@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Microbiology and Immunology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Hancock, Elizabeth Jane"@en ; dcterms:issued "2010-05-02T22:28:44Z"@en, "1983"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """When injected with live P815 tumour cells, DBA/2 mice developed cytotoxic cells reactive to the tumour. In addition, T helper cells from tumour-bearing mice enhanced the iri vitro generation of cytotoxic cells from normal DBA/2 spleen cells. The helper cells had the following properties (1) expression of the Thy-1.2 antigen; (2) resistance to y-radiation; (3) specific enhancement of the cytotoxic response to P815; (4) detectability in P815-bearing mice at the peak of cytotoxic cell activity; (5) activity in the early phase of cytotoxic cell activation. In parallel to the development of helper cell activity, suppressor cells were generated which suppressed the cytotoxic response to P815. These suppressor cells were removed by pre-treating mice with low doses of cyclophosphamide. High doses of cyclophosphamide reduced the cytotoxic response to both P815 and C57B1/6 alloantigens. Cyclophosphamide treatment reduced the frequency of cytotoxic precursor cells directed against P815, and an antigen-reactive helper cell involved in interleukin 2 production. Both interleukin 2 and thymocytes from P815-primed mice, restored the cytotoxic response against P815, to normal levels. Twenty six percent of animals primed with tumour cells cleared a challenge dose of P815 faster than unprimed control mice. Of these, 88% survived longer than the control animals. Eighteen percent of the recipients of cells from tumour-primed mice, cleared a challenge dose of P815 faster than mice injected with normal cells. Of these 53% survived significantly longer than control groups given either normal cells or no cells at all. Cells from mice primed to PPD showed significantly enhanced proliferative responses to soluble and P815-bound PPD, when compared with unprimed animals. However, cells from only a few PPD-primed mice showed enhanced cytotoxicity against P815 tumour cells, and PPD-primed cells either did not alter, or suppressed, the cytotoxic response of normal DBA/2 spleen cells, when stimulated with PPD-coated P815."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/24297?expand=metadata"@en ; skos:note "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) 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). 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) 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-AMF; 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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Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Antigen-specific helper T cells in the responses of DBA/2 mice to a syngeneic tumour, P815"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/24297"@en .