t t r a n s p l a n t m o d e l , i m m u n i z a t i o n w i t h p u r i f i e d T c e l l s 7 days p r i o r t o t r a n s p l a n t has l i t t l e e f f e c t on r e j e c t i o n . When B c e l l s a r e immunized i n t h e same way, g r a f t s u r v i v a l i s p r o l o n g e d . I f t h e c e l l s a r e h e a t e d t o 56°C f o r 30 m i n u t e s t h i s e f f e c t i s r e d u c e d bu t n o t e l i m i n a t e d . T h i s e f f e c t i n d i c a t e s t h a t v d e n a t u r a t i o n o f p r o t e i n HLA a n t i g e n s on t h e p r e s e n t i n g c e l l s u r f a c e reduces t h e e n h a n c i n g e f f e c t o f t h e i n t a c t a n t i g e n s on B c e l l s . By d e n a t u r i n g , t h e p r e s e n t i n g B c e l l p r o t e i n g r a f t enhancement i s s t i l l p r e s e n t , s u g g e s t i n g t h e phenomenon o f g r a f t enhancement i s n o t t o t a l l y dependent on p r o t e i n a n t i g e n s bu t may have a c o n t r i b u t i o n f r o m m u c o p o l y s a c c h a r i d e s o r o t h e r c a r b o h y d r a t e s . Donor s p e c i f i c p u r i f i e d p l a t e l e t p r e t r a n s p l a n t i m m u n i z a t i o n s p roduced no s t a t i s t i c a l l y s i g n i f i c a n t p r o l o n g a t i o n o f e i t h e r PV6 o r h e a r t g r a f t s . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e f i n d i n g s t h a t p u r i f i e d T c e l l i m m u n i z a t i o n do n o t p roduce graft enhancement . v i TABLE OF CONTENTS Page 1 . A b s t r a c t i i i 2 . L i s t o f T a b l e s v i i 3. L i s t o f Graphs v i i 3 . L i s t o f F i g u r e s v i i i 4 . D e d i c a t i o n x 5. Acknowledgements x i 6. I n t r o d u c t i o n - A l l o g r a f t P r o l o n g a t i o n 1 7. Review o f L i t e r a t u r e 5 8 . M a t e r i a l s and Methods 17 E x p e r i m e n t a l Models 17 T e c h n i q u e o f C e l l S e p a r a t i o n 30 S t a t i s t i c a l A n a l y s i s 35 9 . Purpose o f S t u d y 41 10 . E x p e r i m e n t 1 41 E x p e r i m e n t 2 43 E x p e r i m e n t 3 44 3 - 1 45 3-2 45 3-3 45 3- 4 46 E x p e r i m e n t 4 - D e n a t u r e d C e l l i m m u n i z a t i o n 46 4 - 1 46 4 -2 47 4 -3 47 4 - 4 47 E x p e r i m e n t 5 - M ixed C e l l I m m u n i z a t i o n 48 E x p e r i m e n t 6 - P I a t e l e t ' . I m m u n i z a t i o n s 48 6 - 1 49 6-2 50 6-3 50 E x p e r i m e n t 7 - S p e c i f i c i t y C o n t r o l s 50 1 1 . D i s c u s s i o n 52 12 . B i b l i o g r a p h y 92 v i i L IST OF TABLES T a b l e 1 - Model - O r t h o t o p i c h e a r t 59 t r a n s p l a n t t o neck o f r e c i p i e n t . T a b l e 2 - C o n t r o l Rat G r a f t s 60 T a b l e 3 - Unheated Gel 1 I m m u n i z a t i o n s 61 T a b l e 4 - Heated C e l l I m m u n i z a t i o n s (56°C) 62 T a b l e 5 - Heated I m m u n i z a t i o n s (86°C) 63 T a b l e 6 - M ixed C e l l I m m u n i z a t i o n 64 T a b l e 7 - P l a t e l e t I m m u n i z a t i o n 65 T a b l e 8 - S p e c i f i c i t y C o n t r o l s 66 L IST OF GRAPHS Graph 1 - C o n t r o l s 67 Graph 2 - PVG B o r T c e l l i m m u n i z a t i o n F- HT X ^ Graph 3 - PVG B + T c e l l i m m u n i z a t i o n -> 69 DA F j H e a r t T x Graph 4 - F j (B + T c e l l s ) -> DA F j HT X 70 Graph 5 - PVG B c e l l - F j B c e l l i m m u n i z a t i o n 71 Graph 6 - PVG B c e l l i m m u n i z a t i o n 72 Graph 7 - T c e l l i m m u n i z a t i o n F j HT X 73 Graph 8 - B c e l l i m m u n i z a t i o n s F j , HT X 74 Graph 9 - S p e c i f i c i t y C o n t r o l s 75 v i i i L IST OF FIGURES F i g u r e 1 - The c u f f v a s c u l a r a n a s t o m o s i s s e t s i d e by s i d e 76 F i g u r e l a - Th rough t h e p o l y e t h y l e n e c u f f , t h e r e c i p i e n t 76 v e s s e l i s p u l l e d . F i g u r e l b - The r e c i p i e n t v e s s e l i s f o l d e d o v e r t h e c u f f 76 and l i g a t e d i n p l a c e . F i g u r e l c - The c u f f i s i n v a g i n a t e d i n t o t h e d o n o r v e s s e l 76 and l i g a t e d . Note t h a n o n l y i n t i m a i s i n c o n t a c t w i t h b l o o d f l o w . F i g u r e 2 - ECG 77 F i g u r e 3 - Removed r a t h e a r t t r a n s p l a n t a f t e r venous 78 i n f a r c t i o n p o s t - o p Day 2 , s h o w i n g a c u t e venous c l o t . F i g u r e 4 - Removed r a t h e a r t t r a n s p l a n t s h o w i n g a c u t e 79 a r t e r i a l c l o t Day 1 . F i g u r e 5 - Removed r a t h e a r t t r a n s p l a n t Day 6 . Some 80 r e j e c t i o n c h a n g e s - a r e e v i d e n t , b u t t h e an imal d i e d o f pneumon ia . G r a f t was f u n c t i o n i n g a t t h e t i m e o f d e a t h . F i g u r e 6 - Normal h e a r t , no t t r a n s p l a n t e d . 81 F i g u r e 7 - Removed r a t h e a r t t r a n s p l a n t a f t e r 14 d a y s . 82 A c u t e a l l o g r a f t r e j e c t i o n . F i g u r e 8 - Rat h e a r t a l l o g r a f t r e j e c t e d 8 d a y s , 83 removed 60 d a y s . F i g u r e 9 - C e l l S e p a r a t i o n T e c h n i q u e . 84 F i c o l l - I s o p a q u e / n y l o n w o o l . F i g u r e 10a - P e r o x i d a s e S t a i n (T c e l l s ) . T c e l l s - few 85 c e l l s w i t h d a r k h a l o . T c e l l s do n o t s t a i n . Very da rk c e l l s a re d e a d . F i g u r e 10b - P e r o x i d a s e S t a i n (S c e l l s ) . B c e l l s - most 86 have t a k e n up s t a i n . F i g u r e 11a - B c e l l s , phase c o n t r a s t . 87 F i g u r e l i b - P e r o x i d a s e S t a i n (B c e l l s ) . To d e m o n s t r a t e 88 l a c k o f c o n t a m i n a t i o n . ix LIST OF FIGURES ( c o n t ' d ) F i g u r e 12a - Phase c o n t r a s t o f T c e l l s . F i g u r e 12b - F l u o r e s c e i n S t a i n (T c e l l s ) . Images a re dead c e l l s . F i g u r e 13 - B c e l l s i n c u b a t e d a t 85°C . Note t h a t c e l l s a r e i n t a c t . X DEDICATION To Trevor, March 3, 1985 x i ACKNOWLEDGEMENTS I wou ld l i k e t o t h a n k t h o s e members o f t h e D e p a r t m e n t o f S u r g e r y , U n i v e r s i t y o f C a m b r i d g e , f o r t h e i r a i d i n my r e s e a r c h . F i r s t , P r o f e s s o r Roy York Ca ine who was a c o n s t a n t s o u r c e o f i n s p i r a t i o n and an i d e a l r o l e m o d e l . Second , P r o f e s s o r A l l a n D a v i e s w h o ' s g u i d a n c e i n t h e f i e l d o f immunology was o f g r e a t i m p o r t a n c e . T h i r d , Gabsi Brons who p e r f o r m e d t h e c e l l s t a i n i n g and d e m o n s t r a t e d t h e s e p a r a t i o n t e c h n i q u e s . F o u r t h , M i c h a e l B u t t s , Eas t A n g l i a n R e g i o n a l H e a l t h A u t h o r i t y , who p e r f o r m e d t h e s t a t i s t i c a l a n a l y s i s . F i n a l l y , D o c t o r D e r r i c k Wigh t who p e r f o r m e d t h e p a t h o l o g i c a l p r e p a r a t i o n s . - 1 - INTRODUCTION ALLOGRAFT PROLONGATION P r o l o n g a t i o n o f a l l o g r a f t s u r v i v a l i s t h e f u n d a m e n t a l p r o b l e m i n c l i n i c a l t r a n s p l a n t a t i o n . 4 2 ' 6 9 There are t h r e e methods o f p r o l o n g i n g a l l o g r a f t s u r v i v a l : h o s t i m m u n o s u p p r e s s i o n , i n d u c t e d h o s t t o l e r a n c e , i n d u c t i o n o f g r a f t enhancement . S u p p r e s s i o n o f t h e t h e h o s t ' s immune sys tem by c h e m i c a l o r p h y s i c a l means i s t h e c u r r e n t p r a c t i c e . T h i s w o u l d i n c l u d e a g e n t s such as a z a t h i o p r i n e , c o r t i c o s t e r i o d s or c y c l o s p o r i n e . The p r o b l e m w i t h t h e s e agen ts i s t h e y a r e n o n s p e c i f i c and have b r o a d a c t i o n s t h u s mak ing t h e h o s t v u l n e r a b l e t o i n f e c t i o n s and n e o p l a s t i c d e v e l o p m e n t as w e l l as a g r e a t number o f o t h e r u n d e s i r a b l e s i d e e f f e c t s . 7 T o l e r a n c e was f i r s t r e c o g n i z e d i n f r a t e r n a l t w i n c a t t l e who s h a r e d t h e i r f e t a l c i r c u l a t i o n . 4 T h u s , t h e immature immune sys tems s h a r e d c e l l s common t o bo th f e t u s e s . I n a d u l t h o o d t h e s e a n i m a l s wou ld n o t r e j e c t s k i n g r a f t t r a n s f e r s . These were named c h i m e r a s . 1 6 The e x i s t e n c e o f t o l e r a n c e i s v i t a l so t h a t t h e h o s t ' s immune s y s t e m i s t o l e r a n t t o i t s e l f . F a i l u r e o f p h e n o t y p i c s e l f t o l e r a n c e i s one t h e o r y f o r t h e mechanism o f autoimmune d i s o r d e r s . 2 2 S t u d i e s have d e m o n s t r a t e d t h a t t o l e r a n c e i s an a c t i v e p r o c e s s wh ich i n v o l v e s d i r e c t c o n t a c t between s e l f components and s p e c i f i c a n t i g e n - r e a c t i v e c e l l s . 2 l > 4 6 y n e a n t i g e n n a s g e n e r a l l y been t h o u g h t t o be a p r o t e i n . 1 3 , 1 4 , 8 3 There a re two t y p e s o f t o l e r a n c e : c e n t r a l and p e r i p h e r a l . I n t h e c e n t r a l f o rms t h e r e i s no a n t i b o d y p roduced a f t e r a n t i g e n i c c h a l l e n g e , s i n c e - 2 - the responsible immunocytes are missing (clonal deleation) or are silenced directly (clonal anergy).^ In peripheral forms some antibody is produced but because of the expression of the suppression mechanism, antibody production is decreased or masked so that i t is ineffective. For example, the production of certain antibodies simultaneously induces T suppressor cel ls to suppress the immunocompetent c e l l s , the induction of anti-idiotypes or the production of immune compleses which do not have the a b i l i t y to bind to 25 effector ce l l s . 47 49 79 87 The third method of graft prolongation is graft enhancement. ' ' ' After immunization with foreign tissues, antibodies may be formed that prevent graft reject ion. Enhancing antibodies may coat the target graft or combine with released antigen to form immune complexes that react with lymphoid cel ls and prevent the immune response. Suppressor (T lymphocytes) 80 cel ls may be generated by alloantigen stimulation, thus suppressing the host immune system to react with the specific antigen. It has been shown 81 that this is a very specif ic process. A response against the fu l l array of antigens is not necessary.^ In renal al lograft recipients, an important c l in ica l observation has been made: grafts survive longer in patients who received multiple blood transfusions without becoming strongly sensitized. Thus the c l i n i c a l promise of naturally induced graft tolerance has been demonstrated. ' There are three groups of lymphocytes in the peripheral c irculat ion: B lymphocytes (bursa derived), T lymphocytes (thymic derived) and N lymphocytes (null ce l l s or non B or T by antibody staining technique). - 3 - The T lymphocytes are those lymphocytes thought to be responsible for cel l mediated immunity (delayed hypersens i t iv i ty ) .^ T lymphocytes respond to chemical messages ( i . e . interlukens) and processed antigens. They may help the cell mediated response by inducing T cel l conversion (T helper cel l ) to suppress this response (T suppressor ce l l s ) or act d irect ly by destroying 2 antibody coated c e l l s . The B cel ls produce the antibody in response to an antigen (either direct or induction) but usually after the antigen has been processed 19 (antigen presenter ce l l ) by a T lymphocyte. Lymph nodes and spleen contain large numbers of both B and T 33 lymphocytes. Fundamental questions to be addressed here are: - Can enhancement be achieved without sensitizing the host? - Is there a difference between B and T cel l immunization and the production of enhancement? - What substances form the antigens? - Can enhancement be induced by components of lymphocytes rather than the intact functioning ce l l? The purpose of this paper is not to attempt to answer these questions but to add some knowledge towards their solution. - 4 - The u l t i m a t e goa l o f t r a n s p l a n t immunosuppress ion i s t o i n d u c e a s p e c i f i c s t a t e o f s y m b i o s i s w i t h t h e t r a n s p l a n t and t h e h o s t . 7 - 5 - REVIEW OF LITERATURE GRAFT ENHANCEMENT I m m u n o l o g i c a l enhancement a c q u i r e s i t s name f r o m t h e e a r l y o b s e r v a t i o n s o f F l e x n e r and J o b l i n g i n 1 9 0 7 . 4 1 I t was n o t e d t h a t p r e i m m u n i z a t i o n o f an an imal w i t h n o n v i a b l e tumor c e l l s a l l o w e d t h e t u m o r c h a l l e n g e t o grow and k i l l t h e an ima l wh ich w o u l d o t h e r w i s e have r e s i s t e d t h e t u m o r c h a l l e n g e and s u r v i v e d . T h i s o b s e r v a t i o n was c o n t r a r y t o t h e t h o u g h t t h a t p r e i m m u n i z a t i o n o f tumor c e l l s wou ld enhance tumor r e j e c t i o n . T h i s i s i n c o n t r a d i s t i n c t i o n t o t h e o b s e r v a t i o n o f P a s t e u r who n o t e d p r i o r i m m u n i z a t i o n p r o v i d e d i m m u n i t y f r o m i n f e c t i o n . The p a r a d o x rema ined t h a t p r e i m m u n i z i n g a g a i n s t a t r a n s p l a n t i s a way of p r e v e n t i n g i t s r e j e c t i o n . T h i s can be shown by d e m o n s t r a t i n g p a s s i v e enhancement . For e x a m p l e , one can immunize an i n b r e d an ima l o f s t r a i n A u s i n g t h e c e l l s o b t a i n e d f r o m a second s t r a i n B. T h i s r a i s e s a l l o a n t i s e r u m i n an ima l A. T a k i n g t h e serum f r o m an ima l A and i n j e c t i n g i t i n t o a second an imal o f t h e same s t r a i n A]^ w i l l a l l o w A^ t o a c c e p t g r a f t s f r o m s t r a i n B w i t h o u t r e j e c t i o n . The p a s s i v e enhancemen t , i . e . v i a t h e serum, i s m a i n t a i n e d as l o n g as t h e a l l o a n t i s e r u m f r o m s t r a i n A i s i n j e c t e d i n t o A i . 5 6 I n a c t i v e enhancement t h e immunized an imal i t s e l f i s t h e r e c i p i e n t o f t h e g r a f t . For e x a m p l e , i f a s t r a i n -A r a t r e c e i v e s a l i v e r t r a n s p l a n t f r o m s t r a i n B r a t , t h e r a t w i l l r e j e c t t h e s t r a i n B l i v e r ; h o w e v e r , i f immunosuppress ion i s used f o r a p e r i o d o f a p p r o x i m a t e l y t h r e e m o n t h s , t h e immunosuppress i ve a g e n t s can be w i t h d r a w n and t h e l i v e r f r e q u e n t l y m a i n t a i n s i t s f u n c t i o n and i s n o t r e j e c t e d . I f a h e a r t g r a f t f r o m s t r a i n B i s - 6 - p e r f o r m e d on t h i s an ima l wh ich has r e c e i v e d t h e l i v e r t r a n s p l a n t , t h e h e a r t i s no t r e j e c t e d d e s p i t e t h e l a c k o f r e i n s t i t u t i n g i m m u n o s u p p r e s s i o n f o r t h i s second o r g a n . One w o u l d t h i n k t h a t a mere a n a l y s i s o f t h e serum wou ld r e v e a l t h e a c t i v e agent p r o d u c i n g enhancement . U n f o r t u n a t e l y , i n v e s t i g a t i o n s i n t o t h i s a r e a have no t l e a d t o any such a g e n t . I t i s g e n e r a l l y f e l t t h a t t h e agen ts a re l i k e l y p r o t e i n s and may be e x p r e s s i o n s o f t h e human l e u k o c y t e a n t i g e n (HLA) s y s t e m , most p r o b a b l y a n t i b o d i e s . There i s a g r e a t d e a l o f c o n f u s i o n i n t h i s p a r t i c u l a r a r e a o f immunology as e v i d e n c e d by s e v e r a l r e v i e w s on t h i s s u b j e c t , most e m p h a s i z i n g t h e c o m p l e x i t y o f t h e phenomenon. E a r l y work was p e r f o r m e d w i t h o u t g e n e t i c a l l y d e f i n e d a n i m a l s ; n o m e n c l a t u r e was no t s t a n d a r d i z e d . M u l t i p l e i m m u n o l o g i c m a n i p u l a t i o n s were p e r f o r m e d t o p r o d u c e an e x p e c t e d e f f e c t c o n f u s i n g t h e c o n t r i b u t i o n o f each t o produce e n h a n c e m e n t . 7 > 8 , 1 6 , 2 2 , 2 5 , 3 7 , 4 0 , 7 6 , 9 8 , 1 0 0 The d a t a r e l a t e d t o tumor enhancement s h o u l d be a v o i d e d because o f t h e c o m p l e x i t y o f t h e n e o p l a s t i c c e l l and i t s immuno log i c u n i q u e n e s s . I t i s ou r i n t e r e s t t o s t u d y norma l t i s s u e s . The h i s t o r y o f enhancement i s a l o n g o n e , and s e r i o u s c o n s i d e r a t i o n o f t h e method and c o n t e n t o f t r a n s p l a n t s was g r e a t l y d e l a y e d , f i r s t l y , because t h e p r i n c i p l e s t u d i e s were c o n f i n e d t o t h e r e a l m o f t u m o r s , and s e c o n d l y , s k i n g r a f t s a r e p a r t i c u l a r l y d i f f i c u l t t o enhance compared w i t h r e v a s c u l a r i z e d o rgan g r a f t s . ^ 2 Enhancement i n t r a n s p l a n t a t i o n was r e v i e w e d by Wassen i n 1 9 7 1 . Work on v a s c u l a r i z e d o r g a n t r a n s p l a n t s was n o t p o s s i b l e u n t i l g e n e t i c a l l y d e f i n e d a n i m a l s l a r g e enough f o r a p p l i c a t i o n o f m i c r o s u r g i c a l methods became a v a i l a b l e . The i n i t i a l work - 7 - was done on mice w h i c h were g e n e r a l l y t o o s m a l l t o r e l i a b l y p e r f o r m t r a n s p l a n t s o f s o l i d o r g a n s . Once i n b r e d s t r a i n s o f r a t s were i d e n t i f i e d and r e a d i l y a v a i l a b l e , enhancement was shown t o be a s i g n i f i c a n t method o f i m m u n o s u p p r e s s i o n . - ^ 5 Rat s t r a i n s w i t h d i f f e r e n t MHC's ( m a j o r h i s t o c o m p a t i b i l i t y comp lexes ) d e m o n s t r a t e m a r k e d l y d i f f e r e n t r e s p o n s e s t o h i s t o i n c o m p a t i b i l i t i e s . Thus g r a f t s t e n d t o be a c c e p t e d q u i t e r e a d i l y i n c e r t a i n s t r a i n s more t h a n i n o t h e r s . Enhancement t e n d s t o be more d r a m a t i c be tween c e r t a i n s t r a i n s t h a n o t h e r s , and somet imes an e x p e r i m e n t t h a t works i n c o m p a t i b l e s t r a i n s may no t work i n o t h e r s . I t i s a l s o n o t e d t h a t c e r t a i n t y p e s o f g r a f t s r e j e c t e d more r a p i d l y t h a n o t h e r s , f o r e x a m p l e , t h o s e g r a f t s t h a t t e n d t o be c l o s e s t t o t h e e n v i r o n m e n t r e j e c t more r e a d i l y , i . e . s k i n g r a f t s t e n d t o r e j e c t more r a p i d l y t h a n l i v e r g r a f t s . F u r t h e r m o r e h i g h and low r e s p o n s i v e genes a re a l s o a p o s s i b i l i t y ( I R g e n e s ) . The h i e r a r c h y f o r r e j e c t i b i l i t y o f d i f f e r e n t t i s s u e has l ong been r e c o g n i z e d and i s g e n e r a l l y now a s c r i b e d i n p a r t t o t h e i r c o n t e n t o f d e n d r i t i c c e l l s , i . e . t h o s e passenger l e u k o c y t e s and h i s t o c y t e s t h a t respond t o an a n t i g e n i c c h a l l e n g e , and t h e i r c l a s s 1 MHC a n t i g e n d e n s i t y , i . e . 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 complex e x p r e s s i o n a n t i g e n s . Known MHC a n t i g e n s and t h e i r p o s i t i o n on t h e chromsome may a l s o have an e f f e c t . 1 5 There i s o f t e n c o n f u s i o n a r i s i n g between t h e d e f i n i t i o n s o f a c t i v e enhancement and i m m u n o l o g i c a l t o l e r a n c e as d i s c u s s e d i n t h e i n t r o d u c t i o n . T h i s c o n f u s i o n a r i s e s because b o t h a re i n d u c e d by t r e a t m e n t o f a p o t e n t i a l t r a n s p l a n t hos t w i t h d o n o r t y p e a n t i g e n . I n f a c t t h e s e phenomena a re d i s t i n c t . T o l e r a n c e can be i n d u c e d by p r o v i d i n g a n t i g e n i n a t o l e r o g e n i c - 8 - (non immunogen ic ) f o r m . W h i l e enhancement r e q u i r e s immunogen, t o l e r a n c e may be i n d u c e d most e a s i l y i n non-MHC i n c o m p a t i b l e p a r t n e r s h i p s by i n j e c t i o n a t t h e t i m e o f t r a n s p l a n t a t i o n . 8 4 For t h e e s t a b l i s h m e n t o f enhancement , however , t h e immunogen must be p r o v i d e d l o n g enough i n advance t o a l l o w an immune r e s p o n s e t o be e l i c t e d , i . e . one o r two weeks b e f o r e t r a n s p l a n t . Thus t o l e r a n c e i n d u c e d a c r o s s an MHC b a r r i e r i s n o t e a s i l y p r o d u c e d and o f t e n r e q u i r e s an a d d i t i o n a l s e t o f c i r c u m s t a n c e s f o r i t t o be p r o d u c e d , such as a s h o r t c o u r s e o f i m m u n o s u p p r e s s i o n . 1 2 , 5 3 Enhancement was i n d u c e d w i t h humoral a n t i b o d y . T h i s p r o d u c t i o n o f humoral a n t i b o d y i s n o t w i t h o u t h a z a r d . The serum may c o n t a i n a n t i g e n a n t i b o d y complexes where t h e a n t i g e n i s immunogen ic , e x p o s i n g t h e p a t i e n t t o immunocomplex d i s e a s e o r s e n s i t i z i n g t h e i n d i v i d u a l , t h u s e n h a n c i n g r e j e c t i o n r a t h e r t h a n t h e g r a f t . The c o m p l e x i t i e s a r i s e because o f t h e number o f v a r i a b l e s i n t h e c l i n i c a l s i t u a t i o n , such as v a r i o u s an ima l s p e c i e s , s t r a i n s , p r e t r e a t m e n t , t i s s u e g r a f t e d , amount o f m a t e r i a l g r a f t e d , r o u t e s o f i n j e c t i o n , t i m i n g , a n t i g e n r e s p o n s e s , passenger l e u k o c y t e s , degree o f t r a u m a a t t a c h e d t o s u r g i c a l p r o c e d u r e and so o n . The s i m p l e s t c o n c e p t i s t h a t i n t o l e r a n c e a n t i g e n i s p r o v i d e d and an a n t i b o d y a r i s e s l a t e r by an immune response t o t h i s , o r t h e g r a f t , o r b o t h . Donor a n t i g e n and a n t i d o n o r a n t i b o d y a re t h e r e s u l t . I n p a s s i v e enhancement t h e a n t i b o d y t h a t p r o v i d e d a n t i g e n comes f r o m t h e g r a f t . There a re two d i f f e r e n t t y p e s o f t o l e r a n c e : s e l f t o l e r a n c e , t h e e x a m p l e , used w i t h two s e q u e n t i a l g r a f t s ; and p e r i n a t a l t o l e r a n c e , as i n d i z y g o t i c t w i n s . 2 4 I n s t u d y i n g enhancement t h e r e i s a p a r a l l e l be tween enhancement and t h e e f f e c t o f a n t i l e u k o c y t e s e r u m . Enhancement i s a v e r y s p e c i f i c a l l o e f f e c t . - 9 - A n t i l y m p h o c y t i c serum i s a n o n s p e c i f i c x e n o e f f e c t . A n t i l y m p h o c y t i c serum i s made f r o m a n t i t h y m o c y t e g l o b u l i n . Thymocytes a r e t h e b e s t immunogen t o g e n e r a t e a n t i t h y m o c y t e g l o b u l i n . The T c e l l s a re t h e a c t u a l o b s e r v e d t a r g e t a s opposed t o B c e l l s w h i c h a r e n e c e s s a r y f o r immunogenic e n h a n c e m e n t . 2 0 I t i s o f n o t e t h a t B c e l l a n t i b o d y c r o s s r e a c t s w i t h l a a n t i g e n s i m p l y i n g t h a t i f d e n d r i t i c c e l l s i n t h e g r a f t a c t as o l d passenger c e l l s wh ich a re t h e t a r g e t o f c l a s s I I a n t i b o d y in. t h e e s t a b l i s h m e n t o f enhancement , t h e d e n d r i t i c c e l l s a r e n o t t h e f o r e i g n a n t i g e n b u t t h e p r e s e n t e r o f f o r e i g n a n t i g e n c l a s s I . ^ 5 Enhancement e x p e r i m e n t s a r e u s u a l l y c a r r i e d o u t u s i n g h e m i z y g o u s , i . e . F]_ d o n o r , t r a n s p l a n t s because homozygous c a r d i a c and r e n a l g r a f t s a r e r a r e l y s u s c e p t i b l e t o enhancement i n r a t s . 3 5 I t seems t h a t h a l v i n g t h e s t r e n g t h o f t h e i n c o m p a t i b i l i t y h a l v e s t h e power o f r e j e c t i o n and b r i n g s a w i d e r range o f e x p e r i m e n t a l models i n t o p l a y . I t i s i n t e r e s t i n g t h a t t h e f a c t o r o f i m m u n i z a t i o n does n o t seem t o change w i t h t h e dose o f immunogen, f o r examp le , i n mixed l y m p h o c y t e r e a c t i o n s t h e r e i s no a d d i t i v e e f f e c t when more o r l e s s MHC immunogen i s p r e s e n t on a per c e l l b a s i s . ^ T h e r e f o r e t h i s i m p l i e s t h a t e i t h e r a humora l r e s p o n s e i s r e s p o n s i b l e and n o t a c e l l u l a r o n e , o r t h a t i t s r e j e c t i b i 1 i t y i s d i m i n i s h e d . I t i s known t h a t a n t i b o d y complement g roup med ia ted c y t o t o x i c i t y i n v i t r o and h a l v i n g t h e a n t i g e n i c s i t e s does i n d e e d d i m i n i s h t h e r e a c t i o n by l i m i t i n g t h e s i t e and d e n s i t y . ^ The q u e s t i o n o f a n t i - i d i o t y p i c a n t i b o d y i s r a i s e d b y ' - t h i s phenomenon, and t h e i d i o t y p e i s e x p l a i n e d i n t h e f o l l o w i n g way. The e x p e c t a t i o n on i m m u n i z i n g F i h y b r i d r a t w i t h a p a r e n t a l one ( P i one o f t h e p a r e n t a l s t r a i n s ) i s t h a t no i m m u n o l o g i c a l r e s p o n s e w i l l be o b t a i n e d as t h e ? \ has a dose o f what t h e ?i s t r a i n p r e s e n t s , summarized as t h e F\ h a v i n g c o d o m i n a n t t r a n s p l a n t a t i o n a n t i g e n s f r o m - 10 - t h e p a r e n t ; however , t h e F j c o u l d make a n t i - i d i o t y p e a n t i b o d y a g a i n s t r e c e p t o r s , t h i s s p e c i f i c i t y b e i n g f o r b i d d e n by s e l f - t o l e r a n c e i n t h e F^ an ima l i t s e l f . The serum f r o m an F j a n i m a l , h a v i n g d e v e l o p e d t h e s e a n t i b o d i e s , can be i n j e c t e d i n t o a second p a r e n t a l s t r a i n , say P2. T h i s w i l l p r o l o n g t h e s u r v i v a l o f a g r a f t i n P2, h a v i n g F^ g e n e t i c make u p . 2 1 I n r a t c a r d i a c enhancement m o d e l s , a l t h o u g h F^ t r a n s p l a n t s a r e g e n e r a l l y u s e d , t h e c e l l s used f o r p r e i m m u n i z a t i o n i n e i t h e r a c t i v e o r p a s s i v e enhancement e x p e r i m e n t s a r e o f t e n P\ c e l l s . Sometimes F^ c e l l s a re u s e d , bu t i n many cases b o t h P]_ and F j c e l l s a re seen t o be used as t h o u g h t h e y are i n t e r c h a n g e a b l e , t h u s Fj_ c e l l s a re i n c a p a b l e o f e l i c i t i n g an a n t i - i d i o t y p e r e s p o n s e , bu t ?i c e l l s c o u l d do s o , t h u s c o n f u s i n g t h e i s s u e o f enhancement . I t i s known when u s i n g F^ h e t e r o t o p i c c a r d i a c g r a f t s i n P^ r a t s i n an a c t i v e enhancement model (DA X PVG) F]_ i n t o PVG t h a t p r e i m m u n i z a t i o n a week b e f o r e g r a f t i n g u s i n g F]_ c e l l s o r u s i n g P]_ c e l l s g i v e s d i f f e r e n t r e s u l t s . 15 p-̂  c en a r e c a p a b l e o f i n d u c i n g a n t i - i d i o t y p e r e s p o n s e and g i v e b e t t e r g r a f t p r o l o n g a t i o n t h a n a s i m i l a r t r e a t m e n t u s i n g F]^ c e l l s w h i c h a re u n a b l e p roduce a n t i - i d i o t y p e r e s p o n s e i f immunized i n t o an F]_ r e c i p i e n t , B c e l l s possess c l a s s I and c l a s s I I a n t i b o d y whereas n o n - s t i m u l a t e d T c e l l s c o n t a i n c l a s s I o n l y . T h e r e f o r e t h e o r e t i c a l l y i f a n t i - i d i o t y p i c a n t i b o d y was t h e s o l e means o f g r a f t p r o l o n g a t i o n one s h o u l d see s i m i l a r p r o l o n g a t i o n by u s i n g e i t h e r c e l l o r mixed c e l l i m m u n i z a t i o n s p r . e t r a n s p l a n t . The e x i s t i n g e x p e r i m e n t a l e v i d e n c e a t t r i b u t e d t o enhancement has been a n t i - i d i o t y p i c and has no doubt s e r v e d t o compound t h e c o n f u s i o n and c o n t r i b u t e t o m i s q u o t e d d a t a . 1 5 Some s t u d i e s have a t t e m p t e d t o c l a s s i f y t h e agent w h i c h p r o d u c e s e n h a n c e m e n t . 3 0 , 8 9 , 1 0 6 j n e most l i k e l y c a n d i d a t e s f o r enhancement a re G i m m u n o g l o b u l i n ( I g G ) and a r e a b l e t o p roduce c y t o t o x i c i t y . The re seems t o be - 11 - agreement t h a t i m m u n o g l o b u l i n M ( IgM) does no t enhance and t h a t complement b i n d i n g c a p a c i t y i s n o t e s s e n t i a l . 6 4 The f a c t t h a t c e r t a i n p a r e n t s show more enhancement t h a n o t h e r s may be an e f f e c t o f i m m u n i z a t i o n s c h e d u l e s used i n d i f f e r e n t s t u d i e s , u s i n g d i f f e r e n t c e l l s , d i f f e r e n t t i m e s , and r o u t e s , as w e l l as d i f f e r e n t q u a n t i t i e s . I t has been n o t e d , f o r e x a m p l e , t h a t , i n g e n e r a l s k i n , g r a f t i n g g i v e s a p o t e n t e n h a n c i n g a n t i s e r u m w i t h low i n v i t r o c y t o t o x i c i t y . 5 5 The d i f f i c u l t y i n d i s c o v e r i n g t h e c l a s s o f a n t i b o d y s h o u l d be a b l e t o be worked o u t now t h a t m o n o c l o n a l a n t i b o d i e s a re a v a i l a b l e . I t i s g e n e r a l l y t h o u g h t t h a t a n t i b o d y a n t i g e n i n t e r a c t i o n i s h i g h l y s p e c i f i c . - ^ The o b s e r v a t i o n t h a t monoc lona l a n t i b o d i e s a r e no t a b s o l u t e l y s p e c i f i c i s a d i f f i c u l t c o n c e p t t o u n d e r s t a n d . One way o f e x p l a i n i n g t h i s phenomenon i s t o p o s t u l a t e s u b l o c i on t h e MHC. The g e n e r i c e x p r e s s i o n s , i . e . a n t i g e n s , c o e x i s t w i t h a n t i b o d i e s i n d i s t i n g u i s h a b l e f r o m t h o s e used i n t i s s u e t y p i n g . T h i s c o n c e p t was i n i t i a l l y p u t f o r w a r d by Medawar i n 1958 i n o r d e r t o e x p l a i n t h e o b s e r v a t i o n t h a t a n t i g e n s on r o d e n t r e d c e l l s a r e n o t c a p a b l e o f e l i c i t i n g an a l l o i m m u n e r e s p o n s e (as measured by h a s t e n e d s k i n g r a f t r e j e c t i o n ) w h i l e a n t i g e n s on l e u k o c y t e s a re an adequate s e n s i t i z i n g s t i m u l u s by t h e same d e f i n i t i o n . The p r o p o s i t i o n o f H - a n t i g e n s and T - a n t i g e n s a r o s e . ^ A s i m i l a r d i s t i n c t i o n c o u l d be made, i n t h e c a p a c i t y t o s t i m u l a t e mixed l y m p h o c y t e r e a c t i o n s , and w i t h t h i s i n mind a p u t a t i v e new gene p r o d u c t was sough t by a b s o r b i n g a mouse a l l o a n t i s e r u m t o c o m p l e t i o n w i t h r e d c e l l s and t e s t i n g t h e abso rbed s e r a f o r new a n t i b o d y . These abso rbed s e r a r e t a i n c y t o t o x i c i t y o n l y f o r c e r t a i n r e s t r i c t e d t a r g e t c e l l s , and i n v i v o t h e s e r a had u n d i m i n i s h e d c a p a c i t y f o r enhancement h a v i n g no H2 a n t i b o d i e s , i . e . t h e - 12 - known h i s t o c a p a b i 1 i t y a n t i b o d i e s a t t h a t t i m e . i y These enhancement e x p e r i m e n t s used h e t e r o t o p i c c a r d i a c g r a f t s i n t h e r a t (Wag x A r g u s ) F]_ t o Argus c o m b i n a t i o n . Normal r e j e c t i o n t i m e was c o n s i s t e n t l y 7-9 d a y s , bu t t h e o r i g i n a l serum and a b s o r b e d serum gave permanent s u r v i v a l . A b s o r p t i o n w i t h donor s t r a i n l e u k o c y t e s ( b u t n o t r e c i p i e n t s t r a i n l e u k o c y t e s ) removes a l l e n h a n c i n g a c t i v i t y ; t h u s o l d H£ t i s s u e t y p i n g (SD a n t i b o d y now c a l l e d MHC c l a s s I a n t i b o d y ) seemed t o be a t l e a s t u n n e c e s s a r y f o r enhancement and t h a t new n o m e n c l a t u r e Locus D (LD) e q u a l l i n g l a i n t h e mouse o r Locus D s u b l o c u s r ( D r ) i n t h e human (now c a l l e d MHC c l a s s I I a n t i b o d y ) was t h e m e d i a t o r . l ' ' The p o s s i b i l i t y t h a t c l a s s I a n t i g e n a n t i b o d y complexes may have e l u t e d f r o m t h e a b s o r b i n g c e l l s and p l a y e d some r o l e i n t h e p r o c e s s was c o n s i d e r e d u n l i k e l y f r o m t h e d a t a p r e s e n t e d a t t h e t i m e bu t w i l l be r e c o n s i d e r e d b e l o w . O t h e r n o n - m i x e d l y m p h o c y t e r e a c t i o n (MLR) s t i m u l a t i n g m a t e r i a l c o u l d be employed f o r a b s o r p t i o n i n p l a c e o f r e d c e l l s , i . e . p l a t e l e t s . H ' 2 2 These have been w i d e l y used f o r p r e p a r i n g c l a s s I I a n t i s e r a . 3 i n man and p r i m a t e s g e n e r a l l y r e d c e l l s do n o t c a r r y c l a s s I I MHC a n t i g e n s . Enhancement by c l a s s I I a n t i b o d y (B c e l l a n t i b o d y ) has been g e n e r a l l y c o n f i r m e d i n a v a r i e t y o f m o d e l s , e s p e c i a l l y f o r r a t h e a r t and k i d n e y g r a f t s . y 3 » l u ? C o n s i s t e n t w i t h t h i s d a t a Meo e t al showed t h a t c l a s s I I ( l a ) a n t i s e r a i n h i b i t e d t h e s t i m u l a t o r c e l l i n MLR, d e t r a c t i n g f r o m t h e c l a r i t y o f t h e s i t u a t i o n . ^ The re a r e e x p e r i m e n t a l models where c l a s s I a n t i b o d y w i l l e n h a n c e . 3 8 , 4 5 , 9 5 , 9 6 , 9 7 These e x c e p t i o n s may be u n d e r s t o o d i n t h e l i g h t o f an e x p l a n a t i o n o f enhancement i n t h e s i m p l e s i t u a t i o n o f i n d u c t i o n o f c l a s s I I a n t i b o d i e s . I t can be p o s t u l a t e d t h e r e f o r e t h a t s t i m u l a t i o n by p l a t e l e t s - 13 - which do n o t c a r r y c l a s s I I MHC a n t i g e n s c o u l d a c t l i k e pu re T c e l l enhancement w i t h o u t t h e r i s k o f s e n s i t i z i n g t h e d o n o r by s t i m u l a t i n g c l a s s I I a n t i b o d y , h e n c e , t h i s w o u l d have some c l i n i c a l a p p l i c a t i o n . T a r a s a k i has shown t h a t r e d b l o o d c e l l s s t i m u l a t e enhancement i n r e n a l t r a n s p l a n t s bu t t h e c o s t o f t h i s i s s e n s i t i z a t i o n o f some i n d i v i d u a l s . 1 0 1 T h i s s e n s i t i z a t i o n i s g e n e r a l l y t h o u g h t t o be a n t i b o d i e s a g a i n s t c l a s s I I a n t i g e n , and c o u l d be avo ided i f a s i m i l a r e f f e c t c o u l d be o b t a i n e d by p l a t e l e t s w h i c h o b v i o u s l y c o u l d no t i n d u c e c l a s s I I a n t i b o d y and s t i l l p r o d u c e t h e e n h a n c i n g e f f e c t . F u r t h e r e x p e r i m e n t s t h r o w f u r t h e r l i g h t on c e r t a i n c l a s s I and c l a s s I I a n t i b o d y r e a c t i o n s . 6 0 C l a s s I I a n t i b o d y i s t h o u g h t t o be a m e d i a t o r o f enhancement u s i n g c a r d i a c a l l o g r a f t s i n t h e r a t s y s t e m (Wag x A r g u s ) F^ t o Argus r e c i p i e n t s i n a c t i v e enhancement . The e n h a n c i n g c a p a c i t i e s o f B c e l l s and T c e l l s as p r e i m m u n i z i n g a n t i g e n s were c o m p a r e d . T c e l l s j u d g e d t o i n d u c e o n l y c l a s s I a n t i b o d y d i d n o t l e a d t o enhancement . B c e l l s j u d g e d t o i n d u c e c l a s s I a n t i b o d y and c l a s s I I a n t i b o d y d i d l e a d t o enhancement . F u r t h e r c o n f i r m a t i o n was o b t a i n e d i n a n o t h e r s t r a i n c o m b i n a t i o n (DA x PVG) F]_, two PVG r e c i p i e n t s . I n t h i s case B c e l l p r e i m m u n i z a t i o n gave enhancement bu t T c e l l p r e i m m u n i z a t i o n f a i l e d t o p roduce enhancement . The re a r e s e v e r a l k i n d s o f e x p e r i m e n t s and c l i n i c a l d a t a t o show t h e t o x i c i t y f o r c l a s s I a n t i b o d y bu t none f o r any t o x i c i t y f o r c l a s s I I a n t i b o d y ; t h u s , by c o n t i n u i n g i n d u c t i o n o f IgG o r s u b c l a s s e s i t may be t h a t i t i s n e c e s s a r y t o enhance c l a s s I I a n t i b o d y o r d e c r e a s e c l a s s . I a n t i b o d y . 3 7 ' ^ Two.phases o f enhancement have been w i d e l y a c c e p t e d - an i n d u c t i o n phase and a m a i n t e n a n c e p h a s e d The c i r c u l a t i n g a n t i g e n , a n t i b o d y , o r a n t i g e n - a n t i b o d y comp lexes a r e p r e s u m a b l y excess t o r e q u i r e m e n t s i n t h e c o n t e x t o f donor s p e c i f i c i m m u n o s u p r e s s i o n , s i n c e o n l y by i n t e r a c t i o n w i t h - 14 - some c e l l s i s t h e p o t e n t i a l a c t i v i t y t o be r e a l i z e d . A component no t d e t e c t a b l e i n t h e serum may be p r e s e n t o n l y i n s u f f i c i e n t amount t o b l o c k t h i s r e c e p t o r . 3 9 Because c l a s s I I a l l o a n t i b o d y can i n i t i a t e enhancement , i t i s an a t t r a c t i v e p r o p o s i t i o n t h a t t h e c l a s s I I MHC a n t i g e n t o w h i c h i t a t t a c h e s i s t h a t on d o n o r d e n d r i t i c c e l l s , c e l l s w h i c h a re now b e l i e v e d t o p l a y a m a j o r r o l e i n i n i t i a t i n g t h e a l l o i m m u n e r e s p o n s e . 4 8 They a re v e r y r i c h i n c l a s s I I ( l a , D r ) a n t i g e n . Such a mechanism f o r t h e i n i t i a t i o n o f enhancement by m i n i m i z i n g t h e i n i t i a l s e n s i t i z a t i o n f i t s w e l l w i t h t h e o b s e r v a t i o n t h a t s m a l l amounts o f serum somet imes i s s u f f i c i e n t f o r enhancement . T h i s i s an amount t o o s m a l l t o c o v e r up g r a f t a n t i g e n i n t h e sense of m a s k i n g . F ree g r a f t a n t i g e n ( c l a s s I ) however i s c e r t a i n l y exposed on c e l l s o f e n h a n c i n g t r a n s p l a n t s . The e v i d e n c e shows h a s t e n e d r e j e c t i o n by c l a s s I a n t i b o d y and t h a t i t i s r e a l l y c l a s s I I a n t i b o d y r e a c t i n g w i t h d e n d r i t i c c e l l s w h i c h i n i t i a t e enhancement by p r e v e n t i n g t h e a l l o i m m u n e r e s p o n s e f r o m g e t t i n g u n d e r w a y . 7 2 » 7 8 j n e r e - j s e v i d e n c e t h a t c l a s s I complexes d i r e c t l y b l o c k B c e l l d i f f e r e n t i a t i o n t o p lasma c e l l s . 1 0 2 i t i s a l s o c l e a r l y shown t h a t complexes l e a d t o d i f f e r e n t i a t i o n o f a n t i g e n r e a c t i v e c e l l s , and f u r t h e r , t h a t t h i s a c t i v i t y i s r e a l i n p a s s i v e l y enhanced s k i n a l l o g r a f t s i n m i c e . ^ The re were no a n t i g e n r e a c t i v e c e l l s i n m ice c a r r y i n g s k i n g r a f t s as a r e s u l t o f h a v i n g been made t o l e r a n t n e o n a t e l y . . We know t h a t t h e l i v e r p r o v i d e s abundan t c l a s s I a n t i g e n and l i t t l e c l a s s I I a n t i g e n and i s a l a r g e amount o f t i s s u e as a t r a n s p l a n t , 6 3 and l e a s t s u s c e p t i b l e t o r e j e c t i o n . S k i n has abundan t c l a s s t l and l i t t l e c l a s s I a n t i g e n * 6 9 and i s most d i f f i c u l t t o enhance i n t o a m a i n t e n a n c e p h a s e . When serum or lymph f r o m l i v e r t r a n s p l a n t e d r a t s i s t r a n s f e r e d t o new u n t r e a t e d s y n g e n e t i c a n i m a l s wh ich have been s k i n g r a f t e d f r o m t h e same donor t h e g r a f t i s p r e s e r v e d b u t o n l y w h i l e serum i n j e c t i o n s a r e m a i n t a i n e d ; a f t e r t r e a t m e n t t h i s s t o p s . * 8 - 15 - S k i n g r a f t r e j e c t i o n t h e n b e g i n s t o t a k e i t s normal c o u r s e as t h o u g h t h e t r e a t m e n t c o u l d p r o v i d e f o r a m a i n t e n a n c e phase bu t n o t c a r r y o u t t h e i n i t i a t i o n p r o c e s s . More s p e c i f i c a l l y , p e r h a p s , t h e c l a s s I complexes i n t h e l i v e r g r a f t e d r a t serum d i d n o t r e a c t w i t h s k i n g r a f t d e n d r i t i c c e l l s ( L a n g e r h a n s 1 c e l l s ) e f f e c t i v e l y and c l a s s I I a n t i b o d y d i d no t i n a c t i v a t e them under t h e c o n d i t i o n s o f t h e e x p e r i m e n t , a l t h o u g h t h e r e a r e some e x p e r i m e n t a l s i t u a t i o n s , e s p e c i a l l y i n r o d e n t s , where i m m u n o s u p p r e s s i o n can e v e n t u a l l y be w i t h d r a w n w i t h o u t l o s i n g t h e g r a f t even when t h e MHC b a r r i e r i s i n v o l v e d . T h i s i s n o t on r e c o r d i n t h e c l i n i c a l p r a c t i c e . 1 0 3 Long s u r v i v i n g r e n a l t r a n s p l a n t s w i t h e x c e l l e n t f u n c t i o n i n p a t i e n t s on low l e v e l s o f i m m u n o s u p p r e s s i v e d r u g s have r e j e c t i o n e p i s o d e s i f t r e a t m e n t i s w i t h d r a w n . T h i s k i n d o f a d a p t a t i o n i s n o t c o m p l e t e and i t s b a s i s , t h o u g h much d i s c u s s e d , i s no t known. One m i g h t ask w h e t h e r enhancement e n t e r s i n t o t h i s and i n t o some k i n d s o f s a t i s f a c t o r y c l i n i c a l t r a n s p l a n t s i t u a t i o n s , i n c l u d i n g t h o s e r e s u l t i n g f r o m b l o o d t r a n s f u s i o n s . No answer can be g i v e n because t h e end r e s u l t o f an enhanced s i t u a t i o n s t i l l c a n n o t be p r e c i s e l y d e s c r i b e d i n t e r m s o f r e s u l t i n g B c e l l and T c e l l f u n c t i o n a l m o d i f i c a t i o n s . R a t h e r l i t t l e e f f o r t has been made so f a r t o t e s t p o s s i b i l i t i e s o f u s i n g enhancement c l i n i c a l l y . 4 3 Some c l i n i c a l work has been done i n t h e a r e a o f a n t i b o d y enhancement . An a t t e m p t was made t o s t u d y t h i s by t h e use o f p o o l e d B c e l l a n t i b o d y a d m i n i s t e r e d t o r e n a l t r a n s p l a n t p a t i e n t s . ^ I n t h i s t r i a l b l o o d donor v o l u n t e e r s were c r o s s - i m m u n i z e d and t h e r e s u l t i n g a n t i s e r a poo led and absorbed w i t h p l a t e l e t s t o remove t h e c y t o t o x i c c l a s s I a n t i b o d y . The poo l s i z e was j u d g e d adequate f r o m t h e known c r o s s r e a c t i v i t y o f l a s p e c i f i c i t i e s , u n l i k e t h a t o f c l a s s I ( t i s s u e t y p i n g ) s p e c i f i c i t i e s ; b u t , i n r e t r o s p e c t , t h e amount o f c l a s s I I a n t i b o d y i n t h e absorbed s e r a was p r o b a b l y i n s u f f i c i e n t t o be i n f l u e n t i a l and t h e f i n a l outcome was i n c o n c l u s i v e . S t u d i e s have been 92 made i n t h e l o w e r p r i m a t e s w i t h s i m i l a r r e s u l t s . - 16 - I n enhancement b o t h B and T c e l l s a re m o d u l a t e d b u t IgM a n t i b o d y a g a i n s t donor MHC can be made, and T c e l l s a re s t i l l a b l e t o r e a c t i n MLR i n g r a f t v e r s u s h o s t r e a c t i o n s . I t i s i m p o r t a n t t o remember t h a t many o r g a n s i n many MHC c o m b i n a t i o n s c a n n o t be e n h a n c e d , and f a v o u r a b l e e x p e r i m e n t a l d a t a come f r o m t h e c o m b i n a t i o n s p u r p o s e l y chosen t o s t u d y t h e e f f e c t where i t works b e s t . Background i m m u n o s u p p r e s s i o n w i t h d r u g s w i l l a lways be n e e d e d , and t h e s t u d i e s o f what o t h e r methods do and d o . n o t s y n e r g i z e w i t h enhancement a r e i m p o r t a n t . 7 6 I n summary t h e m i n i m a l mechanisms t o s a t i s f y t h e s i m p l e s t s i t u a t i o n seem t o r e q u i r e a means o f n e u t r a l i z i n g ( k i l l i n g ) d e n d r i t i c ( a n t i g e n p r e s e n t i n g , MHC c l a s s I I p o s i t i v e ) c e l l s i n a g r a f t t o p r e v e n t t h e a l l o i m m u n e r e s p o n s e f r o m b u i l d i n g u p , i . e . an i n i t i a t i o n p h a s e , and t h i s o f t e n can be m a i n t a i n e d by c l a s s I a n t i b o d y m e d i a t e d a n t i g e n r e a c t i v e c e l l o p s o n i z a t i o n and c l a s s I complex m e d i a t e d i n h i b i t i o n o f B c e l l m a t u r a t i o n . These do n o t a lways w o r k . 6 4 T h u s , enhancement may be d i f f e r e n t i n d e g r e e , and i t s p r i n c i p l e w i l l a p p l y a c c o r d i n g t o t h e p a r t i c u l a r t i s s u e t r a n s p l a n t e d , power o f t h e i n i t i a t o r , and t h e v u l n e r a b i l i t y o f t h e t a r g e t . - 1 7 - MATERIALS AND METHODS EXPERIMENTAL MODELS: RABBIT KIDNEY ALLOGRAFTS, RAT HEART ALLOGRAFTS, RAT HEART AND LUNG ALLOGRAFTS. REVIEW OF 454 CARDIAC ALLOGRAFTS . I n t r o d u c t i o n In o r d e r t o s t u d y t h e e f f e c t s o f p r e i m m u n i z a t i o n on enhancement i n t r a n s p l a n t r e j e c t i o n , a s u i t a b l e an ima l model had t o be d e v e l o p e d . Requ i remen ts w e r e : ( a ) Ready a v a i l a b i l i t y o f l a r g e numbers o f a n i m a l s . ( b ) N o n - e l a b o r a t e t e c h n i q u e f o r t r a n s p l a n t i n g a v a s c u l a r i z e d g r a f t . ( c ) Low m o r t a l i t y , h i g h success r a t e . ( d ) E a s i l y - d e f i n e d e n d - p o i n t o f g r a f t r e j e c t i o n . (e ) Low v a r i a n c e o f e n d - p o i n t o f r e j e c t i o n , r e d u c i n g numbers o f a n i m a l s n e c e s s a r y i n each e x p e r i m e n t a l g r o u p , i m p l y i n g t h e use o f r e l i a b l y i n b r e d an imal s t r a i n s . A . R a b b i t K i d n e y A l l o g r a f t s I n i t i a l l y r a b b i t k i d n e y a l l o g r a f t s were s t u d i e d . K i d n e y s were t r a n s p l a n t e d f r o m New Zea land w h i t e r a b b i t s t o non-New Zea land w h i t e r a b b i t s o f a h y b r i d s t r a i n , u s i n g t h e t e c h n i q u e o f D u n n . 3 1 Young a d u l t r a b b i t s w e i g h i n g 2 - 3 kg were u s e d . K i d n e y s were t r a n s p l a n t e d o r t h o t o p i c a l l y and r e c i p i e n t s were b i l a t e r a l l y n e p h r e c t o m i z e d a t t h e t i m e o f t r a n s p l a n t a t i o n . Serum c r e a t i n i n e was measured e v e r y two t o t h r e e d a y s . - 18 - The r a b b i t r e n a l a l l o g r a f t model was f o u n d t o be u n s u i t a b l e f o r t h e p l a n n e d e x p e r i m e n t s , f o r t h e f o l l o w i n g r e a s o n s : (a ) A l t h o u g h t h e a n i m a l s were r e a d i l y a v a i l a b l e t h e y a re r e l a t i v e l y e x p e n s i v e t o p u r c h a s e and m a i n t a i n . ( b ) The t e c h n i q u e i s s i m p l e , u s i n g a c u f f t e c h n i q u e f o r t h e a r t e r i a l a n a s t o m o s i s , b u t t h e p e r i o p e r a t i v e m o r t a l i t y was h i g h . Even i n i s o g r a f t s , t h e s u r v i v a l r a t e was l e s s t h a n 50% a t 14 d a y s . Death was due t o s e v e r a l f a c t o r s i n c l u d i n g pneumon ia , h y p o t h e r m i a * r e n a l a r t e r i a l and venous t h r o m b o s i s , and u r e t e r i c b l o c k a g e . ( c ) The e n d - p o i n t o f r e j e c t i o n was n o t w e l l d e f i n e d . Serum c r e a t i n i n e may r i s e because an an imal i s u n w e l l ( f o r example d e v e l o p i n g pneumonia) r a t h e r t h a n r e j e c t i n g t h e t r a n s p l a n t e d k i d n e y . Death may be due t o causes u n r e l a t e d t o r e j e c t i o n , y e t h i s t o l o g y o f an a l l o g r a f t e d k i d n e y w i l l s t i l l show h i s t o l o g i c a l changes o f r e j e c t i o n . ( d ) The e n d - p o i n t o f r e j e c t i o n , b e i n g d i f f i c u l t t o d e f i n e , had a h i g h v a r i a n c e . ( e ) I t was no t p o s s i b l e t o d e v i s e a sys tem f o r c o n t i n u o u s i n t r a v e n o u s i n f u s i o n i n r a b b i t s , w i t h o u t c l o s e c o n f i n e m e n t o f t h e r a b b i t s . The r a b b i t s were a b l e t o d e s t r o y a l l c a n n u l a e by b i t i n g t h e m , o r by s p i n n i n g r a p i d l y a round i.n t h e i r c a g e s . For t h e s e r e a s o n s , a t t e n t i o n was d i r e c t e d t o w a r d s a r a t h e a r t a l l o g r a f t m o d e l . - 19 - B. Rat C a r d i a c A l l o g r a f t s The r a t h e t e r o t o p i c h e a r t g r a f t t e c h n i q u e , m o d i f i e d a f t e r H e r o n 5 1 was u s e d . I n t h i s s y s t e m h e a r t s a re t r a n s p l a n t e d t o t h e necks o f r e c i p i e n t r a t s . T h e i r s u b c u t a n e o u s l o c a t i o n makes them p a r t i c u l a r l y easy t o assess f o r e v i d e n c e o f r e j e c t i o n : c e s s a t i o n o f p a l p a b l e bea t i s u s u a l l y t a k e n as t h e e n d - p o i n t o f r e j e c t i o n . T h i s p r o c e d u r e was f o u n d t o be v e r y s u i t a b l e f o r f u r t h e r p l a n n e d e x p e r i m e n t s , and t h e p r o c e d u r e became t h e s t a n d a r d e x p e r i m e n t a l model f o r a l a r g e p r o p o r t i o n , o f t h e work d e s c r i b e d i n t h i s t h e s i s . I n t h e f o l l o w i n g s e c t i o n , t h e p r o c e d u r e used f o r h e t e r o t o p i c t r a n s p l a n t a t i o n o f r a t h e a r t s i s d e s c r i b e d , and a g e n e r a l r e v i e w o f t h e f i r s t 464 t r a n s p l a n t s p e r f o r m e d by t h e a u t h o r i s made. A l l t h e r e q u i r e m e n t s f o r a s u i t a b l e model f o r f u r t h e r e x p e r i m e n t a t i o n were s a t i s f i e d : (a ) A n i m a l s were r e a d i l y a v a i l a b l e i n l a r g e numbers . " D A t R T l 3 ) 1 and PVG(RT1 C ) r a t s were b r e d by t h e a u t h o r f r o m s t o c k s o b t a i n e d f r o m B a n t i n and K i n g m a n , G r i m s t o n , H u l l . R e g u l a r s y n g e n e i c and a l l o g e n e i c s k i n and h e a r t g r a f t s c o n f i r m e d c o n s i s t e n t p a t t e r n s o f g r a f t a c c e p t a n c e o r r e j e c t i o n i n u n t r e a t e d r a t s , w i t h no e v i d e n c e o f o u t b r e e d i n g . '• ( b ) The t r a n s p l a n t t e c h n i q u e was s i m p l e , and r a p i d l y l e a r n t . ( c ) The o p e r a t i v e m o r t a l i t y was l o w , and t h e o v e r a l l success r a t e , i n c l u d i n g d e a t h due t o pneumonia and g r a f t l o s s due t o a r t e r i a l o r venous t h r o m b o s i s was 78.5%. - 20 - ( d ) The end p o i n t o f g r a f t r e j e c t i o n , t a k e n as c e s s a t i o n o f p a l p a b l e h e a r t bea t was w e l l - d e f i n e d , and o c c u r r e d w i t h i n one o r two h o u r s . ( e ) The v a r i a n c e o f e n d - p o i n t o f r e j e c t i o n , i n c o n t r o l a l l o g r a f t s was l o w . In b o t h male and n o n - p r e g n a n t f e m a l e PVG(RT1 c ) r a t s r e c e i v i n g h e a r t s f r o m D A ( R T l a ) r a t s o f t h e same s e x , t h e mean r e j e c t i o n t i m e i n u n t r e a t e d r a t s was 7 .38 + 0 .77 (SD) d a y s . i . T e c h n i q u e f o r h e t e r o t o p i c t r a n s p l a n t a t i o n o f r a t h e a r t s The method used was t h a t o f H e r o n 6 1 w i t h c e r t a i n m o d i f i c a t i o n s . H e a r t s were removed a f t e r l i g a t i n g a l l v e s s e l s e x c e p t f o r t h e a o r t a and p u l m o n a r y a r t e r y , and t h e stumps o f t h e s e v e s s e l s were j o i n e d t o t h e r e c i p i e n t c a r o t i d a r t e r y and j u g u l a r v e i n , r e s p e c t i v e l y , u s i n g c u f f t e c h n i q u e s ( F i g u r e 1 ) . A r t e r i a l b l o o d t h e r e f o r e p e r f u s e d t h e c o r o n a r y c i r c u l a t i o n , d r a i n e d t o t h e r i g h t s i d e o f t h e h e a r t , and was e j e c t e d f r o m t h e g r a f t p u l m o n a r y a r t e r y i n t o t h e r e c i p i e n t venous c i r c u l a t i o n . The g r a f t b e a t was v i g o r o u s , a n d , i n a l l o g r a f t s , c e s s a t i o n o f p a l p a b l e g r a f t b e a t was t a k e n as t h e e n d - p o i n t o f r e j e c t i o n . T h i s was a d e f i n i t e e n d - p o i n t , and i n u n t r e a t e d a l l o g r a f t s be tween i n b r e d s p e c i e s t h e r e j e c t i o n t i m e had a low v a r i a n c e . - 21 - i i . M a t e r i a l s and methods I n s t r u m e n t s For t h e t e c h n i q u e t o be d e s c r i b e d , t h e f o l l o w i n g i n s t r u m e n t s were u s e d . Most were o b t a i n e d f r o m John Weiss and Son L t d . , 11 Wigmore S t r e e t , London WlH ODN. C a t a l o g u e Number Number ( W e i s s ) I n s t r u m e n t 1 BC 54 D i s s e c t i n g f o r c e p s 1 BC 100 S t r a i g h t Mayo s c i s s o r s 1 BC 37 Mclndoe f o r c e p s 1 B 1047 Sharp s t r a i g h t s c i s s o r s 1 - La rge a r t e r i a l c lamp 2 B 981 C Curved M o s q u i t o f o r c e p s 1 B 1050 Sharp c u r v e d s c i s s o r s 1 - M i c r o s u r g i c a l c o a g u l a t o r 1 B 1201 M a g n i f y i n g l o u p e 1 BC 30 Curved j e w e l l e r ' s f o r c e p s 1 BC 31 5AR S t r a i g h t j e w e l l e r ' s f o r c e p s , d e f l e c t e d t i p 2 - M i c r o s u r g i c a l v e s s e l o c c l u d e r s 1 C 14 M i c r o s u r g i c a l n e e d l e h o l d e r 1 B 981 • S t r a i g h t M o s q u i t o f o r c e p s - 22 - i i i . O the r m a t e r i a l s Cork mat Round headed p i n s 6 cm cube o f m o d e l l i n g c l a y 10 ml s y r i n g e 19 gauge n e e d l e , p o i n t sawn o f f and edge rounded P o r t e x c a n n u l a , 3 F.G. O.D. 1.02 mm ( f o r a r t e r i a l c u f f ) P o r t e x c a n n u l a , 5 F .G. O.D. .1 .65 mm ( f o r venous c u f f ) Razor b l a d e 5'0' b l a c k s i l k - 6'0' b l a c k s i l k 8'0' E t h i c o n s u t u r e ( E t h i c o n n o . W2808) 4 1 0 ' c a t g u t s u t u r e Sodium c h l o r i d e i n j e c t i o n B.P. H e p a r i n E t h e r 10 ml s y r i n g e c o n t a i n e r C o t t o n wool H y p n o r * ( J a n s s e n ) A t r o p i n e C o t t o n wool buds - 23 - P r o c e d u r e : Donor O p e r a t i o n T w e n t y - f i v e ( 2 5 ) ml o f s a l i n e were t r a n s f e r r e d t o a s m a l l p l a s t i c po t s u r r o u n d e d by i c e , and 250 u o f h e p a r i n added. T h i s was f o r p e r f u s i o n and s t o r a g e o f t h e h e a r t t o be r e m o v e d . A d o n o r r a t w e i g h i n g 140 t o 300 gm was a n a e s t h e t i z e d i n an e t h e r j a r u n t i l r e s p i r a t i o n s t a r t e d t o s l o w . The r a t was t h e n p l a c e d s u p i n e on t h e c o r k b o a r d , u s i n g r o u n d headed p i n s t h r o u g h t h e w e b - s k i n o f t h e f e e t t o s e c u r e i t . A n a e s t h e s i a was m a i n t a i n e d by p l a c i n g t h e 10 ml s y r i n g e c o n t a i n e r c o n t a i n i n g c o t t o n - w o o l m o i s t e n e d w i t h e t h e r ove r t h e nose t o t h e l e v e l o f t h e i n c i s o r t e e t h . A m i d l i n e abdomina l i n c i s i o n was made u s i n g f o r c e p s and s t r a i g h t Mayo s c i s s o r s . The s m a l l bowel was d i s p l a c e d upwards and 250 u h e p a r i n i n j e c t e d i n t o t h e i n f e r i o r vena cava u s i n g a 25 -gauge n e e d l e . The x i p h o i d was l i f t e d w i t h f o r c e p s and an i n c i s i o n made w i t h s c i s s o r s a round t h e r i g h t b o r d e r o f t h e d i a p h r a g m . The r i b s were c u t p o s t e r i o r l y , p a r a l l e l t o t h e t h o r a c i c s p i n e on e i t h e r s i d e . A l a r g e a r t e r i a l c lamp was a t t a c h e d t o t h e x i p h o i d , and t h e who le a n t e r i o r t h o r a c i c cage l i f t e d s u p e r i o r l y w h i l e t h e p e r i c a r d i u m was o p e n e d . The h e a r t was c o o l e d w i t h t o p i c a l s a l i n e a t 4 ° C . U s i n g f o r c e p s and s t r a i g h t s c i s s o r s t h e i n f e r i o r vena cava-was m o b i l i z e d , and t h e n t h e r i g h t s u p e r i o r vena c a v a . These v e s s e l s were t h e n l i g a t e d w i t h 5 / 0 s i l k : one end o f each l i g a t u r e was l e f t 2 cm l o n g . - 24 - The t i p o f a c u r v e d M o s q u i t o f o r c e p s was p l a c e d i n t h e c o r o n a r y s i n u s f r o m t h e l e f t t o t h e r i g h t ; t h e a o r t a was m o b i l i z e d and d i v i d e d a c r o s s j u s t be low t h e f i r s t b r a n c h . Ten ( 1 0 ) ml o f h e p a r i n i z e d s a l i n e a t 4°C were c a r e f u l l y i n j e c t e d i n t o t h e a o r t i c s t u m p , w i t h g e n t l e c l o s u r e o f t h e open end o f t n e a o r t a w i t h Mc lndoe f o r c e p s . The p u l m o n a r y a r t e r y was m o b i l i z e d and d i v i d e d j u s t p r o x i m a l t o i t ' s b i f u r c a t i o n w i t h s h a r p c u r v e d s c i s s o r s . A v e r y s m a l l v e s s e l a t t h e a o r t i c r o o t was c o a g u l a t e d . The s u p e r i o r vena cava and i n f e r i o r vena cava were d i v i d e d d i s t a l t o t h e l i g a t u r e s . The h e a r t was t h e n p u l l e d a n t e r i o r l y and a 5 / 0 s i l k mass l i g a t u r e a p p l i e d a r o u n d t h e t i s s u e s p o s t e r i o r t o t h e a t r i a ( m a i n l y p u l m o n a r y v e i n s and l e f t s u p e r i o r vena c a v a ) . The h e a r t was f r e e d by d i v i d i n g t h e t i s s u e s d i s t a l t o t h e mass l i g a t u r e , and t h e h e a r t p l a c e d i n 4 ° C , t o r e m a i n t h e r e w h i l e t h e r e c i p i e n t was p r e p a r e d . v . R e c i p i e n t o p e r a t i o n A r e c i p i e n t r a t w e i g h i n g 150 t o 350 gm was i n j e c t e d i n t r a m u s c u l a r l y w i t h 0 . 5 m l / k g o f Hypnorm. Ten m i n u t e s l a t e r i t was i n j e c t e d s u b c u t a n e o u s l y w i t h 120 p g / k g o f a t r o p i n e and t h e r i g h t s i d e o f t h e neck shaved w i t h c l i p p e r s . The r a t was p i n n e d o u t s u p i n e o n t o t h e c o r k m a t , and t h e upper i n c i s o r t e e t h hooked o v e r a r u b b e r band wh ich e x t e n d e d t h e neck . ••' A v e r t i c a l i n c i s i o n was made s l i g h t l y t o t h e r i g h t o f t h e m i d l i n e f r o m 0 . 5 cm be low t h e f i r s t r i b upwards t o 0 . 5 cm be low t h e m a n d i b l e , u s i n g s c i s s o r s and f o r c e p s . The l a t e r a l s k i n f l a p was m o b i l i s e d w i t h a - 25 - m o i s t c o t t o n - w o o l b u d , and t h e f l a p p i n n e d t o t h e c o r k b o a r d . The f a t o v e r l y i n g p e c t o r a l i s m a j o r was l i f t e d a n t e r i o r l y and m o b i l i z e d upwards u s i n g f i n e sharp s c i s s o r s . One v e s s e l p e n e t r a t i n g p e c t o r a l i s ma jo r was c o a g u l a t e d . When t h e j u g u l a r v e i n was app roached t h i s v e s s e l was c l e a r e d a n t e r i o r l y by b l u n t d i s s e c t i o n t o j u s t above i t s main b i f u r c a t i o n . U s i n g a m a g n i f y i n g l o u p e , and two p a i r s o f j e w e l l e r ' s f o r c e p s f o r d i s s e c t i o n , t h e j u g u l a r v e i n was m o b i l i z e d : a l l b ranches e x c e p t t h e b i f u r c a t i o n were m o b i l i z e d and c o a g u l a t e d . The a n t e r i o r b ranch was t h e n l i g a t e d c l o s e t o t h e main v e s s e l , and t h e n b o t h b ranches were l i g a t e d t o g e t h e r 0 . 5 cm. d i s t a l t o t h e f i r s t l i g a t u r e . The a n t e r i o r b ranch was d i v i d e d between t h e two l i g a t u r e s . The s t e r n o m a s t o i d musc le was now m o b i l i z e d , l i g a t e d w i t h 5 / 0 s i l k as h i g h and low as p o s s i b l e , and t h e c e n t r a l segment e x c i s e d : t h i s gap was used f o r t h e t r a n s p l a n t e d h e a r t t o r e s t i n . The s t e r n o h y o i d musc le was now t e a s e d a p a r t i n t h e m i d d l e o f i t s b e l l y , and t h e common c a r o t i d a r t e r y i d e n t i f i e d . Us ing j e w e l l e r ' s f o r c e p s t h e common c a r o t i d a r t e r y was m o b i l i z e d f r o m t h e vagus n e r v e , and t h e a r t e r y was t h e n l i g a t e d j u s t p r o x i m a l t o i t s b i f u r c a t i o n w i t h 5 / 0 s i l k . A m i c r o s u r g i c a l v e s s e l o c c l u d e r was p l a c e d a c r o s s t h e common c a r o t i d a r t e r y as p r o x i m a l l y as p o s s i b l e , and t h e n t h e v e s s e l was p a r t i a l l y d i v i d e d j u s t p r o x i m a l t o t h e l i g a t u r e . An 8 / 0 E t h i c o n s u t u r e was used t o t r a n s f i x t h e l e f t hand edge o f t h e c a r o t i d a r t e r y , and t h e d i v i s i o n o f t h e a r t e r y c o m p l e t e d . The j u g u l a r v e i n was a l s o o c c l u d e d w i t h a m i c r o s u r g i c a l c l i p , and t h e p o s t e r i o r b r a n c h d i v i d e d f l u s h w i t h t h e b i f u r c a t i o n o f t h e main v e i n . Us ing a s t r a i g h t M o s q u i t o f o r c e p s mounted on a b l o c k o f m o d e l l i n g c l a y , a sma l l c u f f made f r o m t h e 5 F.G. P o r t e x c a n n u l a was mounted i n t h e t i p - 26 - o f t h e f o r c e p s , and p o s i t i o n e d o v e r t h e j u g u l a r v e i n . The j u g u l a r v e i n was t h r e a d e d t h r o u g h t h e lumen o f t h e c u f f and e v e r t e d ove r i t , and h e l d t h e r e w i t h a c i r c u m f e r e n t i a l 5 / 0 b l a c k s i l k l i g a t u r e . The c u f f was s e p a r a t e d f r o m t h e M o s q u i t o c l i p , and t h e p r o c e s s r e p e a t e d w i t h t h e c a r o t i d a r t e r y and a 3 F.G. P o r t e x c u f f , and a 6 / 0 b l a c k s i l k l i g a t u r e . The h e a r t g r a f t was removed f r o m i t s s a l i n e b a t h , and p o s i t i o n e d u p s i d e down i n t h e n e c k . The a o r t i c stump was p l a c e d o v e r t h e c a r o t i d a r t e r y c u f f , and h e l d t h e r e w i t h a 5 / 0 b l a c k s i l k l i g a t u r e . The p u l m o n a r y a r t e r y was p l a c e d o v e r t h e j u g u l a r v e i n c u f f , and h e l d t h e r e w i t h a 5 / 0 b l a c k s i l k l i g a t u r e . The venous and t h e n a r t e r i a l o c c l u d i n g c l i p s were r e m o v e d , and t h e g r a f t p e r f u s e d w i t h b l o o d . The g r a f t s t a r t e d t o b e a t i m m e d i a t e l y , o r a f t e r a few seconds o f v e n t r i c u l a r f i b r i l l a t i o n . The s k i n was c l o s e d w i t h c o n t i n u o u s 4 / 0 c a t g u t , and t h e r a t a l l o w e d t o r e c o v e r . v i . Assessment o f r e j e c t i o n A t t h e t i m e o f c e s s a t i o n o f p a l p a b l e h e a r t b e a t t h e r a t was a n a e s t h e t i s e d w i t h e t h e r and t h e h e a r t e x p o s e d . O c c a s i o n a l l y , a se rous c o l l e c t i o n was d i s c h a r g e d , and t h e u n d e r l y i n g h e a r t was t h e n seen t o b e a t . More u s u a l l y , c e s s a t i o n o f b e a t was c o n f i r m e d and t h e v e s s e l s were examined f o r e v i d e n c e o f venous o r a r t e r i a l t h r o m b o s i s . I n e q u i v o c a l cases h i s t o l o g y was used t o d i f f e r e n t i a t e between venous o r a r t e r i a l t h r o m b o s i s o r r e j e c t i o n . I n i t i a l l y , e l e c t r o c a r d i o g r a p h y was u s e d , b u t l o s s o f p a l p a b l e b e a t a lways c o i n c i d e d w i t h l o s s o f e l e c t r i c a l a c t i v i t y ( F i g u r e 2 ) , and d i r e c t e x a m i n a t i o n o f t h e h e a r t was t h e r e f o r e " s u f f i c i e n t . - 27 - For s u c c e s s f u l g r a f t s u s i n g t h i s t e c h n i q u e , t h e f o l l o w i n g p o i n t s a re v e r y i m p o r t a n t : Donors w e i g h i n g o v e r 300 gm a r e u n s u i t a b l e as t h e h e a r t i s t o o l a r g e , and e a s i l y f l o p s o v e r i n t h e neck c a u s i n g venous c o n g e s t i o n . T i s s u e s must be k e p t m o i s t and h a n d l e d g e n t l y . B lood l o s s must be a v o i d e d . The j u g u l a r v e i n and c a r o t i d a r t e r y s h o u l d be h a n d l e d by c o n t a c t w i t h b r a n c h e s or a d v e n t i t i a whe reve r p o s s i b l e . P o s i t i o n i n g o f t h e h e a r t must a v o i d p r e s s u r e by t h e a t r i a upon t h e p u l m o n a r y v e i n . The a t r i a must n o t be d i s t e n d e d a t t h e t i m e o f c l o s u r e o f t h e s k i n : t h i s i s a s i g n o f excess venous o c c l u s i o n . The r a t ' s nose s h o u l d be k e p t away f r o m sawdust d u r i n g r e c o v e r y , o t h e r w i s e i n h a l a t i o n o f sawdust and a s p h y x i a i s common. C. Review o f 464 Rat H e a r t A l l o g r a f t s The r e s u l t s o f t h e f i r s t 464 r a t h e a r t a l l o g r a f t s p e r f o r m e d by t h e a u t h o r were a n a l y z e d . 97.4% (452 g r a f t s ) were i n i t i a l l y s u c c e s s f u l , t h e g r a f t s b e a t i n g w e l l a t s i x h o u r s ( F i g u r e 6 ) . The 12 o p e r a t i v e f a i l u r e s were due t o s h r e d d i n g o f t h e c a r o t i d a r t e r y d u r i n g p r e p a r a t i o n o f t h e e v e r t e d c u f f (3 g r a f t s ) , heamor rhage f r o m t h e venous a n a s t o m o s i s (6 g r a f t s ) , u n c o r r e c t a b l e venous c o n g e s t i o n (2 g r a f t s ) , o r o m i s s i o n o f h e p a r i n i n p e r f u s i o n f l u i d ( 1 g r a f t ) . Venous t h r o m b o s i s o c c u r r e d i n 5.5% (26 g r a f t s ) and a r t e r i a l t h r o m b o s i s i n 3.5% (16 g r a f t s ) . The t o t a l m e c h a n i c a l f a i l u r e r a t e was 1116%. (a ) (b ) ( c ) (d) ( e ) ( f ) ( 9 ) - 28 - Pneumonia d e v e l o p e d i n 9.9% o f r e c i p i e n t s (46 c a s e s ) and t h i s a lways l e d t o d e a t h o f t h e r a t s . The t o t a l f a i l u r e r a t e ( o r e x c l u s i o n r a t e f r o m e x p e r i m e n t a l g r o u p s ) was 21.5%. Venous t h r o m b o s i s o c c u r r e d most o f t e n on day 1 o r 2 ( F i g u r e 3) w h i l e a r t e r i a l t h r o m b o s i s o c c u r r e d m a i n l y on days 1 , 4 and 5 ( F i g u r e 4 ) . Pneumonia was most common f r o m day 2 t o day 6 ( F i g u r e 5 ) , and was more l i k e l y when r e c i p i e n t s w e i g h i n g l e s s t h a n 200 gm were u s e d . These r e s u l t s compare f a v o u r a b l y w i t h t h o s e o f Heron who d e s c r i b e d a m e c h a n i c a l f a i l u r e r a t e o f 15% and a 10% i n c i d e n c e o f a r t e r i a l t h r o m b o s i s . The second s e r i e s ( T a b l e 1) shows t h e r e s u l t s o f t h e f i r s t e x p e r i m e n t a l model as opposed t o t h e o v e r a l l r e s u l t s . The o v e r a l l m o r t a l i t y was 26.6% i n t h e f i r s t s e r i e s . D. Rat H e a r t and Lung A l l o g r a f t s A t e c h n i q u e f o r h e t e r o t o p i c h e a r t and l u n g a l l o g r a f t t r a n s p l a n t a t i o n i n r a t s was d e v e l o p e d . I n t h e donor o p e r a t i o n , b o t h s u p e r i o r vena cavae were l i g a t e d and d i v i d e d , and t h e i n f e r i o r vena cava was c u t f l u s h w i t h t h e r i g h t a t r i u m . The a o r t a was d i v i d e d and t h e h e a r t and l u n g s p e r f u s e d w i t h h e p a r i n i z e d s a l i n e . The t r a c h e a was d i v i d e d h i g h i n t h e n e c k , and t h e h e a r t and l u n g s removed t o g e t h e r , and s t o r e d i n c o o l e d s a l i n e . - 29 - In t h e r e c i p i e n t o p e r a t i o n , c a r o t i d and j u g u l a r c u f f s were p r e p a r e d as i n t h e h e a r t a l l o g r a f t mode l . The g r a f t a o r t a was j o i n e d t o t h e r e c i p i e n t c a r o t i d a r t e r y , and t h e g r a f t r i g h t a t r i u m was t i e d o v e r t h e r e c i p i e n t j u g u l a r v e i n c u f f . When t h e v a s c u l a r c lamps were r e l e a s e d a r a p i d r e t u r n t o s i n u s r h y t h m o c c u r r e d , w i t h good p e r f u s i o n o f t h e h e a r t and l u n g s . The p a t t e r n o f c o n t r a c t i o n o f t h e h e a r t appeared t o be much more normal t h a n t h a t seen i n h e a r t a l l o g r a f t s , p r o b a b l y because o f m i n i m a l a t r i a l d i s t u r b a n c e . The t r a c h e a was b r o u g h t o u t t o open i n t h e c e n t r e o f t h e neck wound, and t h e l u n g s were p e r i o d i c a l l y i n f l a t e d u s i n g a s y r i n g e . A l t h o u g h t h e i n i t i a l r e s u l t s were g o o d , a l l 10 r e c i p i e n t s d i e d w i t h i n 76 hours due t o r e s p i r a t o r y d i s t r e s s : t h e r e was n o t enough room i n t h e neck f o r t h e h e a r t and two l u n g s t o l i e s a t i s f a c t o r i l y w i t h o u t c a u s i n g venous c o n g e s t i o n and t r a c h e a l c o m p r e s s i o n i n t h e r e c i p i e n t . T h i s t e c h n i q u e was t h e r e f o r e abandoned. - 30 - TECHNIQUE FOR B AND T CELL SEPARATION The B and T c e l l s were s e p a r a t e d u s i n g f i c o l l - i s o p a q u e d e n s i t y g r a d i e n t s as d e s c r i b e d by Loos and R o o s . 6 6 A. P r e p a r a t i o n o f n y l o n co lumn I t i s known t h a t B c e l l s adhere t o f i n e n y l o n s t r a n d s due t o t h e i r pseudopods , where T c e l l s do n o t . T h i s p h y s i o l o g i c a l d i f f e r e n c e between B and T c e l l s i s used t o s e p a r a t e a m i x t u r e o f B and T c e l l s and n u l l c e l l s . For o u r p u r p o s e s , 600 mg o f n y l o n wool i s u s e d . T h i s i s packed i n t o a 10 ml s y r i n g e and soaked w i t h medium w h i c h i s made up o f a phospha te b u f f e r e d s a l i n e and f e t a l c a l f s e r u m . The f o l l o w i n g i s t h e p r e p a r a t i o n f o r m u l a f o r t h i s m i x t u r e : sod ium c h l o r a t e 8 . 0 gm, sod ium hydrogen p h o s p h a t e 1.15 gm, p o t a s s i u m c h l o r i d e 0 . 2 gm, and p o t a s s i u m d i h y d r o g e n p h o s p h a t e 0 . 2 gm. T h i s i s mixed w i t h 1 l i t e r o f d o u b l e d i s t i l l e d w a t e r . Then 0 . 1 gm o f magnesium c h l o r i d e and 0 . 1 gm o f c a l c i u m c h l o r i d e h y d r o u s x 6 i s a d d e d , and t o t h i s m i x t u r e i s added 2 . 5 ml o f f e t a l c a l f serum t o each 100 ml o f PBS ( p h o s p h a t e b u f f e r e d s a l i n e ) . The medium f o r i n c u b a t i o n c o n t a i n s 10% o f f e t a l c a l f serum (FCS) . The medium i s a l l o w e d t o soak t h r o u g h t h e n y l o n w o o l , and i s i n c u b a t e d a t 37°C u n t i l t h e c e l l s have been p r e p a r e d . The e x t r a medium t h a t i s l e f t o v e r f r o m t h e i n c u b a t i o n i s k e p t a t 37°C as w e l l , as i s t h e PBS, FCS 2.5% m i x t u r e . - 31 - B. P r e p a r a t i o n o f lymph node o r s p l e e n c e l l s A s i m i l a r p r o c e d u r e i s done when e i t h e r lymph node o r s p l e e n c e l l s a re u s e d , i . e . t h e same s t e p s a r e c a r r i e d o u t . T e c h n i q u e d e s c r i b e d by E r e m i n , Plemb and Coombs, 1 9 7 6 . 3 3 The lymph nodes o r s p l e e n s a re d i s r u p t e d w i t h f i n e s c i s s o r s and s c a l p e l i n a m i x t u r e o f PBS FCS. T h i s i s p o u r e d i n t o u n i v e r s a l V o r t e x t u b e s t o l e t t h e l a r g e r lumps s e t t l e o u t f o r a p e r i o d o f two m i n u t e s . T h i s s u s p e n s i o n i s d e c a n t e d i n t o f r e s h u n i v e r s a l v e s s e l s . The m i x t u r e i s c e n t r i f u g e d a t 1600 r e v o l u t i o n s per m i n u t e ( rpm) f o r 10 m i n u t e s . The s u p e r n a t e i s d e c a n t e d . The s e d i m e n t i s mixed and f i l l e d a g a i n w i t h PBS FCS. The l a r g e r c lumps a r e a l l o w e d t o s e t t l e and d e c a n t e d a g a i n i n t o equa l t u b e s and f i l l e d t o 10 m l . Us ing a s y r i n g e and s low i n j e c t i o n , f i c o l l - i s o p a q u e i s i n j e c t e d s l o w l y u n t i l abou t 7 -10 ml i s f i l l e d . The c e l l s u s p e n s i o n t h e n r i s e s above t h i s much more dense medium, and t h i s a c t s as a g r a d i e n t , a l l o w i n g h e a v i e r s e d i m e n t d e b r i s t o pass t h r o u g h , bu t n o t a l l o w i n g l y m p h o c y t e s t o pass t h r o u g h , t h u s f o r m i n g an i n t e r p h a s e c o n t a i n i n g t h e l y m p h o c y t e s . The s e d i m e n t o f w h i t e c e l l s and e r y t h r o c y t e s t e n d s t o pass t h r o u g h and i s c a u g h t i n t h e v o r t e x o f t h e u n i v e r s a l . The s u s p e n s i o n i s mixed w i t h PBS FCS and c e n t r i f u g e d aga in a t 1800 rpm f o r 10 m i n u t e s t o wash o u t t h e e x c e s s f i c o l l . The s u p e r n a t e i s t h e n d e c a n t e d and f i l l e d agai-n w i t h PBS FCS, and c e n t r i f u g e d once a g a i n a t 1500 rpm f o r 5 m i n u t e s . The s u p e r n a t e i s a g a i n d e c a n t e d and m i x e d , and 5 ml o f medium w i t h 10% FCS i s m i x e d . The m i x t u r e i s t h e n c o u n t e d i n a Neubauer chamber w h i c h i s a c o u n t i n g chamber u s i n g g r a d a t i o n s , and a l l o w s c o u n t i n g o f t h e number o f c e l l s i n a s p e c i f i c - 32 - vo lume. F i n a l l y , t h e m i x t u r e i s p u t i n t o an E p p e n d o r f t u b e so t h a t t h e c e l l c o n c e n t r a t i o n i s be tween 50 and 100 m i l l i o n , i n 2 ml o f medium p l u s FCS 10%. T h i s i s s u i t a b l e f o r IgG l a b e l l i n g . Now t h a t a l y m p h o c y t e m i x t u r e has been o b t a i n e d , s e p a r a t i o n between t h e T and B c e l l t y p e s i s r e q u i r e d C. S e p a r a t i o n o f t h e B and T C e l l s ( A f t e r G. B r o n s , Ph .D . T h e s i s i n p r o g r e s s , U n i v e r s i t y o f Cambr idge , U .K . ) The n y l o n co lumn i s washed w i t h 5 ml o f warm medium p l u s 10% FCS. The wool m i x t u r e i s compressed s l i g h t l y w i t h t h e p l u n g e r o f t h e s y r i n g e t o pack t h e wool so a l l g r o s s f l u i d i s e x p e l l e d , t h u s a l l o w i n g maximum exposu re t o t h e s u r f a c e o f t h e n y l o n w o o l . The t i p o f t h e s y r i n g e i s p l u g g e d , and d r o p w i s e , 2 ml o f c e l l s u s p e n s i o n i s a l l o w e d t o f a l l i n t o t h e m i d d l e o f t h e n y l o n wool s y r i n g e , and t h e c e l l s a re a l l o w e d t o p e r c o l a t e t h r o u g h t h e n y l o n wool m i x t u r e . One ml o f medium i s added t o t h e t u b e , and t h e r e m a i n i n g c e l l s a r e m i x e d . A f u r t h e r 1 ml i s added t o t h e t o p o f t h e n y l o n w o o l . T h i s m i x t u r e o f c e l l s and medium i s a l l o w e d t o i n c u b a t e f o r one hour a t 37°C. The t o p o f t h e t u b e i s s e a l e d w i t h p a r a f i l m f o r a i r t i g h t n e s s and t o p r e v e n t c o n t a m i n a t i o n by b a c t e r i a . The column i s t h e n opened a t t h e b o t t o m over t h e u n i v e r s a l t u b e . Warm (37°C) PBS FCS i s p o u r e d t h r o u g h t h e co lumn i n t o t h e U n i v e r s a l . A t o t a l o f 25 ml i s r e q u i r e d , so t h a t a i l nonadhered c e l l s a r e washed t h r o u g h . The nonadhered c e l l s a re t h e T c e l l s . One hundred ( 1 0 0 ) ml o f warm PBS FCS m i x t u r e i s p e r c o l a t e d t h r o u g h t h e n y l o n wool and d i s c a r d e d . T h i s removes t h e n u l l c e l l s , i . e . t h e non B, non T c e l l s . - 33 - Ten ( 1 0 ) ml o f warm PBS and FCS 2.5% i s added t o t h e s y r i n g e , and t h e p l u n g e r i s a p p l i e d t o t h e r e m a i n i n g n y l o n w o o l , and t h e n y l o n wool i s compressed w i t h m o d e r a t e f o r c e t o e x p e l t h e a d h e r e n t c e l l s . These a re t h e B c e l l s . T h i s p r o c e d u r e i s done t w i c e t o remove a l l t h e B c e l l s . The c e l l s , once l a b e l l e d , a r e c e n t r i f u g e d a t 1600 RPM f o r 10 m i n u t e s . The s u p e r n a t e i s d i s c a r d e d , t h e s e d i m e n t i s aga in m i x e d , and added t o 5 ml o f PBS FCS. The c e l l s a re t h e n c o u n t e d , and t h e c o n c e n t r a t i o n s a d j u s t e d t o 50 m i l l i o n c e l l s i n 1/2 m l . These c e l l s a r e used f o r i m m u n i z a t i o n s , w h i l e some a r e k e p t f o r T and B c e l l f l u o r e s c e n t a s s a y s . Note f l u o r e s c e n t assays i n F i g u r e 1 2 ; summary o f p r o c e d u r e , F i g u r e 9 . STAINING-OF CELLS C e l l s a re s t a i n e d t o d e m o n s t r a t e t h a t we w e r e , i n d e e d , s e p a r a t i n g t h e B f r o m T c e l l s , and t h a t we were p r o d u c i n g a low c o n t a m i n a t i o n r a t e o f N u l l c e l l s (See F i g u r e s 1 0 , 1 1 and e x p l a n a t i o n b e l o w ) . The c e l l s a re s t a i n e d by two methods : 1) A p e r o x i d a s e s t a i n and 2) A f l u o r e s c e i n s t a i n , b o t h d i r e c t and i n d i r e c t m e t h o d . P e r o x i d a s e S t a i n ( a f t e r H e y d e r m a n 5 2 ) D i r e c t Method C e l l s were f i x e d on t h e s l i d e w i t h m e t h a n o l f o r 10 m i n u t e s . To e l i m i n a t e endogenous p e r o x i d a s e , t h e c e l l s were washed i n a 2 : 1 s o l u t i o n o f me thano l and hydrogen p e r o x i d e f o r 10 m i n u t e s t h e n c l e a r e d w i t h a s a l i n e wash . The - 34 - cel ls are then stained with ant i -rat IgG peroxidase diluted 1:10 for 30 minutes at room temperature (Miles Peroxidase Anti-Rat IgG No. 61-206, specific for rat B cel l surface antigen). The sl ide is washed with saline three times after counter staining with Carrazi s ta in . Fluorescein Stain Direct Method The same process was used as for the peroxidase stain except for the peroxidase wash. Miles FITC Anti-Rat IgG from the Rabbit No. 65-174 (specific for rat B ce l l surface antigen) was used for staining. Indirect Method The B cel ls were fixed on the sl ide with ethanol for 10 minutes, then washed with 2:1 methanol:water solution. The ce l l s were incubated for 30 minutes at room temperature with Anti -rat IgG raised in rabbit used as the intermediate (Miles No. 65-160). Anti-rabbit FITC (fluorescein labeled) raised in the sheep was used as the antibody staining (Miles No. 65-173). - 35 - PREPARATION OF FETAL CALF SERUM ABSORBED WITH SHEEP RED BLOOD CELLS F i v e cc o f s h e e p ' s b l o o d f r e s h l y t a k e n i s spun 250 rpm f o r e i g h t m i n u t e s . The s u p e r n a t e and b u f f y c o a t a re b o t h p i p e t t e d o f f . PBS i s added and m i x e d . T h i s m i x t u r e i s a g a i n c e n t r i f u g e d a t 250 rpm f o r e i g h t m i n u t e s . T h i s p r o c e s s i s r e p e a t e d once more . The t u b e o f f e t a l c a l f serum i s h e a t e d and a c t i v a t e d a t 56°C f o r 30 m i n u t e s . The sheep r e d b l o o d c e l l s a r e t h e n mixed w i t h t h e f e t a l c a l f serum and mixed a g a i n and i n c u b a t e d a t 37°C f o r 30 m i n u t e s , t h e n p l a c e d a t 4°C o v e r n i g h t . Once done t h e m i x t u r e i s c e n t r i f u g e d a t 3000 ^mp f o r 15 m i n u t e s . The s u p e r n a t e i s t h e a b s o r b e d f e t a l c a l f serum and i s p l a c e d i n 2 ml a l i q u o t s f o r f u t u r e use ( 0 . 2 gm o f KC1, 0 . 2 gm K - o r t h o p h o s p h a t e KH2PO4). T h i s i s added t o f u l f i l a vo lume o f 1 l i t r e o f d o u b l e d i s t i l l e d w a t e r . I t i s mixed f o r f i v e m i n u t e s . Under a l a m i n a r f l o w sys tem 0 . 1 mg o f magnesium c h l o r i d e and 0 . 1 mg o f a n h y d r o u s c a l c i u m c h l o r i d e are a d d e d . t o t h i s l i t r e . T h i s i s k e p t a t 4°C and i s changed once a week. STATISTICAL ANALYSIS OF HEART TRANSPLANTS IN RATS S t a t i s t i c a l a n a l y s i s was p e r f o r m e d by M i c h a e l B u t t s , East A n g l i a Cambridge M e d i c a l H e a l t h D i s t r i c t . The mean s u r v i v a l o f t h e PVG h e a r t i n t o a DA r e c i p i e n t was 6 . 8 d a y s . The mean s u r v i v a l o f a (DVG x DA) F^ ' -hear t t r a n s p l a n t i n t o a DA r e c i p i e n t was 8 . 6 days . These two g r o u p s a r e s i g n i f i c a n t l y d i f f e r e n t p < 0 . 0 5 . T h i s d i f f e r e n c e i s e x p e c t e d as F i g r a f t s have h a l f t h e g e n e t i c d i s p a r i t y and t h e r e f o r e t a k e s l i g h t l y l o n g e r t o r e j e c t ( T a b l e 2 ) . - 36 - DA r a t s immunized w i t h n o n - h e a t e d T c e l l s d e r i v e d f r o m PVG r a t s p roduced no d i f f e r e n c e i n g r a f t s u r v i v a l f r o m PVG c o n t r o l s a f t e r h e a r t s a r e t r a n s p l a n t e d . I f (PVG x DA) F]_, T c e l l s a re u s e d , t h e mean i s t h e same d e s p i t e t h e g r e a t e r mean i n t h e F]_ c o n t r o l g r o u p s . D e s p i t e t h i s d i f f e r e n c e t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between t h e two g r o u p s ( T a b l e 3 ) . When B c e l l s a re s u b s t i t u t e d f o r T c e l l s t h e r e i s a s i g n f i c a n t d i f f e r e n c e between n o n - h e a t e d B c e l l s and c o n t r o l s . T h i s d i f f e r e n c e i s m a i n t a i n e d d e s p i t e t h e use o f PVG B c e l l s o r F i B c e l l s ( T a b l e 3 ) . When T c e l l s h e a t e d t o 56"C were used t o immunize t h e r e c i p i e n t t h e mean g r a f t s u r v i v a l f o r t h e PVG T c e l l g roup was 5 . 9 days and f o r F i T c e l l s was 6 . 4 d a y s , a g a i n n o t d i f f e r e n t f r o m c o n t r o l s ( T a b l e 4 ) . I f B c e l l s h e a t e d t o 56°C were s u b s t i t u t e d f o r t h e T c e l l s i m m u n i z a t i o n s t h e mean s u r v i v a l s f o r PVG B c e l l s was 1 3 . 8 days and f o r F]_ B c e l l s was 9 . 8 d a y s . There i s a s i g n i f i c a n t d i f f e r e n c e between t h e PVG g r o u p s b u t n o t t h e F]_ g roup ( T a b l e 4 ) . Compar ing t h e n o n - h e a t e d B c e l l s t o t h e 56°C h e a t e d B c e l l immunized g r o u p , t h e r e i s no d i f f e r e n c e between t h e s e g r o u p s . When PVG o r F]_ T c e l l s were h e a t e d t o 85°C and immunized i n t o DA r e c i p i e n t s no d i f f e r e n c e was o b s e r v e d . When PVG B c e l l s were s u b s t i t u t e d f o r T c e l l s t h e mean s u r v i v a l o f t h e g r a f t was 1 9 . 7 d a y s , s i g n i f i c a n t l y l o n g e r t h a n n o n t r e a t e d h e a r t r e c i p i e n t s . Compar ing t h i s w i t h t h e n o n - h e a t e d B c e l l s t h e r e i s no d i f f e r e n c e i n s u r v i v a l b u t t h e r e i s a d i f f e r e n c e when 85°C h e a t e d B c e l l s a re compared t o t h e 56°C h e a t e d B c e l l s ( T a b l e s 3 , 4 , 5 ) . - 37 - When p l a t e l e t s were p u r i f i e d and immunized p r i o r t o h e a r t t r a n s p l a n t , no s i g n i f i c a n t p r o l o n g a t i o n was o b t a i n e d . Because o f t h e c o m p l e x i t y o f t h e g roups and t h e number o f v a r i a b l e s b e i n g compared , a m u l t i v a r i e d a n a l y s i s was c a r r i e d o u t . The v a r i a b l e s were : 12 e x p e r i m e n t a l g r o u p s , each w i t h a c e r t a i n number o f members. V a r i a b l e s i n c l u d e d PVG c e l l s , Fi_ c e l l s . Of t h e s e s p e c i f i c g roups o f c e l l s , t h e y c o u l d e i t h e r be T c e l l s o r B c e l l s , and a f u r t h e r sub g roup c o u l d be u n h e a t e d , hea ted t o 56°C , o r h e a t e d t o 8 5 ° C . Noted i n an a n a l y s i s o f one v a r i a b l e , a l l main e f f e c t s s i g n i f i c a n t l y a f f e c t g r a f t s u r v i v a l . I n a d d i t i o n , t h e t y p e o f r a t and t h e t y p e o f c e l l a f f e c t s s u r v i v a l . I f a s t a r i s n o t e d by t h e f i g u r e , t h i s i s s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . I f two s t a r s a r e n o t e d by t h e number, i t i s w i t h i n c o n f i d e n c e l i m i t s ( p < 0 . 0 1 ) . An i m p o r t a n t o b s e r v a t i o n n o t e d i n T a b l e 5 i s t h a t l o n g t e r m s u r v i v a l s were o b t a i n e d by i m m u n i z a t i o n w i t h 85°C hea ted PVG B c e l l s . I t appears t o show t h a t some o f t h e t r a n s p l a n t s a r e i n f a c t i n f l u e n c e d d i f f e r e n t l y by t h e i m m u n i z a t i o n t h u s p r o d u c i n g a subgroup w h i c h canno t be compared as a w h o l e . I f t h i s i s t r u e t h e n a s s i g n i n g Rank Sum c o m p a r i s i o n s t a t i s t i c a l methods does no t a p p l y . - 38 - STATISTICS NUMBER OF VARIABLE 1 NUMBER OF CASES 9999 NUMBER OF LEVELS IN A 2 NUMBER OF LEVELS IN B 2 NUMBER OF LEVELS IN C .3 NUMBER OF FORMAT CARDS 1 , DATA INPUT MODE 0 0 = CARDS MISSING DATA MODE 0 0 =' NONE / 1 = FILE / 1 = MISSING DATA 9999 CASES IMPLIES THAT UNEQUAL GROUPS ARE INPUT DATA FORMAT IS ( I O . F O . O ) GROUP 1 ( 9 MEMBERS) PVG, T , UNHEATED GROUP 2 ( 7 MEMBERS) PVG, T , 56 GROUP 3 ( 8 MEMBERS) PVG, T, 85 GROUP 4 (12 MEMBERS) PVG, B, UNHEATED GROUP 5 ( 6 MEMBERS) PVG, B, 56 GROUP 6 ( 8 MEMBERS) PVG, B, 85 GROUP 7 ( 9 MEMBERS) F I , T , UNHEATED GROUP 8 ( 8 MEMBERS) F I , T, 56 GROUP 9 ( 8 MEMBERS) F I , T, 85 GROUP 10 (10 MEMBERS) F I . , B, UNHEATED GROUP 11 (10 MEMBERS) F I , . B > 56 GROUP 12 ( 6 MEMBERS) F I , B, 85 - 39 - ANALYSIS OF VARIANCE SOURCE M.S. TOTAL 34 .589 BETWEEN 2 1 5 . 8 5 2 A = PVG/F1 103 .232 B = T/B 7 7 9 . 8 4 0 C = UNHEATED/56/85 122 .377 AB 119 .957 AC 1.453 BC 5 9 . 5 6 0 ABC 2 . 2 8 1 WITHIN 1 3 . 3 0 9 MEANS FOR ALL EFFECTS O.F F-RATIO PROBABILITY 100. 1 1 . 1 . 7 .7565 0 . 0 0 6 6 * * 1 . 1 3 3 . 7 3 1 1 0 . 0 0 0 0 * * 2 . 9 . 1 9 5 0 0 . 0 0 0 4 * * 1 . 9 .0132 0 . 0 0 3 8 * * 2 . 0 . 1 0 9 1 0 .8964 2. 4 . 4 7 5 2 0 . 0 1 3 9 * 2 . 0 . 1 7 1 4 0 .8438 8 9 . - 40 - ************************************** A MN 1 (PVG) 2 ( F I ) 12 .0817 10 .0204 SPECIFIC COMPARISONS (TUKEY METHOD) FOR ALL CELLS CELL A 1 A 2 4 . 0 2 * * *********************************************************************** B MN 1 (T ) 2 (B) 6 .7715 15 .3306 SPECIFIC COMPARISONS (TUKEY METHOD) FOR ALL CELLS CELL B 1 B 2 - 1 6 . 6 7 * * *********************************************************************** C MN 1 (UNHEATED) 2 ( 5 6 ° ) 3 ( 8 5 ° ) 1 1 . 1 9 8 6 9 . 0 3 7 8 12 .9167 SPECIFIC COMPARISONS (TUKEY METHOD) FOR ALL CELLS CELL C I C 2 3 . 4 1 * C 3 - 2 . 7 1 CELL C 2 C 2 - 6 . 1 2 * * *********************************************************************** A.B 1 (T ) 2 (B) 1 6 . 6 9 1 1 17 .4722 2 6 .8519 1 3 . 1 8 8 9 SPECIFIC COMPARISONS (TUKEY METHOD) FOR ALL CELLS C e l l A 1 B 1 B I B2 A 1 - 1 4 . 8 4 * * A 2 - 0 . 2 2 - 8 . 9 5 * * CELL A 2 B 1 B I B2 A2 - 8 . 9 5 * * CELL A 1 B 2 B I B2 A 2 1 4 . 6 2 * * 5 . 9 0 * * *********************************************************************** - 4 1 - PURPOSE OF STUDY I n t h e f o l l o w i n g e x p e r i m e n t s , t h e a s s u m p t i o n i s made t h a t enhancement i s s i m p l e , by e x a m i n i n g some supposed b e l i e f s . P r e v i o u s w o r k , n o t e d i n t h e i n t r o d u c t i o n , showed t h a t c l a s s I I ( l a ) a n t i b o d i e s can m e d i a t e p a s s i v e enhancement , and s u b s e q u e n t l y t h a t a c t i v e enhancement i s m e d i a t e d by p r e i m m u n i z a t i o n w i t h B c e l l s . We w i s h t o show t h a t t r u e enhancement c a n . b e d i s t i n g u i s h e d f r o m p r o l o n g a t i o n due t o a n t i - i d i o t y p i c a n t i b o d i e s . We a l s o w i s h t o f i n d t h e r o l e o f c l a s s I a l l o - a n t i b o d i e s w h i c h may h a s t e n r e j e c t i o n , and t h e r o l e o f c l a s s I I a n t i b o d i e s wh ich i s l i k e l y c o n f i n e d t o p r o l o n g a t i o n o f g r a f t s u r v i v a l . EXPERIMENT 1 Are T c e l l s a b l e t o p r o v i d e t h e immunogen f o r enhancement? From t h e p r e v i o u s l y d e s c r i b e d work o f L a u c h a r t e t a l , 6 0 , 6 1 t n e y may^ a f t e r s e v e r a l i m m u n i z a t i o n s w i t h m ixed c e l l s , bu t do T c e l l s d e m o n s t r a t e t h e same e f f e c t a f t e r a s i n g l e i m m u n i z a t i o n ? B c e l l s and T c e l l s have i d e n t i f i a b l e s u r f a c e a n t i g e n s , and t h e r e f o r e can be s e p a r a t e d o u t so t h a t i m m u n i z a t i o n can be c a r r i e d ou t u s i n g p u r e T c e l l s o r p u r e B c e l l s . By p r e i m m u n i z i n g a r e c i p i e n t seven days p r i o r t o e n g r a f t m e n t o f a F i h e a r t , one c o u l d o b s e r v e t h e e f f e c t o f e i t h e r p u r e T c e l l o r p u r e B c e l l p r e t r a n s p l a n t i m m u n i z a t i o n . PVG donor c e l l s were o b t a i n e d b y : D i s s e c t i n g t h e lymph n o d e s , wh ich a re r i c h i n T c e l l s , and o b t a i n i n g s e p a r a t e d T c e l l s by t h e method d e s c r i b e d i n C e l l S e p a r a t i o n T e c h n i q u e - 42 - ( F i g u r e 9 ) . The c e l l s , t h u s s e p a r a t e d , were i n j e c t e d i n t r a v e n o u s l y i n t o DA r a t s seven days p r i o r t o e n g r a f t m e n t . The r e c i p i e n t a n i m a l s we ighed between 125 and 250 gms. A t o t a l o f 1 x 1 0 7 c e l l s were i n j e c t e d seven days p r i o r t o e n g r a f t m e n t . The a n i m a l s were k e p t i n i n d i v i d u a l cages and o b s e r v e d f o r t h e seven days t o be c e r t a i n t h a t t h e y d i d n o t d e v e l o p any s e p s i s o r o t h e r i l l e f f e c t s s e c o n d a r y t o t h e i m m u n i z a t i o n . On Day 7 , a (PVG x DA) F]_ h e a r t was h a r v e s t e d by t h e method e n t i t l e d , "Donor O p e r a t i o n " . T h i s h e a r t was t h e n t r a n s p l a n t e d i n t o t h e r e c i p i e n t i n a h e t e r o t o p i c l o c a t i o n , t h u s n o t s u p p o r t i n g c i r c u l a t i o n . (See Method e n t i t l e d , " R e c i p i e n t O p e r a t i o n " ) These a n i m a l s were o b s e r v e d d a i l y u n t i l t h e donor h e a r t r e j e c t i o n was n o t e d t o be t h e t i m e a t wh ich t h e h e a r t b e a t c e a s e d . The an ima l s u r v i v e s a f t e r t h e d o n o r h e a r t i s r e j e c t e d , and t h e h e a r t g e n e r a l l y i n v o l u t e s . Note F i g u r e 7, A c u t e A l l o g r a f t R e j e c t i o n . The r e j e c t e d h e a r t was n o t removed , as t h e an imal s u r v i v e d w i t h t h e r e j e c t e d h e a r t i n p l a c e , and a f t e r a p e r i o d o f t i m e , t h e h e a r t f i b r o s e d down t o a s c a r r e d remnant w h i c h d i d n o t a f f e c t t h e a n i m a l ' s o v e r a l l w e l l b e i n g ( F i g u r e 8 ) . The r e s u l t s o f t h i s e x p e r i m e n t , u t i l i z i n g T c e l l p r e i m m u n i z a t i o n , a r e p r e s e n t e d ( T a b l e 3 ; Graphs 1 , 2 , 3 ) . The o v e r a l l mean s u r v i v a l o f t h e s e a l l o g r a f t s was 6 . 5 d a y s , wh ich i s s h o r t e r t h a n t h e e x p e c t e d r a t e o f s u r v i v a l i n t h e c o n t r o l (PVG x DA) F\ by DA w h i c h had a mean s u r v i v a l o f 8 . 6 days ( T a b l e 2 ; Graph 4 , 5 ) . An a n a l o g o u s e x p e r i m e n t was c a r r i e d o u t u s i n g B c e l l s i n s t e a d o f T c e l l s u s i n g t h e method j u s t d e s c r i b e d . I n s t e a d o f d i s s e c t i n g ou t lymph n o d e s , wh ich a r e r i c h i n T c e l l s , PVG s p l e e n s were r e m o v e d , and t h e s e c e l l s were s e p a r a t e d by t h e method o f L o o s , 6 5 t h u s o b t a i n i n g - 43 - a p p r o x i m a t e l y 1 x 1 0 7 p u r i f i e d B c e l l s , and i m m u n i z i n g them i n t o t h e DA r e c i p i e n t seven days p r i o r t o e n g r a f t m e n t w i t h an F]_ h e a r t . T h i s t i m e , t h e e n g r a f t e d h e a r t s s u r v i v e d a mean o f 1 7 . 6 days ( T a b l e 3 - # 2 ) . EXPERIMENT 2 I s . a n t i - i d i o t y p e i n v o l v e d i n t h i s phenomenon? As m e n t i o n e d i n t h e i n t r o d u c t i o n , enhancement i s l i k e l y d i f f e r e n t f r o m a n t i - i d i o t y p e p r o l o n g a t i o n . I n o r d e r t o d e m o n s t r a t e t h a t a n t i - i d i o t y p e a n t i b o d y p r o d u c t i o n causes t h i s o b s e r v e d phenomenon i n E x p e r i m e n t 1 , an a n a l o g o u s e x p e r i m e n t was c a r r i e d o u t , u t i l i z i n g no t PVG B c e l l s o r T c e l l s , b u t (PVG x DA) .Fi_ T c e l l s o r B c e l l s , and i n j e c t i n g them i n t o DA r e c i p i e n t s , and t h e n e n g r a f t i n g an F^ h e a r t . I f a n t i - i d i o t y p e were t h e s o l e mechanism r e s p o n s i b l e f o r enhancement one wou ld e x p e c t t h a t B c e l l s and T c e l l s wou ld p r o d u c e a s i m i l a r r e s p o n s e . I f a n t i - i d i o t y p e a n t i b o d y was dose dependen t ( i . e . F\ t r a n s p l a n t s s u r v i v e l o n g e r ) PVG B o r T c e l l s s h o u l d p r o d u c e a s h o r t e r p r o l o n g a t i o n t h a n F^ B o r T c e l l s . The r e s u l t s o f t h e e n g r a f t m e n t o f F]_ h e a r t s i n t o DA a n i m a l s p r e v i o u s l y immunized by (PVG x DA) F]_ T c e l l s one week p r i o r t o e n g r a f t m e n t , a r e shown on T a b l e 3 - #3 (Graph 4 ) . The mean s u r v i v a l was 6 . 5 d a y s , s i m i l a r t o t h o s e p r e v i o u s l y immunized w i t h PVG T c e l l s ( L i n e 1 compared t o L i n e 3 - T a b l e 3 ) . When (PVG x DA) Fi_ B c e l l s were immunized one week p r i o r t o e n g r a f t m e n t i n a DA an imal w i t h a (PVG x DA) Fj_ h e a r t , t h e mean s u r v i v a l was 1 4 . 1 d a y s . T h i s i s s i g n i f i c a n t and d i f f e r e n t f r o m L i n e 2 - T a b l e 3 (Graph 5 ) . - 44 - EXPERIMENT 3 C h a r a c t e r i z a t i o n o f t h e immunogen. I s i t a p r o t e i n o r a c a r b o h y d r a t e ? Immunogens a r e n o t e d t o be p r o t e i n s or m u c o p o l y s a c c h a r i d e s . The • c a r b o h y d r a t e p o r t i o n o f m u c o p o l y s a c c h a r i d e s i s a l s o known t o be immunogen ic . A c l a s s i c example o f t h i s i s t h e c a p s u l e o f c e r t a i n v i r u s e s and b a c t e r i a . C a r b o h y d r a t e c a p s u l e s do p r o d u c e a n t i b o d y r e s p o n s e s , and t h e r e f o r e a re immunogen ic . I t i s n o t known what t h e immunogen i s i n e n h a n c e m e n t . 6 2 , 9 4 , 1 0 0 Hav ing d e m o n s t r a t e d t h a t B and T c e l l s have d i f f e r e n t i m m u n i z a t i o n p o t e n t i a l and t h a t B c e l l s d o , i n f a c t , p r o l o n g g r a f t s u r v i v a l by a mechanism o f n o n - i d i o t y p e a c t i v e enhancement , i t i s i m p o r t a n t t o c h a r a c t e r i z e t h e i n i t i a t o r o f t h i s r e s p o n s e . The c e l l membrane i s known t o be immunogen ic , and c o n t a i n s a n t i g e n s . " The c e l l w a l l i s made up o f b o t h p r o t e i n and c a r b o h y d r a t e m o i e t i e s . The i n t e r n a l s t r u c t u r e s o f t h e B o r T c e l l c o n t a i n t h e p r o d u c t s o f n u c l e a r DNA and RNA p r o d u c t i o n and a re p r e d o m i n a n t l y p r o t e i n s . 8 5 To answer t h e q u e s t i o n w h e t h e r o r n o t t h e immunogen i s p r o t e i n o r c a r b o h y d r a t e , i t i s i m p o r t a n t t o d e t e r m i n e w h e t h e r t h e i n t a c t c e l l i s n e c e s s a r y t o p r o d u c e t h e same e f f e c t , o r w h e t h e r d e n a t u r e d c e l l s a l s o i n d u c e g r a f t enhancement by a c t i v e i m m u n i z a t i o n . C e l l s have t h e i r membrane p r o t e i n s d e n a t u r e d by i n c u b a t i o n a t 56°C f o r h a l f an h o u r . T h i s i n c u b a t i o n does n o t d i s r u p t t h e c e l l membrane, b u t t h e p r e s e n t a t i o n (HLA p r o d u c t ) p r o t e i n s a re d e n a t u r e d . C a r b o h y d r a t e s a r e n o t a l t e r e d on t h e membrane a f t e r t h i s i n c u b a t i o n , n o r a re i n t e r i o r p r o t e i n s . r - 45 - EXPERIMENT 3-1 (PVG x DA) F]_ h e a r t s were t r a n s p l a n t e d i n t o DA r e c i p i e n t s a f t e r h a v i n g t h e r e c i p i e n t immunized w i t h PVG T c e l l s s e p a r a t e d and i n c u b a t e d a t 56°C f o r 30 m i n u t e s . The r e s u l t s show a mean s u r v i v a l o f 5 . 9 days ( T a b l e 4 - L i n e 1 ; Graph 7 ) . T h i s r e s u l t i s s i m i l a r t o g r a f t s u s i n g m ixed c e l l s ( T a b l e 6 ) , and s h o r t e r t h a n PVG T c e l l s wh ich a re n o n - h e a t e d . T h i s d i f f e r e n c e i s n o t s t a t i s t i c a l l y s i g n i f i c a n t . EXPERIMENT 3-2 An ana logous p r o c e d u r e was c a r r i e d o u t , u t i l i z i n g (PVG x DA) Fi_ h e a r t s e n g r a f t e d i n t o DA r e c i p i e n t s , f o l l o w i n g PVG B c e l l i m m u n i z a t i o n . T h i s t i m e , t h e B c e l l s were i n c u b a t e d a t 56°C f o r 30 m i n u t e s . The mean s u r v i v a l f o l l o w i n g t h i s i m m u n i z a t i o n was 1 3 . 8 d a y s . T h i s i s l o n g e r t h a n e x p e c t e d , b u t no t as l o n g as t h e u n h e a t e d B c e l l s (Graph 6 ) . EXPERIMENT 3-3 (PVG x DA) F]_ h e a r t s were t r a n s p l a n t e d i n t o DA r e c i p i e n t s . T h i s t i m e t h e r e c i p i e n t was p r e v i o u s l y immunized seven days p r i o r t o e n g r a f t m e n t w i t h F]_ T c e l l s i n c u b a t e d a t 56°C f o r 30 m i n u t e s . The mean s u r v i v a l f o l l o w i n g t h i s e x p e r i m e n t i s 6 . 3 days (Graph 7 ) . T h i s i s no t s i g n i f i c a n t l y d i f f e r e n t f r o m n o n - h e a t e d T - c e l l s . - 46 - EXPERIMENT 3-4 (PVG x DA) ?i h e a r t s were e n g r a f t e d i n t o DA r e c i p i e n t s p r e v i o u s l y immunized w i t h F]_ B c e l l s , i n c u b a t e d a t 56°C f o r 30 m i n u t e s . The mean s u r v i v a l f o l l o w i n g t h i s i m m u n i z a t i o n was 9 . 8 d a y s , as opposed t o 1 4 . 1 days i n t h e nonhea ted B c e l l i m m u n i z a t i o n e x p e r i m e n t s ( T a b l e 4 - L i n e 4 ; Graph 8 ) . EXPERIMENT 4 D e n a t u r e d C e l l I m m u n i z a t i o n C e l l s a r e d e n a t u r e d by i n c u b a t i n g them f o r t e n m i n u t e s a t 85°C ( T a b l e 5 ) . P r o t e i n s w o u l d be d e n a t u r e d and c a r b o h y d r a t e s wou ld s t i l l have t h e i r b i o c h e m i c a l i n t e g r i t y m a i n t a i n e d . The f o l l o w i n g e x p e r i m e n t s were pe r fo rmed t o o b s e r v e t h e e f f e c t o f i m m u n i z a t i o n w i t h c e l l s h a v i n g had t h e i r p r o t e i n s d e n a t u r e d and t h e c a r b o h y d r a t e m o i e t y exposed i n t h e a n a l a g o u s s i t u a t i o n as d e s c r i b e d i n E x p e r i m e n t s 1 , 2 , and 3 . 2 3 , 8 7 T n e c e n s a r e n o t d i s r u p t e d by t h i s p r o c e s s ( F i g u r e 1 3 ) . C e l l s were h e a t e d a t 85° f o r 30 m i n u t e s . The c e l l s t h e m s e l v e s m a i n t a i n e d t h e i r shape b u t became c r e n a t e d a f t e r t h e i n t e r n a l p r o t e i n s were d e n a t u r e d . EXPERIMENT 4 - 1 F]_ h e a r t s were t r a n s p l a n t e d i n t o DA r e c i p i e n t s a f t e r b e i n g p r e v i o u s l y immunized one week p r i o r t o t r a n s p l a n t w i t h PVG T c e l l s i n c u b a t e d a t 85°C f o r 10 m i n u t e s . The r e s u l t o f t h i s e x p e r i m e n t show a mean s u r v i v a l o f 7 .4 d a y s . T h i s i s l o n g e r t h a n t h e nonhea ted and 56°C immunized c e l l s ( T a b l e 5 - L i n e 1 ) . T h i s d i f f e r e n c e i s n o t s i g n i f i c a n t . - 47 - EXPERIMENT 4-2 hearts were transplanted into DA recipients. On this occasion, PVG B cel ls incubated at 85°C for 10 minutes were used as the immunizing agent. The mean graft survival was 19.7 days. This was not s ignif icantly longer than unheated controls or B cel ls heated to 56°C. These groups may not be comparable, however, as a subgroup demonstrating long term survival seems to exist thus rendering comparison invalid (Table 5 - Line 2). EXPERIMENT 4-3 Fj hearts, were transplanted into DA immunized recipients, T ce l l s having been incubated to 85° for 10 minutes. The mean survival was 8.1 days (Table 5 - Line 3). This result was not s ignif icantly different from controls. EXPERIMENT 4-4 Fj donor hearts were transplanted into DA recipients after being previously immunized seven days prior to transplant by Fj B ce l l s incubated at 85°C for 10 minutes. The mean survival was 15.7 days. This approaches but does not reach s ta t i s t i ca l difference from 56°C heated cel l immunizations. These results are summarized on Table 5 (Line 4). - 48 - EXPERIMENT 5 Mixed C e l l I m m u n i z a t i o n To d e t e r m i n e w h e t h e r an e f f e c t o t h e r t h a n t h e s e p a r a t i o n o f t h e B and T c e l l s i s a c t i v e i n t h e E x p e r i m e n t s 1 -4 , c e l l s were s e p a r a t e d by t h e t e c h n i q u e s m e n t i o n e d i n C e l l S e p a r a t i o n T e c h n i q u e and t h e n mixed t o g e t h e r and i n j e c t e d as a mixed B and T c e l l i m m u n i z a t i o n . T h i s e x p e r i m e n t a c t s as a c o n t r o l f o r t h e b i o c h e m i c a l and p h y s i c a l s e p a r a t i o n t e c h n i q u e . F]_ h e a r t s were t r a n s p l a n t e d i n t o DA r e c i p i e n t s f o l l o w i n g t h e r e c i p i e n t b e i n g immunized seven days p r i o r t o t r a n s p l a n t by mixed T and B c e l l s h a v i n g p r e v i o u s l y been s e p a r a t e d . The mean s u r v i v a l f o l l o w i n g t h e s e t r a n s p l a n t s were 6 . 8 days f o r ¥ \ mixed c e l l i m m u n i z a t i o n s and 7 . 3 days f o r t h e PVG mixed c e l l i m m u n i z a t i o n s ( T a b l e 6 ) . There a r e no s i g n i f i c a n t d i f f e r e n c e s f r o m c o n t r o l s . EXPERIMENT 6 P l a t e l e t I m m u n i z a t i o n s I t i s known t h a t c l a s s I I a n t i g e n i s n o t p r e s e n t on p l a t e l e t s and t h e r e f o r e be a s i m i l a r immunogen as p u r i f i e d r e d b l o o d c e l l s wh ich do n o t c a r r y c l a s s I I a n t i g e n e i t n e r . ^ I f e x p o s u r e o f c l a s s I a n t i g e n w i t h o u t exposure t o c l a s s I I a n t i g e n c o u l d i n d u c e enhancement t h e chance o f p r o d u c i n g s e n s i t i z i n g a n t i b o d i e s wou ld be l e s s e n e d . I f p l a t e l e t s p roduce an e f f e c t s i m i l a r i n m a g n i t u d e t o who le b l o o d t h e r e wou ld be a t h e o r e t i c a l advan tage i n - 49 - substituting platelets for whole blood when immunizing patients prior to transplantation. EXPERIMENT 6.1 Four experiments were carried out to see i f platelets could produce the effect noted in the previous experiments using B c e l l s . (PVG x DA) F 1 donor hearts were transplanted into DA recipients. PVG platelets harvested from 10 cc of whole blood treated with EDTA and centrifuged at 1,000 revolutions per minute produced a buffy coat. This buffy coat was aspirated by micropipette and washed s e r i a l l y with 5 cc of saline and u l tra - centrifuged at 10,000 rpm for 10 minutes. This buffy coat contains white blood ce l l s and platelets mixed and is separated from the serum and red blood ce l l s . The ultracentrifugatidn performed with heparinized saline at 10,000 rpm removes the white c e l l s , and leaves the platelets behind. Microscopic analysis shows only one white ce l l per 10,000 platelets , thus the white cel l transfusion effect should not be active in platelet immunizations. By micro-counting, approximately 100,000,000 platelets were injected at intervals prior to transplant, intervals being 21, 14, and 17 days pretransplant. The survival of the (PVG x DA) F^ heart in the DA recipient , having been immunized with serial PVG platelets , showed a mean survival of 9.2 days (Table 7 - Line 1). - 50 - EXPERIMENT 6-2 A (PVG x DA) Fj heart was transplanted into a DA rat using ul tra- centrifuged and purified F^ platelets . The mean survival from this experiment was 10.3 days. This experiment was carried out subsequently with Fj platelets (heated to 56°C for 30 minutes). The survival mean was 8.8 days. This result is not different from nonheated platelets (Table 7 - Lines 2 and 4). EXPERIMENT 6-3 As in 6-2 except PVG platelets were used to immunize the DA recipients by the same treatment schedule. The mean survival of the non-heated platelets was 9.2 days. When platelets were heated to 56°C for 10 minutes, the mean survival was 9.0 days. Neither group's survival was s ignif icantly different from controls (Table 7 - Lines 1-4). EXPERIMENT 7 Specif icity Controls To demonstrate that this effect is not entirely a result of the particular strain combination used, or separation ar t i fac t , three experiments were carried out to demonstrate that the effect was secondary to the immunizations. 1. AO (RT1°) hearts were transplanted to DA recipients and these rejected between seven and eight days. A total of four grafts were carried out. - 51 - EXPERIMENT 7 Continued 2. An AO heart was transplanted into a previously immunized DA rat , and three types of immunizations were carried out: F j , T, and B ce l l s . Again, the mean rejection was 7.5 days. 3. A DA rat was previously immunized by AO T or B c e l l s , and transplanted with an F^ heart, rejected in the same period of time, showing no species pecul iarity . The results are summarized in Table 8. Al l three groups showed no signif icant difference from controls. - 52 - DISCUSSION The d e v e l o p m e n t o f a s u i t a b l e model f o r r e j e c t i o n t o s t u d y an e f f e c t on t h e immuno log i c i n t e g r i t y o f t h e r e c i p i e n t i s n o t s i m p l e . I n o r d e r t o show s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n s u b t l e a l t e r a t i o n s o f g r a f t enhancement a l a r g e number o f t r a n s p l a n t s i s n e c e s s a r y and t h e success r a t e must be h i g h o r t e c h n i c a l f a c t o r s overshadow t h e i m m u n o l o g i c e f f e c t . The p r o c e d u r e must be r a p i d enough so t h a t warm i s c h a e m i c t i m e does n o t become a s i g n i f i c a n t v a r i a b l e . L a s t l y , t h e end p o i n t o f t h e g r a f t s u r v i v a l must be a b r u p t and e a s i l y m e a s u r e d . The r e s u l t s o f t h e 464 r a t h e a r t a l l o g r a f t s show t h a t t h e model i s most s u i t a b l e f o r assessment o f t h e d e s i r e d immuno log ic m a n i p u l a t i o n s . The s u c c e s s r a t e o f 97.4% d e m o n s t r a t e s t h e r e l i a b i l i t y o f t h e model and absence o f a s i g n i f i c a n t t e c h n i c a l componen t . The r a t h e a r t t r a n s p l a n t s a l l had v e r y s i m i l a r and s h o r t warm i s c h a e m i c t i m e s p e r m i t t i n g t h i s be d i s c o u n t e d as a v a r i a b l e . S e p a r a t e e x p e r i m e n t s n o t r e p o r t e d he re showed t h a t a r a t h e a r t k e p t a t 4°C f o r as l o n g as f o u r h o u r s f u n c t i o n e d a f t e r r e p e r f u s i o n . The re i s a d e f i n i t e l e a r n i n g c u r v e as d e m o n s t r a t e d by t h e c o m p l i c a t i o n r a t e i n t h e f i r s t s e r i e s o f 96 r a t s compared t o t h e o v e r a l l r e s u l t s o f 464 r a t h e a r t t r a n s p l a n t s ( T a b l e 1 ) . S e v e r a l m o d i f i c a t i o n s o f t h e h e a r t g r a f t s t e c h n i q u e were adop ted a f t e r t h e p i l o t s t u d i e s and l e a r n i n g phase were c o m p l e t e d . These were m a i n l y i n t h e c u f f t e c h n i q u e o f m i c r o v a s c u l a r a n a s t o m o s i s w h i c h p r o v i d e d an i n t i m a t o i n t i m a c o n t a c t as w e l l as t h e r a p i d i t y o f c o m p l e t i o n t h u s d e c r e a s i n g i s c h a e m i c t i m e and s h o r t e n i n g t h e a n e s t h e t i c ( F i g u r e l ) . 5 1 The end p o i n t o f r e j e c t i o n i s u s u a l l y t a k e n as t h a t p o i n t a t wh ich t h e t r a n s p l a n t e d o r g a n ceases t o f u n c t i o n o r can no l o n g e r m a i n t a i n t h e - 53 - h o m e o s t a s i s e x p e c t e d i n t h e o rgan sys tem o f t h e r e c i p i e n t . For e x a m p l e , i f a k i d n e y i s t r a n s p l a n t e d i n t o a r e c i p i e n t t h e end p o i n t o f r e j e c t i o n i s t h a t t i m e when t h e an ima l d i e s o f r e n a l f a i l u r e , b u t i t c o u l d a l s o be t a k e n t o be t h e p o i n t r e n a l f u n c t i o n has c e a s e d , i . e . r a p i d l y r i s i n g BUN o r c r e a t i n i n e . The animal d i e s some t i m e a f t e r t h e t r a n s p l a n t e d k i d n e y has ceased t o f u n c t i o n and may be i n f l u e n c e d by t h e m e t a b o l i c r a t e as w e l l as o t h e r v a r i a b l e s , t h u s , t h e end p o i n t i s n o t c l e a r . The same can be s a i d f o r p a n c r e a t i c g r a f t s . The end p o i n t i s g e n e r a l l y a c c e p t e d as t h a t p o i n t where exogenous i n s u l i n i s 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 g l u c o s e h o m e o s t a s i s . As h y p e r g l y c e m i a o f t e n i s a s s o c i a t e d w i t h s t r e s s due t o i n f e c t i o n , g r a f t r e j e c t i o n o r p a n c r e a t i t i s , t h e end p o i n t i s o f t e n n o t c l e a r . The h e t e r o t o p i c r a t h e a r t t r a n s p l a n t p r o v i d e s a r e l i a b l e and a c c u r a t e end p o i n t . I t does n o t s u p p o r t c i r c u l a t i o n so t h e an ima l does n o t d i e a t r e j e c t i o n . The t i m e t h a t t h e h e a r t b e a t s t o p s i s t a k e n as t h e end p o i n t and i s t h e same p o i n t as i s o e l e c t r i c ECG t r a c i n g s . The h e a r t b e a t can e a s i l y be n o t e d i n i t s s u b c u t a n e o u s l o c a t i o n i n t h e neck as opposed t o t h e a b d o m e n . 6 3 A o r t i c p u l s a t i o n s may c o n f u s e t h e examiner ( F i g u r e 2). Once t h e h e a r t r e j e c t s , t h e o r g a n i s p h a g o c y t o s e d by t h e r e c i p i e n t l e u k o c y t e s and d i s a p p e a r s . I s o g e n i c h e a r t g r a f t s f u n c t i o n f o r t h e l i f e o f t h e an imal as do a d e q u a t e l y t o l e r a n t immunosuppressed r e c i p i e n t s . Rat h e a r t l u n g t r a n s p l a n t s were a t t e m p t e d b u t t h e r e s u l t s show t h a t t h e t e c h n i q u e i s n o t s u i t a b l e . The b u l k o f t i s s u e was t o o l a r g e t o be - 54 - t r a n s p l a n t e d i n t h e neck and wou ld seem t o o b s c u r e any e x p e r i m e n t a l e f f e c t p r o d u c e d . I t i s c l e a r t h a t g r a f t enhancement i s a r e a l phenomenon as e v i d e n c e d by T a r a s a k i ' s work on p r e - t r a n s p l a n t b l o o d t r a n s f u s i o n s . 5 7 , 5 8 , 1 0 1 I t became e v i d e n t t h a t , i n o r d e r t o p r o d u c e t h e d e s i r e d enhancement , s e v e r a l b l o o d t r a n s f u s i o n s were n e c e s s a r y . 5 T h i s e f f e c t i s d i m i n i s h e d o v e r t i m e and , w i t h t h e adven t o f c y c l o s p o r i n A, t h i s e f f e c t may be l o s t . 7 2 T h i s e v i d e n c e i n d i c a t e s t h a t t h e a b i l i t y t o enhance g r a f t s may n o t be a s t r o n g e f f e c t . 4 I n o r d e r t o r e p r o d u c e enhancement a f u l l HLA mismatch u s u a l l y causes r a p i d r e j e c t i o n and e n h a n c i n g i s d i f f i c u l t . To a l l o w a l e s s r a p i d r e j e c t i o n donor o rgans a re u s e d , t h u s d i l u t i n g t h e HLA i n c o m p a t i b i l i t y by a h a l f . 2 6 ' 2 7 The d i s a d v a n t a g e o f t h i s model when a p p l i e d t o t h e c l i n i c a l s i t u a t i o n i s t h a t , a p a r t f r o m f a m i l y , c a d a v e r d o n o r s a r e n o t HLA m a t c h e d . PVG(RTI C ) and D A ( R T I a ) have been shown t o have a h i g h immune i n c o m p a t i b i l i t y i n p r e v i o u s t r a n s p l a n t e x p e r i m e n t s u s i n g many s t r a i n c o m b i n a t i o n s . 1 ' 3 6 T h i s s t r a i n i n c o m p a t i b i l i t y r e d u c e s t h e chances o f a low r e s p o n s e b e i n g m i s t a k e n f o r an e f f e c t o f e n h a n c e m e n t . 5 7 The p r o l o n g a t i o n o f g r a f t s by p r e - t r a n s p l a n t i m m u n i z a t i o n w i t h B c e l l s c o r r o b o r a t e d t h e e f f e c t shown by L a u c h a r t i n a d i f f e r e n t s t r a i n c o m b i n a t i o n (WAG vs P V G ) . 6 0 , 6 1 M - j x e d l y m p h o c y t e s f a i l e d t o p r o d u c e a s i m i l a r e f f e c t . A l t h o u g h n o t s i g n i f i c a n t , t h e t e n d e n c y t o h a s t e n g r a f t l o s s a f t e r T c e l l i m m u n i z a t i o n i s seem i n some e x p e r i m e n t s . T h i s may become s i g n f i c a n t i f t h e i m m u n i z a t i o n s c h e d u l e were t o change as T c e l l s t e n d t o r e a c t w i t h t h e h o s t more r a p i d l y p r o d u c i n g more r a p i d r e j e c t i o n . 6 7 ' 6 ^ B c e l l s a re t h e s o u r c e o f - 55 - a n t i b o d y p r o d u c t i o n and p r e - t r a n s p l a n t s t i m u l a t i o n o f t h e B c e l l sys tem may p roduce e n h a n c i n g a n t i b o d y . The r e s p o n s e i s s p e c u l a t i v e , b u t a n t i b o d i e s have been i d e n t i f i e d a g a i n s t k i l l e r T c e l l s and a n t i b o d y a g a i n s t a n t i b o d y has been r e c o g n i z e d . 4 4 A f u r t h e r t h e o r y p o s t u l a t e s t h a t t h e a n t i g e n p r e s e n t a t i o n s i t e i n t h e donor may be masked by t h e e n h a n c i n g a n t i b o d y r a i s e d i n t h e immunized h o s t . 1 5 A p o s s i b l e p r o b l e m i n u s i n g a ¥ \ ( f i l i a l ) d o n o r h e a r t i s t h a t a n t i - i d i o t y p e a n t i b o d y w i l l be p roduced when P]_ ( p a r e n t a l ) c e l l a r e used and t o a l e s s e r deg ree when F]_ B and T c e l l s a re used t o i m m u n i z e . No g e n e t i c d i f f e r e n c e s e x i s t be tween t h e s e n s i t i z i n g c e l l s and t h e F]_ d o n o r , h e a r t b u t a n t i - i d i o t y p e a n t i b o d i e s c o u l d be p r e s e n t i n t h e DA r e c i p i e n t p r i o r t o e n g r a f t m e n t . A n t i - i d i o t y p i c a n t i b o d y i s l i k e l y n o t t h e s o l e cause o f enhancement because o f two o b s e r v a t i o n s : o n e , B and T c e l l s b o t h c a p a b l e o f p r o d u c i n g a n t i - i d i o t y p e a n t i b o d y have d i f f e r e n t s u r v i v a l s ; t w o , no d i f f e r e n c e i n s u r v i v a l was c o n s i s t e n t l y n o t e d when p u r e PVG o r Fi_ h y b r i d c e l l s were u s e d . A l t h o u g h F ^ ' s a r e c a p a b l e o f p r o d u c i n g a n t i - i d i o t y p i c a n t i b o d y as a re PVG, PVG wou ld o n l y p a r t i a l l y p r o t e c t t h e g r a f t as a n t i - i d i o t y p i c a n t i b o d y p r o t e c t i n g PVG a n t i g e n s wou ld be p r o d u c e d . I t has g e n e r a l l y been a c c e p t e d t h a t t h e immunogen i s a p r o t e i n p r o d u c t o f t h e HLA t r a n s p l a n t a n t i g e n s y s t e m . 7 7 These a n t i g e n s a r e f o u n d on t h e c e l l s u r f a c e o f B and T c e l l s . I n c u b a t i n g t h e c e l l t o 56°C f o r 30 m i n u t e s d e n a t u r e s t h e s e exposed p r o t e i n s . I f t h e s e a n t i g e n s were c r i t i c a l i n p r o d u c i n g and e n h a n c i n g a n t i b o d y , d e s t r o y i n g t h e s e p r o t e i n s s h o u l d r e v e r s e t h e p r o l o n g a t i o n o b s e r v e d i n E x p e r i m e n t 1 . T h i s i n c u b a t i o n d i d no t d i s r u p t t h e c e l l membrane i n t e g r i t y ( F i g u r e 1 3 ) . - 56 - The i n c u b a t e d PVG B c e l l s (56°C) s t i l l p roduced g r a f t enhancement b u t no t as l o n g as t h e u n h e a t e d B c e l l i m m u n i z a t i o n s . The same o b s e r v a t i o n was no ted when (56°C) h e a t e d B c e l l s were u s e d . S e v e r a l c o n c l u s i o n s c o u l d be made: ( 1 ) t h e d e n a t u r e d HLA p r o t e i n i s a b l e t o p r o d u c e a p a r t i a l r e s p o n s e , ( 2 ) t h e r e a r e s e v e r a l mechanisms p r o d u c i n g a n e t e f f e c t and h e a t i n g t o 56"C removed s e l e c t e d p r o c e s s e s r e d u c i n g t h e e f f e c t o f enhancement , ( 3 ) t h e f u l l e f f e c t c o u l d s t i l l be p r e s e n t bu t t h e r e i s an a l t e r a t i o n i n t h e p r o c e s s i n g o f t h e e n h a n c i n g a n t i g e n by t h e r e c i p i e n t , ( 4 ) t h e p r o c e s s p r o d u c e s t o x i c m a t e r i a l t o t h e h o s t i n t e r f e r i n g w i t h t h e h o s t ' s immune r e s p o n s e . 6 8 , 6 9 To add ress t h e s e p r o b l e m s one wou ld be i n t e r e s t e d i n s e e i n g i f t h e B c e l l s w h i c h a re h e a t e d a r e a b l e t o be i m m u n o l a b e l l e d t o d e t e r m i n e i f t h e a n t i g e n i s s t i l l i n t a c t . The h e a t e d B c e l l s d i d , i n f a c t , s t a i n w i t h b o t h immunoperox idase and a n t i b o d y l a b e l l e d w i t h f l u o r e s c e i n , w h i c h shows t h a t t h e immuno log ic p r e s e n t i n g a n t i g e n i s n o t e n t i r e l y d e s t r o y e d by h e a t i n g t o 56°C o r 85°C ( F i g u r e 1 3 ) . T h u s , a t l e a s t one p r e s e n t i n g a n t i g e n complex on t h e B c e l l i s n o t a p u r e p r o t e i n b u t l i k e l y a p r o t e i n c a r b o h y d r a t e r e n d e r i n g i t h e a t s t a b l e , and some i s p r o t e i n b e i n g a l t e r e d by t h e h e a t i n g and d e c r e a s i n g enhancement once d e s t r o y e d . The T c e l l s , w h e t h e r PVG o r F i d i d n o t seem t o a f f e c t g r a f t enhancement nor h a s t e n i t s r e j e c t i o n . One c o u l d c o n c l u d e f r o m t h i s o b s e r v a t i o n c l a s s I I a n t i b o d y i s n e c e s s a r y t o p r o d u c e enhancement o r t h e i m m u n i z a t i o n s c h e d u l e was a p p r o p r i a t e d f rom t h e B c e l l a n t i g e n i n c l u d i n g c l a s s I I b u t i n a p p r o p r i a t e f o r T c e l l a n t i g e n s . One w o u l d need d i f f e r e n t i m m u n i z a t i o n s c h e d u l e s u s i n g T c e l l s t o see i f t h i s i s an i m p o r t a n t f a c t o r (Graph 7 ) . 2 8 , 2 9 , 7 8 - 57 - The B c e l l s h e a t e d t o 56°C l o s t some o f t h e i r e n h a n c i n g e f f e c t bu t t h i s e f f e c t seemed t o r e t u r n when h e a t e d t o 85°C. T h i s s u g g e s t s t h a t enhancement may be m u l t i f a c t o r i a l and c e r t a i n e f f e c t s are l o s t w h i l e o t h e r s may become unopposed. An i m p o r t a n t o b s e r v a t i o n i s t h a t t h e e n h a n c i n g e f f e c t i s n o t l o s t when t h e c e l l s a r e h e a t e d t o 85°C s u g g e s t i n g a n o n p r o t e i n m o i e t y i s a b l e t o p roduce enhancement . I t i s n o t e w o r t h y t h a t t h e s e l o n g t e r m s u r v i v a l s were g e n e r a t e d by t h e 8 5 ° C . B c e l l i m m u n i z a t i o n s . T h i s may be a s u b p o p u l a t i o n o f a n i m a l s who have r e s p o n d e d t o t h e a n t i g e n n o t d e s t r o y e d by t h e h e a t i n g . T h i s o b s e r v a t i o n i s v e r y e n c o u r a g i n g , and we may be s e e i n g a v e r y p o w e r f u l s t i m u l u s o f g r a f t enhancement . These o b s e r v a t i o n s a r e d o n o r s p e c i e s s p e c i f i c . T h i s i s d e m o n s t r a t e d by i m m u n i z i n g t h e r e c i p i e n t o f a h e a r t g r a f t w i t h B o r T c e l l s f r o m a d i f f e r e n t r a t s t r a i n and does n o t p r o d u c e g r a f t enhancement . F u r t h e r m o r e , i f t h e B and T c e l l s , once s e p a r a t e d a r e r e c o n s t i t u t e d and i m m u n i z e d , t h e e n h a n c i n g e f f e c t o f pu re B c e l l s i s l o s t . T h i s s e r v e s as e v i d e n c e t h a t t h e enhancement i s n o t a b i o c h e m i c a l a r t i f a c t p roduced by t h e s e p a r a t i o n p r o c e s s o r by B c e l l i n g e n e r a l . How have t h e s e e x p e r i m e n t s h e l p e d w i t h c l i n i c a l t r a n s p l a n t a t i o n ? One w ishes t o p r o d u c e p u r e enhancement o f t h e g r a f t i n t h e hos t w i t h o u t s e n s i t i z i n g t h e h o s t i n t h e p r o c e s s . P u r i f i e d B c e l l s w i l l p r o d u c e enhancement o f h e t e r o t o p i c a l l y t r a n s p l a n t e d h e a r t a l l o g r a f t s i n t h e r a t m o d e l . F u r t h e r m o r e by h e a t i n g t h e B c e l l s t o 85°C f o r 10 m i n u t e s , t h e B c e l l s a re n o t d e s t r o y e d and t h e e n h a n c i n g e f f e c t s i s n o t l o s t s u g g e s t i n g t h i s e f f e c t i s n o t p r o d u c e d s o l e l y by p r o t e i n a n t i g e n s . I n t h e case o f a l i v i n g r e l a t e d ( n o n - i d e n t i c a l t w i n ) d o n o r , b l o o d c o u l d be d o n a t e d p r i o r t o - 58 - transplantation and the B ce l l s could be separated and transfused instead of whole blood. There is no assurance that sensit ization would be less l i k e l y to occur. In the above model MLR's could be performed comparing the sensitization potential of whole blood versus B ce l l immunization prior to transplant. Unfortunately, this type of immunization would l ike ly be inconceivable for cadaver derived organs until preservation of the organs for much longer periods is developed. In this particular model, platelet transfusion prior to engraftment did not have a significant enhancing effect. This does suggest that the class II antigen is important in the production of enhancement and is consistent with the observation that T ce l l s do not produce enhancement. - 59 - T a b l e 1 : Model - H e t e r o t o p i c H e a r t T r a n s p l a n t t o Neck o f R e c i p i e n t F i r s t S e r i e s (96 r a t s ) Times (AO) : Donor 13 mi n R e c i p i e n t 72 min W e i g h t s (AO) : Donor 135 gm R e c i p i e n t 267 gm M o r t a l i t y 26. .6% A. T e c h n i c a l F a i l u r e - venous t h r o m b o s i s 8.6% - a r t e r i a l t h r o m b o s i s 4.5% - b l e e d i n g 5.5% B. I n f e c t i o n 8.0% - 60 - T a b l e 2: C o n t r o l Rat G r a f t s * Donor Reci p i e n t S u r v i v a l ( d a y s ) Mean 1 . PVG(RT1 C ) D A ( R T l a ) 6 , 7 , 7 , 6 , 8 , 7 , 7 6 . 8 2 . (PVGxDA)F 1 DA 9 , 9 , 1 0 , 8 , 8 , 6 , 1 0 , 9 8 . 6 * t i m e o f r e j e c t i o n t a k e n as c e s s a t i o n o f h e a r t b e a t - 61 - T a b l e 3: Unheated C e l l I m m u n i z a t i o n s * Donor H e a r t R e c i p i e n t Immuni zed C e l l Type I m m u n i z a t i o n S u r v i v a l ( d a y s ) Mean S u r v i v a l 1 . ( P V G x D A ^ DA (PVG T c e l l s ) 6 , 6 , 7 , 8 , 6 , 8 , 7 , 6 , 5 6 . 5 1 2 . ( P V G x D A ^ DA (PVG B c e l l s ) 1 2 , 1 8 , 1 8 , 1 8 , 2 0 , 1 6 , 1 1 , 1 7 , 2 0 , 1 6 , 2 6 , 1 9 1 7 . 6 3 3. (PvGxDA)F! DA (PVGxDA)F x T c e l l s 6 , 6 , 6 , 8 , 8 , 7 , 7 , 5 , 6 6 . 5 1 4 . (PVGxDA)F x DA (PVGxDA)F x B c e l l s 9 , 1 2 , 1 4 , 1 1 , 1 8 , 1 7 , 1 3 , 1 3 , 1 6 , 1 8 14 .12 1 Not s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s 2 p < 0 . 0 5 3 p < 0 . 0 1 - 62 - T a b l e 4 : Heated C e l l I m m u n i z a t i o n s ( 5 6 ° C ) * Donor R e c i p i e n t Ce l1 Type Mean H e a r t Immunized I m m u n i z a t i o n S u r v i v a l ( d a y s ) S u r v i v a l 1 . (PVGxDA)F 1 DA PVG (T c e l l s ) 6 , 5 , 7 , 4 , 6 , 7 , 6 5 . 9 1 2 . (PVGxDA)F 1 DA PVG (B c e l l s ) 1 5 , 1 0 , 1 8 , 1 2 , 1 2 , 1 6 2 1 3 . 8 3- (PVGxDA)F 1 DA F x (T c e l l s ) 6 , 6 , 5 , 5 , 7 , 8 , 7 , 7 6 . 4 1 4 . (PVGxDA)F 1 DA ?l (B c e l l s ) 9 , 1 0 , 1 2 , 7 , 8 , 6 , 6 , 9 . 8 1 1 2 , 1 3 , 1 5 * 56°C h e a t e d f o r 30 m i n u t e s i n w a t e r b a t h 1 Not s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s 2 p < 0 . 0 5 3 p < 0 . 0 1 - 63 - T a b l e 5: Heated C e l l I m m u n i z a t i o n s ' ( 8 5 ° ) * Donor Reci p i e n t Ce l1 Type Mean H e a r t Immunized I m m u n i z a t i o n S u r v i v a l ( d a y s ) S u r v i v a l 1 . ( P V G x D A ^ DA PVG (T c e l l s ) 4 , 6 , 8 , 8 , 9 , 9 , 8 , 7 . 7 . 4 l 2 . ( P V G x D A ^ DA PVG (B c e l l s ) 1 0 - , 1 6 , 1 0 , 1 2 , 3 0 + , 3 0 + , 3 0 + 1 9 . 7 2 3 . (PVGxDA)F x DA F x (T c e l l s ) 8 , 8 , 9 , 6 , 7 , 9 , 6 , 1 2 8 . l l 4. (PVGxDA)F 1 DA F : (B c e l l s ) 1 0 , 1 6 , 1 9 , 1 8 , 1 9 , 1 2 . 15 .72 * h e a t e d 10 min 85°C w a t e r b a t h Not s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s p < 0 . 0 5 p < 0 . 0 1 1 2 3 - 64 - T a b l e 6: Mixed C e l l I m m u n i z a t i o n Donor Reci p i e n t Ce l1 Type S u r v i v a l Mean 1. (PVGxDA)F! DA ?l (T+B c e l l s ) 6 , 7 , 6 , 8 •6.8 2 . (PVGxDA)F 1 DA PVG (T+B c e l l s ) 7 , 8 , 7 , 7 7 .3 - 65 - T a b l e 7: P l a t e l e t I m m u n i z a t i o n Donor R e c i p i e n t I m m u n i z a t i o n S u r v i v a l Mean . 1 . (PVGxDA)F x DA PVG ( p l a t e l e t s ) 8 , 9 , 1 0 , 1 0 , 1 1 , 9 , 8 9 . 2 1 2 . (PVGxDA)F! DA F x ( p l a t e l e t s ) 1 0 , 1 0 , 1 1 , 1 2 , 1 5 , 6 , 1 0 , 8 1 0 . 3 1 3. (PVGxDA)Fi DA PVG ( p l a t e l e t s 5 6 " C ) * 8 , 1 0 , 9 , 9 9 . 0 1 4 . ( P V G x D A ^ DA Fj_ ( p l a t e l e t s 56°C) 1 0 , 8 , 8 , 9 8 . 8 1 * 30 m i n u t e s 1 Not s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s - 66 - T a b l e 8 : S p e c i f i c i t y C o n t r o l s Donor R e c i p i e n t S u r v i v a l ( d a y s ) Mean 1 . AO(RTIO) DA 7 , 7 , 7 , 8 7 .3 2 . AO D A ( I M M . F 1 T ) * 4 , 7 , 7 , 9 , 8 7 . 0 3 . AO DA( IMM.F 1 B) 7 , 7 , 8 , 8 7 .5 4 . (PVGxDA)F 1 DA(IMM.AOjJ) 8 , 6 , 6 , 7 6 . 8 5 . ( P V G x D A ^ DA(IMM.AOB) 4 , 8 , 8 , 7 , 7 6 . 8 107 c e l l s i n j e c t s 7 days p r i o r t o e n g r a f t m e n t 100 80 D | 60 to | 401 Q_ L PVG 20 •Mixed Cells 0 GRAPH 1 Controls PVG ( H e a r t ) — D A (DA x PVG) F| (Hear t ) -DA F, (Mixed T + B Cells) F, HT X * D A 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 O N GRAPH 2 100 80 o | 60 8 40 20 - 0 PVG B or T Cell Immunization Fi HTY I T-T Cells B-B Cells C-NO Cells 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 GRAPH 3. 100 80 ~ 60 D CO •*— 8 40 Q_ 20 0 M B PVG B + T Cell Immunization + DA F| Heart T x C - (PVG x DA) HT X T - PVG, T Cell Immunizat ion M - M ixed B + T Cell I m m " B- PVG B Cell Immunization 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 GRAPH 4 100 80 o | 60 CO •4— I 40; O 20 0 1 A - A a M 1 B L F| (B + T Cel ls)* DA F,HTX T ( • ) T Cell Immunizat ion M ( o ) Mixed Cells (B + T) C (-A ) Non Immunized B - B Cell Immunizat ion 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 o GRAPH 5 100 80 - o I 60 k_ D CO •4— | 40 0) 20 0 L PVG B Cell - F, B Ce Immunization 1_ PVG h 1 L 1 C - N o Immunizat ion F, -(PVG x DA) F, B Cells PVG - P. (PVG) B Cells 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 GRAPH 6 PVG B Cell Immunization C - Control (No B Cells) B37 - Non heated B Cells B56 - 56°c 30 min B85 - 85°c 10 min D CO B85 e C 8 40 B56 I B37 a> Q_ 20 0 5 10 15 20 25 30 Heterotopic Heart Graft Survival (days) GRAPH 7 100 80 o I 60 CO | 40 O a. 20 0 T Cell Immunization F,HT, F,56 137- Control 37°c F|56 - T| Cells heated to 56 c F| 85 - T| Cells heated to 85° PVG 85 - PVG Cells heated to 8 5 c 85 PVG 85 F, 37 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 oo GRAPH 8 100 80 | 60 i _ D CO -*- | 40 20 0 B Cell Immunizations HTX L - I" : B37 B56 C - Control No l m m n B37- B Cells at 37°c B 5 6 - BCells t reated to 56°c B 8 5 - B Cells t reated to 85°c B 8 5 5 10 15 20 25 Heterotopic Heart Graft Survival (days) 30 --4 -p. 100 80 | 60 D to 8 40 Q_ 20 0 AO GRAPH 9 Specificity Controls C-Cont ro l A O — D A A O - A O H T x — D A (I mm. F, B Cells) F,- F| HT-*- DA ( Imm.AO, B Cells) 5 10 15 20 25 30 Heterotopic Heart Graft Survival (days) cn F I G U R E 1 mi T H E CU F F VA S C U L A R AN A S T O M O S I S S E T S I D E BY S I D E F I G 1A - TH R O U G H T H E P O L Y E T H Y L E N E C U F F / T H E R E C I P I E N T V E S S E L I S P U L L E D , F I G 1B - T H E R E C I P I E N T V E S S E L I S F O L D E D O V E R T H E C U F F A N D L I G A T E D I N P L A C E . F I G l c - TH E C U F F I S I N V A G I N A T E D I N T O T H E DONOR V E S S E L A N D L I G A T E D . NO T E T H A T O N L Y I N T I M A I S I N C O N T A C T W I T H B L O O D F L O W . m i F I G 2 - ECG O F H E A R T A F T E R G R A F T I N G , CH A R T S P E E D 2X N O R M A L . F I G 3 - R E M O V E D R A T H E A R T T R A N S P L A N T A F T E R V E N O U S I N F A R C T I O N P O S T - O P DAY 2, SHOWING A C U T E V E N O U S C L O T . /79/ F I G l\ - R E M O V E D RAT H E A R T T R A N S P L A N T SHOWING A C U T E A R T E R I A L C L O T DAY 1. 7 8 0 / F I G 5 - REMOVED RAT HEART TRANSPLANT DAY 6 . SOME R E J E C T I O N CHANGES ARE E V I D E N T , BUT THE ANIMAL DIED OF PNEUMONIA. GRAFT FUNCTIONING AT T I M E OF DEATH. 781/ mi F I G 7 - REMOVED RAT HEART TRANSPLANT AFTER 1 4 DAYS. ACUTE ALLOGRAFT R E J E C T I O N . 7 8 3 / F I G 8 - RAT HEART ALLOGRAFT R E J E C T E D 8 DAYS, REMOVED 60 DAYS, c n / 8 4 / FIGURE 9 C E L L S E P A R A T I O N TECHNIQUE F I C O L L - I S O P A Q U E / N Y L O N WOOL LYMPH NODES S P L E E N GOO o o O O 6 4- PBS + FCS LYMPHOCYTES F I C O L L DOLYS + RBC'S WASHED WITH MEDIUM ( 3 7 ° C ) ,1 T fi IT COLUMN NYLON, WOOL + MEDIUM + FCS WASHED WITH o MEDIUM (.4 C) T B 785/ F I G 10A - PE R O X I D A S E ST A I N (T C E L L S ) T C E L L S - FEW C E L L S W I T H D A R K H A L O . T C E L L S DO NOT S T A I N . 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Scudamore CH, Freeman HJ: Effects of small bowel transection, resection, or bypass in 1,2-dimethylhydrazine-induced rat intestinal neoplasia. Gastroenterology, 1983, 84:725-31. 6. Scudamore CH: Late b i l i ary tract complications. In: Liver Transplantation. The Cambridge/King's College Hospital Experience, R.Y. Caine (Ed), Chapter 27, Cambridge: Press Oxford, 1983, 327-341. 7. Scudamore CH, Harrison RC, White TT: Management of duodenal d iver t i cu la . Can J Surg 1982, 25(3):311-314. 8. Scudamore CH, Ashmore P: Spontaneous sternal segment dis locat ion. Ped Surg 1982; 17(l):61-63. 9. Sandy JTM, Taylor RH, Christensen RM, Scudamore CH, Leckie P: Pancreatic pseudocyst. Changing concepts in management. Am J . Surg 1981; 141(5):574-575. 10. Opie J , Scudamore CH, Fraser R, Ashmore P: Mobile esophageal manometry in pediatric patients. Am J Ped 1980, 41:211-215. 11. Ping C,Scudamore CH, Cooperberg P: Ultrasound demonstration of radio- log ica l ly obscure small bowel obstruction. Am J Rad, 1979 (Ju l ) , 133:145-146. 12. Scudamore CH: Efferent loop intussusception as a course of upper gastro- intestinal hemorrhage post-partial gastrectomy. San Fran Med, 1976 (Nov). 13. Scudamore CH: The drug abuse arm. B.C. Med, 1974 (June).

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