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Immune responses to syngenetic tumours Kwong, Linda Chu 1983

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IMMUNE RESPONSES TO SYNGENEIC TUMOURS by • • LINDA CBU KWONG .Sc.,. The U n i v e r s i t y o f B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF. MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1983 © Linda Chu Kwong, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f MiCR08'0L06Y  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e Aijusf- -26,  a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s DE-6 (3/81) ABSTRACT The anti-tumour activity of normal C57BL/;6j (B6) spleen cells that were sensitized to the syngeneic tumour, EL4 and expanded in inter-leukin 2 (IL2) were assessed i n vitro and i n vivo. Cultured cells demons-trated strong cytotoxic responses in vitro. However, although the cyto-toxic T lymphocytes (CTL) preferentially k i l l e d EL-» these cells were nonspecific in the sense that they also k i l l e d syngeneic B6 LPS blast as well as the allogeneic DBA/2 (D2) tumour, P815, albeit to a lesser degree. Nonspecific activity appeared as a consequence of expanding the cultures i n IL2-containing medium. The effector cells were shown to + 125 be Thy-1 . In studies which followed the clearance of I-i'ododeoxy-125 uridine- ( IUdR) labelled tumour cells from B6 mice, i t was found that mice treated with B6 anti-EL4- cultured cells survived longer than untrea-ted mice. However, studies with labelled cultured cells indicated that the cultured c e l l s have a short half l i f e i n vivo (-= 10% remaining after 4.8 hr). Thus, the long term effects of cultured cells i n vivo i s most li k e l y due to the activation of the host's immune system. In agree-ment with in vitro results, B6 anti-EL4 cultured donor cells were not absolutely specific for EL4 i n the sense that (C57BL/6J x DBA/2J)F1 (BDF1) 125 mice treated with these cells also showed an enhanced clearance of IUdR P815. However, the mean survival time (MST) to P815 relative to controls was not enhanced. The i n vivo activity of D2 anti-P815 effector cells were also determined. Unlike B6 anti-EL/+. system, these cells specifically enhanced the elimination of P815 tumour cells; however, they failed to prolong the MST of the mice to P815 when compared to controls. Two i i i out of five B6 mice treated with B6 anti-EL4 cultured cells survived a lethal challenge of EL4. The frequency of CTL precursors from the two immune and normal spleen cells were 1/800, 1/950 and 1/4000, respectively. Furthermore, CTL from primed spleen cells were more specific for EL4 than normal spleen c e l l s . When used i n conjunction with cyclophosphamide (CY), B6 anti-EL4 cultured cells were more effective i n conferring protection against EL4 i n B6 mice. CY by i t s e l f also led to a higher rate of EL4 clearance from B6 mice. The use of cultured cells i n immunotherapy against syngeneic tumour i s discussed. iv ACKNOWLEDGEMENTS I am grateful to Dr. Hung-Sia Teh for his encouragement and guidance, to Dr. Douglas Kilburn for his helpful advice during Dr. Tehjs sabbatical leave and to Mrs. Margaret Ho for the prepara-tion of Con A SN and EL4 SN. V TABLE OF CONTENTS Abstract . i i Acknowledgements • iv List of Abbreviations v i i List of Table and Figures v i i i Introduction 1 Materials and Methods U Mice and cell lines 4-Preparation of cells for in vitro culture .... 4 Mixed tumour lymphocyte cultures ............ 4 Sources of IL2 5 Measurement of IL2 activity 5 Assay for CTL activity 6 Cell cultures for limiting dilution 7 LPS blasts 7 1 2 5 I-labelling of tumour cells -8 1 2 5I-labelling of cultured cells 8 Cyclophosphamide • •• 8 Anti-Thy-1 antibody 8 Statistical analysis 9 Results 9 Effect of Con A SN on the anti-EL4 tumour cyto-toxicity in normal B6 spleen cells 9 Specificity of B6 anti-EL4 CTL before and after feeding with Con A SN 12 Thy-1 phenotype of B6 anti-EL4 effector cells following feeding with Con A SN 12 v i E f f e c t o f priming w i t h B6 a n t i - E L 4 c u l t u r e d c e l l s on the r a t e o f e l i m i n a t i o n o f ' 2 5 i u d R - l a b e l l e d E L 4 tumour c e l l s from B6 mice 14 S u r v i v a l of c u l t u r e d c e l l s i n vivo 14 S p e c i f i c i t y o f B6 a n t i - E L 4 c u l t u r e d c e l l s i n v i v o 20 E f f e c t o f combined chemotherapy on the clearance of 1 2 5 I U d R E L 4 2 6 E f f e c t o f pretreatment o f GY on the clearance o f T 2 5 I U d R E L 4 2 6 E f f e c t o f small tumour l o a d on s u r v i v a l 34 Discussion 39 References 46 v i i LIST OF ABBREVIATIONS B6 C57BL/6J BDF1 (G57BL/6J x DBA/2J)F1 C complement CM complete media Con A SN concanavalin A supernatant CTL cytotoxic T lymphocytes CTL-P cytotoxic T lymphocyte precursors CY cyclophosphamide D2 DBA/2J E/T effector to target ratio IL2 interleukin 2 i.p. intraperitoneal IUdR iododeoxyuridine i.v. intravenous LPS lipopolysaccharide MI-IC major histocompatibility complex MST mean survival time SN supernatant TUC helper T cells v i i i LIST OF TABLE AND FIGURES Table I. Specificity of B6 anti-EL4 CTL before and after feeding with Con A SN 13 Figure 1. Effect of Con A SN on the activity of B6 anti-EL4 CTL 11 Figure 2A. Thy-1 phenotype of B6 anti-EL4 effector cells 16 Figure 2B. Thy-1 phenotype of effector cells from cultures of B6 spleen cells stimulated with Con A SN only 17 Figure 3. Effect of priming with B6 anti-EL4 cultured cells on the rate of elimination of '^IUdR-labelled £^4 tumour cells 19 125 Figure 4. Survival of IUdR cultured cells i n vivo ....... 22 Figure 5A. Specificity of B6 anti-EL4 cultured cells in BDF1 mice challenged with 1 2 5 iudR EL4 24 Figure 5B. Specificity of B6 anti-EL4 cultured cells in BDF1 mice challenged with 1 2 5IUdR P815 25 Figure 6A. Specificity of D2 ahti-P815 cultured cells in BDF1 mice challenged with 1 25iudR EL4 28 Figure 6B. Specificity of D2 anti-P815 cultured cells in BDF1 mice challenged with 1 2 5IUdR P815 29 Figure 7. Effect of combined chemotherapy on the clearance of 1 25iudR EL4 31 Figure 8. Effect of pretreatment of CI on the clearance of 1 2 5IUdR EL4 33 Figure 9. CTL-P frequency of normal and B6 anti-EL4 primed B6 mice to EL4 36 Figure 10A. Specificity of CTL from normal B6 spleen c e l l s . . . 38. Figure 10B. Specificity of CTL from primed B6 spleen c e l l s . . . . 38. 1 INTRODUCTION Burnet's immune-surveillance theory (5) i s based on the notion that cells i n the immune system circulate and seek out the presence of foreign antigens, such as bacteria, viruses or tumours i n the body. Once these antigens are detected, lymphocytes eliminate them from the system. However, the effective immune response i n vivo to syngeneic tumours are often weak. As a consequence, the host eventually succumbs. Escape of tumours from surveillance has been ascribed to blocking antibodies or to free antigens shed by tumour cells that inhibit the destruction of tumour cells by effector lymphocytes (43)• Extensive studies also show that T suppressor c e l l s play a significant role i n negatively regulating the immune response i n the course of tumour progression (20,4.3,51,52,65). There are evidence that show that soluble tumour antigens favour the generation of suppressor T cells (65). Takei et. a l . demonstrated that the response to tumours involve an i n i t i a l rise i n cytotoxic T lympho-cytes (CTL) population i n the host followed by the induction of immuno-suppressive cells which promote tumour growth by inhibiting the activa-tion of CTL precursors (CTL-P) specific for the tumour:: (60). However, macrophages have also been implicated to mediate immunosuppression (50). The potential use of cultured lymphocytes for cancer therapy i s well-documented (12,27,30,39,50,55). One expects the adoptively transferred lymphoid cells to suppress tumour growth unt i l such times that the r e c i -pient i s able to develop a strong anti-tumour response. In the past twenty years, there are numerous instances demonstrating that adoptive transfer of specifically sensitized cultured lymphocytes aid the host in 2 tumour destruction (50). Among various c e l l types studied, CTL appear,, to be a prime candidate for immunotherapy because they.. are capable of specifically lysing tumour cells (9). Berke et. a l . (2) suggested that in vitro sensitization of lymphocytes might lead to more potent anti-tumour effect than i n vivo sensitization, since antigens which are found to e l i c i t weak immune response i n vivo are able to stimulate lympho cytes to react strongly in vitr o . In vitro immunization of lymphoid c e l l against different syngeneic tumours have since been studied by many inves tigators i n the hope of using them i n immunotherapy (6,44,54). A c r i t i c a l requirement for the successful intervention of tumour i growth i s that syngeneic lymphocytes and not allogeneic lymphocytes be util i z e d . This i s based on the assumption that tumour cells possess tu-mour associated antigens and major histocompatibility complex (MHC) on their surface (13). To e l i c i t tumour specific response, syngeneic lympho cytes stimulated with the tumour would only be sensitized- to tumour anti-gens. On the other hand, allogeneic lymphocytes would recognize both the tumour and normal MHC antigens of the host. The host can also recognize the strong MHC antigens on the allogeneic cells and eliminate them before any significant tumour destruction has occurred. In favour of this i s the finding that allogeneic cells were less effective than syngeneic c e l l i n tumour rejection, presumably because of the short survival of the for-mer cells (25,26,44). The survival of allogeneic lymphoid cells can be extended by immunosuppression of the tumour-bearing recipient, but this can lead to potentially disastrous graft versus host reaction (27)• Lymphocytes often exhibit weaker response i n vitro towards syngeneic 3 tumour cells than allogeneic lymphocytes. A study on the mechanism by which CTL k i l l syngeneic tumour cells show that the reduced reactivity stems from the low number of CTL capable of k i l l i n g tumour cells; furthermore, the avidity of binding between CTL and syngeneic tumour i s lower than that observed for CTL and allogeneic tumour (31) • Thus, the transfer of a large amount of lymphocytes i s required to g mediate tumour eradication. At least 10 cells were administered per animal before signs of tumour rejection occurred (4,17). The use of in vitro sensitization has been limited by inefficient expansion of effector cells during culture (11). In recent years, the discovery of inter!eukin 2 ( I L 2 ) has made possible the generation of large numbers of tumour-specific syngeneic lymphocytes (34)• I L 2 i s defined as a soluble immunoenhancing factor which i s secreted by helper T cells (Tj^C) upon stimulation with T c e l l mitogens or allogeneic c e l l s . I L 2 can also be produced by stimulation of certain T c e l l lymphomas with phorbol esters (24 ) . Mills et. a l . (48) presented evidence that cultures supplemented with I L 2 yielded a cytotoxic response towards a syngeneic tumour that was as high as the one observed for the allogeneic tumour i n non •IL2-supplemented cultures. IL2 appears to cause antigen-or mitogen-activated T cells to proliferate i n vitro (24)• Tumour-specific CTL has been maintained for periods of more than four months in IL2~supplemented cultures (34)• Culture conditions limiting for allogeneic MHC antigens or syngeneic tumour antigens CTL-P can also be achieved by supplementing the cultures with exogenous I L 2 (46 ,59 ,62) . In this study, conditions for the generation of B6 anti-EL4 reactive T lymphocytes were established. The therapeutic efficacy of the lymphoid 4 125 c e l l s were assessed by following the elimination of I-iododeoxy-u r i d i n e - ( 1 2^IUdR) l a b e l l e d EL4 c e l l s from B6 mice. The data presented here demonstrate that although s e n s i t i z e d lymphocytes are very e f f i c i e n t i n destroying tumour target,-: c e l l s i n v i t r o , they have only a marginally b e n e f i c i a l e f f e c t i n mice bearing large tumour load. The parameters important f o r tumour r e j e c t i o n are discussed. MATERIALS AND METHODS Mice and c e l l l i n e s . C57BL/6J (Bb), (C57BL/6J x DBA/2J)F1 (BDF1) and DBA/2J (D2) were purchased from the Jackson Laboratory, Bar Harbor, Maine). EL4 i s a benzopyrene induced lymphoma c e l l l i n e of B6 o r i g i n . P815 mastocytoma i s syngeneic to D2 mice. These tumours were maintained i n a s c i t i c form by weekly passage i n syngeneic mice. Preparation of c e l l s f o r i n v i t r o c u l t u r e . Spleen c e l l s were teased into s i n g l e c e l l suspension i n complete media (CM), large debris were allowed to s e t t l e and the c e l l s i n suspension were spun, followed by l y -s i s of red blood c e l l s with NH^Cl. C e l l s were then washed three times i n CM. The CM was RPMI 1640 supplemented with 10% f e t a l c a l f serum (Flow), 50 units/ml p e n i c i l l i n , 50 ug/ml streptomycin (GIBCO), 5 x 10~^M 2- mercaptoethanol, 10 mM Hepes buffer, pH 7.2 and 30 ug/ml glutamine, (Sigma Chemical Co.). Mixed tumour lymphocyte c u l t u r e s . B6 spleen c e l l s were s e n s i t i z e d to 6 5 EL4 by c u l t u r i n g 2 x 10 r-normal B6 spleen and 1 x 10 i r r a d i a t e d EL4 (4000 rads from Co source) i n a volume of two ml. CM i n tubes 5 (Falcon # 2057). When larger quantities of lymphocytes were required, 6 x 10^ responder and 3 x 10^ irradiated EL4 (4000 rads) were cultured in flasks (corning # 25100) in a volume of 20 ml. per flask. Sources of IL2. Concanavalin A supernatant (Con A SN) was prepared as previously described (35). Spleen cells from 2 to 3 month old Uistar rats were treated with 0.83% NH.C1 in 0.01 M Tris buffer, pH 7.2 to 4 lyse red c e l l s . The treated spleen cells were then cultured at 1 x 10^ cells/ml with 5 ug/ml Con a (Sigma Chemical Co.) in CM for 48 hr. at 37 C and 5% C0 2 i n a i r . The SN were f i l t e r — s t e r i l i z e d and stored at - 20°C. IL2-containing SN were also prepared from a variant of the murine T lymphoma line, EL4, as described (23). The EL4 variant was grown in CM to a maximum density of 1 x 10^ cells/ml. Large numbers of this variant were obtained by passing the tumour in the asci-t i c form in B6 mice. For production of factor, EL4 cells were cultured at 1 x 10^ cells/ml in medium supplemented with 4% horse serum (GIBCO) and 10 ng/ml phorbol-12-myristate-13-acetate (Sigma) for 24 hr at 37°C The SN was f i l t e r - s t e r i l i z e d and stored at - 20°C. This i s referred to as EL4 supernatant (EL4 SN). The EL4 line was a g i f t from Dr. Verner Paetkau of the Dept of Biochemistry, University of Alberta, Edmonton. Measurement of IL2 activity. The activity of the IL2-containing SN was quantitated by determining the ab i l i t y of SN to maintain growth of an IL2-dependent murine T c e l l l i n e , MTL 2.8.1. This lin e was obtained from Dr. Verner Paekau of the Dept. of Biochemistry, Univ. of Alberta, Edmonton. A:j<unit of IL2 activity is defined as the amount of IL2 required to give 50% of the maximum proliferative response of MTL 2.8.1 cultured at 2.5 x 10^ cells/ml for 2 days; 1 uCi of ^H-TdR was added for 0.20 ml of culture during the last 16 hr of culture period. Typically, crude EL4 and Con A SN have IL2 activity of 5,000 ^ o 10,000 units/ml and 300 to 600 units/ml, respectively. Most of the experiments carried out were performed using EL4 SN since i t contains higher amount of IL2 activity. When Con A SN was supplemented in CM, i t was necessary to add 50 mM ^-methyl-G"* mannoside to neutralize residual Con A present in the SN, thus preventing the polyclonal activation of CTL-P by Con A (3,61). Assay for CTL activity. Briefly, 3 x 10^ target cells were incu-51 o bated with 0.4 mCi of Na2 CrO^ (Amersham Corp) for one hr at 37 C with occasional shaking. Various number of effector cells in duplicate 4 51 were assayed against 1 x 10^ " Cr-labelled EL4 target cells in a quan-tity of 0.2 ml/well in 96 well U-bottom microtiter plate (Flow Lab, cat. # 76-013-05)• The plates were spun at 170 x g for 5 min and incubated for 3 - 4 hr at 37°C and 5% C02 before harvesting. 0.1 ml of cell-free supernatant were transferred to 12 x 75 plastic tubes (Amersham) and counted in a Picker Pace-1 gamma counter. Percent specific lysis was calculated by the following formula: % Specific lysis = test count - spontaneous count maximum releasable count - spontaneous count x 100% 7 The maximum r e l e a s a b l e count was u s u a l l y 90% o f the t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o the t a r g e t c e l l s . The spontaneous r e l e a s e f o r the experiments reported ranged from 15 -30% of the maximum r e l e a s a b l e count. The f r a c t i o n o f t a r g e t c e l l s l y s e d , f , i s f i t t e d to the equa-t i o n f = 1 - e ~ N o < t (4-7). Where N= t o t a l number of s e n s i t i z e d c e l l s , i s the constant p r o p o r t i o n a l to the frequency o f CTL and t i s the assay time i n h r . Under appropriate c o n d i t i o n s , f i s the same as the f r a c -t i o n a l s p e c i f i c l y s i s , p, so t h a t N°<t= - I n (1-p) (4-7). Expressing the data i n N«<t values r e f l e c t the number of CTL i n v o l v e d i n c y t o t o x i c i t y r a t h e r than the number o f t a r g e t c e l l s destroyed. C e l l c u l t u r e s f o r l i m i t i n g d i l u t i o n . A l l c u l t u r e s were set up i n c o n i -cal-bottom m i c r o t i t e r t r a y (Flow Lab, c a t . # 76-023-05) i n 0.2 ml volumes c o n t a i n i n g l i m i t i n g numbers of spleen c e l l s , 1 x 10^" i r r a d i a t e d s t i m u l a t o r c e l l s and 2% EL4 SN. On day 5 o r 6, w e l l s were assayed against the appropriate t a r g e t s . In cases where i n d i v i d u a l 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 a g a i n s t two t a r g e t c e l l s , c u l t u r e s were f i r s t d i v i d e d i n t o halves as p r e v i o u s l y described (61). Contents o f each w e l l s were mixed using a 12 channel m u l t i p i p e t t o r (Flow) and 80 u l dispensed i n t o 2 separate p l a t e s . 120 u l of 3 x 10 3 B6 LPS b l a s t o r EL4 l a b e l l e d t a r g e t s were added. Cultures were spun as before and allowed to i n c u -bate f o r 4 hr before h a r v e s t i n g . A c u l t u r e was scored as p o s i t i v e i f i t s count exceeds the 95% confidence l i m i t o f the spontaneous count. LPS b l a s t s . B6 spleen c e l l s were c u l t u r e d w i t h 20 ug/ml l i p o p o l y s a c c h a r i d e (LPS) (derived from E s c h e r i c h i a c o l i serotype 055:B5) (Sigma) at 1 x 10^ 8 cells/ml in a volume of 10 ml in tissue culture flasks (Falcon #3012, 2 25 cm growth area) kept in an upright position. The cells harvested AB hr later were referred to as B6 LPS blasts. 125 I-labelling of tumour cells. Cells were labelled as previously described (49). 5 x 106 cells/ml of tumour cells and 2 uCi of 1 2 5IUdR /ml (Edmonton Pharmaceutical Center) were incubated at 37°C and 5% COg for 3 hr in CM, following which the cells were washed three times^in phosphate buffered saline. Each mouse was injected intraperitoneally (i.p.) with 1 x 10 IUdR-labelled tumour cells. Counts were moni-tored using a Phillips electronic counter and timer. To reduce thyroid 125 uptake of I, the drinking water was supplemented with 0.1% KI. 125 I-labelling of cultured cells. Normal lymphocytes sensitized with irradiated EL-4 cells and subsequently expanded in EL4 SN were incubated with 0.5 uCi of 125IUdR/ml for an additional 18 hr.(49). 2 x 107 cells were injected i.p. into each mouse. Cyclophosphamide (CY). 200 mg of CI (Bristol Myers of Canada) were dissolved in 10 ml sterile distilled water. Mice were individually weighed and administered intravenously (i.v.) with CY at 100 mg/kg. Anti-Thy-1 antibody. The anti-Thy 1.2 antibody used was a rat anti-murine Thy 1.2 monoclonal antibody. It was a gift of Frank Symington 9 of the Dept, of Pathology, Univ, of Pennsylvania). The a s c i t i c form of t h i s antibody was r a i s e d by i n j e c t i n g 1 - 2 x 10 hybridoma c e l l s into i r r a d i a t e d (600 rads) mice of any s t r a i n which has been in j e c t e d i , p . with 0 . 5 ml pristane 2 weeks before and received 50 u l of rabbit anti-mouse lymphocyte serum (Microbiological Associates) i . p . 3 days before the i n j e c t i o n of hybridoma c e l l s . For treatment with t h i s a n t i -body, the e f f e c t o r c e l l s were incubated with 1 ml of a s c i t i s at a d i l u -t i o n of 1 i n 100 i n RPMI 1640 i n 10mM Hepes, pH 7.2 f o r 30 min at room temperature. The c e l l s were washed once and resuspended i n 1 ml of 1 i n 10 low t o x i c rabbit complement (Cedarlane Lab) and incubated f o r another 30 min at 37°C. The c e l l s were washed twice before use. S t a t i s t i c a l a n a l y s i s . To assess the s i g n i f i c a n c e of the mean s u r v i v a l time (MST), t s t a t i s t i c s f o r the difference between two means were u t i l i z e d (57). rA P value of<0.05 i s considered s i g n i f i c a n t l y d i f f e r e n t . RESULTS E f f e c t of Con A SN on the anti-EL4 tumour c y t o t o x i c i t y i n normal B6  spleen c e l l s . I t has been demonstrated that activated T c e l l s can be expanded i n v i t r o by means of Con A SN (34)• We tested t h i s by stimu-l a t i n g normal B6 spleen c e l l s with or without i r r a d i a t e d EL4 tumour c e l l s f o r 5 days ( F i g . - I ) . EL4-stimulated B6 spleen c e l l cultures gave a small l e v e l of CTL activity,,whereas unstimulated B6 spleen c e l l s did not evoke any anti-EL4 c y t o t o x i c i t y . Following the addition of Con A SN and alpha methyl mannoside, EL4 stimulated B6 cultures showed a marked i n -10 Figure 1. E f f e c t o f Con A SN on the a c t i v i t y o f B6 anti-EL4 CTL 2 x 10° normal spleen c e l l s + 1 x 10 i r r a d i a t e d EL4 ( 4000 rads) were c u l t u r e d i n a t o t a l volume of 2 ml i n tubes (Falcon # 2057) i n CM. Cultures were harvested on day 5»K The harvested c e l l s were r e c u l t u r e d a t 1 x 106 ( • ), 3 x 105 ( O ) or 1 x 105 ( • ) c e l l s per tube supplemented w i t h 25% Con A SN and 50 mM °<-methyl , -D.-' mannoside. C e l l s were c o l l e c t e d and assayed f o r CTL a c t i v i t y on days 1, 2, 3, or 4 days a f t e r a d d i t i o n of Con A SN«; + EL4 (EL4 stimulated c u l t u r e s ) and - EL4 (non-antigen stimulated c u l t u r e s ) . Note: The arrow a t the bottom of the graph i n d i c a t e the l y t i c a c t i v i t y o f c u l t u r e d c e l l s on day 5 assay. IT 1 2 crease in activity which peaked on day 3 after Con A SN addition. The decline in activity on day 4- after Con A SN addition may be due to the depletion of growth factors from the culture medium. A small but highly significant level of cytotoxic activity against EL4- tumour cells was detected in unstimulated B6 cultures. The lytic activity against EL4 of the recovered cells on a per cell basis is about equal, regardless of the starting cell number in each group. Specificity Qf B6 anti-EL4 CTL before and after feeding with Con A SN. Cultures were assessed for specificity by testing against EL4, Bo LPS blasts and P815 (Table I). Feeding with Con A SN increased the per-cent recovery and activity of both stimulated and unstimulated effector cells. The increase in activity towards EL4- targets was concomitant with an increase in nonspecific activity towards B6 LPS blast and P815 targets. However, the extent of lysis of B6 LPS blast and P815 targets is much lower than that of the EL4 targets. Unstimulated B6 spleen cell:cultures also exhibited nonspecific killing towards EL4-» B6 LPS blast and P815 targets. Similar observations were obtained in cultures fed with EL4- SN. The CTL used in a l l subsequent experiments were those from B6 anti-EL4- cultures after one or two 3-day cycle of feeding with IL2-containing SN. Thy-1 phenotype of B6 anti-EL4- effector cells following feeding with  Con A SN. Treatment of effector cells expanded in Con A SN with mono-13 Table I. Specificity of B6 anti-EU CTL before and after feeding with f\ A r-tn T3-Con A SN' Day 5 assay Responder Stimulator Target B6 B6 ec i f i c l y s i s b NoCt/lQ 6 EL4 EL4 B6 LPS blast P815 EL4 B6 LPS blast P815 42.4.4. 5.56 2.16 11. U 3.24 1.4-8 12.90 0.20 0.55 2.20 0.4-5 0 % Recovery 48.03 41.43 Day 3 post f i r s t feed  Presence of EL4 on d.O + Day 3 post second feed  Presence of EL4 on d.O + Target ^Specific l y s i s Nott/10 6 % Recovery EL4 65.40 69.20 53.10 B6 LPS blast 22.10 7.00 EL4 20.95 (2.5/1) 19.60 23.10 B6 LPS blast 37.34 (2.5/1) 24.00 Target ^Specific l y s i s N*t/10 6 % Recovery EL4 78.96 102.40 66.00 B6 LPS blast 24.51 11.60 P815 48.13 35.60 EL4 40.78 73.60 56.58 B6 LPS blast 43.51 33.20 P815 44.62' 65.60 b 5 2 x 10 responder spleen cells and 10 irradiated EL4 tumour cells were cultured i n a total vol of 2 ml i n tubes (Falcon # 2057). Controls did not include EL4 stimulators. On day 5, cells were washed and 10& cultured cells were fed with 25% Con A SN + 50 mM ©<-methyl-D- mannoside in CM. On day 3 after f i r s t feed, B6 anti-EL4 effector cells were seeded at 3 x 10^ and again supplemented with 25% Con A SN + 50 mM oC-methyl-D-- mannoside. Cul-tures were harvested on day 3 post second feed. Cytolytic activity were determined on day 5, day3 post f i r s t and second feed,using 51Cr EL4, B6 LPS blast and P815 targets Unless indicated otherwise % specific l y s i s values were those from effector to target ratio (E/T) of 5/1. clonal anti-Thy-1 antibody and complement (C) resulted in the abro-gation of activity of both EL4 stimulated and unstimulated effector cells (Figs. 2A and 2B). The cytotoxic indices of anti-Thy-1 antibody for B6 spleen cells sensitized initially vith EL4 and unstimulated cultures were 92% and 94%, respectively. Almost a l l of the CTL acti-vity against EL4 or P815 resides with the Thy-1+ population in both groups. Effect of priming with B6 anti-EL4 cultured cells on the rate of elimi-125 nation of IUdR-labelled EL4 tumour cells from B6 mice. To obtain an indication of tumour clearance in vivo, the radioisotopic assay method of Mills et. al. was adapted in which tumour cells were radiolabelled 125 with IUdR and injected into mice ( 4 9 ) . The amount of remaining 125 IUdR-labelled EL4 in B6 and BDF1 mice is shown in Fig. 3. B6 or BDF1 mice treated with cultured donor cells were able to eliminate 125 IUdR-labelled EL4 tumour cells much more rapidly than control mice. This effect is more evident 7-11 days after the transfer of cultured cells. The data also indicate that mice that received B6 anti-EL4 cultured cells had a significantly longer MST than con-trol mice when they were challenged with live EL4 tumour cells. Survival of cultured cells in vivo. One explanation for the longer survival times of B6 mice treated with B6 anti-EL4 cultured cells to challenge with live EL4 cells is that the cultured cells, which are cytolytic for EL4, could survive long enough in vivo to cause the lysis of the injected EL4 tumour cells. To test this possibility, 15 Figure 2. Thy-1 phenotype of effector cells A) Thy-1 phenotype of B6 anti-EM effector cells B) Thy-1 phenotype of effector cells from culture of B6 spleen cells stimulated with Con A SN only. Cells were cultured as in Table I. On day 3 post first feed, cells were collected and treated with C + monoclonal anti-Thy-1 antibody. Treated cells were assayed for CTL activity against 1 x 10^ 51 cr EL4- ( • ) and 1 x 10* P815 ( O ) targets. E / T R A T I O 18 Figure 3. Effect of priming with B6 anti-EL4. cultured cells on the rate of elimination of ^judR-labelled EL4- tumour cells On day -1, 1 x 10<J "^IUdR EL-4 were administered i.p. in B6 and BDF1 mice. Mice were left untreated (control: O for B6 and BDF1 mice, respectively) or treated with 6 x 10° B6 anti-EL4 cultured cells on day 0 (test: • , • for B6 and BDF1 mice, respectively). Clearance of 125j was measured by whole body gamma counting. On day 6, when the radioactive level was low, both control and test mice were injected with another freshly labelled EL4- tumour cells § 1 x 10 /mouse. The data in parentheses indi-cate the MST (days) of mice in each group; (*) indicates that the MST is significantly longer (P~< 0.05) than control',. Each group consists of five mice. 19 D A Y S A F T E R T R A N S F E R O F C U L T U R E D C E L L S 20 B6 anti-EL4 cultured cells were labelled with IUdR and injected into B6 mice. After 4-8 hr, almost 90% of the label was cleared (Fig. 4)« EL4 SN containing IL2 was injected i.p. daily for 6 days after transfer of cultured cells in an effort to prolong their survival. Also, since CY has been reported to reduce the serum level of IL2 :. inhibitor(s), CY was therefore injected in conjunction with daily injection of EL4 SN (64). The results in Fig. 4 show that neither CY, IL2 or CY and IL2 treated group increased the retention of the B6 anti-EL4 cultured c e l l s . Specificity of B6 anti-EL4 cultured cells in vivo. BDF1 mice were challenged with either labelled EL4 or P815 tumour cells and subse-quently injected with B6 anti-EL4 cultured c e l l s . The elimination of 125 IUdR EL4 was greatly enhanced 7 days following transfer of cultured cells i n B6 anti-EL4 immunized mice (Fig. 5A). The injected donor r x' ... cells also aided i n the elimination of P815 tumour cells during the f i r s t week after transfer of cultured cells (Fig. 5B). As found for CTL activity assayed i n vitro, B6 anti-EL4 effectors appear not to be absolutely specific i n i t s action i n vivo. However, B6 anti-EL4 treated mice bearing EL4 tumours did show a significantly longer MST relative to the control. There was no prolongation of MST i n P815 tumour bearing mice that received B6 anti-EL4 cultured c e l l s . In fact, control mice had a significantly longer MST than test mice. In a reciprocal experiment, mice were challenged with either EL4 or P815 tumours and subsequently administered with D2 anti-P815 effector -21 Figure 4-. S u r v i v a l o f 1 2 5 I U d R c u l t u r e d c e l l s i n vivo Cultured c e l l s were generated as i n ma t e r i a l s and methods. Mice were i n j e c t e d w i t h : a) c u l t u r e d c e l l s on day 0 ( • ); b) 100 mg/kg of CY i . v . 6 h r . before challenge w i t h B6 anti~EL4- c u l t u r e d c e l l s on day 0 ( • ); c) c u l t u r e d c e l l s on day 0 and 100 u l EL4- SN (con-t a i n i n g 100 u n i t s o f IL2 a c t i v i t y i . p . d a i l y f o r 6 days ( O.) and d) 100 mg/kg CY i . v . 6 h r . before c u l t u r e d c e l l s were i n j e c t e d on day 0 and 100 u l EL4- SN (c o n t a i n i n g 100 u n i t s o f IL2 a c t i v i t y ) d a i l y f o r 6 days ( Q ) . Each group c o n s i s t s o f f i v e mice. 22 [ I I I l_ I L 1 2 3 4 5 6 • A Y S A F T E R T R A N S F E R O F C U L T U R E D C E L L S 23 Figure 5. Specificity of B6 anti-EL4 cultured cells in vivo* A) .BDF1 mice were challenged on day -1 with I^UdR EL4. Mice were left untreated ( O ) or challenged with 4 x 10^ B6 anti-EL4 cultured cells on day 0 ( . • ). B) BDF1 mice were challenged on day -1 with I^UdR P815. Mice were left untreated ( a ) or challenged with 4 x 107 B6 anti-EL4 cultured cells on day 0( • ). The data in parentheses indicate the MST of mice in each group; (*) indicates that the MST is significantly longer (P<0.05) than control . Each group consists of five mice. C E L L S 25 26 c e l l s . Elimination of IUdR P815 was rapid during the f i r s t week after tumour challenge in D2 anti-P815 immune mice (Fig. 6B). This effect was no longer observed after the second tumour challenge. D2 anti-P815 cultured lymphocytes did not appear to influence the elimi-125 nation of IUdR EL4 (Fig. 6 A ) . These k i l l e r s tend to be more speci-f i c than B6 anti-EL4 cultured cells.„ However, no survival benefit was conferred on BDF1 mice treated with D2 anti-P815 cultured c e l l s . 125 Effect of combined chemotherapy on the clearance of IUdR EL4« The use of cytotoxic drug as an adjunct to T c e l l therapy may improve tumour destruction i n vivo (21,22,27,38,41,50,52). Mice were given either cultured c e l l s , CY or both (Fig. 7). A l l 3 groups of mice retained less label when compared to the control group. Although the rate of elimination of a l l the experimental groups were equally rapid, treatment with combined CY and B6 anti-EL4 cultured cells led to the longest MST. The data also suggest that CY may have been directly cytotoxic to tumours since the tumour cells were eliminated more rapidly i n mice treated with CY alone. Effect of pretreatment of CY on the clearance of 12^IUdR EL4. The importance of timing in the administration of CY i s illustrated in the experiment that follows. Mice were again given either cultured cel l s , CY or both. However, mice were pretreated with CY a day before instead of after tumour inoculation (Fig. 8).. It was found that CY treatment no longer enhances tumour clearance. This i s expected since CY dis—-appears from blood after 6 hr (45) and can, therefore, no longer 27 Figure 6. Specificity of D2 anti-P815 cultured cells in vivo A) BDF1 mice were challenged on day -1 with 12^IUdR EL4. _ Mice were left untreated ( O ) or challenged with 4 x10 D2 anti-P81$ cultured cells on day 0 ( # ). Note: condi-tions for the generation of D2 anti-P815 effector cells were similar to that of B6 anti-EL4 (see materials and methods). B) BDF1 mice were challenged on day -1 with I^UdR P81..5. Mice were left untreated ( a ) or challenged with 4 x 10 D2 anti-P815 cultured cells on day 0( • ). The data in parentheses indicate the MST of mice in each group. ( A ) indicates that the MST is not significantly different from the control i . Each group consists of five mice. 28 RG-. B A 29 D A Y S A F T E R T R A N S F E R O F C U L T U R E D C E L L S F I G . 6 B 30 Figure 7. Effect of combined chemotherapy on the clearance of '^iUdR EL4 All B6 mice received 1,0IUdR EL4 on day -1. The four groups were: a) control - mice were left untreated ( O ); b) CY - mice were injected with 100 mg/kg CY i.v. on day 0 ( D ); c) mice were immunized with B6 anti-EL4 cultured cells ( • ); d) CY + B6 anti-EL4 - mice received 100 mg/kg CY/mouse i.v. 6 hr before 1 x 10° cultured cells were injected ( • ). The data in parentheses indicate the MST (days) of mice in each group; (*) indicates that the MST is significantly longer (P<0.05) than control. Each group consists of five mice. 31 D A Y S A F T E R T R A N S F E R O F C U L T U R E D C E L L S 32 Figure 8. Effect of pretreatment of CY on the clearance of '^IUdR EU B6 mice were divided into four groups: a) control - mice were left untreated ( O )j b) CY - mice received CY i.v. 1 day prior to challenge with tumour ( O );•c) B6 anti-EL4 cultured cells---mice received 1 x 10^ cultured cells on day 0 ( • ); d) B6 anti-EU cultured cells + CY - mice received CY i.v. on day -2, tumour on day -1-and 1 x 10^ B6 anti-EL4 cultured cells i.p. on day 0 ( • )• The data in parentheses indicate the MST of mice in each group. (-"-) indicates that the MST is significantly longer (P 0.05) than the control, (A) indicates that the MST is not significantly different from the control. Each:-group consists of five mice. 33 D A Y S A F T E R T R A N S F E R O F C U L T U R E D C E L L S 34 influence tumour clearance. The pattern of elimination of B6 anti-EL4 treated group were similar. The MST of CY treated group was not significantly different from that of the control. Both B6 anti-EL4 and combined CY and B6 anti-EL4 treated group exhibited longer MST than the control. Effect of small tumour load on survival. Thus far, mice given large tumour doses a l l succumbed to the tumour. To investigate whether aurvivors can be obtained from mice challenged with small tumour load, mice were injected with 1 x 10^ " live EL4 cells via the subcu-taneous route. Survival times of the control group (untreated) and the experimental group (5 x 10^ B6 anti-EL4 cultured cells separately injected i.v. and i.p.) were recorded. It was found that the sur-vival of the control group was 25 + 1 days. However, of the five cultured cells immunized mice, two died on day 25, one died on day 38 and the two remaining mice survived indefinitely. On the 88th day after tumour challenge, the two survivors were sacrificed and the frequency of anti-EL4 CTL-P in their spleen cells were determined (Fig. 9)• Various doses of normal and tumour immune spleen cells were stimulated with " 104 ' EL4 cells in the presence of 2% EL4 SN. The data shows that the two B6 primed spleen cells have a higher precursor frequency (1/800, 1/950) than normal B6 spleen cells (1/4000). In a separate experiment, one out of five mice survived a lethal challenge of EL4 tumours. The spleen cells of the survivor mouse was obtained and different doses of responder spleen cells were again stimulated with EL4 and 2% EL4 SN. On the day of assay, each well was split into 35 Figure 9 . CTL-P frequency o f normal and B6 anti-EL4 primed B6 mice to EL4 D i f f e r e n t c e l l doses o f responder spleen c e l l s were stimulated w i t h 1 x 1CT i r r a d i a t e d EL4 [4D00 Rads) and 2% EL4 SN and assayed on day 5 against 2- x 1CK 5 1 Cr EL4 t a r g e t s . Primed B6 -1 ( O ); Primed Bb- 2 ( « ) and normal aspleen c e l l s ( ) . 3 6 37 Figure 10. S p e c i f i c i t y o f CTL from normal and primed spleen c e l l s A) S p e c i f i c i t y o f CTL from normal B6 spleen c e l l s B) S p e c i f i c i t y o f CTL from primed B6 spleen c e l l s B6 spleen, c e l l s from normal and primed mice were c u l t u r e d a t 2.5 x 10^ c e l l s / w e l l w i t h 1 x 1 0 ^ i r r a d i a t e d EL4 s t i m u l a t o r s and 2% EL4 f a c t o r , 24 r e p l i c a t e s per c e l l dose. S i x days l a t e r , each w e l l was s p l i t two ways and assayed against 3 x 10^ 51cr-l a b e l l e d EL4 or B6 LPS b l a s t s . 39 halves and assayed against EL4 and B6 LPS blast. Results show that CTL derived from B6 anti-EL4 primed spleen cells were more specific for EL4 than CTL derived from unprimed responder spleen cells (Figs. 10A and 10B). DISCUSSION Normal B6 spleen cells were sensitized in vitro with irradiated EL4 tumour cells and were subsequently expanded in Con A or EL4 SN. These B6 anti-EL4 cultured cells were used in immunotherapy against the growth of EL4 in syngeneic B6 mice. Although the cultured cells, especially after subsequent culture in Con A SN or EL4 SN, were highly cytolytic for EL4, they also killed in a nonspecific manner syngeneic B6 LPS blasts and the third party H-2 mastocytoma, P815, albeit to a lesser extent. The induction of nonspecific CTL by IL2-containing culture SN has been previously described ( 4 0 , 6 3 ) . Adminis-tration of B6 anti-EL4 cultured cells into B6 mice did confer some protection against subsequent challenge against EL4; however, in a majority of the cases, the mice eventually/succumbed to the tumour. 125 In IUdR elimination assays, i t was demonstrated that the cultured <. cells were able to enhanced the clearance of the tumours. The high mortality rate observed even after immunotherapy could reflect the limited survival of these cells in vivo. Studies using radiolabelled lymphocytes have revealed that two days after transfer of lymphoid cells, only 10% of the label remained. This is in agreement with the results obtained by Mills et. a l . ( 4 9 ) . The poor viability of these cells may be due to an inherent deficiency of IL2 in vivo. 40 i t i s reasoned that since exogenous IL2 can sustain cultured T lympho-cytes in vitro, IL2 may act in the same way in vivo (12). However, exogenous IL2 has a short half l i f e in vivo; studies by Wagner eW.al. indicate that there are IL2 inhibitors present in the serum and that GY is effective in reducing the level of inhibitors (64). Therefore, daily injections must be carried out to maintain reasonable IL2 titers (12 ,64). Furthermore, a combination of EL4- SN and CY challenge would be expected to prolong the survival of these cells in vivo. Results in Fig. 4 indicate that crude EL4 SN or both CY and EL4 SN failed to increase the level of retention of donor cells in vivo. By contrast, Cheever et. a l . (12) obtained higher cure rate for the FBL-3 tumours when mice were given a combination of cultured cells, CY and daily injections of purified IL2. The discrepancy in our results may be due to the different manner in which donor cells v/ere sensitized and cultured and/or the inherent immunogenicity of the tumours. There are disparities between reports in regard to the presence of serum IL2 inhibitors. In a recent report, Donohue et. a l . (18) suggested that kidney clearance of IL2 and not IL2 inhibitors, may account for the short half l i f e of IL2. In sum the reasons for the short half l i f e of the labelled lymphocytes in vivo are not understood. Analysis of the functional T cell subsets of in vivo primed donor T cells that were secondarily sensitized in vitro have been described by others (8,29,30,38). It was found that the majority of the cells that express the Lyt 1 alloantigen or a rat equivalent of TTrC marker n ' + — t 4* (W3/25 ), and not the Lyt 1~2 3 , mediate in vivo tumour destruction. Moreover, in vivo effects and in vitro assays do not often correlate 41 as these helper cells are not cytotoxic in chromium release assays. Others have observed that after infusion of donor T cells, there was a time delay before regression of established tumours occurred(20,29, 30,52). It was hypothesized that the time delay is caused by the recruitment of other cell types in the recipient by donor TTTC, thereby n giving rise to a secondary CTL response (30). The involvement of the hostjs immune system in the enhanced elimination of EL4 in B6 mice injected with B6 anti-EL4 cultured cells is likely. This is so since the injected cells had a short half time (< 10% remaining at 48 hr) and enhanced clearance of EL4 was detected 7 days later. However, since cultured cells consist of a heterogeneous population of different sub-sets of lymphocytes, i t is not clear whether T^ C, CTL or other T cell subsets are preferentially expanded in the presence of IL2. The Lyt phenotype of the cell subpopulation responsible for tumour destruction in vivo remains to be determined. T cells appear to be the major effector cells in vivo as anti-Thy-1 and C abolishes its effectiveness in tumour therapy (10,14,29)* In our study, nonspecific killer cells induced in vitro appear to fee operative in vivo as well. The work of Orgad et. al. (53) has indicated that there is a potential hazard of generating lymphocytes % that may develop immune response against normal tissue antigens, thereby resulting in autoimmune disease when reinjected into the host. Our result show that although cultured cells lysed B6 LPS blast in vitro, no graft versus host symptoms were observed in mice treated with these cells. Cultured cells sensitized to EL4 that aided in the elimi-nation of P815 failed to prolong the MST of the mice to P815. ' -42 D2 spleen cells stimulated with P815 exhibited nonspecific ki l l ing for EL4 tumours as well. However, specificity against P815 was demons-trated in in vivo elimination assays. The transferred cells no longer enhanced the elimination of P815 7-11 days after adoptive transfer of cultured cells into BDF1 mice. Furthermore, D2 anti~P8l5 treated BDF1 mice did not show increased MST to either EL4- or P815 (Figs. 6A and 6B). Results obtained by Mills et. a l . using tumour immune D2 spleen cells expanded in IL2 showed that these kil lers were similarly nonspecific in vitro but were nonetheless specific in vivo. However, they found a greater enhancement of syngeneic P815 tumour elimination and in some cases, the mice were rendered tumour free ( 4 8 , 4 9 ) • The use of CY as an adjunct to adoptive therapy have proved to be more effective than the transfer of cultured cells alone (21,22,27,38, 41,50,52). CY appears to be directly cytotoxic to tumours in vivo 125 since IUdR-labelled tumour cells were eliminated more rapidly in CY treated mice when compared to control mice (Fig. 7)• Although the elimination profile of mice treated with CY, B6 anti-EL4 cultured cells or combination of CY and anti-EL4 cultured cells were similar, CY plus cultured cells treated animals' allowed for the longest MST. A number of investigators have employed donor cells from mice that received prior in vivo priming before secondary sensitization in vitro. This approach may have generated a large number of effector cells with an enhanced in vivo anti-tumour reactivity in view of the higher rate of survival observed in mice administered with cultured cells alone (28-30) 43 or in conjunction with CY (11,12,21,27,38,39). It is also argued that direct killing of tumour cells may not be the sole function of CY in vivo in that unlike other tumours, Meth A tumours are relatively resis-tant to CY treatment; tumour regression was only observed in mice trea-ted with both CY and immune cells. It was propsed that CY treatment may have resulted in the depletion of suppressor T cells leading to an enhanced immune response of sufficient magnitude to effect tumour eradi-cation. Two out of five B6 -mice treated with B6 anti-EL4 cultured cells survived a lethal challenge of EL4 tumour cells. In these mice, the CTL-P frequency against EL4 was five times higher than uhprimed mice. Furthermore, the CTL obtained from the immune mice were more specific for EL4 as compared to CTL obtained from normal mice. Thus, one expla-nation for the greater resistance to tumour challenge as a result of immunization with B6 anti-EL4 cultured cells may be due to an increase in the frequency, and possibly, the affinity of CTL-P to EL4. The discovery of IL2 allows for the continued propagation of antigen reactive T cell clnes in vitro (34)• Since T cell clones are homogeneous, they facilitate the analysis of their function. Attempts were also made to generate B6 anti-EL4 CTL clones. Normal B6 lymphocytes stimulated with EL4 were maintained in the presence of EL4 SN with weekly addition of irradiated EL4 stimulator and irradiated B6 feeder spleen cells. However, these long term cultures became increasingly nonspecific in their cytolytic activity and attempts to clone CTL specific for EL4 also failed. Giorgi et. a l . (36) have been more successful in maintaining CTL lines reactive against Balb/C 43 or in conjunction with CY (11,12,21,27,38,39). It is also argued that direct killing of tumour cells may not be the sole function of CY in vivo in that unlike other tumours, Meth A tumours are relatively resis-tant to CY treatment; Tumour regression was only observed in mice trea-ted with both CY and immune cells. It was proposed that CY treatment may have resulted in the depletion of supprespar T cells leading to an enhanced immune response of sufficient magnitude to effect tumour eradi-cation. Two out of five B6 mice treated with B6 anti-EL4 cultured cells survived a lethal challenge of EL4 tumour cells. In these mice, the CTL-P frequency against EL4 was five times higher than unprimed mice. Furthermore, the CTL obtained from the immune mice were more specific for EL4 as compared to CTL obtained from normal mice. Thus, one expla-nation for the greater resistance to tumour challenge as a result of immunization with B6 anti-EL4 cultured cells may 'be due to an increase in the frequency, and possibly, the affinity of CTL-P to EL4» The discovery of IL2 allows for the continued propagation of antigen reactive T cell clones in vitro c ( 3 4 )• Since T cell clones are homogeneous, they facilitate the analysis of their function. Attempts were also made to generate B6 anti-EL4 CTL clones. Normal B6 lymphocytes stimulated with EL4 were maintained in the presence of EL4 SN with weekly addition of irradiated EL4 stimulator and irradiated B6 feeder spleen cells. However, these long term cultures became increasingly nonspecific in their cytolytic activity and attempts to clone CTL specific for EL4 also failed. Giorgi et. a l . (36) have been more successful in maintaining CTL lines reactive against Balb/C AA plasmacytoma MPC-11 for one year in Con A SN. The CTL lines were more effective when a mixture of tumour cells and clones were administered (Winn assay) and not when injected i.v. four days after tumour adminis-tration. Another CTL clone (1E4) specific for abelson virus induced lymphoma was found to survive for long period of time without antigen or culture SN both in vitro and in vivo (16). Multiple or single injection of T cell clones in animals given a low dose of tumour cells resulted in 50-80% long term survivors. However, they also observed \i that i.v. injection of CTL clones were less effective than i f the clones were injected directly at the tumour site. It was postulated that cytotoxic clones were unable to reach the site of the tumour when injected i.v. Evidence of the latter is supported by the demonstration that clones lose their surface receptors for high endothelial venules - receptors that are necessary for normal, lymphocyte:-migration (15). Moreover, cloned IL2-dependent CTL and TTJC show poor localization to n recipient lymphoid tissue (7)• Lymphocytes recirculate from blood to lymph via high endothelial venules into lymph nodes and Peyer's patches. Dailey et. al. (15) suggested that lymphocyte migration is important in the immune response in that i t permits cell to cell interaction bet-ween lymphocytes of different effector functions. Clearly, clones would have limited use in immunotherapy unless these can survive long in vivo and traffic to sites of tumour growth. The potential use of clones to study the interaction between different subsets of lymphocytes should be investigated further. Future studies should be directed towards the use of autologous 4 5 lymphocytes as a source of i n vivo primed cells since this alleviate the problem of finding histocompatible donors (44)• However, the concurrent expansion of suppressor cells i n vitro may pose a signi-ficant obstacle to tumour therapy (37)• This i s exemplified by Sharma and Terasaki's findings (56) that i t was more d i f f i c u l t to sensitize-the lymphocytes of cancer patients to tumours than i t was to sensitize ' the lymphocytes of normals. Thus, measures must be taken to remove suppressor cells from the donor population. This may be achieved by anti-I-J and .'C treatment (19,50) or removal of glass adherent cells (30,50). It should also be pointed out that lymphocytes cultivated in vitro be free of tumour c e l l s . Mills et. a l . obtained promising results when adoptive transfer of cultured lymphoid cells derived from terminal tumour bearers were performed (49) • To date, there has been no documentation on the use of autologous cultured cells i n the treatment of human cancer. In vitro study of lymphocytes from melanoma patients showed that cytotoxic activity were generated against autologous and allogeneic melanoma cells when cultured i n phytohemaglu-tin i n SN containing IL2 (42). Other methods for i n vitro generation of cytotoxic activity from different cancer patients have been esta-blished (58,66). However, i t i s clear that further work in animal model systems are necessary to determine the mechanics of immune regu-lation concomitant with the adoptive transfer of culture-immune c e l l s . • 46 REFERENCES 1. Baldwin, R.W. 1981• Mechanism of immunity in cancer. Pathobiol Annual 11: 155. 2. Berke, G., W.R. Clark and M. Feldman. 1971. In vitro induction of a heterograft reaction. 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