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Antigen-specific helper T cells in the responses of DBA/2 mice to a syngeneic tumour, P815 Hancock, Elizabeth Jane 1983

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ANTIGEN-SPECIFIC HELPER T CELLS IN THE RESPONSES OF DBA/2 MICE TO A SYNGENEIC TUMOUR, P815 by ELIZABETH JANE HANCOCK B.Sc. U n i v e r s i t y of Adelaide, 1974 B.Sc. (Hons) U n i v e r s i t y of Adelaide, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s t h e s i s as conforming to the req u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia October 1983 l i z a b e t h Jane Hancock, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publi c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of MICROBIOI OfiY  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date OCTOBER 14th 1983 DE-6 (3/81) ABSTRACT When i n j e c t e d w i t h l i v e P815 tumour c e l l s , DBA/2 mice developed c y t o t o x i c c e l l s r e a c t i v e to the tumour. In a d d i t i o n , T helper c e l l s from tumour-bearing mice enhanced the ir i v i t r o generation of c y t o t o x i c c e l l s from normal DBA/2 spleen c e l l s . The helper c e l l s had the f o l l o w i n g p r o p e r t i e s (1) expression of the Thy-1.2 antigen; (2) r e s i s t a n c e to y - r a d i a t i o n ; (3) s p e c i f i c enhancement of the c y t o t o x i c response to P815; (4) d e t e c t a b i l i t y i n P815-bearing mice at the peak of c y t o t o x i c c e l l a c t i v i t y ; (5) a c t i v i t y i n the e a r l y phase of c y t o t o x i c c e l l a c t i v a t i o n . In p a r a l l e l to the development of helper c e l l a c t i v i t y , suppressor c e l l s were generated which suppressed the c y t o t o x i c response to P815. These suppressor c e l l s were removed by p r e - t r e a t i n g mice with low doses of cyclophosphamide. High doses of cyclophosphamide reduced the c y t o t o x i c response to both P815 and C57B1/6 a l l o a n t i g e n s . Cyclophosphamide treatment reduced the frequency of c y t o t o x i c precursor c e l l s d i r e c t e d against P815, and an a n t i g e n - r e a c t i v e helper c e l l i n volved i n i n t e r l e u k i n 2 production. Both i n t e r l e u k i n 2 and thymocytes from P815-primed mice, r e s t o r e d the c y t o t o x i c response against P815, to normal l e v e l s . Twenty s i x percent of animals primed with tumour c e l l s c l e a r e d a challenge dose of P815 f a s t e r than unprimed c o n t r o l mice. Of these, 88% survived longer than the c o n t r o l animals. Eighteen percent of the r e c i p i e n t s of c e l l s from tumour-primed mice, c l e a r e d a challenge dose of P815 f a s t e r than mice i n j e c t e d w i t h normal c e l l s . Of these 53% survived s i g n i f i c a n t l y longer than c o n t r o l groups given e i t h e r normal c e l l s or no c e l l s at a l l . C e l l s from mice primed to PPD showed s i g n i f i c a n t l y enhanced p r o l i f e r a t i v e responses to s o l u b l e and P815-bound PPD, when compared with unprimed animals. However, c e l l s from only a few PPD-primed mice showed enhanced c y t o t o x i c i t y against P815 tumour c e l l s , and PPD-primed c e l l s e i t h e r d i d not a l t e r , or suppressed, the c y t o t o x i c response of normal DBA/2 spleen c e l l s , when sti m u l a t e d w i t h PPD-coated P815. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v L i s t of Tables i x L i s t of Figures x i ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 1. Background 1 2. T Regulator C e l l s 3 3. Regulation of Cyt o t o x i c Responses 4 4. Separation of Regulator T c e l l s from other T c e l l Populations 5 (A) S e n s i t i v i t y to Rad i a t i o n 5 (B) S e n s i t i v i t y to Drugs ( i ) C o r t i c o s t e r o i d s 7 ( i i ) Cyclophosphamide 7 (C) C e l l Surface Markers ( i ) l a antigens 8 ( i i ) L yt antigens 8 5. Adherent C e l l s and H-2 R e s t r i c t i o n 9 6. The Mode of A c t i o n of Regulator C e l l s 10 7. Models of Cy t o t o x i c T C e l l A c t i v a t i o n 14 (A) The Two-Signal Model 14 (B) A P o s t u l a t e d Model of Cy t o t o x i c C e l l A c t i v a t i o n 19 In v o l v i n g A d d i t i o n a l C e l l s and Factors V Page 8. Anti-Tumour Responses 25 9. Regulation of the C y t o t o x i c Response to a Syngeneic 29 Tumour, P815 10. The Objectives of t h i s I n v e s t i g a t i o n 29 MATERIALS AND METHODS 1. Mice 33 2. Tumours a. Source 33 b. Cloning 34 c. Maintenance 34 d. I n j e c t i o n 35 3. Medium a. RPMI 1640 35 b. Phosphate bu f f e r e d s a l i n e 36 4. C e l l preparations a. S i n g l e c e l l suspensions 36 b. Lympholyte M 37 c. I r r a d i a t i o n 38 d. Treatment with anti-Thy 1.2 serum plus complement 38 r e. Mitomycin C 38 5. In v i t r o Generation and Assay of Cy t o t o x i c C e l l s a. Generation of c y t o t o x i c i t y against syngeneic 39 tumours b. C y t o t o x i c i t y against minor h i s t o c o m p a t i b i l i t y 39 antigens c. C y t o t o x i c i t y against major h i s t o c o m p a t i b i l i t y 41 antigens d. Helper assays e. C y t o t o x i c i t y assays 6. L i m i t i n g D i l u t i o n Cultures a. Cultures f o r es t i m a t i n g c y t o t o x i c c e l l precursor frequency b. S p l i t c u l t u r e s assayed on two tar g e t s c. C a l c u l a t i o n of precursor frequency 7. Pre p a r a t i o n and Assay of I n t e r l e u k i n 2 a. Bulk, preparation from r a t and mouse spleen c e l l s b. Antigen and mitogen induced i n t e r l e u k i n 2 a c t i v i t y from mouse spleen c e l l s c. Preparation from the EL4.IL2 c e l l l i n e d. Thymocyte p r o l i f e r a t i o n assay f o r 112 a c t i v i t y e. Assay on an i n t e r l e u k i n 2-dependent c e l l l i n e 8. Cyclophosphamide I n j e c t i o n 9. Assaying Helper A c t i v i t y i n v i v o a. L a b e l l i n g tumour c e l l s w i t h l ^ I . I o d o d e o x y u r i d i n e b. Measuring tumour c e l l clearance 10. Helper Determinants a. Priming mice against tuberculin-PPD b. Coupling PPD to P815 c e l l s c. P r o l i f e r a t i o n assays d. C y t o t o x i c i t y assays RESULTS CHAPTER I . The C y t o t o x i c Response of DBA/2 Mice to the Syngeneic Tumour P815 v i i Page 1. In v i v o and i j i v i t r o Generation of C y t o t o x i c i t y 54 Against P815 2. C e l l s from Tumour-bearing Mice Enhance the Primary 60 i n v i t r o C y t o t o x i c Response 3. The S u s c e p t i b i l i t y of Enhancing and C y t o t o x i c A c t i v i t i e s 71 to Y-Radi ati° n 4. The Nature of the Helper C e l l s 78 5. The Time of Appearance of Helper C e l l s i n the Thymus 81 6. The Antigen S p e c i f i c i t y of Helper C e l l s 81 7. The Timing of Helper C e l l A c t i v i t y i n the i j i v i t r o 84 Cy t o t o x i c Response to P815 8. Summary 90 CHAPTER I I : The E f f e c t s of Cyclophosphamide on the Cy t o t o x i c 91 Response of DBA/2 Mice to P815 1. The E f f e c t of Cyclophosphamide on C y t o t o x i c Responses 91 of DBA/2 Mice 2. The Influence of Cyclophosphamide on I n t e r l e u k i n 2 94 Production 3. The E f f e c t of Helper C e l l s on the Cyclophosphamide- 98 Depleted C y t o t o x i c Response 4. The E f f e c t of Cyclophosphamide on C y t o t o x i c 104 Precursor C e l l s 5. Summary 114 CHAPTER I I I . The In Vivo A c t i v i t y of P815-Induced Helper C e l l s 118 1. Clearance of P815 Tumour C e l l s by DBA/2 Mice 118 Primed to P815 2. Clearance of P815 Tumour C e l l s by DBA/2 Mice I n j e c t e d 129 wi t h C e l l s from P815 Primed Animals 3. Summary 135 v i i i Page CHAPTER IV: Enhancing the In V i t r o C y t o t o x i c Response to 136 P81S Using an Exogenous Helper Determinant 1. Coupling T u b e r c u l i n PPD to P815 Tumour C e l l s 136 2. Lymph Node P r o l i f e r a t i v e Responses of PPD-Primed 137 DBA/2 Mice to PPD and PPD-Coupled P815 C e l l s 3. In v i t r o C y t o t o x i c Responses of PPD-Primed Animals 144 to P815 4. Summary 147 DISCUSSION 1. Evidence f o r Helper T C e l l s A c t i v e i n the Primary 148 In V i t r o C y t o t o x i c Response to P815. 2. E f f e c t s of Pre-Treatment of Mice with Cyclophosphamide 151 3. The In v i v o Primary Response to P815 154 4. The In v i t r o C y t o t o x i c Response to PPD-Modified 156 P815 Tumour C e l l s 5. Prospectives 157 6. Summary 159 LITERATURE CITED 161 i x LIST OF TABLES Page I. The p r o p e r t i e s of T helper c e l l subclasses 23 invo l v e d i n B c e l l a c t i v a t i o n I I . The coexistence of helper and suppressive a c t i v i t i e s 77 i n thymocytes from normal and tumour-bearing mice I I I . The antigen s p e c i f i c i t y of helper c e l l s 85 IV. The antigen s p e c i f i c i t y of i n v i t r o generated 86 c y t o t o x i c c e l l s V. The timed a d d i t i o n of helper c e l l s to c y t o t o x i c c e l l s 88 VI. The time of a c t i v i t y of helper c e l l s i n the i i i v i t r o 89 c y t o t o x i c response to P815 V I I . The e f f e c t of cyclophosphamide i n j e c t i o n upon spleen 92 c e l l recovery V I I I . The e f f e c t of cyclophosphamide i n j e c t i o n on the 93 i n v i t r o c y t o t o x i c responses of DBA/2 mice IX. Concanavalin A-induced i n t e r l e u k i n 2 from spleen 97 c e l l s of normal and cyclophosphamide-treated mice X. Antigen-induced i n t e r l e u k i n 2 from spleen c e l l s of 99 normal and cyclophosphamide-injected mice XI. The frequency of c y t o t o x i c precursors i n DBA/2 109 spleen c e l l s d i r e c t e d against P815 X I I . The frequency of anti-P815 c y t o t o x i c precursor c e l l s 112 in DBA/2 mice i n j e c t e d w i t h cyclophosphamide X I I I . The dependence of precursor c y t o t o x i c c e l l frequency 113 c a l c u l a t e d by l i m i t i n g d i l u t i o n a n a l y s i s , on the l e v e l s of i n t e r l e u k i n 2 i n c u l t u r e . XIV. Clearance of P815 tumour c e l l s by DBA/2 mice 120 primed against P815 XV. Clearance of P815 by DBA/2 mice i n j e c t e d w i t h 130 c e l l s from tumour-primed mice XVI. Coupling 1 2 5 I - l a b e l l e d - P P D to P815 c e l l s w i t h ECDI 138 XVII. Priming of DBA/2 mice to t u b e r c u l i n p u r i f i e d p r o t e i n 139 d e r i v a t i v e (PPD) X Page X V I I I . The p r o l i f e r a t i v e responses of lymph node c e l l s from 141 PPD-primed DBA/2 mice XIX. I l l v i t r o c y t o t o x i c responses of PPD-primed DBA/2 mice 145 to P815 tumour c e l l s x i LIST OF FIGURES Page 1. The 2 - s i g n a l model of c y t o t o x i c T c e l l a c t i v a t i o n 15 2. A proposed model of c y t o t o x i c T c e l l a c t i v a t i o n 20 3. The pa t t e r n of tumour growth and appearance of 30 c y t o t o x i c i t y i n DBA/2 mice i n j e c t e d w i t h P815 tumour c e l l s 4. The assay system f o r generating c y t o t o x i c c e l l s against 40 d i f f e r e n t antigens 5. The assay system f o r r e g u l a t o r c e l l s i n the primary i j i v i t r o 42 c y t o t o x i c response to P815 6. In v i t r o generated c y t o t o x i c a c t i v i t y by spleen c e l l s from 56 normal or tumour-bearing mice 7. In v i t r o generated c y t o t o x i c a c t i v i t y by lymph node c e l l s 58 from normal or tumour-bearing mice 8. In v i t r o generated c y t o t o x i c i t y by thymocytes from normal 61 and tumor-bearing mice 9. The a d d i t i o n of spleen c e l l s from normal or tumour-bearing 64 mice to c u l t u r e s generating c y t o t o x i c i t y against P815 10. The a d d i t i o n of lymph node c e l l s from normal or 66 tumour-bearing mice to c u l t u r e s generating c y t o t o x i c i t y against P815 11. The a d d i t i o n of thymocytes from normal or tumour-bearing 69 mice to c u l t u r e s generating c y t o t o x i c i t y against P815 12. The y - r a d i a t i o n s e n s i t i v i t y of c y t o t o x i c responder 72 c e l l s 13. The Y - r a d i a t i o n s e n s i t i v i t y of c y t o t o x i c a m p l i f e r 74 c e l l s 14. Pretreatment of a m p l i f i e r c e l l s w i t h anti-Thy 1.2 79 antibody plus complement 15. The time of appearance of helper c e l l s i n the thymus 82 a f t e r i n j e c t i o n of P815 16. The e f f e c t of i n t e r l e u k i n 2 on the cyclophosphamide- 95 depleted c y t o t o x i c response of DBA/2 mice to P815 x i i Page 17. The e f f e c t of cyclophosphmaide upon the c y t o t o x i c 100 response of DBA/2 mice to P815 and upon P815-induced i n t e r l e u k i n 2 production 18. The a d d i t i o n of DBA/2 thymocytes r e s t o r e d the 102 cyclophosphamide-depleted c y t o t o x i c response to B6 a l l o a n t i g e n s 19. Normal DBA/2 thymocytes d i d not r e s t o r e the 105 cyclophosphamide-depleted c y t o t o x i c response to P815 20. R e s t o r a t i o n of the cyclophosphamide-depleted c y t o t o x i c 107 response to P815 with thymocytes from normal or tumour-bearing mice 21. The antigen s p e c i f i c i t y of c y t o t o x i c c e l l s generated 115 i n l i m i t i n g d i l u t i o n c u l t u r e s 22. Clearance of P815 by DBA/2 mice primed w i t h two 122 i n j e c t i o n s of P21 tumour c e l l s 23. Clearance of P815 by DBA/2 mice primed wit h two 125 i n j e c t i o n s of P911 tumour c e l l s 24. Clearance of P815 by DBA/2 mice primed wit h P815 127 tumour c e l l s 25. Clearance of P815 by DBA/2 mice i n j e c t e d w i t h thymocytes from P815-bearing mice 133 ACKNOWLEDGEMENTS I am indebted to Dr. D.G. K i l b u r n f o r h i s generous support, constant encouragement and f r i e n d s h i p over the past 5 years. I acknowledge the f i n a n c i a l support of the N a t i o n a l Cancer I n s t i t u t e of Canada during most of my s t u d i e s . I wish to thank my supervisory committee, members of the Microb i o l o g y Department and f r i e n d s i n the u n i v e r s i t y community f o r t h e i r continued patience, support and understanding during t h i s work. Thanks to Rosario Bauzon f o r her s k i l l f u l t y p i n g of t h i s manuscript and i t s many and v a r i e d d r a f t s . S p e c i a l thanks to Bob, whose support and f a i t h made a l l t h i s hard work bearable and encouraged me to continue. x i v DEDICATION This t h e s i s i s dedicated to my parents who i n s p i r e d and supported me f o r so long; and to Bob who has given me so much of himself and shares my triumphs and defeats w i t h me. 1 INTRODUCTION 1. Background Immune responses were o r i g i n a l l y p o s t u l a t e d to occur as a r e s u l t of the simple i n t e r a c t i o n of antigen w i t h immunocompetent c e l l s . These c e l l s were compartmentalized i n t o those i n v o l v e d i n cell-mediated immunity and those i n v o l v e d i n humoral immunity. I t i s now recognized that immune responses are a r e s u l t of the complex i n t e r a c t i o n s of many d i f f e r e n t c e l l p o p u l a t i o n s , w i t h an important r o l e f o r r e g u l a t o r c e l l s i n the i n t e r a c t i o n s l e a d i n g to both humoral and c e l l u l a r immunity. E a r l y studies showed that small c i r c u l a t i n g lymphocytes are important i n the immune response. Gowans et^ a l (1962) depleted the c i r c u l a t i n g pool of small lymphocytes by t h o r a c i c duct cannulation and observed severe immunodeficiency. Immune f u n c t i o n could be r e s t o r e d w i t h syngeneic t h o r a c i c duct c e l l s . They were al s o able to show passive t r a n s f e r of immunological memory by t r a n s f e r of small lymphocytes from immunized syngeneic donors i n t o h e a v i l y i r r a d i a t e d r a t s (Gowans and Uhr, 1966). Not a l l small lymphocytes are f u n c t i o n a l l y e q u i v a l e n t , since removal of the Bursa of Fabricus from newly hatched ch i c k s r e s u l t s i n a decrease i n antibody responses, but has l i t t l e e f f e c t on cell-m e d i a t e d responses such as g r a f t r e j e c t i o n ( G l i c k ej: a l . , 1956; Warner, 1965). In mammals, neonatal thymectomy not only prevents the development of cel l - m e d i a t e d immunity such as r e j e c t i o n of a l l o g e n e i c s k i n g r a f t s , but als o diminishes the antibody response to many antigens such as sheep red 2 blood c e l l s and f o r e i g n serum p r o t e i n s ( M i l l e r et a l . , 1962; Papermaster et a l . , 1962). Responsiveness can be r e s t o r e d by implantation of a thymus g r a f t , and c e l l s from the g r a f t undergo antigen-induced m i t o s i s w i t h i n the spleen of the r e c i p i e n t animal (Dalmasso et a l . , 1962; M i l l e r , 1963). L e t h a l l y i r r a d i a t e d mice can be r e c o n s t i t u t e d w i t h respect to hemopoietic f u n c t i o n by syngeneic bone marrow c e l l s (which are b e l i e v e d to represent the mammalian equivalent of Bursa-derived c e l l s ) but the thymus needs to be present f o r f u l l immunocompetence. Animals which are thymectomized before i r r a d i a t i o n cannot respond to most antigens. Thus, i t appears that c e l l s from d i f f e r e n t organs are important i n both humoral and c e l l u l a r immune responses. The f i r s t evidence of the i n t e r a c t i o n between these d i f f e r e n t c e l l populations was the work of Claman et^ a l . (1966) on the antibody response to sheep red blood c e l l s . L e t h a l l y i r r a d i a t e d mice were i n j e c t e d i n t r a v e n o u s l y w i t h v a r y i n g numbers of spleen, thymus and/or bone marrow c e l l s from normal or immune syngeneic donors. A f t e r antigen challenge, the spleens of r e c i p i e n t s were assayed f o r antibody production. I n j e c t i o n of e i t h e r marrow or thymus c e l l s alone gave no response. However, when thymus and marrow c e l l s were i n j e c t e d together, the antibody responses were equivalent to those when spleen c e l l s were i n j e c t e d . The synergy between thymus and bone marrow was r e l a t e d to the number of each c e l l type i n j e c t e d and the authors suggested that the marrow population provides the e f f e c t o r c e l l s which produce antibody only i n the presence of a u x i l i a r y c e l l s from the thymus. Studies of Davies et. a l . (1967) and M i l l e r and M i t c h e l l (1968, 1969) showed d i r e c t l y that the bone marrow provides precursors of antibody forming c e l l s , and that 3 thymocyte populations (and t h o r a c i c duct lymphocytes) provide the " a u x i l i a r y c e l l s " e s s e n t i a l f o r the a c t i v a t i o n of antibody forming c e l l s . Hosier (1967) separated spleen c e l l s i n t o adherent and non-adherent populations and could show a requirement f o r both c e l l types i n the i n  v i t r o antibody response. Exposure of the adherent population to antigen f o r a short p e r i o d of time before a d d i t i o n of the non-adherent c e l l s was s u f f i c i e n t to i n i t i a t e a maximal response. By c u l t u r i n g l i m i t i n g numbers of one c e l l type w i t h an excess of the other, they suggested that one adherent c e l l and at l e a s t two non-adherent c e l l s are r e q u i r e d to i n t e r a c t f o r an immune response to develop (Mosier and Coppleson, 1968). They a l s o showed that the c e l l u l a r d e f i c i t i n thymectomized mice i s i n the non-adherent population (Mosier et a l . , 1970). The non-adherent (lymphocyte) population has subsequently been separated i n t o two major c l a s s e s on the basis of ontogeny and c e l l surface antigens (1) thymus-dependent (T) lymphocytes which bear the Thy-1 antigen ( R e i f and A l l e n , 1966); and (2) thymus-independent, bone marrow-derived (B) lymphocytes which bear surface immunoglobulin as a receptor f o r antigen, and are the precursors of antibody-producing c e l l s (reviewed i n Warner, 1974). 2. T Regulator C e l l s The T c e l l p o pulation provides both the precursors and e f f e c t o r c e l l s f o r c e l l - m e d i a t e d immune responses and a l s o r e g u l a t o r T c e l l p o p ulations. These r e g u l a t o r T c e l l s include a m p l i f i e r (helper) T c e l l populations and suppressor T c e l l s which n e g a t i v e l y r e g u l a t e responses (Gershon, 1974; 4 1980; Benacerraf and Germain, 1979). The response to an a n t i g e n i c stimulus r e f l e c t s the balance between the e f f e c t s of the suppressive and enhancing r e g u l a t o r c e l l populations (Eardley and Sercarz, 1976). I n i t i a l l y , the importance of r e g u l a t o r c e l l s was demonstrated i n the antibody response but now a l a r g e body of evidence has accumulated to show that c e l l - m e d i a t e d responses are al s o r e g u l a t e d by suppressor and helper T c e l l s (Katz, 1977). These c e l l u l a r responses include g r a f t - v e r s u s - h o s t r e a c t i o n s (Cantor and Asofsky, 1972); the development of delayed-type h y p e r s e n s i t i v i t y (Askenase, 1980; M i l l e r e t a l . , 1981; Wright and Ramshaw, 1983); a l l o g r a f t r e j e c t i o n (Ascher et a l . , 1983); and the generation of c y t o t o x i c T c e l l s ( E i j s v o o g e l et a l . , 1973; Schendl et a l . , 1973; Wagner, 1973; Cantor and Boyse, 1975; A l t e r et a l . , 1976; Bach et a l . , 1976; P i l a r s k i , 1977). These r e a c t i o n s appear to be mediated by d i f f e r e n t T c e l l populations (Katz, 1977) but whether or not the r e g u l a t o r c e l l s i n v o l v e d i n these d i v e r s e i n t e r a c t i o n s are the same i s unclear. However, d i s t i n c t populations (both suppressor and helper) have been i m p l i c a t e d i n r e g u l a t i o n of the IgG and IgE response to the same antigen (Suemura et a l . , 1983). Furthermore, human humoral and c y t o t o x i c T c e l l responses appear to be regulated by d i s t i n c t T helper c e l l s (Friedman and Thompson, 1983) . 3. Regulation of Cy t o t o x i c Responses The r o l e of a m p l i f i e r and suppressor c e l l s i n c y t o t o x i c responses has now been stud i e d i n a v a r i e t y of systems. Wagner (1973) observed synergy between T c e l l s from d i f f e r e n t anatomical l o c a t i o n s i n the c y t o t o x i c 5 response to major h i s t o c o m p a t i b i l i t y antigens. Cantor and Boyse (1975(b)) showed that Lyt 1 + helper c e l l s augment the primary c y t o t o x i c T c e l l response to a l l o a n t i g e n s . P i l a r s k i (1977) then described the absolute requirement f o r T helper c e l l s i n the i n d u c t i o n of c y t o t o x i c i t y from thymocyte precursors, Many groups have now described a T-helper c e l l mechanism i n the i n d u c t i o n of c y t o t o x i c T c e l l responses to a l l o a n t i g e n s (Corley et a l . , 1980; Glasebrook and F i t c h , 1980; Wagner et a l . , 1980; Li v n a t and Corley, 1983); minor h i s t o c o m p a t i b i l i t y antigens (von Boehmer and Haas, 1979); haptens ( B u t l e r and B a t t i s t o , 1979; Cooley and Schmitt-Verhulst, 1979; Hamaoka et a l . , 1979); v i r u s e s (e.g. Zinkernagel et a l . , 1978); and tumours (Stutman e_t a l . , 1977 ; Hancock et a l . , 1981). Woodward et a l . (1979) and Ig a r a s h i et a l . (1979), reported the a m p l i f y i n g a c t i v i t y of macrophages and t h e i r products i n the c y t o t o x i c response to d i f f e r e n t syngeneic tumours. 4. Separation of Regulator T c e l l s from other T c e l l Populations The apparent r o l e of r e g u l a t o r T c e l l s i n the generation of c y t o t o x i c e f f e c t o r c e l l s i n d i c a t e s the need to develop ways i n which the balance between the r e g u l a t o r and c y t o t o x i c c e l l s can be manipulated. Regulator T c e l l s have been d i s t i n g u i s h e d not only on the basis of a c t i v i t y i n enhancing or suppressing responses but a l s o by a number of p h y s i c a l c r i t e r i a (Katz, 1977). (a) S e n s i t i v i t y to R a d i a t i o n . The s e l e c t i v e e f f e c t s of r a d i a t i o n on r e g u l a t o r y and e f f e c t o r T c e l l and B c e l l populations have been reviewed by Doria et a l . (1982). In 6 whole animals, r a d i a t i o n a f f e c t s the antibody response more profoundly than c e l l - m e d i a t e d responses such as delayed-type h y p e r s e n s i t i v i t y . The i n d u c t i v e phase of the humoral response i s more s e n s i t i v e than the e f f e c t o r phase. I r r a d i a t i o n a f t e r antigen-presentation may a c t u a l l y cause enhanced antibody production. G e n e r a l l y , the f u n c t i o n a l r o l e of antigen-presenting c e l l s such as macrophages and d e n d r i t i c c e l l s i s unaffected by X - i r r a d i a t i o n (Roseman, 1969). The r e l a t i v e r e s i s t a n c e of T helper c e l l s to r a d i a t i o n was f i r s t e s t a b l i s h e d by Kettman and Dutton (1971). These T c e l l s must be a c t i v a t e d by antigen p r i o r to i r r a d i a t i o n to show r a d i o - r e s i s t a n c e suggesting that the p r o l i f e r a t i v e phase during which helper clones are increased i s r a d i o - s e n s i t i v e but the helper e f f e c t o r c e l l i s r e s i s t a n t to i r r a d i a t i o n . The r a d i o s e n s i t i v i t y of suppressor T c e l l s has been thoroughly i n v e s t i g a t e d and they are i n general much more r a d i o - s e n s i t i v e than T helper c e l l s (Eardley and Sercarz, 1977; Dor i a et al., 1982). As w i t h helper c e l l s , a c t i v a t e d suppressor c e l l s are more r a d i o r e s i s t a n t than t h e i r precursors (Rich and Ri c h , 1975). The c e l l s i nvolved i n ce l l - m e d i a t e d immune r e a c t i o n s have a l s o been shown to be r a d i o - r e s i s t a n t . Delayed-type h y p e r s e n s i t i v i t y can be t r a n s f e r r e d w i t h i r r a d i a t e d c e l l s (Asherson and Loewi, 1967) and low doses of i r r a d i a t i o n ( l e s s than 500R) have l i t t l e e f f e c t on the development of delayed-type h y p e r s e n s i t i v i t y i n v i v o (Ettringham and Weissman, 1971). C y t o t o x i c e f f e c t o r c e l l s do not appear to be s e n s i t i v e to i r r a d i a t i o n (Deluca et a l . , 1975), although X - r a d i a t i o n i n h i b i t s the ca p a c i t y of u n s e n s i t i z e d c e l l s to develop i n t o c y t o t o x i c T lymphocytes (Sprent et a l . , 1974). 7 (b) S e n s i t i v i t y to Drugs. ( i ) C o r t i c o s t e r o i d s have a g e n e r a l l y suppressive e f f e c t on immune responses and cause d e p l e t i o n of c e l l s i n many organs p a r t i c u l a r l y i n the thymus. C o r t i s o n e - r e s i s t a n t thymic populations seem p a r t i c u l a r l y e f f e c t i v e i n generating g r a f t - v e r s u s - h o s t r e a c t i o n s (Cohen et a l . , 1970). B c e l l s are more s e n s i t i v e than most T c e l l s to c o r t i s o n e (Cohen and Claman, 1971). The r e s i s t a n c e to c o r t i s o n e depends on the s t a t e of a c t i v a t i o n of the T c e l l population (Segal et a l . , 1972). P e r i p h e r a l T c e l l s capable of helper f u n c t i o n are c o r t i s o n e - s e n s i t i v e p r i o r to antigen s e n s i t i z a t i o n and develop r e s i s t a n c e f o l l o w i n g immunization. C o r t i s o n e - s e n s i t i v i t y p a r a l l e l s the r e s i s t a n c e to r a d i a t i o n of primed T c e l l s and r a d i a t i o n - s e n s i t i v i t y of precursor c e l l s , discussed above. ( i i ) Cyclophosphamide i s a n o n - s p e c i f i c , DNA a l k y l a t i n g agent which acts upon a c t i v e l y r e p l i c a t i n g c e l l s , and has been widely used i n the chemotherapy of tumours (Gershwin §_t a l . , 1974; Shand, 1979). Cyclophosphamide has been shown to be immunosuppressive w i t h p r e f e r e n t i a l e f f e c t s upon the B c e l l compartment although T c e l l subpopulations are a l s o a f f e c t e d (Turk et a l . , 1972; Stockman et^ a l . , 1973; M i l t o n et a l . , 1976). Suppressor T c e l l s have been shown to be more s e n s i t i v e to cyclophosphamide than hel p e r s . This probably e x p l a i n s the enhancing e f f e c t s of low doses of cyclophosphamide on c e l l u l a r immune responses ( M i l t o n et a l . , 1976; R o l l i n g h o f f et a l . , 1977). Higher doses of cyclophosphamide w i l l g e n e r a l l y suppress the same responses ( M i l t o n e_t a l . , 1976). Several groups have shown diminished c y t o t o x i c responses by animals t r e a t e d w i t h high doses of cyclophosphamide (Taswell et a l . , 1979; 8 Hancock and K i l b u r n , 1982; V a r k i l a and Hurme, 1983). The depleted responses could be r e s t o r e d by the a d d i t i o n of i n t e r l e u k i n 2 or normal thymocytes: more s p e c i f i c a l l y by Lyt 1 + , 2 T helper c e l l s ( M e r l u z z i et a l . , 1980; 1981 ( a ) ) . V a r k i l a and Hurme (1983) could r e s t o r e the response i n v i t r o w i t h a n t i g e n - s p e c i f i c helper c e l l s . These r e s u l t s suggest that cyclophosphamide may a f f e c t helper c e l l populations i n v o l v e d i n the c y t o t o x i c response more than the c y t o t o x i c precursor c e l l s , (c) C e l l Surface Markers ( i ) I -region coded antigens ( l a ) have been used to d i s t i n g u i s h f u n c t i o n a l subpopulations of T lymphocytes (reviewed i n McDevitt et a l . . 1976). There i s s t i l l controversy whether T helper c e l l s express l a , but suppressor T c e l l s have g e n e r a l l y been found to be l a ( I - J ) p o s i t i v e (McDevitt et a l . , 1977; Thomas et a l . , 1977). Vadas et a l . (1976) found that helper T c e l l s i n the antibody response are l a - p o s i t i v e a f t e r priming but l a - n e g a t i v e before s e n s i t i z a t i o n . P l a t e (1976) showed that helper c e l l s i n v o l v e d i n the generation of c y t o t o x i c T c e l l s are a l s o l a p o s i t i v e . Tada e_t a l . (1978) have reported two d i s t i n c t helper c e l l populations i n the secondary antibody response, one i s l a - p o s i t i v e , the other negative. ( i i ) Helper c e l l s have a l s o been separated from other T c e l l f u n c t i o n a l subclasses on the b a s i s of t h e i r Lyt phenotype. The Lyt d i f f e r e n t i a t i o n antigens were f i r s t d e scribed as markers of d i s t i n c t populations i n ijn v i t r o c e l l - m e d i a t e d c y t o t o x i c responses (Cantor and Boyse, 1975; Shiku e t a l . , 1975). In the appropriate s t r a i n s of mice, helper c e l l s are L y t - l + 2 , 3 whereas c y t o t o x i c c e l l s are Lyt-1 2,3 + 9 or i n some cases L y t - l + 2 , 3 + . Suppressor T c e l l s are al s o L yt-1~2,3 + (Maier e t a l . , 1980). C e l l s mediating delayed-type h y p e r s e n s i t i v i t y are L y t - 1 + and those i n v o l v e d i n generating 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 v i t r o are from the L y t - 1 + and L y t - 2 , 3 + populations ( K i s i e l o w et a l . , 1975). Recent f i n d i n g s i n d i c a t e that some of these d i f f e r e n c e s are q u a n t i t a t i v e r a t h e r than q u a l i t a t i v e (Beverley et a l . , 1976; Nakayama et a l . , 1979; Ledbetter et a l . , 1980) Thomas and Calderon (1982) have shown that T helper c e l l s can change t h e i r Lyt-1,2 phenotype during the course of the immune response. Swain (1980) has proposed that the Lyt phenotype of c e l l s r e f l e c t s t h e i r antigen s p e c i f i c i t y and t h e i r f u n c t i o n a l r e s t r i c t i o n by products of the major h i s t o c o m p a t i b i l i t y complex ra t h e r than being a f u n c t i o n a l c l a s s i f i c a t i o n . I t i s now h e l d t h a t most, i f not a l l T c e l l s express Lyt-1 to some extent (Teh and Teh, 1980) although not a l l c e l l s express the Lyt-2 antigen ( C e r o t t i n i , 1980). 5. Adherent C e l l s and H-2 R e s t r i c t i o n The r o l e of the adherent c e l l appears to be processing and pre s e n t a t i o n of antigen to a n t i g e n - r e a c t i v e lymphocytes (Unanue and C e r o t t i n i , 1970; Lee et a l . , 1979) and a l s o r e g u l a t i o n of lymphocyte responses ( p o s i t i v e and negative) (Erb and Feldmann, 1975; Hetzger et a l . , 1980). The importance of the adherent c e l l p opulation (macrophage-like or d e n d r i t i c c e l l s ) has been reviewed by Unanue (1981). T helper c e l l s have been shown to recognize macrophage-bound antigen i n a s s o c i a t i o n w i t h I-region coded determinants (the c l a s s I I , l a antigens of the major 10 h i s t o c o m p a t i b i l i t y complex) on the macrophage (Erb and Feldmann, 1975; P i e r c e et a l . , 1976; Rosenthal et a l . , 1978). The process of a c t i v a t i o n of T c e l l s by antigen-presenting accessory c e l l s i s dependent on the presence of l a antigens on the accessory c e l l , and may i n v o l v e macrophage-secreted f a c t o r s (Gery and Waksman, 1972; Schrader, 1973). This i s the b a s i s of H-2 r e s t r i c t i o n of T c e l l - B c e l l and T c e l l - T c e l l i n t e r a c t i o n s where the i n t e r a c t i o n between T c e l l and antigen presenting c e l l i s r e s t r i c t e d by the I-region coded determinants of the responding animal ( P i e r c e et a l . , 1976; McDougal and Cort, 1978; Erb et a l . , 1979). E f f e c t o r T c e l l s (such as c y t o t o x i c c e l l s ) appear to be r e s t r i c t e d to recognize antigen i n a s s o c i a t i o n w i t h the c l a s s I molecules of the major h i s t o c o m p a t i b i l i t y complex ( i . e . H-2K/D products) (Bach et a l . , 1976; Zinkernagel and Doherty, 1979). As w i t h antibody responses, helper c e l l s i n c e l l u l a r responses r e q u i r e i n t e r a c t i o n s w i t h macrophages or t h e i r products, and the i n t e r a c t i o n w i t h the macrophage i s r e s t r i c t e d by I-region products on the macrophage (Wagner et a l . , 1972). 6. The Mode of A c t i o n of Regulator C e l l s Some understanding of the nature of the i n t e r a c t i o n between T helper c e l l s and B c e l l s came from studies w i t h haptenated-protein antigens. Ovary and Benacerraf (1963) had shown that animals primed w i t h d i n i t r o p h e n y l a t e d - b o v i n e serum albumin (DNP-BSA) show a secondary response ( i . e . greater response, sooner a f t e r i n j e c t i o n ) to the DNP-hapten when challenged w i t h DNP-BSA but not w i t h DNP-ovalbumin (OVA). Independent studies by Rajewsky et a l . (1969); Raff (1970) and M i t c h i s o n (1971 (a) 11 (b)) showed that T c e l l s recognize c a r r i e r - p r o t e i n determinants ( i . e . OVA or 8SA above) and amplify the response of B c e l l s r e c o g n i z i n g haptenic-determinants ( i . e . DNP above). The c a r r i e r and hapten determinants must be p h y s i c a l l y l i n k e d . This concept of p h y s i c a l l i n k a g e b r i n g i n g about a s s o c i a t i v e r e c o g n i t i o n of B c e l l - d i r e c t e d (or more g e n e r a l l y e f f e c t o r - d i r e c t e d ) a n t i g e n i c determinants and T h e l p e r - d i r e c t e d determinants appears to be a general phenomenon, although under some s p e c i a l i z e d circumstances, i t has been shown that p h y s i c a l l i n k a g e i s not e s s e n t i a l ( S u l i c a et a l . , 1971; Hamaoka et a l . , 1973). T helper c e l l s are not e s s e n t i a l f o r the antibody response to a small group of antigens known as T-independent antigens, however, they may s t i l l i n f l u e n c e these a n t i g e n - a c t i v a t e d B c e l l s (Katz, 1977; Mond et a l . , 1980). T helper c e l l s enhance not only the magnitude of the antibody response but a l s o the c l a s s of the antibodies secreted (Herzenberg et^ a l . , 1976; Rosenberg and C h i l l e r , 1979); the a f f i n i t y of the antibodies produced (Gershon and P a u l , 1971); and memory r e c a l l upon r e s t i m u l a t i o n (Okumura et a l . , 1976). I d i o t y p e - s p e c i f i c , i s o t y p e - s p e c i f i c and a l l o t y p e - s p e c i f i c T c e l l populations have been described (Bottomly et^ a l . , 1979; Bona and P a u l , 1979; Herzenberg et a l . , 1979; Janeway et a l . , 1977). Immunoglobulin-specific helper T c e l l s s e l e c t i v e l y i n t e r a c t w i t h a n t i g e n - a c t i v a t e d B c e l l s bearing the appropriate immunoglobulin molecules and enhance the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of those c e l l s thus r e g u l a t i n g the response w i t h respect to a l l o t y p e , c l a s s or i d i o t y p e . Herzenberg et a l . (1980) have proposed a model to e x p l a i n the a f f i n i t y maturation of the antibody response i n which helper T c e l l s s e l e c t B c e l l s 12 w i t h high a f f i n i t y - r e c e p t o r s and suppressor T c e l l s i n h i b i t the a c t i v a t i o n of B c e l l s bearing low a f f i n i t y r e c e p t o r s . A v a r i e t y of evidence, from a number of l a b o r a t o r i e s , now e x i s t s f o r there being d i f f e r e n t T c e l l subpopulations i n v o l v e d i n both the i n d u c t i o n and expression of helper T c e l l a c t i v i t y . These subpopulations have been i d e n t i f i e d by d i f f e r e n t c r i t e r i a i n c l u d i n g (1) i d i o t y p i c or a n t i - i d i o t y p i c determinants (Woodland and Cantor, 1978; A d o r i n i et a l . , 1979; Bottomly and Mosier, 1979); (2) H-2 r e s t r i c t i o n (Bottomly and Mosier, 1981); (3) nylon-wool adherence (Tada e t a l . , 1978; Agarossi et a l . , 1981); (4) r a d i o - s e n s i t i v i t y (Tada et a l . , 1978; Agarossi et a l . , 1981) or g l u c o - c o r t i c o s t e r o i d s e n s i t i v i t y (Bradley and M i s h e l l , 1982); (5) Lyt and l a phenotype (Feldmann et a l . , 1977; Tada et a l . , 1978; Janeway et a l . , 1980); and (6) mode of a c t i o n w i t h B c e l l s ( l i n k e d or u n - l i n k e d r e c o g n i t i o n ) (Tada et a l . , 1978; A d o r i n i e_t a l . , 1983). These r e s u l t s a l l support the idea of B c e l l a c t i v a t i o n by d i s t i n c t helper T c e l l subpopulations which mediate d i f f e r e n t forms of T c e l l - B c e l l cooperation. One subpopulation (T helper-1) recognizes c a r r i e r -determinants of the priming antigen i n a s s o c i a t i o n w i t h H-2 determinants (I region products). These c e l l s then i n t e r a c t w i t h hapten-reactive B c e l l s i n a process i n v o l v i n g h a p t e n - c a r r i e r l i n k a g e , and most c l o s e l y resemble the c e l l s described by M i t c h i s o n (1971 (b)) and others. The second c l a s s of c a r r i e r - p r i m e d helper c e l l s (T helper-2) recognize i d i o t y p i c or other immunoglobulin determinants on the B c e l l . These c e l l s are not H-2 r e s t r i c t e d and do not r e q u i r e l i n k a g e of c a r r i e r and hapten determinants. Both helper T c e l l types are a c t i v a t e d by, and bind to 13 c a r r i e r a n t i g e n i c determinants and, both appear to be necessary and act s y n e r g i s t i c a l l y f o r optimal B c e l l a c t i v a t i o n . Many of the i n t e r a c t i o n s between a n t i g e n - a c t i v a t e d T c e l l s (both suppressive and helper subpopulations) are mediated by s o l u b l e T c e l l products known as T c e l l f a c t o r s . These f a c t o r s act i n e i t h e r an a n t i g e n - s p e c i f i c fashion (reviewed by Tada and Okumura, 1979; K i l b u r n and Levy, 1980; Taussig, 1980); or i n a n o n - s p e c i f i c manner (Schimpl et a l . , 1980; Swain et a l . , 1982). The a n t i g e n - s p e c i f i c f a c t o r s produced by antigen-stimulated T c e l l s presumably bind and focus antigen f o r prese n t a t i o n to B c e l l s or precursor e f f e c t o r T c e l l s ( c y t o t o x i c T c e l l s and T c e l l s mediating delayed-type h y p e r s e n s i t i v i t y ) , e i t h e r on the T c e l l surface or on macrophage-like antigen-presenting c e l l s . Many s p e c i f i c f a c t o r s w i l l f u n c t i o n when T c e l l s are l i m i t i n g although macrophages are re q u i r e d (Kindred and Corley, 1977) and those which depend on T c e l l s to f u n c t i o n may r e c r u i t helper T c e l l s (Tada and Okumura, 1979) . Non-sp e c i f i c f a c t o r s a l s o produced by a n t i g e n - a c t i v a t e d T c e l l s , are invo l v e d i n the p r o l i f e r a t i o n and maturation of other a n t i g e n - a c t i v a t e d T c e l l s and B c e l l s (Swain et a l . , 1982). K e l l e r et a l . (1980) have proposed that the antigen s p e c i f i c f a c t o r s are secreted by the T helper-1 c e l l population and n o n - s p e c i f i c f a c t o r s by a second helper c e l l p opulation (see a l s o Taussig, 1980; Fathman and F i t c h , 1982). Suppressor c e l l s have al s o been d i v i d e d i n t o d i s t i n c t subpopulations and a c i r c u i t of suppressor c e l l a c t i v i t i e s proposed (Cantor and Gershon, 1979). The L y t - 1 + helper T c e l l which induces feedback i n h i b i t i o n (Cantor ejt a l . , 1978) i s at l e a s t one of the points of i n t e r a c t i o n between 14 the helper and suppressor c e l l c i r c u i t s . Helper c e l l s are the most probable t a r g e t s of suppressor T c e l l s (Herzenberg et a l . , 1976; Hamaoka et a l . , 1977; Cantor and Gershon, 1979). Soluble T c e l l and macrophage products (both s p e c i f i c and n o n - s p e c i f i c ) have been shown to play an important r o l e i n the c y t o t o x i c response ( P l a t e , 1976; G i l l i s and Smith, 1977; Wagner and R o l l i n g h o f f , 1978; F a r r a r et a l , , 1980 ( b ) ; 1982; K i l b u r n and Levy, 1980; Wagner et a l . , 1980. Here again a number of d i f f e r e n t r e g u l a t o r c e l l s have been i m p l i c a t e d . 7. Models of C y t o t o x i c T C e l l A c t i v a t i o n (a) The Two-Signal Model The f i r s t models f o r the i n v i t r o a c t i v a t i o n of c y t o t o x i c T c e l l s were based on the Bretscher and Cohn (1969, 1970) model f o r B c e l l a c t i v a t i o n . Their model.proposed that the i n d u c t i o n of the response re q u i r e s the r e c o g n i t i o n of two a n t i g e n i c determinants, one by "receptor antibody" on a n t i g e n - s e n s i t i v e c e l l s (B lymphocytes) and the other by " c a r r i e r antibody" (now recognized to be the helper T c e l l r e c o g n i t i o n u n i t ) . From t h i s a two-signal model of c y t o t o x i c c e l l a c t i v a t i o n was developed (Wagner, 1973; Cantor and Boyse, 1975; Bach e_t a l . , 1976; M o l l e r , 1977; 1980; 1982; Symington and Teh, 1980; Teh and Teh, 1980; Wagner et a l . , 1980) (Figure 1). The f i r s t s i g n a l (the d i f f e r e n t i a t i v e s i g n a l ) involves the i n t e r a c t i o n of a c y t o t o x i c T c e l l precursor (CTLp) wi t h a s p e c i f i c antigen. In responses to f o r e i g n major h i s t o c o m p a t i b i l i t y antigens ( a l l o a n t i g e n s ) the c y t o t o x i c T c e l l s p r i m a r i l y recognize the 15 Figure 1: The 2 - s i g n a l model of c y t o t o x i c T c e l l a c t i v a t i o n . ( M o d i f i e d from Wagner et a l . , 1980). The f i r s t s i g n a l (1) involves the i n t e r a c t i o n of c y t o t o x i c T c e l l precursors (CTLp) w i t h a s p e c i f i c antigen (Ag) on the s t i m u l a t o r c e l l s . The precursor c e l l s then d i f f e r e n t i a t e i n t o "poised" c y t o t o x i c precursors (CTLpoi). The second s i g n a l (2) comes from helper T lymphocytes (Th). These helper c e l l s secrete a no n - a n t i g e n - s p e c i f i c helper f a c t o r i n t e r l e u k i n 2 (IL2) i n response to two s t i m u l i - the i n t e r a c t i o n w i t h antigen (Ag) on the s t i m u l a t o r c e l l s and the antigen-induced n o n - s p e c i f i c macrophage (M<t>) product i n t e r l e u k i n 1 ( I L 1 ) . I n t e r l e u k i n 2 ( S i g n a l 2) st i m u l a t e s p r o l i f e r a t i o n of the "poised" precursor and the development of e f f e c t o r c y t o t o x i c T lymphocytes (CTLe). 17 c l a s s I products (the H-2K/D antigens) (Bach et a l . , 1976). When sti m u l a t e d by non-alloantigens such as those on haptenated c e l l s , v i r u s e s or tumour c e l l s , the response of the c y t o t o x i c precursor c e l l i s r e s t r i c t e d by the c l a s s I products of the major h i s t o c o m p a t i b i l i t y complex. That i s , the c y t o t o x i c precursor c e l l s recognize the f o r e i g n a n t i g e n i c determinants i n a s s o c i a t i o n w i t h " s e l f " c l a s s I antigens (Zinkernagel and Doherty, 1979) S i g n a l 2 (the p r o l i f e r a t i v e s i g n a l ) to the a n t i g e n - a c t i v a t e d "poised" precursor c e l l (CTLpoi) comes from helper T lymphocytes (Th). The Lyt l + 2 helper T c e l l s recognize c l a s s I I products of the major h i s t o c o m p a t i b i l i t y complex ( l a antigens) on the a l l o g e n e i c s t i m u l a t o r c e l l (Bach et a l . , 1976; B e l l g r a u , 1983). The response of helper T c e l l s to non-alloantigens such as v i r u s antigens i s r e s t r i c t e d by the c l a s s I I molecules of the responder c e l l haplotype ( S n e l l , 1978; Unanue, 1981). S i g n a l 2 can be provided by an a c t i v a t e d s t i m u l a t o r c e l l p o pulation or the so l u b l e product of antigen or mitogen-stimulated Lyt l + 2 helper T c e l l s ( P l a t e , 1976; M o l l e r , 1980; 1982). This s o l u b l e product i s the non - a n t i g e n - s p e c i f i c lymphokine known as i n t e r l e u k i n 2 (Aarden e_t a l . , 1979) which a l s o supports the long-term growth of a n t i g e n - s p e c i f i c T helper T c e l l s (Watson, 1979), and sti m u l a t e s thymocyte p r o l i f e r a t i o n i n the presence of mitogen or antigen (Chen and d i Sabato, 1976; Paetkau et a l . . 1976; F a r r a r et a l . , 1978). The a n t i g e n - s p e c i f i c Lyt l + 2 helper T c e l l s , shown to be re q u i r e d f o r the a c t i v a t i o n of c y t o t o x i c precursors ( P i l a r s k i , 1977; Von Boehmer and Haas, 1979), produce i n t e r l e u k i n 2 i n response to s p e c i f i c antigen (Wagner and R b l l i n g h o f f , 1978; F a r r a r et a l . , 18 1978; Okada et a l . , 1979). The s p e c i f i c i t y of the response i s maintained by l i m i t a t i o n of the i n t e r a c t i o n of i n t e r l e u k i n 2 to c e l l s bearing the appropriate receptor (Smith, et a l . , 1980), which i s expressed on c y t o t o x i c precursors and other predominantly Lyt 1 2,3 + T c e l l s only a f t e r antigen or mitogen s t i m u l a t i o n (Lalande e t a l . , 1980; Robb et a l . , 1981; G u l l b e r g and Larsson, 1983). Macrophages or the s o l u b l e product known as i n t e r l e u k i n 1 ( M i z e l . , 1979; F a r r a r et a l . , 1980; Symington and Teh, 1980) are e s s e n t i a l f o r the generation of c y t o t o x i c e f f e c t o r c e l l s (Shaw et a l . , 1980; Wagner et a l . , 1980). Although i n t e r l e u k i n 1 does not promote the p r o l i f e r a t i o n of a n t i g e n - a c t i v a t e d c e l l s d i r e c t l y , i t i n f l u e n c e s the production of i n t e r l e u k i n 2 by Lyt 1 + T c e l l s (Larsson et a l . , 1980; Smith et a l . , 1980); and a l s o the expression of i n t e r l e u k i n 2 responsiveness ( G u l l b e r g and Larsson, 1983; Hiinig et a l . , 1983). Macrophage-like c e l l s may a l s o be important i n antigen-processing and p r e s e n t a t i o n to the helper T c e l l s or c y t o t o x i c precursor c e l l since helper c e l l s have been shown to recognize antigen on the surface of antigen-presenting c e l l s (Thomas et a l . , 1979; Schwartz et a l . , 1977; Woodward et a l . , 1979; Weinberger et a l . , 1980; B e l l g r a u , 1983). I t i s known that a v i a b l e s t i m u l a t o r c e l l i s re q u i r e d to induce a primary response whereas the secondary c y t o t o x i c response can be t r i g g e r e d by i n a c t i v a t e d c e l l s or s o l u b l e antigens i f i n t e r l e u k i n 2 i s present (Bach et a l . , 1976; P l a t e , 1976; Talmage et a l . , 1977; Wagner and R o l l i n g h o f f , 1978; F a r r a r et a l . , 1981). I t has been proposed, that m e t a b o l i c a l l y -i n a c t i v e s t i m u l a t o r c e l l s ( i . e . UV-treated or glu t a r a l d e h y d e - f i x e d ) f a i l 19 to a c t i v a t e i n t e r l e u k i n 2-producing helper T c e l l s (Schendel and Bach, 1975; Talmage et a l . , 1977). These treatments may block the p r e s e n t a t i o n of antigen by accessory c e l l s or abrogate cytokine ( i . e . lymphocyte and macrophage s o l u b l e products) production ( L e t v i n et a l . , 1980). Droge et a l . (1983) showed that m e t a b o l i c a l l y - a c t i v e s t i m u l a t o r c e l l s are not only r e q u i r e d to a c t i v a t e the L y t l + 2 i n t e r l e u k i n 2-producing c e l l but a l s o f o r s t i m u l a t i o n of the c y t o t o x i c c e l l precursor. (b) A P o s t u l a t e d Model of C y t o t o x i c C e l l A c t i v a t i o n I n v o l v i n g  A d d i t i o n a l C e l l s and F a c t o r s . These 2 - s i g n a l models of T c e l l a c t i v a t i o n described above, do not include the other more r e c e n t l y i m p l i c a t e d c e l l s and f a c t o r s such as a n t i g e n - s p e c i f i c f a c t o r s or f - i n t e r f e r o n . Some of these have been included i n an expanded 2 - s i g n a l model (modified from F a r r a r §_t a l . , 1982; Figure 2) and discussed below. For convenience, I have separated the a c t i v a t i o n process i n t o 5 stages which are proposed to represent the time course of t h i s process. Stage one of t h i s model represents antigen i n t e r a c t i o n w i t h at l e a s t four subclasses of T c e l l s , the Lyt 1 + helper c e l l c l a s s e s 1 (Thl) and 2 (Th2), Lyt 2 + c e l l s , and the c y t o t o x i c precursor c e l l s (CTLp). The helper c e l l s recognize antigen i n a s s o c i a t i o n w i t h I region products of the major h i s t o c o m p a t i b i l i t y antigen on the surface of macrophage-like antigen-presenting c e l l s (Erb and Feldmann, 1975; P i e r c e et a l . , 1976; Rosenthal et a l . , 1978). The i n c l u s i o n of 2 h y p o t h e t i c a l c l a s s e s of T helper c e l l s i n t h i s model i s based on the assumption that s i m i l a r helper c e l l mechanisms are i n v o l v e d i n both a n t i -body and c e l l - m e d i a t e d immunity. Keene and Forman (1982) and Schwartz et a l . 20 Figure 2: A proposed model of c y t o t o x i c T c e l l a c t i v a t i o n . Stage one (1) represents the i n t e r a c t i o n of antigens (Ag) on the s t i m u l a t o r c e l l s (Stim. C e l l ) w i t h at l e a s t 4 c l a s s e s of T c e l l s . L y t - 1 + T helper c e l l s ( T . l - see Table I) recognize antigens (Ag) presented by macrophage-like n accessory c e l l s (M<J>). They secrete a n t i g e n - s p e c i f i c helper f a c t o r (Hf) and n o n - s p e c i f i c f a c t o r s such as colony s t i m u l a t i n g f a c t o r (CSF) (stage 2). These f a c t o r s s t i m u l a t e the macrophage (M<t>) to produce the n o n - s p e c i f i c f a c t o r s i n t e r l u e k i n 1 (IL1) and activated-macrophage f a c t o r (AM((>F) (stage 3). L y t - 1 + T helper c e l l s (T h2 - see Table I) are s t i m u l a t e d by antigen, again presented by macrophages (stage 1) and the macrophage products IL1 and AM<|>F, and i n turn secrete a v a r i e t y of n o n - s p e c i f i c f a c t o r s - i n t e r l e u k i n 2 (IL2) and c y t o t o x i c i t y - i n d u c i n g d i f f e r e n t i a t i o n f a c t o r s ( e a r l y - , CTL-DFE, and l a t e - , CTL-DFL, a c t i n g ) (stage 4,5). The c y t o t o x i c precursor c e l l (CTLp) i s st i m u l a t e d by antigen (Ag) (stage 1) and d i f f e r e n t i a t e s to a "poised" precursor (CTLpoi). This process i s enhanced by the e a r l y - a c t i n g d i f f e r e n t i a t i o n f a c t o r (CTL-DFE) produced by the T.2 c e l l . I n t e r l e u k i n 2 (IL2) enhances the n p r o l i f e r a t i o n of the "poised" precursor (CTLpoi) and other a n t i g e n - a c t i v a t e d L y t - 2 + T c e l l s by i n t e r a c t i o n w i t h a s p e c i f i c IL2 receptor ( -C) expressed on- the c e l l surface a f t e r antigen s t i m u l a t i o n . Antigen-primed L y t - 1 + T c e l l s produce y - i n t e r f e r o n (y-IFN) (stage 5) when s t i m u l a t e d by i n t e r l e u k i n 2. Y-I n t e r f e r o n and the l a t e - a c t i n g d i f f e r e n t i a t i o n f a c t o r (CTL-DFL) promote the d i f f e r e n t i a t i o n of the i n t e r l e u k i n 2-stimulated p r o l i f e r a t i n g "poised" precursors i n t o e f f e c t o r c y t o t o x i c c e l l s (CTLe). 21 ,2 \ \ 1 JCSF / / / 3 22 (1982) have a l s o proposed a r o l e f o r these two d i s t i n c t helper c e l l populations i n c y t o t o x i c T c e l l a c t i v a t i o n . The p r o p e r t i e s of the helper T c e l l s , as d e l i n e a t e d mainly from s t u d i e s on B c e l l a c t i v a t i o n , are summarized i n Table I . The c y t o t o x i c precursor c e l l s (CTLp) and other Lyt 2 + c e l l s (these two populations may be i d e n t i c a l ) recognize antigen i n a s s o c i a t i o n w i t h the K/D antigens of the major h i s t o c o m p a t i b i l i t y complex (Bevan, 1975; Cantor and Boyse, 1975; Bach et a l . , 1976; Zinkernagel and Doherty, 1979; Nabholz and MacDonald, 1983). Subsequent to antigen s t i m u l a t i o n (Stage 1 ) , the T helper 1 (Thl) c e l l s produce colony s t i m u l a t i n g f a c t o r (CSF) ( F a r r a r et a l . , 1980 ( b ) ; Moore et a l . , 1981) and may produce an a n t i g e n - s p e c i f i c helper f a c t o r (Hf) (Stage 2 ) , both of which act upon the macrophage-like accessory c e l l s (M<|>) ( F a r r a r et a l . , 1980 ( c ) ) . These c e l l s i n turn secrete i n t e r l e u k i n 1 ( I L 1 ) , and other s o l u b l e n o n - s p e c i f i c f a c t o r s (designated as a c t i v a t e d macrophage factors-AM<t>F; Finke et a l . , 1981; Kawase et a l . , 1983) (Stage 3 ) . I n t e r l e u k i n 1 s t i m u l a t e s the a n t i g e n - a c t i v a t e d T helper 2 c e l l s (Th2) to secrete a v a r i e t y on n o n - s p e c i f i c helper f a c t o r s (Finke et a l . , 1981; P l a t e et a l . , 1982; Raulet and Bevan, 1982; Wagner et a l . , 1982; Mannel et a l . , 1983). These f a c t o r s include i n t e r l e u k i n 2 (IL2) (Raulet and Bevan, 1982) and an e a r l y a c t i n g c y t o t o x i c i t y - i n d u c i n g d i f f e r e n t i a t i o n f a c t o r (CTL-DFE; Reddehase et a l . , 1982; F a l k et a l . , 1983; Mannel et a l . , 1983) (Stage 4 ) . The "poised" c y t o t o x i c precursor c e l l s a r i s e from the c y t o t o x i c precursor population (CTLp) upon antigen s t i m u l a t i o n (Stage 1 ) , and the a n t i g e n - s p e c i f i c helper f a c t o r (Hf) produced by the T helper 1 23 Table I : The p r o p e r t i e s of T helper c e l l subclasses involved i n B c e l l a c t i v a t i o n PROPERTY HELPER CELL CLASS T h2 REFERENCES* Lyt phenotype Lyt-1+2,3" Lyt-1+2,3" A,B,G,H,K,L,0,R,U,Z l a phenotype Ia p o s i t i v e I a negative K,0 Nylon wool non-adherent adherent C,E,H,K,N,0,U,W adherence Radiation r e s i s t a n t s e n s i t i v e K,0 s e n s i t i v i t y s e n s i t i v e s e n s i t i v e A,B,G,R s e n s i t i v e s e n s i t i v e H,U Antigen c a r r i e r c a r r i e r H,U,Z recognition' 3 c a r r i e r c a r r i e r + Ig A,B,C,E,G,I,J,L,M,N P.R.S.V.X c a r r i e r n o n - s p e c i f i c D I n t e r a c t i o n w i t h l i n k e d - un-1inked K,M,N,0,V,Z hapten-primed B c e l l c r e c o g n i t i o n r e c o g n i t i o n Factors produced by antigen- n o n - s p e c i f i c C,E,J,N,P,S,T,W,Y an t i g e n - a c t i v a t e d s p e c i f i c helper T c e l l s aA. Janeway, 1975(a); B. Janeway, 1975(b); C. Marrack and Kappler, 1975; D. Herzenberg et a l . , 1976; E. Marrack and Kappler, 1976; F. Okumura et a l . , 1976; G. Janeway et a l . , 1977; H. Agarossi et a l . , 1978; H. Hetzelberger and Eichmann, 1978; J . Swierkosz et a l . , 1978; K. Tada e_t a l . , 1978; L. Woodland and Cantor, 1978; M. Bottomly and Mosier, 1978; N. Marrack et a l . , 1979; 0. Okumura et a l . , 1979; P. Swierkosz et a l . , 1979; Q. Eichmann et a l . , 1980; R. Janeway §_t a l . , 1980; S. K i l l e r et a l . , 1980; T. Taussig, 1980; U. Agarossi et a l . , 1981; V. Bottomly and Mosier, 1981; W. Kappler et a l . , 1981; X. Rosenberg and Asofsky, 1981; Y. Fathman and F i t c h , 1982; Z. A d o r i n i et a l . , 1983. ^Helper c e l l s recognize c a r r i e r a n t i g e n i c determinants. Some c e l l s recognize c a r r i e r a n t i g e n i c determinants and immunoglobulin determinants (Ig) i n c l u d i n g i d i o t y p i c determinants, on the surface of a n t i g e n - a c t i v a t e d B c e l l s . c H e l p e r c e l l s , primed to recognize c a r r i e r a n t i g e n i c determinants, amplify the response of hapten-primed B c e l l s . The hapten and c a r r i e r determinants can be p h y s i c a l l y l i n k e d or un-linked. 24 c e l l s (Thl) may a l s o be i n v o l v e d d i r e c t l y i n t h i s d i f f e r e n t i a t i v e process (Keene and Forman, 1982). I n t e r l e u k i n 2 s t i m u l a t e s not only the p r o l i f e r a t i o n of "poised" c y t o t o x i c precursor c e l l s (CTLp) but a l s o the f o u r t h species of a n t i g e n - a c t i v a t e d c e l l s , the Lyt 2 + T c e l l s , which secrete ^ - i n t e r f e r o n (y~IFN) (Stage 5). Under s p e c i f i c c o n d i t i o n s , Y - i n t e r f e r o n i s able to augment a l l o - s p e c i f i c c y t o t o x i c T c e l l responses i n v i t r o ( L i n d a h l et a l . , 1972; Simon et a l . , 1979; Hsu et a l . , 1981). I n t e r l e u k i n 2 enhances c y t o t o x i c T c e l l development and Y - i n t e r f e r o n production (by Lyt 2 + c e l l s ) w i t h s i m i l a r dose response curves (Morris et a l . , 1980; F a r r a r et a l . , 1981). Both of these events r e q u i r e an a n t i g e n - a c t i v a t i o n s i g n a l i n a d d i t i o n to i n t e r l e u k i n 2, which may cause p r o l i f e r a t i v e expansion of the c y t o t o x i c T c e l l precursors and Y-interferon-producing c e l l s , r e s p e c t i v e l y . I n t e r l e u k i n 2 i n t e r a c t s w i t h c e l l s bearing the i n t e r l e u k i n 2 receptor. This i s expressed on c y t o t o x i c "poised" precursor c e l l s and Lyt 2 + c e l l s a f t e r antigen s t i m u l a t i o n (Stage 1) (Lalande et a l . , 1980; Robb et a l . , 1981; G u l l b e r g and Larsson, 1983). Y-I n t e r f e r o n may be important i n the development of i n t e r l e u k i n 2 receptors s i n c e , p u l s i n g r e s t i n g T c e l l s (normally unresponsive to i n t e r l e u k i n 2) w i t h Y - i n t e r f e r o n r e s u l t e d i n t h e i r p r o l i f e r a t i o n i n response to subsequently added i n t e r l e u k i n 2 ( F a r r a r et a l . , 1981). The T helper 2 c e l l s (Th2) a l s o secrete a l a t e - a c t i n g d i f f e r e n t i a t i o n f a c t o r (CTL-DFL; Raulet and Bevan, 1982; Wagner et a l . , 1982; Mannel et a l . , 1983). CTL-DFL and y-IFN then act on the IL2-driven expanded clones of poised c y t o t o x i c T lymphocyte precursors and induce t h e i r d i f f e r e n t i a t i o n i n t o c y t o t o x i c e f f e c t o r T c e l l s (CTLe) (Stage 5). 25 8. Anti-Tumour Responses A number of groups have reported evidence of an jin v i v o immune response to a developing tumour although i n the m a j o r i t y of cases, the tumour grows d e s p i t e the immune response (Old, 1964; C e r o t t i n i and Brunner, 1974; H e l l s t r o m , 1974; Johnson et a l . , 1975; Fujimoto et a l . , 1976; Benjamini et a l . , 1977; M i t c h i s o n , 1977; North and K i r s t e i n , 1977; Stutman et a l . , 1977; I g a r a s h i e t a l . , 1979; Kedar et a l . , 1979; Schechter and Feldman, 1979; Shimizu and Shen, 1979; Woodward et a l . , 1979; Yamauchi et a l . , 1979; Boon et a l . , 1980; G l a s e r , 1980; M i l l s and Paetkau, 1980; Weiss, 1977; 1980). G e n e r a l l y , c e l l - m e d i a t e d responses, and i n p a r t i c u l a r , c y t o t o x i c responses are induced by tumours ( C e r o t t i n i and Brunner, 1974), although antibodies against the tumour are a l s o induced ( K l e i n and Sjogren, 1960; K l e i n , 1966; H e l l s t r o m and H e l l s t r o m , 1974). The l a c k of e f f e c t i v e n e s s of the immune response i n c o n t r o l l i n g tumour growth and m e t a s t a s i s , may r e f l e c t weak immunogenicity of tumours i n t h e i r syngeneic hosts (Lindenmann and K l e i n , 1967; M i t c h i s o n , 1970; Herbermann et a l . , 1976; Weiss, 1977; 1980), and/or the development of supressive a c t i v i t y (Fujimoto et a l . , 1976.; 1978; Hodes, 1976; M i l l e r , 1976; Takei et a l . , 1976; 1977; 1978; Greene et a l . , 1977; Perry et a l . , 1978; Hakim, 1979; Yefenof, 1980; Berendt and North, 1980; Dye and North, 1981; P l a t e r et a l . , 1981; T i l k i n et a l . , 1981; Howie and McBride, 1982). Rotter and T r a i n i n (1975) depleted mice of suppressor T c e l l s and saw an i n h i b i t i o n of tumour growth i n these mice which had both adequate T helper c e l l f u n c t i o n , and normal numbers of c e l l s i n v o l v e d i n a l l o g r a f t r e j e c t i o n . Tumour c e l l products have f r e q u e n t l y been shown to suppress the immune 26 response (Cheung, 1979; F a v i l a et a l . , 1978; Huchet and L h e r i t i e r , 1976; Green et^ a l . , 1977) as w i l l " b l o c k i n g a n t i b o d i e s " against the tumour ( K l e i n and Sjogren, 1960; Hellstrom and He l l s t r o m , 1969; 1974; Bansal et a l . , 1972; Baldwin, 1973). Several groups have reported the emergence of an t i g e n i c v a r i a n t s of tumours ( g e n e r a l l y seen as the l o s s of a n t i g e n i c determinants from the tumour) as the tumour progresses, against which the host i s unable to mount an immune response (Biddison and Palmer, 1977; Maryanski and Boon, 1982; Maryanski et a l . , 1982; 1983; Urban et a l . , 1982; Uyttenhove et a l . , 1983; Wortzel et a l . , 1983). Important goals of the studies of immunity to tumours have been the enhancement of the immune response to favour tumour r e j e c t i o n and the use of tumour-reactive c e l l s i n immunotherapy. Svet-Moldavsky and Hamburg (1967) o r i g i n a l l y suggested " h e t e r o g e n i z a t i o n " ( i . e chemical or v i r u s m o d i f i c a t i o n ) of tumours to make them more f o r e i g n to the host and increase the response against the tumour. M i t c h i s o n (1970) a l s o proposed the enhancement of anti-tumour responses by coupling exogenous a n t i g e n i c determinants to the tumour. He suggested that i n j e c t i o n of antigen-coupled tumour c e l l s i n t o a p p r o p r i a t e l y antigen-primed mice, should r e s u l t i n an enhanced response to the l i n k e d tumour antigens s i m i l a r to the c l a s s i c a l h a p t e n - c a r r i e r cooperative e f f e c t s seen i n the antibody response ( M i t c h i s o n , 1971 ( b ) ) . This approach has been u t i l i z e d i n a number of tumour systems and g e n e r a l l y the response to e x t r a a n t i g e n i c determinants has r e s u l t e d i n enhanced immunogenicity of the tumour and i n some cases r e j e c t i o n of the tumour (Czajkowski et a l . , 1966; Shohan et a l . , 1970; Martin et a l . , 1971; 27 Boone et a l . , 1974; G a l i l i et a l . , 1976; Hashimoto et a l . , 1976; Naor and G a l i l i , 1977; Lachmann and S i k o r a , 1978; Altman e_t a l . , 1979; Hamaoka ejt a l . . 1979; Takatsu et a l . , 1980). This was g e n e r a l l y assayed by i n j e c t i n g antigen-coupled, i n a c t i v a t e d tumour c e l l s i n t o antigen-primed mice and measuring the growth of subsequently i n j e c t e d uncoupled, l i v e tumour c e l l s . This concept termed he t e r o g e n i z a t i o n has been reviewed i n d e t a i l i n Kobayashi (1979). The major models of immunotherapy have in v o l v e d the t r a n s f e r of tumour-sensitized c e l l s and/or sera i n t o animals with p r o g r e s s i v e l y growing tumours (reviewed by Weiss, 1980; Fefer and G o l d s t e i n , 1982; M i h i c h , 1982). The r e s u l t s of such t r a n s f e r s vary from a c c e l e r a t e d r e j e c t i o n of the tumour to enhancement of i t s growth ( M i t c h i s o n , 1953; 1955; S n e l l et a l . , 1961; Winn, 1961; Hutchin e t a l , , 1967; F e f e r , 1969; 1974; I r v i n and Eustace, 1970; Borberg et a^. , 1972; Porteus and Munro, 1972; Treves and Cohen, 1973; Umiel and T r a i n i n , 1974; Rotter and T r a i n i n , 1975; Treves et a l . , 1975; Carlson and Ter r e s , 1976; 1978; Fefer et a l . , 1976 ( a ) , ( b ) ; 1979; Small and T r a i n i n , 1976; Burton and Warner, 1977; Cheever et a l . , 1977; 1978; 1980; 1981 ( a ) , ( b ) ; Fernandez-Cruz et a l . , 1979; M i l l s e t a l . , 1980; Greenberg et a l . , 1981). Several of these reports ( M i t c h i s o n , 1953; 1955; Winn, 1961; F e f e r , 1969; I r v i n and Eustace, 1970; Porteous and Munro, 1972; Rotter and T r a i n i n , 1975; Treves et a l . , 1975; Small and T r a i n i n , 1976; Burton and Warner, 1977) showed that c e l l s s e n s i t i z e d f o r a short p e r i o d of time i r i v i t r o or i n v i v o , w i l l cause enhanced clearance of that tumour by a r e c i p i e n t animal. C e l l s s e n s i t i z e d f o r longer periods of time g e n e r a l l y cause enhancement of the 28 tumour as measured by growth and m e t a s t a t i c a b i l i t y (Winn, 1961; I r v i n and Eustace, 1970; Porteous and Munro, 1972; Umiel and T r a i n i n , 1974; Treves et a l . , 1975; Small and T r a i n i n , 1976). The c e l l s r e s p o n s i b l e f o r both enhancement and i n h i b i t i o n of tumour growth, are g e n e r a l l y T lymphocytes and Treves and Cohen (1973) showed that the i n j e c t e d s e n s i t i z e d T c e l l s r e c r u i t an e f f e c t o r T c e l l population i n the r e c i p i e n t mice which mediates r e j e c t i o n of the tumour. T helper lymphocytes may a l s o be a c t i v a t e d which can amplify the antibody response to the tumour (Hutchin et a]L., 1967; Carlson and Terres, 1976; 1978; Greenberg et a l . , 1981). In a l l , these data suggest that c e l l s which a r i s e e a r l y i n the response to a tumour enhance the r e j e c t i o n of that tumour i n syngeneic animals. However, c e l l s a r i s i n g l a t e r i n tumour development (probably suppressor c e l l s ) enhance tumour growth probably by a c t i n g upon the e a r l y tumour-reactive c e l l s . This technique has been used to examine the i n  v i v o e f f e c t of s e l e c t e d c e l l populations such as c y t o t o x i c lymphocytes ( M i l l s et a l . , 1980) and c e l l l i n e s maintained i n long-term c u l t u r e (Fefer and G o l d s t e i n , 1982; M i h i c h , 1982; Fathman and F i t c h , 1982). The most s i g n i f i c a n t e f f e c t s of t h i s type of therapy are seen i n combination w i t h more conventional types of therapy such as surgery, i r r a d i a t i o n and chemotherapy ( F e f e r , 1974; Fefer et a l . , 1976; Berendt and North, 1980; Fernandez-Cruz et a l . , 1980; M i l l s et a l . , 1980; Greenberg et a l . , 1981). These other m o d a l i t i e s serve to reduce tumour l o a d and, i n the case of the l a t t e r two, a l s o to deplete suppressor c e l l s and t h e i r precursors from the tumour-bearing animal. 29 9. Regulation of the Cy t o t o x i c Response to a Syngeneic Tumour, P815 As discussed above, many groups have reported t r a n s i e n t or i n e f f e c t i v e responses to a p r o g r e s s i v e l y growing tumour. A model of r e g u l a t i o n of anti-tumour c y t o t o x i c T c e l l responses has been developed i n t h i s l a b o r a t o r y . Takei et a l . (1976) showed that i n DBA/2 mice i n j e c t e d w i t h 4 1 x 10 syngeneic mastocytoma P815 c e l l s , the tumour grew r a p i d l y f o r approximately 12 days and then regressed temporarily. Subsequently, at 16-18 days, i t once again grew r a p i d l y , metastasized and u l t i m a t e l y k i l l e d the animal between 25 and 35 days (Figure 3A) . Spleen c e l l s from tumour-bearing animals showed c y t o t o x i c a c t i v i t y against P815 c e l l s ( Figure 3B). They detected maximal c y t o t o x i c i t y i n the spleen between 12 and 18 days a f t e r tumour i n j e c t i o n , during the pe r i o d of tumour r e g r e s s i o n . The c y t o t o x i c a c t i v i t y of spleen c e l l s decreased as the tumour r e - i n i t i a t e d growth. This p a t t e r n c o r r e l a t e d with the appearance, i n the l a t e r phase of tumour growth, of a n t i g e n - s p e c i f i c suppressor c e l l s able to suppress the in_ v i t r o c y t o t o x i c response to P815 (Takei et a l . , 1976; 1977; 1978; Levy et a l . , 1979; Maier et a l . , 1980). K i l b u r n et a l . (1979; 1981) were able to i s o l a t e an a n t i g e n - s p e c i f i c helper f a c t o r from the spleens of animals e a r l y i n the course of tumour-growth. 10. The Objectives of This I n v e s t i g a t i o n The aim of the present study was to examine the r o l e of helper c e l l populations i n the primary c y t o t o x i c response to P815 both i_n v i v o and i n  v i t r o . This i n v o l v e d the c h a r a c t e r i z a t i o n of a m p l i f i e r c e l l s w i t h respect to organ l o c a l i z a t i o n , c e l l surface phenotype and d i s t i n c t i o n from 30 Figure 3: The pattern of tumour growth and appearance of c y t o t o x i c i t y i n DBA/2 mice i n j e c t e d w i t h P815 tumour c e l l s . Reprinted from Takei et a l , 1976. Groups of 6 mice were i n j e c t e d 4 subcutaneously w i t h 1 x 10 l i v e P815 tumour c e l l s . S o l i d tumours were detectable 8 days a f t e r tumour i n j e c t i o n . Tumours were e x c i s e d and weighed, and mean weight (± S.E.M.) p l o t t e d (A). Spleen c e l l s of those animals were c u l t u r e d f o r 18 hours with 5 1 C r labelled-P815 t a r g e t c e l l s at a r a t i o of 200 spleen c e l l s per tar g e t c e l l . Percent c y t o t o x i c i t y was c a l c u l a t e d from 5 1 C r rele a s e d and p l o t t e d as mean percent c y t o t o x i c i t y (± S.E.M.) of the 6 animals t e s t e d each day (B). 32 c y t o t o x i c and suppressor c e l l s , time of appearance and, mode of a c t i o n w i t h respect to c y t o t o x i c c e l l s (Results - Chapters I and I I ) . The i n  v i v o a c t i v i t y of these c e l l s was then examined with the aim of using them i n immunotherapy against the tumour (Results - Chapter I I I ) . F i n a l l y , h e t e r o g e n i z a t i o n (with t u b e r c u l i n PPD) of the tumour c e l l s to enhance immunogenicity and the c y t o t o x i c response against P81S, was a l s o i n v e s t i g a t e d as a model of immunotherapy and tumour r e j e c t i o n (Results -Chapter I V ) . 33 MATERIALS AND METHODS 1. Mice. Four to eigh t week o l d female mice of the l i s t e d s t r a i n s were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained i n our animal u n i t on a d i e t of Pu r i n a Mouse S t r a i n Rodent Chow and Abb r e v i a t i o n water ad l i b i t u m . H-2 Haplotype DBA/2J D2 d C57B1/6J B6 b CBA/J CBA k C57Bl/10.DBA/2/nSnJ B10.D2 d (congenic on H-2** background) 2. Tumours. a. Source. P815-X2, a s u b - l i n e of a methylcholanthrene-induced mastocytoma of DBA/2 mice (Dunn and P o t t e r , 1957) and L1210, a methylcholanthrene-induced acute s t e m - c e l l leukemia from DBA/2 mice (Law et a l . , 1949) were obtained from Dr. B. Smith, I n s t i t u t e f o r Cancer Research, P h i l a d e l p h i a , PA. EL4, a 9:10-dimethyl-l:2-benzanthracene-induced T - c e l l leukemia from C57B1 mice (Gorer, 1938) was obtained from Dr. H.-S. Teh, Department of M i c r o b i o l o g y , UBC, Vancouver, B.C. EL4-IL2, a v a r i a n t s u b - l i n e of EL4 ( F a r r a r et a l . , 1980; Shimizu et a l . , 1980) was obtained from Dr. V. Paetkau, Department of Biochemistry, U n i v e r s i t y of A l b e r t a , Edmonton, A l b e r t a . P21 and P911, 34 two N-methyl-N'-nitro-N-nitrosoguanadine (NTG)-induced v a r i a n t cloned l i n e s d erived from P815-X2 (Boon et a l . , 1980; Uyttenhove et a l . , 1980) were recei v e d from Dr. T. Boon, Ludwig I n s t i t u t e f o r Cancer Research, B r u s s e l s , Belgium. b. Cloning. Due to v a r i a b i l i t y with respect to growth c h a r a c t e r i s t i c s i_n v i v o and i j i v i t r o , P815 and EL4 were cloned by l i m i t i n g d i l u t i o n . Clones P815-2006 and EL4-5 were i s o l a t e d , grown up i n DBA/2 and C57B1/6 mice, r e s p e c t i v e l y , and used as stock tumour sources f o r a l l experiments a f t e r November, 1980. c. Maintenance. Tumours were maintained i n a s c i t e s form i n syngeneic animals, i . e . P815 and L1210 i n DBA/2 and EL4 i n C57B1/6 mice, r e s p e c t i v e l y . The c e l l s were passaged every 5-7 days by withdrawing a s c i t e s from the peritoneum, using a 21G. needle, washing with PBS and i n j e c t i o n of 1 x 10 7 c e l l s i n l m l p e r i t o n e a l l y using a 26G. needle. EL4-IL2 was maintained i n c u l t u r e i n complete medium plus 5% f e t a l c a l f serum. The P21 and P911 turn v a r i a n t s of P815 grew normally ijn v i t r o but were r e j e c t e d by normal syngeneic DBA/2 mice (Uyttenhove et a l . , 1980) so they were passaged i n s u b - l e t h a l l y i r r a d i a t e d mice (700 Rads gamma-radiation). A l l tumours were stored i n l i q u i d n i t r o g e n at 10 7 c e l l s per l m l v i a l i n 10% dimethyl s u l f o x i d e (DMSO, Sigma 5879, St. L o u i s , MO.) plus 90% c a l f serum, or i n complete medium plus 10% DMSO and 20% f e t a l c a l f serum. New v i a l s of P815 35 were thawed every 4-5 months, EL4 l e s s f r e q u e n t l y , d. I n j e c t i o n . Tumour c e l l s were f r e s h l y removed from mice as i n 2 c , and i n j e c t e d i n t o mice as f o l l o w s : ( i ) 0.2-10 x 10 i n 0.1ml PBS subcutaneously i n the r i g h t hind f l a n k w i t h a 26G. needle ( s c ) ; 4 ( i i ) 0.2-10 x 10 i n 50ul PBS subcutaneously i n each hind footpad wit h a 27G. needle ( f p ) ; ( i i i ) 0.2-20 x 1 0 4 i n 50yl PBS with 20ug demecolcine (N-desacetyl-N-methylcolchicine, Sigma D3785) subcutaneously at the base of the t a i l ( b t ) . Priming i n the footpad s t i m u l a t e s c e l l s i n the d r a i n i n g p o p l i t e a l and i n g u i n a l nodes (Taub et a l . , 1970), and at the base of the t a i l s t i m u l a t e s c e l l s i n the p e r i - a o r t i c nodes (Ruddle, 1974; Corradin et a l . , 1977). 3. Medium. a. RPMI 1640. Powdered RPMI 1640 (Flow Labs 10-601-24, Mississauga, O n t a r i o ) , a balanced s a l t and amino a c i d mixture c o n t a i n i n g L-glutamine, glucose, reduced-glutathione and phenol red (pH i n d i c a t o r ) but without bicarbonate, was d i s s o l v e d at 10.38g per l i t r e d i s t i l l e d water i n 4-8 l i t r e batches. 2.39g/l (lOmM) N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic a c i d (HEPES, Sigma H-3375) and 2 g / l (2.38mM) sodium bicarbonate ( F i s h e r S233, Vancouver, B.C.) were added, the mixture s t e r i l i z e d by f i l t r a t i o n through a 0.2um f i l t e r ( S c h l e i c h e r and S c h u e l l 1121-142mm, Keene, NH), and o stored i n the dark at 4 C i n 450ml a l i q u o t s f o r a maximum of three 36 weeks. Before use, medium was supplemented with ( i ) 5ml/500ml lOOx a n t i b i o t i c - a n t i m y c o t i c mixture (Grand I s l a n d B i o l o g i c a l Company (GIBCO) 600-5240, B u r l i n g t o n , Ontario) to give f i n a l concentrations of 100 Un i t s P e n i c i l l i n G/ml; 0.25ug Fungizone/ml (Amphotericin B); and lOOyg Streptomycin S u l f a t e / m l . ( i i ) 0.5ml/500ml of 0.05M 2-mercaptoethanol (2me, Sigma M6520) to give a f i n a l c oncentration of 5 x 10 - 5M. ( i i i ) 50ml/500ml f e t a l c a l f serum (10% FCS) (Flow Labs; GIBCO; Animal Health Labs, Toronto, Ontario; p r e t e s t e d batches g i v i n g low backgrounds but which supported the generation of s p e c i f i c c y t o t o x i c i t y ) . FCS was stored frozen at -20°C and heat i n a c t i v a t e d f o r 30 minutes at 56°C before use. This supplemented mixture was c a l l e d complete medium plus 10% FCS. b. Phosphate buf f e r e d s a l i n e (PBS). Phosphate b u f f e r e d s a l i n e pH 7.2 was made up i n d i s t i l l e d water with Phenol Red ( F i s h e r S c i e n t i f i c 755061) as a pH i n d i c a t o r and f i l t e r e d as f o r medium (3a). I t contained 8 g / l sodium c h l o r i d e (NaCl), 0.2g/l potassium c h l o r i d e (KC1), 0.2g/l potassium phosphate monobasic (KH 2P0 4) and 2.17g/l sodium phosphate d i b a s i c 7-hydrate (Na^O^.7H 20). 4. C e l l P r e p arations. a. S i n g l e c e l l suspensions. Spleens or thymuses were c a r e f u l l y d i s e c t e d out, a l l f a t and attached lymph nodes removed, minced and gently pressed through a 37 s t a i n l e s s s t e e l mesh (60-80 mesh) i n t o medium without serum. P e r i p h e r a l lymph nodes ( p o p l i t e a l , i n g u i n a l , p e r i a o r t i c , a x i l l a r ) were teased apart, using 18G. needles, i n t o medium without serum. Where appr o p r i a t e , c e l l s were spun through c a l f serum to remove de b r i s and f a t . A f t e r suspension i n medium w i t h or without serum, c e l l s were counted by the trypan e x c l u s i o n method ( D i r e c t Blue 14, 0.07% i n s a l i n e , Hatheson, Coleman and B e l l TX1580, Norwwod, Ohio) i n a Speers-Levy E o s i n o p h i l Counter (2/10mm deep x 1/16 square mm, CA Mausser and Son, P h i l a d e l p h i a , PA). b. Lympholyte M. Lympholyte M i s a d e n s i t y separation medium (d = 1.087-1.088) used f o r lymphocyte separation w i t h mouse c e l l s (Cedarlane Labs, CL5030, Hornby, O n t a r i o ) . In some experiments red blood c e l l s were l y s e d w i t h T r i s - b u f f e r e d ammonium c h l o r i d e [0.16M ammonium c h l o r i d e (NH^Cl, Sigma A4514) i n 0.17M tris-(hydroxymethyl)-aminomethane (TRIZMA base, Sigma T1503), pH 7.65] and the l y s e d c e l l s removed on lympholyte M. When b l a s t c e l l s were used as t a r g e t s f o r c y t o t o x i c i t y assays (5e), smaller lymphocytes were removed before l a b e l l i n g . I f c e l l suspensions contained many dead c e l l s these were removed on lympholyte M. The c e l l suspension at 2 x 10^c/ml i n complete medium was l a y e r e d over an equal volume of lympholyte M i n a p l a s t i c tube and c e n t r i f u g e d f o r 20 minutes at 500xg at 20°C. Lymphocytes or b l a s t c e l l s were c o l l e c t e d from the i n t e r f a c e between lympholyte M and l i g h t e r medium, dead c e l l s and red blood c e l l s formed a p e l l e t below the lympholyte M. 38 c. I r r a d i a t i o n . 60 C e l l s i n c o l d medium without serum were exposed to a Co-gamma-r a d i a t i o n source at a r a t e of 19-32 Rads/second (Gammacell 220, AECL, Ottawa) ( i n the Department of Chemistry, UBC). They were washed at l e a s t once before c u l t u r e or i n j e c t i o n . d. Treatment w i t h anti-Thy 1.2 serum plus complement. C e l l s were resuspended at 3 x 10 7/ml i n PBS co n t a i n i n g a 1/1000 d i l u t i o n of monoclonal anti-Thy 1.2 (a g i f t of Dr. P. Lake, Tumour Immunology U n i t , U n i v e r s i t y C o l l e g e , London) and 1/10 d i l u t i o n of r a b b i t complement (Cedarlane, Lo-tox-M, CL3051 preabsorbed with mouse lymphocytes). Co n t r o l s were resuspended i n complement alone. A f t e r i ncubation at 37°C f o r 1 hour with o c c a s i o n a l shaking, the c e l l s were washed 3 times w i t h PBS. Anti-Thy 1.2 plus complement k i l l e d 95% of DBA/2 thymocytes, complement alone k i l l e d <5%. e. Mitomycin C. Mitomycin C from Streptomyces caespitosus causes DNA c r o s s - l i n k i n g and i n h i b i t s c e l l d i v i s i o n , and was used to prevent tumour c e l l growth i n v i v o or ijn v i t r o when tumour c e l l s were used as s t i m u l a t o r c e l l s . F r e s h l y 6 prepared tumour c e l l s at 5 x 10 c/ml were t r e a t e d w i t h 50ug mitomycin C/ml (Sigma M0503) (stock s o l u t i o n of 0.5mg/ml i n medium without serum) f o r 1 hour at 37°C i n complete medium w i t h o c c a s i o n a l mixing. Treated c e l l s were washed 3 times with complete medium or PBS. 39 5. In v i t r o Generation and Assay of C y t o t o x i c C e l l s (Figure 4 ) . a. Generation of c y t o t o x i c i t y against syngeneic tumours. S p e c i f i c c y t o t o x i c i t y against the DBA/2 tumours P815 and L1210 was generated i r i v i t r o using m o d i f i c a t i o n s of the method reported by Takei §_t a l . (1977; K i l b u r n §_t a l . , 1979). I n i t i a l l y , t h e i r system was f o l l o w e d i n 6 which 5-10 x 10 spleen c e l l s pooled from normal or tumour-bearing DBA/2 mice were c u l t u r e d w i t h 5 x 10"* mitomycin C-treated tumour c e l l s i n t r i p l i c a t e 2.5ml c u l t u r e s i n f l a t - b o t t o m p l a t e s (LINBRO 76-033-05, Flow Labs) f o r 5 days at 37°C, 5% CO^. This was subsequently modified to 5 4 c u l t u r i n g 3 x 10 spleen c e l l s w i t h 1 x 10 tumour c e l l s i n 0.2ml i n 48 w e l l s of a 96-well V-bottom p l a t e (LINBRO, 76-023-05, Flow Labs) f o r 4 days at 37°C i n a 5% C 0 2 > 95% Rh incubator (Hancock et a l . , 1981). This m o d i f i c a t i o n s l i g h t l y reduced the number of c e l l s r e q u i r e d per assay and the l e v e l of c y t o t o x i c i t y generated a f t e r 5 days, but i t decreased the v a r i a b i l i t y between r e p l i c a t e c u l t u r e s , the major reason i t was used i n most of the experiments reported here. S i m i l a r l e v e l s of c y t o t o x i c a c t i v i t y were generated w i t h the two tumours. b. C y t o t o x i c i t y against C57B1/10 minor h i s t o c o m p a t i b i l i t y antigens. B10.D2 mice are i d e n t i c a l to DBA/2 mice at the major d h i s t o c o m p a t i b i l i t y complex ( i . e . H-2 haplotype) but d i f f e r i n many other l o c i and minor h i s t o c o m p a t i b i l i t y antigens d e r i v e d from C57B1/10 mice ( i . e . background genes). Very l i t t l e i j i v i t r o primary c y t o t o x i c a c t i v i t y was generated against B10.D2 c e l l s but high l e v e l s of secondary response could be generated (Bevan, 1975). C y t o t o x i c i t y was generated in. 40 Figure 4: The assay system f or generating cytotoxic c e l l s against d i f f e r e n t antigens. NORMAL DBA/2 NORMAL OR P815-INJECTED DBA/2. (1) IRRADIATED C57B1/6 SPLEEN CELLS (2) IRRADIATED B10.D2 SPLEEN CELLS (3) MITOMYCIN C-TREATED P815 (4) MITOMYCIN C-TRBATED L1210 SPLEEN CELLS RESPONDER CELLS THYMOCYTES £ HELPER CELLS JL STIMULATOR CELLS culture 4-5 days v : CYTOTOXIC EFFECTOR CELLS (varied numbers) INCUBATE 4-18 HOURS MEASURE 5 1 C r RELEASE 41 v i t r o using DBA/2 mice primed 3-4 weeks p r e v i o u s l y with 2-5 x 10° B10.D2 spleen c e l l s i n t r a p e r i t o n e a l l y . 3 x 10"* spleen c e l l s were c u l t u r e d w i t h 4 1 x 10 i r r a d i a t e d (2000 Rad) B10.D2 spleen c e l l s i n 0.2 ml V-bottom p l a t e s f o r 5 days at 37°C. c. C y t o t o x i c i t y against C57B1/6 major h i s t o c o m p a t i b i l i t y antigens. C57B1/6 mice d i f f e r from DBA/2 mice with respect to major and minor h i s t o c o m p a t i b i l i t y antigens and a strong primary in. v i t r o c y t o t o x i c response was generated i n 4 day c u l t u r e s . C y t o t o x i c i t y was generated by c u l t u r i n g 3 x 10^ normal DBA/2 spleen c e l l s w i t h 1-3 x 10^ i r r a d i a t e d (2000 Rad) C57B1/6 spleen c e l l s f o r 4 days at 37°C i n 0.2ml V-bottom p l a t e s . d. Helper assays (Figure 5). DBA/2 mice were i n j e c t e d w i t h P815 tumour c e l l s (see 2d) and thymocytes, spleen c e l l s or lymph node c e l l s used as helper c e l l s by mixing 1-3 x 10 5 w i t h 1-3 x 10 5 normal spleen responder c e l l s i n c u l t u r e s generating c y t o t o x i c i t y against the d i f f e r e n t s t i m u l a t o r s described i n 5a-c. Normal thymocytes, spleen c e l l s or lymph node c e l l s were added as c o n t r o l s as appropriate i n a l l experiments. e. C y t o t o x i c i t y assays. A f t e r 4-5 days of c u l t u r e , groups of 3 or 48 w e l l s were pooled, c e l l s counted and used as e f f e c t o r c e l l s i n c y t o t o x i c i t y assays w i t h 5 * C r - l a b e l l e d t a r g e t c e l l s (Brunner et a l . , 1968; C e r o t t i n i and 42 Figure 5: The assay system f o r regulator c e l l s i n the primary i n v i t r o cytotoxic response to P815. NORMAL DBA/2 SPLEEN CELLS 5* RESPONDER CELLS (CYTOTOXIC PRECURSOR CELLS) P815-INJECTBD (TUMOUR-BEARING OR IMMUNE MICE) SPLEEN; THYMUS; LYMPH NODE CELLS : it : REGULATOR CELLS (AMPLIFIER/HELPER CELLS; SUPPRESSOR CELLS) P815 TUMOUR CELLS /mitomycin C-treated STIMULATOR CELLS co-culture for 5 days EFFECTOR CELLS (CYTOTOXIC CELLS) varied numbers P815 TUMOUR CELLS la b e l with ^Cr-sodium chromate TARGET CELLS ( 5 1 C r LABELLED-P815) fixe d numbers INCUBATE FOR 18 HOURS MEASURE 5 1 C r RELEASE 43 Brunner, 1974). This assay was i d e n t i c a l whether e f f e c t o r s were generated i n 3 x 2.5ml or 48 x 0.2ml c u l t u r e s . P815 and L1210 were used as t a r g e t s i n assaying responses to syngeneic tumours, EL4 i n responses to B6 major h i s t o c o m p a t i b i l i t y antigens, and B10.D2 b l a s t s f o r responses to BIO minor h i s t o c o m p a t i b i l i t y antigens. B10.D2 b l a s t s were generated by incubating 1 x 10 7 B10.D2 spleen c e l l s / m l w i t h 2ug/ml concanavalin A ( i n 0.2M sodium acetate, 0.2H sodium c h l o r i d e b u f f e r , pH 5.2) f o r 2 days at 37°C, 5% C0 2. B l a s t c e l l s were separated on lympholyte M (4b). Target c e l l s were l a b e l l e d at 7 51 10 c/ml wi t h 0.25 - 2.5 mCi Cr-sodium chromate/ml (300-7OOmCi/mg) (New England Nuclear (NEN) NEZ-030S, Lachine, Quebec; Amersham CJS.4V, o O a k v i l l e , Ontario) i n complete medium f o r 60 - 90 minutes at 37 C wi t h o c c a s i o n a l mixing (Goodman, 1961). C e l l s were washed and P815 and L1210 o were f u r t h e r incubated f o r 1 hour at 37 C to reduce the spontaneous 4 rele a s e i n 18 hour assays. Target c e l l s were adjusted to 5-10 x 10 c/ml. The i_n v i t r o a c t i v a t e d e f f e c t o r c e l l s were resuspended to 5-10 x 10 6c/ml, 0.1ml dispensed i n t o round-bottom p l a t e s (LINBRO 76-013-05, Flow Labs or NUNCLON 163320, Gibco) i n t r i p l i c a t e and s e r i a l l y d i l u t e d i n 2 - f o l d d i l u t i o n s 4-8 times. 0.1ml of l a b e l l e d t a r g e t c e l l s 3 (5-10 x 10 c/well) were added, the c e l l s were sedimented gently (5 o minutes at 150-200xg) and the p l a t e s were incubated at 37 C, 5% CO^. A f t e r 4 hours (EL4 or B10.D2 b l a s t s ) or 18 hours (P815 or L1210), 0.1ml of the c e l l - f r e e supernatant was removed and counted i n a gamma counter ( P i c k e r S p e c t r o s c a l a r 4R; LKB 80,000 Wallac; Beckman Biogamma; or 44 Micromedic Systems I s o f l e x 28122). Percent s p e c i f i c c y t o t o x i c i t y was c a l c u l a t e d according to the formula: % S p e c i f i c C y t o t o x i c i t y = Test Release - Spontaneous Release x 100 Maximum Release - Spontaneous Release Spontaneous r e l e a s e was from target c e l l s c u l t u r e d i n 0.2 ml medium alone; maximum rel e a s e from c e l l s l y s e d w i t h 5% T r i t o n X-100 (Sigma T6878). The 18-hour incubation of e f f e c t o r c y t o t o x i c c e l l s and syngeneic tumour ta r g e t c e l l s was necessary to e f f e c t s i g n i f i c a n t l e v e l s of "^Cr r e l e a s e . This showed the weak c y t o t o x i c responses generated i n c u l t u r e against syngeneic tumours. Spontaneous release over 18-hours d i d not exceed 20-25%. Percent c y t o t o x i c i t y was p l o t t e d against the l o g e f f e c t o r c e l l to t a r g e t c e l l r a t i o (E:T r a t i o ) . Data was f i t t e d by l i n e a r r e g r e s s i o n to a s t r a i g h t l i n e . The c o r r e l a t i o n c o e f f i c i e n t was g e n e r a l l y greater than 0.92. L y t i c u n i t s were c a l c u l a t e d from t h i s l i n e w i t h 1 l y t i c u n i t 4 defined as the number of e f f e c t o r c e l l s to give 25% l y s i s of 1 x 10 t a r g e t c e l l s i n the 4-hour or 18-hour assays ( M i l l e r and Dunkley, 1974). Results were expressed as the l y t i c u n i t s per 10 6 c e l l s or the t o t a l l y t i c u n i t s per c u l t u r e c a l c u l a t e d from the number of e f f e c t o r c e l l s recovered a f t e r 4 or 5 day c u l t u r e s . Student's t - t e s t s t a t i s t i c s were used to compare data generated from these experiments. 6. L i m i t i n g D i l u t i o n C u l t u r e s . a. Cultures f o r e s t i m a t i n g c y t o t o x i c c e l l precursor frequency. A s i m i l a r technique to that of Teh et a l . (1977(a); 1981) was used. 0.2ml m i c r o c u l t u r e s i n V-bottom 96 w e l l p l a t e s c o n t a i n i n g s e r i a l 45 d i l u t i o n s of responder spleen c e l l s , a constant number of mitomycin C-treated P815 s t i m u l a t o r c e l l s and a constant d i l u t i o n of an i n t e r l e u k i n o 2 source (Methods 7a, c ) , were incubated f o r 5 days at 37 C, 5% CO^. 50-100ul c e l l - f r e e supernatant was removed and replaced w i t h 50-100ul 3 51 medium co n t a i n i n g 2 x 10 C r - l a b e l l e d P815 ta r g e t c e l l s . P l a t e s were c e n t r i f u g e d f o r 5 minutes at 120-200xg and incubated f o r 18 hours at 37°C. lOOul supernatant was removed and counted as i n 5e. Percentage l y s i s was c a l c u l a t e d and expressed as mean ± S.E.M. f o r 12-24 r e p l i c a t e w e l l s . b. S p l i t c u l t u r e s assayed on two t a r g e t s . C u l t u r e s were set up as i n 6a, but a f t e r 5 days, e f f e c t o r s were assayed on two d i f f e r e n t t a r g e t s (Teh et a l . , 1977(b)). Each 200ul w e l l 3 51 was s p l i t i n t o 2 x l 0 0 u l i n V-bottom p l a t e s , 2 x 10 C r - l a b e l l e d t a r g e t c e l l s ( e i t h e r P815 and DBA/2 concanavalin A-spleen c e l l b l a s t s or P815 and EL4) added i n lOOyl to these w e l l s and then t r e a t e d as i n 6a. Percent l y s i s of each w e l l against the two ta r g e t s was c a l c u l a t e d and p l o t t e d on one f i g u r e . I f the c u l t u r e s were s p l i t e q u a l l y and then the h a l f - w e l l s assayed on the same t a r g e t , the points ( l y s i s of i n d i v i d u a l w e l l s ) should be s c a t t e r e d about a s t r a i g h t l i n e from the o r i g i n and diagonal to the x and y axes i . e . the h a l f w e l l s should l y s e the t a r g e t s e q u a l l y . c. C a l c u l a t i o n of precursor frequency. Test w e l l s were scored as p o s i t i v e i f the counts re l e a s e d exceeded the mean spontaneous re l e a s e by 2.2 standard d e v i a t i o n s 46 (p<0.05). This value u s u a l l y represented s p e c i f i c l y s i s of >4%. Cy t o t o x i c c e l l precursor frequencies were c a l c u l a t e d using a computer program set up by T. Buican, (Department of P h y s i c s , UBC) based on the maximum l i k e l i h o o d method of Po r t e r and Berry (1963). P l o t t i n g the negative In of the f r a c t i o n of negative c u l t u r e s (Fo) (-InFo) against the number of responding c e l l s r e s u l t e d i n a s t r a i g h t l i n e (zero order term of the Poisson d i s t r i b u t i o n ) . The frequency was c a l c u l a t e d from t h i s l i n e as the r e c i p r o c a l of the number of responding c e l l s per w e l l when the f r a c t i o n of negative c u l t u r e s (Fo) was 0.37. The range represents the 95% 2 confidence l i m i t s and x approximates to the consistency of f i t of the data to the l i n e . Both the method and c a l c u l a t i o n s are discussed i n d e t a i l i n L e f k o v i t s and Waldmann (1979). 7. Pre p a r a t i o n and Assay of I n t e r l e u k i n 2 a. Bulk p r e p a r a t i o n from r a t and mouse spleen c e l l s . I n t e r l e u k i n 2 (Aarden et a l . , 1979) was prepared from spleen c e l l s as f o l l o w s : Spleen c e l l s Number Concanavalin A Supernatant Reference C o l l e c t e d  mouse 6 x l 0 6 / m l l-2ug/ml 18-24 hours Watson et a l . 1979 r a t l x l 0 6 / m l 4ug/ml 24-48 hours G i l l i s et a l . 1978 Concanavalin A was removed by absorption w i t h Sephadex G25 (Pharmacia, Uppsala, Sweden) (Glasebrook and F i t c h , 1980) by s t i r r i n g lOg dry beads o per l i t r e of supernatant at 20 C f o r 2 hours. The supernatant was 47 p a r t l y - p u r i f i e d by 80% ammonium sulphate p r e c i p i t a t i o n (Baker JT0792-7, Western S c i e n t i f i c ) and f i l t r a t i o n on Sephadex G150 (Pharmacia). A c t i v e f r a c t i o n s (see Methods 7d) were pooled f o r use as a source of i n t e r l e u k i n 2 (Watson et a l . , 1979). b. Antigen and mitogen induced i n t e r l e u k i n 2 a c t i v i t y from mouse  spleen c e l l s . C u l t u r e s were set up i n 50ml t i s s u e c u l t u r e f l a s k s (Costar 3050, John's S c i e n t i f i c , Toronto, Ontario) c o n t a i n i n g e i t h e r 1 x 10^ spleen 6 c e l l s / m l w i t h 4ug Concanavalm A/ml or 2 x 10 spleen c e l l s / m l w i t h 1 x 10^ mitomycin C-treated P815/ml and incubated u p r i g h t at 37°C, 5% C0 2. Supernatants were c o l l e c t e d a f t e r 24 hours (mitogen c u l t u r e s ) or 1-5 days (antigen c u l t u r e s ) . a-Methylmannoside (Sigma M6882) was added to mitogen c u l t u r e supernatants to complex remaining concanavalin A and these d i d not need concentration before assaying i n t e r l e u k i n 2 as i n 7d,e. Antigen-induced supernatants were concentrated up to 1 6 - f o l d using Minicon B15 Macrosolute Concentrators (15,000 MW c u t - o f f ) (Amicon, O a k v i l l e , Ontario) and s t e r i l i z e d through a 0.2um f i l t e r (Gelman Acrodisc 4192, Montreal, Quebec) before assaying i n 7d,e (Baker et a l . . 1978; G i l l i s et a l . , 1978). c. P r e p a r a t i o n from the EL4.IL2 c e l l l i n e . EL4.IL2 ( F a r r a r et a l . , 1980 ( a ) ; Shimizu et a l . , 1980) was 6 resuspended i n complete medium plus 5% horse serum at 10 c/ml, and lOng/ml 4B-Phorbol-12-myristate-13-acetate (TPA, Sigma P8139) was added f o r 18-24 hrs at 37°C. The c e l l - f r e e supernatant was p r e c i p i t a t e d w i t h 48 80% ammonium s u l f a t e and d i a l y s e d against HEPES buff e r e d s a l i n e pH 7.4. This preparation was used as a source of i n t e r l e u k i n 2 without f u r t h e r p u r i f i c a t i o n and stored at -20°C. d. Thymocyte p r o l i f e r a t i o n assay f o r 112 a c t i v i t y . 10 5 CBA/J thymocytes were c u l t u r e d i n t r i p l i c a t e i n 0.2ml complete medium i n f l a t - b o t t o m p l a t e s (Linbro 76-003-05, Flow Labs) with d i l u t i o n s of the i n t e r l e u k i n 2 sample e i t h e r w i t h or without 2ug concanavalin A/ml f o r 72 hours at 37°C, 5% CO^ (Watson et a l . , 1979). 3 0.5uCi H-Thymidine (NEN, NET-027A) was added f o r the l a s t 6-12 hours to assess p r o l i f e r a t i o n . Cultures were harvested w i t h a 12-channel harvester onto g l a s s - f i b r e f i l t e r s (Gelman 61638), c e l l s were l y s e d and the f i l t e r s washed with d i s t i l l e d water and f i l t e r s d r i e d . F i l t e r s were then placed i n t o 3ml s c i n t i l l a t i o n f l u i d ( 4 g / l 2 , 5 - d i p h e n y l o x a z o l e / l , 4 - b i s -(2-methyl-stryryl)-benzene (PPO/BIS MSB) i n toluene) i n s c i n t i l l a t i o n v i a l s (Kimble-Solvent Saver 66022-387, Western S c i e n t i f i c ) and counted i n a s c i n t i l l a t i o n counter (Nuclear Chicago, Unilux I I , Searle Instrumentation; Tracor A n a l y t i c 6892, NCS Instrumentation, Richmond, BC). Results were expressed as Acpm (cpm with ConA - cpm without ConA) and s t i m u l a t i o n index (cpm without ConA ). I n t e r l e u k i n 2 u n i t s were c a l c u l a t e d as the d i l u t i o n of c o s t i m u l a t o r preparation to give 37% maximal s t i m u l a t i o n of thymocytes with concanavalin A from a p l o t of l o g c o s t i m u l a t o r a c t i v i t y (as f r a c t i o n maximal s t i m u l a t i o n ) against d i l u t i o n of c o s t i m u l a t o r p r e p a r a t i o n (Shaw e_t ajl. , 1978; 1980). 49 e. Assay on an i n t e r l e u k i n 2-dependent c e l l l i n e . MTL 2.8.1 i s an i n t e r l e u k i n 2-dependent c y t o t o x i c T - c e l l l i n e r e c e i v e d from Dr. V. Paetkau, Department of Biochemistry ( U n i v e r s i t y of A l b e r t a , Edmonton, A l b e r t a ) (B l e a c k l e y et a l . , 1982; G i l l i s et a l . , 1978). I t was maintained i n complete medium with 10% FCS plus an optimal d i l u t i o n of IL2 (1/100 d i l u t i o n of i n t e r l e u k i n 2 prepared from EL4-IL2 c e l l l i n e prep #SC9-5 ( 7 c ) ) . The c e l l s were adherent and detached from f l a s k s w i t h PBS c o n t a i n i n g 0.02M et h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) (Sigma ED2SS) and r e c u l t u r e d every 3-4 days at 5 x 10"* c e l l s / l O m l i n 50ml t i s s u e c u l t u r e f l a s k s (Costar) incubated on t h e i r sides at 37°C, 5% C0 2. The assay was set up i n f l a t - b o t t o m p l a t e s with 0.2 ml complete medium plus 10% FCS (but without added i n t e r l e u k i n 2) per w e l l c o n t a i n i n g 4 a d i l u t i o n of the i n t e r l e u k i n 2 sample and 1-5 x 10 MTL 281. A f t e r 24 hours at 37°C, 5% C0 2 the c e l l s were l a b e l l e d with 0.5-1.0uCi 3 H-Thymidine (NEN, NET-027A, or Amersham TRA-310) or 0.1-0.5uCi 125 I-5-Iodo-2'-deoxyuridine (I.UdR) (Edmonton Radiopharmaceutical Centre, Edmonton, A l b e r t a ) i n 50ul of a l a b e l mix c o n t a i n i n g 25ug/ml 5-fluorodeoxyuridine (FUdR, Sigma F0503) (5ug/ml i n c u l t u r e ) and 0.5mM 2'-deoxyinosine (dlno, Sigma D9875) (O.lmM i n c u l t u r e ) . 12-24 hours l a t e r the c e l l s were harvested and counted as i n 7d. (Negative c o n t r o l s contained medium alone and p o s i t i v e c o n t r o l s contained d i l u t i o n s of i n t e r l e u k i n 2 prepared from the EL4-IL2 c e l l l i n e ( 7 c ) ) . Results were expressed as Acpm (cpm t e s t sample - cpm without i n t e r l e u k i n 2) and s t i m u l a t i o n index (cpm t e s t sample/cpm without i n t e r l e u k i n 2). I n t e r l e u k i n 2 u n i t s were c a l c u l a t e d from maximal s t i m u l a t i o n w i t h the p o s i t i v e c o n t r o l as discussed above. 50 8. Cyclophosphamide I n j e c t i o n . 200mg Cyclophosphamide ( c o n t a i n i n g NaCl) (Cytoxan, B r i s t o l Labs, Quebec) was d i s s o l v e d i n 10ml water (20mg/ml and then d i l u t e d 1/2 - 1/8 i n PBS. DBA/2 mice were weighed and i n j e c t e d w i t h 10-200mg cyclophosphamide/ kg body weight i n t r a p e r i t o n e a l l y . Most of the mice weighed between 15 and 30g so t h i s represented 0.15-6mg cyclophosphamide per mouse. 2-48 Hours a f t e r treatment the spleen c e l l s of these mice were used as responders i n c u l t u r e s generating c y t o t o x i c i t y (5a,c). G e n e r a l l y , spleens were used at 24 hours since t h i s gave b e t t e r spleen c e l l recovery and higher y i e l d s of e f f e c t o r c e l l s a f t e r 5 days i n v i t r o than at 48 hours. 9. Assaying Helper A c t i v i t y i n v i v o . Priming p r o t o c o l s and d e t a i l s of adoptive t r a n s f e r experiments are l i s t e d i n the R e s u l t s : Tables XIV and XV. Some of these experiments were performed under s u p e r v i s i o n , by E. Schmieg w h i l s t she was an undergraduate-project student i n D. K i l b u r n ' s l a b o r a t o r y . 125 a. L a b e l l i n g tumour c e l l s w i t h I-Iodo-deoxvundine. 6 F r e s h l y removed P815 c e l l s were l a b e l l e d at 5 x 10 c/ml w i t h 125 125 2uCi/ml I-5-Iodo-2'-deoxyuridine ( I.UdR, ERPC) f o r three hours at 37°C with frequent mixing (Hofer et a l . , 1969; Forman et a l . , 1972; F i d l e r , 1970). C e l l s were washed at l e a s t three times and resuspended to 1 x 10 7c/ml i n PBS by assuming 10% l o s s during l a b e l l i n g and washing. 125 131 C e l l s were l a b e l l e d w i t h I.UdR r a t h e r than I.UdR ( M i l l s et a l . , 125 1980) due to the longer h a l f - l i f e of the I isotope i . e . 60 days 51 compared to 8.1 days so, r a d i o a c t i v e decay was not a c o m p l i c a t i n g f a c t o r i n the measurement of clearance of tumour c e l l s . b. Measuring tumour c e l l clearance. 6 125 Mice were 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 1 x 10 I -l a b e l l e d P815 tumour c e l l s i n 0.1ml ( M i l l s et a l . , 1980), and t h e i r whole body counts recorded on a P h i l i p s 4032 E l e c t r o n i c Counter (set up by M. Morley, Department of M i c r o b i o l o g y , UBC). Mice were ear-marked when i n j e c t e d so that the clearance r a t e s of i n d i v i d u a l mice could be determined. D u p l i c a t e counts were taken f o r each mouse over 4-5 days and recorded as a percentage of the o r i g i n a l count f o r that mouse. Hofer et 125 a l . (1969) have shown that Iodo-deoxyurldine l e v e l s i n mice i n j e c t e d w i t h l a b e l l e d tumour c e l l s r e f l e c t the number of l i v e c e l l s i n the mouse. The isotope i s r e l e a s e d as c e l l s d i e and breakdown but i s r a p i d l y excreted and not r e - u t i l i z e d . The s u r v i v a l time of each mouse a f t e r tumour i n j e c t i o n was recorded; c o n t r o l animals g e n e r a l l y survived 10-14 days a f t e r i n j e c t i o n of 1 x 10 6 P815 i . p . 10. Helper Determinants. a. Priming mice against t u b e r c u l i n PPD. DBA/2 mice were primed against the t u b e r c u l i n p u r i f i e d p r o t e i n d e r i v a t i v e (PPD) as l i s t e d i n Table XVI ( R e s u l t s ) . b. Coupling PPD to P815 c e l l s . PPD was coupled to P815 tumour c e l l s i n two ways: ( i ) Using a 52 PPD-Concanavalin A conjugate from Dr. P. Lachmann, MRC Group on Mechanisms i n Tumour Immunology (Cambridge, England) as described i n Lachmann and S i k o r a (1978). Mitomycin C-treated P815 c e l l s were incubated at 5 x 10 6c/ml w i t h a 1/3000 d i l u t i o n of the PPD-ConA conjugate i n medium without serum, f o r 30 minutes at 20°C with shaking. C e l l s were then washed three times before a d d i t i o n i n t o c u l t u r e , ( i i ) Using 1 - e t h y l -3(3-dimethylaminopropyl)carbodiimide (ECDI) (Sigma E7750) to c r o s s - l i n k PPD and P815 membrane p r o t e i n s (Golub et a l . , 1968; Henney, 1970; Takatsu et a l . , 1978). Mitomycin C-treated P815 c e l l s were suspended to 1 x lO^c/ml i n PBS c o n t a i n i n g 0.5mg/ml ECDI (a f r e s h l y made s o l u t i o n i n PBS) and 0.1-5.0 mg/ml PPD (Connaught CT68). This mixture was incubated at 20°C f o r 1 hour w i t h o c c a s i o n a l mixing and the c e l l s washed 3 times before i n j e c t i o n or a d d i t i o n to c u l t u r e . C e l l s were greater than 90% v i a b l e a f t e r treatment with ECDI. c. P r o l i f e r a t i o n assays. The p r o l i f e r a t i v e responses of primed animals were assayed using lymph node c e l l s . D r a i n i n g lymph nodes were used from footpad i n j e c t e d mice i n a m o d i f i c a t i o n of the method developed by Lee et^ a l . (1979; S i k o r a and Levy, 1980). The assay was set up i n complete medium without 2-mercaptoethanol and supplemented w i t h 4.4mM sodium bicarbonate (to increase the b u f f e r i n g c a p a c i t y i n a 10% CO^ incubator) and 10% human serum (from Dr. A. C o o p e r - W i l l i s , Department of M i c r o b i o l o g y , UBC; Red Cross e x p i r e d plasma was d e f i b r i n a t e d w i t h calcium c h l o r i d e and heat i n a c t i v a t e d f o r 30 minutes at 56°C). Cul t u r e s were set up i n t r i p l i c a t e 53 w i t h 0.25ml medium c o n t a i n i n g 1 x 10" lymph node c e l l s per w e l l plus 4 e i t h e r 50ug/ml s o l u b l e antigen (PPD or CGG) or 5 x 10 mitomycin C-treated P815 (uncoated or coated w i t h PPD) per w e l l , incubated at o 3 37 C, 10% C0 2 f o r 4 days, pulsed w i t h 0.5-luCi H-Thymidine i n 50ul medium (Amersham TRA-310) f o r a f u r t h e r 24 hours, harvested and counted as i n 7d. Results were expressed as mean cpm f o r r e p l i c a t e c u l t u r e s without antigen and Acpm (mean cpm w i t h antigen - mean cpm without antigen) f o r a l l antigens t e s t e d . The advantages of the lymph node p r o l i f e r a t i o n assay include s p e c i f i c i t y , low p r o l i f e r a t i o n of c e l l s without antigen and low responses of unprimed animals to antigen i n v i t r o . d. C y t o t o x i c i t y assays. Assays f o r s t i m u l a t i o n of c y t o t o x i c c e l l s were set up as i n 5a, where 3 x 10 5 spleen c e l l s (from unprimed or PPD primed animals) i n 0.2ml V-bottom p l a t e s were st i m u l a t e d w i t h 1 x 10 mitomycin C-treated o P815 (uncoated or coated w i t h 0-5mg PPD/ml) f o r 5 days at 37 C. C y t o t o x i c i t y was assayed against uncoated 5 1 C r - l a b e l l e d P815. Helper assays were designed as i n (5e) by c u l t u r i n g spleen or lymph node c e l l s from (unprimed or) PPD primed animals as helpers w i t h normal spleen c e l l s and uncoated or PPD coated mitomycin C-treated P815 and assaying c y t o t o x i c i t y w i t h 5 1 C r - l a b e l l e d uncoated P815 t a r g e t s . 54 RESULTS CHAPTER I . THE CYTOTOXIC RESPONSE OF DBA/2 MICE TO THE  SYNGENEIC TUMOUR P815 1. In v i v o and i n v i t r o generation of c y t o t o x i c i t y against P815. Takei et a l . (1976) o r i g i n a l l y d e scribed the presence of c y t o t o x i c a c t i v i t y i n the spleens of tumour-bearing mice and assayed these c e l l s d i r e c t l y by c u l t u r i n g them f o r 18 hours w i t h " ^ C r - l a b e l l e d t a r g e t c e l l s . T h e i r subsequent work involved the i n v i t r o a c t i v a t i o n of c y t o t o x i c T c e l l s against P815. Spleen c e l l s from normal or tumour-bearing mice were c u l t u r e d f o r 5 days with mitomycin C-treated P815 s t i m u l a t o r c e l l s and c y t o t o x i c i t y assayed as before, using ^ C r - l a b e l l e d P815 t a r g e t c e l l s . This approach of i n v i t r o a c t i v a t i o n of c y t o t o x i c c e l l s i n e i t h e r a primary-(spleen c e l l s from normal mice) or secondary-spleen c e l l s from tumour-bearing mice) response generated much higher l e v e l s of c y t o t o x i c i t y and was used i n the m a j o r i t y of experiments described i n t h i s t h e s i s . F i r s t , the t i s s u e d i s t r i b u t i o n of c y t o t o x i c a c t i v i t y was examined. DBA/2 mice were i n j e c t e d subcutaneously with 3 2-50 x 10 l i v e P815 c e l l s , t h e i r spleen, thymus or p e r i p h e r a l lymph nodes removed 6-10 days l a t e r , and used as responder c e l l s i n v i t r o to generate c y t o t o x i c i t y against P815. These c e l l s were c u l t u r e d f o r 5 days w i t h mitomycin C-treated P815 c e l l s and the c y t o t o x i c a c t i v i t y of c u l t u r e s 55 assayed i n an 18 hour "'"'"Cr-release assay against P815 t a r g e t c e l l s . Figure 6 shows the c y t o t o x i c a c t i v i t y generated i t i v i t r o u s i n g spleen c e l l s from normal or tumour-bearing mice. Both 7 and 10 days a f t e r i n j e c t i o n of l i v e tumour c e l l s , increased l e v e l s of c y t o t o x i c i t y were measured when compared to normal mice. This was r e f l e c t e d i n the p o s i t i o n of l y s i s curves and the t o t a l c y t o t o x i c i t y ( l y t i c u n i t s ) generated per c u l t u r e . The c y t o t o x i c a c t i v i t y generated i t i v i t r o using spleen c e l l s from mice 7 days a f t e r P815 i n j e c t i o n was higher than a f t e r 10 days. G e n e r a l l y , c e l l s from tumour-bearing animals produced more c y t o t o x i c i t y than c e l l s from normal mice, and c y t o t o x i c a c t i v i t y was higher at 7-12 days than at 14-17 days a f t e r i n j e c t i o n of the tumour. Although Takei et a l . (Figure 3) observed peak c y t o t o x i c a c t i v i t y 14-20 days a f t e r i n j e c t i o n of the tumour, they measured the d i r e c t l y t i c a c t i v i t y of spleen c e l l s whereas these experiments involved a 5 day c u l t u r e p e r i o d to generate c y t o t o x i c c e l l s . When lymph node c e l l s were used as responder c e l l s and s t i m u l a t e d i n  v i t r o w i t h mitomycin C-treated P815, high c y t o t o x i c responses were generated from both normal and 6-day tumour bearing animals (Figure 7). As observed w i t h spleen c e l l s , lymph node c e l l s from tumour-bearing mice generated more c y t o t o x i c a c t i v i t y than c e l l s from normal mice. In t h i s experiment, the c o n t r o l s were pooled c e l l s from 4 normal mice, the c y t o t o x i c a c t i v i t y generated from the c e l l s of the 4 i n d i v i d u a l tumour-bearing mice was from 2.5-5 times greater than the c o n t r o l . In repeats of t h i s experiment, the tumour-injected mice c o n s i s t e n t l y showed 56 Figure 6: In v i t r o generated c y t o t o x i c a c t i v i t y by spleen c e l l s from normal or tumour-bearing mice. 5 x 10^ responder spleen c e l l s pooled from 3 normal (0 0) or 7 day (A A) and 10-day (• •) tumour-bearing mice (1 x 10 P815, subcutaneous i n j e c t i o n ) were c u l t u r e d w i t h 5 x 10 5 mitomycin C-treated P815 s t i m u l a t o r c e l l s . A f t e r 5 days, r e p l i c a t e c u l t u r e s were pooled and assayed f o r c y t o t o x i c i t y against ' ^ C r - l a b e l l e d P815 ta r g e t c e l l s . Results were p l o t t e d as mean percent c y t o t o x i c i t y (± S.D.) against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) . T o t a l l y t i c u n i t s per c u l t u r e (LU) were c a l c u l a t e d . E:T ratio 58 Figure 7: In v i t r o generated c y t o t o x i c i t y by lymph node c e l l s from normal 5 or tumour-bearing mice. 3 x 10 lymph node c e l l s pooled from 4 normal (0 0) or from 4 i n d i v i d u a l 6-day 4 tumour-bearing mice (A,•,#,?) (1 x 10 P815 i n j e c t e d 4 subcutaneously) were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s . R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and assayed f o r c y t o t o x i c i t y against 5''"Cr-labelled P815 t a r g e t c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . % Cytotoxicity 60 more a c t i v i t y than u n i n j e c t e d c o n t r o l s although the degree of enhanced a c t i v i t y v a r i e d between animals. The generation of c y t o t o x i c c e l l s from thymocytes from normal and 8-day tumour-bearing mice i s shown i n Figure 8. Neither of these c e l l populations generated appreciable c y t o t o x i c a c t i v i t y when st i m u l a t e d w i t h P815. However, when i n t e r l e u k i n 2-containing supernatants were added to c u l t u r e s of thymocytes and P815 s t i m u l a t o r c e l l s , s i g n i f i c a n t l e v e l s of c y t o t o x i c i t y were observed. Again, the c e l l s from P815-injected mice generated more c y t o t o x i c a c t i v i t y to P815, than c e l l s from normal mice. In summary, spleen, lymph node and thymus c e l l s of tumour-bearing mice were a l l good sources of responder c e l l s f o r the generation of anti-tumour c y t o t o x i c a c t i v i t y i j i v i t r o . C e l l s taken from mice 5-10 days a f t e r i n j e c t i o n of the tumour appeared to be the best sources of responder c e l l s , although normal and l a t e r tumour-bearing populations a l s o produced s i g n i f i c a n t e f f e c t o r a c t i v i t y . These r e s u l t s could be i n t e r p r e t e d as an increased number of c y t o t o x i c precursor c e l l s and/or the presence of a m p l i f i e r c e l l s i n tumour-bearing mice. The assay system used at t h i s stage could not d i s t i n g u i s h between these a l t e r n a t i v e s . 2. C e l l s from Tumour-bearing Mice Enhance the Primary i n v i t r o C y t o t o x i c  Response. Takei §_t a l . (1976;1977) showed that c e l l s from tumour-bearing mice suppress the i r i v i t r o c y t o t o x i c response generated by normal spleen c e l l s to P815. A m p l i f i e r c e l l s were s t u d i e d using s i m i l a r mixing experiments. A general o u t l i n e of the experimental p r o t o c o l i s shown i n 61 Figure 8; In v i t r o generated c y t o t o x i c i t y by thymocytes from normal and tumour-bearing mice. 3 x 10"* thymocytes pooled from 3 normal (0,«) or 8 day P815-bearing mice (A,A) (1 x 10* P815-4 subcutaneous i n j e c t i o n ) were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0,A) or with a 1/200 d i l u t i o n of supernatant from concanavalin A-stimulated r a t spleen c e l l s as a source of i n t e r l e u k i n 2 ( t , i ) . A f t e r 5 days, r e p l i c a t e c u l t u r e s were pooled and assayed against ^"''Cr-labelled P815 t a r g e t c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against the l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . 63 Figure 5 (Methods 5d). C e l l s from normal or tumour-bearing mice were c u l t u r e d w i t h normal spleen c e l l s and mitomycin C-treated P815 s t i m u l a t o r c e l l s f o r 5 days. C y t o t o x i c i t y was then assayed on 5 ^ C r labelled-P815 t a r g e t c e l l s . Since spleen thymus and lymph node c e l l s of tumour-bearing animals had a l l shown increased c y t o t o x i c a c t i v i t y when compared w i t h the equivalent c e l l s from normal animals, the a m p l i f i e r a c t i v i t y of these three c e l l populations was assayed. Spleen c e l l s from normal or tumour-bearing mice 7 or 8 days a f t e r tumour i n j e c t i o n were added to equal numbers of normal spleen responder c e l l s and c y t o t o x i c i t y assayed a f t e r 5 days against "'"''Cr-labelled P815 t a r g e t c e l l s (Figure 9). The a d d i t i o n of normal spleen c e l l s or immune spleen c e l l s (from tumour-bearing mice) r e s u l t e d i n enhanced c y t o t o x i c i t y to P815, with c e l l s from tumour-bearing mice producing gr e a t e r enhancement. There was a small d i f f e r e n c e between immune spleen c e l l s from mice 7 and 8 days a f t e r i n j e c t i o n of tumour although t h i s was not always as obvious. This data r e l a t e d w e l l to the experiments where spleen c e l l s from normal or tumour-bearing mice were c u l t u r e d as responder c e l l s w i t h mitomycin C-treated P815 c e l l s (Figure 6 ) , although the normal spleen c e l l s alone were more a c t i v e i n t h i s experiment. When lymph node c e l l s from normal and tumour-bearing mice were c u l t u r e d w i t h normal spleen c e l l s and mitomycin C-treated P815 s t i m u l a t o r c e l l s , enhancement of the c y t o t o x i c response was seen (Figure 10). Normal lymph node c e l l s had only a minimal e f f e c t on the c y t o t o x i c a c t i v i t y of normal spleen c e l l s but c e l l s from tumour-bearing mice were very e f f e c t i v e . Two d i f f e r e n t animals were t e s t e d i n t h i s experiment and the 64 Figure 9: The a d d i t i o n of spleen c e l l s from normal or tumour-bearing mice to c u l t u r e s generating c y t o t o x i c i t y against P815. 5 3 x 10 normal spleen responder c e l l s were c u l t u r e d with 1 x 4 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0 0) (A A, 7 day tumour-bearing spleen alone) or with 3 x 10^ spleen c e l l s from normal (•———•) or 7 and 8 day tumour-bearing mice (A A, • •) (1 x 10 P815-subcutaneous i n j e c t i o n ) . C y t o t o x i c i t y was assayed a f t e r 5 days against ^ C r - l a b e l l e d P815 target c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . 80-1 12-5:1 25*1 507l 100:1 E:T ratio 66 Figure 10: The a d d i t i o n of lymph node c e l l s from normal or tumour-bearing mice to c u l t u r e s generating c y t o t o x i c i t y against P815. 3 x 10"* normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0 0) or with 3 x 10 5 lymph node c e l l s from normal (0 •) or 6 day tumour-bearing mice (• • , 4 • •) (1 x 10 P815 - subcutaneous i n j e c t i o n ) . R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and assayed 51 against C r - l a b e l l e d P815 t a r g e t c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . 12-5:1 25-1 5o7l 100:1 E:T ratio 68 v a r i a b l e e f f e c t s of i n d i v i d u a l animals was a c o n s i s t e n t observation. Regulator c e l l a c t i v i t y would vary from no d i f f e r e n c e between normal and i n j e c t e d mice to even greater a c t i v i t y than animal number 2 shown here. These d i f f e r e n c e s i n d i c a t e d d i f f e r e n t priming of DBA/2 to the P815 tumour antigens and may be due to v a r i a b i l i t y of the tumour i t s e l f w i t h respect to growth r a t e s and surface antigen expression observed i n t h i s and other l a b o r a t o r i e s . Thymocytes were a l s o e f f e c t i v e as r e g u l a t o r c e l l s i n the response to P815 (Figure 11). Normal thymocytes g e n e r a l l y had l i t t l e e f f e c t or were s l i g h t l y suppressive as seen i n t h i s experiment. However, thymocytes from 5 or 7 day immune mice ( a f t e r i n j e c t i o n of l i v e tumour c e l l s ) were very e f f e c t i v e at a m p l i f y i n g the response to P815. As shown f o r lymph node c e l l s , thymocytes from i n d i v i d u a l tumour-bearing mice showed q u i t e d i f f e r e n t e f f e c t s but u s u a l l y a m p l i f i e d the response s i g n i f i c a n t l y . Thymocytes of some animals suppressed the c y t o t o x i c response to P815 (e.g. normal thymocytes i n Figure 11). This was probably due to suppressor c e l l s present i n normal mice or generated under these c u l t u r e c o n d i t i o n s . In c o n c l u s i o n , spleen, thymus and lymph node c e l l s from tumour-bearing mice enhanced the primary i n v i t r o c y t o t o x i c response of spleen c e l l s to P815. Normal thymocytes and lymph node c e l l s were l e s s e f f e c t i v e than normal spleen c e l l s i n enhancing the c y t o t o x i c response. These mixing experiments may measure the presence of e i t h e r an a m p l i f i e r c e l l or an increased number of c y t o t o x i c precursor c e l l s i n the primed populations. Since normal spleen, thymus and lymph node c e l l s generated c y t o t o x i c a c t i v i t y against P815 i n c u l t u r e , i t was necessary to remove the c y t o t o x i c 69 Figure 11 The a d d i t i o n of thymocytes from normal or tumour-bearing mice to c u l t u r e s generating c y t o t o x i c i t y against P815. 3 x 10 5 4 normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (A A) or with 3 x 10** thymocytes from normal (• •) or 5 4 (A 4) or 7 (• •) day immune mice (1 x 10 P815, subcutaneous i n j e c t i o n ) . R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and assayed f o r c y t o t o x i c a c t i v i t y against """"""Cr-labelled P815 ta r g e t c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E.T r a t i o ) and the t o t a l l y t i c u n i t s per c u l t u r e (LU) were c a l c u l a t e d . 71 a c t i v i t y from the r e g u l a t o r population added i n mixing experiments. 3. The S u s c e p t i b i l i t y of Enhancing and C y t o t o x i c A c t i v i t i e s to  YY-Radiation. Many workers (reviewed by Katz, 1977, p 315-325) have shown that c y t o t o x i c and suppressor T c e l l s are more s e n s i t i v e to Y - r a d i a t i o n than helper T lymphocytes. A low dose of r a d i a t i o n (100-500R) i n a c t i v a t e s both c y t o t o x i c precursor and e f f e c t o r c e l l s but does not a f f e c t a l l helper c e l l subpopulations, although r a d i a t i o n - s e n s i t i v e helper c e l l s have been described (Agarossi et a l . , 1978). The d i f f e r e n c e i n r a d i a t i o n s e n s i t i v i t y o f f e r s a way to d i s t i n g u i s h between helper c e l l s and/or increased numbers of c y t o t o x i c precursor c e l l s i n the c e l l populations from tumour-bearing mice which enhance the c y t o t o x i c response of normal spleen c e l l s i n mixing experiments. When spleen c e l l s from normal mice were exposed to 1000 Rads y - r a d i a t i o n , t h e i r c y t o t o x i c response to P815 was completely removed (Figure 12). S i m i l a r l y , the high l e v e l of c y t o t o x i c i t y generated from spleen c e l l s of 7-day P815-bearing mice was l o s t when spleen c e l l s were i r r a d i a t e d before c u l t u r e . As p r e v i o u s l y shown, the n o n - i r r a d i a t e d spleen c e l l s from tumour-bearing mice were more a c t i v e than n o n - i r r a d i a t e d normal spleen c e l l s . I f the enhanced c y t o t o x i c a c t i v i t y seen i n mixing experiments w i t h c e l l s from P815-bearing mice was due to the presence of a P815-induced a m p l i f i e r p o p u l a t i o n , i r r a d i a t i o n might not a f f e c t the a c t i v i t y . As shown i n Figure 13, thymocytes from normal or tumour-bearing mice were t e s t e d 72 Figure 12: The y - r a d i a t i o n s e n s i t i v i t y of cytotoxic-responder c e l l s . 5 x 1 0 6 untreated ( t ^ •, M •) or i r r a d i a t e d (1000 Rads, ^Co source) (0 0, • •) spleen c e l l s from 4 normal (#,0) or 7 day tumour-bearing mice (1 x 10 P815 subcutaneous) (•,&) were c u l t u r e d w i t h 5 x 10"* mitomycin C-treated P815 s t i m u l a t o r c e l l s . A f t e r 5 days, r e p l i c a t e c u l t u r e s were pooled and assayed f o r c y t o t o x i c i t y against 5 1 C r - l a b e l l e d P815 target c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . 73 12 5:1 25-1 50il IOC)-! E:T ratio 74 Figure 13: The y - r a d i a t i o n s e n s i t i v i t y of c y t o t o x i c - a m p l i f i e r c e l l s . 3 x 10 5 normal spleen responder c e l l s were c u l t u r e d w i t h 4 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s and 3 x 10 5 thymocyte a m p l i f i e r c e l l s . Thymocytes from normal (0,») or 6 day tumour-bearing mice (A, A; •, •) (1 x 10 P815 i n j e c t e d subcutaneously) were added e i t h e r untreated (0,A,D) or a f t e r exposure to 1000 Rads y - r a d i a t i o n (<'0Co source) (•,!,•). R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and c y t o t o x i c i t y assayed against 5 1 C r - l a b e l l e d P815 t a r g e t c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t r a t i o (E:T r a t i o ) and l y t i c u n i t s (LU) were c a l c u l a t e d . 12-5:1 25:1 50:1 E:T ratio 100:1 76 f o r a m p l i f i e r c e l l s i n t h i s way. The a m p l i f i e r a c t i v i t y of normal thymocytes was enhanced by 1000 Rads i r r a d i a t i o n perhaps due to the i n a c t i v a t i o n of suppressor c e l l s present i n the thymus. Untreated 6-day immune thymocytes showed very l i t t l e enhancing a c t i v i t y when compared w i t h normal c e l l s and one mouse ( 0 0 ) showed suppressive a c t i v i t y . A f t e r i r r a d i a t i o n , the thymocyters from tumour-bearing animals showed 2-3.5 times more a m p l i f i c a t i o n than i r r a d i a t e d normal thymocytes. The increase i n helper a c t i v i t y f o l l o w i n g i r r a d i a t i o n was not always observed perhaps due to d i f f e r e n c e s i n the number of suppressor c e l l s . That the suppressive a c t i v i t y of thymocytes from immune mice (e.g. Figure 13, 0 • ) could be reversed to helper a c t i v i t y by i r r a d i a t i o n , suggested the coexistence of suppressor and helper populations a f t e r tumour i n j e c t i o n . These mixing experiments measured the net a c t i v i t y of suppressor c e l l s and helper c e l l s upon the c y t o t o x i c response. This can be seen i n experiments where the a m p l i f i e r c e l l s were exposed to d i f f e r e n t doses of r a d i a t i o n before they were c u l t u r e d w i t h responder and s t i m u l a t o r c e l l s (Table I I ) . Treatment of normal and immune thymocytes with 500R y - r a d i a t i o n r e s u l t e d i n enhanced l y s i s i n most cases when compared to n o n - i r r a d i a t e d c e l l s . Further increases i n r a d i a t i o n dose increased t h i s enhancement by immune c e l l s but not normal c e l l s . The l a c k of a c t i v i t y seen with c e l l s from tumour-bearing mouse #1 suggested e i t h e r the presence of a r a d i a t i o n - s e n s i t i v e helper c e l l or more l i k e l y the f a i l u r e of the tumour to s t i m u l a t e helper c e l l s . T o t a l l a c k of a c t i v i t y such as t h i s was r a r e l y seen although the degree of enhancement v a r i e d widely. The r e s u l t s presented i n t h i s s e c t i o n showed that c y t o t o x i c precursor TABLE I I : The coexistence of helper and suppressive a c t i v i t i e s i n thymocytes from normal and tumour-bearing mice. TOTAL LYTIC UNITS PER CULTURE'3 AMPLIFIER RADIATION DOSE (Rads) c THYMOCYTESa OR 500R 1000R 150QR 2000R normal 0, .1 6, .9* 5 .8* 5, .6* 10, .8* 6 day tumour-bearing 91 0. 9** N, ,T. 0 .7 N .T. N, ,T. 6 day tumour-bearing 92 5. . 2** 14. .7* 8, .9* 14. .1* 20, .6* 6 day tumour-bearing 93 2. ,0** 4, .9* 16, .0* 20, .9* N, • T. 6 day tumour-bearing #4 7. .8** 12. .3* 2 .3* 22, .4* 4, .7* a 3 x 10 3 normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10^ mitomycin C-treated P815 s t i m u l a t o r c e l l s and 3 x 10 5 thymocytes from normal or 6 day immune mice ( i n j e c t e d w i t h 1 x 10^ P815 subcutaneously). b R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and c y t o t o x i c i t y assayed against ^•'•Cr-labelled P815 t a r g e t c e l l s . Percent l y s i s was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o and l y t i c u n i t s c a l c u l a t e d as the r e c i p r o c a l number of e f f e c t o r c e l l s to give 25% l y s i s of 1 x 1 0 4 c e l l s i n 18 hours. T o t a l u n i t s per c u l t u r e were c a l c u l a t e d from the recovery of e f f e c t o r c e l l s a f t e r 5 days i n c u l t u r e . c Thymocytes were exposed to a 6 0 C o source at 20-30 Rads/second and washed once before a d d i t i o n to c u l t u r e . * S i g n i f i c a n t l y d i f f e r e n t from same thymocytes, u n i r r a d i a t e d , p<0.05. ** S i g n i f i c a n t l y d i f f e r e n t from normal thymocytes, u n i r r a d i a t e d , p<0.05. 78 c e l l s which were a c t i v a t e d i n c u l t u r e s w i t h P815, were s e n s i t i v e to Y - r a d i a t i o n . In c o n t r a s t , a m p l i f i e r c e l l s from normal or tumour-bearing mice were r e s i s t a n t to Y _ r & d i a t i o n . When c e l l s from tumour-bearing animals were used as responder c e l l s or a m p l i f e r c e l l s (by mixing w i t h normal spleen c e l l s ) i n c u l t u r e s s t i m u l a t e d w i t h P815, more c y t o t o x i c i t y was generated than when c e l l s from normal mice were added. Since the a m p l i f i e r e f f e c t was s t i l l seen a f t e r i r r a d i a t i o n of the r e g u l a t o r c e l l p o p u l a t i o n s , the increased c y t o t o x i c i t y from immune mice was l a r g e l y due to the presence of a m p l i f e r c e l l s . 4. The Nature of the Helper C e l l s . As mentioned p r e v i o u s l y a m p l i f i e r a c t i v i t y i n c y t o t o x i c responses has been a t t r i b u t e d to both T c e l l s and macrophages. The nature of the am p l i f e r c e l l s i n the c y t o t o x i c response to P815 was determined by p r e t r e a t i n g the a m p l i f e r c e l l p o pulation w i t h a n t i Thy 1.2 antibody plus complement to remove a l l T c e l l s (Thy 1.2 p o s i t i v e c e l l s ) . As a c o n t r o l , c e l l s were t r e a t e d w i t h complement alone. Figure 14 shows that the helper a c t i v i t y of thymocytes from tumour-bearing mice was abrogated by anti-Thy 1.2 plus complement i n d i c a t i n g that the helper a c t i v i t y was due to a T lymphocyte. Although the c y t o t o x i c a m p l i f i e r c e l l s detected i n tumour-bearing mice were T lymphocytes, macrophages or other antigen presenting c e l l s are e s s e n t i a l f o r a c y t o t o x i c response (Symington and Teh, 1980). These c e l l s would be present i n high numbers i n the spleen c e l l responder populations used i n the mixing experiments. 79 Figure 14: Pretreatment of a m p l i f i e r c e l l s w i t h anti-Thy 1.2 antibody plus complement. 3 x 10 5 normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s and thymocytes from normal (0,t) or 5 day tumour-bearing 4 mice (A,A) (1 x 10 P815 i n j e c t e d subcutaneously). The thymocytes were p r e t r e a t e d w i t h complement alone (0;A) or anti-Thy 1.2 antibody plus complement ( t ; A ) . A f t e r 5 days, c u l t u r e s were assayed f o r c y t o t o x i c i t y against " ^ C r - l a b e l l e d P815 c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and l y t i c u n i t s (LU) c a l c u l a t e d . 5-4 LU 125:1 2r}<1 LU 25:1 50:1 E:T ratio 100:1 81 5. The Time of Appearance of Helper C e l l s i n the Thymus. I t has been shown that P815-bearing DBA/2 mice e x h i b i t e d a peak of c y t o t o x i c a c t i v i t y during the per i o d of slowed growth or re g r e s s i o n of the tumour (Figure 3). Helper a c t i v i t y i n these mice might be expected to c o r r e l a t e i n some way w i t h the expression of c y t o t o x i c a c t i v i t y . Therefore, animals were t e s t e d f o r helper a c t i v i t y at d i f f e r e n t times a f t e r the i n j e c t i o n of l i v e P815 tumour c e l l s (Figure 15). Thymocytes from normal or tumour-bearing mice were mixed with normal spleen responder c e l l s and P815 s t i m u l a t o r c e l l s . The response obtained with thymocytes from tumour-bearing mice was expressed as a percentage of the response w i t h normal thymocytes to standardize the v a r i a b l e l y t i c a c t i v i t y of c u l t u r e s set up on d i f f e r e n t days. S i g n i f i c a n t helper a c t i v i t y (1.5 -2.5 times c o n t r o l a c t i v i t y measured as l y t i c u n i t s ) was seen between 4 and 12 days a f t e r tumour i n j e c t i o n and suppressive a c t i v i t y was evident a f t e r 12 days. The peak of helper a c t i v i t y p a r a l l e l e d that of c y t o t o x i c a c t i v i t y seen i n tumour-bearing mice. 6. The Antigen S p e c i f i c i t y of Helper C e l l s . In most other systems stu d i e d helper c e l l s have been shown to i n t e r a c t w i t h c y t o t o x i c precursor c e l l s i n an a n t i g e n - s p e c i f i c manner. The a n t i g e n - s p e c i f i c i t y of the P815 induced helper c e l l s was assayed i n two ways. F i r s t l y , thymocytes from normal or P815-bearing mice were assayed f o r helper a c t i v i t y i n the c y t o t o x i c response of DBA/2 spleen c e l l s against d i f f e r e n t s t i m u l a t o r s (See Figure 4) - (1) C57B1/6 major 82 Figure 15: The time of appearance of helper c e l l s i n the thymus a f t e r i n j e c t i o n of P815. 3 x 10 5 normal spleen responder c e l l s 4 were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s and 3 x 10 5 thymocytes from normal or 4 tumour-bearing animals (1 x 10 P815 - subcutaneous i n j e c t i o n ; thymocytes taken on d i f f e r e n t days a f t e r tumour i n j e c t i o n ) . R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days, and 51 c y t o t o x i c i t y assayed against C r - l a b e l l e d P815 ta r g e t c e l l s . Mean percent c y t o t o x i c i t y was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o and l y t i c u n i t s c a l c u l a t e d as the number of e f f e c t o r c e l l s g i v i n g 25% l y s i s of 1 x 10 t a r g e t c e l l s i n 18 hours. L y t i c u n i t s per c u l t u r e w i t h immune thymocytes were c a l c u l a t e d as a percentage of l y t i c u n i t s per c u l t u r e w i t h normal thymocytes ( r e l a t i v e c y t o t o x i c i t y %) and the mean percentage (± S.E.M.) p l o t t e d f o r the 3 immune animals t e s t e d on each day. 83 1 T 1— 18 -1 r 2 -J-4 - i r 6 8 — i — 10 i 1— 12 Days after P815 injection H 16 84 h i s t o c o m p a t i b i l i t y antigens; (2) C57B1/10 minor h i s t o c o m p a t i b i l i t y antigen; (3) P815 syngeneic tumour antigens, and (4) L1210 syngeneic tumour antigens (Table I I I ) . High l e v e l s of c y t o t o x i c a c t i v i t y were generated against a l l 4 s t i m u l a t o r s . Thymocytes from 5-day tumour-bearing mice #'s 1 and 3 s i g n i f i c a n t l y enhanced the c y t o t o x i c a c t i v i t y against P815 but not the other 3 s t i m u l a t o r s . Thymocytes from tumour-bearing mouse #2 suppressed the response against P815 but a l s o against B6 a l l o a n t i g e n s and L1210. Secondly, thymocytes from normal and tumour-bearing mice were mixed w i t h normal spleen responder c e l l s and P815 s t i m u l a t o r c e l l s but c y t o t o x i c a c t i v i t y was assayed against 4 d i f f e r e n t t a r g e t c e l l s (Table I V ) . Thymocytes from normal or tumour-bearing mice d i d not increase the l e v e l s of n o n - s p e c i f i c c y t o t o x i c i t y generated i n these c u l t u r e s ( i . e . c y t o t o x i c i t y against t a r g e t s d i f f e r e n t from the s t i m u l a t o r c e l l ) but thymocytes from tumour-bearing mice d i d enhance the response against P815. In c o n c l u s i o n , the helper c e l l s induced i n P815-bearing mice were antigen s p e c i f i c . They enhanced the i n v i t r o generation of c y t o t o x i c i t y against P815 but not other r e l a t e d or u n r e l a t e d s t i m u l a t o r c e l l s and d i d not enhance the generation of n o n - s p e c i f i c c y t o t o x i c c e l l s . 7. The Timing of Helper C e l l A c t i v i t y i n the i n v i t r o C y t o t o x i c Response  to P815. Helper c e l l a c t i v i t y and c y t o t o x i c precursor c e l l s were detected simultaneously i n mice bearing P815 tumours. The helper c e l l s s p e c i f i c a l l y enhanced the i n v i t r o generation of c y t o t o x i c a c t i v i t y 85 TABLE I I I : The antigen s p e c i f i c i t y of helper c e l l s . TOTAL LYTIC UNITS PER CULTURE13 STIMULATOR ANTIGENSC HELPER THYMOCYTESa B6 ALLOANTIGENS BIO MINOR ANTIGENS P815 L1210 normal 92.4 152.2 33.9 44.4 5 day tumour-bearing #1 94.4 136.7 50.8* 46.4 5 day tumour-bearing #2 36.7* 126.5 19.7* 31.7* 5 day tumour-bearing #3 99.3 126.1 51.4* 39.7 3 x 10 3 normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10* to 3 x 10-* s t i m u l a t o r c e l l s and 3 x 10 5 thymocytes from normal or 5 day immune mice (1 x 10* P815, subcutaneous i n j e c t i o n ) . R e p l i c a t e c u l t u r e s were pooled a f t e r 4-5 days and assayed f o r c y t o t o x i c i t y w i t h - ^ C r - l a b e l l e d target c e l l s . Percent l y s i s was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o and l y t i c u n i t s c a l c u l a t e d as the r e c i p r o c a l number of e f f e c t o r c e l l s to give 25% l y s i s of 1 x 10* c e l l s i n 4 or 18 hours. T o t a l l y t i c u n i t s were c a l c u l a t e d from the number of e f f e c t o r c e l l s recovered a f t e r 4 or 5 days of c u l t u r e . See Figure 2 f o r explanation of s t i m u l a t o r and ta r g e t c e l l s used. S i g n i f i c a n t l y d i f f e r e n t from the l y s i s w i t h normal thymocytes as helper c e l l s i n that p a r t i c u l a r antigen system, p< 0.05. A l l others not s i g n i f i c a n t l y d i f f e r e n t from normal thymocytes p>0.2. 86 TABLE IV: The antigen s p e c i f i c i t y of i n v i t r o generated c y t o t o x i c c e l l s . TOTAL LYTIC UNITS PER CULTURE13  TARGET CELL  HELPER THYMOCYTES'1 P815 L1210 D2 SPLEEN BLASTS EL4 Normal 4.01 0.41 0.1 0.1 7 day tumour-bearing 15.07* 0.33 0.1 0.1 3 x 10^ normal spleen responder c e l l s were c u l t u r e d w i t h 1 x 10 4 mitomycin C-treated P815 s t i m u l a t o r c e l l s and 3 x 10 5 thymocytes from normal or 7 day tumour-bearing mice (1 x 10^ P815, subcutaneous i n j e c t i o n ) . R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and assayed f o r c y t o t o x i c i t y against - ^ C r - l a b e l l e d t a r g e t c e l l s (see t e x t ) . Percent l y s i s was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o and l y t i c u n i t s c a l c u l a t e d as the r e c i p r o c a l number of e f f e c t o r c e l l s to give 25% l y s i s of 1 x 10 4 t a r g e t c e l l s i n 18 hours. T o t a l l y t i c u n i t s were c a l c u l a t e d from the number of e f f e c t o r c e l l s recovered a f t e r 5 days of c u l t u r e . S i g n i f i c a n t l y d i f f e r e n t from c u l t u r e w i t h normal thymocytes p< 0.05. A l l others not s i g n i f i c a n t l y d i f f e r e n t from normal thymocytes p>0.2. 87 against P815. The mechanism of helper c e l l a c t i v i t y i n the Ln v i t r o a c t i v a t i o n of c y t o t o x i c c e l l s was t e s t e d by the a d d i t i o n of helper c e l l s at d i f f e r e n t times to c u l t u r e s generating c y t o t o x i c a c t i v i t y . Two s l i g h t l y d i f f e r e n t experimental p r o t o c o l s were used to compensate f o r the v a r i a b i l i t y observed both i n priming DBA/2 mice f o r helper c e l l s and i n the ijn v i t r o generation of c y t o t o x i c c e l l s . F i r s t l y , thymocytes from normal or 5-day primed DBA/2 mice were added as helper c e l l s on day 0, 1 or 2 to one group of 5-day c u l t u r e s (Table V). When immune thymocytes were added on day 0 or 1 of 5-day c u l t u r e s they showed s i g n i f i c a n t helper a c t i v i t y when compared to normal thymocytes added at the same time. However, when added on day 2, no s i g n i f i c a n t a m p l i f i c a t i o n was seen. When set up i n t h i s way a l l c u l t u r e s should have generated e q u i v a l e n t l y t i c a c t i v i t y and d i f f e r e n c e s would be due to the added thymocyte populations. As was shown i n previous experiments, the degree of helper a c t i v i t y v a r i e d between i n j e c t e d DBA/2 mice so the l a c k of enhancement by c e l l s added on day 2 could have been due to reduced priming i n those animals. Secondly, one group of mice were i n j e c t e d and used f o r helper c e l l s . Thymocytes from normal or 6-day tumour-bearing mice were added to c u l t u r e s set up on d i f f e r e n t days so that again the helper c e l l s were present from day 0, 1, 2 or 3 of 5-day c u l t u r e s (Table V I ) . The l y t i c a c t i v i t y of c u l t u r e s c o n t a i n i n g thymocytes from tumour-bearing mice were expressed as a percentage of a c t i v i t y i n c u l t u r e s w i t h normal thymocytes. C e l l s added on day 0 or 1 ( i . e . at the beginning of c u l t u r e s ) s i g n i f i c a n t l y enhanced the c y t o t o x i c response to P815. G e n e r a l l y , c e l l s added l a t e r i n the 88 TABLE V; The timed a d d i t i o n of helper c e l l s to c y t o t o x i c c e l l s . TOTAL LYTIC UNITS PER CULTURE HELPER THYMOCYTES DAY HELPERS ADDED TO 5 DAY CULTURES Normal 5.1 4.9 3.8 5 day Tumour-bearing 22.8* 11.3* 5.3 P r o t o c o l : DAY 0: Set up c u l t u r e s of 3 x 10"* normal spleen responder c e l l s 4 w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s . DAY 0, 1, 2: Added 3 x 10"* thymocytes pooled from normal or 4 5 day-tumour-bearing mice (1 x 10 P815, subcutaneous i n j e c t i o n ) . DAY 5: Pooled r e p l i c a t e c u l t u r e s and assayed against 5 1 C r - l a b e l l e d P815 ta r g e t c e l l s . Percent c y t o t o x i c i t y p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o and t o t a l l y t i c u n i t s c a l c u l a t e d . * Results s i g n i f i c a n t l y d i f f e r e n t from c u l t u r e s w i t h normal thymocytes p<0.05. A l l others not s i g n i f i c a n t l y d i f f e r e n t p>0.2. TABLE VI; The time of a c t i v i t y of helper c e l l s i n the i n v i t r o c y t o t o x i c response to P815. TOTAL LYTIC UNITS PER CULTURE (% WITH NORMAL THYMOCYTES) HELPER THYMOCYTES DAY HELPERS ADDED TO 5 DAY CULTURES 89 Expt. 1. Normal 6 day immune #1 6 day immune #2 6 day immune #3 4.0 16.6(415)* 6.0(150)* 8.5(214)* 1.2 4.7 4.3(358)* 5.6(119) 3.1(258)* 3.7 (79) 1.1 (92) 28.3(602)* 38.4 18.5 (48)* 41.9(109) 31.0 (81) Expt. 2 Normal 5 day immune #1 5 day immune #2 23.0 4.9 5.6 9.5 58.1(253)* 12.6(257)* 7.3(130)* 10.5(111) 31.7(138)* 10.3(210)* 5.1 (91) 9.6(101) P r o t o c o l : DAY-3,-2,-1,0: Set up c u l t u r e s of 3 x 10^ normal spleen responder c e l l s w i t h 1 x 10* mitomycin C-treated P815 s t i m u l a t o r c e l l s . DAY 0: Add 3 x 10-> thymocytes pooled from normal or i n d i v i d u a l immune mice (1 x 10* P815, subcutaneous i n j e c t i o n ) . DAY 2,3,4,5: i . e . 5 days a f t e r each c u l t u r e set up, r e p l i c a t e c u l t u r e s were pooled and assayed against " C r -l a b e l l e d P815 t a r g e t c e l l s . Percent c y t o t o x i c i t y was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o to determine t o t a l l y t i c u n i t s per c u l t u r e . * S i g n i f i c a n t l y d i f f e r e n t from c u l t u r e s w i t h normal thymus p<0.05. A l l others not s i g n i f i c a n t l y d i f f e r e n t p>0.2. 90 c u l t u r e p e r i o d (days 2 or 3) d i d not enhance the c y t o t o x i c response although o c c a s i o n a l l y (see animal #3, Expt. 1, Table V I ) , helper c e l l s were a c t i v e when added l a t e r i n the c u l t u r e p e r i o d . This was seen w i t h only two animals i n a t o t a l of 5 experiments and 18 i n d i v i d u a l mice. In c o n c l u s i o n , helper c e l l s from tumour-bearing mice were a c t i v e i n the e a r l y phase of in. v i t r o a c t i v a t i o n of c y t o t o x i c c e l l s against P815. 8. Summary. DBA/2 mice i n j e c t e d w i t h l i v e P815 syngeneic tumour c e l l s developed c y t o t o x i c precursor c e l l s r e a c t i v e to the tumour. These c e l l s were a c t i v a t e d by c u l t u r i n g spleen, thymus, or lymph node c e l l s of tumour-bearing mice with P815 s t i m u l a t o r c e l l s (Figures 6;7;8). C e l l s taken from tumour-bearing mice enhanced the i j i v i t r o generation of c y t o t o x i c c e l l s from normal DBA/2 spleen c e l l s (Figures 9;10;11). The c y t o t o x i c precursor c e l l s were s e n s i t i v e to Y- radiati° n (Figure 12) whereas the a m p l i f y i n g a c t i v i t y of c e l l s from tumour-bearing mice was r a d i a t i o n r e s i s t a n t (Figure 13). The helper c e l l s a m p l i f i e d the c y t o t o x i c response d i r e c t e d against the priming P815 tumour but not c y t o t o x i c responses to other antigens (Table I I I ) . The enhanced response to P815 was not accompanied by an increase i n the generation of n o n s p e c i f i c c y t o t o x i c i t y (Table I V ) . The helper c e l l s were detected i n P815-bearing mice at the same time as the peak i n c y t o t o x i c c e l l a c t i v i t y (Figure 15) and were a c t i v e ijn v i t r o only when added at the beginning of c u l t u r e s generating c y t o t o x i c a c t i v i t y (Tables V, V I ) . 91 CHAPTER I I : THE EFFECTS OF CYCLOPHOSPHAMIDE ON THE CYTOTOXIC RESPONSE OF DBA/2 MICE TO P815 1. The E f f e c t of Cyclophosphamide on C y t o t o x i c Responses of DBA/2 Mice. The d i f f e r e n t i a l s e n s i t i v i t y to cyclophosphamide of helper and c y t o t o x i c precursor c e l l s ( M e r l u z z i e_t a l . , 1979) suggested a way to examine the i n t e r a c t i o n of these two c e l l p opulations. DBA/2 mice were 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 0-200mg cyclophosphamide per kg body weight and t h e i r spleens were removed a f t e r 24-48 hours. Cyclophosphamide reduced the number of spleen c e l l s recovered 24 hours a f t e r i n j e c t i o n , i n a dose-dependent f a s h i o n (Table V I I ) . The reduction i n spleen c e l l numbers was g r e a t e r a f t e r 48 hours. The spleen c e l l s from DBA/2 mice 24 hours a f t e r i n j e c t i o n of 0-200mg/kg cyclophosphamide were c u l t u r e d as responder c e l l s w i t h e i t h e r i r r a d i a t e d B6 spleen c e l l s or mitomycin C-treated P815 tumour c e l l s as s t i m u l a t o r c e l l s . C y t o t o x i c i t y was assayed using 5 1 C r - l a b e l l e d EL4 and P815 t a r g e t c e l l s , r e s p e c t i v e l y (Table V I I I ) . Up to 50mg/kg cyclophosphamide enhanced the c y t o t o x i c response to both B6 a l l o a n t i g e n s and P815 tumour antigens. This was probably due to removal of cyclophosphamide-sensitive suppressor c e l l s ( G l a s e r , 1979). Higher doses of cyclophosphamide (75-200mg/kg) reduced or e l i m i n a t e d the c y t o t o x i c response to both antigens. This experiment was repeated numerous times and the dose-dependent decrease i n c y t o t o x i c response to both P815 and B6 a l l o a n t i g e n s was always observed. The enhancement of the cytotoxic-response by low doses of 92 TABLE V I I ; The e f f e c t of cyclophosphamide i n j e c t i o n upon spleen c e l l recovery DOSE OF CYCLOPHOSPHAMIDE* % SPLEEN CELL RECOVERY15 NUMBER OF EXPERIMENTS (mg/kg body weight) mean + S.E.M. 10 91 + 14 2 25 96 + 14 6 50 73 + 4 30 75 51 + 7 8 100 49 + 3 35 150 41 + 13 4 200 30 + 7 5 a Mice were weighed and then 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 0.1 - 0.4 ml of a cyclophosphamide s o l u t i o n . b Spleen removed 24 hours a f t e r i n j e c t i o n . % Spleen c e l l recovery = number spleen c e l l s per i n j e c t e d mouse x 100 number spleen c e l l s per u n i n j e c t e d mouse 93 TABLE V I I I : The e f f e c t of cyclophosphamide i n j e c t i o n on the i n v i t r o c y t o t o x i c responses of DBA/2 mice. LYTIC UNITS (TOTAL LYTIC UNITS PER CULTURE) d DOSE OF CYCLOPHOSPHAMIDE STIMULATING ANTIGENS (mg/kg body w e i g h t ) a  B6 ALLOANTIGENSb P815 c 0 8 .2 (66, .9) 1.0(6.5) 25 15, .7(113, .0)* 1.8(8.8)* 50 17, .4(116, .6)* 2.4(9.8)* 75 6, .3 (38, .4)* 0.5(2.1)* 100 2, .3 (13. ,3)* 0.4(1.8)* 150 - -200 - -DBA/2 mice were 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 cyclophosphamide and spleen c e l l s used 24 hours l a t e r as responder c e l l s . 3 x 10^ responder spleen c e l l s from normal or cyclophosphamide-i n j e c t e d mice were c u l t u r e d with 3 x 10-> i r r a d i a t e d B6 spleen c e l l s f o r 4 days. C y t o t o x i c i t y was assayed against ^ C r - l a b e l l e d EL4 t a r g e t c e l l s i n a 4 hour -^Cr-release assay. 3 x 10^ responder spleen c e l l s from normal or cyclophosphamide-i n j e c t e d mice were c u l t u r e d with 1 x 10* mitomycin C-treated P815 s t i m u l a t o r c e l l s f o r 5 days. C y t o t o x i c i t y was assayed against 5 1 C r - l a b e l l e d P815 t a r g e t c e l l s i n an 18 hour 5 1 C r r e l e a s e assay. Percent c y t o t o x i c i t y was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o to determine l y t i c u n i t s . One l y t i c u n i t was defined as the r e c i p r o c a l number of e f f e c t o r c e l l s to give 25% l y s i s of 1 x 10* t a r g e t c e l l s i n 4 or 18 hours. T o t a l l y t i c u n i t s were c a l c u l a t e d from the number of e f f e c t o r c e l l s recovered at end of 4-5 day c u l t u r e s . S i g n i f i c a n t l y d i f f e r e n t from untreated animals p<0.05. 94 cyclophosphamide was l e s s repeatable. Animals t r e a t e d w i t h 50mg/kg cyclophosphamide o f t e n generated s l i g h t l y lower c y t o t o x i c responses than untreated animals but never as low as animals i n j e c t e d w i t h lOOmg/kg. In subsequent experiments, DBA/2 mice were i n j e c t e d w i t h 50 or lOOmg/kg cyclophosphamide. 2. The Influence of Cyclophosphamide on I n t e r l e u k i n 2 Production. I n t e r l e u k i n 2 has been i m p l i c a t e d i n the generation of c y t o t o x i c T c e l l s (Wagner §_t a l . , 1980) and i t s a d d i t i o n to c u l t u r e s permitted the generation of c y t o t o x i c T c e l l s from thymocyte precursors (Figure 8 ) . DBA/2 mice i n j e c t e d w i t h 50 or lOOmg/kg cyclophosphamide showed a reduced c y t o t o x i c response to P815 but t h i s was r e s t o r e d by the a d d i t i o n of i n t e r l e u k i n 2 c o n t a i n i n g supernatants (Figure 16). Because i n t e r l e u k i n 2 i s important i n the generation of c y t o t o x i c c e l l s and r e s t o r e d the cyclophosphamide-depleted c y t o t o x i c response to P815, the primary defect i n cyclophosphamide-treated mice could have been a reduction i n the number of i n t e r l e u k i n 2-producing T c e l l s (Lattime et a l . , 1981). However, when the l e v e l s of i n t e r l e u k i n 2 induced by concanavalin A - s t i m u l a t i o n of spleen c e l l s were measured, cyclophosphamide i n j e c t i o n had no e f f e c t except at 200mg/kg (Table I X ) . At t h i s high dosage very few spleen c e l l s were recovered from i n j e c t e d mice. Thus, i t appeared cyclophosphamide treatment had not caused a s e l e c t i v e decrease i n the number of i n t e r l e u k i n 2-secreting helper c e l l s . Antigen-induced l e v e l s of i n t e r l e u k i n 2 were a l s o measured from c u l t u r e s of spleen c e l l s from cyclophosphamide-treated mice and e i t h e r 95 Figure 16: The e f f e c t of i n t e r l e u k i n 2 on the cyclophosphamide-depleted c y t o t o x i c response of DBA/2 mice to P815. 3 x 10 5 responder spleen c e l l s from untreated (0,9) or 50 mg/kg (•,•) and 100 mg/kg (A,A) cyclosphosphamide-injected mice were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0,0,A) or with the supernatant from the EL4-IL2 c e l l l i n e as a source of i n t e r l e u k i n 2 (•,I,A). A f t e r 5 days, r e p l i c a t e c u l t u r e s were pooled and assayed 51 against C r - l a b e l l e d P815 target c e l l s . Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) c a l c u l a t e d . 97 TABLE IX: Concanavalin A-induced i n t e r l e u k i n 2 from spleen c e l l s of normal and cyclophosphamide-treated mice. DOSE OF CYCLOPHOSPHAMIDE* INTERLEUKIN 2 UNITS b mg/kg BODY WEIGHT  Expt. 1 0 13.6 25 13.5 50 14.5 75 12.0 100 11.0 Expt. 2 0 17.6 50 22.5 100 19.2 150 14.0 200 5.0* a DBA/2 mice were 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 cyclophosphamide 24 hours before spleen removal. b Spleen c e l l s at 1 x 10 7/ml were c u l t u r e d w i t h 4 ug concanavalin A/ml f o r 24 hours. C e l l f r e e supernatant was harvested and assayed i n the thymocyte s t i m u l a t i o n assay w i t h or without concanavalin A (Methods 7d). A c t i v i t y was c a l c u l a t e d by the d i f f e r e n c e i n cpm w i t h and without concanavalin A and c a l c u l a t e d as a f r a c t i o n of the maximum i n c o r p o r a t i o n . This was p l o t t e d against the d i l u t i o n of supernatant. One i n t e r l e u k i n 2 u n i t was de f i n e d as the d i l u t i o n of sample g i v i n g 37% maximal a c t i v i t y . * S i g n i f i c a n t l y d i f f e r e n t from untreated mice p< 0.05. 98 P815 or C57B1/6 spleen s t i m u l a t o r c e l l s (Table X). The l e v e l s of i n t e r l e u k i n 2 produced i n response to B6 a l l o a n t i g e n s by spleen c e l l s from untreated or cyclophosphamide-injected mice were the same. In c o n t r a s t , cyclophosphamide i n j e c t i o n reduced the l e v e l of i n t e r l e u k i n 2 induced i n response to P815 tumour antigens. The reduction i n i n t e r l e u k i n 2 production p a r a l l e l e d decreased c y t o t o x i c responses of cyclophosphamide-i n j e c t e d mice (Figure 17). Cultures were set up of spleen c e l l s from 0, 50, lOOmg/kg cyclophosphamide-injected mice and mitomycin C-treated P815 s t i m u l a t o r c e l l s . A f t e r 5 days, i n t e r l e u k i n 2 was assayed i n c u l t u r e supernatants and the c y t o t o x i c a c t i v i t y generated i n c u l t u r e was assayed against "^Cr l a b e l l e d t a r g e t c e l l s . These r e s u l t s i n d i c a t e d that the c e l l depleted i n spleen c e l l populations from cyclophosphamide-treated mice was probably not the i n t e r l e u k i n 2-producing c e l l but another c e l l i n v o l v e d i n i n t e r l e u k i n 2 production upon antigen s t i m u l a t i o n . The c e l l s s t i m u l a t e d by the syngeneic tumour c e l l s were e i t h e r present at a lower frequency than c e l l s responding to a l l o a n t i g e n s , or were more s e n s i t i v e to cyclophosphamide i n j e c t i o n . 3. The E f f e c t of Helper C e l l s on the Cyclophosphamide-Depleted C y t o t o x i c  Response. M e r l u z z i et a l . (1979;1980) and V a r k i l a and Hurme (1983) suggest that cyclophosphamide i n h i b i t s the i n d u c t i o n or a c t i v i t y of helper T c e l l s . They could r e s t o r e the c y t o t o x i c responses of cyclophosphamide t r e a t e d mice w i t h normal thymus helper c e l l s . Figure 18 shows that the c y t o t o x i c 99 TABLE X: Antigen-induced i n t e r l e u k i n 2 from spleen c e l l s of normal and cyclophosphamide-injected mice. INTERLEUKIN 2 UNITS DOSE OF CYCLOPHOSPHAMIDE4 ANTIGEN TO STIMULATE SPLEEN CELLS b (mg/kg body weight) CONCANAVALIN A B6 ALLOANTIGENS P815 0 27.6 6.4 7.2 50 25.2 8.0 4.8* 100 24.0 9.2 0.3* DBA/2 mice were i n j e c t e d w i t h cyclophosphamide i n t r a p e r i t o n e a l l y 24 hours before spleens were removed. Spleen c e l l s were c u l t u r e d at 2 x 10^c/ml with 1 x 10-Vml mitomycin-C t r e a t e d P815 or with 2 x 10 6/ml i r r a d i a t e d B6 spleen c e l l s f o r 4 days. Spleen c e l l s at 1 x 10^/ml were c u l t u r e d w i t h 4 yg concanavalin A/ml f o r 24 hours. C e l l - f r e e supernatants were assayed by the supported growth of an i n t e r l e u k i n 2-dependent c e l l l i n e (Methods 7e). One i n t e r l e u k i n 2 u n i t was the d i l u t i o n of supernatant to give 37% maximal a c t i v i t y (with a standard p r e p a r a t i o n ) . No i n t e r l e u k i n 2 a c t i v i t y was detected i n supernatants of c e l l s c u l t u r e d without antigen or mitogen. S i g n i f i c a n t l y d i f f e r e n t from untreated mice p<0.05. 100 Figure 17: The e f f e c t of cyclophosphamide upon the c y t o t o x i c response of DBA/2 mice to P815 and upon P815-induced i n t e r l e u k i n 2 production. 3 x 10^ responder spleen c e l l s from untreated ( 0 ) , 50 mg/kg (A), or 100 mg/kg (!) cyclophosphamide-4 i n j e c t e d DBA/2 mice were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s . R e p l i c a t e c u l t u r e s were pooled and c e l l s assayed f o r c y t o t o x i c i t y against 5 1 C r - l a b e l l e d t a r g e t c e l l s (A). Percent c y t o t o x i c i t y was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s were c a l c u l a t e d (0 mg/kg = 11.4; 50 mg/kg = 10.2; 100 mg/kg =<0.1). Supernatants were assayed f o r i n t e r l e u k i n 2 a c t i v i t y by the supported growth of an 3 i n t e r l e u k i n 2-dependent c e l l l i n e (B). H-Thymidine uptake was p l o t t e d against d i l u t i o n of supernatant and u n i t s of i n t e r l e u k i n 2 a c t i v i t y were c a l c u l a t e d (0 mg/kg = 4.8; 50 mg/kg = 3.2; 100 mg/kg =<0.1). 101 102 Figure 18: The a d d i t i o n of DBA/2 thymocytes r e s t o r e d the cyclophosphamide-depleted c y t o t o x i c response to B6 a l l o a n t i g e n s . 3 x 105 responder spleen c e l l s from normal (0,*) and 100 mg/kg cyclophosphamide-injected DBA/2 mice (A,A) were c u l t u r e d w i t h 3 x 105 i r r a d i a t e d B6 spleen s t i m u l a t o r c e l l s alone (0,A) or wi t h 3 x 105 normal DBA/2 thymocytes (*,A). A f t e r 4 days, r e p l i c a t e c u l t u r e s were pooled and assayed against EL4 tar g e t c e l l s i n a 4-hour ^ C r - r e l e a s e assay. Mean percent c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and t o t a l l y t i c u n i t s (LU) were c a l c u l a t e d . 80 233 LU 9.4:1 18-8:1 37-5:1 75-1 E:T ratio 104 response of spleen c e l l s from cyclophosphamide t r e a t e d DBA/2 mice to C57B1/6 a l l o a n t i g e n s was a l s o r e s t o r e d w i t h normal DBA/2 thymocytes. However, the same normal thymocytes d i d not increase the c y t o t o x i c response to P815 tumour antigens (Figure 19). Figure 11 showed that thymocytes from P815-bearing mice showed more helper a c t i v i t y than normal thymocytes i n the primary iri v i t r o generation of c y t o t o x i c i t y against P815. When thymocytes from tumour-bearing mice were added to spleen c e l l s from cyclophosphamide-injected mice, much higher l e v e l s of c y t o t o x i c i t y were generated than when normal thymocytes were added (Figure 20). Low helper a c t i v i t y i n thymocytes from tumour-bearing mice (as i n Figure 18 comparing the e f f e c t of normal and immune thymocytes with normal spleen c e l l s ) c o i n c i d e d w i t h incomplete r e s t o r a t i o n of the response by spleen c e l l s from cyclophosphamide-treated mice. Thus, the l a c k of e f f e c t of normal thymocytes on the response to P815 (Figure 19) was probably due to low numbers of helper c e l l s i n unprimed animals. 4. The E f f e c t of Cyclophosphamide on C y t o t o x i c Precursor C e l l s . Thymocytes from P815-bearing mice provided s i g n i f i c a n t helper a c t i v i t y to spleen c e l l s from cyclophosphamide-treated mice although the c y t o t o x i c a c t i v i t y generated d i d not reach the l e v e l s seen w i t h spleen c e l l s from untreated mice. The frequency of c y t o t o x i c precursors to P815 i n spleen c e l l populations from untreated and cyclophosphamide-injected mice was determined by l i m i t i n g d i l u t i o n assays (Teh et a l . , 1981). Table XI shows the r e s u l t s of one experiment. The frequency of c y t o t o x i c precursor c e l l s detected i n t h i s assay was s i g n i f i c a n t l y reduced by i n j e c t i o n of cyclophosphamide. 105 Figure 19: Normal DBA/2 thymocytes d i d not r e s t o r e the cyclophosphamide-depleted c y t o t o x i c response to P815. 3 x 10"* responder spleen c e l l s from normal (0,#) or 100 mg/kg cyclophosphamide 4 i n j e c t e d DBA/2 mice (A,A) were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0,A) or wit h 3 x 10 5 normal DBA/2 thymocytes (•.A). R e p l i c a t e c u l t u r e s were pooled a f t e r 5 days and assayed against 51 C r - l a b e l l e d P815 t a r g e t s . Percent c y t o t o x i c i t y (mean ± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and l y t i c u n i t s (LU) c a l c u l a t e d . 107 Figure 20: Re s t o r a t i o n of the cyclophosphamide-depleted c y t o t o x i c response to P815 w i t h thymocytes from normal or tumour-bearing mice. 3 x 10"* responder c e l l s from normal (0,A,D) or 100 mg/kg cyclophosphamide-injected DBA/2 mice (1,1,1) 4 were c u l t u r e d w i t h 1 x 10 mitomycin C-treated P815 s t i m u l a t o r c e l l s alone (0,t) or w i t h 3 x 10 5 DBA/2 thymocytes from normal (A,A) or 6 day tumour-bearing mice (•,•). R e p l i c a t e c u l t u r e s were pooled a f t e r S days and assayed f o r c y t o t o x i c i t y against 5 1 C r - l a b e l l e d P815 ta r g e t c e l l s . Mean percentage c y t o t o x i c i t y (± S.D.) was p l o t t e d against l o g e f f e c t o r : t a r g e t c e l l r a t i o (E:T r a t i o ) and l y t i c u n i t s (LU) were c a l c u l a t e d . % Cytotoxicity rvj .t- cr> o o o o I I I I 1 1 L o 00 109 TABLE XI: The frequency of c y t o t o x i c precursor c e l l s i n DBA/2 spleen c e l l s d i r e c t e d against P815. DOSE OF CYCLOPHOSPHAMIDE* (mg/kg body weight) NUMBER OF RESPONDING SPLEEN CELLS PER WELL0 PERCENT LYSIS C OF P815 TARGETS NUMBER OF RESPONDING WELLS d 51200 25600 12800 6400 3200 1600 22.5 22.9 14.2 2.6 2.8 0.2 14.8 16.2 9. 1, 1. 0, .8 .6 .1 .7 15/24 12/24 12/24 4/24 3/24 1/24 50 51200 25600 12800 6400 3200 1600 13.4 + 9.8 5.2 + 2.4 1.2 + 2.1 10/24 6/24 1/24 1/24 0/24 0/24 100 51200 25600 12800 6400 3200 1600 3.0 + 1.5 1.4 + 1.2 6/24 4/24 3/24 2/24 0/24 0/24 Mice were i n j e c t e d 24 hours before spleens were removed. Responder spleen c e l l s were c u l t u r e d w i t h 25,600 mitomycin C-treated P815 s t i m u l a t o r c e l l s and a 1/100 d i l u t i o n of i n t e r l e u k i n 2 (supernatant from EL4-IL2 c e l l l i n e ) f o r 5 days and assayed i n s p l i t h a l f - w e l l s f o r l y s i s of 5 1 C r - l a b e l l e d P815 and DBA/2 spleen c e l l b l a s t t a r g e t s . Mean percent l y s i s (+ S.E.M.) f o r 24 w e l l s at each responder c e l l d i l u t i o n . 110 TABLE XI; Continued FREQUENCY6 CHI' PERCENT LYSIS C OF D2 BLAST TARGETS NUMBER OF RESPONDING WELLS1 FREQUENCY6 CHI 2 124 (1/8100) 2.45 2.1 + 1.8 5.0 + 3.2 1.1 + 1.0 3/24 7/24 2/24 1/24 0/24 2/24 1.9 2.83 109 2.51 - 1/24 (1/9200) - 0/24 0/24 1/24 0/24 1/24 18.4 (1/54,300) 3.16 1/24 0/24 1/24 1/24 0/24 0/24 • > ±Cr re l e a s e d exceeded the mean spontaneous r e l e a s e by 2.2 standard d e v i a t i o n s (8.1%P815; 7.9% b l a s t s ) . Frequency (per 10^ spleen c e l l s ) was c a l c u l a t e d by computer using the zero-order term of the Poisson d i s t r i b u t i o n . CHI 2 i n d i c a t e s goodness of f i t of l i n e c a l c u l a t e d from the f r a c t i o n of non-responding w e l l s p l o t t e d against the number of responding spleen c e l l s per w e l l . I l l The absolute frequency of precursor c y t o t o x i c c e l l s d i r e c t e d against P815, v a r i e d between experiments but the cyclophosphamide-induced reduction of precursor frequency was observed i n 15 out of 16 experiments. The mean frequency of c y t o t o x i c c e l l precursors i n cyclophosphamide-injected mice, c a l c u l a t e d from those 16 experiments was expressed as a percentage of the frequency c a l c u l a t e d f o r normal spleen c e l l s (Table X I I ) . The i n j e c t i o n of lOOmg/kg cyclophosphamide s i g n i f i c a n t l y reduced the frequency of c y t o t o x i c c e l l precursors to 20% of tha t measured f o r untreated mice. One f a c t o r l e a d i n g to t h i s v a r i a t i o n i n c a l c u l a t e d precursor frequency was the source and l e v e l of i n t e r l e u k i n 2 added i n t o l i m i t i n g d i l u t i o n c u l t u r e s (Table X I I I ) . G e n e r a l l y , the d i l u t i o n of i n t e r l e u k i n 2-containing supernatant used i n l i m i t i n g d i l u t i o n assays corresponded with that g i v i n g optimal i n t e r l e u k i n 2 a c t i v i t y i n e i t h e r thymocyte s t i m u l a t i o n assays or T c e l l growth f a c t o r assays. Supernatants from concanavalin A-stimulated spleen c e l l s c o n t a i n a v a r i e t y of s t i m u l a t o r y f a c t o r s p o s s i b l y d i s t i n c t from those secreted by the EL4.IL2 c e l l l i n e ( F a r r a r et a l . , 1980; Harwell et a l . , 1980; Shimizu et a l . , 1980). One p o s s i b i l i t y to e x p l a i n the higher frequency of c y t o t o x i c c e l l precursors seen with spleen c e l l s from untreated mice was due to the a c t i v a t i o n of n o n s p e c i f i c c y t o t o x i c i t y by the i n t e r l e u k i n 2 pr e p a r a t i o n . N o n - s p e c i f i c c y t o t o x i c i t y was assayed by d i v i d i n g each c u l t u r e w e l l and then assaying the h a l f w e l l s against d i f f e r e n t 5"""Cr-labelled t a r g e t c e l l s (Teh et a l . , 1981). P815 t a r g e t c e l l s were used to measure s p e c i f i c c y t o t o x i c i t y and DBA/2 spleen c e l l b l a s t s were used as t a r g e t c e l l s to assay the 112 TABLE X I I : The frequency of anti-P815 c y t o t o x i c precursor c e l l s i n DBA/2 mice i n j e c t e d w i t h cyclophosphamide. DOSE OF CYCLOPHOSPHAMIDE FREQUENCY PER 10 6 PERCENTAGE NUMBER (mg/kg body weight) SPLEEN CELLS OF OF mean + S.E.M. NORMAL SPLEEN EXPERIMENTS 0 50 100 528 + 215 302 + 178 70 + 29 100.0 67.1 18.6 16 11 12 L i m i t i n g numbers of responder spleen c e l l s from normal and cyclophosphamide-treated mice were c u l t u r e d w i t h mitomycin C-treated P815 s t i m u l a t o r c e l l s and a source of i n t e r l e u k i n 2. Cul t u r e s were assayed a f t e r 5 days against ^•'•Cr-labelled P815 t a r g e t c e l l s . The frequency of c y t o t o x i c precursor c e l l s was assayed as o u t l i n e d i n M a t e r i a l s and Methods 6a and c. TABLE X I I I : The dependence of precursor c y t o t o x i c c e l l frequency, c a l c u l a t e d by l i m i t i n g d i l u t i o n a n a l y s i s , on the l e v e l s of i n t e r l e u k i n 2 i n c u l t u r e . DILUTION OF INTERLEUKIN 2 CALCULATED FREQUENCY INTERLEUKIN 2 a ACTIVITY (U/ml) b PER 10 6 SPLEEN CELLS none - 23 1/8 0.50(45.0) 83 1/16 0.84(22.5) 140 1/32 1.0 (11.3) 207 1/64 0.95 (5.6) 81 1/128 0.64 (2.8) 81 1/256 0.4 (1.4) 67 1/512 0.24 (0.7) 38 I n t e r l e u k i n 2 was a p a r t i a l l y p u r i f i e d supernatant from the (EL4-IL2) c e l l l i n e (Methods, 7c). I n t e r l e u k i n 2 a c t i v i t y was assayed i n the thymocyte s t i m u l a t i o n assay (Methods, 7d) and a c t i v i t y of each d i l u t i o n was expressed as a f r a c t i o n of the maximal response (with a 1/32 d i l u t i o n ) . This i n t e r l e u k i n 2 prepa r a t i o n was estimated at 360 uni t s / m l so the units/ m l were determined from the d i l u t i o n of preparation used. Spleen c e l l s from normal DBA/2 mice c u l t u r e d at l i m i t i n g d i l u t i o n w i t h n o n - l i m i t i n g numbers of mitomycin C-treated P815 s t i m u l a t o r s and the appropriate d i l u t i o n of i n t e r l e u k i n 2. C y t o t o x i c i t y was assayed a f t e r 5 days with - ^ C r - l a b e l l e d P815 tar g e t c e l l s . Frequency was c a l c u l a t e d as i n Methods, 6c. 114 n o n - s p e c i f i c c y t o t o x i c i t y (Table X). The percentage l y s i s by each w e l l against the two ta r g e t s was then p l o t t e d on one f i g u r e (Figure 21). Only P 8 1 5 - s p e c i f i c c y t o t o x i c i t y was generated i n these c u l t u r e s as shown by the s c a t t e r of points near the y axi s w i t h few w e l l s showing p o s i t i v e l y s i s of both w e l l s or DBA/2 spleen c e l l b l a s t s alone. No s i g n i f i c a n t c y t o t o x i c i t y was detected i n w e l l s without any added i n t e r l e u k i n 2. A t o t a l of 61% of w e l l s were p o s i t i v e f o r e i t h e r t arget of which 16% were p o s i t i v e against both, 73% p o s i t i v e on P815 alone, and 11% were p o s i t i v e against DBA/2 b l a s t s only. S i m i l a r r e s u l t s were obtained w i t h EL4 tumour t a r g e t c e l l s to assay n o n s p e c i f i c c y t o t o x i c i t y . When h a l f - w e l l a n a l y s i s was done using responder spleen c e l l s from cyclophosphamide-injected mice, s i m i l a r low l e v e l s of n o n - s p e c i f i c c y t o t o x i c i t y were detected (Table X I ) . This data was not p l o t t e d . These r e s u l t s showed that the higher frequency of c y t o t o x i c precursor c e l l s measured i n normal spleen c e l l s when compared w i t h c e l l s from cyclophosphamide-injected mice was not due to high l e v e l s of n o n - s p e c i f i c c y t o t o x i c i t y but rat h e r to a reduction i n precursor frequency a f t e r cyclophosphamide i n j e c t i o n . 5. Summary DBA/2 mice i n j e c t e d w i t h v a r y i n g doses of cyclophosphamide showed enhanced or decreased in v i t r o c y t o t o x i c responses against B6 a l l o a n t i g e n s and P815 tumour antigens depending upon the dose i n j e c t e d (Table V I I I ) . The cyclophosphamide depleted c y t o t o x i c response to a l l o a n t i g e n s could be re s t o r e d to the l e v e l observed i n normal mice by the a d d i t i o n of normal thymocyte helper c e l l s (Figure 18). The response to P815 tumour-antigens 115 Figure 21: The antigen s p e c i f i c i t y of c y t o t o x i c c e l l s generated i n l i m i t i n g d i l u t i o n c u l t u r e s . L i m i t i n g numbers of spleen c e l l s (51,200 •; 25600 •; 12 , 8 0 0 f ) from normal DBA/2 mice were c u l t u r e d w i t h n o n l i m i t i n g numbers of mitomycin C-treated P815 st i m u l a t o r s and a 1/250 d i l u t i o n of i n t e r l e u k i n 2 (from the EL4-IL2 c e l l l i n e ) . A f t e r 5 days each w e l l was d i v i d e d i n two 3 51 and the h a l f - w e l l s were assayed against 2 x 10 Cr-l a b e l l e d P815 and DBA/2 concanavalin A-stimulated spleen c e l l b l a s t s as target c e l l s . C y t o t o x i c i t y was c a l c u l a t e d f o r each h a l f w e l l and c y t o t o x i c i t y w i t h the P815 t a r g e t p l o t t e d against c y t o t o x i c i t y w i t h the D2 b l a s t s t a r g e t so that each poin t represents one c u l t u r e w e l l . Wells were scored as p o s i t i v e where the 5 1 C r rel e a s e exceded the spontaneous rel e a s e by 2.2 standard d e v i a t i o n s (8.1% l y s i s of P815 ( ); 7.9% l y s i s of D2 b l a s t s ( ). Frequencies are recorded i n Table XI. 116 100i 80H 60H LO 5 • Q_ 40-• • 20i 20 30 AO 50 100 % Lysis of D2 blasts 117 was p a r t i a l l y r e s t o r e d by helper c e l l s present i n P815 tumour-bearing mice but only i n low numbers i n normal mice (Figures 19,20). I n t e r l e u k i n 2 al s o r e s t o r e d the c y t o t o x i c response to the P815 syngeneic tumour (Figure 14). DBA/2 spleen c e l l s could be st i m u l a t e d w i t h mitogen or antigen to produce i n t e r l e u k i n 2. Cyclophosphamide-treated spleen c e l l s produced l e s s i n t e r l e u k i n 2 a f t e r s t i m u l a t i o n w i t h P815 c e l l s than d i d normal spleen c e l l s , but s i m i l a r l e v e l s were produced a f t e r mitogen s t i m u l a t i o n (Tables IX, X; Figure 17). Thus, cyclophosphamide appeared to a f f e c t an an t i g e n - r e a c t i v e c e l l i n v o l v e d i n i n t e r l e u k i n 2 production. Cyclophosphamide a l s o reduced the frequency of c y t o t o x i c precursor c e l l s d i r e c t e d against P815 (Tables X I , X I I ) . Hence, the i n j e c t i o n of cyclophosphamide r e s u l t e d i n at l e a s t two l e s i o n s i n DBA/2 mice -redu c t i o n of the number of c y t o t o x i c precursor c e l l s d i r e c t e d against a syngeneic tumour and reduced a c t i v i t y of an a n t i g e n - r e a c t i v e helper c e l l i n v o l v e d i n the production of i n t e r l e u k i n 2 a f t e r antigen s t i m u l a t i o n . 118 CHAPTER I I I : THE IN VIVO ACTIVITY OF P815-INDUCED HELPER CELLS 1. Clearance of P815 Tumour C e l l s by DBA/2 Mice Primed to P815. In the preceding chapters, evidence was presented f o r r a d i a t i o n - r e s i s t a n t , a n t i g e n - s p e c i f i c helper T c e l l s i n v o l v e d i n the c y t o t o x i c response to the DBA/2 syngeneic tumour P815. These experiments examined the i n v i t r o a c t i v i t y of a n t i g e n - s p e c i f i c helper c e l l s but gave no i n s i g h t i n t o the p o s s i b l e r o l e f o r helper c e l l s i n ir i v i v o responses of DBA mice to the P815 tumour. The t r a n s f e r of s e n s i t i z e d c e l l s (mostly T c e l l s ) and immune sera has r e s u l t e d i n both a c c e l e r a t e d r e j e c t i o n and enhanced growth of tumour a l l o g r a f t s i n the r e c i p i e n t animal i n a number of host tumour combinations (Weiss, 1980; Fefer and G o l d s t e i n , 1982). The data suggest that c e l l s which a r i s e e a r l y i n the response to a tumour ( i n v i v o and in. v i t r o ) enhance the r e j e c t i o n of the tumour whereas c e l l s a r i s i n g l a t e r (presumably suppressor c e l l s ) enhanced tumour growth. In most experiments, c e l l s were t r a n s f e r r e d i n t o the r e c i p i e n t animal e i t h e r before or a f t e r tumour t r a n s p l a n t a t i o n and the growth of the tumour was measured (Takatsu et a l . , 1980; Cheever et a l . , 1981; Fernandez-Cruz et a l . , 1982; M i l l s and North, 1983). Clearance of l a b e l l e d tumour c e l l s has a l s o been used to assess the e f f e c t s of t r a n s p l a n t e d c e l l populations (Hofer et a l . , 1969; Carlson and Terres, 1978; M i l l s et a l . , 1980). In the studies reported here, DBA/2 mice were primed w i t h P815 to 125 induce helper T c e l l s , and then challenged w i t h I - l a b e l l e d P815 119 tumour c e l l s . The clearance of the challenge dose was fol l o w e d by measurement of r a d i o a c t i v i t y remaining i n the mouse which r e f l e c t s the number of l i v e tumour c e l l s p e r s i s t i n g i n the animal (Hofer e_t a l . , 1969). Since even low doses of P815 were l e t h a l to DBA/2 mice, a modified form of the tumour was used to prime mice before challenge with the pa r e n t a l tumour. Dr. T. Boon's l a b o r a t o r y have generated v a r i a n t s of the P815-X2 tumour by NTG mutagenesis (Uyttenhove et a l . , 1980; Boon et a l . , 1980). These v a r i a n t s grow normally i n v i t r o but are r e j e c t e d by syngeneic mice and w i l l grow only i n s u b l e t h a l l y i r r a d i a t e d DBA/2 mice and have thus, been termed turn . I t has been proposed that the r e s i s t a n c e of normal mice to growth of these tumours r e l a t e s to increased immunogenicity of the v a r i a n t (Uyttenhove et^ a l . , 1980). We reasoned that one way i n which the immunogenicity of the tumour might be increased would be by a p r e f e r e n t i a l a c t i v a t i o n of T helper c e l l s over T suppressor c e l l s . These experiments were designed to t e s t t h i s premise. DBA/2 mice were primed to P815 with a v a r i e t y of pr o t o c o l s as d e t a i l e d i n Table XIV. Unprimed animals served as c o n t r o l s . Four to twelve days a f t e r the priming i n j e c t i o n , mice were challenged w i t h 1 x 10^ 125 I - l a b e l l e d P815 c e l l s i . p . and tumour clearance measured as the 125 percentage of I remaining over 4-6 days. Figures 22 - 24 show the r e s u l t s from a r e p r e s e n t a t i v e experiment (Table XIV, No. 3 ) . Groups of 4 mice were e i t h e r unprimed or i n j e c t e d subcutaneously w i t h P815 tumour 6 125 c e l l s . Eleven days l a t e r they were a l l given l x 10 I - l a b e l l e d P815 c e l l s i . p . and tumour clearance measured. When mice were primed with 2 i n j e c t i o n s of P21 (a turn v a r i a n t ) (Figure 22), 2 out of 4 animals 120 TABLE XIV: Clearance of P815 tumour c e l l s by DBA/2 mice primed against P815 PRIMING PROTOCOL TIME OF EXPT. • 1 2 5 I.P815 C NO. ANTIGEN* DOSE INJECTIONS ROUTE0 INJECTION (DAYS AFTER PRIMING) 1 a P21 1 x 10 4 1 sc 6 b P21 1 x 10 4 1 fp 6 c P21 1 x 10 4 1 bt 6 2 a P21 1 x 10 4 1 sc 4 b P21 1 x 10 4 1 sc 6 c P21 1 x 10 4 1 sc 8 d P21 1 x 10 4 1 sc 10 e P21 1 x 10 4 1 sc 12 3 a P815 1 x 10 4 1 sc 11 b P21 2 x 10 5 2 sc 11 c P911 2 x 10 5 2 sc 11 4 a P21 1 x 10 4 3 sc 6 b P21 1 x 10 4 2 sc 6 c P21 1 x 1 0 4 1 sc 6 5 a P21 1 x 10 3 1 bt 6 b P21 1 x 10 4 1 bt 6 c P21 1 x 10 5 1 bt 6 d P21 1 x 10 6 1 bt 6 a P815 i s the parental . tumour c e l l l i n e ; P21 and P911 are P815 turn -v a r i a n t s (Uyttenhove et a l (1980) which do not grow i n DBA/2 mice. sc = subcutaneous i n the r i g h t hind flank., f p = i n t r a d e r m a l l y i n both hind footpads, bt = subcutaneous at the base of the t a i l . 121 TABLE XIV: Continued. ANIMALS CONTROL ANIMALS CLEARING 1 2 5 I . P 8 1 5 SURVIVAL CLEARING 1 2 5 I . P 8 1 5 SURVIVAL EXPT. AT SAME RATE TIME FASTER THAN CONTROLS TIMES NO. AS CONTROLS** (DAYS) e (DAYS) 1 a 6 b 6 12.0+0.6 c 3 3 19;26;28 2 a 8 b 4 4 14;14;16;17 c 7 12.2+0.2 1 14 d 8 - -e 8 - -3 a 2 2 22;25 b 2 12.8 + 0.8 2 31;55 c 3 1 66 4 a 3 3 19;19;17 b 5 13.9 + 1.5 4 14;15;17;24 c 6 5 a 4 2 25;31 b 5 17.3 + 3.1 1 21 c 4 2 20;22 d 3 3 22;37;32 c 1 x 10 6 1 2 5 I - l a b e l l e d P815 c e l l s i n j e c t e d i n 0.1 ml i n t r a p e r i t o n e a l l y . An equal number of unprimed mice as i n each t e s t group were used as c o n t r o l s . e Mean + S.E.M. 122 Figure 22: Clearance of P815 by DBA/2 mice primed w i t h two i n j e c t i o n s of P21 tumour c e l l s . Four DBA/2 mice were primed with 2 subcutaneous i n j e c t i o n s of 2 x 10? P21 tumour c e l l s over a 3 week per i o d . Eleven days a f t e r the second i n j e c t i o n , mice 6 125 were challenged w i t h 1 x 10 , I - l a b e l l e d P815 c e l l s . T o t a l body counts were measured on 4 consecutive days and 125 p l o t t e d as percentage I remaining f o r i n d i v i d u a l mice (• • ) . Four unprimed mice served as c o n t r o l s (• • ) . S u r v i v a l time f o r each mouse was recorded and mean s u r v i v a l time (± S.E.M.) f o r c o n t r o l s was c a l c u l a t e d . See Table XIV, Experiment 3b. 124 c l e a r e d the tumour much f a s t e r than the unprimed c o n t r o l s and survived longer (31 and 55 days compared with 12.8). When mice were primed w i t h 2 i n j e c t i o n s of P911, a second turn v a r i a n t (Figure 23), 1 out of 4 animals c l e a r e d the tumour f a s t e r than the c o n t r o l animals and a l s o s u r v i v e d longer (66 days compared with 12) than the unprimed mice. When mice were primed w i t h one i n j e c t i o n of the parent P815 tumour, 2 out of 4 animals c l e a r e d the tumour f a s t e r and sur v i v e d longer (22 and 25 days compared w i t h 12) than unprimed mice (Figure 24). When only those primed mice which r a p i d l y c l e a r e d the tumour were considered, priming with the turn v a r i a n t s P21 and P911 r e s u l t e d i n longer s u r v i v a l times than priming w i t h the parent tumour. Death i n the mice primed w i t h p a r e n t a l P815 may have r e s u l t e d from e i t h e r the priming i n j e c t i o n or challenge dose since the p a r e n t a l P815 l i n e grows i n DBA/2 mice. When s u r v i v a l i n those mice was c a l c u l a t e d from the priming i n j e c t i o n of P815, the s u r v i v a l times were comparable to those of mice primed w i t h P21 or P911. Results obtained i n other experiments were s i m i l a r to these (Table XIV). In a l l 5 experiments, the c o n t r o l , unprimed animals c l e a r e d the tumour at a s i m i l a r r a t e to those shown i n Figures 22, 23, 24. The s u r v i v a l times of these animals were al s o very s i m i l a r w i t h a mean (± SE) of 13.6 ± 1.9 days f o r the 36 c o n t r o l mice i n j e c t e d . Twenty nine of one hundred and f i f t e e n primed mice showed some degree of enhanced clearance of the challenge dose of P815, and prolonged s u r v i v a l when compared with c o n t r o l animals. Not a l l immunization p r o t o c o l s were s u c c e s s f u l , and only some animals were s i g n i f i c a n t l y primed by any one p r o t o c o l . One subcutaneous i n j e c t i o n of tumour ( e i t h e r i n the f l a n k or at 125 Figure 23: Clearance of P815 by DBA/2 mice primed w i t h 2 i n j e c t i o n s of P911 tumour c e l l s . Four DBA/2 mice were primed with 2 subcutaneous i n j e c t i o n s of 2 x 10"* P911 tumour c e l l s over a 3 week pe r i o d . Eleven days a f t e r the second i n j e c t i o n , mice 6 125 were challenged with 1 x 10 , I - l a b e l l e d P815 c e l l s . T o t a l body counts were measured on 4 consecutive days and 125 p l o t t e d as percentage I-remaining f o r i n d i v i d u a l mice (• • ) . Four unprimed mice served as c o n t r o l s (• • ) . S u r v i v a l time f o r each mouse was recorded and mean s u r v i v a l time (± S.E.M.) f o r c o n t r o l s c a l c u l a t e d . See Table XIV, Experiment 3c. 127 Figure 24: Clearance of P815 by DBA/2 mice primed w i t h P815 tumour c e l l s . Four DBA/2 mice were primed with a subcutaneous i n j e c t i o n of 4 1 x 10 P815 tumour c e l l s . Eleven days l a t e r , mice were 6 125 challenged w i t h 1 x 10 , I l a b e l l e d P815 c e l l s . T o t a l body counts were measured on 4 consecutive days and p l o t t e d as 125 percentage I remaining f o r each mouse (• • ) . Four unprimed mice served as c o n t r o l s (• • ) . S u r v i v a l time f o r each mouse was recorded and the mean s u r v i v a l of c o n t r o l s (± S.E.M.) was c a l c u l a t e d . See Table XIV, Experiment 3a. 128 1 — i 1 r 0 1 2 3 A Days 129 the base of the t a i l ) 6 days before challenge, was the most s u c c e s s f u l priming p r o t o c o l both w i t h respect to the number of animals showing enhanced tumour clearance and the s u r v i v a l time of the primed mice. This was s i m i l a r to the i n j e c t i o n used to generate helper c e l l s w i t h i n v i t r o a c t i v i t y as discussed i n Chapters I and I I . 2. Clearance of P815 Tumour C e l l s by DBA/2 Mice I n j e c t e d w i t h C e l l s from  P815 Primed Animals. The second group of experiments performed examined the e f f e c t of P815 induced helper c e l l s on the i n v i v o response to P815 i n an adoptive t r a n s f e r system using syngeneic r e c i p i e n t s . Thymus, lymph node or spleen c e l l s from DBA/2 mice w i t h small tumour loads were t r a n s f e r r e d w i t h or without mitomycin C-treated P815 c e l l s i n t o normal DBA/2 mice. Controls r e c e i v e d normal thymus, lymph node or spleen c e l l s w i t h or without P815 c e l l s . One to e i g h t days l a t e r , mice were challenged w i t h 1 x 10 6 125 I - l a b e l l e d P815 c e l l s and the tumour clearance again measured as 125 percentage I remaining i n the i n j e c t e d mouse and i t s s u r v i v a l time a f t e r challenge (Table XV). Figure 25 shows the r e s u l t s of one r e p r e s e n t a t i v e experiment (Table XV, Number 6). Groups of 8 mice were i n j e c t e d w i t h 1 x 10"* mitomycin C-treated P815 alone or w i t h 2.5 x 10 7 thymocytes from normal or P815 primed mice. Four days l a t e r they r e c e i v e d 6 125 a challenge dose of 1 x 10 l i v e I - l a b e l l e d P815 c e l l s . Animals i n j e c t e d w i t h tumour c e l l s alone or tumour c e l l s w i t h normal thymocytes, c l e a r e d the tumour at the same rate and s u r v i v e d e q u a l l y long (Figure 25). Two of e i g h t mice i n j e c t e d w i t h immune thymocytes and P815 c l e a r e d 130 TABLE XV: Clearance of P815 by DBA/2 mice i n j e c t e d w i t h c e l l s from tumour-primed mice. CELLS TRANSFERRED INTO DBA/2 MICE EXPT. DAY OF PRIMING 4 CELL 0 TUMOUR CELLS 0 TYPE (NO.) 1 a 5d P815 Spleen (4 X 10 7) b 5d P815 Thymus (3 X 10 7) — 2 a 6d P815 Spleen (4 X 10 7) b 6d P815 Thymus (3 X 10 7) — 3 a 6d P815 Spleen (4 X 10 7) P815 b 6d P815 Thymus (4 X 10 7) P815 c 6d P815 Lymph Node (2 x 10 7) P815 4 a 21d P21 Thymus (2 X 10 7) P21 b 21d P21 Lymph Node (2 x 10 7) P21 c 21d P911 Thymus (2 X 10 7) P911 d 21d P911 Lymph Node (2 x 10 7) P911 5 a 6d P815 Thymus (2 X 10 7) P815 b 6d P815 Lymph Node (2 x 10 7) P815 6 6d P815 Thymus (2. 5 x 10 7) P815 7 a 6d P815 Thymus (2 X 10 7) P815 b 6d P815 Thymus (2 X 10 7) — 8 a 6d P815 Thymus (0. 5 x 10 7) P815 b 6d P815 Thymus (1 X 10 7) P815 c 6d P815 Thymus (2. 5 x 10 7) P815 9 a 6d P815 Thymus (1 X 10 7) P815 b 6d P815 Thymus (1 X 10 7) P815 c 6d P815 Thymus (1 X 10 7) P815 d 6d P815 Thymus (1 X 10 7) P815 131 TABLE XV: Continued. TIME (DAYS) 1 2 5 I . P 8 1 5 EXPT. INJECTED AFTER CELL TRANSFER** ANIMALS CLEARING 1 2 5 I . P 8 1 5 AT SAME RATE AS CONTROLS CONTROL*5 SURVIVAL TIME (DAYS) ANIMALS CLEARING SURVIVAL 1 2 5 I . P 8 1 5 TIME FASTER THAN (DAYS) CONTROLS 1 a b 6 3 16.5 +0.8 18.5 + 0.6 2 1 20; 23 20 2 a b 1 1 6 4 16.8 + 1.0 17.0 + 1.4 15; 18 3 a b c 6 6 3 13.0 + 0.7 14.4 + 1.0 14.2 + 0.7 12; 13 4 a b c d 4 4 4 4 6 6 5 3 17.5 + 1.0 13.7 + 0.7 17.8 + 0.7 15.7 + 0.3 1 3 16 16; 16;17 5 a b 4 6 15.5 + 1.6 17.0 + 0.2 14; 17 12.4 + 1.0 23; 36 7 a b 3 2 15.5 + 0.5 14.5 + 0.4 1 2 14 16;13 8 a b c 4 4 4 15.0 + 0.2 17.0 + 0.8 16.0 + 1.0 9 a b c d 1 2 4 8 4 2 2 3 16.1 + 0.4 18.5 + 0.3 19.5 + 0.3 16.5 + 1.3 2 18;18 2 20;34 1 29 TABLE XV: Legend P815 i s the pa r e n t a l tumour c e l l l i n e . P21, P911 are turn" v a r i a n t s (Uyttenhove e_t a l , 1980) . A l l animals were i n j e c t e d subcutaneously w i t h 1 x 10 4 c e l l s . C o n t r o l mice recei v e d the equivalent dose of normal c e l l s . A l l mice were 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 lymphoid c e l l s ± tumour c e l l s . 1 x 10^ mitomycin C-treated c e l l s . Mice challenged i n t r a p e r i t o n e a l l y w i t h 1 x 10 6, 1 2 5 I - l a b e l l e d tumour c e l l s i n 0.1 ml. Mean ± S.E.M. f o r c o n t r o l mice. Same number of c o n t r o l mice i n j e c t e d as i n experimental groups. Clearance r a t e w i t h i n 2x S.E.M. of c o n t r o l mean considered to be at same rate as c o n t r o l s . 133 25: Clearance of P815 by DBA/2 mice i n j e c t e d w i t h thymocytes from P815-bearing mice. Groups of 8 mice were i n j e c t e d w i t h 1 x 10 5 mitomycin C-treated P815 alone (• •) or w i t h 2.5 x 10 7 thymocytes from normal (A A) or 6 day 4 P815-bearing mice (•—•) (1 x 10 P815 subcutaneous i n j e c t i o n ) . Four days l a t e r they were challenged w i t h 6 125 1 x 10 , I - l a b e l l e d P815 c e l l s . Tumour clearance was 125 p l o t t e d as percentage I-remaining on 4 consecutive days. (Mean of 8 c o n t r o l animals). S u r v i v a l time a f t e r challenge 125 w i t h I-P815 was recorded. (Mean ± S.E.M. f o r each 8 c o n t r o l animals). See Table XV, Experiment 6. 134 I 1 1 -T r 0 1 2 3 4 Days 135 the tumour much f a s t e r than e i t h e r c o n t r o l group, and survived longer (23 and 36 days compared with 12.4 ± 13.3 days). Three other mice c l e a r e d the tumour somewhat f a s t e r than the c o n t r o l s given normal thymocytes although t h e i r s u r v i v a l time was not longer than the c o n t r o l mice. Results obtained i n other experiments were s i m i l a r to these (Table XV). Again the c o n t r o l s c l e a r e d the tumour at s i m i l a r r a t e s i n a l l 9 experiments and mean s u r v i v a l times f o r c o n t r o l s were s i m i l a r (16.0 ± 1.8 days). Twenty one of one hundred and twenty animals i n j e c t e d w i t h primed c e l l s showed some degree of enhanced clearance of the l a b e l l e d c e l l s when compared with c o n t r o l mice i n j e c t e d w i t h normal c e l l s . Many of these a l s o survived longer than the c o n t r o l mice. 3. Summary The in. v i v o a c t i v i t y of helper c e l l s a c t i v e i n the _ v i t r o c y t o t o x i c response to P815 was measured i n two ways. F i r s t l y , mice were primed w i t h a low dose of a turn v a r i a n t of P815, and then challenged w i t h a l a r g e r 125 dose of I - l a b e l l e d P815 c e l l s (Table XIV; Figures 22-24). Twenty s i x percent of animals primed to P815 c l e a r e d the challenge dose of P815 f a s t e r than unprimed c o n t r o l animals, and of these 88 percent s u r v i v e d longer than the c o n t r o l mice. Secondly, when c e l l s from tumour-bearing mice were t r a n s f e r r e d i n t o normal mice (Table XV; Figure 25), 18 percent of the r e c i p i e n t s c l e a r e d a challenge dose of P815 c e l l s f a s t e r than mice i n j e c t e d w i t h normal c e l l s . Only 53 percent of these mice survived s i g n i f i c a n t l y longer than c o n t r o l groups of mice i n j e c t e d w i t h normal c e l l s or no c e l l s at a l l . 136 CHAPTER IV: ENHANCING THE IN VITRO CYTOTOXIC RESPONSE TO  P815 USING AN EXOGENOUS HELPER DETERMINANT The primary c y t o t o x i c responses of DBA/2 mice to the syngeneic tumour P815, were extremely v a r i a b l e , and although c y t o t o x i c and helper a c t i v i t i e s were detected i n tumour-bearing mice, suppression of the c y t o t o x i c response occurred as tumour growth progressed. M i t c h i s o n (1970) f i r s t suggested the p o s s i b i l i t y of enhancing the response to tumours by presenting the tumour to the immune system i n a s s o c i a t i o n w i t h a strong antigen. I f the animal was already immune to the e x t r a a n t i g e n i c determinants, helper c e l l s may be able to enhance the response to the as s o c i a t e d tumour antigen. This approach was extended to the i_n v i t r o response to P815 by s t i m u l a t i n g c e l l s from tuberculin-primed mice with PPD-coupled tumour c e l l s and assaying c y t o t o x i c a c t i v i t y against uncoupled P815 tumour c e l l s . 1. Coupling T u b e r c u l i n PPD to P815 Tumour C e l l s . PPD was coupled to P815 tumour c e l l s w i t h e i t h e r the PPD-concanavalin A conjugate of Lachmann and S i k o r a (1978) (Methods 10b) or using a carbodiimide reagent ECDI (Methods 10b). To q u a n t i t a t e the degree of 125 c o u p l i n g of PPD to the tumour c e l l s w i t h ECDI, I - l a b e l l e d PPD was 125 bound to P815. The I - l a b e l l e d PPD was prepared by H. Neyndorff using a standard chloramine T method. A f i x e d number of TCA - p r e c i p i t a b l e counts 125 of I - l a b e l l e d PPD was mixed with d i f f e r e n t amounts of u n l a b e l l e d PPD 137 and incubated w i t h mitomycin C-treated P815 c e l l s . The """[-PPD s p e c i f i c a l l y bound to the c e l l s was c a l c u l a t e d from the counts bound e i t h e r i n the presence or absence of the coupling reagent ECDI (Table XVI). Approximately 2-5 percent of the added PPD was bound to the tumour c e l l s . This compares w e l l w i t h the r e s u l t s of Takatsu e_t a l . (1980) who measure about 5% of PPD bound to X5563 tumour c e l l s w i t h ECDI. 2. Lymph Node P r o l i f e r a t i v e Responses of PPD-Primed DBA/2 Mice to PPD  and PPD-Coupled P815. The object of t h i s set of experiments was to determine i f primed T helper c e l l s could recognize PPD when i t was bound to P815 c e l l s . I t has been suggested that the predominant c e l l type responding to antigen i n i n  v i t r o p r o l i f e r a t i v e assays are of the helper c e l l c l a s s (Cantor and Boyse, 1975; Yano et a l . , 1977), so priming was assessed i n the lymph node p r o l i f e r a t i v e assay (Lee §_t a l . , 1979; S i k o r a and Levy, 1980). Lymph node c e l l s from unprimed mice and PPD primed mice (see Table XVII) (some were given low doses of cyclophosphamide before priming i n an e f f o r t to remove suppressor c e l l s ) showed very l i t t l e p r o l i f e r a t i v e a c t i v i t y when c u l t u r e d without antigen. The only exception was immune mouse 1 i n experiment 1 where the lymph node c e l l s p r o l i f e r a t e d very w e l l without antigen (Table X V I I I ) . This may r e f l e c t the i t i v i t r o s t i m u l a t i o n of lymph node c e l l s by serum p r o t e i n or n o n - s p e c i f i c a c t i v a t i o n in, v i v o (e.g. by i n f e c t i o n ) of c e l l s which then p r o l i f e r a t e d i r i v i t r o . Such high l e v e l s of n o n - s p e c i f i c p r o l i f e r a t i o n may have masked s p e c i f i c a c t i v a t i o n of lymph node c e l l s by antigen i n c u l t u r e . When c e l l s from unprimed or PPD-primed animals were 138 TABLE XVI: Coupling 1 2 5 I - l a b e l l e d - P P D to P815 c e l l s w i t h ECDI. EXPERIMENT P815m a PPD INPUT CPM % BOUND NUMBER mg/ml 1 2 5 I - P P D b 1 2 5 I - P P D C 5 x 10 6 5.0 25,000 1.7 1.0 7.0 0.1 6.3 0.0 7.1 x 10 6 5.0 29,000 4.0 1.0 4.2 0.5 5.0 0.1 5.4 0.01 6.1 0.0 6.7 5 x 10 6 5.0 800,000 0.5 1.0 1.5 0.5 2.1 0.1 2.7 0.01 3.3 0.0 2.9 Mean % bound (± SD) 5.0 2.1 ± 1.8 1.0 4.2 ± 2.8 0.5 3.6 ± 2.1 0.1 4.3 ± 1.8 0.01 4.7 ± 2.0 0.0 5.6 ± 2.3 a Mitomycin C-treated P815 (at 10 7 per ml). Reaction set up with or without ECDI at 0.5 mg/ml i n 0.5 ml PBS, pH 7.2. b TCA p r e c i p i t a b l e counts of input: Expt 1, 2 = 45%; Expt 3 = 81%. c % bound = (cpm + ECDI) - (cpm - ECDI) x 100 input cpm t 139 TABLE XVII: P r o t o c o l s . 1 L i v e BCG: B a c i l l u s Calmette-Guerin f r e e z e - d r i e d organisms were r e c o n s t i t u t e d i n PBS. (Connaught 075132, Willowdale, Ontario) (Glaxo PL004/0247, Greenford, England). 2 One tenth of a human dose = lOug and approximately 2 x 10 5 v i a b l e organisms. 3 sc = subcutaneously i n the hind f l a n k s , o f t e n i n m u l t i p l e s i t e s , h.k. = heat k i l l e d 4 l i v e BCG were t r e a t e d i n a b o i l i n g water bath f o r 30 minutes. 5 i n t r a d e r m a l l y and subcutaneously i n the hind footpads - 50ul per f o o t . ^ Antigen was e m u l s i f i e d i n complete Freund's adjuvant (CFA) ( D i f c o 0638-60, D e t r o i t , Michigan). CFA contained a suspension of 5 mg Mycobacterium butyricum i n 10ml Bayol F ( p a r a f f i n o i l ) and A r a c e l A ( e m u l s i f y i n g agent). 7 Incomplete Freund's adjuvant (IFA) ( D i f c o 0639-60) contained Bayol F and A r a c e l only. 8 Animals were given two separate i n j e c t i o n s of 500ug over a 6 week pe r i o d . 9 Aluminium potassium sulphate (alum) p r e c i p i t a t e of antigen or b a c t e r i a was resuspended i n PBS and i n j e c t e d . 1 0 Chicken gamma g l o b u l i n (CGG) (freeze d r i e d ) (Cappel Labs 16240, C o c h r a n v i l l e , Pa) i n t r a p e r i t o n e a l . T u b e r c u l i n p u r i f i e d p r o t e i n d e r i v a t i v e (PPD). The ammonium sulphate p r e c i p i t a t e of a b o i l e d f i l t r a t e from Mycobacterium t u b e r c u l o s i s c u l t u r e s (Connaught PPD CT68 s u p p l i e d at 2 mg/ml i n PBS). *3 Acetone powder of h e a t - k i l l e d Mycobacterium t u b e r c u l o s i s s t r a i n H37Rv (Tbc) r e c e i v e d from Dr. T. Hamaoka, Osaka U n i v e r s i t y Medical School Osaka, Japan. 1^ 500ug acetone powdered Tbc e m u l s i f i e d i n 0.2 ml p a r a f f i n o i l . A v i r u l e n t s t r a i n H37Ra of Mycobacterium t u b e r c u l o s i s (American Type Tissue C u l t u r e C o l l e c t i o n 1t 25177). Complete adjuvant H37Ra(Difco) contains 10 mg k i l l e d and d r i e d Tbc H37Ra i n 10 ml Bayol F and A r a c e l A. 1 6 50ug adjuvant H37Ra i n 10 ml Bayol F and A r a c e l A. 50ul adjuvant H37Ra e m u l s i f i e d w i t h 50ul PBS and 50uml of t h i s i n j e c t e d i n t o each footpad or t o t a l 100 u l i . p . 140 TABLE XVII: Priming DBA/2 mice to t u b e r c u l i n p u r i f i e d p r o t e i n d e r i v a t i v e (PPD). PROTOCOL ANTIGEN DOSE ROUTE CONTROLS NUMBER OF No. OF INJECTION EXPERIMENTS 1 l i v e BCG 1 0, ,01-lOug 2 s c 3 no i n j e c t i o n 3 2 h.k. BCG 4 200ug f p 5 no i n j e c t i o n 2 3 BCG i n CFA 6 O.lug sc no i n j e c t i o n 1 4 h.k. BCG i n IFA 7 2 x 500ug 8 sc no i n j e c t i o n 1 5 BCG-alum 9 lOOug fp lOOyg CGG 1 0-alum fp 1 6 BCG-alum lOOug i p l l lOOyg CGG-alum i p 1 7 PPD i n CFA 1 2 lOOug sc no i n j e c t i o n 2 8 PPD i n IFA lOOug fp no i n j e c t i o n 2 9 h.k. T b c 1 3 i n o i l 1 4 2 x 500ug sc no i n j e c t i o n 1 LO H37Ra T b c 1 5 50ug 1 6 f p 50yl IFA fp 9 11 H37Ra Tbc lOOyl i p 100 y l IFA i p 1 141 TABLE X V I I I : The p r o l i f e r a t i v e responses of lymph node c e l l s from PPD-primed DBA/2 mice. EXPT. FOOTPAD PRIMING OF MICE d TIME AFTER NO. ANTIGEN INJECTED SOURCE OF LYMPH NODE IMMUNIZATION (NO. OF MICE) (DAYS) 1 normal # 1 — normal 0 2 H37Ra 1 0 immune 0 1 8 H37Ra 1 0 immune 0 2 8 25 rag Cy/kg c;H37Ra 1 0 immune 0 3 8 25 mg Cy/kg; H37Ra 1 0 immune 0 4 8 2 normal pool (5) H37Ra 1 0 immune pool (10) 7 25 mg Cy/kg; H37Ra 1 0 immune pool (10) 7 3 normal pool (2) CGG-alum5 immune pool (2) 7 BCG-alum 5 immune pool (2) 7 H37Ra 1 0 immune pool (2) 7 4 I F A 1 0 normal pool (14) 7 H37Ra 1 0 immune pool (14) 7 a S u p e r s c r i p t numbers r e f e r to the p r o t o c o l s i n Table XVI. b CGG and PPD 50ug/ml(12.5ug/well); P815 and PPD-coated P815 2 x 10 5/ml (2 x 1 0 4 / w e l l ) ; PPD-coated P815 at approximately 0.15ug PPD/well. - Not determined Cy - 25 mg/kg Cyclophosphamide i n j e c t e d i p 24 hours before antigen i n j e c t i o n . 142 TABLE X V I I I : Continued. BACKGROUND RESPONSE TO ANTIGEN IN CULTURE" RESPONSE (BACKGROUND SUBSTRACTED) (X ± S.E.M.) CGG PPD P815 PPD coated P815 387 ± 30 0 0 3516 13 785 + 108 99 397 1233 979 44821 + 1155 0 59373 608 14315 7741 ± 1456 0 93146 0 14739 1949 + 115 442 9832 4030 9972 3353 + 140 0 103104 7836 12010 235 ± 5 2 22 0 308 3151 ± 243 - 61099 5386 0 615 ± 184 - 111414 266 0 598 ± 4 8 1 0 927 1113 647 ± 185 0 0 0 0 308 ± 15 0 187139 142 525 347 ± 85 238 240379 476 2657 197 ± 64 1021 ± 151 238 204565 295 59 0 0 143 c u l t u r e d w i t h chicken gamma g l o b u l i n (CGG), no s t i m u l a t i o n of the p r o l i f e r a t i v e response was seen, i . e . the thymidine uptake of w e l l s w i t h antigen was equal to or below those without antigen (Table X V I I I ) . The s p e c i f i c i t y of the lymph node p r o l i f e r a t i v e assay was obvious when the antigen i n c u l t u r e was the same as that used to prime the mice i . e . PPD. C e l l s from unprimed mice showed l i t t l e or no p r o l i f e r a t i o n i n response to s o l u b l e PPD, whereas c e l l s from PPD-primed mice a l l responded w e l l to PPD, although to d i f f e r i n g degrees. This v a r i a t i o n was observed both between mice t e s t e d together and i n experiments performed at d i f f e r e n t times. Most of these experiments were done using lymph node c e l l s pooled from 2-14 mice. Some but not a l l mice (both unprimed and PPD primed) responded to mitomycin C-treated P815 tumour c e l l s i n c u l t u r e . When the P815 c e l l s were coated w i t h PPD however, only PPD primed animals responded b e t t e r to the PPD coupled c e l l s than to uncoupled c e l l s . Not a l l lymph node c e l l s from PPD-primed animals responded b e t t e r to PPD-coupled tumour c e l l s than to uncoated P815, and none responded as w e l l to PPD on P815 as to s o l u b l e PPD. The concentration of PPD present alone or bound to P815 was very d i f f e r e n t (12.5ug per w e l l and a maximum of 0.25ug per w e l l , r e s p e c t i v e l y ) . The l e v e l of PPD coupled to P815 should be increased to present more PPD to the helper c e l l s . However, at very high l e v e l s of PPD on P815, a l l tumour antigens may be masked which would prevent s t i m u l a t i o n of other c e l l s (e.g. c y t o t o x i c precursor c e l l s ) responsive to the tumour antigens and remove the e f f e c t i v i t y of the l i n k e d r e c o g n i t i o n d e s i r e d i n these experiments. These r e s u l t s showed that animals could be primed to PPD and when 144 primed, t h e i r c e l l s were able to respond to both s o l u b l e PPD and PPD presented on the surface of P815 c e l l s . 3. In V i t r o C y t o t o x i c Responses of PPD-Primed Animals to P815. Spleen c e l l s from normal or PPD-primed DBA/2 mice were s t i m u l a t e d i n  v i t r o w i t h e i t h e r PPD-coated or uncoated mitomycin C-treated P815 c e l l s . C y t o t o x i c a c t i v i t y was then assessed against "'"'"Cr-labelled, uncoated P815 t a r g e t c e l l s . I f PPD priming had induced a population of helper c e l l s able to enhance the c y t o t o x i c response to the tumour, the response of c e l l s from primed mice st i m u l a t e d w i t h coated P815 should have been higher than the response of c e l l s s t i m u l a t e d w i t h uncoated P815. C e l l s from unprimed mice should respond e q u a l l y w e l l when sti m u l a t e d w i t h coated or uncoated P815 c e l l s . Eleven experiments were set up using responder spleen c e l l s from normal or PPD-primed mice st i m u l a t e d f o r 5 days with PPD-coated or uncoated mitomycin C-treated P815 c e l l s and assayed against uncoated P815 t a r g e t s . Two experiments are shown i n Table XIX. In experiment 1, c u l t u r e s of normal spleen c e l l s s t i m u l a t e d w i t h uncoated P815 generated more c y t o t o x i c i t y than when sti m u l a t e d w i t h PPD-coated P815. This was s i m i l a r to the e f f e c t seen i n the lymph node p r o l i f e r a t i v e assay (Table X V I I I ) . Only one animal primed to PPD (No. 2) showed enhanced c y t o t o x i c i t y a f t e r s t i m u l a t i o n w i t h PPD-coated P815 when compared to the response to uncoated P815. A l l three primed animals i n t h i s experiment were t e s t e d f o r immunity by delayed-type h y p e r s e n s i t i v i t y . Mice were i n j e c t e d i n one hind f o o t w i t h lOyg PPD. Footpad s w e l l i n g was measured TABLE XIX: In v i t r o c y t o t o x i c responses of PPD-primed DBA/2 mice to P815 tumour c e l l s . EXPT. PPD PRIMING* TIME RESPONDER PLYSIS OF P815 TARGETS1 NO. ANTIGEN DOSE ROUTE AFTER SPLEEN STIMULATOR PRIMING CELLS P815 P815 + PPD (DAYS) BCG h.k. 2 x 500yg sc BCG h.k. 2 x 500yg sc BCG h.k. 2 x 500yg sc - normal pool 27 immune 9 1 27 immune 9 2 27 immune 9 3 11.9 2.7** 11.6 10.0 12.0 19.1** 7.7* 6.9** 2 - - • normal 10.6 11.4 BCG l i v e lOyg sc 26 immune 9 1 6.1* 4.8 BCG l i v e l y g sc 26 immune 9 2 11.2 5.6** BCG l i v e O.lyg sc 26 immune # 3 4.1* 5.1 BCG l i v e O.Olyg sc 26 immune # 4 12.1 16.3** * BCG = B a c i l l u s calmette-guerin (see Table XV) sc= subcutaneous i n m u l t i p l e s i t e s . D 3 x 10 5 spleen c e l l s from normal or PPD-primed DBA/2 mice c u l t u r e d w i t h 1 x 10 4 mitomycin C-treated P815 s t i m u l a t o r c e l l s (uncoated or PPD-coated at 0.5 mg per 10 7 c e l l s ) f o r 5 days. Cu l t u r e s were harvested and assayed against uncoated 5 1 C r - l a b e l l e d P815 ta r g e t c e l l s . T o t a l l y t i c u n i t s c a l c u l a t e d from the e f f e c t o r c e l l recovery a f t e r 5 days. c Spleen c e l l s from 3 normal mice were pooled. * S i g n i f i c a n t l y d i f f e r e n t from l y s i s by normal spleen c e l l s s t i m u l a t e d w i t h uncoated P815 p<0.05. ** S i g n i f i c a n t l y d i f f e r e n t from l y s i s by same spleen c e l l s i n c u l t u r e s w i t h uncoated P815 p<0.05. A l l others not s i g n i f i c a n t l y d i f f e r e n t p >0.2. 146 a f t e r 24-48 hours and compared to the u n i n j e c t e d f o o t . Although a l l three primed mice gave p o s i t i v e delayed 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 (greater than l.S f o l d increase i n footpad t h i c k n e s s ) , only one showed an enhanced c y t o t o x i c response i j i v i t r o . In experiment 2, normal spleen c e l l s responded e q u a l l y to both PPD-coated and uncoated P815 s t i m u l a t o r s . C e l l s from PPD-primed mice #1 and #3 were n o n - s p e c i f i c a l l y suppressed and responded poorly to both uncoated and PPD-coated P815. PPD-primed mouse #2 was s p e c i f i c a l l y suppressed and responded as w e l l as the normal mouse to uncoated P81S but l e s s to PPD coated P81S. Mouse #4 was the only mouse to show some enhanced response to PPD-coated P815 when compared to the response to uncoated P815. In most experiments, spleen c e l l s from mice primed to PPD d i d not show enhanced c y t o t o x i c i t y to P815 when st i m u l a t e d w i t h PPD-coated P815 c e l l s . Both n o n - s p e c i f i c and s p e c i f i c suppression was detectable i n the primed mice. C e l l s from n o n - s p e c i f i c a l l y suppressed mice responded l e s s to both uncoated and PPD-coated P815 than normal c e l l s whereas c e l l s from s p e c i f i c a l l y - s u p p r e s s e d mice responded as w e l l as normal c e l l s to uncoated P815 but l e s s to PPD-coated P815 c e l l s than c e l l s from normal mice. In e i g h t f u r t h e r experiments, the helper a c t i v i t y of c e l l s from PPD primed mice was assessed. I r r a d i a t e d thymus, lymph node or spleen helper c e l l s from normal or PPD-primed mice were c u l t u r e d w i t h normal spleen responder c e l l s and uncoated or PPd-coated, mitomycin C-treated P815 s t i m u l a t o r c e l l s . C y t o t o x i c i t y was assayed a f t e r S days against ^ C r - l a b e l l e d P815 t a r g e t c e l l s . In none of these experiments was a s i g n i f i c a n t helper a c t i v i t y of the c y t o t o x i c response against P815 147 detected. Again, c e l l s from many of the PPD-primed animals suppressed the c y t o t o x i c response to P815 i n c u l t u r e s w i t h uncoated or PPD-coated P815 s t i m u l a t o r c e l l s . 4. Summary. DBA/2 mice were primed to PPD using a v a r i e t y of pr o t o c o l s (Table XVII) . The lymph node c e l l s from primed mice showed s i g n i f i c a n t l y enhanced p r o l i f e r a t i v e responses both to s o l u b l e and P815-bound PPD (Table XVIII) . However, the spleen c e l l s from only a few PPD-primed mice showed enhanced c y t o t o x i c a c t i v i t y against P815 tumour c e l l s a f t e r i n v i t r o s t i m u l a t i o n w i t h PPD-coated P815 tumour c e l l s (Table XIX). When c e l l s from PPD-primed mice were c o - c u l t u r e d w i t h normal spleen responder c e l l s and PPD-coated P815 s t i m u l a t o r c e l l s , no s i g n i f i c a n t helper a c t i v i t y was detected. The predominant e f f e c t of the PPD priming p r o t o c o l s used was suppression ( s p e c i f i c and n o n - s p e c i f i c ) of the i i i v i t r o c y t o t o x i c response against P815. 148 DISCUSSION The work presented i n t h i s t h e s i s , examined the r o l e of tumour-specific helper T c e l l s i n the i n v i t r o and i n v i v o responses to a syngeneic tumour P815. DBA/2 mice i n j e c t e d w i t h l i v e P815 tumour c e l l s developed c y t o t o x i c precursor c e l l s r e a c t i v e to the tumour. These c e l l s were a c t i v a t e d by c u l t u r i n g spleen, thymus, or lymph node c e l l s of tumour-bearing mice with P815 s t i m u l a t o r c e l l s (Figures 6; 7; 8 ) . Other c e l l s (helper c e l l s ) taken from tumour-bearing mice enhanced the i n v i t r o generation of c y t o t o x i c c e l l s from normal DBA/2 spleen c e l l s (Figures 9; 10; 11). 1• Evidence f o r Helper T C e l l s A c t i v e i n the Primary i n v i t r o C y t o t o x i c  Response to P815 Previous studies have demonstrated a need f o r helper c e l l s i n secondary anti-tumour responses (Stutman et a l . , 1977; Altman ejt a l . , 1979; Green et a l . , 1979; Igara s h i et a l . , 1979; Woodward et a l . , 1979) but t h i s was the f i r s t report of T helpers a c t i v e i n a primary anti-tumour response (Hancock et a l . , 1981). The d i s t i n c t i o n between the primary and secondary response may be important i f the requirements f o r help d i f f e r i n these responses. For instance, a v i a b l e s t i m u l a t o r c e l l i s u s u a l l y r e q u i r e d to induce the primary response, whereas the secondary c y t o t o x i c response can be t r i g g e r e d by i n a c t i v a t e d c e l l s or s o l u b l e antigens ( R o l l i n g h o f f and Wagner, 1975; Talmage et a l . , 1977). The need f o r the 149 v i a b l e s t i m u l a t o r c e l l s can be circumvented by the a d d i t i o n of i n a c t i v a t e d c e l l s and i n t e r l e u k i n 2 (Talmage et a l . , 1977). I t was shown here that DBA/2 spleen c e l l s s t i m u l a t e d in. v i t r o w i t h P815 tumour c e l l s , produced s i g n i f i c a n t l e v e l s of i n t e r l e u k i n 2 (Figure 17; Table X) w h i l s t added i n t e r l e u k i n 2 r e s t o r e d the c y t o t o x i c response against P815, a f t e r d e p l e t i o n by cyclophosphamide treatment (Figure 16). The helper c e l l s had the f o l l o w i n g p r o p e r t i e s . Whereas the c y t o t o x i c precursor c e l l s were s e n s i t i v e to y - r a d i a t i o n (Figure 12), the am p l i f y i n g a c t i v i t y of c e l l s from tumour-bearing mice was r a d i a t i o n r e s i s t a n t (Figure 13). Furthermore, the helper c e l l s a m p l i f i e d the c y t o t o x i c response d i r e c t e d against the priming P815 tumour but not the c y t o t o x i c responses to other antigens (Table I I I ) . The enhanced response to P815 was not accompanied by an increase i n the generation of n o n s p e c i f i c c y t o t o x i c i t y (Table I V ) . The helper c e l l s were detected i n P815-bearing mice at the same time as the peak i n c y t o t o x i c c e l l a c t i v i t y i n v i v o (Figure 15) and were a c t i v e i r i v i t r o only when added at the beginning of c u l t u r e s generating c y t o t o x i c a c t i v i t y (Tables V, V I ) . This observation suggested that they were i n v o l v e d i n an e a r l y stage i n the in d u c t i o n of the response r a t h e r than i n the expansion of t r i g g e r e d clones of c y t o t o x i c precursors. The helper c e l l s d e scribed here are probably of the T helper c e l l 1 subclass (Table I ; Figure 2) because of the k i n e t i c s of t h e i r a c t i v i t y in v i t r o , and t h e i r e f f e c t on the cyclophosphamide-depleted c y t o t o x i c response to P815 (see s e c t i o n 2 below). I t was not c l e a r l y demonstrated whether these helper c e l l s produce i n t e r l e u k i n 2 themselves or induce i t s production by other T helper c e l l s i n the 150 responder c e l l p o p u l a t i o n . This would r e q u i r e the a n a l y s i s of f a c t o r s produced by c l o n a l populations of T helper c e l l s a f t e r s t i m u l a t i o n w i t h P815 c e l l s . Pretreatment of mice with cyclophosphamide r e s u l t e d i n decreased l e v e l s of i n t e r l e u k i n 2 produced f o l l o w i n g i n v i t r o s t i m u l a t i o n w i t h P815, although mitogen-induced l e v e l s were unaltered (Tables IX, X). In these experiments i n t e r l e u k i n 2 l e v e l s were measured from bulk c u l t u r e s of responder c e l l s s t i m u l a t e d w i t h antigen or mitogen. Under these circumstances, a small clone of c e l l s might expand to produce the same l e v e l s of i n t e r l e u k i n 2 as would many d i f f e r e n t clones. The decreased l e v e l s of i n t e r l e u k i n 2 produced, i m p l i e d that cyclophosphamide depleted a c e l l i n v o l v e d i n i n d u c t i o n of i n t e r l e u k i n 2 by antigen r a t h e r than the i n t e r l e u k i n 2 producing c e l l d i r e c t l y . I n t e r l e u k i n 2 production occurs a f t e r the Lyt 1 + T helper, c e l l p o pulation has been s t i m u l a t e d by antigen and i n t e r l e u k i n 1 ( F a r r a r et a l . , 1978; Wagner and R o l l i n g h o f f , 1978). I n t e r l e u k i n 1 production i s a l s o regulated by a population of T helper c e l l s ( F a r r a r et a l . , 1980), and the P815 s p e c i f i c helper c e l l s discussed here may be i n v o l v e d at t h i s e a r l y stage of i n t e r l e u k i n 2 production. K i l b u r n et a l . (1979; 1981) have i s o l a t e d an a n t i g e n - s p e c i f i c helper f a c t o r a c t i v e i n the primary in. v i t r o c y t o t o x i c response to P815. This f a c t o r i s s i m i l a r to the a n t i g e n - s p e c i f i c f a c t o r s i n v o l v e d i n the a m p l i f i c a t i o n of the antibody response (Tada and Okumura, 1979; K i l b u r n and Levy, 1980; Taussig, 1980). I t bears I region coded-antigenic determinants but not immunoglobulin constant region determinants, and i t binds to P815 antigens. The f a c t o r i s not i d e n t i c a l to i n t e r l e u k i n 2 as i t f a i l s to enhance the c y t o t o x i c response of thymocytes and w i l l not support 151 growth of an i n t e r l e u k i n 2-dependent T c e l l l i n e . They a l s o showed an e a r l y phase e f f e c t of i n t e r l e u k i n 2 i n c y t o t o x i c T c e l l i n d u c t i o n whereas the a n t i g e n - s p e c i f i c helper f a c t o r acted o p t i m a l l y when added l a t e i n t o c u l t u r e s . As discussed above, the a n t i g e n - s p e c i f i c helper c e l l s described here were e f f e c t i v e only when added e a r l y i n t o c u l t u r e s generating a primary c y t o t o x i c response (Tables V and V I ) . They may f u n c t i o n by e l a b o r a t i n g the a n t i g e n - s p e c i f i c helper f a c t o r described by K i l b u r n et a l . , which expands a c t i v a t e d c y t o t o x i c T c e l l clones. Since washed, i n t a c t c e l l s were added as helper c e l l s i n these experiments, and K i l b u r n et a l . p u r i f i e d t h e i r f a c t o r from d i s r u p t e d c e l l s , i t seems u n l i k e l y that the r e s u l t s reported here were due to the a d d i t i o n of contaminating helper f a c t o r . The d i f f e r e n t k i n e t i c s of a c t i o n of helper c e l l s support t h i s c o n c l u s i o n . Since P815 does not express l a antigens ( F r e l i n g e r et a l . , 1974) i t was presumed i n t h i s study, that l a p o s i t i v e antigen-presenting c e l l s i n the responder spleen c e l l p o pulation presented processed P815 tumour antigens i n a s s o c i a t i o n w i t h l a molecules, to the helper c e l l p o p u l a t i o n . The a n t i g e n - s p e c i f i c f a c t o r of K i l b u r n et a l . (1981) bears I-region coded determinants and may serve to focus s o l u b i l i z e d P815 antigens on the macrophage surface to the T helper 2 c e l l and hence induce i n t e r l e u k i n 2 s e c r e t i o n . 2. E f f e c t s of Pretreatment of Mice with Cyclophosphamide Mice bearing small tumour loads (up to 12 days a f t e r i n j e c t i o n of l i v e P815 c e l l s ) had increased numbers of c y t o t o x i c precursor c e l l s i n spleen, thymus and lymph node. Spleen, thymus, and lymph nodes a l s o contained 152 P 8 1 5 - s p e c i f i c helper c e l l s which a m p l i f i e d the i n v i t r o primary c y t o t o x i c response to P815 (Figures 8, 9, 10, 14). In p a r a l l e l w i t h the development of helper c e l l s , suppressor c e l l s could be detected which suppressed the c y t o t o x i c response to P815 (Table I I ) . The response against P815 both i n  v i v o and in. v i t r o appears to r e f l e c t the balance between these two c e l l p o pulations. Our own group as w e l l as a number of others have described the development of suppressor c e l l s during tumour progression as was seen w i t h P815 DBA/2 mice (Takei et a l . , 1976; 1977; 1978; Fujimoto et a l . , 1978; Perry et a l . , 1979; Hakim, 1979; Dye and North, 1981; Maier, 1981; P l a t e r et a l . , 1981). This provides an added l e v e l of complexity i n c o n s i d e r a t i o n of the experiments described h e r e i n . I attempted to remove the suppressor c e l l s and n a t u r a l l y o c c u r r i n g helper c e l l s present i n normal spleen c e l l s by p r e t r e a t i n g responder animals wit h cyclophosphamide. There have been frequent reports of the use of animals so t r e a t e d to study c y t o t o x i c responses (Bonavida, 1977; R o l l i n g h o f f et a l . , 1977; G l a s e r , 1979; Hurme, 1979). Low doses of cyclophosphamide appear to s e l e c t i v e l y i n a c t i v a t e suppressor T c e l l s (Lagrange et a l . , 1974; R o l l i n g h o f f et a l . , 1977; Gl a s e r , 1979; Shand and Liew, 1980), while high doses depress or e l i m i n a t e the generation of c y t o t o x i c T c e l l s ( G l a s e r , 1979; M e r l u z z i et a l . , 1979). M e r l u z z i et a l . (1979) have shown that doses of 100 - 200 mg/kg e l i m i n a t e the CTL response to a l l o a n t i g e n but that t h i s response can be r e s t o r e d by adding normal thymocytes. I t h e r e f o r e attempted a s i m i l a r pretreatment of responder animals wit h cyclophosphamide before s t i m u l a t i o n i r i v i t r o w i t h syngeneic tumour c e l l s . As a c o n t r o l the response of these mice to C57B1/6 153 a l l o a n t i g e n s was als o t e s t e d . In Table V I I I i t was shown that the response to both the syngeneic tumour, and to a l l o a n t i g e n s , was depressed or e l i m i n a t e d by pretreatment of DBA/2 mice with 100 mg cyclophosphamide per kg, 24 hours before c u l t u r e s were i n i t i a t e d . In agreement w i t h the work of M e r l u z z i et a l . (1979) the a d d i t i o n of normal thymocytes r e s o r t e d the response to the a l l o a n t i g e n s . Although i n the experiments of M e r l u z z i et a l . (1979) the thymocytes d i d not appear to provide c y t o t o x i c precursor c e l l s (CTLp - Figures 1, 2) f o r the a l l o g e n e i c response, the work of P i l a r s k i (1977) c l e a r l y shows that t h i s precursor population i s present i n normal thymus at low frequency. In the presence of i n t e r l e u k i n 2, anti-P815 c y t o t o x i c precursors i n the thymuses of DBA/2 mice could be a c t i v a t e d (Figure 8). Normal DBA/2 thymocytes, however, could not r e s t o r e the cyclophosphamide depleted anti-tumour response (Figure 19). This may have been r e l a t e d to low numbers of helper c e l l s f o r the anti-tumor response i n the thymuses of unprimed animals. Thymocytes from animals bearing P815 were able to p a r t i a l l y r e s t o r e the response (Figure 20) . The a b i l i t y to r e s t o r e the allo-response by adding thymocytes suggested to M e r l u z z i et a l . (1980) that cyclophosphamide d i d not deplete the precursor pool but merely an a m p l i f y i n g thymocyte population. The r e s u l t s of l i m i t i n g d i l u t i o n experiments i n Table XI disagree w i t h t h i s i n t e r p r e t a t i o n of the data, and i n d i c a t e that at l e a s t f o r the anti-tumour response, the precursor frequency was markedly reduced. This observed decrease i n the anti-tumour c y t o t o x i c c e l l precursor frequency may be of prime importance when cons i d e r i n g the use of cyclophosphamide i n cancer chemotherapy. An a l t e r n a t i v e i n t e r p r e t a t i o n i s supported by our 154 observation that the a d d i t i o n of i n t e r l e u k i n 2 to c u l t u r e s r e s t o r e d the anti-tumour response s a t i s f a c t o r i l y (Figure 6). The c a p a c i t y of c e l l s to produce i n t e r l e u k i n 2 when measured a f t e r mitogen s t i m u l a t i o n (Table IX) was unaffected by cyclophosphamide but was reduced a f t e r antigen s t i m u l a t i o n (Table X ) . The treatment w i t h cyclophosphamide apparently blocked the normal process of generation of i n t e r l e u k i n 2, perhaps by e l i m i n a t i o n of an antigen s p e c i f i c helper c e l l subclass ( T h l , see Figure 2). In any case the a d d i t i o n of concanavalin A can apparently bypass t h i s block and permit the a c t i v a t i o n of i n t e r l e u k i n 2-producing c e l l s . Thus these r e s u l t s i n d i c a t e d that the treatment of animals w i t h high doses of cyclophosphamide introduced at l e a s t two l e s i o n s i n the t r i g g e r i n g sequence of c y t o t o x i c T c e l l s . One was at the l e v e l of i n t e r l e u k i n 2 production and the second was i n the number of c y t o t o x i c T lymphocyte precursor present. 3. The i n v i v o Primary Response to P815 As the tumour progressed, suppressor c e l l a c t i v i t y became predominant (Figure 15). These observations may p a r t i a l l y e x p l a i n the v a r i a b l e success of the i r i v i v o experiments described i n Chapter I I I . In a d d i t i o n , a number of groups (Biddison and Palmer, 1977; Uyttenhove et a l . , 1983; Wortzel et. a l . , 1983) have now described the emergence of tumour c e l l v a r i a n t s i n the course of tumour growth. These were g e n e r a l l y detected as mutants which had l o s t tumour antigens and thus against which no immune response was evident. Boon's group (Boon et a l . , 1980; Uyttenhove et a l . , 1980; Maryanski et. a l . , 1982) have generated a number of tumour v a r i a n t s 155 (by NTG mutagenesis) which appear to be more immunogenic than the p a r e n t a l tumour. When these were used to prime mice, s i g n i f i c a n t l y enhanced clearance and prolonged s u r v i v a l a f t e r tumour challenge were observed (Figures 22, 23, 24, Table XIV) i n 25 percent of animals. In 88% of animals showing r a p i d clearance of tumour c e l l s , s u r v i v a l was markedly enhanced. This suggested that the priming p r o t o c o l generated an e f f e c t o r c e l l population ( p o s s i b l y helper c e l l s ) r e s p o n s i b l e f o r these e f f e c t s . C l a s s i c a l models of tumour immunotherapy have involved i n j e c t i o n of tumour-primed c e l l populations i n t o animals e i t h e r together w i t h or soon a f t e r i n j e c t i o n of l i v e tumour c e l l s . In our experiments the i n j e c t i o n of P815-induced helper c e l l s caused enhanced clearance of a tumour c e l l challenge but only i n 18% of the r e c i p i e n t mice (Figure 25, Table X I I ) . Of these, 55% s u r v i v e d longer than animals i n j e c t e d w i t h c o n t r o l c e l l s . Although these r e s u l t s were d i s a p p o i n t i n g , i t should be noted that none of the animals i n j e c t e d w i t h c o n t r o l c e l l s showed enhanced a b i l i t y to c l e a r P815. As discussed above, e i t h e r the s e l e c t i o n of tumour v a r i a n t s or the generation of suppressor c e l l s could account f o r the f a i l u r e to p r o t e c t these mice. Several groups have shown greater success i n tumour p r o t e c t i o n studies by removing suppressor c e l l s or t h e i r precursors before c e l l t r a n s f e r (Fefer et a l . , 1976; Berendt and North, 1980; M i l l s et a l . , 1980; Greenberg §_t a l . , 1981). The c e l l s u t i l i z e d i n many of those reports were c y t o t o x i c e f f e c t o r c e l l s which were often expanded i n i n t e r l e u k i n 2-containing medium before i n j e c t i o n , w i t h an exogenous i n t e r l e u k i n 2 source (Fefer and G o l d s t e i n , 1982; M i h i c h , 1982). 156 4. The i n v i t r o C y t o t o x i c Response to PPD-Modified P815 Tumour C e l l s The f i n a l s e c t i o n of t h i s t h e s i s ( R e s u l t s , Chapter IV) examined the a m p l i f i c a t i o n of the c y t o t o x i c response to P815 using helper c e l l s d i r e c t e d against the exogenous antigen, t u b e r c u l i n - p u r i f i e d p r o t e i n d e r i v a t i v e (PPD). This u t i l i z e d the concept of h e t e r o g e n i z a t i o n (Svet-Moldavsky and Hamburg, 1967; M i t c h i s o n , 1970; Kobayashi, 1979), i n which tumour c e l l s are modified chemically or by v i r a l i n f e c t i o n and i n j e c t e d i n t o a p p r o p r i a t e l y primed r e c i p i e n t s . Heterogenization might enhance the anti-tumour response by i n t r o d u c i n g a new, strong a n t i g e n i c determinant which acts to amplify or prolong the anti-tumour response (perhaps by f a v o r i n g T helper or suppressor c e l l i n d u c t i o n ) . The tumour c e l l mutants of Boon et a l . (1980) are thought to f u n c t i o n t h i s way and are p o s s i b l y the best examples of converting a "non-immunogenic" tumour to an immunogenic form. A l t e r n a t i v e l y , the m o d i f i c a t i o n of the tumour may introduce a new determinant which l i n k s the anti-tumour immune response, to the p r e - e x i s t i n g immunity against the added determinant. This i n e f f e c t produces a secondary response to the tumour. The PPD system t e s t e d by Lachmann and S i k o r a (1978) and Takatsu e_t a l . (1980) e x e m p l i f i e s t h i s approach. S i g n i f i c a n t p r o l i f e r a t i v e responses could be measured w i t h c e l l s from tuberculin-primed animals against PPD presented e i t h e r i n s o l u b l e form or on the surface of the P815 tumour c e l l (Table X V I I I ) . This i n d i c a t e d that the new determinant on P815 was indeed being seen by a T c e l l p opulation i n the primed animals. However, no c o n s i s t e n t enhancement of anti-P815 c y t o t o x i c i t y was observed w i t h c e l l s from PPD-primed animals (Table XIX), and suppressed responses were f r e q u e n t l y 157 seen. The reasons f o r these f i n d i n g s are not c l e a r but probably r e l a t e to the general i l l h e l a t h and suppressed immunity i n our DBA mice h e l d under overcrowded c o n d i t i o n s i n the o l d animal f a c i l i t y at the time these p a r t i c u l a r s tudies were conducted. This explanation i s supported by the observation that soon a f t e r a r r i v a l i n the u n i t , DBA/2 mice uniformly developed splenomegaly. This c o n d i t i o n i s not observed i n the new animal u n i t . 5. Prospectives Future experiments i n t h i s model system should be designed to d e l i n e a t e the helper T c e l l mechanism i n the in, v i t r o c y t o t o x i c response to P815. The c e l l s should be examined f o r t h e i r Lyt and l a phenotype by both p o s i t i v e - ( p a n n i n g techniques) and negative-(antiserum plus complement l y s i s ) s e l e c t i o n methods. This would a i d to assign the c e l l s to the Thl or Th2 c l a s s as o u t l i n e d i n Table I . In most s t r a i n s of mice the T h l c e l l s have been de f i n e d as Lyt l + 2 , I a + and the Th2 c e l l s as Lyt l + 2 ~ I a ~ . The c y t o t o x i c T c e l l s i n the DBA/2 response to P815, were shown to be Lyt l + 2 + (Maier §_t a l . , 1980) not Lyt 1~2 + as u s u a l l y reported, so the helper c e l l s may al s o d i f f e r i n t h i s s t r a i n . The production of a n t i g e n - s p e c i f i c and n o n - s p e c i f i c f a c t o r s by the helper c e l l s should be examined as w e l l as t h e i r mode of i n t e r a c t i o n w i t h c y t o t o x i c c e l l s , i . e by d i r e c t contact or through s o l u b l e f a c t o r s and by l i n k e d or un- l i n k e d r e c o g n i t i o n of a n t i g e n i c determinants (see Table I -mode of i n t e r a c t i o n of Thl and Th2 c e l l s w i t h B c e l l s i n the antibody response). The development of s p e c i f i c helper T c e l l clones ( e i t h e r by 158 f u s i o n w i t h tumour c e l l l i n e s or by development of i n t e r l e u k i n 2-dependent c e l l l i n e s ) would g r e a t l y f a c i l i t a t e these s t u d i e s . The work, i n t h i s t h e s i s describes experiments with heterogenous c e l l p o pulations. I t d i d not define an absolute requirement f o r the helper c e l l s i n the c y t o t o x i c response. I attempted to d e r i v e P 8 1 5 - s p e c i f i c helper T c e l l l i n e s dependent on T c e l l growth f a c t o r s but was unsuccessful. With the development of such l i n e s , t h e i r d i r e c t i n t e r a c t i o n s w i t h c y t o t o x i c T c e l l p r e c u r s o r s , macrophages, and suppressor c e l l s i n l i m i t i n g d i l u t i o n c u l t u r e s could be examined. As cyclophosphamide can be a c t i v a t e d i n v i t r o , the d i r e c t e f f e c t s of t h i s drug on the helper c e l l population (and s o l u b l e f a c t o r production) could be examined using cloned l i n e s . A second important area f o r f u r t h e r study i s to examine the r o l e of T helper c e l l s i n tumour r e j e c t i o n . Again, cloned l i n e s would permit d e f i n i t i o n of the r o l e of the helper c e l l and/or the involvement of other c e l l s which the helper c e l l s may amplify i n r e c i p i e n t animals. From other r e p o r t s , i t i s important to remove suppressor c e l l s from r e c i p i e n t mice before t r a n s f e r r i n g s e n s i t i z e d helper c e l l s . This could be done n o n s p e c i f i c a l l y e.g. w i t h low doses of cyclophosphamide or more d i r e c t l y w i t h a n t i s e r a d i r e c t e d against antigens on the suppressor c e l l , e.g. Lyt or l a antigens, should these be d i s t i n c t from those on the helper c e l l s . The experiments with PPD-modified tumour c e l l s to prime mice to subsequent challenge w i t h tumour c e l l s were p r e l i m i n a r y i n nature and should be expanded. Again, removal of suppressor c e l l s i s important. S i g n i f i c a n t p r o l i f e r a t i v e responses were measured i n c e l l s from PPD primed mice and 159 these c e l l s may enhance _ v i v o tumour clearance, although in v i t r o c y t o t o x i c i t y was not enhanced. PPD provides a more defined a n t i g e n i c (helper) determinant f o r these experiments than e.g. using the tumour v a r i a n t s of Boon e_t a l . (1980) and would be a p p l i c a b l e to a number of tumour systems. I d e a l l y , animals bearing small tumour loads could have t h e i r tumour e x c i s e d , be primed with t u b e r c u l i n , boosted with PPD-coupled, i n a c t i v a t e d tumour c e l l s and then r e j e c t f u r t h e r challenges w i t h v i a b l e tumour c e l l s . 6. Summary I examined the _ v i v o and ijn v i t r o responses of DBA/2 mice to the syngeneic t r a n s p l a n t a b l e mastocytoma P815. Mice i n j e c t e d w i t h low doses of P815 showed enhanced c y t o t o x i c responses against P815 a f t e r r e s t i m u l a t i o n i n v i t r o w i t h tumour c e l l s . A n t i g e n - s p e c i f i c r a d i o - r e s i s t a n t helper T c e l l s were found i n mice bearing small tumour loads. These c e l l s a m p l i f i e d the primary _ v i t r o c y t o t o x i c response of DBA/2 spleen c e l l s against P815, functioned o p t i m a l l y during the e a r l y stages of c y t o t o x i c T c e l l a c t i v a t i o n and can be t e n t a t i v e l y placed i n the Thl c l a s s . The primary _ v i t r o c y t o t o x i c responses of DBA/2 mice to both C57B1/6 a l l o a n t i g e n s and P815-tumour antigens were reduced by cyclophosphamide pretreatment. These responses could be r e s t o r e d by the a d d i t i o n of exogenous i n t e r l e u k i n 2. Cyclophosphamide reduced the production of i n t e r l e u k i n 2 a f t e r antigen s t i m u l a t i o n but such a reduction was not observed i n the l e v e l s of i n t e r l e u k i n 2 produced a f t e r mitogen s t i m u l a t i o n . This suggested that cyclophosphamide reduced an 160 a n t i g e n - r e a c t i v e T c e l l population i n v o l v e d i n i n t e r l e u k i n 2 production but not the i n t e r l e u k i n 2-secreting c e l l . Thus cyclophosphamide appears to a f f e c t the Thl c e l l c l a s s rather than the Th2 c e l l (discussed i n Table I ) . This co n c l u s i o n was supported by experiments where thymocytes were added to c u l t u r e s of spleen c e l l s from cyclophosphamide-treated mice with antigen. Normal thymocytes r e s t o r e d the cyclophosphamide-depleted c y t o t o x i c response to a l l o a n t i g e n s but not to P815 tumour antigens. This may be due to the low numbers of anti-tumour helper c e l l s i n normal thymocytes, since thymocytes from P815-injected mice d i d r e s t o r e the c y t o t o x i c response to P815. This was f u r t h e r support f o r the c l a s s i f i c a t i o n of P815-reactive T helper c e l l s as Thl c e l l s . Cyclophosphamide treatment a l s o reduced the number of c y t o t o x i c precursor c e l l s which could be a c t i v a t e d i n v i t r o by P815 s t i m u l a t o r c e l l s . Although suppression of the response to P815 appeared to a r i s e during the course of tumour growth, DBA/2 mice could be primed against P815 to show both enhanced clearance of tumour c e l l s and prolonged s u r v i v a l a f t e r tumour challenge. C e l l s taken from P815-primed animals and t r a n s f e r r e d i n t o normal DBA/2 mice caused enhanced clearance of tumour c e l l s by the r e c i p i e n t animals, when compared w i t h animals r e c e i v i n g normal DBA/2 c e l l s . C e l l s from t u b e r c u l i n - p u r i f i e d p r o t e i n d e r i v a t i v e (PPD)-primed mice p r o l i f e r a t e d i n v i t r o i n response to both s o l u b l e PPD and PPD bound to the surface of P815 c e l l s . 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