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Non specific splenic suppressor cells in tumor-bearing mice Pope, Barbara Lynn 1978

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NON SPECIFIC SPLENIC SUPPRESSOR CELLS IN TUMOR-BEARING MICE by BARBARA LYNN POPE B.Sc. 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 , 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPY IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We 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 UNIVERSITY OF BRITISH COLUMBIA J a n u a r y , 1978 © B a r b a r a Lynn Pope, 1978 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 representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 wesbrook Place Vancouver, Canada V6T 1W5 Date /y / / / 7 ^ i i ABSTRACT The p r o g r e s s i v e growth o f tumors i n human c a n c e r p a t i e n t s and e x p e r i m e n t -a l a n i m a l s has f r e q u e n t l y been a s s o c i a t e d w i t h a g e n e r a l i z e d d e p r e s s i o n o f i m m u n o l o g i c a l r e s p o n s i v e n e s s . S u p p r e s s o r c e l l s have been i m p l i c a t e d as m e d i a t o r s o f t u m o r - a s s o c i a t e d immunosuppression, b u t t h e i d e n t i t i e s o f t h e c e l l s c a u s i n g s u p p r e s s i o n and t h e mechanisms by w h i c h t h e y a c t have been u n c l e a r . The o b j e c t o f t h i s t h e s i s was th u s t o d e t e r m i n e : i f s u p p r e s s o r c e l l s c a p a b l e o f non s p e c i f i c a l l y s u p p r e s s i n g immune r e s p o n s e s were p r e s e n t i n a n e r g i c mice b e a r i n g m e t h y l c h o l a n t h r e n e - i n d u c e d sarcomas; t h e c e l l t y p e s r e s p o n s i b l e f o r s u p p r e s s i o n ; and t h e mechanisms by w h i c h s u p p r e s s i o n o c c u r s . The s p l e e n s o f mice w i t h l a r g e tumors were found t o c o n t a i n two d i s t i n c t p o p u l a t i o n s o f non s p e c i f i c s u p p r e s s o r c e l l s . One p o p u l a t i o n i n h i b i t e d t h e p r o l i f e r a t i v e r e s p o n s e s o f n o r m a l l y m p h o c y t e s t o t h e T c e l l m i t o g e n , C o n c a n a v a l i n A (Con A) and t h e B c e l l m i t o g e n , l i p o p o l y s a c c h a r i d e ( L P S ) . These c e l l s a l s o i n h i b i t e d t h e g e n e r a t i o n o f a n t i b o d y f o r m i n g c e l l s by normal l y m p h o i d c e l l s s t i m u l a t e d i n v i t r o w i t h t h e T c e l l dependent a n t i g e n , sheep r e d b l o o d c e l l s (SRBC) and t h e T c e l l i n d e p e n d e n t a n t i g e n , d i n i t r o p h e n y l a t e d -l i p o p o l y s a c c h a r i d e (DNP-LPS). These s u p p r e s s o r c e l l s appeared t o be from t h e macrophage/monocyte l i n e s i n c e t h e y adhered t o p l a s t i c and n y l o n w o o l , were removed by c a r b o n y l i r o n and magnet, and were i n a c t i v a t e d by c a r r a g h e e n a n t r e a t m e n t , b u t were n o t removed by a n t i - T h y - 1 o r anti-mouse i m m u n o g l o b u l i n s e r a p l u s complement. They were among t h e l e s s dense s p l e e n c e l l s s i n c e t h e y were r e t a i n e d i n t h e l i g h t f r a c t i o n a f t e r c e n t r i f u g a t i o n on h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08 and d i d n o t appear t o r e q u i r e c e l l d i v i s i o n i n o r d e r t o suppress- s i n c e m i t o m y c i n C t r e a t m e n t d i d n o t i n a c t i v a t e them. C e l l - c e l l c o n t a c t appeared t o be e s s e n t i a l f o r s u p p r e s s i o n . i i i The second p o p u l a t i o n o f s u p p r e s s o r c e l l s , w h i c h p e l l e t e d t o t h e b o t t o m o f a h y p a q u e - f i c o l l g r a d i e n t , i n h i b i t e d o n l y t h e g e n e r a t i o n o f p l a q u e f o r m i n g c e l l s t o t h e T c e l l dependent a n t i g e n , SRBC. These c e l l s appeared t o be T c e l l s s i n c e t h e y were non a d h e r e n t t o p l a s t i c o r n y l o n w o o l , were n o t removed by c a r b o n y l i r o n and magnet, b u t were removed by a n t i - T h y - 1 serum p l u s complement. C e l l d i v i s i o n was n e c e s s a r y s i n c e s u p p r e s s i v e a c t i v i t y was t o t a l l y removed by m i t o m y c i n C t r e a t m e n t . S u p p r e s s i o n by t h i s c e l l t y p e appeared t o be m e d i a t e d by a s o l u b l e f a c t o r w i t h a m o l e c u l a r w e i g h t o f about 3,500 t o 12,000. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES x LIST OF ABBREVIATIONS x i INTRODUCTION I . The Immune Response and Cancer 1 I I . S p e c i f i c Immunosuppression i n Tumor-Bearers 2 I I I . Mechanisms of S p e c i f i c Immunosuppression i n Tumor-Bearers 3 IV. G e n e r a l i z e d Immunosuppression i n Tumor-Bearers 6 V. Mechanisms of G e n e r a l i z e d Immunosuppression i n Tumor-Bearers 8 V I . S u p p r e s s o r C e l l s i n Immune R e g u l a t i o n 11 V I I . S p e c i f i c S u p p r e s s o r C e l l s 12 V I I I . Non S p e c i f i c S u p p r e s s o r C e l l s 15 IX. S o l u b l e F a c t o r s From Non S p e c i f i c S u p p r e s s o r C e l l s 23 X. T h e s i s O b j e c t i v e s 27 MATERIALS AND METHODS I . M i c e 28 I I . Tumor L i n e s 28 I I I . M a i n t e n a n c e of Tumor L i n e s 28 IV. Tumor-Bearing M i c e Used f o r S u p p r e s s i o n E x p e r i m e n t s 29 V. Growth C h a r a c t e r i s t i c s o f M - l Tumors I n V i v o 30 V I . C h a r a c t e r i s t i c s of S p l e e n s i n Tumor-bearing M i c e 30 V I I . I m m u n o g e n i c i t y of t h e M - l Tumor 33 V I I I . Immune M i c e 33 V TABLE OF CONTENTS ( c o n t i n u e d ) Page MATERIALS AND METHODS IX. P r e p a r a t i o n of Lymphoid C e l l S u s p e n s i o n s . 33 X. M i t o g e n S t i m u l a t i o n 34 X I C u l t u r e System f o r t h e I n V i t r o G e n e r a t i o n of P l a q u e Forming C e l l s (PFC) 35 X I I . G e n e r a t i o n of PFC i n Marbrook Chambers 35 X I I I . PFC A s s a y 38 XIV. E s t i m a t i o n of C e l l V i a b i l i t i e s 39 XV. E s t i m a t i o n of P h a g o c y t i c C e l l s 39 XVI. H y p a q u e - F i c o l l S e p a r a t i o n 40 X V I I . Method f o r D e p l e t i n g C e l l S u s p e n s i o n s of B c e l l s 43 X V I I I . Method f o r D e p l e t i n g C e l l S u s p e n s i o n s o f T C e l l s 45 XIX. Methods f o r D e p l e t i n g C e l l S u s p e n s i o n s of P h a g o c y t i c C e l l s . 47 XX. M i t o m y c i n C Treatment 54 X X I . D a t a P r e s e n t a t i o n 54 RESULTS I . Immunocompetence o f Tumor-Bearers 56 I I . S u p p r e s s o r C e l l s i n Tumor-Bearer S p l e e n s : S u p p r e s s i o n of M i t o g e n Responses 59 I I I . H y p a q u e - F i c o l l E n r i c h m e n t of S u p p r e s s o r C e l l s 61 IV. B a s i c P a r a m e t e r s of t h e M i t o g e n A s s a y 64 V. E f f e c t of Tumor Load on S u p p r e s s o r C e l l A c t i v i t y 68 V I . Lymph Node C e l l s f r o m Tumor-Bearers 71 V I I . C h a r a c t e r i z a t i o n of S u p p r e s s o r C e l l s 73 V I I I . S u p p r e s s i o n of the I n V i t r o G e n e r a t i o n of PFC. 78 IX. C h a r a c t e r i z a t i o n of S u p p r e s s o r C e l l s 85 v i TABLE OF CONTENTS ( c o n t i n u e d ) Page RESULTS X. S u p p r e s s o r C e l l A c t i v i t y i n M a r b r o o k Chambers 96 DISCUSSION 106 LITERATURE CITED 114 v i i L I ST OF TABLES T a b l e T i t l e Page I . Tumor and s p l e e n w e i g h t s i n tumor-b e a r i n g m i c e 31 I I . Lymphoid c e l l and p h a g o c y t i c c e l l r e c o v e r y f r o m s p l e e n s of t u m o r - b e a r e r s 32 I I I . E f f e c t of h y p a q u e - f i c o l l s e p a r a t i o n on t h e r e s p o n s e s o f s p l e e n c e l l s 42 IV. E f f e c t of a n t i - i m m u n o g l o b u l i n serum on the r e s p o n s e s of s p l e e n c e l l s 44 V. E f f e c t of a n t i - T h y - 1 serum on the r e s p o n s e s of s p l e e n c e l l s 46 V I . E f f e c t of n y l o n w o o l adherence on the r e s p o n s e s o f s p l e e n c e l l s 51 V I I . E f f e c t of c a r b o n y l iron/magnet and c a r r a g h e e n a n t r e a t m e n t on t h e r e s p o n s e s of s p l e e n c e l l s 52 V I I I . Responses of s p l e e n c e l l f r a c t i o n s o b t a i n e d a f t e r p e t r i d i s h a d herence 53 IX. M i t o g e n r e s p o n s e s of s p l e e n c e l l s f rom t u m o r - b e a r i n g mice 57 X. M i t o g e n r e s p o n s e s o f m i c e b e a r i n g a v a r i e t y of tumor t y p e s 58 X I . S u p p r e s s i v e e f f e c t s of t u m o r - b e a r e r s p l e e n c e l l s on t h e m i t o g e n r e s p o n s e s of n o r m a l . l y m p h o i d c e l l s 60 X I I . S u p p r e s s i v e e f f e c t s of u n f r a c t i o n a t e d tumor-b e a r e r s p l e e n c e l l s and h y p a q u e - f i c o l l f r a c t i o n s . . . 63 X I I I . S u p p r e s s i v e e f f e c t s of s p l e e n c e l l s f r o m t u m o r - r e s e c t e d m i c e 69 XIV. E f f e c t s of tumor immune s p l e e n p o p u l a t i o n s on n o r m a l s p l e e n c e l l s 70 XV. E f f e c t s of t u m o r - b e a r e r lymph node and s p l e e n c e l l s on m i t o g e n r e s p o n s e s of n o r m a l l y m p h o i d c e l l s 72 v i i i L IST OF TABLES ( c o n t i n u e d ) T a b l e T i t l e Page X V I . E f f e c t of m i t o m y c i n C t r e a t m e n t on s u p p r e s s o r c e l l a c t i v i t y 75 X V I I . E f f e c t s o f a n t i - T h y - 1 and a n t i - M I g on s u p p r e s s o r c e l l a c t i v i t y 76 X V I I I . E f f e c t s of t r e a t m e n t s w h i c h remove a d h e r e n t and p h a g o c y t i c c e l l s on s u p p r e s s o r c e l l a c t i v i t y 77 XIX. V i a b i l i t y of c u l t u r e d s p l e e n c e l l s 82 XX. E f f e c t of h y p a q u e - f i c o l l s e p a r a t i o n and p e t r i d i s h adherence on s u p p r e s s o r c e l l a c t i v i t y 89 X X I . E f f e c t of n y l o n w o o l adherence on s u p p r e s s o r c e l l a c t i v i t y 90 X X I I . E f f e c t of c a r b o n y l i r o n and magnet t r e a t m e n t on s u p p r e s s o r a c t i v i t y 91 X X I I I . E f f e c t of c a r r a g h e e n a n t r e a t m e n t on s u p p r e s s o r c e l l a c t i v i t y 92 XXIV. E f f e c t of a n t i - M I g t r e a t m e n t on s u p p r e s s o r c e l l a c t i v i t y 93 XXV. E f f e c t of a n t i - T h y - 1 t r e a t m e n t on t h e s u p p r e s s i v e a c t i v i t y of hypaque- * f i c o l l f r a c t i o n s 94 XXVI. E f f e c t of m i t o m y c i n C t r e a t m e n t on s u p p r e s s o r c e l l a c t i v i t y . . . . . . . — . . . . . . . . . . . 95 X X V I I . T i t r a t i o n o f s u p p r e s s o r c e l l a c t i v i t y i n Marbrook chambers 100 X X V I I I . V i a b i l i t y of c e l l s c u l t u r e d i n Marbrook chambers 101 XXIX. E f f e c t of h y p a q u e - f i c o l l f r a c t i o n a t i o n on s u p p r e s s o r c e l l a c t i v i t y i n Marbrook chambers 102 XXX. E f f e c t of p e t r i d i s h adherence on s u p p r e s s o r c e l l a c t i v i t y i n Marbrook chambers 103 i x L I S T OF TABLES ( c o n t i n u e d ) T a b l e T i t l e Page XXXI. E f f e c t o f c a r b o n y l i r o n and n y l o n w o o l t r e a t m e n t s on s u p p r e s s o r c e l l a c t i v i t y 104 X X X I I . A b i l i t y o f s u p p r e s s o r c e l l f a c t o r t o pass t h r o u g h d i f f e r e n t membranes 105 LIST OF FIGURES F i g u r e T i t l e Page 1. Double chamber Ma r b r o o k v e s s e l 37 2. F r a c t i o n a t i o n of s p l e e n c e l l s on hypaque-f i c o l l o f d i f f e r e n t s p e c i f i c g r a v i t i e s 62 3. The m i t o g e n r e s p o n s e - o f n o r m a l s p l e e n c e l l s c o - c u l t u r e d w i t h v a r y i n g numbers of n o r m a l o r t u m o r - b e a r e r s p l e e n c e l l s 65 4. K i n e t i c s of t h e m i t o g e n r e s p o n s e 66 5. The m i t o g e n r e s p o n s e w i t h d i f f e r e n t doses of Con A 67 6. The ^ n v i t r o PFC r e s p o n s e of n o r m a l c e l l s c o - c u l t u r e d w i t h v a r y i n g numbers of c o n t r o l o r t u m o r - b e a r e r s p l e e n c e l l s 81 7. K i n e t i c s of t h e i n v i t r o PFC r e s p o n s e 83 8. The i n v i t r o PFC r e s p o n s e w i t h d i f f e r e n t a n t i g e n doses 84 x i L I ST OF ABBREVIATIONS ALS : A n t i l y m p h o c y t e serum BCG : B a c i l l u s - C a l m e t t e - G u e r i n BGG : B o v i n e gamma g l o b u l i n CFA : Complete Freund's a d j u v a n t Con A : . . C o n c a n a v a l i n A CP : C o r y n e b a c t e r i u m parvum DNCB : D i n i t r o c h l o r o b e n z e n e DNP-Asc : D i n i t r o p h e n y l a t e d A s c a r i s p r o t e i n e x t r a c t DNP-LPS : D i n i t r o p h e n y l a t e d l i p o p o l y s a c c h a r i d e FCS : F o e t a l c a l f serum GAT : L - g l u t a m i c a c i d - L - a l a n i n e - L - t y r o s i n e GVH : G r a f t - v e r s u s - h o s t r e a c t i o n HRBC : Horse r e d b l o o d c e l l s IDP : I n h i b i t o r o f DNA p o l y m e r a s e IDS : I n h i b i t o r of DNA s y n t h e s i s IG : Immunoglobulin IRA : Immunoregulatory a l p h a g l o b u l i n KLH : K e y h o l e l i m p e t haemocyanin LPS : L i p o p o l y s a c c h a r i d e MIF : Macrophage i n h i b i t i o n f a c t o r MIg : Mouse i m m u n o g l o b u l i n MSV : M u r i n e Sarcoma V i r u s NIP : Normal immunosuppressive p r o t e i n PBS : Phosphate b u f f e r e d s a l i n e PFC : P l a q u e f o r m i n g c e l l L I S T OF ABBREVIATIONS ( c o n t i n u e d ) PHA RPMI 1640 SIRS SRBC S S S - I I I Phy tohaemagglut i n i n Medium number 1640 f r o m t h e R o s w e l l P a r k M e m o r i a l I n s t i t u t e S o l u b l e immune r e s p o n s e s u p p r e s s o r Sheep r e d b l o o d c e l l s Type I I I pneumococcal p o l y s a c c h a r i d e 1 INTRODUCTION I THE IMMUNE RESPONSE AND CANCER The i m m u n o l o g i c a l s u r v e i l l a n c e t h e o r y o f B u r n e t (20) p r e d i c t s t h a t one o f t h e f u n c t i o n s o f t h e immune sys t e m i s t o r e c o g n i z e n a s c e n t tumor c e l l s as ' f o r e i g n ' and t o e l i m i n a t e them as t h e y a r i s e . From work c a r r i e d o ut u s i n g e x p e r i m e n t a l tumor systems (30,50,52,62,108,134), i t i s now w i d e l y a c c e p t e d t h a t : 1. tumor s p e c i f i c a n t i g e n s do e x i s t and a r e e x p r e s s e d as n e o a n t i g e n s o r m o d i f i e d h i s t o c o m p a t i b i l i t y a n t i g e n s on tumor c e l l membranes, and 2. immune r e s p o n s e s t o tumor a n t i g e n s do o c c u r . C o n v i n c i n g e v i d e n c e f o r tumor s p e c i f i c immunity was o b t a i n e d as e a r l y as 1957 by P r e h n and M a i n ( 1 0 8 ) . They showed t h a t s u r g i c a l r e m o v a l o f c h e m i c a l l y -i n d u c e d tumors i n mice r e s u l t e d i n immunity w h i c h was s p e c i f i c f o r t h e tumor l i n e and d i d n o t c r o s s - r e a c t w i t h normal t i s s u e . S i n c e t h a t t i m e , s p e c i f i c immunity has been o b s e r v e d i n e x p e r i m e n t a l tumor systems u s i n g a v a r i e t y o f i n v i v o and i n v i t r o a s s a y s . M i c e w i t h c h e m i c a l l y - i n d u c e d (30) o r v i r a l l y -i n d u c e d (62) tumors a r e , f o r example, c a p a b l e o f r e j e c t i n g tumor c e l l s i n j e c t e d a t a second s i t e . Lymphoid c e l l s f rom t u m o r - b e a r i n g mice s p e c i f i c a l l y i n -a c t i v a t e tumor c e l l s when t h e two a r e admixed b e f o r e i n j e c t i o n i n t o n o r m a l m i c e whereas c e l l s f r o m n o r m a l s y n g e n e i c a n i m a l s o r mice b e a r i n g u n r e l a t e d tumors have no s u c h p r o t e c t i v e e f f e c t ( 3 0 , 6 2 ) . I n v i t r o a s s a y s o f immunity i n c l u d e ; p r o d u c t i o n of macrophage i n h i b i t i o n f a c t o r (MIF) o r p r o l i f e r a t i o n by tumor-b e a r e r l y m p h o i d c e l l s i n t h e p r e s e n c e o f tumor a n t i g e n ( 5 0 ) , k i l l i n g o f "^Cr l a b e l l e d tumor c e l l s by T l y m p h o c y t e s f r o m t u m o r - b e a r i n g mice ( 5 2 ) , and t h e c o l o n y i n h i b i t i o n a s s a y ( 1 3 4 ) . The e v i d e n c e f o r s p e c i f i c tumor immunity i n human c a n c e r p a t i e n t s i s l e s s c o n v i n c i n g a l t h o u g h s p e c i f i c immune r e s p o n s e s t o c u l t u r e d tumor c e l l s have been d e m o n s t r a t e d i n v i t r o . F o r example, p e r i p h e r a l b l o o d l y m p h o c y t e s f r o m p a t i e n t s 2 w i t h m a l i g n a n t melanoma (58) and u r i n a r y b l a d d e r c a r c i n o m a ( 1 0 0 ) c o u l d k i l l tumor c e l l s o f t h e same h i s t o l o g i c a l t y p e but c o u l d n o t k i l l e i t h e r n o r m a l c e l l s o r tumor c e l l s o f a d i f f e r e n t t y p e . However, i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s c o m p l i c a t e d by t h e f a c t t h a t l y m p h o c y t e s from many nor m a l p e o p l e a l s o k i l l c u l t u r e d c e l l s i n v i t r o . A l t h o u g h i t i s p o s s i b l e t h a t s i m i l a r mechanisms of immunity o c c u r i n human c a n c e r p a t i e n t s and e x p e r i m e n t a l tumor-b e a r i n g a n i m a l s , no good e v i d e n c e has been p r e s e n t e d t o s u p p o r t t h i s c o n t e n t -i o n . I n s p i t e o f t h e e v i d e n c e t h a t immune r e s p o n s e s t o tumor a n t i g e n s do o c c u r , c a n c e r s grow and u l t i m a t e l y k i l l t h e i r h o s t s . Many d i f f e r e n t t h e o r i e s have been p r o p o s e d t o e x p l a i n t h i s 'escape' f r o m i m m u n o l o g i c a l s u r v e i l l a n c e . Many of them h i n g e on t h e f a c t t h a t p r o g r e s s i v e tumor growth i s u s u a l l y a s s o c i a t e d w i t h a l o s s of immunity t o t h e tumor and, i n l a t e r s t a g e s , a gen-e r a l i z e d l o s s of immune r e s p o n s i v e n e s s . I I SPECIFIC IMMUNOSUPPRESSION IN TUMOR-BEARERS The s p e c i f i c a n t i - t u m o r immunity o b s e r v e d i n t u m o r - b e a r e r s o f t e n d e c l i n e s as tumors grow. F o r example, H e l l s t r o m et^ a l . (58) found t h a t a l t h o u g h p e r i p h e r a l b l o o d l y m p h o c y t e s f r o m p a t i e n t s w i t h e a r l y m a l i g n a n t melanoma k i l l e d c u l t u r e d melanoma c e l l s , l y m p h o c y t e s f r o m p a t i e n t s w i t h l a r g e tumor l o a d s d i d n o t . S i m i l a r l y , O'Toole ejt a l . (100) showed t h a t 88% of p a t i e n t s w i t h l o c a l i z e d b l a d d e r c a r c i n o m a s , , b u t o n l y 41% of p a t i e n t s w i t h i n v a s i v e tumors, had l y m p h o c y t e s c a p a b l e of k i l l i n g tumor c e l l s . U s i n g m e t h y l c h o l a n t h r e n e - i n d u c e d sarcomas i n m i c e , D e c k e r s e t a l . (30) and Whitney e t a l . (133) showed t h a t s p e c i f i c tumor immunity c o u l d be demons-t r a t e d by a v a r i e t y of i n v i v o and i n v i t r o t e s t s . I n b o t h c a s e s , t h e immunity d e c l i n e d as t h e tumors grew i n s i z e . V i r a l l y - i n d u c e d tumors, s t u d i e d by 3 H o w e l l e t a l . ( 6 2 ) , gave a s i m i l a r p a t t e r n of immunity. I l l MECHANISMS OF SPECIFIC IMMUNOSUPPRESSION IN TUMOR-BEARERS S u p p r e s s i o n o f t h e s p e c i f i c immunity t o tumors has been a t t r i b u t e d t o humoral f a c t o r s s u c h as b l o c k i n g a n t i b o d y , c i r c u l a t i n g tumor a n t i g e n s , o r a n t i g e n - a n t i b o d y complexes. More r e c e n t l y , s u p p r e s s o r c e l l s w h i c h s p e c i f i c -a l l y i n h i b i t t h e immune r e s p o n s e t o tumor a n t i g e n s have a l s o been o b s e r v e d i n t u m o r - b e a r e r s . 1. Humoral F a c t o r s H e l l s t r b m e t a l . (57,58) proposed t h a t t h e d e c l i n e o f s p e c i f i c immunity o b s e r v e d i n human melanoma p a t i e n t s was due t o tumor s p e c i f i c b l o c k i n g a n t i b o d i e s w h i c h c o a t e d tumor c e l l s and p r e v e n t e d t h e i r d e s t r u c t -i o n by immune l y m p h o i d c e l l s . I n s u p p o r t of t h i s t h e o r y , t h e y showed t h a t s e r a f r o m p a t i e n t s w i t h a c t i v e l y g r o w i n g tumors c o u l d i n d e e d a d s o r b onto tumor c e l l t a r g e t s and b l o c k k i l l i n g by immune c e l l s . However, S j o g r e n e t a l . (117) l a t e r found t h a t b l o c k i n g a c t i v i t y e l u t e d f r o m tumor c e l l s o b t a i n e d f r o m human a s c i t e s o r p l e u r a l e f f u s i o n s was s e p a r a b l e i n t o 2 components o f h i g h o r low m o l e c u l a r w e i g h t s . N e i t h e r f r a c t i o n c o u l d b l o c k w i t h o u t t h e o t h e r and b l o c k i n g o c c u r r e d o n l y i f tumor c e l l s were exposed t o t h e h i g h m o l e c u l a r w e i g h t component b e f o r e c e l l s were washed and t h e low m o l e c u l a r w e i g h t component was added. I t a p p e a r e d , t h e r e f o r e , t h a t a n t i g e n - a n t i b o d y complexes were r e s p o n s i b l e f o r b l o c k i n g t h e k i l l i n g o f tumor c e l l s by l y m p h o c y t e s . C u r r i e (28) found t h a t t h e a b i l i t y o f lymp h o c y t e s f r o m melanoma p a t i e n t s t o k i l l tumor c e l l s c o u l d be i n c r e a s e d by washing o f f b l o c k i n g s u b s t a n c e s assumed t o be a n t i g e n . These f a c t o r s and t h o s e found i n the serum o f t h e same p a t i e n t s had m o l e c u l a r w e i g h t s o f 20,000 t o 40,000 as a s s e s s e d by g e l f i l t r a t i o n on Sephadex G-100 4 and b l o c k e d o n l y i f p r e - i n c u b a t e d w i t h t h e l y m p h o c y t e s . B l o c k i n g of s p e c i f i c immune r e s p o n s e s has a l s o been o b s e r v e d i n a number of d i f f e r e n t mouse tumor systems. Bhatnagar e t a l . (16) were a b l e t o d e m o n s t r a t e a l a r g e m o l e c u l a r w e i g h t p r o t e i n i n t h e a s c i t i c f l u i d o f m i c e i n o c u l a t e d w i t h a m e t h y l c h o l a n t h r e n e - i n d u c e d f i b r o s a r c o m a . T h i s p r o t e i n b l o c k e d the p r o d u c t i o n of MIF and the b l a s t o g e n i c r e s p o n s e o f s p l e e n c e l l s i n t h e p r e s e n c e of tumor a n t i g e n . G o r c z y n s k i a n d K n i g h t (52) found t h a t l a t e p r o g r e s s o r or e a r l y r e g r e s s o r s e r a f r o m M u r i n e Sarcoma V i r u s (MSV)-induced t u m o r - b e a r e r s were c a p a b l e of b l o c k i n g t h e c y t o t o x i c r e s p o n s e of T c e l l s t o tumor c e l l s . They assumed t h a t b l o c k i n g was due t o a n t i g e n - a n t i b o d y complexes i n a n t i g e n e x c e s s s i n c e o n l y p a r t of the b l o c k i n g a c t i v i t y was a d s o r b e d by anti-mouse i m m u n o g l o b u l i n (MIg). Youn e t a l . (137) showed t h a t t h e i n h i b i t i o n o f growth of tumor c o l o n i e s by immune l y m p h o i d c e l l s c o u l d be b l o c k e d by s e r a f r o m advanced tumor-b e a r e r s . They assumed t h a t b l o c k i n g was due t o complexes s i n c e lymph-o c y t e s , not tumor c e l l s , a d s o r b e d t h e f a c t o r . S i m i l a r o b s e r v a t i o n s have been made i n r a t s . B a l d w i n et: a l . (8,9) f o u n d t h a t s e r a f r o m r a t s b e a r i n g c h e m i c a l l y i n d u c e d hepatomas i n h i b i t e d t h e lymph node c e l l s f r o m immune r a t s when i n c u b a t e d w i t h e f f e c t o r b u t not t a r g e t c e l l s . C u r r i e e t a l . (29) i m p l i c a t e d f r e e a n t i g e n as the b l o c k i n g f a c t o r i n r a t s b e a r i n g a sarcoma. Humoral b l o c k i n g f a c t o r s have a l s o been shown t o have an e f f e c t i n v i v o . W e x l e r e t a l . (131) have shown t h a t b l o o d f r o m m i c e b e a r i n g a v a r i e t y of c h e m i c a l l y and v i r a l l y - i n d u c e d tumors c o n t a i n e d f r e e c i r c u l a t -i n g tumor c e l l s w h i c h i n d u c e d l u n g m e t a s t a s e s i n n o r m a l mice when i n j e c t e d i n t r a v e n o u s l y . The number of m e t a s t a s e s i n d u c e d was enhanced when b l o o d c e l l s were suspended i n a u t o l o g o u s t u m o r - b e a r e r serum s u g g e s t i n g t h a t 5 b l o c k i n g f a c t o r s enhanced t h e s u r v i v a l of t h e tumor c e l l s . Fewer m e t a s t a s e s were formed when b l o o d c e l l s were suspended i n s e r a f r o m m i c e t h a t were n o r m a l o r b o r e a d i f f e r e n t tumor t y p e . A l t h o u g h tumor a n t i g e n s and complexes of a n t i g e n and a n t i b o d y c e r t a i n l y do appear t o b l o c k c e l l - m e d i a t e d immunity t o tumor c e l l s i n v i t r o , t h e s u p p r e s s i o n may not be e n t i r e l y a n t i g e n s p e c i f i c . G o r c z y n s k i et al (53) have pr o p o s e d t h a t complexes a r e c a p a b l e of b l o c k i n g a n t i g e n s p e c i f i c T c e l l s by b l o c k i n g r e c e p t o r s w i t h a n t i g e n . However, l y m p h o c y t e s b e a r i n g F c r e c e p t o r s may a l s o b i n d complexes v i a the Fc p o r t i o n o f complexed i m m u n o g l o b u l i n s and thus have t h e i r s p e c i f i c a n t i g e n r e c e p t o r s b l o c k e d by s t e r i c h i n d r a n c e o r i m m o b i l i z a t i o n . I t does ap p e a r , however, t h a t s p e c i f i c l y m p h o c y t e s may be b l o c k e d a t lo w e r c o n c e n t r a t i o n s of the complex. 2. S p e c i f i c S u p p r e s s o r C e l l s S u p p r e s s o r c e l l s have been o b s e r v e d i n mice b e a r i n g P815 mastocytoma. T a k e i e t a l . (124,125) have shown t h a t thymus, s p l e e n , and lymph nodes f r o m m i c e w i t h advanced tumors c o n t a i n e d c e l l s w h i c h s p e c i f i c a l l y s u p p r e s s -ed t h e i n v i t r o g e n e r a t i o n o f a n t i - P 8 1 5 c y t o t o x i c T c e l l s . S u p p r e s s o r c e l l s were T c e l l s and c o u l d be s e p a r a t e d f r o m t h e k i l l e r c e l l s on hypaque-f i c o l l g r a d i e n t s . Fuj.imoto j i t a l . (41,42) have a l s o d e m o n s t r a t e d s u p p r e s s o r c e l l s i n m i c e b e a r i n g m e t h y l c h o l a n t h r e n e - i n d u c e d sarcomas. These c e l l s i n h i b i t e d t h e r e j e c t i o n of tumor c e l l s by immune m i c e , were r e s i s t e n t t o c o r t i s o n e , had l o w d e n s i t y on f i c o l l g r a d i e n t s , and were T c e l l s . 6 IV GENERALIZED IMMUNOSUPPRESSION IN TUMOR-BEARERS P r o g r e s s i v e m a l i g n a n t growth i s o f t e n a s s o c i a t e d , n o t o n l y w i t h a d e p r e s s i o n o f t h e s p e c i f i c immune r e s p o n s e t o tumor a n t i g e n s , but a l s o w i t h a g e n e r a l i z e d immunosuppression. T h i s has been o b s e r v e d w i t h a v a r i e t y o f tumor t y p e s i n human c a n c e r p a t i e n t s and i n e x p e r i m e n t a l tumor systems. A number of d i f f e r e n t jLn v i t r o and i j i v i v o t e s t s o f i m m u n o l o g i c a l r e s p o n s i v e n e s s have been employed t o d e m o n s t r a t e t h e anergy a s s o c i a t e d w i t h c a n c e r i n humans. F o r example, p e r i p h e r a l b l o o d l y m p h o c y t e s have been a s s a y e d i n v i t r o f o r t h e i r p r o l i f e r a t i v e r e s p o n s e t o t h e m i t o g e n p h y t o h a e m a g g l u t i n i n (PHA). In v i v o t e s t s have i n c l u d e d t h e d e l a y e d h y p e r s e n s i t i v i t y r e s p o n s e s t o d i n i t r o c h l o r o b e n z e n e (DNCB) o r K e y h o l e l i m p e t haemocyanin (KLH) a f t e r i m m u n i z a t i o n and c h a l l e n g e , and t h e e s t a b l i s h e d d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s t o r e c a l l a n t i g e n s . A number o f f a i r l y e x t e n s i v e s t u d i e s have d e m o n s t r a t e d d e p r e s s i o n o f m i t o g e n and d e l a y e d h y p e r s e n s i t i v i t y r e s p o n s e s i n c a n c e r p a t i e n t s w i t h a v a r i e t y o f non-lymphoid tumors (35,36,87,97,99,115) as w e l l as i n r e t i c u l o -e n d o t h e l i a l c a n c e r s s u c h as myelogenous l e u k e m i a ( 5 9 ) . I n a d d i t i o n t o a d e c r e a s e i n immune r e s p o n s i v e n e s s , t h e number o f T c e l l s i n t h e b l o o d o f c a n c e r p a t i e n t s has a l s o been found t o be low. F o r example, T c e l l r o s e t t e s formed w i t h sheep r e d b l o o d c e l l s (SRBC) have been found t o be d e p r e s s e d i n p a t i e n t s w i t h g a s t r i c (99) and l u n g c a n c e r s ( 9 7 ) . A g e n e r a l t r e n d o b s e r v e d i n t h e s e s t u d i e s i s t h a t the deg r e e o f immuno-s u p p r e s s i o n found i n c a n c e r p a t i e n t s i s d i r e c t l y r e l a t e d t o t h e c l i n i c a l s t a g e of t h e d i s e a s e (35,87,115). A n e r g i c p a t i e n t s t e n d t o have advanced d i s e a s e and a h i g h f r e q u e n c y o f m e t a s t a s e s and r e c u r r e n c e s (87,115). They do n o t g e n e r a l l y respond t o chemotherapy and t h e i r p r o g n o s i s i s poor (97,115). Thus t h e s t u d y of immune r e s p o n s i v e n e s s t o common a n t i g e n s and m i t o g e n s may h e l p 7 the p h y s i c i a n e v a l u a t e t h e p a t i e n t ' s c l i n i c a l s t a t u s and d e c i d e on a t h e r a p y s c h e d u l e . I n one s t u d y o f p a t i e n t s w i t h c a r c i n o m a o f t h e l u n g , the number of SRBC r o s e t t e s i n p a t i e n t b l o o d l y m p h o c y t e s dropped 3 months b e f o r e r e c u r r e n t d i s e a s e was o b s e r v e d ( 9 7 ) . T h e r e f o r e , i f t e s t s a p p r o p r i a t e f o r a p a r t i c u l a r c a n c e r a r e c a r r i e d out on a r e g u l a r b a s i s , i t may be p o s s i b l e t o p r e d i c t t h e c l i n i c a l c o u r s e o f a g i v e n p a t i e n t . S i m i l a r l y , t h e p r o l i f e r a t i v e r e s p o n s e s of mouse s p l e e n c e l l s t o b o t h B and T c e l l m i t o g e n s d e c l i n e w i t h a d v a n c i n g tumor s i z e . The r e s p o n s e s o f s p l e e n c e l l s t o t h e m i t o g e n s PHA and c o n c a n a v a l i n A (Con A) have been found t o be d e p r e s s e d i n mi c e b e a r i n g m e t h y l c h o l a n t h r e n e - i n d u c e d sarcomas (1,46) and MSV-i n d u c e d (71) tumors. I n most c a s e s , t h e r e s p o n s e s t o B c e l l m i t o g e n s s u c h as l i p o p o l y s a c c h a r i d e (LPS) o r pokeweed m i t o g e n a r e l e s s a f f e c t e d ( 4 6 , 7 1 ) . A d e p r e s s i o n i n a n t i b o d y r e s p o n s e t o h o r s e r e d b l o o d c e l l s (HRBC), LPS, and 4-h y d r o x y - 3 - i o d o - 5 - n i t r o p h e n y l a c e t i c a c i d (NIP) c o u p l e d t o b o v i n e gamma-globulin (BGG) i n m i c e w i t h mammary tumors was found by Rowland e t a l . (113) t o be more s e v e r e when t h e immune r e s p o n s e e x h i b i t e d a g r e a t e r T c e l l dependence. Thus i t a p p e a r s t h a t T c e l l s may be more s e n s i t i v e t o t h e immunosuppressive e f f e c t s o f tumor growth t h a n B c e l l s . S i n c e T c e l l s a ppear t o be i n v o l v e d i n a n t i -tumor immunity, a g e n e r a l i z e d l o s s o f T c e l l f u n c t i o n may e l i m i n a t e t h i s means of c o n t r o l l i n g tumor growth. T h i s p a t t e r n o f s e n s i t i v i t y may v a r y , however, between d i f f e r e n t tumor t y p e s . Immunosuppression seen i n t u m o r - b e a r i n g m i c e appears t o be r e s t r i c t e d t o s p l e e n c e l l s . Lymph node c e l l s i n a s p l e e n - s u p p r e s s e d a n i m a l o f t e n have normal immune r e s p o n s i v e n e s s when t e s t e d i n v i t r o ( 4 6 ) . The s e v e r i t y o f the s u p p r e s s -i o n seen i n s p l e e n c e l l s i n c r e a s e s w i t h a d v a n c i n g tumor growth (46) and r e v -e r s e s a f t e r r e m i s s i o n (71) o r s u r g i c a l r e m o v a l o f t h e tumor ( 4 6 ) . I t i s of p o t e n t i a l i m p o r t a n c e t o u n d e r s t a n d t h e mechanisms by w h i c h tumor 8 development d e p r e s s e s immune r e s p o n s i v e n e s s s i n c e t h e outcome o f c a n c e r t h e r a p y may be i n t i m a t e l y r e l a t e d t o t h e s t a t u s of the immune system. F o r example, L e v y e t a l . ' (82) found t h a t immunotherapy w i t h BCG ( B a c i l l u s - C a l m e t t e - G u e r i n ) and s o l u b l e e x t r a c t s o f tumor c e l l s was e f f e c t i v e i n r e d u c i n g tumor r e c u r r e n c e a f t e r s u r g e r y o n l y when the a n i m a l s had competent immune systems a t the t i m e o f s u r g e r y . V MECHANISMS OF GENERALIZED IMMUNOSUPPRESSION IN TUMOR-BEARERS A number o f d i f f e r e n t mechanisms by w h i c h g e n e r a l i z e d immunosuppression o c c u r s i n t u m o r - b e a r e r s have been p r o p o s e d . A l t h o u g h immunosuppression a p p e a r s t o be me d i a t e d d i f f e r e n t l y i n d i s t i n c t tumor t y p e s , a l l o f t h e d i f f e r -e n t mechanisms d e s c r i b e d may o p e r a t e t o v a r y i n g d e grees and a t d i f f e r e n t t i m e s d u r i n g tumor development. However, an u n d e r s t a n d i n g of t h e i r r e l a t i o n s h i p i s c o m p l i c a t e d by t h e d i f f e r e n t a s s a y s used t o s t u d y immunosuppression. 1. Tumor P r o d u c t s Tumor c e l l s t h e m s e l v e s have been o b s e r v e d t o be immunosuppressive. F o r example, P l e s c i a e t a l . (106) p r o p o s e d t h a t some m e t h y l c h o l a n t h r e n e -i n d u c e d tumors i n mi c e produce and r e l e a s e p r o s t a g l a n d i n s w h i c h d e p r e s s t h e r e s p o n s e s o f ly m p h o i d c e l l s i n t h e m i c r o - e n v i r o n m e n t o f t h e tumor i t s e l f and l a t e r i n t h e whole a n i m a l . T h e i r p r o p o s a l has been s u p p o r t e d by the e v i d e n c e t h a t t h e In v i t r o s u p p r e s s i v e a c t i o n o f such tumor c e l l s on t h e PHA r e s p o n s e o f normal s p l e e n c e l l s was p a r t i a l l y b l o c k e d by p r o s t a g l a n d i n i n h i b i t o r s . S i m i l a r l y , plasmacytoma and mastocytoma tumor c e l l s o r e x t r a c t s were shown by F r i e d m a n ej; a l , (40) t o be s u p p r e s s i v e f o r i n v i t r o a n t i b o d y p r o d u c t i o n by no r m a l l y m p h o i d c e l l s . A s c i t e s f r o m tumors grown i n t r a p e r i t o n e a l l y was a l s o s u p p r e s s i v e f o r a n t i b o d y p r o d u c t -i o n i n v i t r o (40) and i n v i v o ( 5 5 ) . F o r example, Hamaoka e t a l . (56) 9 showed t h a t m i c e i n j e c t e d w i t h a s c i t i c f l u i d f r o m E h r l i c h tumors-bearers had d e p r e s s e d a n t i b o d y p r o d u c t i o n t o T c e l l dependent a n t i g e n s . However, i n t h i s c a s e , t h e y s u g g e s t e d t h a t t h e a s c i t i c f l u i d may p r e v e n t T c e l l s f r o m r e a c h i n g l y m p h o i d o r g a n s where a n t i b o d y i s s y n t h e s i z e d . Most of t h e s e s t u d i e s a r e ambiguous, s i n c e tumors and a s c i t i c f l u i d s a r e i n f i l t r a t e d by h o s t l y m p h o i d c e l l s w h i c h may be t h e s o u r c e o f immuno-s u p p r e s s i v e s u b s t a n c e s . 2. Humoral F a c t o r s Many d i f f e r e n t k i n d s o f non s p e c i f i c a l l y immunosuppressive humoral f a c t o r s have been d e s c r i b e d i n human c a n c e r p a t i e n t s and t u m o r - b e a r i n g a n i m a l s . These f a c t o r s have been i d e n t i f i e d as a - g l o b u l i n s , a n t i g e n -a n t i b o d y complexes, i m m u n o g l o b u l i n s or i m m u n o g l o b u l i n * - a s s o c i a t e d f a c t o r s , and s m a l l p e p t i d e s . S e r a f r o m human c a n c e r p a t i e n t s w i t h a v a r i e t y of non-lymphoid tumors have been f o u n d t o be s u p p r e s s i v e f o r normal l y m p h o i d r e s p o n s e s t o PHA and a l l o g e n e i c c e l l s (18,120) i n v i t r o . I n a s t u d y o f p a t i e n t s w i t h i n t r a c r a n i a l tumors by B r o o k s e t a l . ( 1 8 ) , t h e i m m u n o g l o b u l i n f r a c t i o n f r o m p a t i e n t s e r a was found t o s u p p r e s s PHA and mixed l e u k o c y t e r e a c t i o n s i n v i t r o . The s u p p r e s s i o n c o r r e l a t e d w i t h the in v i v o s u p p r e s s i o n o f t h e DNCB r e s p o n s e . Immunosuppressive f a c t o r s have been found i n t h e o ^ - g l o b u l i n p o r t i o n of n o r m a l human serum. T h i s f a c t o r , c a l l e d n o r m a l immunosuppressive p r o t e i n (NIP) or im m u n o r e g u l a t o r y a l p h a g l o b u l i n ( I R A ) , i s s u p p r e s s i v e f o r T c e l l dependent immune r e s p o n s e s s u c h as a n t i b o d y p r o d u c t i o n t o SRBC (4) and c e l l m e d i a t e d c y t o t o x i c i t y . F o r example, G l a s e r e t a l . (48) fo u n d t h a t human IRA i n h i b i t s c e l l m e d i a t e d c y t o t o x i c i t y a g a i n s t M u r i n e F r i e n d V i r u s - i n d u c e d l e u k e m i a . T h e r e i s some e v i d e n c e t h a t t h i s f a c t o r may 10 p r o v i d e an i m m u n o r e g u l a t o r y f u n c t i o n s i n c e i t i n c r e a s e s d u r i n g a n t i g e n i c s t i m u l a t i o n (19,129). C o r r e l a t e d w i t h t h i s , i s t h e f a c t t h a t s e v e r a l tumors assumed t o be s t r o n g l y a n t i g e n i c a r e a s s o c i a t e d w i t h i n c r e a s e d serum a - g l o b u l i n s ( 1 7 ) . Nimberg and Glasgow e t a l . (49,91) have shown t h a t serum f r o m human c a n c e r p a t i e n t s c o n t a i n s n o t o n l y t h e a - g l o b u l i n s u p p r e s s i v e f a c t o r b u t a s m a l l e r s u p p r e s s i v e p e p t i d e o f m o l e c u l a r w e i g h t l e s s t h a n 10,000. T h i s p e p t i d e , w h i c h has been s e p a r a t e d f r o m a - g l o b u l i n s by DEAE c e l l u l o s e chromatography and d i a f i l t r a t i o n , i s s i m i l a r t o IRA i n t h a t i t s u p p r e s s e s T c e l l f u n c t i o n s . I t has been s u g g e s t e d t h a t t h e p e p t i d e i s c a r r i e d by a - g l o b u l i n s n o r m a l l y and i s found i n a non-bound s t a t e o n l y when i n e x c e s s ( 9 1 ) . Humoral f a c t o r s have a l s o been p r o p o s e d as m e d i a t o r s of immuno-s u p p r e s s i o n i n e x p e r i m e n t a l a n i m a l s . Bogden a t a l . (17) were a b l e t o d i f f e r e n t i a t e h i s t o l o g i c a l l y s i m i l a r m e t h y l c h o l a n t h r e n e - i n d u c e d tumors i n r a t s by t h e e l e c t r o p h o r e t i c a l l y d i s t i n c t tumor s p e c i f i c a-2 g l y c o -p r o t e i n s i n t h e serum of t u m o r - b e a r e r s . Tumors were i d e n t i f i e d as s t r o n g , weak or n o n - s e c r e t o r s . Tumors a s s o c i a t e d w i t h s t r o n g s e c r e t i o n s grew f a s t e r s u g g e s t i n g t h a t t h e g l y c o p r o t e i n may have a s u b v e r s i v e e f f e c t on t h e immune system. However, i t i s n o t known i f t h i s f a c t o r was produced by the tumor o r by h o s t l i v e r c e l l s . A n t i g e n - a n t i b o d y complexes i n t u m o r - b e a r e r serum have been pr o p o s e d as m e d i a t o r s of non s p e c i f i c s u p p r e s s i o n . G o r c z y n s k i et^ a l . (54) have s u g g e s t e d t h a t complexes a r e a b l e t o non s p e c i f i c a l l y b l o c k T c e l l f u n c t i o n s by b i n d i n g t o F c r e c e p t o r s . K i l b u r n e_t aJL. (69) have a l s o s u g g e s t e d t h a t complexes of a g g r e g a t e d i m m u n o g l o b u l i n s may f o c u s i n h i b i t -o r s f r o m t u m o r - b e a r e r s e r a onto l y m p h o i d c e l l s s i n c e immune complexes 11 s y n e r g i z e w i t h t u m o r - b e a r e r serum i n t h e s u p p r e s s i o n o f m i t o g e n i c r e s p o n s e s t o Con A and LPS, However, i f tumor a n t i g e n and a n t i g e n -a n t i b o d y complexes do p l a y a r o l e i n non s p e c i f i c immunosuppression i n v i v o , t h e n t h e r e s h o u l d be l a r g e q u a n t i t i e s o f a n t i g e n i n the b l o o d of t u m o r - b e a r e r s . I f t h i s i s t h e c a s e , t h e n i t i s odd t h a t t h e r e have n o t been r e p o r t s o f t u m o r - s p e c i f i c a n t i g e n s i s o l a t e d f r o m t h i s s o u r c e and t h a t c a n c e r p a t i e n t s do n o t d i e f r e q u e n t l y of k i d n e y f a i l u r e . L e v y and Whitney e t a l . (84,132) have d e m o n s t r a t e d an i m m u n o g l o b u l i n -a s s o c i a t e d s u p p r e s s i v e f a c t o r i n t h e serum of mi c e b e a r i n g advanced m e t h y l c h o l a n t h r e n e - i n d u c e d tumors. T h i s f a c t o r , w h i c h s u p p r e s s e s a v a r i e t y of T c e l l dependent r e s p o n s e s , i s removed by a n t i b o d i e s t o t h e K, Y and u but n o t t h e a c h a i n o f mouse i m m u n o g l o b u l i n s ( 8 3 ) . V e r y l i t t l e i s known f o r c e r t a i n about the o r i g i n o f humoral f a c t o r s o r t h e way i n w h i c h t h e y o p e r a t e . The i m m u n o g l o b u l i n - a s s o c i a t e d f a c t o r s d e s c r i b e d by Whitney e_t a l . (135) f o r example, a r e produced even i n s p l e n e c t o m i z e d t u m o r - b e a r e r s known t o have non s p e c i f i c s u p p r e s s o r c e l l s i n s p l e e n s b u t not lymph nodes. Humoral f a c t o r s may t h e r e f o r e be q u i t e i n d e p e n d e n t o f s u p p r e s s o r c e l l s i n t h e same a n i m a l . 3. S u p p r e s s o r C e l l s More r e c e n t l y , s u p p r e s s o r c e l l s have been r e p o r t e d i n a v a r i e t y of ly m p h o i d and non-lymphoid tumor systems i n humans and e x p e r i m e n t a l a n i m a l s . However, t h e y w i l l be d i s c u s s e d l a t e r i n s e c t i o n V I I I i n t h e c o n t e x t of non s p e c i f i c s u p p r e s s o r c e l l s . V I SUPPRESSOR CELLS IN IMMUNE REGULATION The c o n c e p t of s u p p r e s s o r c e l l s has been i n e x i s t e n c e f o r l e s s t h a n t e n 12 y e a r s and y e t t h e volume of l i t e r a t u r e on the s u b j e c t i s s u b s t a n t i a l ( 1 3 0 ) . S u p p r e s s o r c e l l s have been o b s e r v e d i n e s s e n t i a l l y e v e r y k i n d o f immune r e s p o n s e . They a r e , f o r example, i n v o l v e d i n maintenance of i m m u n o l o g i c a l t o l e r a n c e , l i m i t a t i o n of humoral r e s p o n s e s , c o n t a c t s e n s i t i v i t y and d e l a y e d h y p e r s e n s i t i v i t y , c e l l m e d i a t e d r e s p o n s e s t o a l l o a n t i g e n s , g e n e t i c c o n t r o l o f t h e immune r e s p o n s e , a n t i g e n c o m p e t i t i o n , and a l l o t y p e s u p p r e s s i o n . No one t y p e o f l y m p h o i d c e l l i s r e s p o n s i b l e f o r s u p p r e s s i o n and t h e r e i s no u n i v e r s a l mechanism by w h i c h s u p p r e s s o r c e l l s a c t . However, s e v e r a l g e n e r a l s t a t e m e n t s can be made: 1. S u p p r e s s o r c e l l s may be s p e c i f i c o r non s p e c i f i c i n a c t i o n . S p e c i f i c s u p p r e s s o r c e l l s i n h i b i t o n l y t h e r e s p o n s e t o t h e . a n t i g e n t o w h i c h t h e y were i n d u c e d whereas non s p e c i f i c c e l l s i n h i b i t t h e r e s p o n s e s t o a v a r i e t y o f a n t i g e n s . 2. Most s u p p r e s s o r c e l l s appear t o be i n v o l v e d w i t h some a s p e c t of i m m u n o r e g u l a t i o n and an e x c e s s i v e number of s u p p r e s s o r c e l l s i n v i v o u s u a l l y r e f l e c t s a d i s o r d e r i n r e g u l a t i o n . A l t h o u g h s u p p r e s s o r c e l l s may p r o v i d e an e s s e n t i a l r e g u l a t o r y r o l e i n th e g e n e r a l immune r e s p o n s e , t h e y appear t o be d e t r i m e n t a l i n t h e case o f m a l i g n a n c y when a p o t e n t i a l l y b e n e f i c i a l immune r e s p o n s e may be s p e c i f i c a l l y o r non s p e c i f i c a l l y a b o r t e d by s u p p r e s s o r c e l l s . Thus, i t i s hoped t h a t the s t u d y of s u p p r e s s o r c e l l s w i l l n ot o n l y f u r t h e r t h e u n d e r s t a n d i n g o f t h e immune r e s p o n s e i n g e n e r a l b u t w i l l p r o v i d e i n s i g h t s i n t o t h e t h e r a p y and management of c a n c e r p a t i e n t s . V I I SPECIFIC SUPPRESSOR CELLS S u p p r e s s o r c e l l s have been found t o p l a y a r o l e i n t h e r e g u l a t i o n o f a w i d e v a r i e t y o f immune r e s p o n s e s t o s p e c i f i c a n t i g e n s . I n a l l c a s e s i n w h i c h t h e s u p p r e s s i o n has been shown t o be a n t i g e n s p e c i f i c , t h e s u p p r e s s o r c e l l s have been i d e n t i f i e d as T c e l l s . 13 S u p p r e s s o r T c e l l s h ave, f o r example, been . i d e n t i f i e d as one of t h e m e d i a t o r s of i m m u n o l o g i c a l t o l e r a n c e . Gershon and Kondo (43) d e s c r i b e d a s t a t e of ' i n f e c t i o u s i m m u n o l o g i c a l t o l e r a n c e ' i n w h i c h n o n r e s p o n s i v e n e s s f o r a s p e c i f i c a n t i g e n c o u l d be produced i n i r r a d i a t e d , thymectomized m i c e by t h e t r a n s f e r of s p l e e n c e l l s f rom t o l e r a n t m i c e . Low zone t o l e r a n c e produced by m u l t i p l e i n j e c t i o n s of phage FD has been shown by K o l s c h ej: a l . (75) t o be due t o h y d r o c o r t i s o n e r e s i s t a n t s u p p r e s s o r T c e l l s . T o l e r a n c e , w h i c h c o u l d be t r a n s f e r r e d w i t h s p l e e n c e l l s , was o b s e r v e d as d e p r e s s e d IgG p r o d u c t i o n and d i d n o t o c c u r when s p l e e n c e l l s were t r a n s f e r r e d t o a n t i g e n primed m i c e . They s u g g e s t e d t h a t t h e s u p p r e s s o r c e l l s p r e v e n t e d a B c e l l s w i t c h f r o m IgM t o IgG p r o d u c t i o n . Low zone t o l e r a n c e was a l s o o b s e r v e d by Baker (7) i n t h e r e s p o n s e o f m i c e t o t h e T c e l l i n d e p e n d e n t a n t i g e n Type I I I pneumococcal p o l y s a c c h a r i d e ( S S S - I I I ) . I n t h i s c a s e , i t was n o t p o s s i b l e t o i n d u c e t o l e r a n c e i n c o n g e n i t a l l y a t h y m i c m i c e showing t h a t T c e l l s were i n d e e d r e s p o n s i b l e f o r t h e i n d u c t i o n o f t o l e r a n c e . S u p p r e s s o r T c e l l s have a l s o been found t o be r e s p o n s i b l e f o r t h e t o l e r a n c e i n d u c e d when mi c e a r e i n j e c t e d w i t h d e a g g r e g a t e d p r e p a r a t i o n s o f f o w l gamma g l o b u l i n . B a s t e n el: a l . (14) found t h a t t h e c e l l s w h i c h t r a n s f e r t h i s t o l e r a n c e c o u l d be removed by i m m u n o l o g i c a l 125 s u i c i d e p r o c e d u r e s . That i s , t r e a t m e n t o f s p l e e n c e l l s w i t h I l a b e l l e d a n t i g e n b e f o r e a d o p t i v e t r a n s f e r p r e v e n t e d the t r a n s f e r of t o l e r a n c e . S u p p r e s s o r T c e l l s a r e i n v o l v e d i n t h e r e g u l a t i o n o f normal humoral r e s p o n s e s t o b o t h T c e l l dependent and i n d e p e n d e n t a n t i g e n s . Baker (7) showed t h a t t h e r e s p o n s e t o S S S - I I I a n t i g e n was enhanced by t r e a t m e n t of m i c e w i t h a n t i - l y m p h o c y t e serum ( A L S ) , a t r e a t m e n t w h i c h i s known t o k i l l T c e l l s . Tada e t a l . (122) d e m o n s t r a t e d t h a t t h e magnitude and d u r a t i o n o f IgE a n t i b o d y r e s p o n s e s t o t h e h a p t e n d i n i t r o p h e n y l c o u p l e d t o A s c a r i s p r o t e i n e x t r a c t (DNP-ASC) i s l i m i t e d by s u p p r e s s o r T c e l l s . The d u r a t i o n o f IgE p r o d u c t i o n i s 14 i n c r e a s e d by i r r a d i a t i o n , thymectomy, s p l e n e c t o m y and ALS t r e a t m e n t . S u p p r e s s -o r c e l l s i n t h i s system a r e c a r r i e r s p e c i f i c and may be r e p l a c e d by a s o l u b l e e x t r a c t o b t a i n e d by r e p e a t e d f r e e z e - t h a w i n g or s o n i c a t i o n o f s p l e e n c e l l s or thymocytes f r o m hyperimmunized a n i m a l s . The a c t i v e e x t r a c t component i s a p r o t e i n of m o l e c u l a r w e i g h t 35,000 t o 60,000 w h i c h i s a d s o r b e d by c a r r i e r and removed by a n t i - t h y m o c y t e serum. The e x t r a c t and c e l l s a l s o e x p r e s s an a n t i g e n coded f o r by t h e I - J r e g i o n on the M a j o r H i s t o c o m p a t i b i l i t y Complex (MHC) i n m i c e (121 ) . S u p p r e s s o r T c e l l s have a l s o been i m p l i c a t e d i n t h e g e n e t i c c o n t r o l o f s p e c i f i c immune r e s p o n s e s . B e n a c e r r a f e t a l . (15) were a b l e t o show t h a t i n b r e d mice t h a t a r e n o n r e s p o n d e r s t o t h e s y n t h e t i c polymer o f L - g l u t a m i c a c i d , L - a l a n i n e and L - t y r o s i n e (GAT) produced s u p p r e s s o r c e l l s when immunized w i t h GAT. These s u p p r e s s o r c e l l s were r a d i o s e n s i t i v e T c e l l s w h i c h i n h i b i t e d the r e s p o n s e t o GAT complexed to m e t h y l a t e d b o v i n e serum a l b u m i n , a c o m b i n a t i o n w h i c h g e n e r a l l y i n i t i a t e d an immune r e s p o n s e i n t h e s e m i c e . C h r o n i c a l l o t y p e s u p p r e s s i o n , o b s e r v e d i n a t l e a s t one s t r a i n o f mouse, a p p e a r s t o be m e d i a t e d by s u p p r e s s o r T c e l l s . E x p r e s s i o n o f t h e I g - l b i m m u n o g l o b u l i n a l l o t y p e i s s u p p r e s s e d i n t h e s e mice by p r e n a t a l e x p o s u r e t o a n t i - a l l o t y p e serum. C e l l s c a r r y i n g the I g - l b a l l o t y p e a r e p r e s e n t i n t h e mice a l t h o u g h t h e y do not mature i n t o i m m u n o g l o b u l i n s e c r e t i n g c e l l s . Sup-p r e s s i o n can be a d o p t i v e l y t r a n s f e r r e d and has been shown t o be due t o T c e l l s b e a r i n g I r e g i o n d e t e r m i n a n t s coded by the I - J r e g i o n ( 8 9 , 9 5 ) . S i m i l a r l y , Eichmann (34) has f o u n d t h a t t h e i n j e c t i o n o f low doses o f a n t i b o d i e s d i r e c t e d t o a s p e c i f i c i d i o t y p e produced a s t a t e o f c h r o n i c sup-p r e s s i o n of t h a t i d i o t y p e . The s u p p r e s s i o n c o u l d be a d o p t i v e l y t r a n s f e r r e d by T c e l l s w h i c h were adso r b e d by h i s t a m i n e c o a t e d beads. S u p p r e s s o r T c e l l s a l s o p l a y a r o l e i n the r e g u l a t i o n o f c e l l m e d i a t e d 15 immune r e s p o n s e s . I f , f o r example, m i c e a r e s e n s i t i z e d t o a l l o a n t i g e n s by t h e i n j e c t i o n o f a l l o g e n e i c l y m p h o c y t e s , t h e s p l e e n s of t h e s e a n i m a l s c o n t a i n s u p p r e s s o r T c e l l s w h i c h can r e g u l a t e the p r o l i f e r a t i v e r e s p o n s e and t h e g e n e r a t i o n o f c y t o t o x i c l y m p h o c y t e s i n t h e p r e s e n c e of a l l o a n t i g e n i n v i t r o (111,128). R i c h e t a l . (112) have shown t h a t i n v i v o a c t i v a t e d s u p p r e s s o r T c e l l s , when r e s t i m u l a t e d by s p e c i f i c a n t i g e n i n v i t r o , w i l l r e l e a s e a s o l u b l e f a c t o r w h i c h non s p e c i f i c a l l y i n h i b i t s p r o l i f e r a t i o n i n mixed l e u k o c y t e r e a c t i o n s . A s i m i l a r p a t t e r n i s o b s e r v e d i n t h e s u p p r e s s i o n o f c o n t a c t s e n s i t i v i t y . M i c e i n j e c t e d w i t h p i c r y l s u l f o n i c a c i d d e v e l o p s u p p r e s s o r T c e l l s w h i c h s p e c i f i c a l l y i n h i b i t c o n t a c t s e n s i t i v i t y t o p i c r y l c h l o r i d e . S p e c i f i c s u p p r e s s o r c e l l s can be a d o p t i v e l y t r a n s f e r r e d w i t h lymph node c e l l s f rom s u p p r e s s e d mice. However, A s h e r s o n and Zembala (3) have shown t h a t s u p p r e s s o r T c e l l s can r e l e a s e a s o l u b l e s u p p r e s s o r f a c t o r w h i c h i s a d s o r b e d on t o macrophage s u r f a c e s . Armed macrophages can t h e n s u p p r e s s non s p e c i f i c a l l y upon a d o p t i v e t r a n s f e r p r o v i d i n g t h e s p e c i f i c a n t i g e n i s p r e s e n t . R e c e n t l y i t has been p o s s i b l e t o show t h a t s p e c i f i c s u p p r e s s o r c e l l s g e n e r a t e d d u r i n g a n t i g e n s e n s i t i z a t i o n i n v i v o (24) on i n v i t r o (37) e x p r e s s L y 1 2 + 3 + a l l o a n t i g e n s on t h e i r s u r f a c e . T h i s has p e r m i t t e d t h e s e p a r a t i o n o f h e l p e r and s u p p r e s s o r c e l l s s t u d i e d i n t h e s e s y s t e m s . V I I I NON SPECIFIC SUPPRESSOR CELLS Non s p e c i f i c s u p p r e s s o r c e l l s have been o b s e r v e d i n a w i d e v a r i e t y of i n v i v o and i n v i t r o i m m u n o l o g i c a l s i t u a t i o n s . However, u n l i k e s p e c i f i c s u p p r e s s o r c e l l s , w h i c h g e n e r a l l y a r e T c e l l s , non s p e c i f i c s u p p r e s s o r c e l l s have been d e s c r i b e d as macrophages, T c e l l s , and B c e l l s . 16 1. Macrophages Normal r a t s p l e e n s have been shown t o c o n t a i n macrophages w h i c h e x e r t a s u p p r e s s i v e e f f e c t when s p l e e n c e l l s a r e s t i m u l a t e d i n v i t r o . F o r example, O e h l e r et^ a l . (94) d e m o n s t r a t e d non s p e c i f i c s u p p r e s s o r c e l l s i n n o r m a l W/Fu r a t s p l e e n s w h i c h s u p p r e s s e d p r o l i f e r a t i o n and c y t o t o x i c r e s p o n s e s t o a l l o a n t i g e n s i n v i t r o . The c e l l s were assumed t o be macro-phages s i n c e t h e y were a d h e r e n t , p h a g o c y t i c , and r e s i s t a n t t o i r r a d i a t i o n , a n t i - t h y m o c y t e serum, and m i t o m y c i n C t r e a t m e n t . Macrophages from a non-r e s p o n d e r r a t s t r a i n (BN) have been shown by R a f f and H i n r i c h (109) t o s u p p r e s s t h e p r o l i f e r a t i v e r e s p o n s e s o f r e s p o n d e r r a t (LE) l y m p h o c y t e s t o Con A o r the a n t i g e n , egg a l b u m i n . These f i n d i n g s g i v e an e x p l a n a t i o n f o r p r e v i o u s o b s e r v a t i o n s t h a t s p l e e n c e l l s f r o m r a t s were n o t as r e -s p o n s i v e in v i t r o as t h o s e f r o m mice. P e r i t o n e a l and s p l e e n c e l l s f r o m m i c e i n j e c t e d i n t r a p e r i t o n e a l l y w i t h h e a t k i l l e d C o r y n e b a c t e r i u m parvum (CP) c o n t a i n non s p e c i f i c s u p p r e s s o r c e l l s w h i c h appear t o be macrophages. F o r example, Wing and Remington (136) showed t h a t p e r i t o n e a l c e l l s f r o m CP a c t i v a t e d m i c e i n h i b i t e d 3 32 H - t h y m i d i n e and P - p h o s p h o r i c a c i d u p t a k e by l y m p h o c y t e s s t i m u l a t e d by Con A. S u p p r e s s i o n o c c u r r e d a f t e r m i t o m y c i n C t r e a t m e n t b u t not h e a t k i l l i n g , t h u s DNA s y n t h e s i s was n o t r e q u i r e d b u t v i a b i l i t y was. Lee and B e r r y (79) d e m o n s t r a t e d t h a t p e r i t o n e a l c e l l s f r o m CP i n j e c t e d m i c e c o n t a i n e d two d i s t i n c t p o p u l a t i o n s of a d h e r e n t c e l l s , one o f w h i c h was an a c c e s s o r y c e l l w h i c h promoted humoral immune r e s p o n s e s and one w h i c h was c y t o s t a t i c f o r L1210 l e u k e m i a c e l l s . K i r c h n e r e t a l . (70) a l s o f o u n d t h a t s p l e e n c e l l s f r o m CP a c t i v a t e d m i c e s u p p r e s s e d t h e p r o l i f e r -a t i o n of lymphoma c e l l s and the p r o l i f e r a t i v e r e s p o n s e s o f PHA o r Con A s t i m u l a t e d s p l e e n c e l l s . S i m i l a r i l y , s p l e e n c e l l s f r o m m i c e primed w i t h 17 L i s t e r i a monocytogenes were shown by Kongshavn e t a l . (76) t o s u p p r e s s the ±n v i t r o n o r m a l a n t i b o d y r e s p o n s e t o SRBC. T h i s s u p p r e s s i o n o c c u r r e d even when s u p p r e s s o r c e l l p o p u l a t i o n s were d e p l e t e d of T c e l l s . B a i r d and K a p l a n (5) found t h a t the i n j e c t i o n o f m i c e w i t h p y r a n copolymer, a known r e t i c u l o e n d o t h e l i a l system s t i m u l a t o r , i n c r e a s e d the n o r m a l i n h i b i t o r y e f f e c t of a d h e r e n t c e l l s f r o m the s p l e e n and p e r i t o n e a l c a v i t y . P y r a n a c t i v a t e d c e l l s s u p p r e s s e d the b l a s t o g e n i c r e s p o n s e of s p l e e n c e l l s t o b o t h B and T c e l l m i t o g e n s . S i m i l a r l y , t h i o g l y c o l l a t e i n d u c e d p e r i t o n e a l exudate c e l l s were shown by F e r n b a c h e t a l . (38) t o i n h i b i t p r o l i f e r a t i o n and c e l l m e d i a t e d l y m p h o l y s i s i n t h e mixed l e u c o c y t e c u l t u r e . .Macrophages have a l s o been i m p l i c a t e d i n t h e r e g u l a t i o n o f the g r a f t -v e r s u s - h o s t (GVH) r e a c t i o n . S j o b e r g (116) f o u n d t h a t s u p p r e s s o r c e l l s w h i c h i n h i b i t e d the p r o d u c t i o n of p l a q u e f o r m i n g c e l l s (PFC) t o SRBC and LPS by n o r m a l l y m p h o i d c e l l s c u l t u r e d ija v i t r o were p r e s e n t i n t h e s p l e e n s of F^ m i c e i n j e c t e d w i t h p a r e n t a l l y m p h o i d c e l l s . These s u p p r e s s o r c e l l s were n o t s e n s i t i v e t o a n t i - T h y - 1 serum and p h a g o c y t i z e d i r o n f i l i n g s . The growth o f a v a r i e t y o f d i f f e r e n t t y p e s o f tumors a l s o appear t o i n d u c e s u p p r e s s o r macrophages. F o r example, A r g y r i s and H o r o w i t z (2) showed t h a t s p l e n i c macrophages f r o m m i c e b e a r i n g a l l o g e n e i c P815 or EL4 tumors i n h i b i t e d the c y t o t o x i c i t y o f p e r i t o n e a l exudate c e l l s . These s u p p r e s s o r c e l l s were d e s t r o y e d by a n t i - m a c r o p h a g e serum. K o l b e t a l . (74) d e m o n s t r a t e d r a d i o r e s i s t a n t , , n y l o n w o o l and p l a s t i c a d h e r e n t sup-p r e s s o r c e l l s i n t h e s p l e e n and p e r i t o n e a l c a v i t y o f mice w i t h p l a s m a -cytomas. These c e l l s s u p p r e s s e d t h e p r o d u c t i o n o f a n t i b o d i e s t o SRBC by n o r m a l c e l l s i n c u l t u r e . K r a k a u e r jst a l . (77) d e m o n s t r a t e d s i m i l a r g l a s s a d h e r e n t , p h a g o c y t i c s u p p r e s s o r c e l l s i n a s c i t e s of m i c e b e a r i n g 18 plasmacytomas. K i r c h n e r et^ a l . (72,73) have d e s c r i b e d macrophage s u p p r e s s o r c e l l s i n t h e s p l e e n s o f m i c e b e a r i n g p r o g r e s s o r MSV-induced tumors. These c e l l s , w h i c h i n h i b i t e d the p r o l i f e r a t i v e r e s p o n s e s of n o r m a l , s y n g e n e i c s p l e e n c e l l s t o PHA, and the DNA s y n t h e s i s of c u l t u r e d lymphoma c e l l s , were n y l o n w o o l a d h e r e n t , r a d i o r e s i s t a n t , p h a g o c y t i c , i n a c t i v a t e d by c a r r a g -heenan t r e a t m e n t , and r e s i s t a n t t o a n t i - T h y - 1 serum p l u s complement. G l a s e r e t a l . (47) and O e h l e r et_ aJL. (93) found s i m i l a r s u p p r e s s i v e macrophages i n r a t s b e a r i n g p r o g r e s s o r - G r o s s Leukemia V i r u s - i n d u c e d tumors. S u p p r e s s o r c e l l s , p o s s i b l y macrophages, have a l s o been o b s e r v e d i n one c h e m i c a l l y - i n d u c e d m urine tumor system. Eggers and W u n d e r l i c h (33) found t h a t n y l o n w o o l a d h e r e n t s p l e e n c e l l s f r o m m i c e b e a r i n g m e t h y l -c h o l a n t h r e n e - i n d u c e d tumors i n h i b i t e d t h e i n d u c t i o n of c y t o t o x i c c e l l s a f t e r i n v i t r o i m m u n i z a t i o n a g a i n s t a l l o a n t i g e n s . Human c a n c e r p a t i e n t s a l s o appear t o have non s p e c i f i c s u p p r e s s o r c e l l s . Zembala e t a l . (138) showed t h a t a d h e r e n t , non-SRBC r o s e t t e f o r m i n g , e s t e r a s e - p o s i t i v e , p h a g o c y t i c p e r i p h e r a l b l o o d c e l l s s u p p r e s s e d t h e r e s p o n s e of n o r m a l c e l l s t o m i t o g e n s . They assumed t h a t t h e s e s u p p r e s s o r c e l l s , w h i c h c o n s t i t u t e d about 8 t o 10% of t h e o r i g i n a l p o p u l a t i o n , were c i r c u l a t i n g monocytes. 2. T C e l l s S p l e e n c e l l s f r o m r a t s g i v e n l a r g e i n t r a v e n o u s doses of o v a l b u m i n or SRBC have s u p p r e s s e d r e s p o n s e s t o the m i t o g e n PHA. Bash e t a l . (11,13) s u g g e s t e d t h a t b o t h macrophages and T c e l l s were r e s p o n s i b l e f o r t h e s u p p r e s s i o n s i n c e c a r r a g h e e n a n and low doses of c y c l o p h o s p h a m i d e p a r t i a l l y r e s t o r e d the r e s p o n s e . F o l c h and Waksman (39) have a l s o s t u d i e d the r e s p o n s e s of c e l l s f r o m 19 n o r m a l r a t s p l e e n s . U n l i k e O e h l e r e t d . (94) t h e y have c o n c l u d e d t h a t t h e c e l l s r e s p o n s i b l e f o r r e d u c i n g the r e s p o n s i v e n e s s of r a t c e l l s i n v i t r o a r e g l a s s w o o l a d h e r e n t T c e l l s . They found t h a t r e m o v a l of t h e s e " . c e l l s on g l a s s w o o l columns enhanced the r e s p o n s e o f r a t s p l e e n c e l l s i n t h e mixed l e u c o c y t e c u l t u r e . They c o n c l u d e d t h a t t h e s u p p r e s s o r c e l l s were T c e l l s b ecause c y c l o h e x i m i d e t r e a t m e n t and thymectomy p r e v e n t e d s u p p r e s s i o n . These s u p p r e s s o r c e l l s d i d n o t r e q u i r e DNA s y n t h e s i s b ut d i d r e q u i r e p r o t e i n s y n t h e s i s , F u r t h e r e v i d e n c e was o b t a i n e d by Bash et a l . (12) who f o u n d t h a t s p l e e n c e l l s f r o m a d u l t thymectomized r a t s had s e v e r e l y d e p r e s s e d r e s p o n s e s t o PHA w h i c h c o u l d be r e s t o r e d by r e m o v a l of g l a s s w o o l a d h e r e n t c e l l s or by a t h y m i c f a c t o r . They h y p o t h e s i z e d t h a t the r a t s p l e e n c o n t a i n s T c e l l s w h i c h a r e non s p e c i f i c a l l y s u p p r e s s i v e u n t i l t h e y d i f f e r e n t i a t e , under t h e i n f l u e n c e o f t h y m i c hormones, i n t o more mature c e l l s . I t i s p o s s i b l e , however, t h a t t h e a c t u a l s u p p r e s s i o n by t h i s t y p e of T c e l l i n v o l v e s an i n t e r a c t i o n w i t h macrophages. Burns &t a l . (21) have d e s c r i b e d t h e i n d u c t i o n of s u p p r e s s o r T c e l l s f r o m mouse s p l e e n c e l l s c u l t u r e d f o r 4 days i n medium c o n t a i n i n g f o e t a l c a l f serum (FCS). These ' s p o n t a n e o u s l y i n d u c e d 1 s u p p r e s s o r c e l l s s u p p r e s s e d t h e a n t i b o d y r e s p o n s e of f r e s h l y p r e p a r e d n o r m a l s p l e e n c e l l s when s t i m u l a t e d by a range of T dependent and i n d e p e n d e n t a n t i g e n s . R e c e n t l y , P a r i s h (101) has shown t h a t t h e s e s u p p r e s s o r c e l l s have t h e Ly l + 2 l a phenotype. He has s u g g e s t e d t h a t s u p p r e s s o r c e l l s r e s p o n d t o FCS a n t i g e n s and t h a t they s u b s e q u e n t l y i n a c t i v a t e B c e l l s or a c c e s s o r y c e l l s r e q u i r e d f o r a n t i b o d y f o r m a t i o n w h i c h have p a s s i v e l y a d s o r b e d t h e s e a n t i g e n s . S p l e e n c e l l s f r o m m i c e i n f e c t e d w i t h Trypanosoma b r u c e i have been 20 shown by E a r d l e y and Jayawardena (32) t o c o n t a i n a d h e r e n t T s u p p r e s s o r c e l l s w h i c h i n h i b i t the i n v i t r o a n t i b o d y r e s p o n s e t o SRBC and D N P - F i c o l l as w e l l as t h e p r o l i f e r a t i v e r e s p o n s e s t o B and T c e l l m i t o g e n s . S u p p r e s s -i o n was p a r t l y r e d u c e d by t r e a t m e n t o f c e l l s w i t h a n t i - T h y - 1 serum p l u s complement b u t was n o t r e d u c e d by a n t i - M I g serum p l u s complement. They c o n c l u d e d t h a t s u p p r e s s i o n i s due t o T c e l l s w h i c h have s m a l l amounts of Thy-1 a n t i g e n on t h e i r s u r f a c e and w h i c h a r e n o t s e n s i t i v e t o ALS. They a l s o s u g g e s t e d t h a t macrophages may be i n v o l v e d i n t h e s u p p r e s s i o n s i n c e r e m o v a l o f T h y - 1 + c e l l s d i d n o t remove a l l o f the s u p p r e s s i o n , whereas a ^ c o m b i n a t i o n of n y l o n w o o l adherence and a n t i - T h y - 1 serum t r e a t m e n t d i d . Non s p e c i f i c a l l y s u p p r e s s i v e T c e l l s have been o b s e r v e d i n GVH m i c e . Shand (114) found t h a t s p l e e n c e l l s f r o m GVH mice s u p p r e s s e d t h e i n v i t r o a n t i b o d y r e s p o n s e of n o r m a l s y n g e n e i c c e l l s t o T c e l l dependent and i n d e p e n d e n t a n t i g e n s and d e p r e s s e d B c e l l p r o l i f e r a t i o n . He f o u n d t h a t t h e s u p p r e s s o r c e l l s were g e n e r a t e d f r o m g r a f t e d c e l l s . P i c k e l and Hoffman (104,105) extended t h e s e o b s e r v a t i o n s by showing t h a t t h e sup-+ + p r e s s o r c e l l s were r a d i a t i o n s e n s i t i v e T c e l l s w i t h the Ly 1 2 phenotype. Gershon et_ ad. (44) have s u g g e s t e d t h a t thymocytes l o c a l i z i n g i n t h e s p l e e n s o f m i c e p r o v i d e an i m m u n o r e g u l a t o r y f u n c t i o n . They o b s e r v e d t h a t s p l e n e c t o m y of m i c e , 3 h o u r s a f t e r i n j e c t i o n o f p a r e n t a l t h y m o c y t e s , a l t e r e d the r e s p o n s e i n the lymph nodes. Splenectomy enhanced t h e DNA s y n t h e s i s n o r m a l l y s e e n i n lymph nodes and r e - i n j e c t i o n o f t h e s p l e e n c e l l s s u p p r e s s e d the r e s p o n s e a g a i n . Hodes ejt a l . (60) f o u n d t h a t s p l e e n c e l l s p r i med _in v i t r o w i t h a l l o -a n t i g e n g e n e r a t e d not o n l y r a d i o r e s i s t a n t s p e c i f i c T s u p p r e s s o r c e l l s , b u t r a d i o s e n s i t i v e non s p e c i f i c s u p p r e s s o r c e l l s . These c e l l s i n h i b i t e d mixed l e u c o c y t e c u l t u r e s and c y t o t o x i c l y m p h o c y t e g e n e r a t i o n . However, 21 i t i s p o s s i b l e i n t h i s c a s e , t h a t t h e non s p e c i f i c s u p p r e s s o r c e l l s were i n d u c e d by FCS a n t i g e n s as d e s c r i b e d by P a r i s h ( 1 0 1 ) . The m i t o g e n Con A has been shown by a number o f w o r k e r s t o s t i m u l a t e s u p p r e s s o r T c e l l s w h i c h a r e non s p e c i f i c i n e f f e c t . However, t h i s t e c h n i q u e may a c t u a l l y r e f l e c t a p o l y c l o n a l a c t i v a t i o n of s p e c i f i c sup-p r e s s o r c e l l s . J a c o b s (64) f o u n d t h a t Con A a c t i v a t e s b o t h s t i m u l a t o r and i n h i b i t o r c e l l s w h i c h she assumes a r e d i f f e r e n t s u b p o p u l a t i o n s o f T c e l l s . The s u p p r e s s o r c e l l i s p r o b a b l y a T c e l l s i n c e s u p p r e s s i o n does no t o c c u r i n a d u l t thymectomized m i c e and the t a r g e t i s t h e B c e l l s i n c e r e s p o n s e s t o T c e l l i n d e p e n d e n t a n t i g e n s a r e s u p p r e s s e d . Con A has a l s o been shown by Markham e_t a l . (86) t o d e p r e s s the ±n v i v o a n t i b o d y r e s p o n s e t o S S S - I I I . T h i s s u p p r e s s i o n i s n o t seen i n nude m i c e and i s p a r t l y e l i m i n a t e d by ALS. The k i n e t i c s of s u p p r e s s i o n p a r a l l e l t h a t f o r low dose p a r a l y s i s w i t h S S S - I I I . T h e r e f o r e , i t appears t h a t Con A c a n i n d u c e s u p p r e s s o r T c e l l s i n v i v o w h i c h s u p p r e s s a T c e l l i n d e p e n d e n t humoral r e s p o n s e . Non s p e c i f i c s u p p r e s s o r T c e l l s have n o t been commonly o b s e r v e d i n t u m o r - b e a r e r s . However, T r e v e s e t a l . (127) showed t h a t i n v i t r o a c t i v a t i o n o f l y m p h o c y t e s a g a i n s t s y n g e n e i c f i b r o b l a s t s o r tumor c e l l s p roduced s e n s i t i z e d T c e l l s w h i c h were c y t o t o x i c i n v i t r o b u t enhanced t h e growth of L e w i s Lung Ca r c i n o m a i n v i v o . . S i n c e a n t i - T h y - 1 serum removed t h e e n h a n c i n g c e l l s , t h e y s u g g e s t e d t h a t T s u p p r e s s o r c e l l s were g e n e r a t e d jLn v i t r o . Enhancement o c c u r r e d when i n v i t r o a c t i v a t e d c e l l s were i n j e c t e d f i v e days b e f o r e tumor. T h e r e f o r e t h e y s u g g e s t e d t h a t t h e s u p p r e s s o r c e l l s d e p r e s s e d h o s t r e s i s t a n c e t o t h e tumor. Cerny and S t i l l e r (26,118) have a l s o f o u n d a s u p p r e s s o r c e l l i n t h e s p l e e n s of m i c e w i t h Moloney Leukemia w h i c h s u p p r e s s e s t h e PFC r e s p o n s e t o SRBC. They 22 t h i n k t h a t t h i s c e l l may be a T c e l l s i n c e i t i s found i n t h e T c e l l r i c h n onadherent c e l l s , b u t th e y were not a b l e t o remove i t w i t h a n t i - T h y - 1 serum. Zembala e t a l . (138) a l s o found t h a t the b l o o d o f two human p a t i e n t s w i t h c a n c e r c o n t a i n e d s u p p r e s s o r c e l l s t h a t appeared t o be T c e l l s s i n c e t h e y formed SRBC r o s e t t e s . These c e l l s s u p p r e s s e d t h e m i t o g e n r e s p o n s e o f no r m a l p e r i p h e r a l b l o o d l e u c o c y t e s . A l l i n a l l , t he e v i d e n c e f o r non s p e c i f i c s u p p r e s s o r T c e l l s i s v e r y c o n f u s i n g . However, i t does appear t h a t s u c h c e l l s may e x i s t in. v i v o . They may be of some i m p o r t a n c e i n s i t u a t i o n s i n v o l v i n g a n t i g e n i c c o m p e t i t -i o n and may be due t o the u n u s u a l p r o l i f e r a t i o n o f u n d i f f e r e n t i a t e d , immature t h y m i c c e l l s . They may t h e r e f o r e be o f i m p o r t a n c e i n tumor immunity i f the tumor i s a n t i g e n i c and t h e immune r e s p o n s e t o t h e tumor s t i m u l a t e s t h e p r o d u c t i o n o f t h e s e i m m u n o r e g u l a t o r y c e l l s . 3. B. C e l l s P e r s s o n (102) has de m o n s t r a t e d t h e i n d u c t i o n o f s u p p r e s s o r c e l l s by i n v i v o i n j e c t i o n o f LPS i n t o m i c e . S p l e e n c e l l s f r o m mice i n j e c t e d w i t h h i g h doses of LPS s u p p r e s s IgM and IgG a n t i b o d y r e s p o n s e s t o t h e T c e l l dependent a n t i g e n SRBC In v i t r o . The s u p p r e s s o r c e l l s were assumed t o be B c e l l s s i n c e they were a c t i v a t e d by LPS, and s i n c e a n t i - T h y - 1 serum and c a r b o n y l i r o n t r e a t m e n t s w h i c h remove T c e l l s and macrophages d i d n o t a f f e c t the s u p p r e s s i o n . The mechanism of s u p p r e s s i o n p r o p o s e d i s t h a t LPS a c t i v a t e d B c e l l s r e l e a s e a n t i b o d i e s w h i c h b i n d t o F c r e c e p t o r s on T c e l l s and i n some way s u p p r e s s them. The non s p e c i f i c n a t u r e o f the sup-p r e s s i o n c o u l d be due t o t h e p o l y c l o n a l a c t i v a t i o n o f B c e l l s by LPS. B c e l l s have a l s o been pr o p o s e d t o be s u p p r e s s o r c e l l s i n one tumor system. K i l b u r n e t a l . (68) and G o r c z y n s k i . ( 5 1 ) have d e m o n s t r a t e d t h a t s p l e e n c e l l s f r o m m i c e b e a r i n g s e v e r a l l y v i r a l l y - i n d u c e d and one 23 c h e m i c a l l y - i n d u c e d tumor non s p e c i f i c a l l y s u p p r e s s e d T c e l l r e s p o n s e s such as MIF p r o d u c t i o n t o tumor a n t i g e n and s t i m u l a t i o n by PHA. These s u p p r e s s o r c e l l s were i n a c t i v a t e d by a n t i - M I g serum p l u s complement. They s u g g e s t e d t h a t t h e s e s u p p r e s s o r c e l l s may a c t by r e l e a s i n g a n t i -b o d i e s w h i c h form complexes w i t h tumor a n t i g e n . The complexes may t h e n b i n d t o F c r e c e p t o r s on T c e l l s and i n a c t i v a t e them. I X SOLUBLE FACTORS FROM NON SPECIFIC SUPPRESSOR CELLS I t i s p o s s i b l e t o r e p l a c e s p e c i f i c s u p p r e s s o r T c e l l s i n most a s s a y systems by e i t h e r a s o l u b l e p r o d u c t r e l e a s e d f r o m t h e c e l l o r by a s o l u b l e c e l l membrane e x t r a c t . S i m i l a r i l y , some of t h e non s p e c i f i c s u p p r e s s o r c e l l s appear t o s u p p r e s s v i a a s o l u b l e f a c t o r . 1. Macrophages. Non s p e c i f i c s u p p r e s s o r macrophages do not g e n e r a l l y appear t o s u p p r e s s t h r o u g h a s o l u b l e f a c t o r and seem t o r e q u i r e c e l l c o n t a c t . C a l d e r o n e t a l . (22) f o u n d t h a t c u l t u r e s o f mouse p e r i t o n e a l e x u d a t e c e l l s r e l e a s e d a f a c t o r i n t o t h e s u p e r n a t a n t w i t h a m o l e c u l a r w e i g h t 3 s m a l l e r t h a n 1,400 w h i c h i n h i b i t e d H - t h y m i d i n e i n c o r p o r a t i o n by EL4 l e u k e m i a c e l l s , 3T3 c e l l s and m i t o g e n s t i m u l a t e d l y m p h o c y t e s . However, F e r n b a c h e t a l . (38) showed t h a t the s u p e r n a t a n t had no e f f e c t on b l a s t c e l l c o u n t s or c e l l m e d i a t e d l y m p h o l y s i s and t h a t the a p p a r e n t i n h i b i t i o n 3 c o u l d be r e v e r s e d by u s i n g a low a c t i v i t y H - t h y m i d i n e p r e p a r a t i o n . O p i t z e t a l . ( 9 8 ) , s t u d y i n g the same phenomenon:,, found t h a t RNA and p r o t e i n s y n t h e s i s were n o t i n h i b i t e d . They c o n c l u d e d t h a t t h e a p p a r e n t s u p p r e s s -i o n was due t o a c o m p e t i t i o n between l a b e l l e d t h y m i d i n e and c o l d t h y m i d -i n e r e l e a s e d f r o m d y i n g and p h a g o c y t i z e d c e l l s . 24 B a i r d and K a p l a n (6) d e m o n s t r a t e d t h a t p y r a n a c t i v a t e d macrophages r e q u i r e d c e l l c o n t a c t i n o r d e r t o s u p p r e s s b l a s t f o r m a t i o n . Treatment o f c e l l s w i t h n e u r a m i n i d a s e removed t h e s u p p r e s s i o n s u g g e s t i n g t h a t a g l y -c o p r o t e i n on macrophage s u r f a c e s was n e c e s s a r y f o r i n h i b i t i o n t o o c c u r . H o e s s l i et_ a l _ . (61) d e m o n s t r a t e d a d i a l y s a b l e i n h i b i t o r f r o m t h e s u p e r n a t a n t o f c u l t u r e d p e r i t o n e a l c e l l s f rom mice i n j e c t e d w i t h p r o t e a s e peptone. At l o w c o n c e n t r a t i o n s t h i s s u p e r n a t a n t d e p r e s s e d t h e uptake o f 3 H - t h y m i d i n e , u r i d i n e , and l e u c i n e b u t not t h e a c t u a l s y n t h e s i s o f DNA, p r o t e i n o r RNA. However, a t h i g h e r c o n c e n t r a t i o n s of s u p e r n a t a n t , b o t h u p t a k e o f p r e c u r s o r s and s y n t h e s i s were i n h i b i t e d . They s u g g e s t e d t h a t t h i s c o u l d be due t o t h e p r e s e n c e o f h y d r o l a s e s b r e a k i n g down c e l l s o r c o u l d be due t o a second f a c t o r r e l e a s e d from a minor p o p u l a t i o n o f s u p p r e s s o r T c e l l s i n t h e p e r i t o n e u m . S u p p r e s s o r macrophages from mice primed w i t h L i s t e r i a monocytogenes (76) o r b e a r i n g a m e t h y l c h o l a n t h r e n e - i n d u c e d tumor (33) d i d n o t appear t o p roduce s o l u b l e f a c t o r s . However, K r a k a u e r e t a l . (77) found t h a t a c e l l e x t r a c t o f m o l e c u l a r w e i g h t l e s s t h a n 40,000 from t h e macrophage s u p p r e s s o r c e l l i n t h e a s c i t e s o f mice b e a r i n g plasmacytomas was s u p p r e s s i v e . Tumor n o d u l e e x t r a c t s were n o t s u p p r e s s i v e i n t h i s c a s e , t h u s r u l i n g out t h e p o s s i b i l i t y t h a t t h e e x t r a c t was s o l u b l e tumor a n t i g e n . I t a p p e ars t h a t a l t h o u g h s u p p r e s s i v e macrophages do n o t g e n e r a l l y r e l e a s e a s o l u b l e s u p p r e s s i v e f a c t o r i n t o t h e medium, t h e y may have on t h e i r s u r f a c e a s u p p r e s s i v e component w h i c h i s produced by t h e macrophage i t s e l f o r a d s o r b e d from T c e l l s . T h i s component may, however, s u p p r e s s DNA s y n t h e s i s a f t e r i t has been r e l e a s e d i n a s o l u b l e form d u r i n g t h e p r e p a r a t i o n o f c e l l e x t r a c t s . 25 2. T C e l l s Non s p e c i f i c s u p p r e s s o r f a c t o r s p r o duced by s u p p r e s s o r T c e l l s have been d e m o n s t r a t e d i n a number o f d i f f e r e n t s ystems. However, a l t h o u g h t h e s o l u b l e f a c t o r s t h e m s e l v e s s u p p r e s s non s p e c i f i c a l l y , the T c e l l s sometimes r e q u i r e c o n t a c t w i t h s p e c i f i c a n t i g e n b e f o r e t h e f a c t o r s a r e r e l e a s e d . I t may be t h a t s o l u b l e non s p e c i f i c s u p p r e s s o r f a c t o r s a r e produced by b o t h s p e c i f i c and a p p a r e n t l y non s p e c i f i c s u p p r e s s o r T c e l l s . D o u g l a s and R u b i n (31) d e m o n s t r a t e d t h a t s p l e e n c e l l s f r o m m i c e immunized w i t h HRBC r e l e a s e d an 'antibody i n i t i a t i o n s u p p r e s s o r f a c t o r ' i n t o t h e medium when r e - s t i m u l a t e d w i t h HRBC. T h i s s o l u b l e f a c t o r , w h i c h had a m o l e c u l a r w e i g h t of about 34,000 a f t e r g e l f i l t r a t i o n , s u p p r e s s e d i n v i t r o p r o d u c t i o n o f IgM a n t i b o d y t o SRBC. Thomas e t a l . (126) o b s e r v e d a s i m i l a r f a c t o r when s p l e e n c e l l s f r o m ovalbumin-immune mice were s p e c i f i c a l l y r e - s t i m u l a t e d i n v i t r o . They found t h a t t h e f a c t o r , r e l e a s e d by r a d i o s e n s i t i v e T c e l l s , had a m o l e c u l a r w e i g h t o f 55,000 t o 60,000. T h i s i s r e m i n i s c e n t of the non s p e c i f i c f a c t o r r e l e a s e d by t h e s p e c i f i c s u p p r e s s o r T c e l l s o f A s h e r s o n and Zembala (3) w h i c h non s p e c i f i c -a l l y i n h i b i t s c o n t a c t s e n s i t i v i t y a f t e r macrophage a r m i n g . Con A a c t i v a t i o n o f s u p p r e s s o r T c e l l s a l s o l e a d s t o t h e p r o d u c t i o n of s o l u b l e s u p p r e s s o r f a c t o r s . Tadakuma ejt a l . (123) have o b s e r v e d a g l y c o p r o t e i n o f m o l e c u l a r w e i g h t 48,000 t o 67,000 i n t h e s u p e r n a t a n t of Con A a c t i v a t e d mouse s p l e e n c e l l s . T h i s f a c t o r , w h i c h s u p p r e s s e s a n t i -body p r o d u c t i o n ixi v i t r o , i s d e s t r o y e d by t r y p s i n o r c h y m o t r y p s i n b u t n o t by DNase o r RNase and does n o t have a n t i g e n or s p e c i e s s p e c i f i c i t y . They have named t h i s f a c t o r ' s o l u b l e immune r e s p o n s e s u p p r e s s o r ' o r SIRS. R e i n e r t s e n and S t e i n b e r g (110) have d e m o n s t r a t e d a low m o l e c u l a r w e i g h t ( l e s s t h a n 10,000) f a c t o r i n t h e s u p e r n a t a n t of Con A a c t i v a t e d mouse 26 s p l e e n c e l l s w h i c h s u p p r e s s e d i n " v i t r o and i n v i v o p r i m a r y and s e c o n d a r y a n t i b o d y r e s p o n s e s . They s u g g e s t e d . t h a t t h i s f a c t o r c o u l d be t h e a c t i v e p o r t i o n o f SIRS. Namba e t a l . (90) have o b s e r v e d a s o l u b l e f a c t o r , w h i c h t h e y c a l l ' i n h i b i t o r o f DNA s y n t h e s i s ' o r IDS, i n t h e s u p e r n a t a n t o f Con A a c t i v a t e d r a t spleen,, lymph node o r thymus c e l l s . They s u g g e s t e d t h a t c o r t i c a l thymocytes produced IDS s i n c e thymocytes f r o m r a t s t r e a t e d w i t h h y d r o c o r t i s o n e 48 h o u r s b e f o r e , d i d not r e l e a s e t h e f a c t o r . S p l e e n c e l l s r e l e a s i n g IDS were g l a s s w o o l a d h e r e n t as were t h e non s p e c i f i c s u p p r e s s o r T c e l l s d e s c r i b e d by t h i s group ( 3 9 ) . DNA s y n t h e s i s i s r e q u i r e d f o r IDS p r o d u c t i o n s i n c e no s u p p r e s s i v e f a c t o r was r e l e a s e d by m i t o m y c i n C t r e a t e d c e l l s . A s i m i l a r f a c t o r was a l s o r e l e a s e d by s p l e e n c e l l s f r o m r a t s immunized w i t h h i g h doses o f a n t i g e n . No f a c t o r was r e l e a s e d i f a n i m a l s were i n j e c t e d w i t h b r o m o d e o x y u r i d i n e up t o 12 h o u r s a f t e r i n j e c t i o n w i t h a n t i g e n . PHA s t i m u l a t i o n o f human t o n s i l c e l l s has a l s o been shown by Lee and 3 Luc a s (81) t o i n d u c e s o l u b l e f a c t o r s w h i c h i n h i b i t H - t h y m i d i n e i n c o r p -o r a t i o n by m i t o g e n s t i m u l a t e d l y m p h o c y t e s and c u l t u r e d human c e l l l i n e s . They found t h a t . 2 f a c t o r s o f m o l e c u l a r w e i g h t s 40,000 and 80,000 b o t h i n h i b i t c a l f thymus DNA p o l y m e r a s e a. They s u g g e s t t h a t t h e i r f a c t o r s , w h i c h t h e y c a l l ' i n h i b i t o r o f DNA p o l y m e r a s e ' or IDP a r e s i m i l a r t o IDS. I t a p p e a r s , t h e r e f o r e , t h a t s u p p r e s s o r T c e l l s may r e l e a s e non s p e c i f i c s o l u b l e s u p p r e s s o r f a c t o r s i n t h r e e d i f f e r e n t s i t u a t i o n s : 1: S u p p r e s s o r T c e l l s g e n e r a t e d d u r i n g i m m u n i z a t i o n ^ n v i v o may r e l e a s e f a c t o r s upon s e c o n d a r y s t i m u l a t i o n i n v i t r o . 2. S u p p r e s s o r T c e l l s may be a p o l y c l o n a l l y a c t i v a t e d i n v i t r o by Con A o r PHA. 3. Non s p e c i f i c s u p p r e s s o r T c e l l s , g e n e r a t e d by i n v i v o i n j e c t i o n o f l a r g e doses o f a n t i g e n , may r e l e a s e s o l u b l e f a c t o r s i n v i t r o w i t h o u t s p e c i f i c 27 r e - s t i m u l a t i o n by a n t i g e n . These f a c t o r s may p r o v i d e an e x p l a n a t i o n f o r the, phenomenon of a n t i g e n i c c o m p e t i t i o n . 3. B- C e l l s P e r s s o n et_ a l . (103) have d e s c r i b e d 2 f a c t o r s r e l e a s e d i n t o t h e s u p e r -n a t a n t s of c u l t u r e d n o r m a l s p l e e n c e l l s w h i c h s u p p r e s s t h e DNA s y n t h e s i s and b l a s t f o r m a t i o n by l y m p h o c y t e s s t i m u l a t e d by p o l y c l o n a l B and T c e l l a c t i v a t o r s and the p r i m a r y a n t i b o d y r e s p o n s e t o SRBC i n v i t r o . These f a c t o r s , one of w h i c h i s l e s s t h a n 10,000 and the o t h e r g r e a t e r t h a n 30,000 i n m o l e c u l a r w e i g h t , a r e p r oduced by non a d h e r e n t s p l e e n c e l l s and bone marrow c e l l s b u t n o t by t h y m o c y t e s . They h y p o t h e s i z e t h a t t h e s e f a c t o r s a r e r e l e a s e d by B c e l l s b u t have n o t o f f e r e d any c o m p e l l i n g argument t o t h a t e f f e c t . X THESIS OBJECTIVES Le v y and Whitney e t a l . (83,84,132,133) d e m o n s t r a t e d t h a t m i c e b e a r i n g l a r g e m e t h y l c h o l a n t h r e n e - i n d u c e d tumors a r e a n e r g i c and t h a t humoral f a c t o r s f r o m t h e s e m i c e non s p e c i f i c a l l y s u p p r e s s n o r m a l l y m p h o i d c e l l s v i t r o . The main o b j e c t i v e of t h i s t h e s i s was t o e x t e n d t h e i r work by i n v e s t i g a t i n g the p o s s i b i l i t y t h a t non s p e c i f i c s u p p r e s s o r c e l l s a r e a l s o i n v o l v e d i n t h e g e n e r a l -i z e d immunosuppression a s s o c i a t e d w i t h tumor growth. The g o a l s were t h e r e f o r e : 1. To d e t e r m i n e whether or not non s p e c i f i c s u p p r e s s o r c e l l s a r e p r e s e n t i n t h e s p l e e n s of m i c e w i t h g r o w i n g c h e m i c a l l y - i n d u c e d tumors. 2. To d e t e r m i n e w h i c h c e l l t y p e i s r e s p o n s i b l e f o r t h e s u p p r e s s i o n . 3. To d e t e r m i n e the mechanism of s u p p r e s s i o n and i n p a r t i c u l a r whether s o l u b l e f a c t o r s a r e r e l e a s e d from s u p p r e s s o r c e l l s . 28 MATERIALS AND METHODS 1 MICE Female DBA/2J or BALB/CJ m i c e were used i n a l l e x p e r i m e n t s . They were p u r c h a s e d f r o m t h e J a c k s o n L a b o r a t o r y (Bar H a r b o r , Maine) and were used a t 2 t o 4 months o f age. I I TUMOR LINES Four t r a n s p l a n t a b l e tumors were used i n t h e s e e x p e r i m e n t s . None of them m e t a s t a s i z e d r e a d i l y and i n no i n s t a n c e s have tumor c e l l s been o b s e r v e d i n s p l e e n o r lymph node t i s s u e s . 1. The M - l tumor l i n e i s a 3 - m e t h y l c h o l a n t h r e n e i n d u c e d sarcoma o f DBA/ 23 m i c e w h i c h was o r i g i n a l l y p u r c h a s e d f r o m t h e J a c k s o n L a b o r a t o r y . T h i s tumor has been c h a r a c t e r i z e d more e x t e n s i v e l y t h a n t h e o t h e r t h r e e and was used i n a l l e x p e r i m e n t s u n l e s s o t h e r w i s e s t a t e d . 2. The M-2 tumor was a sarcoma i n d u c e d i n a DBA/2J mouse by i n j e c t i n g 0.5 mg of 3 - m e t h y l c h o l a n t h r e n e i n 0. 1 m l t r i o c t a n o i n i n t r a m u s c u l a r l y i n a h i n d l e g . 3. The MA tumor l i n e was a spontaneous mammary adenocarcinoma s y n g e n e i c t o DBA/2J m i c e . I t was p u r c h a s e d f r o m t h e J a c k s o n L a b o r a t o r y . 4. The B - l tumor was a 3 - m e t h y l c h o l a n t h r e n e i n d u c e d sarcoma w h i c h was p r e p a r e d i n a BALB/CJ mouse i n t h e same manner as th e M-2 tumor. I I I MAINTENANCE OF TUMOR LINES 1. In v i v o P a s s a g e . Tumors w i t h a mass of about 1.0 g were a s e p t i c a l l y e x c i s e d and the s k i n s u r r o u n d i n g t h e tumor mass was removed. The tumor was p l a c e d 29 i n 5 m l ph o s p h a t e b u f f e r e d s a l i n e (PBS) and c u t i n t o f i n e p i e c e s w i t h s c i s s o r s . T h r e e t o f i v e m i c e were each i n j e c t e d s u b c u t a n e o u s l y i n t h e r i g h t a b d o m i n a l s i d e w i t h 0.5 ml of minced tumor. 2. I_n v i t r o C u l t u r e . Tumors of about 1.0 g were e x c i s e d and c u t i n t o s m a l l p i e c e s as d e s c r i b e d above. Ten ml of tumor f r a g m e n t s were i n c u b a t e d w i t h 10 ml o f 0.25% t r y p s i n (Grand I s l a n d B i o l o g i c a l Co. (GIBCO); Grand I s l a n d , New Y o r k ) i n c i t r a t e s a l i n e . The i n c u b a t i o n was c a r r i e d o u t f o r 20 min a t room t e m p e r a t u r e w i t h c o n s t a n t s t i r r i n g . One ml h e a t -i n a c t i v a t e d (56°C f o r 30 min) FCS was added t o s t o p the a c t i o n o f t h e t r y p s i n , and t h e s u p e r n a t a n t c o n t a i n i n g f r e e tumor c e l l s was t r a n s f e r r e d t o a t e s t t u b e . C e l l s were c e n t r i f u g e d f o r 4 min a t 190 x G and re s u s p e n d e d i n 10 ml RPMI 1640 medium supplemented w i t h ,50 yg/ml g e n t a m i c i n and 10% FCS (RPMI 1640 and FCS were o b t a i n e d f r o m GIBCO and g e n t a m i c i n s u l f a t e was p u r c h a s e d f r o m Sigma). Tumor c e l l s were c u l t u r e d i n 100 x 20 mm p l a s t i c p e t r i d i s h e s ( F a l c o n //3003) and were m a i n t a i n e d a t 37°C i n a h u m i d i f i e d atmosphere o f 5% CO2 i n a i r . C u l t u r e s were passaged e v e r y 3 t o 6 days, C e l l s were removed f r o m the p e t r i d i s h by a 5 m i n i n c u b a t i o n w i t h 0.25% t r y p s i n , s u s -pended i n 10 m l RPMI 1640 w i t h 10% FCS, c e n t r i f u g e d f o r 4 min a t 190 x G, re s u s p e n d e d i n 10 ml RPMI 1640 w i t h 10% FCS, and t r a n s f e r r e d t o new p l a t e s . I V TUMOR-BEARING MICE USED FOR SUPPRESSION EXPERIMENTS I n o r d e r t o o b t a i n m i c e w i t h r e l a t i v e l y u n i f o r m tumor l o a d s , tumor c e l l s f o r i n o c u l a t i o n were t a k e n f r o m c u l t u r e s w h i c h had been passaged in v i t r o 2 t o 4 t i m e s . C u l t u r e d c e l l s were removed f r o m the p e t r i d i s h by t r y p s i n i z a t i o n , 30 washed w i t h medium c o n t a i n i n g FCS, and r e s u s p e n d e d i n PBS. V i a b l e tumor c e l l s were counted i n a haemocytometer u s i n g t r y p a n b l u e e x c l u s i o n as a c r i t e r i o n o f v i a b i l i t y and were suspended i n PBS a t a c o n c e n t r a t i o n o f 5 x 10~Vml. Each mouse was i n j e c t e d s u b c u t a n e o u s l y on t h e r i g h t a b d o m i n a l s i d e w i t h 0.2 m l s u s p e n s i o n c o n t a i n i n g 10^ tumor c e l l s . V GROWTH CHARACTERISTICS OF M - l TUMORS IN VIVO Dose r e s p o n s e e x p e r i m e n t s showed t h a t tumors d e v e l o p e d i n 70% o f a n i m a l s 3 4 i n o c u l a t e d w i t h 10 tumor c e l l s and i n 100% o f a n i m a l s g i v e > 10 c e l l s . P a l p a b l e tumors were o b s e r v e d 7 t o 14 days a f t e r i n j e c t i o n o f 10^ c e l l s . Tumors grew u n i f o r m l y u n t i l t h e y were 6 g o r more i n mass ( T a b l e I ) and d e a t h u s u a l l y o c c u r r e d a t about 8 weeks a f t e r i n o c u l a t i o n of 10^ tumor c e l l s . M i c e were used as s o u r c e s of s u p p r e s s o r c e l l s 4 t o 6 weeks a f t e r tumor c e l l i n j e c t -i o n . A t t h i s t i m e tumors were 2 t o 6 g and a n i m a l s were s t i l l r e l a t i v e l y h e a l t h y . V I . CHARACTERISTICS OF SPLEENS IN TUMOR-BEARING MICE As the tumors grew i n t h e m i c e , t h e s p l e e n w e i g h t s i n c r e a s e d c o r r e s p o n d -i n g l y ( T a b l e I ) . The number of l y m p h o i d c e l l s and t h e p e r c e n t a g e o f phago-c y t i c c e l l s i n s p l e e n s tended t o i n c r e a s e i n p r o p o r t i o n t o t h e s i z e o f the s p l e e n ( T a b l e I I ) . A n i m a l s used as s o u r c e s of s u p p r e s s o r c e l l s u s u a l l y had s p l e e n s w h i c h weighed between 0.40 and 0.65 g. T h e i r s p l e e n s c o n t a i n e d f r o m 1.1 t o 2.4 t i m e s more p h a g o c y t i c c e l l s t h a n t h o s e of n o r m a l m i c e . The p e r -c e n t a g e of B and T c e l l s i n t u m o r - b e a r e r s p l e e n s was d e c r e a s e d . T h i s was i n d i c a t e d by an i n c r e a s e i n c e l l r e c o v e r y a f t e r t r e a t m e n t w i t h a n t i - M I g or a n t i - T h y - 1 s e r a p l u s complement ( d e s c r i b e d i n s e c t i o n s X V I I and X V I I I "of M a t e r i a l s and M e t h o d s ) . 31 TABLE I TUMOR AND SPLEEN WEIGHTS IN TUMOR - BEARING MICE Time of Tumor ^ Tumor Weight (g) S p l e e n Weight (g) Growth (weeks) I not p a l p a b l e 0.17 + .03 I I 0.07 + .02 0.28 + .08 I I I 0.57 + .15 0.22 + .03 IV 1.52 + .38 0.40 + .03 V 2.55 + .77 0.44 + .06 V I 3.33 + .74 0.63 + .18 V I I 5.08 + .12 0.80 + .12 M i c e were i n j e c t e d w i t h 10 M - l tumor c e l l s . R e s u l t s a r e e x p r e s s e d as t h e mean w e i g h t o f tumors o r s p l e e n s f r o m t h r e e m i c e p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean. 32 TABLE I I LYMPHOID CELL AND PHAGOCYTIC CELL RECOVERY FROM SPLEENS OF TUMOR-BEARERS S p l e e n No. C e l l s % Weight (g) Recovered P h a g o c y t i c ( x 1 0 8 ) C e l l s 3 Normal DBA/2J m i c e 1 0.11 - 0.18 0.8 - 1.0 9 — 10 M - l T u m o r - B e a r e r s 2 0.45 3.25 14 0.47 3.20 11 0.50 3.00 14 0.60 4.80 22 0.60 5.50 24 0.65 4.75 20 0.80 5.40 22 Ranges of v a l u e s f o r normal DBA/2J m i c e a r e g i v e n . D a t a f o r i n d i v i d u a l 4 t o 6 week M - l t u m o r - b e a r e r s a r e g i v e n . The p e r c e n t a g e of p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g t h e number o f c e l l s p h a g o c y t i z i n g l a t e x beads. 33 V I I IMMUNOGENICITY OF THE M - l TUMOR The I m munogenicity o f t h i s tumor was e s t a b l i s h e d i n two ways: 1. M i c e w i t h s u r g i c a l l y r e s e c t e d tumors were a b l e t o r e j e c t c h a l l e n g e s of tumor c e l l s a t d o s e s 100 t i m e s g r e a t e r t h a n t h a t dose w h i c h i n d u c e s tumors 100% of the t i m e i n normal m i c e . 2. S p l e e n o r lymph node c e l l s f rom a r e s e c t e d mouse were i n c u b a t e d i n v i t r o f o r 2 h a t a r a t i o o f 300:1 w i t h tumor c e l l s and t h e m i x t u r e was i n j e c t e d s u b c u t a n e o u s l y i n t o n o r m a l m i c e . Tumor c e l l s mixed w i t h non-immune l y m p h o i d c e l l s produced tumors 100% o f t h e tim e b u t t h e tumor i n c i d e n c e was s i g n i f i c a n t l y r e d u c e d when tumor c e l l s were mixed w i t h s p l e e n o r lymph node c e l l s f r o m r e s e c t e d m i c e ( 1 3 4 ) . V I I I IMMUNE MICE Tumors of about 1.0 g were r e s e c t e d s u r g i c a l l y . A n i m a l s not d e v e l o p i n g r e c u r r e n c e s were c h a l l e n g e d w i t h 10^ v i a b l e tumor c e l l s and t h o s e r e s i s t i n g t h e c h a l l e n g e were c o n s i d e r e d immune. IX PREPARATION OF LYMPHOID CELL SUSPENSIONS S p l e e n s o r lymph nodes were removed a s e p t i c a l l y and p l a c e d i n 10 ml of PBS supplemented w i t h 5% FCS. The t i s s u e was c u t i n t o s m a l l p i e c e s and f r a g m e n t s were g e n t l y p r e s s e d a g a i n s t 60 mesh s t a i n l e s s s t e e l s c r e e n s . The c e l l s r e l e a s e d i n t o t h e PBS were t r a n s f e r r e d t o a t e s t t u b e , c e n t r i f u g e d (4 min a t 200 x G ) , re s u s p e n d e d f o r 3 min i n 10 m l 0.85% NH,C1 ( i n 0.01 M PO, 4 4 b u f f e r ) , c e n t r i f u g e d , and washed once i n PBS. F i n a l p r e p a r a t i o n s were c o u n t e d and suspended a t the d e s i r e d c o n c e n t r a t i o n i n RPMI 1640 a d j u s t e d t o pH 7 w i t h 1 N H C l . 34 X MITOGEN STIMULATION S p l e e n o r lymph node c e l l s were d i s p e n s e d i n t o w e l l s o f a m i c r o t i t e r 5 5 t r a y ( L i n b r o IS-FB-96 TC) a t a c o n c e n t r a t i o n of f r o m 5 x 10 t o 8 x 10 c e l l s i n 0.2 m l RPMI 1640 supplemented w i t h 5% ECS. The v a r i o u s c e l l p o p u l a t i o n s were t e s t e d s e p a r a t e l y o r mixed i n t h e p r o p o r t i o n s i n d i c a t e d . N o r m a l l y when t e s t i n g f o r s u p p r e s s o r a c t i v i t y , 5 x 10^ n o r m a l s p l e e n c e l l s were c u l t u r e d w i t h 3 x 10^ c e l l s o f the t e s t p o p u l a t i o n . M i t o g e n s were added t o w e l l s i n 0.05 ml volumes t o the f o l l o w i n g f i n a l c o n c e n t r a t i o n s : Con A a t 4 yg/ml and LPS a t 25 yg/ml. LPS-W, fr o m S_. t y p h i m u r i u m ( D i f c o , D e t r o i t , M i c h . ) , was d i s s o l v e d i n PBS a t 10 mg/ml, a d j u s t e d t o pH 8.0, h e a t e d f o r 30 m i n i n a b o i l i n g w a t e r b a t h , and s t o r e d f r o z e n a t 500 yg/ml i n PBS. A w o r k i n g s o l u t i o n o f 125 yg/ml was s t o r e d a t 4°C f o r use i n a s s a y s . Con A (Sigma, S t . L o u i s , Mo.) was d i s s o l v e d i n PBS a t a c o n c e n t r a t i o n o f 100 yg/ml and f r o z e n . A w o r k i n g s o l u t i o n o f 20 yg/ml was k e p t a t 4°C. C u l t u r e s were i n c u b a t e d f o r 2 days a t 37°C i n a h u m i d i f e d atmosphere 3 of 5% CO2 a f t e r w h i c h m e t h y l H - t h y m i d i n e ( s p e c i f i c a c t i v i t y 2 Ci/mmole, New E n g l a n d N u c l e a r Canada, L a c h i n e , Que.) was added a t a c o n c e n t r a t i o n o f 1.0 y C i / w e l l . A f t e r a f u r t h e r 16 h i n c u b a t i o n , c u l t u r e s were h a r v e s t e d w i t h a m i c r o p r e c i p i t a t o r a p p a r a t u s w h i c h a s p i r a t e d the c o n t e n t s o f 1 row of 12 w e l l s and d e p o s i t e d t h e c o n t e n t s i n d i s c r e t e s p o t s on a g l a s s f i b r e s t r i p . W e l l s were washed t h r e e t i m e s w i t h w a t e r and once w i t h m e t h a n o l . Each s p o t was c u t o u t , p l a c e d i n a g l a s s s c i n t i l l a t i o n v i a l , d r i e d , and mixed w i t h 3 m l t o l u e n e based f l u i d (4 g O m n i f l u o r / 1 t o l u e n e ) . V i a l s were counted w i t h a N u c l e a r C h i c a g o U n i l u x I s c i n t i l l a t o r . 35 XI CULTURE SYSTEM FOR THE IN VITRO GENERATION OF PLAQUE FORMING CELLS (PFC) One m i l l i o n s p l e e n c e l l s f r o m n o r m a l s y n g e n e i c m i c e were c u l t u r e d i n each w e l l of a m i c r o t i t e r t r a y ( L i n b r o IS-FB-96TC). V a r y i n g numbers o f s p l e e n c e l l s f r o m n o r m a l c o n t r o l o r t u m o r - b e a r i n g m i c e were added t o w e l l s as i n d i c a t e d . N o r m a l l y , when t e s t i n g f o r s u p p r e s s o r a c t i v i t y , 10^ no r m a l s p l e e n c e l l s were mixed w i t h 5 x 10^ c e l l s f rom the t e s t p o p u l a t i o n . Each w e l l was a d j u s t e d w i t h RPMI 1640 t o g i v e a f i n a l volume of 0.3 ml and f i n a l c o n c e n t -— S fi r a t i o n s of 10% FCS, 5 x 10 M 2 - m e r c a p t o e t h a n o l , and e i t h e r 2.5 x 10 SRBC per m l o r 0.1 yg d i n i t r o p h e n y l a t e d - l i p o p o l y s a c c h a r i d e (DNP-LPS) p e r m l . C u l t u r e s were i n c u b a t e d on a r o c k e r p l a t f o r m ( B e l l c o G l a s s , V i n e l a n d , N.J.) a t 37°C i n a h u m i d i f i e d atmosphere of 5% C 0 2 f o r 3 days ( w i t h DNP-LPS) o r 4 days ( w i t h SRBC). SRBC were c o l l e c t e d a s e p t i c a l l y i n c i t r a t e phosphate d e x t r o s e , washed g i n PBS, and co u n t e d i n a haemocytometer. A s t o c k s o l u t i o n o f 10 SRBC/ml was s t o r e d a t 4°C and used f o r 2 t o 3 weeks. DNP-LPS was p r e p a r e d by t h e method of J a c o b s e t a l . ( 6 5 ) . One hundred mg LPS was d i s s o l v e d i n 5 m l c a c o d y l a t e b u f f e r , pH 6.9. The pH was a d j u s t e d t o 11.5 w i t h NaOH and 60 mg 2 , 4 - d i n i t r o b e n z e n e s u l f o n i c a c i d (Eastman Kodak Co., R o c h e s t e r , N.Y.) i n 5 ml b u f f e r were added. The r e a c t i o n m i x t u r e was i n c u b a t e d a t room t e m p e r a t u r e f o r 2 h and t h e n d i a l y s e d a g a i n s t s a l i n e o v e r -n i g h t . X I I GENERATION OF PFC IN MARBR00K CHAMBERS Double p e r i s c o p i c Marbrook chambers ( B i o r e s e a r c h G l a s s , V i n e l a n d , N.J.) were f i t t e d w i t h membranes as i l l u s t r a t e d i n F i g u r e 1. The o u t e r chamber was s e p a r a t e d f r o m t h e o u t s i d e v e s s e l by s t a n d a r d d i a l y s i s t u b i n g (A.H. Thomas Lab., P h i l a d e l p h i a , Pa. #3787-D12) w h i c h was a t t a c h e d f i r m l y t o t h e o u t e r 36 chamber by a r i n g o f s u r g i c a l t u b i n g (Tygon f l e x i b l e p l a s t i c t u b i n g ) . The r i n g was a d j u s t e d so t h a t i t was above the l e v e l of t h e medium i n t h e o u t s i d e v e s s e l . N u c l e p o r e membranes ( N u c l e p o r e Co., P l e a s a n t o n , Ca.) w i t h p o r e s i z e s o f 0.8 y were g l u e d t o t h e bottom of the i n n e r chamber w i t h s i l i c o n e r u b b e r g l u e (RTV 108 C l e a r A d h e s i v e S e a l a n t , C a n a d i a n G e n e r a l E l e c t r i c Co., T o r o n t o , O n t . ) . I n some e x p e r i m e n t s d i a l y s i s t u b i n g was a t t a c h e d t o t h e bottom of t h e I n n e r chamber. I n t h e s e i n s t a n c e s , t h e d i a l y s i s t u b i n g was t i e d on w i t h c o t t o n t h r e a d a t a p o s i t i o n above t h e l e v e l o f medium i n t h e o u t e r chamber. The g l u e was a i r d r i e d a t room t e m p e r a t u r e o v e r n i g h t and a l l membranes were soaked f o r a minimum o f 48 h i n d i s t i l l e d w a t e r w h i c h was changed a t l e a s t once. V e s s e l s were a u t o c l a v e d w i t h the f o l l o w i n g volumes of PBS i n t h e chambers; 20 m l i n t h e o u t e r v e s s e l , 2 m l i n t h e o u t e r chamber, and 1 ml i n t h e i n n e r chamber. The PBS was poured out of the chambers j u s t b e f o r e t h e i r u s e i n e x p e r i m e n t s . S u p p r e s s i o n e x p e r i m e n t s were c a r r i e d out as f o l l o w s . The i n n e r chamber c o n t a i n e d 5 x 10^ s p l e e n c e l l s f r o m n o r m a l s y n g e n e i c m i c e . They were c u l t u r e d i n 1 m l RPMI 1640 supplemented w i t h 10% FCS, 5 x 1 0 ~ 5 M 2-mercapto-6 7 e t h a n o l , and 2.5 x 10 SRBC. The o u t e r chamber c o n t a i n e d 10 n o r m a l c o n t r o l or t u m o r - b e a r e r s p l e e n c e l l s i n 2 ml medium supplemented as above. The o u t s i d e v e s s e l c o n t a i n e d 20 m l RPMI 1640 w i t h 5 x 10 ~* M 2 - m e r c a p t o e t h a n o l as a n u t r i t i o n a l supplement. C u l t u r e s were i n c u b a t e d a t 37°C i n a h u m i d i f i e d atmosphere of 5% C 0 9 f o r 4 d a y s . 37 M A R B R O O K V E S S E L O U T E R C H A M B E R A, N N E R C H A M B E R . O U T S I D E V E S S E L N U C L E P O R E M E M B R A N E D I A L Y S I S T U B I N G F i g u r e !• Double chamber Marbrook v e s s e l . 38 X I I I PFC ASSAY The a s s a y s y s t e m used was a m o d i f i c a t i o n o f t h a t o f Cunningham et^ a l . ( 2 7 ) . C u l t u r e d c e l l s were removed from q u a d r u p l i c a t e w e l l s o f a m i c r o t i t e r t r a y w i t h a p a s t e u r p i p e t t e o r c u l t u r e s were removed from i n d i v i d u a l Marbrook chambers u s i n g a p a s t e u r p i p e t t e w i t h a r u b b e r t i p t o p r e v e n t membrane b r e a k -age. C e l l s u s p e n s i o n s were c e n t r i f u g e d (4 min a t 200 x G) and resuspended i n 0.6 ml RPMI 1640 medium b u f f e r e d w i t h 35 mM HEPES b u f f e r and supplemented w i t h 5% FCS. Volumes o f 0.1 t o 0.3 ml c e l l s u s p e n s i o n were mixed w i t h 0.1 ml 20% (v/v) SRBC ( u n t r e a t e d o r D N P - l a b e l l e d ) p l u s 0.5 ml g u i n e a p i g serum (as a s o u r c e o f complement) and made up t o 0.5 ml w i t h medium. O n e - f i f t h o f t h i s m i x t u r e was t e s t e d i n each o f 3 m i c r o s c o p e s l i d e chambers. A s s a y s were i n -c u b a t e d f o r 1 h a t 37°C and p l a q u e s were enumerated by v i e w i n g t h e chambers t h r o u g h a m a g n i f y i n g g l a s s a g a i n s t a f l u o r e s c e n t l i g h t . PFC s p e c i f i c f o r DNP were enumerated u s i n g d i n i t r o p h e n y l a t e d Fab' fr a g m e n t s from r a b b i t a n t i-SRBC a n t i b o d i e s . R a b b i t a n t i-SRBC Fab' fra g m e n t s were p r e p a r e d as f o l l o w s . A t o t a l volume o f 1 ml o f a 50% s u s p e n s i o n o f SRBC i n PBS e m u l s i f i e d w i t h 25% (v/ v ) Complete Freund's A d j u v a n t (CFA) was i n j e c t e d i n t o f o u r s i t e s i n t r a m u s c u l a r l y . A n i m a l s were b o o s t e d m o n t h l y w i t h 1 ml 50% SRBC i n PBS i n t r a v e n o u s l y and t h e n i n t r a m u s c u l a r l y , and were b l e d 1 week a f t e r i n j e c t i o n . The i m m u n o g l o b u l i n f r a c t i o n o f t h e immune serum was p r e p a r e d by ammonium s u l f a t e p r e c i p i t a t i o n (40% s a t u r a t i o n ) f o l l o w e d by d i a l y s i s a g a i n s t s a l i n e and i o n exchange chromatography on DEAE c e l l u l o s e . Fab' f r a g m e n t s were p r e p a r e d by p a p a i n d i g e s t i o n u s i n g t h e method o f P o r t e r ( 1 0 7 ) . Fab' f r a g m e n t s were d i n i t r o p h e n y l a t e d u s i n g t h e method o f S t r a u s b a u c h et_ a l . ( 1 1 9 ) . Fab' f r a g m e n t s a t 6 mg/ml were i n c u b a t e d w i t h 10 mg/ml D N P - s u l f o n i c a c i d and sodium c a r b o n a t e a t 10 mg/ml. The f i n a l pH was a d j u s t e d 39 t o 9.3 and t h e r e a c t i o n m i x t u r e was l e f t a t room t e m p e r a t u r e f o r 24 h. The m i x t u r e was d i a l y s e d a g a i n s t s a l i n e f o r 24 h a t 4°c. SRBC were c o a t e d w i t h DNP-Fab' by i n c u b a t i o n o f 1 ml d f a 20% s u s p e n s i o n o f SRBC w i t h 0.05 mg DNP-Fab' f o r 1 h a t 37°C. The s e n s i t i z e d SRBC were washed 3 t i m e s w i t h PBS and r e s u s p e n d e d i n HEPES b u f f e r e d RPMI 1640 w i t h 5% FCS. The number o f s p e c i f i c PFC t o DNP was c a l c u l a t e d by s u b t r a c t i n g t h e number o f PFC w i t h SRBC (background) from t h e number of PFC w i t h DNP-Fab' coupled-SRBC. Background v a l u e s were g e n e r a l l y 5% t o 10% o f t h o s e f o r s e n s i t i z e d SRBC. F r e s h g u i n e a p i g complement was ads o r b e d w i t h DNP-SRBC and DBA/2J s p l e e n c e l l s (1 ml packed DNP-SRBC and 10 s p l e e n c e l l s p e r 5 ml g u i n e a p i g serum) f o r 1 h a t 4°C. S e r a were t e s t e d f o r c y t o t o x i c i t y t o SRBC o r s p l e e n c e l l s under t h e a s s a y c o n d i t i o n s b e f o r e t h e y were use d . They were f r o z e n i n s m a l l a l i q u o t s a t -70°C. XIV ESTIMATION OF CELL V I A B I L I T I E S C e l l s u s p e n s i o n s were d i l u t e d 1:1 w i t h 0.07% t r y p a n b l u e ( D i r e c t b l u e 14, Matheson, Coleman, and B e l l , Norwood, Ohio) and c o u n t e d i n a haemocytometer; Dead c e l l s were s t a i n e d b l u e by t h i s method. XV ESTIMATION OF PHAGOCYTIC CELLS One m i l l i o n c e l l s were i n c u b a t e d i n 1 ml RPMI 1640 medium w i t h 10% FCS and about 0.05 ml 2.5% l a t e x beads ( P o l y s t y r e n e M o n o d i s p e r s e L a t e x , P o l y -s c i e n c e s , W a r r i n g t o n , Pa. #7311) f o r 1 t o 2 h a t 37°C. S u s p e n s i o n s were washed 4 t i m e s t o remove u n p h a g o c y t i z e d beads. C e l l s were re s u s p e n d e d i n 0.5 ml PBS and c o u n t e d i n a haemocytometer. C e l l s c o n t a i n i n g more t h a n 5 40 beads were c o n s i d e r e d t o be p h a g o c y t i c . Thymus c e l l s d i d n o t p h a g o c y t i z e beads and p e r i t o n e a l e x u d a t e c e l l s were r i c h i n p h a g o c y t i c c e l l s . About 10% of n o r m a l s p l e e n c e l l s were p h a g o c y t i c by t h i s c r i t e r i o n . X VI HYPAQUE-FICOLL SEPARATION H y p a q u e - f i c o l l s o l u t i o n s were p r e p a r e d from hypaque and f i c o l l s t o c k s o l u t i o n s o f known s p e c i f i c g r a v i t i e s . The s t o c k f i c o l l was a 7.33% s o l u t i o n o f f i c o l l 400 ( P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , Sweden) i n d i s t i l l e d w a t e r . The s p e c i f i c g r a v i t y was d e t e r m i n e d by w e i g h i n g 1 ml o f s o l u t i o n and d i v i d i n g by t h e w e i g h t o f 1 ml d i s t i l l e d w a t e r a t t h e same t e m p e r a t u r e . The s p e c i f i c g r a v i t y o f hypaque sodium ( s t e r i l e aqueous i n j e c t i o n 50% w/v, W i n t h r o p L a b -o r a t o r i e s , A u r o r a , O n t a r i o ) was d e t e r m i n e d and a s t o c k s o l u t i o n o f s p e c i f i c g r a v i t y 1.2 was p r e p a r e d by t h e a d d i t i o n o f d i s t i l l e d w a t e r . The hypaque and f i c o l l s t o c k s o l u t i o n s were n o r m a l l y mixed i n a r a t i o p r o v i d i n g a f i n a l s p e c i f i c g r a v i t y o f 1.08 u n l e s s o t h e r w i s e s t a t e d . S p l e e n c e l l s f rom n o r m a l o r t u m o r - b e a r i n g mice were suspended a t a c o n -c e n t r a t i o n o f 20 x 10 c e l l s / m l i n 5 ml PBS. T h i s s u s p e n s i o n was c a r e f u l l y l a y e r e d on 4 ml o f h y p a q u e - f i c o l l i n a 17 x 100 mm p l a s t i c t u b e ( F a l c o n #2001). The tu b e was c e n t r i f u g e d f o r 30 min a t a speed g i v i n g a c e n t r i f u g a l f o r c e o f 400 x G a t t h e i n t e r f a c e o f t h e medium and h y p a q u e - f i c o l l . The c e l l s r e m a i n i n g a t t h e i n t e r f a c e ( l i g h t f r a c t i o n ) and t h o s e s e d i m e n t i n g t o t h e b o t t o m o f t h e tu b e (dense f r a c t i o n ) were removed w i t h p a s t e u r p i p e t t e s i n t o s e p a r a t e t u b e s , r e s u s p e n d e d i n 10 ml PBS, c e n t r i f u g e d f o r 10 min a t 200 x G, and resuspended i n RPMI 1640. S p l e e n c e l l s r e t a i n e d i n t h e l i g h t f r a c t i o n a f t e r h y p a q u e - f i c o l l s e p a r a t i o n c o n t a i n e d b o t h T and B c e l l s as i n d i c a t e d by good p r o l i f e r a t i v e r e s p o n s e s t o Con A and LPS and i n c r e a s e d in_ v i t r o g e n e r a t i o n o r PFC t o SRBC 41 and DNP-LPS ( T a b l e I I I ) . There was a l s o a s l i g h t e n r i c h m e n t f o r p h a g o c y t i c c e l l s . S p l e e n c e l l s w h i c h sedimented t o t h e dense f r a c t i o n i n a hypaque-f i c o l l g r a d i e n t had re d u c e d numbers o f B c e l l s and p h a g o c y t i c c e l l s when t h e same c r i t e r i a were a p p l i e d . T c e l l s were r e t a i n e d s i n c e dense c e l l s r e s p o n d e d t o Con A. 42 TABLE III EFFECT OF HYPAQUE-FICOLL SEPARATION ON THE RESPONSES OF SPLEEN CELLS Fra c t i o n Tested"*" Untreated Light Dense % Viable C e l l Recovery 2 Mitogen Response 25 - 30 60 - 70 None LPS Con A 4 90 197 6 82 145 2 9 119 Ih v i t r o PFC~ DNP--LPS SRBC 280 ± 42 311 ± 75 521 ± 64 431 ± 47 35 19 6 4 % Phagocytic C e l l s 10 15 Spleen c e l l s from a normal DBA/2J mouse were separated into l i g h t and dense f r a c t i o n s on hypaque-ficoll, s p e c i f i c gravity 1.08. 3 Mitogen responses are expressed as the mean cpm (x 10 ) of t r i p l i c a t e cultures stimulated by PBS, LPS or Con A. Results are expressed as the number of PFC per well plus or minus the standard deviation from the mean of t r i p l i c a t e assays. The percentage of phagocytic c e l l s was estimated by counting the number of c e l l s phagocytizing latex beads. 43 X V I I METHOD FOR DEPLETING CELL SUSPENSIONS OF B CELLS I n o r d e r t o k i l l B c e l l s , 10^ s p l e e n c e l l s were i n c u b a t e d i n 0.1 ml a n t i - M I g serum a t a 1:4 d i l u t i o n p l u s 0.05 m l g u i n e a p i g serum and 0.05 ml RPMI 1640 w i t h 10% FCS f o r 60 min a t 37°C. C e l l s u s p e n s i o n s were washed t h r e e t i m e s w i t h PBS and v i a b l e c e l l s were c o u n t e d . Sheep a n t i - M I g serum was p r e p a r e d by i n j e c t i n g mouse i m m u n o g l o b u l i n l i g h t c h a i n s i n t o sheep. L i g h t c h a i n d i m e r s were p u r i f i e d f r o m the a s c i t i c f l u i d o f a mouse Kappa myeloma by s e p a r a t i o n on a Sephadex G^-150 g e l f i l t r a t i o n column. Sheep were immunized by 2 i n t r a m u s c u l a r i n j e c t i o n s of 1 mg l i g h t c h a i n d i m e r s i n 1 ml 50% CFA 2 weeks a p a r t . Sheep were b l e d 2 weeks a f t e r 8 t h e f i n a l i n j e c t i o n . The a n t i s e r u m (10 ml) was ads o r b e d w i t h 10 DBA/2J thymus c e l l s f o r 30 min a t room t e m p e r a t u r e . I t was h e a t i n a c t i v a t e d f o r 30 m i n a t 56°C, f i l t e r s t e r i l i z e d , and s t o r e d a t -20°C. T h i s a n t i s e r u m has been e x t e n s i v e l y t e s t e d and was shown t o be s p e c i f i c f o r B c e l l s by c y t o t o x i c i t y and a u t o r a d i o g r a p h y e x p e r i m e n t s ( 1 3 4 ) . As can be s e e n by the r e s u l t s i n T a b l e I V , t r e a t m e n t o f s p l e e n c e l l s w i t h g u i n e a p i g complement a l o n e does n o t a f f e c t t h e r e s p o n s e t o m i t o g e n s o r the a b i l i t y o f c e l l s t o p r o d u c e PFC o r p h a g o c y t i z e beads. C e l l s t r e a t e d w i t h a n t i - M I g serum p l u s complement had a reduced p r o l i f e r a t i v e r e s p o n s e t o LPS but responded n o r m a l l y t o Con A. However, a l t h o u g h the PFC r e s p o n s e t o SRBC i n v i t r o was o f t e n d e p r e s s e d i t was u s u a l l y i m p o s s i b l e t o r e d u c e t h e r e s p o n s e t o DNP-LPS. I t would a p p e a r , f r o m t h e s e d a t a , t h a t t h e B c e l l s r e s p o n d i n g t o LPS and t h o s e p r o d u c i n g PFC t o SRBC may be more s u s c e p t i b l e t o k i l l i n g by a n t i - M I g serum p l u s complement t h a n the B c e l l s r e s p o n s i b l e f o r t h e p r i m a r y r e s p o n s e t o DNP-LPS. Treatment of s p l e e n c e l l s w i t h a n t i - M I g p l u s complement d o e s , however, i n c r e a s e t h e p e r c e n t a g e of p h a g o c y t i c c e l l s and may a l s o enhance t h e number of T c e l l s i n t h e p o p u l a t i o n r e m a i n i n g . 44 TABLE IV EFFECT OF ANTI-IMMUNOGLOBULIN SERUM ON THE RESPONSES OF SPLEEN CELLS Treatment of S p l e e n C e l l s ^ A n t i - M I g + U n t r e a t e d Complement Complement % V i a b l e C e l l R e c o v e r y 95 50 1 2 M i t o g e n Response None 7 3 3 LPS 74 70 19 • Con A 89 102 118 3 I n v i t r o PFC DNP-LPS 280 ± 42 325 ± 11 248 ± 11 SRBC 311 ± 75 281 ± 33 130 ± 25 % P h a g o c y t i c C e l l s 4 10 11 15 Normal DBA/2J s p l e e n c e l l s were t r e a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. 3 M i t o g e n r e s p o n s e s a r e e x p r e s s e d as t h e mean cpm (x 10 ) of t r i p l i c a t e c u l t u r e s s t i m u l a t e d by PBS, LPS o r Con A. R e s u l t s a r e e x p r e s s e d as the number o f PFC p e r w e l l p l u s o r minus the s t a n d a r d d e v i a t i o n f r o m the mean of t r i p l i c a t e a s s a y s . The p e r c e n t a g e of p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g the number of c e l l s p h a g o c y t i z i n g l a t e x beads. 45 X V I I I METHOD FOR DEPLETING CELL SUSPENSIONS OF T CELLS R a b b i t a n t i - m o u s e b r a i n a s s o c i a t e d Thy-1 serum was p r e p a r e d as d e s c r i b e d by B a r k e r jet a l . ( 1 0 ) . B r i e f l y , b r a i n s were removed f r o m C3H m i c e and were homogenized i n 2.5 m l c o l d b a l a n c e d s a l t s o l u t i o n . T h i s homogenate was mixed 1:1 w i t h Complete Freund's A d j u v a n t , e m u l s i f i e d , and i n j e c t e d sub-c u t a n e o u s l y i n a r a b b i t . The a n i m a l was b o o s t e d 7 days l a t e r and b l e d on day 15. The serum was adsorbed w i t h homogenates of mouse l i v e r a t a r a t i o o f 6 ml a n t i - s e r u m t o 1.5 m l l i v e r t i s s u e f o r 30 min a t room t e m p e r a t u r e . \ I t was i n a c t i v a t e d a t 56°C f o r 30 min, s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n , and f r o z e n a t -20°C. T h i s a n t i s e r u m has been e x t e n s i v e l y t e s t e d and i t has been shown by c y t o t o x i c i t y t e s t i n g and a u t o r a d i o g r a p h y t h a t i t i s h i g h l y c y t o t o x i c f o r t h y -mus c e l l s and does n o t a f f e c t B c e l l s ( 1 3 4 ) . T h i s i s f u r t h e r d e m o n s t r a t e d by t h e d a t a i n T a b l e V. Treatment o f s p l e e n c e l l s w i t h a n t i - T h y - 1 serum p l u s complement i n c r e a s e s t h e p r o p o r t i o n of B c e l l s i n t h e s u s p e n s i o n s i n c e the p r o l i f e r a t i v e r e s p o n s e t o LPS and the PFC r e s p o n s e t o DNP-LPS a r e b o t h i n c r e a s e d . I t can be seen t h a t T c e l l s a r e k i l l e d however, s i n c e t h e Con A r e s p o n s e i s d e c r e a s e d and the number of PFC t o t h e T dependent a n t i g e n , SRBC, i s a l s o d e p r e s s e d . T h i s t r e a t m e n t a l s o i n c r e a s e s t h e p e r c e n t a g e o f p h a g o c y t i c c e l l s . 46 TABLE V EFFECT OF ANTI-THY-1 SERUM ON THE RESPONSES OF SPLEEN CELLS Treatment of C e l l s 1 A n t i - T h y - 1 + U n t r e a t e d Complement Complement % V i a b l e C e l l R e c o v e r y 92 67 2 M i t o g e n Response None 7 4 9 LPS 72 79 83 Con A 114 96 22 3 I n v i t r o PFC DNP-LPS 430 ± 58 393 ± 18 461 ± 53 SRBC 548 ± 11 451 ± 34 117 ± 25 % P h a g o c y t i c C e l l s 4 10 11 15 Normal DBA/2J s p l e e n c e l l s were t r e a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. 3 M i t o g e n r e s p o n s e s a r e e x p r e s s e d as t h e mean cpm (x 10 ) of t r i p l i c a t e c u l t u r e s s t i m u l a t e d by PBS, LPS or Con A. R e s u l t s a r e e x p r e s s e d as the number of PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean of t r i p l i c a t e a s s a y s . The p e r c e n t a g e of p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g the number of c e l l s p h a g o c y t i z i n g l a t e x beads. 47 X I X METHODS FOR DEPLETING CELL SUSPENSIONS OF PHAGOCYTIC CELLS 1. N y l o n Wool Adherence N y l o n w o o l p u r i f i e d c e l l s were p r e p a r e d u s i n g t h e method of J u l i u s e t a l . ( 6 7 ) . N y l o n w o o l (Leuco Pak, L e u c o c y t e f i l t e r , F enwal Lab, M o r t o n Grove, I l l i n o i s ) was soaked i n 3 changes of d i s t i l l e d w a t e r and d r i e d . Columns were p r e p a r e d by f i l l i n g 12 m l p l a s t i c s y r i n g e s up t o t h e 6 ml mark w i t h 0.6 g n y l o n w o o l . A f t e r a u t o c l a v i n g , columns were washed w i t h p RPMI 1640 and i n c u b a t e d f o r 1 h a t 37°C. S p l e e n c e l l s (10 c e l l s i n 2 m l RPMI 1640 p l u s 10% FCS) were l o a d e d on t o t h e top of the column w i t h a p a s t e u r p i p e t t e and a l l o w e d t o pass s l o w l y i n t o t h e column. A f u r t h e r 1 m l medium was r u n i n t o t h e n y l o n w o o l and columns were i n c u b a t e d f o r 45 min a t 37°C. Columns were s l o w l y washed w i t h 25 ml medium and t h e e l u a n t was c o l l e c t e d . The c e l l s e l u t e d f r o m n y l o n w o o l columns a r e e x t r e m e l y e n r i c h e d f o r T c e l l s . T h i s may be o b s e r v e d as an i n c r e a s e i n Con A r e s p o n s i v e n e s s ( T a b l e V I ) arid by t h e f a c t t h a t a p p r o x i m a t e l y 95% of n y l o n w o o l non-a d h e r e n t c e l l s a r e k i l l e d by a n t i - T h y - 1 serum p l u s complement. T h i s p r o c e d u r e appears t o remove a l a r g e p r o p o r t i o n of t h e B c e l l s s i n c e b o t h t h e p r o l i f e r a t i v e r e s p o n s e t o LPS and PFC r e s p o n s e s were r e d u c e d . However, t h e p o p u l a t i o n r e s p o n d i n g t o t h e T c e l l dependent a n t i g e n appeared t o be l e s s a d h e r e n t t o t h e column t h a n t h e p o p u l a t i o n r e s p o n d i n g t o t h e T c e l l i n d e p e n d e n t a n t i g e n s i n c e t h e PFC r e s p o n s e t o DNP-LPS was r e d u c e d t o a g r e a t e r e x t e n t t h a n t h e r e s p o n s e t o SRBC. P h a g o c y t i c c e l l s a r e d e p l e t e d t o a g r e a t e x t e n t by t h i s t e c h n i q u e as may be o b s e r v e d by t h e d e c r e a s e i n c e l l s p h a g o c y t i z i n g l a t e x beads. A l t h o u g h PFC r e s p o n s e s t o T c e l l i n d e p e n d e n t a n t i g e n s a r e g e n e r a l l y c o n s i d e r e d t o be l e s s macrophage dependent t h a n r e s p o n s e s t o T c e l l 48 dependent a n t i g e n s , i t has been shown by I s h i z a k a _et a l . (63) t h a t r e s p o n s e s t o b o t h TNP-LPS and SRBC a r e h i g h l y macrophage dependent i n v i v o . T h e r e f o r e , a l t h o u g h 2 - m e r c a p t o e t h a n o l was i n c l u d e d i n t h e c u l t u r e s t o r e p l a c e macrophage f u n c t i o n , i t i s p o s s i b l e t h a t t h e d e c r e a s e i n PFC r e s p o n s e was due t o macrophage and n o t B c e l l d e p l e t i o n . 2. C a r b o n y l I r o n and Magnet S p l e e n c e l l s (5 x 1 0 ? i n 3 m l RPMI 1640 p l u s 10% FCS) were i n c u b a t e d i n a 17 x 100 mm p l a s t i c t e s t tube ( F a l c o n #2001) w i t h 0.5 g c a r b o n y l i r o n powder ( G e n e r a l A n i l i n e and F i l m Co., New Y o r k , N.Y.) f o r 45 min i n a s h a k i n g 37°C w a t e r b a t h . The i r o n was e x t r a c t e d by p l a c i n g a magnet a t t h e b o t t o m of t h e tube and removing t h e s u p e r n a t a n t . The p r o c e s s was r e p e a t e d f i v e t i m e s and s u p e r n a t a n t c e l l s were washed t w i c e w i t h PBS. T h i s p r o c e d u r e removed p h a g o c y t i c c e l l s e f f e c t i v e l y s i n c e t h e p e r c e n t a g e o f c e l l s p h a g o c y t i z i n g l a t e x beads was r e d u c e d f r o m 10% t o about 1% ( T a b l e V I I ) . C e l l s t r e a t e d w i t h c a r b o n y l i r o n had r e d u c e d r e s p o n s e s t o LPS and Con A and produced fewer PFC t o DNP-LPS. I t i s no t p o s s i b l e t o d e t e r m i n e f r o m t h e d a t a i n T a b l e V I I whether t h i s d e c r e a s e d r e s p o n s i v e n e s s was due t o a d e c r e a s e i n B o r T c e l l s o r t o a d e c r e a s e i n macrophages r e q u i r e d f o r t h e r e s p o n s e s . However, T a k e i e t a l . (125) have shown t h a t t h e p e r c e n t a g e of c e l l s b i n d i n g f l u o r e s c e n t a n t i - m o u s e I g i n c r e a s e d f r o m 32% t o 36% a f t e r c a r b o n y l i r o n t r e a t m e n t . A l s o , Lee e t a l . (80) found t h a t t h i s p r o c e d u r e d i d n o t d e l e t e T o r B c e l l s as i d e n t i f i e d by c e l l s u r f a c e Thy-1 a n t i g e n or i m m u n o g l o b u l i n ( I g ) • They a l s o f o u n d t h a t t h e immune r e s p o n s e s of t r e a t e d c e l l s c o u l d be r e s t o r e d by i r r a d i a t e d s p l e n i c o r p e r i t o n e a l macrophages. 49 3. Carragheenan Twenty m i l l i o n s p l e e n c e l l s were i n c u b a t e d i n 1 m l RPMI 1640 w i t h 10% FCS and 500 y g c a r r a g h e e n a n (Ca S a l t s , Sigma, S t . L o u i s , Mo.) f o r 4 h a t 37°C. C e l l s were washed 3 t i m e s w i t h PBS, c o u n t e d , and r e s u s p e n d e d i n RPMI 1640. Treatment of no r m a l u n s t i m u l a t e d s p l e e n c e l l s w i t h 500 yg c a r r h a g e e -nan f o r 4 h d i d n o t r e d u c e the p e r c e n t a g e of c e l l s p h a g o c y t i z i n g l a t e x beads ( T a b l e V I I ) . However, i d e n t i c a l t r e a t m e n t of s p l e e n c e l l s f r o m a t u m o r - b e a r i n g mouse r e d u c e d t h e p e r c e n t a g e o f p h a g o c y t i c c e l l s f r o m 14% to 4%. T h i s i s i n agreement w i t h C a t a n z a r o e t a l . (25) who found t h a t u n s t i m u l a t e d mouse macrophages were n o t k i l l e d s u b s t a n t i a l l y by c a r r a g h e e n a n u n t i l 24 h a f t e r t r e a t m e n t , whereas s t i m u l a t e d macrophages were k i l l e d e a r l i e r . C a r r a g h e e n a n t r e a t m e n t has been shown t o have no t o x i c e f f e c t s on l y m p h o c y t e s ( 2 5 ) . R e d u c t i o n s i n Con A and PFC r e s p o n s e s were p r o b a b l y due i n p a r t t o t h e r e m o v a l of macrophages. 4. P e t r i D i s h Adherence g I n o r d e r t o s e p a r a t e c e l l s by adherence t o p l a s t i c , 10 s p l e e n c e l l s were i n c u b a t e d i n 10 ml RPMI 1640 medium w i t h 10% FCS i n 100 x 20 mm p l a s t i c p e t r i d i s h e s ( F a l c o n #3003) f o r 2 h a t 37°C. The non a d h e r e n t c e l l s were removed w i t h a p a s t e u r p i p e t t e . The a d h e r e n t c e l l s were washed f r e e o f non a d h e r e n t c e l l s w i t h 5 ml medium and were s c r a p e d o f f w i t h a r u b b e r p o l i c e m a n . T h i s p r o c e d u r e was r e p e a t e d once. Each pop-u l a t i o n was washed w i t h PBS, c o u n t e d and suspended i n medium f o r t e s t i n g . T h i s method does n o t g i v e a d e f i n i t i v e s e p a r a t i o n o f l y m p h o i d pop-u l a t i o n s . The a d h e r e n t p o p u l a t i o n tended t o have an i n c r e a s e i n t h e number o f p h a g o c y t i c c e l l s and B c e l l s ( T a b l e V I I I ) . The non a d h e r e n t 50 c e l l s were e n r i c h e d f o r T c e l l s and had fewer B c e l l s and p h a g o c y t i c c e l l s . Here a g a i n , t h e B c e l l s r e s p o n d i n g t o SRBC may have a d i f f e r e n t d i s t r i b u t i o n t h a n t h a t o f t h e B c e l l s r e s p o n d i n g t o DNP-LPS s i n c e t h e r e s p o n s e t o SRBC was g r e a t e r w i t h non a d h e r e n t c e l l s . However, i t i s no t p o s s i b l e t o make a c o n c l u s i o n f r o m t h i s d a t a s i n c e the i n c r e a s e i n T h e l p e r c e l l s i n t h e non a d h e r e n t p o p u l a t i o n would t e n d t o s t i m u l a t e t h e r e s p o n s e t o SRBC. 51 TABLE V I EFFECT OF NYLON WOOL ADHERENCE ON THE RESPONSES OF SPLEEN CELLS Treatment of C e l l s U n t r e a t e d N y l o n Wool Columns % V i a b l e C e l l R e c o v e r y 2 M i t o g e n Responses None LPS Con A 5 47 47 4 10 83 I n v i t r o PFC DNP-LPS SRBC 280 ± 42 311 ± 75 15 ± 8 98 ± 4 %:.'Phagocytic C e l l s 10 0.5 - 1.5 ± Normal DBA/2J s p l e e n c e l l s were i n c u b a t e d i n n y l o n w o o l columns and non a d h e r e n t c e l l s were e l u t e d . 2 3 M i t o g e n r e s p o n s e s a r e e x p r e s s e d as t h e mean cpm (x 10 ) of t r i p l i c a t e c u l t u r e s s t i m u l a t e d by PBS, LPS or Con A. 3 R e s u l t s a r e e x p r e s s e d as t h e mean number o f PFC per w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from the mean of t r i p l i c a t e a s s a y s . 4 The p e r c e n t a g e of p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g t h e number o f c e l l s p h a g o c y t i z i n g l a t e x beads. 52 TABLE V I I EFFECT OF CARBONYL IRON/MAGNET AND CARRAGHEENAN TREATMENT ON THE RESPONSES OF SPLEEN CELLS Treatment of C e l l s ' ^ C a r b o n y l U n t r e a t e d I r o n C arragheenan % V i a b l e C e l l R e c o v e r y 60 70 2 M i t o g e n Responses None 10 11 1 LPS 38 7 22 Con A 84 24 27 3 I n v i t r o PFC DNP-LPS 168 ± 1 9 72 ± 8 80 ± 12 4 % P h a g o c y t i c C e l l s 10 0 . 5 - 1 . 5 10 S p l e e n c e l l s were t r e a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. 3 M i t o g e n r e s p o n s e s a r e e x p r e s s e d as t h e mean cpm (x 10 ) o f t r i p l i c a t e c u l t u r e s s t i m u l a t e d by PBS, LPS or Con A. R e s u l t s a r e e x p r e s s e d as the mean number o f PFC per w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean of t r i p l i c a t e a s s a y s . The . p e r c e n t a g e o f p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g the number o f c e l l s p h a g o c y t i z i n g l a t e x beads. 53 TABLE V I I I RESPONSES OF SPLEEN CELL FRACTIONS OBTAINED AFTER PETRI DISH ADHERENCE F r a c t i o n T e s t e d 1 P e t r i D i s h P e t r i D i s h U n t r e a t e d A d h e r e n t Non A d h e r e n t % V i a b l e C e l l R e c o v e r y 2 M i t o g e n Respones None LPS Con A 4 90 197 30 2 86 29 60 3 63 184 I n v i t r o PFC DNP-LPS SRBC 280 ± 42 311 ± 75 420 ± 34 196 ± 72 126 ± 21 356 ± 64 % P h a g o c y t i c C e l l s 10 20 Normal DBA/2J s p l e e n c e l l s were adhered t o p l a s t i c p e t r i d i s h e s as d e s c r i b e d . 3 M i t o g e n r e s p o n s e s a r e e x p r e s s e d as t h e mean cpm (x 10 ) o f t r i p l i c a t e c u l t u r e s s t i m u l a t e d by PBS, LPS o r Con A. R e s u l t s a r e e x p r e s s e d as t h e mean number o f PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean.of t r i p l i c a t e a s s a y s . The p e r c e n t a g e o f p h a g o c y t i c c e l l s was e s t i m a t e d by c o u n t i n g t h e number o f c e l l s p h a g o c y t i z i n g l a t e x beads. 54 XX MITOMYCIN C TREATMENT M i t o m y c i n C f r o m S t r e p t o m y c e s c a e s p i t o s u s (SIGMA) was d i s s o l v e d i n w a t e r a t 500 ug/ml and s t o r e d a t 4°C i n the d a r k . S p l e e n c e l l s were i n c u b a t e d a t a c o n c e n t r a t i o n o f 10^ p e r m l w i t h 25, 50, o r 100 yg m i t o m y c i n C f o r 1 h a t 37°C. C e l l s u s p e n s i o n s were washed 3 t i m e s b e f o r e use. A p p r o x i m a t e l y 90% of^ t h e t r e a t e d c e l l s were r e c o v e r e d . Treatment o f c e l l s w i t h 25 yg o r more of m i t o m y c i n C t o t a l l y removed the r e s p o n s e t o T and B c e l l m i t o g e n s as w e l l as t h e PFC r e s p o n s e t o DNP-LPS and SRBC. XXI DATA PRESENTATION 1. M i t o g e n r e s p o n s e s were e x p r e s s e d as t h e mean c o u n t s p e r min 3 (cpm x 10 ) o f t r i p l i c a t e c u l t u r e s . I n a l l c a s e s the s t a n d a r d d e v i a t i o n s were l e s s t h a n 10%. 2. The p e r c e n t a g e s u p p r e s s i o n of m i t o g e n r e s p o n s e s was e s t i m a t e d by the f o l l o w i n g f o r m u l a : ( cpm f o r 5 x 10^ n o r m a l + 3 x 10^ tumor-bearer c e l l s » _ . j x 1 0 0 % cpm f o r 8 x 10 normal c e l l s T h i s was c o n s i d e r e d t o be a v a l i d method o f c a l c u l a t i n g s u p p r e s s i o n s i n c e t h e r e s p o n s e f o r 8 x 10^ n o r m a l c e l l s was n o t g e n e r a l l y s i g n i f i c a n t l y h i g h e r , and was f r e q u e n t l y l o w e r t h a n t h a t f o r 5 x 10^ n o r m a l c e l l s . T h i s f o r m u l a g e n e r a l l y gave a m i n i m a l e s t i m a t e of s u p p r e s s i o n . 3. The PFC r e s p o n s e d a t a was p r e s e n t e d as the number of PFC.per c u l t u r e p l u s or minus t h e s t a n d a r d d e v i a t i o n from the mean o f t r i p l i c a t e a s s a y s . 55 The p e r c e n t a g e s u p p r e s s i o n f o r PFC c u l t u r e s i n t h e m i c r o t i t e r t r a y s was c a l c u l a t e d as f o l l o w s : i PFC f o r 106 n o r m a l + 5 x 105 t u m o r ^ b e a r e r c e l l s * I n t h i s c a s e , t h e a d d i t i o n o f normal c e l l s t o w e l l s c o n t a i n i n g 10" n o r m a l c e l l s o f t e n i n c r e a s e s t h e number of PFC by i n c r e a s i n g t h e number o f p o t e n t i a l r e s p o n d i n g B c e l l s and T h e l p e r c e l l s , whereas th e t u m o r - b e a r e r c e l l s a r e n o t immunocompetent and do n o t add t o t h e r e s p o n s e o f t h e normal c e l l s . S u p p r e s s i o n i s t h u s c a l c u l a t e d by comparing t h e r e s p o n s e t o t h a t seen w i t h 10 n o r m a l c e l l s a l o n e . T h i s g i v e s t h e most c o n s e r v a t i v e e s t i m a t e o f s u p p r e s s i o n . The s u p p r e s s i o n o f the g e n e r a t i o n of PFC by n o r m a l c e l l s i n t h e i n n e r chamber (IC) of Marbrook Chambers was c a l c u l a t e d as f o l l o w s : 1 - X 100% PFC f o r w e l l s w i t h 10 n o r m a l c e l l s 6 PFC/IC w i t h 10 t u m o r - b e a r e r c e l l s i n OC X 100% PFC/IC w i t h 10 n o r m a l c e l l s i n OC (OC = o u t e r chamber) 56 RESULTS I IMMUNOCOMPETENCE OF TUMOR-BEARERS The g e n e r a l immunocompetence o f t u m o r - b e a r i n g m i c e was e s t i m a t e d a t w e e k l y i n t e r v a l s a f t e r t h e i n j e c t i o n o f 10^ M - l tumor c e l l s . A t t h e s e t i m e s , s p l e e n c e l l s f r o m m i c e were t e s t e d f o r t h e i r p r o l i f e r a t i v e r e s p o n s e s t o t h e T c e l l m i t o g e n , Con A, and the B c e l l m i t o g e n , LPS. Three tumor-bearers, were t e s t e d i n d i v i d u a l l y and s p l e e n c e l l s f r o m t h r e e n o r m a l s y n g e n e i c m i c e were p o o l e d b e f o r e t e s t i n g . The a v e r a g e r e s p o n s e s f o r t u m o r - b e a r e r s were compared t o t h e r e s p o n s e s f o r p o o l e d n o r m a l s p l e e n c e l l s t e s t e d under t h e same e x p e r i m e n t a l c o n d i t i o n s . The p r o l i f e r a t i v e r e s p o n s e s t o b o t h Con A and LPS d e c r e a s e d s t e a d i l y w i t h tumor growth ( T a b l e I X ) . A f t e r 4 weeks of tumor g r o w t h , the r e s p o n s e s of m i c e t o b o t h m i t o g e n s were l e s s t h a n 50% of t h a t seen w i t h n o r m a l mouse s p l e e n c e l l s . I n o r d e r t o show t h a t t h e d e p r e s s i o n o f m i t o g e n r e s p o n s e s was n o t r e s t r i c t e d t o mice b e a r i n g t h e M - l tumor, t h e r e s p o n s e s of mic e b e a r i n g a v a r i e t y o f tumor t y p e s were t e s t e d ( T a b l e X ) . The M - l and M-2 tumor l i n e s a r e two d i f f e r e n t m e t h y l c h o l a n t h r e n e - i n d u c e d sarcomas and MA i s a spontaneous mammary adenoca r c i n o m a . A l l t h r e e a r e s y n g e n e i c t o DBA/2J m i c e . The B - l l i n e i s a m e t h y l c h o l a n t h r e n e - i n d u c e d tumor s y n g e n e i c t o BALB/CJ m i c e . Immunocompetence o f mic e 4 t o 6 weeks a f t e r tumor c e l l i n o c u l a t i o n was t e s t e d by m e a s u r i n g the p r o l i f e r a t i v e r e s p o n s e s o f t h e i r s p l e e n c e l l s t o Con A and LPS. S p l e e n c e l l s f r o m mice b e a r i n g a l l 4 tumor t y p e s and f r o m b o t h s t r a i n s o f m i c e were immunosuppressed. I t would a p p e a r , f r o m t h e s e d a t a , t h a t t he d e p r e s s i o n o b s e r v e d d u r i n g tumor g r o w t h , i s a phenomenon t h a t o c c u r s i n a number o f d i f f e r e n t t r a n s -p l a n t a b l e tumor l i n e s i n mice. 57 TABLE IX MITOGEN RESPONSES OF SPLEEN CELLS FROM TUMOR-BEARING MICE 3 3 3 Time of S p l e e n H-Thymidine I n c o r p o r a t i o n (cpm x 10 ) Tumor Growth C e l l s — — ( w e e k s ) 1 C u l t u r e d 2 None Con A LPS I I N 13 T 10 ± I I I N 5 T 3 ± IV N 10 T 6 ± V N 9 T 7 ± V I N 14 T 9 ± V I I N 5 T 3 ± 170 28 1 176 ± 29(103) 15 ± 2(52) 75 19 1 70 ± 10(93) 14 ± 4(74) 123 38 1 54 ± 14(44) 10 ± 4(26) 137 56 1 76 ± 24(55) 13 ± 5(24) 164 66 4 60 ± 3(37) 10 ± 2(15) 222 60 1 61 ± 30(28) 7 ± 2(12) M i c e were i n j e c t e d w i t h 10^ M - l tumor c e l l s . 8 x 10^ p o o l e d - s p l e e n c e l l s f rom t h r e e normal (N) s y n g e n e i c m i c e o r 8 x 10 c e l l s f r o m i n d i v i d u a l t u m o r - b e a r e r s (T) were c u l t u r e d . R e s u l t s f o r t u m o r - b e a r e r s a r e e x p r e s s e d as the mean of t h e cpm f o r t h r e e i n d i v i d u a l mice p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean. The r e s u l t s a r e a l s o e x p r e s s e d ( i n b r a c k e t s ) as a p e r c e n t a g e of the s t i m u l a t i o n l e v e l f o r t h e c o r r e s p o n d i n g n o r m a l s p l e e n c e l l s . 58 TABLE X MITOGEN RESPONSES OF MICE BEARING A VARIETY OF TUMOR TYPES So u r c e o f Tumor 3 T T m, . , . . . -,^3.2 ^ H-Tnymidine I n c o r p o r a t i o n (cpm x 10 ) S p l e e n C e l l s Type — None Con A LPS N T N T M - l M-2 19 14 7 14 189 57(30) 163 27(17) 95 24(25) 69 31(45) N T MA 21 16 145 2(1) 72 14(19) N T B - l 17 10 130 34(26) 62 12(19) S p l e e n c e l l s f r o m i n d i v i d u a l t u m o r - b e a r i n g (T) mi c e were t e s t e d f o u r t o s i x weeks a f t e r i n j e c t i o n o f 10 tumor c e l l s . S p l e e n c e l l s f r o m t h r e e n o r m a l (N) s y n g e n e i c m i c e were p o o l e d b e f o r e t e s t i n g . R e s u l t s a r e e x p r e s s e d as t h e mean cpm of t r i p l i c a t e c u l t u r e s each c o n t a i n -i n g 5 x 10^ c e l l s . R e s u l t s f o r t u m o r - b e a r e r s a r e a l s o e x p r e s s e d ( i n b r a c k e t s ) as t h e p e r c e n t a g e o f t h e s t i m u l a t i o n l e v e l f o r t h e c o r r e s p o n d i n g n o r m a l s y n g e n e i c c o n t r o l c e l l s . 59 I I SUPPRESSOR CELLS IN TUMOR-BEARER SPLEENS: SUPPRESSION OF MITOGEN  RESPONSES S p l e e n c e l l s f r o m m i c e i n j e c t e d w i t h 10^ M ^ l c e l l s were t e s t e d w e e k l y f o r t h e a b i l i t y t o s u p p r e s s the p r o l i f e r a t i v e r e s p o n s e of s p l e e n c e l l s f r o m n o r m a l s y n g e n e i c m i c e s t i m u l a t e d by t h e m i t o g e n s Con A and LPS. F o r t h e s e e x p e r i m e n t s , 5 x 10^ n o r m a l s p l e e n c e l l s were c o - c u l t u r e d w i t h 3 x 10^ n o r m a l or t u m o r - b e a r e r s p l e e n c e l l s . S u p p r e s s i o n was c a l c u l a t e d by comparing the r e s p o n s e o f n o r m a l c e l l s c o - c u l t u r e d w i t h t u m o r - b e a r e r c e l l s t o t h a t f o r 8 x 10^ n o r m a l c e l l s . T h i s i s c o n s i d e r e d t o be a v a l i d e s t i m a t e o f s u p p r e s s -i o n s i n c e the p r o l i f e r a t i v e r e s p o n s e o f 8 x 10^ n o r m a l s p l e e n c e l l s i s g e n e r a l l y v e r y s i m i l a r t o t h a t f o r 5 x 10^ n o r m a l c e l l s . A r e d u c t i o n o f t h i s r e s p o n s e s h o u l d t h e r e f o r e r e f l e c t a c t i v e s u p p r e s s i o n . S p l e e n c e l l s f r o m M - l t u m o r - b e a r e r s were c a p a b l e , a f t e r 4 weeks o f tumor g r o w t h , of s u p p r e s s i n g t h e r e s p o n s e t o b o t h m i t o g e n s ( T a b l e X I ) . The d e g r e e of s u p p r e s s i o n d i d not change g r e a t l y when t h e tumors grew l a r g e r e x c e p t t h a t t h e s u p p r e s s i v e a b i l i t y o f t e n d e c l i n e d a f t e r 7 weeks o f g r o w t h . S u p p r e s s i o n c o u l d n o t have been due t o tumor c e l l s i n t h e s p l e e n s i n c e M - l tumor c e l l s do n o t m e t a s t a s i z e t o t h e s p l e e n a t any t i m e d u r i n g tumor growth. Tumor n o d u l e s have n e v e r been o b s e r v e d i n s p l e e n s and tumor c e l l s have n e v e r been f o u n d i n s p l e e n c e l l p r e p a r a t i o n s f r o m t u m o r - b e a r i n g m i c e . When s p l e e n c e l l s were c u l t u r e d i n v i t r o under c o n d i t i o n s i n w h i c h M - l tumor c e l l s a r e known t o grow, no tumor c e l l s were o b s e r v e d . 60 TABLE X I SUPPRESSIVE EFFECTS OF TUMOR-BEARER SPLEEN CELLS ON THE MITOGEN RESPONSE OF NORMAL LYMPHOID CELLS Time o f S p l e e n c e l l s 3 3 3 H-Thymidine I n c o r p o r a t i o n (cpm x 10 ) Tumor Growth (weeks) Added t o ^ C u l t u r e s None Con A LPS I I N 13 170 28 T 12 + 2 154 + 15(9) 23 ± 3(8) I I I N 5 75 19 T 5 + 2 67 + 12(11) 11 ± 4(42) IV N 10 123 38 ft T 8 + 2 62 + 8(50) " 19 ± 9(50) V N 8 137 ft 56 ft T 9 + 2 82 + 11(40) 34 ±10(49) V I N 14 164 ft 66 ft T 9 + 3 99 + 15(40) 31 ± 5(53) V I I N 5 222 60 T 4 + 1 121 + 34(45) 48 ± 10(20) M i c e were i n j e c t e d w i t h 10"' M - l tumor c e l l s . C u l t u r e s c o n s i s t e d o f 5 x 10^ p o o l e d s p l e e n c e l l s f r o m t h r e e normal m i c e p l u s 3 x 10 n o r m a l (N) or t u m o r - b e a r e r (T) c e l l s . R e s u l t s f o r n o r m a l c o n t r o l c u l t u r e s a r e e x p r e s s e d as mean cpm of t r i p l i c a t e c u l t u r e s . R e s u l t s f o r t u m o r - b e a r e r s a r e e x p r e s s e d as the mean cpm f o r t h r e e i n d i v i d u a l m i c e p l u s or minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean. S t i m u l a t i o n l e v e l s f o r t u m o r - b e a r e r s a r e a l s o g i v e n as a p e r c e n t a g e s u p p r e s s i o n o f t h e r e s p o n s e by n o r m a l c e l l s ( i n b r a c k e t s ) . The * i n d i c a t e s t h a t t h e 95% c o n f i d e n c e l i m i t s f o r t h e r e s p o n s e s o f tumor-b e a r e r s do n o t i n c l u d e t h e r e s p o n s e s f o r t h e c o r r e s p o n d i n g normal c o n t r o l c e l l s (p < 0.05). 61 I I I HYPAQUE-FICOLL ENRICHMENT OF SUPPRESSOR CELLS. S p l e e n c e l l s f r o m n o r m a l or tumors-bearing m i c e were l a y e r e d on 5 i n d i v i d u a l g r a d i e n t s o f h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t i e s r a n g i n g f r o m 1.06 t o 1.09. A f t e r c e n t r i f u g a t i o n of the g r a d i e n t s , t h e l i g h t f r a c t i o n s , c o n t a i n i n g c e l l s r e t a i n e d a t t h e i n t e r f a c e o f t h e h y p a q u e - f i c o l l and medium, and t h e dense f r a c t i o n s , c o n t a i n i n g c e l l s w h i c h p e l l e t e d t o t h e bottom o f the t u b e s , were c o l l e c t e d . A l l f r a c t i o n s were t e s t e d f o r the a b i l i t y t o s u p p r e s s the m i t o g e n i c r e s p o n s e o f c o - c u l t u r e d n o r m a l s p l e e n c e l l s ( F i g u r e 2 ) . None o f t h e l i g h t o r dense f r a c t i o n s o f n o r m a l s p l e e n c e l l s were s u p p r e s s i v e . I n t h e e x p e r i m e n t shown, u n f r a c t i o n a t e d t u m o r - b e a r e r s p l e e n c e l l s d e m o n s t r a t e d l i t t l e s u p p r e s s i v e a c t i v i t y (about 2 0 % ) . A f t e r c e n t r i f u g a t i o n on hypaque-f i c o l l o f s p e c i f i c g r a v i t i e s 1.08 o r 1.07, t h e c e l l s i n t h e l i g h t f r a c t i o n were h i g h l y s u p p r e s s i v e and produced about 80% i n h i b i t i o n . None of the dense f r a c t i o n s of t u m o r - b e a r e r c e l l s were s u p p r e s s i v e . S p l e e n c e l l s f r o m m i c e b e a r i n g f o u r d i f f e r e n t tumor t y p e s were c e n t r i c f u g e d on h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08 ( T a b l e X I I ) . The d i f f e r e n t s p l e e n s t e s t e d had v a r y i n g d e g r e e s o f s u p p r e s s o r c e l l a c t i v i t y , b u t i n a l l c a s e s t h e l i g h t f r a c t i o n was more s u p p r e s s i v e t h a n u n f r a c t i o n a t e d s p l e e n c e l l s and the dense f r a c t i o n was l e s s s u p p r e s s i v e . T h e r e f o r e , t h e s p l e e n c e l l s o f m i c e b e a r i n g s e v e r a l d i f f e r e n t t y p e s o f tumors c o n t a i n e d s u p p r e s s o r c e l l s w h i c h were, i n a l l c a s e s , amongst the l e a s t dense c e l l s i n the s p l e e n . 62 i i i i i 1 1.090 1.085 1.080 1.070 1.060 r S P E C I F I C G R A V I T Y OF H Y P A Q U E - Fl COL L F i g u r e 2. F r a c t i o n a t i o n o f S p l e e n C e l l s on H y p a q u e - F i c o l l of D i f f e r e n t S p e c i f i c G r a v i t i e s . • • , N + N ; O — • , N + N ; • — • , Li [) N + T ; O—O , N + T . 5 x 10^ u n f r a c t i o n a t e d normal (N) c e l l s . Li U were c u l t u r e d w i t h 3 x 10^ normal (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s f rom t h e l i g h t (L) o r dense (D) f r a c t i o n s from each hypaque-f i c o l l s p e c i f i c g r a v i t y . R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e o f t h e r e s p o n s e o f 8 x 10^ u n f r a c t i o n a t e d normal s p l e e n c e l l s a l o n e . 63 TABLE X I I SUPPRESSIVE EFFECTS OF UNFRACTIONATED TUMOR-BEARER SPLEEN CELLS AND HYPAQUE-FICOLL FRACTIONS C o m p o s i t i o n C u l t u r e of Tumor Type 3 H-Thymidine I n c o r p o r a t i o n 3 2 (cpm x 10 ) 5 x 1 0 5 3 x 1 0 5 None Con A LPS N N N N N N T T L T D M - l 19 27 19 14 20 189 156 104(33) 3(98) 157(0) 95 115 44(62) 6(96) 57(50) N N N N N N T T L T D M-2 7 16 23 20 20 163 139 77(44) 4(97) 106(24) 69 101 49(51) 20(80) 67(34) N N N N N N T T L TD MA 21! 31 25 24 30 145 144 16(89) 3(98) 80(45) 72 55 25(54) 23(58) 46(16) N N N N N N T T L TD B - l 9 16 12 9 14 141 83 3(96) 2(98) 36(57) 27 40 26(35) 4(90) 32(20) C u l t u r e s c o n s i s t e d o f t h e i n d i c a t e d numbers of normal (N) and u n f r a c t i o n a t e d t u m o r - b e a r e r (T) s p l e e n c e l l s o r t h e dense (D) o r l i g h t (L) tu m o r - b e a r e r s p l e e n f r a c t i o n s f r o m a h y p a q u e - f i c o l l s e p a r a t i o n a t a s p e c i f i c g r a v i t y o f 1.08. R e s u l t s a r e e x p r e s s e d as t h e mean cpm of t r i p l i c a t e c u l t u r e s . The p e r c e n t -age s u p p r e s s i o n i s shown i n b r a c k e t s . 64 IV BASIC PARAMETERS OF THE MITOGEN ASSAY 1. T i t r a t i o n o f S u p p r e s s o r C e l l s Normal s p l e e n c e l l s were c o ^ c u l t u r e d w i t h d i f f e r e n t q u a n t i t i e s o f n o r m a l or t u m o r - b e a r e r s p l e e n c e l l s ( F i g u r e 3 ) . The degree of s u p p r e s s -i o n by t u m o r — b e a r e r c e l l s was dependent on t h e number o f c e l l s added t o t h e c u l t u r e s . I n t h i s e x p e r i m e n t , t h e s u p p r e s s i o n by 10^ t u m o r - b e a r e r c e l l s was g r e a t e r t h a n t h a t seen when 5 t i m e s more no r m a l s p l e e n c e l l s were added. T h i s was the c a s e when c u l t u r e s were s t i m u l a t e d by Con A or LPS. 2. K i n e t i c s o f t h e M i t o g e n Response The k i n e t i c s o f the r e s p o n s e o f 5 x 10^ n o r m a l s p l e e n c e l l s t o Con A and LPS was t e s t e d when t h e c e l l s were c u l t u r e d a l o n e o r w i t h an add-i t i o n a l 3 x 10^ normal o r t u m o r - b e a r e r c e l l s ( F i g u r e 4 ) . The c e l l s added t o t h e c u l t u r e s were o b t a i n e d f r o m t h e l i g h t f r a c t i o n a f t e r a 3 h y p a q u e - f i c o l l s e p a r a t i o n . C u l t u r e s a r e n o r m a l l y a s s a y e d f o r H-thy-m i d i n e i n c o r p o r a t i o n a f t e r 3 days i n c u l t u r e . However, s u p p r e s s i o n was s t i l l e v i d e n t when c u l t u r e s were h a r v e s t e d a f t e r 2 o r 4 days. S u p p r e s s -i o n seen on day 3 i s n o t t h e r e f o r e due t o a change i n t h e k i n e t i c s of th e r e s p o n s e . 3. Con A Dose Response Curve The r e s p o n s e o f no r m a l s p l e e n c e l l s t o d i f f e r e n t doses of Con A was t e s t e d when t h e c e l l s were c u l t u r e d a l o n e o r w i t h t h e h y p a q u e - f i c o l l l i g h t f r a c t i o n s o f no r m a l or t u m o r - b e a r e r c e l l s ( F i g u r e 5 ) . S u p p r e s s i o n o c c u r r e d a t a l l doses o f Con A t e s t e d and i s not t h e r e f o r e due t o a change i n t h e dose r e s p o n s e c u r v e . N U M B E R O F C E L L S A D D E D ( X 1 0 ) F i g u r e 3. The m i t o g e n r e s p o n s e of n o r m a l s p l e e n c e l l s c o - c u l t u r e d w i t h v a r y i n g numbers of n o r m a l or tumor-bearer s p l e e n c e l l s . Each w e l l c o n t a i n e d 5 x 10^ n o r m a l p l u s t h e i n d i c a t e d numbers o f : | | , n o r m a l s p l e e n c e l l s ; o r js^j , tumor-bearer s p l e e n c e l l s . C u l t u r e s were i n c u b a t e d w i t h 0.05 ml PBS as a c o n t r o l , 4 yg Con A/ml, or 125 yg LPS/ml. 66 C O N A L P S o s a. u 15 -14 -13 -1 2 -1 1 -10 -9 -8 -7-6-5-4-3 -2 -1 -0 — ! i ! -8 ! 1 Is Is D A Y S C U L T U R E F i g u r e 4. K i n e t i c s of t h e m i t o g e n r e s p o n s e . Each w e l l c o n t a i n e d 5 x 10^ nor m a l s p l e e n c e l l s p l u s ; , RPMI 1640; , 3 x 10"* no r m a l l i g h t c e l l s ; o r [•] , 3 x 10^ t u m o r - b e a r e r l i g h t s p l e e n c e l l s . C u l t u r e s were i n c u b a t e d w i t h 4 yg Con A/ml o r 125 yg LPS/ml f o r the i n d i c a t e d number of days. L i g h t f r a c t i o n s were o b t a i n e d by f r a c t i o n a t i o n or h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08. 67 18 -17-16 -15 -14 -13-12-1 1 -10 -9 -8 -7-6 -5-4 -3 -2-1 -ol— L Is 1 0^ 1 r! l 5 L 1 6 C O N A l u g / m l ) F i g u r e 5. The m i t o g e n r e s p o n s e w i t h d i f f e r e n t doses o f Con A. Each w e l l c o n t a i n e d 5 x 10^ n o r m a l c e l l s p l u s ; | | , RPMI 1640; 3 x 10^ normal l i g h t c e l l s ; o r [ » J , 3 x 10^ t u m o r - b e a r e r l i g h t c e l l s . C u l t u r e s were i t i c u b a t e d f o r 3 days. L i g h t f r a c t i o n s were o b t a i n e d by f r a c t i o n a t i o n on h y p a q u e - f i c o l l of s p e c i f i c g r a v i t y 1.08. 68 V EFFECT OF TUMOR LOAD ON SUPPRESSOR CELL ACTIVITY 1. S p l e e n C e l l s From M i c e W i t h S u r g i c a l l y R e s e c t e d Tumors F i v e weeks a f t e r i n j e c t i o n o f 10^ M - l c e l l s , tumors of 1,60 t o 3.58 g were s u r g i c a l l y r e s e c t e d f r o m m i c e . S p l e e n c e l l s were t e s t e d f o r t h e a b i l i t y t o s u p p r e s s t h e m i t o g e n r e s p o n s e s o f n o r m a l s p l e e n c e l l s on v a r i o u s days a f t e r r e s e c t i o n ( T a b l e X I I I ) . S p l e e n s f r o m m i c e had r e t -u r n e d t o n o r m a l w e i g h t (see T a b l e I ) by 13 days a f t e r r e s e c t i o n . C o r r e l a t e d w i t h t h i s was a l o s s of s u p p r e s s o r c e l l a c t i v i t y . By day 13, s p l e e n c e l l s f r o m r e s e c t e d m i c e were e i t h e r n o t s u p p r e s s i v e o r , i n f a c t , enhanced t h e r e s p o n s e o f n o r m a l c e l l s t o b o t h Con A and LPS. 2. S p l e e n C e l l s From Immune M i c e M i c e were made immune t o t h e M - l tumor by s u r g i c a l r e s e c t i o n of tumors w i t h a mass of about 1.0 g. Two weeks a f t e r r e s e c t i o n of tumors, m i c e were a b l e t o r e j e c t doses of tumor c e l l s 100 t i m e s t h a t w h i c h i n d u c e s tumors 100% of the t i m e i n normal m i c e . U n t r e a t e d s p l e e n c e l l s and h y p a q u e - f i c o l l f r a c t i o n s of s p l e e n c e l l s f r o m t h e s e a n i m a l s were n o t c a p a b l e o f s u p p r e s s i n g t h e r e s p o n s e of n o r m a l s p l e e n c e l l s t o Con A and LPS ( T a b l e X I V ) . T h e r e f o r e , i t a p p e a r s f r o m t h e s e d a t a , t h a t the p r e s e n c e of a l a r g e tumor mass i n m i c e i s r e q u i r e d f o r t h e p r o d u c t i o n and maintenance of s p l e n i c s u p p r e s s o r c e l l s . 69 TABLE X I I I SUPPRESSIVE EFFECTS OF SPLEEN CELLS FROM TUMOR-RESECTED MICE 3 A £ J - o i TT • U4-2 % S u p p r e s s i o n Days A f t e r S p l e e n Weight R e s e c t i o n 1 (g) Con A LPS 0 0.44 + .06 40 + *4 8 4 39 + 16 1 0.41 + .06 14 + 6 33 + 6 4 0.35 + .18 17 + 12 38 + 28 8 0.21 + .05 18 + * 6 32 + 19 13 0.17 + .01 -20 + 49 -18 + 44 F i v e weeks a f t e r i n j e c t i o n o f 10 M - l c e l l s , tumors of 1.60 t o 3.58 g were s u r g i c a l l y r e s e c t e d . S p l e e n w e i g h t r e f e r s t o t h e a v e r a g e w e i g h t of s p l e e n s f r o m 3 r e s e c t e d m i c e p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean. 5 x 10^ p o o l e d s p l e e n c e l l s f r o m 3 n o r m a l s y n g e n e i c m i c e were Rested f o r t h e i r r e s p o n s e s t o m i t o g e n s when c u l t u r e d a l o n e o r w i t h 3 x 10 s p l e e n c e l l s f r o m r e s e c t e d m i c e . P e r c e n t a g e s u p p r e s s i o n r e f e r s t o t h e a v e r a g e s u p p r e s s i o n ( p l u s t h e s t a n d a r d d e v i a t i o n from t h e mean) of the r e s p o n s e f o r n o r m a l c e l l s c u l t u r e d i n d i v i d u a l l y w i t h c e l l s f r o m r e s e c t e d m i c e . The 95% c o n f i d e n c e l i m i t s f o r t h e r e s p o n s e s o f c o - c u l t u r e s of c e l l s f r o m n o r m a l p l u s r e s e c t e d m i c e were compared t o t h e r e s p o n s e s f o r c u l t u r e s w i t h n o r m a l c e l l s a l o n e . R e s u l t s i n w h i c h the upper c o n f i d e n c e l i m i t was l o w e r t h a n t h e n o r m a l r e s p o n s e a r e marked w i t h an * (p < 0.05). TABLE X I V EFFECTS OF TUMOR IMMUNE SPLEEN POPULATIONS ON NORMAL SPLEEN CELLS 3 3 2 C o m p o s i t i o n o f H-Thymidine I n c o r p o r a t i o n (cpm x 10 ) C u l t u r e 1 -5 x 1 0 5 3 x 1 0 5 None Con A LPS 25 152 57 I N N N I I D 22 15 27 24 142 145 140 152 60 52 57 60 S p l e e n c e l l s were t a k e n f r o m n o r m a l (N) o r i n d i v i d u a l tumor immune ( I ) mic e . Immune s p l e e n c e l l s were s e p a r a t e d i n t o dense (D) and l i g h t (L) f r a c t i o n s on h y p a q u e - f i c o l l h a v i n g a s p e c i f i c g r a v i t y o f 1.08. 3 R e s u l t s a r e e x p r e s s e d as mean cpm x 10 o f t r i p l i c a t e c u l t u r e s . 71 V I LYMPH NODE CELLS FROM TUMOR-BEARERS Whereas t u m o r - b e a r e r s p l e e n c e l l s were n o r m a l l y immunosuppressed and e x e r t e d s u p p r e s s i v e a c t i v i t y on n o r m a l s p l e e n c e l l s , t u m o r - b e a r e r lymph node c e l l s r esponded t o m i t o g e n s as w e l l as d i d n o r m a l lymph node c e l l s , a l t h o u g h t h e y d i d have a c o n s i d e r a b l y h i g h e r u n s t i m u l a t e d i s o t o p e i n c o r p o r a t i o n 3 ( T a b l e X V ) . C o n s i d e r i n g t h e a b s o l u t e v a l u e s of H - t h y m i d i n e i n c o r p o r a t i o n , lymph node r e s p o n s e s t o m i t o g e n s were e q u i v a l e n t t o n o r m a l i n t u m o r - b e a r i n g mice whose s p l e e n c e l l s were a l m o s t e n t i r e l y s u p p r e s s e d . However, s i n c e u n s t i m u l a t e d i s o t o p e i n c o r p o r a t i o n was c o n s i d e r a b l y g r e a t e r i n t u m o r - b e a r e r lymph node c e l l s , p r e s e n t a t i o n o f d a t a as r a t i o s of i n c o r p o r a t i o n would i n d i c a t e t h a t t h e r e was i n d e e d s u p p r e s s i o n . S i n c e a n t i - T h y - 1 and complement t r e a t m e n t a b o l i s h e s t h e Con A r e s p o n s e but does n o t i n h i b i t t h e u n s t i m u l a t e d i n c o r p o r a t i o n ( T a b l e V ) , i t i s p r o b a b l e t h a t t h e c e l l s p r o d u c i n g t h e un-s t i m u l a t e d i n c o r p o r a t i o n a r e n o t t h e same c e l l s r e s p o n d i n g t o t h e m i t o g e n . Thus, t h e y s h o u l d n o t be c o n s i d e r e d i n r e l a t i o n t o t h e m i t o g e n - s t i m u l a t e d r e s u l t s . Tumor-bearer lymph node c e l l s had no s u p p r e s s i v e e f f e c t s on e i t h e r n o r m a l lymph node o r s p l e e n c e l l s . U n f r a c t i o n a t e d s p l e e n c e l l s f rom t h e same t u m o r - b e a r e r , however, s u p p r e s s e d n o r m a l lymph node c e l l s . Hypaque-f i c o l l f r a c t i o n s o f s p l e e n c e l l s behaved as b e f o r e : c e l l s f rom t h e l i g h t f r a c t i o n s u p p r e s s e d n o r m a l lymph node c e l l s whereas t h e dense c e l l s d i d n o t . S u p p r e s s o r c e l l s were found i n t h e s p l e e n s but n o t lymph nodes of t u m o r - b e a r i n g m i c e . S p l e n i c s u p p r e s s o r c e l l s were, however, c a p a b l e of s u p p r e s s i n g : t h e m i t o g e n r e s p o n s e s o f lymph node c e l l s w h i c h were t h e m s e l v e s immunocompetent. 72 TABLE XV EFFECTS OF TUMOR-BEARER LYMPH NODE AND SPLEEN CELLS ON MITOGEN RESPONSES OF NORMAL LYMPHOID CELLS C o m p o s i t i o n 3 H-Thymidine I n c o r p o r a t i o n 3 2 (cpm x 10 ) o f C u l t u r e 5 x 105 3 x 105 None Con A LPS N L N N L N 6 122 59 T LN T L N 22 146 69 N L N T L N 7 135(-10) 63(-6) N L N T s 22 46(62) 45(24) N L N T SL 25 23(81) 36(39) N L N TSD 19 140(-14) 56(-5) N s N s 38 141 63 T s T s 16 1 14 N s N s T s 31 30(79) 38(40) T L N 32 137(3) 72(-14) C u l t u r e s c o n t a i n e d e i t h e r 8 x 10"* n o r m a l (N) or M - l t u m o r - b e a r e r (T) lymph node (LN) oj: s p l e e n (S) c e l l s o r 5 x 10 n o r m a l s p l e e n o r lymph node c e l l s p l u s 3 x 10 t u m o r - b e a r e r s p l e e n o r lymph node c e l l s . S p l e e n c e l l f r a c t i o n s , l i g h t ,.(.L) and dense (D) were o b t a i n e d by c e n t r i f u g a t i o n on hypaque-f i c o l l w i t h a s p e c i f i c g r a v i t y o f 1.08. R e s u l t s a r e e x p r e s s e d as mean cpm of t r i p l i c a t e c u l t u r e s and p e r c e n t a g e s u p p r e s s i o n ( i n b r a c k e t s ) . 73 V I I CHARACTERIZATION OF SUPPRESSOR CELLS 1. M i t o m y c i n C Treatment o f S u p p r e s s o r C e l l s Tumor-bearer s p l e e n c e l l s f rom t h e l i g h t f r a c t i o n a f t e r c e n t r i -f u g a t i o n on h y p a q u e - f i c o l l were t r e a t e d w i t h 25, 50, o r 100 yg/ml o f m i t o m y c i n C b e f o r e c o - c u l t u r i n g w i t h n o r m a l s p l e e n c e l l s . The r e s p o n s e s o f n o r m a l s p l e e n c e l l s t o Con A and LPS were s u p p r e s s e d by t u m o r - b e a r e r s p l e e n c e l l s w h i c h had been t r e a t e d w i t h up t o 100 yg/ml m i t o m y c i n C ( T a b l e X V I ) . A dose of 100 yg/ml has been shown t o i n h i b i t RNA and p r o t e i n s y n t h e s i s as w e l l as DNA s y n t h e s i s ( 8 5 ) . Doses as low as 25 3 yg/ml p r e v e n t e d t h e H - t h y m i d i n e i n c o r p o r a t i o n a s s o c i a t e d w i t h t h e p r o l i f e r a t i v e r e s p o n s e t o Con A and LPS. The s p l e e n c e l l s r e s p o n s i b l e f o r s u p p r e s s i o n of t h e m i t o g e n r e s p o n s e d i d n o t t h e r e f o r e r e q u i r e DNA s y n t h e s i s i n o r d e r t o s u p p r e s s . A l s o , s i n c e h i g h e r doses o f m i t o m y c i n C d i d n o t remove s u p p r e s s i o n , RNA and p r o t e i n s y n t h e s i s may a l s o n o t be n e c e s s a r y . 2. Removal o f T and B C e l l s The h y p a q u e - f i c o l l l i g h t f r a c t i o n o f t u m o r - b e a r e r s p l e e n c e l l s was t r e a t e d w i t h a n t i - T h y - 1 o r a n t i - M I g s e r a p l u s complement t o remove T o r B c e l l s . T r e a t e d c e l l s were t h e n c o - c u l t u r e d w i t h n o r m a l c e l l s t o d e t e r m i n e whether o r n o t s u p p r e s s o r c e l l a c t i v i t y had been l o s t a f t e r t h i s t r e a t m e n t ( T a b l e X V I I ) . N e i t h e r of t h e s e t r e a t m e n t s removed s u p p r e s s i v e a c t i v i t y t o a s i g n i f i c a n t e x t e n t s u g g e s t i n g t h a t t h e sup-p r e s s o r c e l l was n o t a T or B c e l l . 3. Removal of P h a g o c y t i c C e l l s S p l e e n c e l l s from .tumor-bearers were d e p l e t e d of p h a g o c y t i c c e l l s by c a r b o n y l iron/magnet t r e a t m e n t o r passage t h r o u g h a n y l o n w o o l column. These two t r e a t m e n t s , b o t h of w h i c h r e d u c e t h e p e r c e n t a g e 74 o f c e l l s p h a g o c y t i z i n g l a t e x beads t o 1% o r l e s s , c o m p l e t e l y removed t h e s u p p r e s s i v e a c t i v i t y f rom t h e s p l e e n c e l l s ( T a b l e X V I I I ) . Adherence o f s p l e e n c e l l s t o p l a s t i c c r e a t e d two f r a c t i o n s o f c e l l s : t h o s e a d h e r e n t ;and t h o s e non a d h e r e n t t o p l a s t i c . The adhe r e n t c e l l s , w h i c h c o n t a i n e d a h i g h e r p e r c e n t a g e o f p h a g o c y t i c c e l l s t h a n t h e non a d h e r e n t f r a c t i o n , were more s u p p r e s s i v e t h a n t h e non adhe r e n t c e l l s , b u t some o f t h e s u p p r e s s o r c e l l s remained i n t h e non adhe r e n t f r a c t i o n . I t t h e r e f o r e appeared t h a t t h e s u p p r e s s o r c e l l s were from t h e macrophage/monocyte l i n e s i n c e t h e s u p p r e s s o r a c t i v i t y was removed by t r e a t m e n t s w h i c h d e p l e t e d t h e s p l e e n o f p h a g o c y t i c c e l l s . A l s o t h e s u p p r e s s o r c e l l s t ended t o adhere t o p l a s t i c . TABLE XVI EFFECT OF MITOMYCIN C TREATMENT ON SUPPRESSOR CELL ACTIVITY Compositon 3 3 2 H-Thymidine I n c o r p o r a t i o n (cpm x 10 ) 5 x 10 o f C u l t u r e 5 3 x 10 _ None Con A LPS 19 155 54 23 174 47 23 2(99) 2(96) 13 165 38 7 13(92) 4(90) 12 140 24 7 19(86) 7.(71) 9 150 32 6 54(64) 12.(63) 1 3 5 1 6 2 1 1 1 1 2 1 1 2 1 1 1 1 N N N N N N N N N N T N L 25 y g N L 50 y g N L 100 y g N L 25 y g N L 50 y g N L 100 y g T L 25 y g T L 50 y g T L 100 y g C u l t u r e s c o n s i s t e d o f t h e i n d i c a t e d numbers o f n o r m a l s p l e e n c e l l s p l u s l i g h t (L) n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n f r a c t i o n s a f t e r hypaque-f i c o l l s e p a r a t i o n a t a s p e c i f i c g r a v i t y o f 1.08. L i g h t f r a c t i o n s were t r e a t e d w i t h 25, 50, o r 100 y g m i t o m y c i n C per ml as i n d i c a t e d . R e s u l t s a r e e x p r e s s e d as mean cpm o f t r i p l i c a t e c u l t u r e s and p e r c e n t a g e s u p p r e s s i o n ( i n b r a c k e t s ) . 76 TABLE X V I I EFFECTS OF ANTI-THY-1 AND ANTI-MIg ON SUPPRESSOR CELL ACTIVITY 3 2 C o m p o s i t i o n Treatment H-Thymidine I n c o r p o r a t i o n 1 (cpm x 10 ) o f C u l t u r e 5 x 1 0 5 3 x 1 0 5 None Con A LPS 1 N 20 133 53 W N.. N t 7 16 101 57 w W N w T L none 23 48(52) 1(98) N w T L a n t i - T h y - 1 14 53(48) 11(79) N w T L a n t i - M I g 18 67(34) 4(92) N a n t i - T h y - 1 10 23 69 N a n t i - M I g 8 105 16 C u l t u r e s c o n s i s t e d o f t h e i n d i c a t e d numbers o f n o r m a l (N) and t u m o r - b e a r e r (T) s p l e e n c e l l s e i t h e r u n t r e a t e d (W) o r t r e a t e d w i t h a n t i - T h y - 1 o r a n t i -MIg s e r a p l u s complement. S u p p r e s s o r c e l l s (T^) were f i r s t c e n t r i f u g e d on h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08. R e s u l t s a r e e x p r e s s e d as mean cpm o f t r i p l i c a t e c u l t u r e s and p e r c e n t a g e s u p p r e s s i o n ( i n b r a c k e t s ) . 77 Q TABLE X V I I I EFFECTS OF TREATMENTS WHICH REMOVE ADHERENT AND PHAGOCYTIC CELLS ON SUPPRESSOR CELL ACTIVITY 3 2 C o m p o s i t i o n Treatment H-Thymidine I n c o r p o r a t i o n 1 (cpm x 10 ) o f C u l t u r e 5 x 10^ 3 x 10^ „ „ . T None Con A LPS N N N none N T N N p e t r i d i s h N T a d h e r e n t N N p e t r i d i s h N T non a d h e r e n t N N c a r b o n y l N T I r o n N N n y l o n w o o l N T 24 115 64 28 128 67 23 3 0 ( 7 7 ) 3 14(79) 26 188 69 14 13(93) 23(67) 20 136 58 20 55(60) 41(29) 13 136 21 15 137(0) 67(0) 22 75 59 32 96(0) 65(0) C u l t u r e s c o n s i s t e d o f 5 x 10D n o r m a l (N) o r t u m o r - b e a r e r (T) p l u s 3 x lO""" n o r m a l or t u m o r - b e a r e r c e l l s f rom t h e l i g h t f r a c t i o n o b t a i n e d from hypaque-f i c o l l and t r e a t e d i n :the i n d i c a t e d manners. R e s u l t s a r e e x p r e s s e d as mean cpm o f t r i p l i c a t e c u l t u r e s . P e r c e n t a g e s u p p r e s s i o n ( i n b r a c k e t s ) was c a l c u l a t e d by c o m p a r i n g t h e r e s p o n s e o f n o r m a l c e l l s c o - c u l t u r e d w i t h t u m o r - b e a r e r c e l l s t o t h a t when a p p r o p r i a t e l y t r e a t e d n o r m a l c e l l s were added t o c u l t u r e s . 78 V I I I SUPPRESSION OF THE IN VITRO GENERATION OF PFC 3 I t was u n l i k e l y t h a t t h e d e c r e a s e i n H - t h y m i d i n e u p t a k e by m i t o g e n s t i m u l a t e d c e l l s was s i m p l y due t o c o l d t h y m i d i n e r e l e a s e d from d y i n g tumor-3 b e a r e r s p l e e n c e l l s s i n c e l ow a c t i v i t y H - t h y m i d i n e was used i n t h e s e s t u d i e s . However, i n o r d e r t o be c e r t a i n t h a t t h e s u p p r e s s o r c e l l s were a c t u a l l y c a p a b l e o f i n h i b i t i n g an e f f e c t o r f u n c t i o n o f l y m p h o c y t e s , t u m o r - b e a r e r s p l e e n c e l l s were t e s t e d f o r t h e a b i l i t y t o s u p p r e s s t h e g e n e r a t i o n o f PFC by no r m a l c e l l s i n v i t r o . A s s a y s were c a r r i e d out u s i n g t h e T c e l l dependent a n t i g e n , SRBC, and t h e T c e l l i n d e p e n d e n t a n t i g e n , DNP-LPS. 1. T i t r a t i o n o f S u p p r e s s o r C e l l s S p l e e n c e l l s f rom n o r m a l m i c e were c u l t u r e d w i t h v a r y i n g numbers o f s p l e e n c e l l s f rom e i t h e r n o r m a l s y n g e n e i c o r t u m o r - b e a r i n g m i c e . I n th e p r e s e n c e o f SRBC, a T c e l l dependent a n t i g e n , t h e number o f PFC g e n e r a t e d i n e a c h c u l t u r e was i n c r e a s e d upon a d d i t i o n o f normal c o n t r o l c e l l s b u t was d e c r e a s e d when t u m o r - b e a r e r c e l l s were added ( F i g u r e 6 ) . I n t h i s a s s a y t h e number o f c e l l s p er c u l t u r e i s s m a l l and t h e r e f o r e t h e number o f T h e l p e r c e l l s i s l i m i t i n g ( 6 4 ) . The a d d i t i o n o f n o r m a l c e l l s t e n d s t o i n c r e a s e b o t h T h e l p e r c e l l s and PFC p r e c u r s o r s and t h u s t h e number o f PFC g e n e r a t e d i s i n c r e a s e d . The a d d i t i o n o f t u m o r - b e a r e r c e l l s n o t o n l y f a i l e d t o enhance t h e g e n e r a t i o n o f PFC b u t , i n f a c t , s u p p r e s s e d t h i s r e s p o n s e . The degr e e o f s u p p r e s s i o n i n c r e a s e d i n p r o -p o r t i o n t o t h e number of t u m o r - b e a r e r c e l l s added. W i t h DNP-LPS, a T c e l l i n d e p e n d e n t a n t i g e n , t h e PFC r e s p o n s e was s u p p r e s s e d by t u m o r - b e a r e r c e l l s when compared t o t h e r e s p o n s e o f n o r m a l c o n t r o l s . The degr e e o f s u p p r e s s i o n was,.however, n o t as g r e a t as t h a t seen w i t h SRBC u n l e s s h i g h numbers o f s u p p r e s s o r c e l l s were p r e s e n t . 79 2. V i a b i l i t y o f C u l t u r e d C e l l s I n o r d e r t o c o n f i r m t h a t t h e s u p p r e s s i o n was n o t s i m p l y due t o a non s p e c i f i c l o s s o f c e l l s i n w e l l s c o n t a i n i n g t u m o r - b e a r e r c e l l s , t h e number o f v i a b l e c e l l s p e r c u l t u r e was d e t e r m i n e d a t the t i m e t h e PFC a s s a y was c a r r i e d o u t . I t can be seen from t h e d a t a i n T a b l e X I X t h a t t h e number o f v i a b l e c e l l s was n e v e r l o w e r i n t h e c u l t u r e s c o n t a i n i n g t u m o r - b e a r e r c e l l s t h a n i n t h e c o r r e s p o n d i n g c u l t u r e s c o n t a i n i n g o n l y n o r m a l c e l l s . The s u p p r e s s i o n seen i n t h e p r e s e n c e o f t u m o r - b e a r e r c e l l s c a n n o t , t h e r e f o r e , be a t t r i b u t e d t o a c y t o t o x i c e f f e c t o f tumor-b e a r e r c e l l s . 3. K i n e t i c s o f t h e I n v i t r o G e n e r a t i o n o f PFC The k i n e t i c s o f t h e PFC r e s p o n s e s i n c u l t u r e s c o n t a i n i n g n o r m a l o r m i x t u r e s o f no r m a l p l u s t u m o r - b e a r e r c e l l s were compared i n o r d e r t o make c e r t a i n t h a t t h e s u p p r e s s i o n was n o t s i m p l y due t o an a l t e r a t i o n i n t h e k i n e t i c s o f t h e r e s p o n s e . C u l t u r e s c o n t a i n i n g 10 no r m a l c e l l s p l u s 5 x 10^ c o n t r o l o r t u m o r - b e a r e r s p l e e n c e l l s were a s s a y e d f o r PFC a t v a r i o u s t i m e s from c u l t u r e s w i t h e i t h e r 2.5 x 10 SRBC p e r ml o r 0.1 y g DNP-LPS p e r ml ( F i g u r e 7 ) . S u p p r e s s i o n was seen on a l l days t e s t e d and tended t o i n c r e a s e w i t h t i m e i n c u l t u r e . The c u l t u r e s a r e n o r m a l l y a s s a y e d a f t e r 3 days ( f o r DNP-LPS) o r 4 days ( f o r SRBC). At t h i s t i m e good n o r m a l r e s p o n s e s t o t h e a n t i g e n s were seen b u t t h e r e was l e s s s u p p r e s s i o n t h a n t h a t o b s e r v e d a f t e r l o n g e r i n c u b a t i o n . 4. Dose Response C u r v e s One m i l l i o n n o r m a l c e l l s were c u l t u r e d w i t h 5 x 10^ no r m a l o r tumor-b e a r e r s p l e e n c e l l s i n t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f t h e t e s t a n t i g e n s . C u l t u r e s were a s s a y e d f o r PFC on day 3 ( f o r DNP-LPS) o r on day 4 ( f o r SRBC) ( F i g u r e 8 ) . The k i n e t i c s o f t h e r e s p o n s e s were 80 s i m i l a r i n t h e p r e s e n c e o f e i t h e r n o r m a l o r t u m o r - b e a r e r c e l l s . A l t h o u g h t h e g r e a t e s t n o r m a l r e s p o n s e t o SRBC was seen w i t h a c o n c e n t -r a t i o n o f 0.5 x 10 SRBC per m l , t h e r e l a t i v e s u p p r e s s i o n by t u m o r - b e a r e r c e l l s was a p p r o x i m a t e l y t h e same when a n t i g e n doses o f 0.5 x 10 t o 12.5 x 10 p e r ml were used. The o p t i m a l r e s p o n s e t o DNP-LPS was seen a t a c o n c e n t r a t i o n o f 0.1 yg/ml b u t i n t h i s c a s e t h e degree o f s u p p r e s s -i o n i n c r e a s e d as t h e a n t i g e n dose was r a i s e d . The s u p p r e s s i o n seen i n c u l t u r e s c o n t a i n i n g t u m o r - b e a r e r c e l l s was n o t t h e r e f o r e s i m p l y due t o a change i n t h e a n t i g e n dose r e q u i r e d t o g i v e a good PFC r e s p o n s e . 81 700 600 500 400 300 200 100 SRBC l l DNP - LPS .5 .25 NUMBER OF C E L L S A D D E D T O WELLS ( X 10* ) F i g u r e 6. The i n v i t r o PFC r e s p o n s e o f nor m a l c e l l s c o - c u l t u r e d w i t h v a r y i n g numbers o f c o n t r o l o r t u m o r - b e a r e r s p l e e n c e l l s . Each w e l l c o n t a i n e d 10^ n o r m a l s y n g e n e i c s p l e e n c e l l s p l u s : | | , norm a l s p l e e n c e l l s ; o r £ , tu m o r - b e a r e r s p l e e n c e l l s . C u l t u r e s were i n c u b a t e d w i t h 2.5 x 10 SRBC p e r ml f o r 4 days o r w i t h 0.1 Ug DNP-LPS per ml f o r 3 da y s . 82 TABLE XIX V I A B I L I T Y OF CULTURED SPLEEN CELLS No. V i a b l e C e l l s p e r w e l l ( x l O ) SRBC PFC • DNP-LPS PFC Normal Tumor-bearer Normal Tumor-bearer 0 2.5 3.3 1.25 3.3 3.5 3.3 3.5 2.5 3.5 4.5 4.5 4.5 5.0 4.5 4.5 5.0 5.0 10.0 4.8 5.0 5.3 6.3 No. o f S p l e e n C e l l s Added t o W e l l s ( x l O 5 ) Each w e l l c o n t a i n e d 10 n o r m a l s p l e e n c e l l s . The number of v i a b l e c e l l s was d e t e r m i n e d a f t e r 4 days i n c u l t u r e ( f o r SRBC) o r 3 days i n c u l t u r e ( f o r DNP-LPS). C e l l s were counted i n a haemocytometer a f t e r t r y p a n b l u e u p t a k e . 83 DAYS IN CULTURE F i g u r e 7. K i n e t i c s o f t h e i n v i t r o PFC r e s p o n s e . Each w e l l c o n t a i n e d 10 n o r m a l s y n g e n e i c s p l e e n c e l l s p l u s : | | , RPMI 1640; ^  5 x 10 n o r m a l s p l e e n c e l l s ; o r J | , 5 x 10 t u m o r - b e a r e r s p l e e n c e l l s . C u l t u r e s were i n c u b a t e d w i t h 2.5 x 10 SRBC o r 0.1 ug DNP-LPS p e r m l . 84 700r 600 500h 400 300h 200h 1001 SRBC (X 106/ ml ) DNP-LPS ( pg / ml) F i g u r e 8. The i n v i t r o PFC r e s p o n s e w i t h d i f f e r e n t a n t i g e n d o s e s . Each w e l l c o n t a i n e d 1 0 6 n o r m a l c e l l s p l u s : Q , RPMI 1640; ^ , 5 x 10 n o r m a l s p l e e n c e l l s ; o r ^ | , 5 x 10 t u m o r - b e a r e r s p l e e n c e l l s . C u l t u r e s were i n c u b a t e d f o r 4 days w i t h SRBC and 3 days w i t h DNP-LPS. 85 I X CHARACTERIZATION OF SUPPRESSOR CELLS I n o r d e r t o d e t e r m i n e w h i c h p o p u l a t i o n o f t u m o r - b e a r e r s p l e e n c e l l s was r e s p o n s i b l e f o r t h e s u p p r e s s i o n , c e l l s were s e p a r a t e d i n t o d i f f e r e n t p o p u l -a t i o n s on t h e b a s i s o f d e n s i t y , a b i l i t y t o adhere t o p l a s t i c o r n y l o n w o o l , p h a g o c y t o s i s and c e l l s u r f a c e a n t i g e n s . 1. H y p a q u e - F i c o l l F r a c t i o n a t i o n S p l e e n c e l l s from n o r m a l o r t u m o r - b e a r i n g mice were s e p a r a t e d i n t o two f r a c t i o n s on h y p a q u e - f i c o l l ( s p e c i f i c g r a v i t y 1.08) g r a d i e n t s and t h e f r a c t i o n s were t e s t e d s e p a r a t e l y f o r t h e a b i l i t y t o s u p p r e s s PFC t o DNP-LPS and SRBC ( T a b l e X X ) . Tumor-bearer c e l l s r e t a i n e d a t t h e i n t e r f a c e o f h y p a q u e - f i c o l l ( l i g h t f r a c t i o n ) had t h e a b i l i t y t o s u p p r e s s t h e r e s p o n s e t o b o t h a n t i g e n s . However, t h e c e l l s p e l l e t i n g t o t h e bot t o m o f t h e g r a d i e n t (dense f r a c t i o n ) s u p p r e s s e d t h e r e s p o n s e t o SRBC but d i d n o t a f f e c t t h e r e s p o n s e t o DNP-LPS. I t a p p e a r e d , t h e r e f o r e , t h a t t h e r e were two p o p u l a t i o n s o f s u p p r e s s o r c e l l s : a low d e n s i t y p o p u l a t i o n c a p a b l e o f s u p p r e s s i n g PFC r e s p o n s e s t o b o t h T c e l l dependent and i n d e p e n d e n t a n t i g e n s and a h i g h e r d e n s i t y p o p u l a t i o n s u p p r e s s i n g o n l y t h e r e s p o n s e t o t h e T c e l l dependent a n t i g e n . 2. Adherence t o P l a s t i c S p l e e n c e l l s were s e p a r a t e d i n t o a d h e r e n t and non adh e r e n t f r a c t i o n s a f t e r i n c u b a t i o n i n p l a s t i c p e t r i d i s h e s and t h e s e p a r a t e f r a c t i o n s were t e s t e d f o r t h e a b i l i t y t o s u p p r e s s ( T a b l e X X ) . Adherent t u m o r - b e a r e r s p l e e n c e l l s s u p p r e s s e d t h e no r m a l PFC r e s p o n s e t o b o t h DNP-LPS and SRBC. Non adh e r e n t c e l l s s u p p r e s s e d o n l y t h e r e s p o n s e t o SRBC. One p o p u l a t i o n o f s u p p r e s s o r c e l l s appeared t h e r e f o r e t o be p l a s t i c a d h e r e n t w h i l e t h e o t h e r was n o t . 86 3. N y l o n Wool Columns The passage o f t u m o r - b e a r e r c e l l s t h r o u g h n y l o n w o o l columns removed the c e l l s r e s p o n s i b l e f o r s u p p r e s s i o n o f t h e r e s p o n s e t o DNP-LPS ( T a b l e X X I ) . The c e l l s e l u t e d from t h e column were, however, c a p a b l e o f s u p p r e s s i n g t h e r e s p o n s e t o SRBC. N y l o n w o o l columns i n our hands, removed 90% o f t h e c e l l s from a n o r m a l s p l e e n . T h i s t e c h n i q u e r e d u c e d t h e number o f c e l l s p h a g o c y t i z i n g l a t e x beads from 10% t o 1%; B c e l l s were r e d u c e d s i n c e t h e e l u t e d c e l l s had r e d u c e d r e s p o n s e s t o LPS and produced fewer PFC; and T c e l l s were e n r i c h e d s i n c e t h e r e s p o n s e o f e l u t e d c e l l s t o Con A was enhanced (see T a b l e V I ) . 4. C a r b o n y l I r o n and Magnet I n c u b a t i o n o f t u m o r - b e a r e r s p l e e n c e l l s w i t h c a r b o n y l i r o n removed th e p o p u l a t i o n o f s u p p r e s s o r c e l l s w h i c h i n h i b i t e d t h e r e s p o n s e t o DNP-LPS. The c e l l s w h i c h remained were s t i l l c a p a b l e o f s u p p r e s s i n g t h e r e s p o n s e t o SRBC ( T a b l e X X I I ) . C a r b o n y l i r o n and magnet t r e a t m e n t r e d u c e d t h e p e r c e n t a g e of c e l l s p h a g o c y t i z i n g l a t e x beads t o 1%. The p o p u l a t i o n r e s p o n s i b l e f o r s u p p r e s s i o n of t h e PFC r e s p o n s e s t o b o t h T c e l l dependent and i n d e p e n d e n t a n t i g e n s t h u s appeared t o be p h a g o c y t i c s i n c e c a r b o n y l i r o n and magnet o r n y l o n wool adherence removed t h e c e l l s c a p a b l e o f s u p p r e s s i n g t h e PFC r e s p o n s e t o DNP-LPS. The c e l l s s u p p r e s s -i n g o n l y t h e T c e l l dependent r e s p o n s e were n o t , however, p h a g o c y t i c when t h e same t e c h n i q u e s were u s e d . 5. C a r r a g h e e n a n Treatment The s u p p r e s s o r c e l l s w h i c h i n h i b i t e d t h e r e s p o n s e t o DNP-LPS appeared t o be o f t h e macrophage/monocyte l i n e s i n c e t h e y were a d h e r e n t t o p l a s t i c and n y l o n w o o l and p h a g o c y t i z e d c a r b o n y l i r o n . I n o r d e r t o more s p e c i f i c a l l y remove macrophages, t h e s p l e e n c e l l s were t r e a t e d w i t h 87 c a r r a g h e e n a n b e f o r e t e s t i n g f o r t h e a b i l i t y t o s u p p r e s s t h e PFC t o DNP-LPS ( T a b l e X X I I I ) . Carragheenan t r e a t m e n t of t u m o r - b e a r e r c e l l s d i d i n a c t i v a t e t h e s u p p r e s s o r c e l l s i n t h i s a s s a y . S i n c e c a r r a g -heenan t r e a t m e n t has been shown t o be non t o x i c f o r l y m p h o c y t e s ( 2 5 ) , i t i s l i k e l y t h a t t h e s u p p r e s s o r c e l l s i n h i b i t i n g t h e PFC r e s p o n s e t o DNP-LPS were macrophages. 6. Removal o f B C e l l s S i n c e B c e l l s t e n d t o be removed by many of t h e t r e a t m e n t s used t o remove macrophages, h y p a q u e - f i c o l l l i g h t s p l e e n c e l l s were t r e a t e d w i t h a n t i - M I g serum p l u s complement t o s p e c i f i c a l l y k i l l B c e l l s b e f o r e t e s t i n g f o r t h e a b i l i t y t o s u p p r e s s t h e r e s p o n s e t o DNP-LPS ( T a b l e X X I V ) . S p l e e n c e l l s d e p l e t e d o f B c e l l s i n t h i s manner were s t i l l s u p p r e s s i v e i n t h i s a s s a y . I t s h o u l d be n o t e d , however t h a t t h i s p r o c e d u r e does n o t appear t o k i l l a l l o f t h e B c e l l s p r e s e n t i n t h e s p l e e n s i n c e t r e a t e d c e l l s were a b l e t o produce PFC (see T a b l e I V ) . 7. Removal o f T C e l l s The t r e a t m e n t o f t u m o r - b e a r e r s p l e e n c e l l s w i t h a n t i - T h y - 1 serum p l u s complement d i d n o t remove t h e s u p p r e s s o r c e l l s and, i n f a c t , i n c r e a s e d t h e d e g r e e o f s u p p r e s s i o n w i t h b o t h a n t i g e n s ( T a b l e XXV) . When c e l l s were f i r s t s e p a r a t e d i n t o l i g h t and dense f r a c t i o n s on h y p a q u e - f i c o l l and t h e n i n d i v i d u a l f r a c t i o n s were t r e a t e d w i t h a n t i -Thy-1 serum p l u s complement, a d i f f e r e n t r e s u l t was o b t a i n e d . The s u p p r e s s i o n by t h e l i g h t f r a c t i o n was enhanced by t h i s t r e a t m e n t . On t h e o t h e r hand, t h e s u p p r e s s i o n o f t h e SRBC r e s p o n s e by t h e dense c e l l s was d e c r e a s e d a f t e r a n t i - T h y - 1 serum t r e a t m e n t . I t a p p e a r s , t h e r e f o r e , t h a t t h e dense s u p p r e s s o r c e l l c o u l d be a T c e l l w h i l e t h e l i g h t s u p p r e s s o r c e l l i s n o t . T h i s e x p e r i m e n t has been r e p e a t e d t h r e e t i m e s . 88 I n t h e s e e x p e r i m e n t s t h e same p a t t e r n was o b s e r v e d b u t t h e degree o f re m o v a l o f t h e s u p p r e s s i o n by a n t i - T h y - 1 serum was n o t as c o m p l e t e . A p o s s i b l e e x p l a n a t i o n f o r t h e d i f f i c u l t i e s e n c o u n t e r e d i n t h e r e p r o d u c -i b i l i t y o f t h e s e d a t a i s t h a t t h e h y p a q u e - f i c o l l s e p a r a t i o n does n o t c o m p l e t e l y s e p a r a t e t h e two s u b p o p u l a t i o n s o f s u p p r e s s o r c e l l s . 8. M i t o m y c i n C t r e a t m e n t Tumor-bearer c e l l s were s e p a r a t e d i n t o l i g h t and dense f r a c t i o n s w h i c h were t h e n t r e a t e d w i t h m i t o m y c i n C t o p r e v e n t DNA s y n t h e s i s ( T a b l e X X V I ) . The l i g h t s u p p r e s s o r c e l l s were not a f f e c t e d by any o f t h e dos e s o f m i t o m y c i n C use d . The dense s u p p r e s s o r c e l l s l o s t t h e i r a b i l i t y t o s u p p r e s s SRBC when t r e a t e d w i t h as l i t t l e as 25 y g m i t o m y c i n C per m l . I n t h i s e x p e r i m e n t , s i n c e t h e a d d i t i o n m i t o m y c i n C t r e a t e d n o r m a l c e l l s d i d n o t i n c r e a s e the r e s p o n s e , p e r c e n t s u p p r e s s i o n was c a l c u l a t e d by co m p a r i n g t h e r e s p o n s e when t u m o r - b e a r e r c e l l s were added t o t h a t when e q u i v a l e n t n o r m a l f r a c t i o n s were added t o t h e 10^ no r m a l c e l l s . I t appeared from t h e s e d a t a , t h a t t h e r e were two d i s t i n c t p o p u l a t i o n s o f s u p p r e s s o r c e l l s . The l i g h t , adherent,, p h a g o c y t i c c e l l s d i d n o t r e q u i r e DNA s y n t h e s i s i n o r d e r t o s u p p r e s s PFC r e s p o n s e s t o e i t h e r DNP-LPS o r SRBC. The dense, non a d h e r e n t , non p h a g o c y t i c c e l l s d i d r e q u i r e DNA s y n t h e s i s f o r s u p p r e s s i o n o f PFC r e s p o n s e s t o SRBC. 89 TABLE XX EFFECT OF HYPAQUE-FICOLL SEPARATION AND PETRI DISH ADHERENCE ON SUPPRESSOR CELL ACTIVITY Ad d i t i o n s " 1 ' Treatment^" DNP-PFC' S u p p r e s s i o n SRBC-PFC S u p p r e s s i o n None . . 299 ± 75 .1012 ±..117 N None 298 ± 42 1398 ± 156 T 146 ± 44 51 146 ± 29 86 N l i g h t 310 ± 36 1628 ± 221 T 4 ± 5 99 5 ± 3 99 N dense 368 ± 69 1078 ± 9 T 290 ± 6 3 503 ±. 41 51 N a d h e r e n t 428 ± 53 843 ± 83 T 71 ± 34 76 245 ± 56 76 N non a d h e r e n t 390 ± 23 908 ± 143 T 275 ± 18 8 611 ± 46 40 Each c u l t u r e c o n t a i n e d 10° n o r m a l s p l e e n c e l l s . 5 x 1 0 3 n o r m a l (N) or t u m o r - b e a r e r (T) s p l e e n c e l l s were added t o c u l t u r e s as i n d i c a t e d . C e l l s were s e p a r a t e d i n t o l i g h t and dense f r a c t i o n s on h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08 o r were a d h e r e n t o r non-adherent t o p l a s t i c p e t r i d i s h e s . PFC a r e e x p r e s s e d as t h e number o f PFC per w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean of t r i p l i c a t e a s s a y s . 99 TABLE X X I EFFECT OF NYLON WOOL ADHERENCE ON SUPPRESSOR CELL ACTIVITY A d d i t i o n s 1 T r e a t m e n t DNP-PFC J % S u p p r e s s i o n SRBC-PFC % S u p p r e s s i o n None 268 ± 84 660 ± 20 N none 410 ± 53 618 ± 70 T 20 ± 10 92 51 ± 6 92 N n y l o n w o o l 254 ± 35 630 ± 72 T 299 ± 8 4 0 164 ± 1 75 1 6 5 C u l t u r e s c o n s i s t e d o f 10 n o r m a l p l u s 5 x 10 n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s . 2 C e l l s were passed t h r o u g h a n y l o n w o o l column. PFC a r e e x p r e s s e d as t h e number o f PFC per w e l l p l u s or minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean o f t r i p l i c a t e a s s a y s . 91 TABLE X X I I EFFECT OF CARBONYL IRON AND MAGNET TREATMENT ON SUPPRESSOR ACTIVITY 1 ? 3 . A d d i t i o n s Treatment DNP-LPS S u p p r e s s i o n SRBC-PFC S u p p r e s s i o n None 197 ± 27 .150 ± 13 N T None 245 ± 30 34 ± 6 86 133 ± 9 40 ± 3 73 N T C a r b o n y l i r o n 237 ± 23 202 ± 26 405 ± 15 70 ± 10 53 1 6 5 C u l t u r e s c o n s i s t e d o f 10 n o r m a l p l u s 5 x 10 no r m a l (N) or t u m o r - b e a r e r (T) c e l l s . 2 C e l l s were i n c u b a t e d w i t h c a r b o n y l i r o n p a r t i c l e s and p h a g o c y t i c c e l l s were removed w i t h a magnet. 3 PFC a r e e x p r e s s e d as t h e number o f PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean o f t r i p l i c a t e a s s a y s . TABLE X X I I I EFFECT OF CARRAGHEENAN TREATMENT ON SUPPRESSOR CELL ACTIVITY DNP-PFC % S u p p r e s s i o n 290 ± 17 395 ± 19 148 ± 7 49 375 ± 42 281 ± 50 A d d i t i o n s Treatment None N T None N T c a r r a g h e e n a n C u l t u r e s c o n s i s t e d o f 10° normal p l u s 5 x 1 0 J n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s . C e l l s were i n c u b a t e d w i t h c a r r a g h e e n a n b e f o r e t e s t i n g . PFC a r e e x p r e s s e d as t h e number o f PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean o f t r i p l i c a t e a s s a y s . 93 TABLE XXIV EFFECT OF ANTI-MIg TREATMENT ON SUPPRESSOR CELL ACTIVITY i ? 3 A d d i t i o n s Treatment DNP-PFC % S u p p r e s s i o n None 290 ± 17 N None 395 ± 19 T 148 ± 7 49 N L None 315 ± 38 T 145 ± 29 50 L N L a n t i - M I g 303 ± 4? 73 T L 77 ± 21 1 C u l t u r e s c o n s i s t e d o f 10^ n o r m a l p l u s 5 x 10~* n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s . N^ and T^ d e s i g n a t e c e l l s i n t h e l i g h t f r a c t i o n a f t e r h y p a q u e - f i c o l l f r a c t i o n a t i o n . 2 C e l l s f r o m t h e l i g h t f r a c t i o n were t r e a t e d w i t h a n t i - M I g p l u s complement. 3 PFC a r e e x p r e s s e d as t h e number of PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean o f t r i p l i c a t e a s s a y s . 94 TABLE XXV EFFECT OF ANTI-THY-1 TREATMENT ON THE SUPPRESSIVE ACTIVITY OF HYPAQUE-FICOLL FRACTIONS 7 % 1 2 3 A d d i t i o n s Treatment DNP-PFC S u p p r e s s i o n SRBC-PFC S u p p r e s s i o n None N T N T N T None a n t i - T h y - 1 None 173 ± 40 188 ± 26 98 ± 42 208 ± 106 68 ± 36 207 ± 16 69 ± 14 43 61 60 528 ± 37 1505 ± 240 214 ± 39 1283 ± 195 54 ± 39 1263 ± 109 85 ± 48 60 90 84 N T N„ N„ a n t i - T h y - 1 .188 ± 47 44 ± 22 None 184 ± 112 149 ± 13 a n t l - T h y - 1 120 ± 39 134 ± 8 75 14 13 1408 ± 187 21 ± 9 1100 ± 64 340 ± 105 833 ± 101 523 ± 33 96 36 C u l t u r e s c o n s i s t e d o f 10° n o r m a l p l u s 5 x 1 0 J u n t r e a t e d , o r t r e a t e d n o r m a l o r t u m o r - b e a r e r s p l e e n c e l l s . N^ and T d e s i g n a t e c e l l s w i t h s p e c i f i c g r a v i t y < 1.08; N^ ahd Tp a r e t h o s e w i t h s p e c i f i c g r a v i t y > 1.08. U n f r a c t i o n a t e d o r h y p a q u e - f i c o l l f r a c t i o n a t e d s p l e e n c e l l s were t r e a t e d w i t h a n t i - T h y - 1 p l u s complement. PFC a r e e x p r e s s e d as t h e number o f PFC p e r w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n from t h e mean o f t r i p l i c a t e a s s a y s . 95 TABLE XXVI EFFECT OF MITOMYCIN C TREATMENT ON SUPPRESSOR CELL ACTIVITY % % A d d i t i o n s 1 2 Treatment DNP-PFC3 S u p p r e s s i o n SRBC-PFC S u p p r e s s i o n None 277 ± 33 138 ± 11 N T None 260 ± 19 47 ± 6 82 240 ± 8 7 ± 2 97 L None 261 ± 11 28 ± 10 89 150 ± 20 5 ± 3 97 25 y g 207 ± 5 17 ± 10 92 155 ± 35 9 ± 1 94 50 y g 207 ± 23 36 ± 18 83 148 ± 45 8 ± 3 95 \ 100 y g 185 ± 46 73 ± 8 61 143 ± 27 24 ± 2 83 None 214 ± 12 209 ± 27 2 145 ± 49 8 ± 2 94 • D 25 y g 177 ± 55 179 ± 42 0 145 ± 16 135 ± 8 7 50 y g 217 ± 23 290 ± 16 0 145 ± 9 133 ± 16 8 100 y g 229 ± 12 232 ± 13 0 145 ± 12 130 ± 9 10 C u l t u r e s c o n s i s t e d o f 10 n o r m a l p l u s 5 x 10 n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s . N^ and T^ d e s i g n a t e c e l l s w i t h s p e c i f i c g r a v i t y < 1.08 and N and T t h o s e w i t h s p e c i f i c g r a v i t y > 1.08. C e l l s were f r a c t i o n a t e d on h y p a q u e - f i c o l l s p e c i f i c g r a v i t y 1.08 and t h e n t r e a t e d w i t h 25, 50 o r 100 y g m i t o m y c i n C p e r ml. R e s u l t s are., e x p r e s s e d as t h e number of PFC per w e l l p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean o f t r i p l i c a t e a s s a y s . 96 X SUPPRESSOR CELL ACTIVITY IN MARBROOK CHAMBERS S p l e e n c e l l s f r o m t u m o r — b e a r i n g m i c e were t e s t e d f o r t h e a b i l i t y t o pro d u c e a s o l u b l e f a c t o r w h i c h c o u l d s u p p r e s s t h e PFC r e s p o n s e o f normal s p l e e n c e l l s . I n t h e s e e x p e r i m e n t s t h e n o r m a l c e l l s were c u l t u r e d i n t h e i n n e r chamber o f Marbrook v e s s e l s a t a c o n c e n t r a t i o n o f 5 x 10^ c e l l s i n 1 m l medium. They were s e p a r a t e d by an 0.8 u n u c l e p o r e membrane fr o m t h e o u t e r chamber w h i c h c o n t a i n e d e i t h e r n o r m a l c o n t r o l o r t u m o r - b e a r e r s p l e e n c e l l s . The n o r m a l c e l l s were c u l t u r e d i n t h e i n n e r chamber f o r 3 r e a s o n s . T h i s arrangement used a minimum number of c e l l s and t h e r e f o r e a s s u r e d adequate n u t r i t i o n . R e l i a b l e numbers o f PFC ( w i t h v e r y l i t t l e v a r i a t i o n between v e s s e l s ) were g e n e r a t e d by c u l t u r e s i n a g i v e n e x p e r i m e n t . A l s o , s i n c e t h e t u m o r - b e a r e r c e l l s were c u l t u r e d i n t h e bottom chamber, t h e p o s s i b i l i t y t h a t pseudopods f r o m s u p p r e s s o r c e l l s c o u l d p a ss t h r o u g h the membrane and p h y s i c -a l l y c o n t a c t t h e normal c e l l s was r e d u c e d . 1. T i t r a t i o n of S u p p r e s s o r C e l l s The number of PFC g e n e r a t e d by normal c e l l s c u l t u r e d i n t h e o u t e r chamber i n c r e a s e d i n p r o p o r t i o n t o t h e number of c e l l s c u l t u r e d ( T a b l e X X V I I ) . These d a t a i n d i c a t e t h a t t h e chambers were n o t overcrowded s i n c e 3 x 10^ n o r m a l c e l l s (or 3 t i m e s the number used i n s u p p r e s s o r e x p e r i m e n t s ) produced good PFC r e s p o n s e s . On the o t h e r hand, tumor-b e a r i n g s p l e e n c e l l s were n o t immunocompetent and d i d n o t r e s p o n d t o SRBC a t any of the c o n c e n t r a t i o n s t e s t e d . Normal c e l l s c u l t u r e d i n the i n n e r chamber were c a p a b l e of g e n e r a t i n g PFC t o SRBC when t h e r e were no c e l l s c u l t u r e d i n the chamber below. They d i d n o t t h e r e f o r e r e q u i r e h e l p f r o m a d d i t i o n a l c u l t u r e d c e l l s and any d e c r e a s e i n the number of PFC produced was p r o b a b l y due t o a sup-p r e s s i v e f a c t o r r e l e a s e d by c e l l s i n t h e o u t e r chamber. Low numbers of 97 n o r m a l c e l l s sometimes enhanced the r e s p o n s e , p r e s u m a b l y by p r o d u c i n g h e l p e r f a c t o r s , b u t d e n s i t i e s o f up t o 3 x 10^ normal c e l l s p e r o u t e r chamber were n o t h i g h l y s u p p r e s s i v e . On the o t h e r hand, t h e normal PFC r e s p o n s e was s u p p r e s s e d by as few as 7.5 x 10 t u m o r - b e a r e r c e l l s and t h e d e g r e e o f s u p p r e s s i o n i n c r e a s e d w i t h t h e number of s u p p r e s s o r c e l l s c u l t u r e d . T h e r e f o r e , i t appeared t h a t t u m o r - b e a r e r s p l e e n c e l l s d i d p r o d u c e a s o l u b l e f a c t o r w h i c h c o u l d s u p p r e s s a c r o s s a c e l l impermeable membrane. 2. V i a b i l i t y o f C u l t u r e d C e l l s S u p p r e s s i o n was n o t due t o a non s p e c i f i c k i l l i n g o f the n o r m a l c e l l s ( T a b l e X X V I I I ) . The v i a b i l i t y of c e l l s i n t h e i n n e r chamber d i d not v a r y s i g n i f i c a n t l y when th e o u t e r chamber c o n t a i n e d medium, 7.5 x 10^ t o 15 x 10 n o r m a l c e l l s , o r e q u i v a l e n t numbers of t u m o r — b e a r e r c e l l s . The v i a b i l i t y dropped when h i g h e r numbers of c e l l s were t e s t e d f o r s u p p r e s s -i o n b u t t h e r e was no d i f f e r e n c e between c u l t u r e s c o n t a i n i n g normal o r t u m o r - b e a r e r c e l l s . The number o f v i a b l e c e l l s i n t h e o u t e r chamber i n c r e a s e d i n p r o p o r t i o n t o t h e number c u l t u r e d a l t h o u g h c e l l s f r o m tumor-b e a r e r s t h e m s e l v e s were n o t as v i a b l e . 3. H y p a q u e - F i c o l l F r a c t i o n a t i o n S p l e e n c e l l s were s e p a r a t e d i n t o two f r a c t i o n s a c c o r d i n g t o t h e i r s e d i m e n t a t i o n on h y p a q u e - f i c o l l ( s p e c i f i c g r a v i t y 1.08) and t h e f r a c t i o n s were t e s t e d s e p a r a t e l y f o r the a b i l i t y t o s u p p r e s s ( T a b l e XXIX), The c e l l s r e t a i n e d above t h e h y p a q u e - f i c o l l ( l i g h t f r a c t i o n ) d i d n o t p r o d u c e a s o l u b l e s u p p r e s s o r f a c t o r i n t h e system. The c e l l s p e l l e t i n g t o t h e b ottom of t h e tube (dense f r a c t i o n ) were, however, h i g h l y s u p p r e s s i v e and t h u s d i d p r o d u c e a f a c t o r w h i c h c o u l d s u p p r e s s a c r o s s an 0,8 y n u c l e -p o r e membrane. 98 4. Adherence t o P l a s t i c Tumor-bearer s p l e e n c e l l s w h i c h were a d h e r e n t t o p l a s t i c d i d n o t s u p p r e s s i n t h i s sytem b u t non a d h e r e n t c e l l s d i d p r o d u c e a f a c t o r w h i c h s u p p r e s s e d a c r o s s a n u c l e p o r e membrane ( T a b l e XXX), The c e l l s p r o d u c i n g t h e f a c t o r t h e r e f o r e appeared t o be among th e most dense, non a d h e r e n t c e l l s . 5. N y l o n Wool Columns Normal c o n t r o l and t u m o r - b e a r e r s p l e e n c e l l s were d e p l e t e d o f p h a g o c y t i c c e l l s by passage t h r o u g h a n y l o n w o o l column and by c a r b o n y l i r o n and magnet t r e a t m e n t ( T a b l e X X X I ) . The r e m o v a l of p h a g o c y t i c c e l l s by c a r b o n y l i r o n and magnet d i d n o t remove s u p p r e s s o r c e l l s . The c e l l s e l u t e d f r o m a n y l o n w o o l column ( r e p r e s e n t i n g about 9% o f t h e o r i g i n a l p o p u l a t i o n ) were s u p p r e s s i v e , but l e s s s o t h a n .the u n t r e a t e d c e l l s . The c e l l s p r o d u c i n g t h e s o l u b l e s u p p r e s s i v e f a c t o r d i d n o t t h e r e f o r e appear t o be p h a g o c y t i c b u t may have been s l i g h t l y a d h e r e n t t o n y l o n w o o l . 6. Membranes of D i f f e r e n t P o r e S i z e s The i n n e r and o u t e r chambers were s e p a r a t e d by membranes of d i f f e r e n t p o r e s i z e s ( T a b l e X X X I I ) . S u p p r e s s i o n was seen when the chambers were s e p a r a t e d by t h e 0.8 u n u c l e p o r e membrane or by s t a n d a r d d i a l y s i s t u b i n g ( w h i c h a l l o w e d s u b s t a n c e s w i t h m o l e c u l a r w e i g h t s of; l e s s t h a n 12,000 t o p a s s ) . When a low p o r o s i t y d i a l y s i s t u b i n g (which a l l o w e d s u b s t a n c e s o f m o l e c u l a r w e i g h t s l e s s t h a n 3,500 t o p a s s ) was u s e d , s o l u b l e f a c t o r s were n o t a b l e t o p a ss i n t o t h e i n n e r chamber. Thus the s u p p r e s s i v e f a c t o r a p p e ars t o have a m o l e c u l a r w e i g h t of between 3,500 t o 12,000. The PFC r e s p o n s e o f n o r m a l s p l e e n c e l l s t o t h e T c e l l dependent a n t i g e n , SRBC, c o u l d t h e r e f o r e be s u p p r e s s e d a c r o s s a c e l l impermeable membrane by t h e h y p a q u e - f i c o l l l i g h t , p l a s t i c and n y l o n w o o l non a d h e r e n t , non p h a g o c y t i c 99 t u m o r - b e a r e r s p l e e n c e l l s . The c e l l s p r o d u c i n g the s o l u b l e f a c t o r were p r e s e n t i n t h e same p o p u l a t i o n as the s u p p r e s s o r c e l l s , p r e s umably T c e l l s , w h i c h were c a p a b l e o f s u p p r e s s i n g o n l y the SRBC r e s p o n s e i n t h e c o - c u l t u r e s . A l t h o u g h DNP-PFC a s s a y s were n o t c a r r i e d o u t i n t h e Marbrook system, i t i s p r o b a b l e t h a t s u p p r e s s i o n would n o t be seen i n t h i s c a s e . 100 TABLE X X V I I TITRATION OF SUPPRESSOR CELL ACTIVITY IN MARBROOK CHAMBERS Number of C e l l s i n Outer Chamber Type of C e l l s i n SRBC - PFC SRBC - PFC Outer Chamber ( I n n e r Chamber) (Outer Chamber) None 1900 ± 300 7.5 x 10 Normal Tumor-bearer 2834 ± 3 1 4 669 ± 67 417 ± 202 10 ± 10 15 x 10 Normal Tumor-bearer 1265 ± 151 52 ± 18 1633 ± 375 2 ± 4 30 x 10 Normal Tumor-bearer 1253 ± 95 21 ± 1 5033 ± 431 0 ± 0 The i n n e r chamber c o n t a i n e d 5 x 10 n o r m a l s p l e e n c e l l s . The two chambers were s e p a r a t e d by an 0.8 y n u c l e p o r e membrane. R e s u l t s a r e e x p r e s s e d as t h e number of PFC p e r c u l t u r e p l u s o r minus t h e s t a n d a r d d e v i a t i o n f r o m t h e mean o f t r i p l i c a t e a s s a y s . 101 TABLE X X V I I I VIABILITY OF CELLS CULTURED IN MARBROOK CHAMBERS C o n t e n t s of Marbrook Chamber No. V i a b l e C e l l s p e r C u l t u r e ( x l O 6 ) O u t e r Chamber I n n e r Chamber Outer Chamber I n n e r Chamber None 5 X i o 6 N - 1.7 7.5 x 10 6 N 5 X i o 6 N 3.8 1.9 7.5 x 10 6 T 5 X i o 6 N 2.3 1.8 15 x 10 6 N 5 X i o 6 N 8.2 1.9 15 x 10 6 T 5 X i o 6 N 4.2 1.7 30 x 10 6 N 5 X i o 6 N 12.4 1.3 30 x 10 6 T 5 X i o 6 N. 8.8 1.2 Chambers were s e p a r a t e d by an 0.8 u n u c l e p o r e membrane. Outer chambers c o n t a i n e d v a r y i n g numbers o f no r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s . V i a b l e c e l l s were counted i n a haemocytometer u s i n g t r y p a n b l u e e x c l u s i o n a f t e r 4 days i n c u l t u r e . 102 TABLE XXIX EFFECT OF HYPAQUE-FICOLL FRACTIONATION ON SUPPRESSOR CELL ACTIVITY IN MARBROOK CHAMBERS SRBC - PFC 7 10 Treatment of C e l l s C e l l s i n o^o^ - r.co 2 i n Outer Chamber Outer Chamber. . . .( I n n e r Chamber). S u p p r e s s i o n None N T 2588 ± 218 438 ± 58 83 H y p a q u e - F i c o l l ( l i g h t ) N T 1850 ± 282 1843 ± 304 H y p a q u e - F i c o l l (dense) N T 2791 ± 687 205 ± 109 93 1 Outer chambers c o n t a i n e d 10^ n o r m a l (N) o r t u m o r - b e a r e r (T) s p l e e n c e l l s f r a c t i o n a t e d on h y p a q u e - f i c o l l o f s p e c i f i c g r a v i t y 1.08. 2 6 The i n n e r chamber c o n t a i n e d 5 x 10 n o r m a l s p l e e n c e l l s . Chambers were s e p a r a t e d by an 0.8 u n u c l e p o r e membrane. R e s u l t s a r e e x p r e s s e d as t h e number of PFC per c u l t u r e p l u s or minus the s t a n d a r d d e v i a t i o n from t h e mean of t r i p l i c a t e a s s a y s . 103: TABLE XXX EFFECT OF PETRI DISH ADHERENCE ON SUPPRESSOR CELL ACTIVITY IN MARBROOK CHAMBERS Treatment of C e l l s C e l l s i n SRBC - PFC 2 % i n O uter Chamber 1 Outer Chamber ( I n n e r Chamber) S u p p r e s s i o n None N 758 ± 126 T 162 ± 29 79 A d h e r e n t N 917 ± 288 T 833 ± 244 9 Non-adherent N 767 ± 210 T 276 1 86 64 1 C e l l s were adhered t o p l a s t i c p e t r i d i s h e s and l o ' a d h e r e n t o r non-adherent normal (N) o r t u m o r - b e a r e r (T) c e l l s were c u l t u r e d i n t h e o u t e r chamber. 2 6 The i n n e r chamber c o n t a i n e d 5 x 10 n o r m a l s p l e e n c e l l s . The two chambers were s e p a r a t e d by an 0.8 u n u c l e p o r e membrane. R e s u l t s a r e e x p r e s s e d as th e PFC p e r c u l t u r e p l u s o r minus the s t a n d a r d d e v i a t i o n f r o m t h e mean o f t r i p l i c a t e a s s a y s . 104 TABLE XXXI EFFECT OF CARBONYL IRON AND NYLON WOOL TREATMENTS ON SUPPRESSOR CELL ACTIVITY Treatment o f C e l l s i n O uter Chambers-C e l l s i n Out e r Chamber SRBC ^ PFC ( I n n e r Chamber)' % S u p p r e s s i o n None N T 917 ± 38 50 ± 0 95 N y l o n Wool N T 1083 ± 104 433 ± 132 60 C a r b o n y l I r o n N T 1333 ± 281 48 ± 8 97 O u t e r chambers c o n t a i n e d 10 no r m a l (N) o r t u m o r - b e a r e r (T) c e l l s w h i c h were u n t r e a t e d , passed t h r o u g h n y l o n w o o l columns o r t r e a t e d w i t h c a r b o n y l i r o n . 2 6 I n n e r chambers c o n t a i n e d 5 x 10 n o r m a l s p l e e n c e l l s . The two chambers were s e p a r a t e d by 0.8 u n u c l e p o r e membranes. R e s u l t s a r e e x p r e s s e d as t h e PFC p e r c u l t u r e p l u s o r minus the s t a n d a r d d e v i a t i o n from t h e mean o f t r i p l i c a t e a s s a y s . 105 TABLE X X X I I ABILITY OF SUPPRESSOR CELL FACTOR TO PASS THROUGH DIFFERENT MEMBRANES Type of Membrane C e l l s i n SRBC - PFC Outer Chamber ( I n n e r Chamber) S u p p r e s s i o n 0.8 u n u c l e p o r e N T 2847 ± 310 467 ± 38 84 S t a n d a r d d i a l y s i s t u b i n g (mw <12,000) N T 4133 ± 483 100 ± 50 98 Low p o r o s i t y d i a l y s i s t u b i n g (mw <3,500) N T 4220 ± 418 3130 ± 161 26 1 O u t e r chambers c o n t a i n e d 1 0 7 n o r m a l (N) o r tum o r - b e a r e r (T) s p l e e n c e l l s . ? 6 I n n e r chambers c o n t a i n e d 5 x 10 n o r m a l s p l e e n c e l l s . 106 DISCUSSION Non s p e c i f i c s u p p r e s s o r c e l l s have been o b s e r v e d i n a number of d i f f e r e n t tumor systems; however, t h e c e l l t y p e s i n v o l v e d and the mechanisms o f sup-p r e s s i o n a r e n o t c l e a r . F o r example, m o n o c y t e - l i k e c e l l s f r o m t h e b l o o d of human c a n c e r p a t i e n t s w i t h a v a r i e t y of tumor t y p e s a r e c a p a b l e o f s u p p r e s s i n g t h e p r o t e i n s y n t h e s i s o f n o r m a l l y m p h o c y t e s s t i m u l a t e d by PHA ( 1 3 8 ) . Sup-p r e s s o r c e l l s c a p a b l e o f i n h i b i t i n g the m i t o g e n r e s p o n s e (26,51,68,71 ?72) and t h e ija v i t r o g e n e r a t i o n o f PFC (26,40) have been o b s e r v e d i n t h e s p l e e n s of m i c e w i t h a number of v i r a l l y - i n d u c e d tumors. These s u p p r e s s o r c e l l s have been assumed by t h e d i f f e r e n t groups t o be macrophages ( 7 2 ) , B c e l l s ( 5 1 , 6 8 ) , and p o s s i b l y T c e l l s (26) . N y l o n w o o l a d h e r e n t s p l e e n c e l l s p o s s i b l y macro-phages, f r o m m i c e b e a r i n g m e t h y l c h o l a n t h r e n e - i n d u c e d tumors have been found t o s u p p r e s s the g e n e r a t i o n of c y t o t o x i c c e l l s by normal c e l l s s t i m u l a t e d i n v i t r o by a l l o a n t i g e n s ( 3 3 ) . The d a t a i n t h i s t h e s i s show t h a t t h e s p l e e n s of m i c e b e a r i n g l a r g e m e t h y l c h o l a n t h r e n e - i n d u c e d tumors c o n t a i n two d i s t i n c t p o p u l a t i o n s o f non s p e c i f i c s u p p r e s s o r c e l l s . One t y p e a p p e a r s t o be o f t h e macrophage/monocyte l i n e w h i l e the o t h e r may be a T c e l l . The s u p p r e s s i v e a c t i v i t y of t h e s e c e l l s has been de m o n s t r a t e d by: 3 1. i n h i b i t i o n o f H - t h y m i d i n e u p t a k e by n o r m a l , s y n g e n e i c s p l e e n o r lymph node c e l l s s t i m u l a t e d by the B c e l l m i t o g e n , LPS, o r t h e T c e l l m i t o g e n , Con A and 2. i n h i b i t i o n o f a n t i b o d y p r o d u c t i o n by n o r m a l , s y n g e n e i c s p l e e n c e l l s s t i m u l a t e d ±n v i t r o by the T c e l l i n d e p e n d e n t a n t i g e n , DNP-LPS, or t h e T c e l l dependent a n t i g e n , SRBC. The p r o l i f e r a t i v e r e s p o n s e s t o m i t o g e n s and t h e g e n e r a t i o n o f PFC i n v i t r o a r e v e r y s e n s i t i v e t o c u l t u r e c o n d i t i o n s s u c h as c e l l d e n s i t y , a n t i g e n d o s e , and i n c u b a t i o n t i m e . Thus i t was i m p o r t a n t , d u r i n g t h e 107 e s t a b l i s h m e n t of t h e s e a s s a y s y s t e m s , t o t e s t a l l of t h e s e p a r a m e t e r s i n c u l t u r e s c o n t a i n i n g e i t h e r n o r m a l or m i x t u r e s o f n o r m a l and t u m o r ^ b e a r e r c e l l s i n o r d e r t o c o n f i r m t h a t t h e s u p p r e s s i o n was n o t s i m p l y due t o a change i n t h e k i n e t i c s o f t h e r e s p o n s e when t u m o r - b e a r e r c e l l s were p r e s e n t . The s u p p r e s s i o n i n b o t h a s s a y systems was o b s e r v e d on a l l d ays and w i t h a l l a n t i g e n o r m i t o g e n doses t e s t e d . The c o n d i t i o n s chosen f o r t h e a s s a y s i n d u c e d good n o r m a l r e s p o n s e s and o f t e n gave the l o w e s t v a l u e s f o r s u p p r e s s i o n . A l s o , s u p p r e s s i o n d i d n o t appear t o be due t o a non s p e c i f i c k i l l i n g o f n o r m a l c e l l s s i n c e v i a b l e c e l l c o u n t s d i d n o t drop when t u m o r - b e a r e r c e l l s were p r e s e n t i n t h e c u l t u r e s . Thus s u p p r e s s i o n o b s e r v e d i n t h e s e two a s s a y systems was n o t s i m p l y an a r t i f a c t of t h e chosen c u l t u r e c o n d i t i o n s . U s i n g t h e two a s s a y s and v a r i o u s p r o c e d u r e s f o r s e p a r a t i n g p o p u l a t i o n s o f s p l e e n c e l l s , i t was p o s s i b l e t o d e m o n s t r a t e two s e p a r a t e p o p u l a t i o n s of s u p p r e s s o r c e l l s . The 'macrophage s u p p r e s s o r c e l l s ' i n h i b i t e d m i t o g e n i n d u c e d p r o l i f e r a t i o n and t h e jLn v i t r o PFC r e s p o n s e s t o b o t h T c e l l dependent and i n d e p e n d e n t a n t i g e n s . The 'T s u p p r e s s o r c e l l ' s u p p r e s s e d o n l y t h e PFC r e s p o n s e t o SRBC. 1. Macrophage - A s s o c i a t e d S u p p r e s s o r C e l l s Macrophages have been i m p l i c a t e d as non s p e c i f i c s u p p r e s s o r c e l l s i n b o t h e x p e r i m e n t a l a n i m a l (72) ahd human (138) t u m o r - b e a r e r s as w e l l as d u r i n g 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 n m i c e ( 1 1 6 ) . More s p e c i f i c a l l y , s p l e e n c e l l s o f m i c e w h i c h have been i n j e c t e d w i t h known macrophage a c t i v a t o r s s u c h as C o r y n e b a c t e r i u m parvum (70,79,136) o r p y r a n copolymer (5) have been f o u n d t o i n h i b i t b l a s t o g e n e s i s . A l s o , a c t i v a t e d macro-phages f r o m t h e p e r i t o n e a l c a v i t y o f m i c e were h i g h l y s u p p r e s s i v e i n a v a r i e t y of a s s a y s m e a s u r i n g p r o l i f e r a t i v e r e s p o n s e s ( 3 8 ) . T h i s t y p e o f s u p p r e s s o r c e l l has g e n e r a l l y been seen t o be a d h e r e n t , l a r g e b u t n o t 108 dense, and n o t a f f e c t e d by m i t o m y c i n C. I n t h i s s t u d y , t h e macrophage s u p p r e s s o r c e l l s were r e t a i n e d m a i n l y i n t h e h y p a q u e - f i c o l l l i g h t f r a c t i o n c o n t a i n i n g t h e l e s s dense c e l l s , were a d h e r e n t t o p l a s t i c and n y l o n w o o l and were removed by c a r b o n y l i r o n / magnet t r e a t m e n t . These p r o c e d u r e s d i d n o t r u l e out the p o s s i b i l i t y t h a t B c e l l s a r e s u p p r e s s o r c e l l s s i n c e the h y p a q u e - f i c o l l l i g h t f r a c t i o n i s r i c h i n B c e l l s and s i n c e a d h e r e n c e t e c h n i q u e s remove B c e l l s as w e l l as macrophages. However, t r e a t m e n t o f t u m o r ^ b e a r e r s p l e e n c e l l s w i t h c a r r a g h e e n a n i n a c t i v a t e d t h e c e l l s w h i c h s u p p r e s s e d t h e PFC r e s p o n s e t o DNP-LPS. S i n c e c a r r a g h e e n a n t r e a t m e n t s p e c i f i c a l l y i n a c t i v a t e s macro-phages, i t i s c l e a r t h a t macrophages a r e i n v o l v e d i n s u p p r e s s i o n of t h e r e s p o n s e t o DNP-LPS. A l s o , t r e a t m e n t of c e l l s w i t h a n t i - T h y - 1 and a n t i -MIg s e r a p l u s complement d i d not remove the s u p p r e s s i o n o f t h e m i t o g e n r e s p o n s e s o r t h e PFC r e s p o n s e t o DNP-LPS showing t h a t B o r T c e l l s a r e n o t r e s p o n s i b l e f o r t h i s s u p p r e s s i o n . Macrophage s u p p r e s s o r c e l l s do n o t r e q u i r e the s y n t h e s i s of macro-m o l e c u l e s i n o r d e r t o s u p p r e s s . These c e l l s d i d n o t l o s e t h e i r sup-p r e s s i v e a b i l i t y when t r e a t e d w i t h up t o 100 yg/ml m i t o m y c i n C, doses w h i c h a r e known t o i n h i b i t RNA and p r o t e i n as w e l l as DNA s y n t h e s i s ( 8 5 ) . The t a r g e t o f s u p p r e s s i o n a p p e a r s t o be b o t h T and B l y m p h o c y t e s s i n c e the p r o l i f e r a t i v e r e s p o n s e s t o T and B c e l l m i t o g e n s and t h e PFC r e s p o n s e s t o b o t h T c e l l dependent and i n d e p e n d e n t a n t i g e n s were a l l i n h i b i t e d . Macrophage s u p p r e s s o r c e l l s were f o u n d i n t h e s p l e e n but n o t lymph nodes of t u m o r - b e a r e r s . R e c e n t l y , G i l l e t t e (45) has s u g g e s t e d t h a t t h e r e a r e two t y p e s of a d h e r e n t c e l l s i n m i c e ; t y p e L, found i n s p l e e n and 109 lymph nodes, w h i c h s t i m u l a t e s DNA s y n t h e s i s and t y p e S, found o n l y i n th e s p l e e n , w h i c h s u p p r e s s e s DNA s y n t h e s i s . The macrophage s u p p r e s s o r c e l l s c o u l d t h e r e f o r e r e f l e c t an i n c r e a s e i n t y p e S a d h e r e n t c e l l s . S u p p r e s s i o n i n t h i s c a s e may a l s o be due t o a c t i v a t e d macrophages. Fo r example, t h e r e appears t o be a q u a l i t a t i v e as w e l l a s a q u a n t i t a t i v e d i f f e r e n c e between n o r m a l and tu m o r — b e a r e r macrophages s i n c e even when p o p u l a t i o n s o f s p l e e n c e l l s f r o m b o t h groups c o n t a i n e d e q u i v a l e n t numbers of c e l l s c a p a b l e o f p h a g o c y t i z i n g l a t e x beads, n o r m a l c e l l s were n o t as s u p p r e s s i v e as tu m o r - b e a r e r c e l l s . A d i f f e r e n c e between n o r m a l and t u m o r - b e a r e r p h a g o c y t i c c e l l s was a l s o o b s e r v e d a f t e r c a r r a g h e e n a n t r e a t m e n t . F o l l o w i n g i n c u b a t i o n w i t h c a r r a g h e e n a n , 10% of nor m a l s p l e e n c e l l s s t i l l p h a g o c y t i z e d l a t e x beads whereas t h e p e r c e n t a g e of p h a g o c y t i c t u m o r - b e a r e r c e l l s was red u c e d f r o m 14% t o 4%. I t has been o b s e r v e d by o t h e r s t h a t a c t i v a t e d macrophages t a k e up c a r r a g h e e n a n and a r e i n a c t i v a t e d by i t more q u i c k l y t h a n normal macrophages (25). Thus t h e macrophage s u p p r e s s o r c e l l may be s i m i l a r t o t h o s e seen i n mice w h i c h have been i n j e c t e d w i t h known macrophage a c t i v a t o r s . I t i s a l s o p o s s i b l e , however, t h a t t h e macrophages a r e armed by a T c e l l p r o d u c t as i s seen i n c o n t a c t s e n s i t i v i t y (3). 2. T S u p p r e s s o r C e l l s T c e l l s have been i m p l i c a t e d as non s p e c i f i c s u p p r e s s o r c e l l s i n a number o f d i f f e r e n t s i t u a t i o n s . G l a s s w o o l a d h e r e n t T c e l l s f r o m t h e s p l e e n s o f normal and a n t i g e n primed r a t s s u p p r e s s t h e PHA s t i m u l a t e d b l a s t o g e n i c r e s p o n s e of r a t l y m p h o i d c e l l s (11,13,39). T c e l l s i n t h e s p l e e n s o f mic e s e n s i t i z e d t o a l l o a n t i g e h s non s p e c i f i c a l l y s u p p r e s s t h e mixed l y m p h o c y t e r e a c t i o n (60). A l s o , t he s p l e e n s o f m i c e u n d e r g o i n g g r a f t - v e r s u s - h o s t r e a c t i o n s (104,105,114) o r i n f e c t e d w i t h p a r a s i t e s 110 (32) c o n t a i n T c e l l s w h i c h s u p p r e s s humoral r e s p o n s e s by l y m p h o c y t e s . The T s u p p r e s s o r c e l l s i n t h i s t h e s i s s e d i mented a l o n g w i t h t h e dense s p l e e n c e l l s on a h y p a q u e - f i c o l l g r a d i e n t . They were non a d h e r e n t t o p l a s t i c , non a d h e r e n t o r p a r t i a l l y a d h e r e n t t o n y l o n w o o l , and were n o t removed by c a r b o n y l iron/magnet t r e a t m e n t . The t a r g e t of t h e s e s u p p r e s s o r c e l l s a ppears t o be t h e T h e l p e r c e l l o r a T c e l l dependent s u b p o p u l a t i o n of B c e l l s (23,66) s i n c e o n l y t h e PFC r e s p o n s e t o SRBC i s a f f e c t e d . I t was d i f f i c u l t t o show u n e q u i v o c a l l y t h a t t h i s s u p p r e s s o r c e l l i s a T c e l l by r e m o v a l w i t h a n t i - T h y - 1 serum p l u s complement. The d i f f i c u l t y c o u l d have been due t o our i n a b i l i t y t o c o m p l e t e l y s e p a r a t e s u p p r e s s i v e macrophages f r o m t h e T c e l l p o p u l a t i o n o r c o u l d r e f l e c t a low c o n c e n t -r a t i o n o f the Thy-1 a n t i g e n on the membrane of t h e dense s u p p r e s s o r c e l l s . I f c o u l d be p o s s i b l e t o a r g u e t h a t the r e s u l t s a r e due t o a s i n g l e p o p u l a t i o n o f macrophage s u p p r e s s o r c e l l s o n l y p a r t i a l l y s e p a r a b l e by h y p a q u e - f i c o l l and adherence t e c h n i q u e s , and t h a t the PFC r e s p o n s e t o SRBC i s more s e n s i t i v e t o s u p p r e s s i o n . However, f u r t h e r e v i d e n c e f o r two p o p u l a t i o n s o f s u p p r e s s o r c e l l s comes f r o m the f a c t t h a t t h e macro-phage t y p e was u n a f f e c t e d by doses of 100 yg/ml of m i t o m y c i n C w h i l e t h e p u t a t i v e T s u p p r e s s o r c e l l was t o t a l l y i n a c t i v a t e d by 25 yg/ml of m i t o m y c i n C. Thus DNA s y n t h e s i s was n e c e s s a r y f o r t h e T s u p p r e s s o r c e l l , b u t n o t t h e macrophage s u p p r e s s o r c e l l a c t i v i t y . The macrophage s u p p r e s s o r c e l l s were found i n t h e s p l e e n s of a l l advanced t u m o r - b e a r e r s , b u t t h e p r e s e n c e of t h e T s u p p r e s s o r c e l l s was more v a r i a b l e . I f m i c e were i n j e c t e d w i t h tumor c e l l s w h i c h had been c u l t u r e d i ^ i v i t r o f o r l o n g p e r i o d s o f t i m e , t h e macrophage s u p p r e s s o r c e l l s p r e d o m i n a t e d . I f , however, m i c e were i n j e c t e d w i t h tumor c e l l s I l l that had been passaged p r i m a r i l y jin vivo, both macrophage and T sup-pressor c e l l types were generally found. The a n t i g e n i c i t y of tumors induced by methylcholanthrene has been shown by 01d~ et a l . C96) to be stable a f t e r 25 to 30 i n vivo passages. However, i t i s possible that the a n t i g e n i c i t y of tumor c e l l s decreases when they have been cultured for long periods of time i n v i t r o before i n j e c t i o n into mice. The T suppressor c e l l may be generated only when the tumor c e l l s injected are highly antigenic whereas macrophage suppressor c e l l s appear to be present during the growth of a l l M-l tumors. It i s also possible that T suppressor c e l l s are induced only when mice are injected with cultured tumor c e l l s which s t i l l contain lymphoid c e l l s . In v i t r o cultures are established from small tumors which contain lymphoid c e l l s and macrophages. Several passages of i n v i t r o cultures are required to completely r i d cultures of 'passenger' c e l l s . When tumor c e l l s from cultures which have only been passaged two or three times i n v i t r o are injected into mice, T suppressor c e l l s may be stimulated by the presence of immune c e l l s i n the inoculum. If this i s the case, then mice injected with tumor c e l l s which have been passaged enough times i n v i t r o to remove a l l immune c e l l s would produce fewer T suppressor c e l l s . - Although' thymuses from tumor-bearers were not weighed and thymocyte suspensions were not prepared, i t was observed that the thymuses from mice which had T suppressor c e l l s tended to be small whereas larger thymuses appeared to be present i n mice with only the macrophage sup-pressor c e l l s . I t i s possible then that spleens of mice from the former category had large quantities of immature, non s p e c i f i c a l l y suppressive T c e l l s which could not d i f f e r e n t i a t e into more mature forms because 112 of a d e f i c i e n c y i n t h y m i c hormone as was seen by Bash e t a l . (12) i n a t h y m i c r a t s . T h e r e i s , however, no good e v i d e n c e t o s u p p o r t t h i s t h e o r y . 3. S o l u b l e F a c t o r s The macrophage s u p p r e s s o r c e l l d i d n o t appear t o s u p p r e s s by p r o d u c i n g a s o l u b l e f a c t o r s i n c e c e l l s a d h e r e n t t o p l a s t i c and t h o s e i n t h e l i g h t f r a c t i o n a f t e r h y p a q u e - f i c o l l c e n t r i f u g a t i o n d i d n o t s u p p r e s s ±n v i t r o g e n e r a t i o n of PFC i n a Marbrook chamber. A l s o , c a r b o n y l / i r o n magnet t r e a t m e n t and n y l o n w o o l adherence d i d n o t remove t h e c e l l s p r o d u c i n g a f a c t o r i n t h i s s t u d y . These d a t a a r e c o n s i s t e n t w i t h t h e r e s u l t s seen i n o t h e r systems i n w h i c h macrophages have been i m p l i c a t e d as s u p p r e s s o r c e l l s . I n most c a s e s , c o n t a c t of macrophages w i t h t h e i r t a r g e t has been r e q u i r e d f o r s u p p r e s s i o n t o o c c u r ( 6 , 3 3 , 7 6 ) . S u p p r e s s i o n by s p l e e n c e l l s f r o m m i c e b e a r i n g a v a r i e t y o f tumor t y p e s has been seen t o r e q u i r e c o n t a c t (33, 118). S u p p r e s s i o n by the T s u p p r e s s o r c e l l a p p e a rs t o be m e d i a t e d by a s o l u b l e f a c t o r s i n c e h y p a q u e - f i c o l l dense, p l a s t i c non a d h e r e n t , and c a r b o n y l i r o n t r e a t e d c e l l s a l l s u p p r e s s e d a c r o s s a c e l l impermeable membrane. I t i s p o s s i b l e t h a t t h e c e l l p r o d u c i n g t h e s o l u b l e f a c t o r i s s l i g h t l y a d h e r e n t t o n y l o n w o o l s i n c e t h e c e l l s e l u t e d f r o m a column were l e s s s u p p r e s s i v e t h a n u n t r e a t e d c e l l s . The f a c t o r was a b l e t o pass t h r o u g h s t a n d a r d d i a l y s i s t u b i n g (which r e t a i n s m o l e c u l a r w e i g h t s of 12,000 or h i g h e r ) b u t d i d n o t p a s s t h r o u g h low p o r o s i t y d i a l y s i s t u b i n g (which r e t a i n s m o l e c u l a r w e i g h t s of 3,500 or h i g h e r ) . Thus, the s o l u b l e f a c t o r r e l e a s e d f r o m s u p p r e s s o r c e l l s a p p e ars t o have a m o l e c u l a r w e i g h t of about 3,500 t o 12,000. 113 Non s p e c i f i c s u p p r e s s i v e f a c t o r s have been found t o be r e l e a s e d f r o m T c e l l s d u r i n g a n t i g e n c o m p e t i t o n i n m i c e (31,126), a f t e r i m m u n i z a t i o n of r a t s w i t h l a r g e doses o f a n t i g e n ( 9 0 ) , and a f t e r Con A (90,110,123) or PHA (81) a c t i v a t i o n . The f a c t o r s were found t o have a v a r i e t y o f m o l e c u l a r w e i g h t s r a n g i n g f r o m 10,000 t o 67,000. However, one of t h e f a c t o r s r e l e a s e d f r o m Con A a c t i v a t e d c e l l s had a m o l e c u l a r w e i g h t of l e s s t h a n 10,000 arid was c a p a b l e o f s u p p r e s s i n g t h e I n v i t r o PFC r e s p o n s e t o SRBC ( 1 1 0 ) . T h i s f a c t o r may be r e l a t e d t o t h e s o l u b l e f a c t o r produced by s u p p r e s s o r c e l l s i n t h i s r e p o r t . I t i s p o s s i b l e t h a t t h e s e s m a l l f a c t o r s a r e a c t i v e p o r t i o n s of the l a r g e r f a c t o r s d e s c r i b e d by o t h e r s . The s o l u b l e s u p p r e s s i v e f a c t o r was n o t produced by s p l e e n c e l l s f r o m a l l t u m o r - b e a r i n g m i c e t e s t e d . I t was produced o n l y by s p l e e n c e l l s of mic e w h i c h c o n t a i n e d t h e T s u p p r e s s o r c e l l s w h i c h s u p p r e s s e d o n l y t h e i n v i t r o g e n e r a t i o n of PFC t o SRBC by c o - c u l t u r e d n o r m a l c e l l s . I t may be t h a t t h i s f o r m o f s u p p r e s s i o n i s due t o a n t i g e n i c c o m p e t i t i o n and t h a t T s u p p r e s s o r c e l l s , and t h e i r s o l u b l e p r o d u c t s , a r e produced o n l y d u r i n g s t r o n g a n t i g e n i c s t i m u l a t i o n . The d a t a p r e s e n t e d i n t h i s t h e s i s have shown t h a t two d i s t i n c t p o p u l a t i o n s o f non s p e c i f i c s u p p r e s s o r c e l l s a r e p r e s e n t i n t h e s p l e e n s o f mi c e b e a r i n g l a r g e c h e m i c a l l y - i n d u c e d tumors. One ty p e of s u p p r e s s o r c e l l , f r o m t h e macrophage/ monocyte l i n e , i s p r e s e n t i n a l l m i c e b e a r i n g l a r g e tumors. Both B and T c e l l s appear t o be s u p p r e s s e d when t h e y come i n t o p h y s i c a l c o n t a c t w i t h t h i s t y p e of s u p p r e s s o r c e l l . 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