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Characterization of T cell clones derived from a mixed lymphocyte reaction Kwong, Pearl C. 1982

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CHARACTERIZATION OF T CELL CLONES DERIVED FROM A MIXED LYMPHOCYTE REACTION by PEARL C. KWONG B.Sc, The University of 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 IN THE FACULTY OF GRADUATE STUDIES i n THE DEPARTMENT OF MICROBIOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September,1982 (c) Pearl C. Kwong 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 available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of tli Uv&Jn^lo The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date QC£«LAS»V IZ.lWO-DE-6 (3/81) ABSTRACT Monoclonal T cells with distinct function and specificity were isolated and characterized. The method used to obtain monoclonal T cells involved four steps: First, C57BL/6 spleen cells were cultured with i r r a -diated D-BA/2 spleen cells in vitro. Second, the activated C57BL/6 cells were maintained for several months in a medium containing T Cell Growth Factor(TCGF) , irradia'ted DBA/2 and C57BL/6 spleen c e l l s . Third, the activated cells were cloned in microtiter wells by limiting dilution. Fourth, wells detected to contain growth were expanded and tested for their functional a c t i v i t i e s . Using this method, three classes of clones were derived. The f i r s t class of clones were cytotoxic clones, presumably of phenotype, which have different specificities against foreign histocompatibility d + + antigens. The second class of clones was an 11—2 , Thy 1 , Lyt 2 and Lyt 1 low cytotoxic clone whose specificity was against cells carrying H-2D antigens. The supernatant derived from this clone nonspecifically supressed CTL generation and mitogen activation, but was weakly toxic 51 d in the Cr release assay. The third class of clones was an H-2 , Thy 1 Lyt 1 and Lyt 2~ clone which helped augment the CTL responses of cells carrying the H-2Db antigens. This clone required accessory cells for i t s helper function and was effective only when added during the early phase of the CTL response. The supernatant derived from this clone exhibited the same properties as the clone and acted in synergy with TCGF in the augmentation of CTL responses. i i i TABLE OF CONTENTS PAGE Abstract • • • i i L i s t of Tables v L i s t of Figures • v i Acknowledgements v i i Introduction 1 Materials and Methods I. Animals 6 I I . Culture Medium - . 6 I I I . TCGF 6 IV. Generation of Long Term Cultures and the Maintenance of Clones 6 V. S p e c i f i c i t y of Cytolytic Clones 9 VI. Target Cells 10 VII. Assay for Helper A c t i v i t y 10 VIII. Assay f o r Suppressor A c t i v i t y 10 IX. P r o l i f e r a t i o n Assays 11 J X. Supernatant Production 11 XI. C e l l Surface Marker Analysis 11 XII. Freezing of Clones 12 XIII. Production of EL4 IL2 13 Results I. Source of T C e l l Clones 14-I I . Characterization of Clones 14-, 23, 25, 27 14 A. P r o l i f e r a t i o n Requirements 14-B. S p e c i f i c i t y of the Cytolytic. Clones 14-C. Fine S p e c i f i c i t y of the Cytolytic Clones 17 i v PAGE I I I . C h a r a c t e r i s t i c s o f Clone 13 17 A. P r o l i f e r a t i o n Requirements o f Clone 13 17 B. S p e c i f i c i t y of Clone 13 17 C. Fine S p e c i f i c i t y o f Clone 13 20 D. Phenotypic Markers.of Clone 13 20 E. C h a r a c t e r i s t i c s o f Clone 13 Supernatant 29 IV. C h a r a c t e r i s t i c s o f Clone 9 29 A. C e l l Surface Karders o f Clone 9 29 B. P r o l i f e r a t i o n C h a r a c t e r i s t i c s of Clone 9............ 29 C. Helper A c t i v i t y o f Clone 9 29 D. S p e c i f i c i t y o f Clone 9 44 E. K i n e t i c s of the A c t i o n o f Clone 9 44-F. Requirements o f Accessory C e l l s f o r Clone 9 Function 49 G. Clone 9 Acts through .Soluble Mediators 49 Ii. A c t i v a t i o n of CTL i n the Presence o f Both Clone 9 SN and TCGF 49 Discu s s i o n 56 Abb r e v i a t i o n 60 Bibl i o g r a p h y .„ 61 LIST OF TABLES TABLE TITLE PAGE I P r o l i f e r a t i o n Requirements, of Clones 14, 23, 25, '27 15 I I S p e c i f i c i t y of Clones 14, 23, 25, 27 -|6 I I I Fine S p e c i f i c i t y of Clones 14-, 23, 25, 27 >|3 IV P r o l i f e r a t i o n Requirements of Clone 13 '19 V(a) S p e c i f i c i t y of Clone 13 i n the CTL Assay 21 51 V(b) S p e c i f i c i t y of Clone 13 i n the Cr Release Assay 22 VI Fine S p e c i f i c i t y of Clone 13 23 VII H-2 Phenotype of Clone 13 '24. VIII Suppressive A c t i v i t y of Clone 13 SN 30 IX S p e c i f i c i t y Tests on Clone 13 SN 31 X Suppression by Clone 13 Cannot be Reversed by IL2 32 XI Comparison of A c t i v i t i e s of Clone 13 Cells and Clone 13 SN i n the Cr-51 Release Assay 33 XII Suppression of Con A and LPS activation by Clone 13 SN 34-XIII II-2 Phenotype of Clone 9 35 XIV P r o l i f e r a t i o n Requirements of Clone 9 42 XV Helper A c t i v i t y of Clone 9 .4-3 XVI S p e c i f i c i t y of Clone 9 45 XVII Fine S p e c i f i c i t y of Clone 9 and Clone 9 SN 4-6 XVIII S p e c i f i c i t y of CTL Activation by Clone 9 and Clone 9 SN '47 XIX Kinetics of the Action of Clone 9 48 XX Requirement of Accessory Cells for Clone'9 Function 50 XXI Kinetics of the Action of Clone 9 SN - 5 1 A c t i v i t y of Clone 9 SN i n the Thymocyte P r o l i f e r a t i o n Assay 52 XXIII A c t i v i t y of Clone 9 SN i n the Clonal P r o l i f e r a t i o n Assay XXIV Activation of CTL i n the Presence of Both Clone 9 SN and TCGF LIST OF FIGURES FIGURE TITLE PAGE 1 Flow Chart of the Establishment and Maintenance of Clones y 2A Thy 1.2 Markers on Clone 13 25 2B Lyt Markers on Clone 13 27 3A Thy 1.2 Markers on Clone 9 26 3B Lyt 1 Markers on Clone 9 3g. 3C Lyt 2 Markers on Clone 9 ^0. ACKNOWLEDGEMENT I would l i k e to express my si n c e r e thanks to Drs. Hung-Sia Teh and D.G. K i l b u r n f o r t h e i r e x c e l l e n t guidance and support i n t h i s p r o j e c t . Many thanks to Miss Margaret Yu who provided a l l the TCGF f o r t h i s p r o j e c t . I am also indebted to Tudor Buican f o r performing the FACS a n a l y s i s o f the clones. 1 INTRODUCTION The activation of T c e l l s requires complex interactions between T c e l l subsets, accessory c e l l s and t h e i r soluble factors. Numerous studies had been done to elucidate the mechanism of T c e l l activation (20, 4 3 , 50, 51). Experimental data gathered so f a r have led investigators to postulate the i n t e r l e u k i n model of T c e l l activation ( For review, see reference 4-2 ) . In t h i s model, resting T c e l l s are activated into mature effector c e l l s v i a two signals. Signal 1 i s i n the form of antigen. Within a few hours of interacting with antigen or mitogen, resting T c e l l s express receptors for interleukin 2 (IL2) (20 , 4 2 , 4 3 ) . Interleukin 2 i s a generic name given to a class of nonspecific f a c t o r ( s ) , usually prepared by stimulating spleen c e l l s with the T c e l l mitogen, Concanavalin A (4-4,49,58). Upon binding of IL2 to IL2 receptors, 'poised' T c e l l s are stimulated into p r o l i f e r a t i o n and matu-ratio n . However, t h i s model i s f a r from complete. For example, the exact number of interleukins required f o r T c e l l activation i s s t i l l undefined and preliminary worksin several laboratories have suggested that i n addition to IL2, T c e l l d i f f e r e n t i a t i o n factors are required for the activation of T c e l l s (50,51). Studies to map the events leading to T c e l l activation had always u t i l i z e d mixtures of c e l l populations or populations enriched by conven-t i o n a l separation techniques (56). The use of such heterogeneous T c e l l populations i n the study of T c e l l function has certain major l i m i t a t i o n s : I t i s d i f f i c u l t to use t h i s approach to determine the function of each c e l l type to any great d e t a i l , and lymphokine studies are complicated since i t i s often d i f f i c u l t to attribute the lymphokine(s) i n question to the p a r t i -cular c e l l type that i s producing i t . F i n a l l y , i t i s hard to define the •. number or the nature of the- c e l l types participating i n the mediation of a par t i c u l a r function. 2 One approach to resolve these problems i s to i s o l a t e a n t i g e n - s p e c i f i c clones. In order f o r these clones to be of use f o r biochemical and genetic studies, the clones must not only have the c a p a b i l i t y to expand c o n t i -nuously i n culture, but they must also r e t a i n stable function and s p e c i f i c i t y . Recently, several strategies vere used to e s t a b l i s h continuous growing T c e l l clones. One of the. f i r s t methods t r i e d was the somatic c e l l h y b r i d i z a t i o n technique which was used su c c e s s f u l l y f o r producing B c e l l hybridomas secreting monospecific antibodies ( 5 7 ) . Normal antigen-primed T c e l l s can also be fused with a T c e l l lymphoma (39)• Hybrids are screened by using s e l e c t i v e conditions such that only hybrids can survive i n the cul t u r e . These hybrids are subsequently cloned and are tested f o r f u n c t i o n . Using t h i s approach, hybrids that suppress antibody responses (39) or help antibody and CTL responses (1,4-7) were obtained. These clones were found to secrete factors which mimic the action of the c e l l s . One advantage associated with t h i s method is' that the clones have a high growth p o t e n t i a l . They can be grown i n v i t r o or adapted f o r a s c i t i c growth i n vivo. However, t h i s technique i s only useful i f hybrids can be e a s i l y obtained; the frequen-cy of getting f u n c t i o n a l hybrids i s very low. Hybrids often lose chromosomes and subsequently immunologic a c t i v i t y . Furthermore, the b i o l o g i c a l l y active materials are only synthesized i n minute qu a n t i t i e s by T c e l l hybrids (39)• A d i f f e r e n t approach was reported by Finn et a l . (9) and R i c c i a r d i -Castagnoli et a l . (28). Thymic lymphomas were induced i n animals Injected intrathymically with r a d i a t i o n leukemia v i r u s - i n f e c t e d immune spleen c e l l s . The lymphomas were established as permanent c e l l l i n e s and were shown to mediate ant i g e n - s p e c i f i c helper or suppressor function. This approach had not proved useful mainly because of the low frequency of obtaining f u n c t i o -nal c e l l s and v a r i a t i o n i n v i r u s preparations always leads to the f a i l u r e of the induction of thymic lymphomas i n r e c i p i e n t mice. 3 Another, method was introduced by Dennert et a l . (5,6,7). They showed that alloreactive T c e l l s from a d anti-k MLR'can be maintained i n tissue culture i n d e f i n i t e l y by regular feeding with stimulator c e l l s . The c e l l l i n e obtained was shown to mediate both helper and c y t o l y t i c function. This c e l l l i n e was .subsequently cloned and they were able to obtain.cytolytic clones which were reactive against Ia" antigens. This approach was also employed by Fathman et al.(8) to produce alloreactive clones and p r o l i f e r a -t i n g T c e l l clones s p e c i f i c for soluble antigens. A major problem with t h i s approach i s the c e l l l i n e s usually lose t h e i r function and eventually die. Rubin et al.(54) had attempted using the Dennert culture technique to produce c e l l l i n e s reactive to various H-2 determinants. I t was observed that i n most cases the c e l l l i n e s l o s t t h e i r functional a c t i v i t y and died a f t e r three to four restimulations. However, such occurences can be prevented i f irradiated spleen c e l l s were added to the culture. In 1976, Morgan, Ruscetti.and Gallo (48) showed that human periphe-r a l blood lymphocytes can be maintained i n v i t r o f o r long periods of time i n TCGF ( T C e l l Growth Factor )-conditioned medium obtained from phyto-hemagglutinin stimulated lymphoid c e l l s . TCGF or IL2 has an apparent molecuar weight of 30,000 i n the mouse (49). I t was subsequently found as . an agent responsible f o r the p r o l i f e r a t i o n of most T c e l l s bearing TCGF receptors(44.,49) • On the basis of these findings, several groups had used TCGF to propagate.cell l i n e s or clones with c y t o l y t i c (10,23,27,30,31,35), helper(25) and suppressor(25) function. The general method used by these workers involves enriching f o r antigen-specific c e l l s by repeated antigen stimulation and propagation of the c e l l s i n TCGF followed by cloning i n soft agar or l i m i t i n g d i l u t i o n . The main problem with t h i s method was encountered i n the cloning step (34)• Haas et a l . (34) observed that cloning from short term cultures usually leads to f a i l u r e i n obtaining clones. The cloning efficiency only increased i f the c e l l s cloned have been grown i n TCGF for a long period. They also improved the cloning effi c i e n c y of c e l l s from short term culture by adding feeder c e l l s to the cloning wells. One major drawback i n adapting the c e l l s i n TCGF before cloning i s that selection i s not focused on antigen-specific clones but rather on clones which have the greatest a b i l i t y to adapt to TCGF. In an e f f o r t to obtain functionally d i s t i n c t and antigen-specific clones i n high frequencies,Glasebrook et a l . (18) obtained alloreactive clones by stimulating responder spleen c e l l s once or several times with stimulator c e l l s i n v i t r o , cloning the c e l l s immediately and thereafter maintaining the c e l l s by periodic feeding with TCGF, stimulators and feeder c e l l s . The clones thus derived are c y t o l y t i c clones reactive towards d i f f e -rent H-2 determinants and a helper clone reactive to Mis determinant. This method was also employed by other workers to derive clones reactive to antigens such as sheep red blood c e l l s (36) and soluble antigens (29). The role of feeder c e l l s , usually i n the form of irradiated syngeneic spleen c e l l s , i s s t i l l unclear. Sredni et a l . (29) mentioned that they added feeder c e l l s i n the propagation of soluble antigen-reactive clones because they can act as a source of antigen presenting c e l l s . I t i s also possible that- the irradiated spleen c e l l s may provide a signal(s) or a factor(s) other than TCGF which i s required for the p r o l i f e r a t i o n of cloned c e l l s . Lutz et a l . (16) showed that the c y t o l y t i c clones derived by Glasebrook et a l . (14) were adherent c e l l dependent for growth and that t h i s dependence can be attributed to the requirement f o r protease sensitive factors produced by macrophages. The clones obtained by t h i s method were-usually found to pr o l i f e r a t e best i n the presence of both antigen and^TCGF. Clones propagated i n TCGF tend to lose chromosomes more readily than those propagated i n TCGF and stimulator c e l l s or feeder c e l l s (13,31,32,38). 5 The method used to derive T c e l l clones i n t h i s project made use of conditions established i n other laboratories. Responder spleen c e l l s were stimulated twice with i r r a d i a t e d stimulator spleen c e l l s i n v i t r o . The c e l l s were then adapted to growth i n medium supplemented with TCGF, stimula-tor and feeder c e l l s f o r 3 months. Clones were obtained from these long term cultures by l i m i t i n g d i l u t i o n and were maintained i n TCGF, stimulator and feeder c e l l s . Using t h i s protocol, c y t o l y t i c and helper clones were derived and characterized. In the accompanying sections some novel proper-t i e s of these clones are described. 6 MATERIALS AND METHODS I. Animals C57BL/6, DBA/2, AKR and SJL mice were purchased from The Jackson L a b o r a t o r i e s , Bar Harbor, Maine. B10, B10.A, B10.A(2R), B10.A(3R), B10.A(4R) and B10.D2 were bred i n the animal f a c i l i t y i n t h i s department. Wistar r a t s were obtained from the Animal Care Center at the U n i v e r s i t y of B r i t i s h Colum-b i a . I I . C ulture Medium The c u l t u r e medium used was RPMI I64O supplemented w i t h 25mM NaHCO^, 10/S f e t a l c a l f serum (Animal Health L a b o r a t o r i e s ) , 10 mM HEPES b u f f e r (pH 7.2), 5 X 10~'Vi 2-mercaptoethanol, 50 units/ m l of p e n i c i l l i n - s t r e p t o m y c i n and 30 ug/ml glutamine. A l l c e l l c u l t u r e s were incubated at 37°C i n 5% C0 2 i n a i r . I I I . T C e l l Growth Factor (TCGF) TCGF was prepared according to the method o f G i l l i s et a l . (4-4-) • B r i e f l y , NH.C1 t r e a t e d spleen c e l l s from 2-3 months o l d Wistar r a t s were 4 c u l t u r e d a t 10^ c e l l s / m l w i t h 5 ug/ml Con A f o r 48 hours i n c u l t u r e d medium. The c u l t u r e supernatants were harvested, f i l t e r s t e r i l i z e d and stored at -o - 2 0 C u n t i l use. The a c t i v i t y of each p r e p a r a t i o n was c a l i b r a t e d by t e s t i n g f o r i t s a b i l i t y to promote the p r o l i f e r a t i o n o f Con A stimulated thymocytes c u l t u r e d at low c e l l d e n s i t y ( 4 5 ) . IV. Generation of Long Term Cultures and the Maintenance of Clones The general procedure f o r the generation o f long term c u l t u r e s i s n o u t l i n e d i n Figure 1. Primary MLR was performed by mixing 4 X 10 C57BL/6 60 responding spleen c e l l s and the same amount o f 2000R i r r a d i a t e d ( Co source GammaCell 220, Atomic Energy of Canada, Ltd.) DBA/2 spleen c e l l s i n a t o t a l volume of 50 ml i n Falcon 3024 f l a s k s . Four days l a t e r , the c u l t u r e s 7 were harvested and 5 X 10 primed responder c e l l s were r e s t i m u l a t e d w i t h the same number o f i r r a d i a t e d DBA/2 spleen c e l l s . A f t e r three days o f 7 Figure 1. Flow Chart of the Establishment and Maintenance of Clones. Refer to Materials and Methods Section f o r Full. D e t a i l s . rt CJ PRIMARY MLR : 4 X 10 ' C51YM,/b SPLEEN CELLS 4 X 1 0 7 DBA/2 IRRADIATED SPLEEN CELLS 5 0 ML COMPLETE MEDIUM, 3024 FLASK 4 DAYS IN 5% C0 2, 37°C SECONDARY MLR : '5 X 1 0 7 PRIMED SPLEEN CELLS 5 X 10 7 DBA/2 IRRADIATED SPLEEN CELLS 50 ML COMPLETE MEDIUM, 3024 FLASK 3 DAYS IN 5% C0 2, 37°C MAINTENANCE AND FEEDING SCHEDULE #1: 10 5 MULTIPLE STIMULATED SPLEEN CELLS 10 6 DBA/2 IRRADIATED SPLEEN CELLS 1 ML 25 % TCGF-50mM ALPHA METHYL MANKOSIDE -COMPLETE MEDIUM ( TCGF-CM ) FALCON 2057 TUBES FEED WITH 1 ML TCGF-CM EVERY 3-4 DAYS RE3TIMULATE WITH 1 0 6 DBA/2 IRRADIATED SPLEEN CELLS EVERY WEEK DURATION : .1 MONTH FEEDING SCHEDULE #2: DOSE OF DBA/2 IRRADIATED SPLEEN CELLS INCREASED TO 2.5 X 1 0 6 PER TUBE. 1 X 10 6 C57BL/6 IRRADIATED SPLEEN CELLS WERE ALSO ADDED WEEKLY TO CULTURES AS FEEDER CELLS. DURATION : 2 MONTHS CLONING: 0.3 CELLS PER WELL 1 X I O 6 DBA/2 IRRADIATED SPLEEN CELLS PER WELL FUNCTIONAL ASSAYS: 5 1CR RELEASE ASSAY HELPER ASSAY SUPPRESSOR ASSAY 9 i n c u b a t i o n , 10 secondary st i m u l a t e d MLR b l a s t s were c u l t u r e d i n Falcon 2057 tubes c o n t a i n i n g 1 ml volume of TCGF-CM ( 25% TCGF, 50 mM alpha methyl mannoside and complete medium). Cultures were f e d with TCGF-CM every 3 -4 days and were r e s t i m u l a t e d w i t h 10^ i r r a d i a t e d DBA/2 spleen c e l l s weekly. Cultures were reseeded when the c u l t u r e d e n s i t y approached 10 . One month a f t e r the i n i t i a t i o n of the c u l t u r e s the dose of DBA/2 s t i m u l a t o r c e l l s 6 6 added weekly was increased to 2 .5 X 10 c e l l s per tube and 10 2000R i r r a d i a t e d C57BL/6 (B6) spleen c e l l s were also added weekly to serve as accessory c e l l s . A f t e r two more months i n c u l t u r e , the long term c e l l s were cloned by l i m i t i n g d i l u t i o n i n 96 w e l l Costar f l a t bottom w e l l s . The c e l l s to be cloned were d i l u t e d to an average number of 0 . 3 c e l l s per w e l l . 6 1 X 10 i r r a d i a t e d DBA/2 spleen c e l l s were added to each w e l l . The w e l l s were fed on days 5 and 10 w i t h TCGF-CM. On day 14 i n d i v i d u a l w e l l s were 51 screened f o r c y t o l y t i c f u n c t i o n using the Cr r e l e a s e assay and w e l l s w i t h growth were i d e n t i f i e d by observation under an i n v e r t e d microscope. N o n - c y t o l y t i c clones were t e s t e d f o r t h e i r a b i l i t i e s to help o r suppress a primary C57BL/6 anti-DBA/2 (B6 anti-D2) c y t o t o x i c response. Clones w i t h detectable helper o r supressor f u n c t i o n s were maintained i n tubes by c u l -t u r i n g a t i n i t i a l d e n s i t i e s o f 1 X 10^ - 5 X 10^ c e l l s per tube i n the presence o f TCGF-CM together w i t h 2 .5 X 10 6 DBA/2 (D2) i r r a d i a t e d spleen c e l l s and 10^ B6 i r r a d i a t e d spleen c e l l s . Clones maintained i n f l a s k s 5 (Falcon 3012) were seeded weekly at 10 c e l l s per f l a s k i n 10 ml of medium 7 7 con t a i n i n g TCGF-CM, 2 .5 X 10 D2 i r r a d i a t e d spleen c e l l s and 10 B6 i r r a d i a -ted spleen c e l l s . V. S p e c i f i c i t y o f C y t o l y t i c Clones P815 mastocytoma c e l l s (H-2 ) or D2 LPS b l a s t s were l a b e l l e d w i t h 100 uCi Na 2 5 1Cr0^(New England Nuclear) at 37°C f o r 1 hour. 1 X 10^ l a b e l l e d c e l l s were added to round bottom w e l l s c o n t a i n i n g twofold d i l u t i o n s o f the 10 c y t o l y t i c clones, at a ra t i o of 2:1 or 1:1 cloned c e l l s to targets. The p l a -tes were then centrifuged at 170 X g for 5 minutes and incubated f o r 4 hours at 37°C. After incubation 100 u l of supernatant from each well were c o l l e c -ted and counted for release of r a d i o a c t i v i t y i n a Picker Pace I gamma coun-t e r . The percent s p e c i f i c cytotoxicity was calculated according to the formula : % s p e c i f i c l y s i s = ( experimental CPM - spontaneous CPM / maxi-mum CPM - spontaneous CPM ) X 100. Spontaneous release i n a 4 hour assay varied from 10-15 % f o r P815 and 12-35 % f o r LPS blasts. VI. Target Cells P815 mastocytoma c e l l s (H-2 ) were maintained by weekly i n t r a p e r i t o -7 neal passage through DBA/2 mice. LPS blasts were prepared by incubating 10 spleen c e l l s with 10 ug/ml LPS (Sigma) i n 10 ml complete medium f o r 2-3 days. VII. Assay for Helper A c t i v i t y Clones were washed thoroughly with prewarmed medium and 10^ 2000R •3 irradiated clones were mixed.with 3 X 10 B6 responder spleen or lymph 5 node (LN) c e l l s and 10 ir r a d i a t e d D2 stimulator spleen c e l l s . These c e l l s were cultured i n 96 well V bottom microtiter trays i n a volume of 0.2 ml at 37°C i n humid 5% COg. On day 5 100 u l of supernatant from each well were 3 51 withdrawn and 2 X 10 Cr lab e l l e d targets were added to each we l l . Trays were centrifuged and incubated f o r 4 to 6 hours at 37°C. 100 u l culture supernatant were withdrawn and counted f o r r a d i o a c t i v i t y . VIII. Assay for Suppressor A c t i v i t y . 1 X 10^ irr a d i a t e d cloned c e l l s were mixed with 2 X 10^ B6 responder 5 spleen c e l l s and 10 irradiated D2 stimulator spleen c e l l s i n 96 well V bottom microtiter trays i n a volume of 0.2 ml per well at 37°C. Five days 51 l a t e r , the same J Cr release assay as described for the helper assay was performed. 11 IX. C e l l P r o l i f e r a t i o n Assays 3 4 A. Clonal P r o l i f e r a t i o n Assay. 5 X 10 - 1 X 10 eloned c e l l s were c u l -tured i n round bottom wells i n the presence of 25% TCGF, 50 mM alpha methyl mannoside and 5 X 10 ir r a d i a t e d D2 spleen c e l l s or B6 spleen c e l l s . The volume of the culture i n each well was 0.2 ml. After 48 hours of incubation at 37 C, the cultures were pulsed with 1 uCi methyl H Thymidine (New Eng-land Nuclear) for 6 hours. The c e l l s were harvested using a c e l l harvester onto glass f i b e r f i l t e r s . The r a d i o a c t i v i t y was counted on a Searle l i q u i d s c i n t i l l a t i o n counter. B. Thymocyte P r o l i f e r a t i o n Assay. The procedure has been described pre-viously (45). B r i e f l y , 10 thymocytes were cultured with 2 ug/ml Con A and the appropriate d i l u t i o n of supernatant i n question i n round bottom wells 3. f o r 72 hours. At the end of 72 hours, the cultures were pulsed with H Thy-midine f o r 6 hours, harvested and counted i n a l i q u i d s c i n t i l l a t i o n counter. X. Supernatant (SN) Production A. Clone 13 Supernatant Production. Clone 13 c e l l s were washed thrice and incubated f o r 48 hours at 4 X 10^ c e l l s i n 2 ml complete medium i n Falcon 2057 tubes. Supernatants were harvested and stored at -20°C u n t i l use. B. Clone 9 Supernatant Production. Thrice washed clone 9 c e l l s were incu-bated at 2 X 10^ c e l l s i n 2 ml complete medium i n Falcon 2057 tubes. Super-natants were harvested at 48 hours and concentrated 10 times by UM10 Amicon u l t r a f i l t r a t i o n . The concentrated material was active at a d i l u t i o n of 1 i n 10. XI. C e l l Surface Markers Analysis The H-2 phenotypes of clones 13 and 9 were analysed by antibody-depen-dent complement-mediated cytotoxicity assay. Anti-D^( (B10.AKM X A.SW) a n t i -ATH ), anti-K d( ( B 1 0 X A) a n t i - B10.D2 ), anti-D b( (A X B10.D2) a n t i - B10.A (5R) ) and anti-K b( (B10.A(5R) X A/SN) anti-B10.A ) sera were kindly provided 1. 12 by Dr. D.G. Kilburn, who obtained them from the National I n s t i t u t e of A l l e r -gy and Infectious Diseases, Bethesda, Maryland. For treatment of c e l l s with 5 these antisere, 5 X 10 c e l l s were incubated i n Falcon 2058 tubes with 0.1 ml of the appropriate antisera f o r 30 minutes at room temperature. The c e l l s were then washed twice and incubated with 0.1 ml of low tox rabbi t complement ( Cedarlane Laboratories, Ontario ), d i l u t e d 8 times with RPMI 1640 + 10 mM HEPES, f o r 40 minutes at 37°C. The c e l l s were washed once and the numbers of l i v e and dead c e l l s were scored; A t o t a l of 200 c e l l s vere counted. The cytotoxic index was calculated as: % k i l l e d i n experimental group - % k i l l e d i n complement group / 100 - % k i l l e d i n complement group. The Thy 1, Lyt 1 and Lyt 2 markers were analysed by using the f l u o r e s -cence activated c e l l sorter (FACS IV,. Becton Dickinson FACS Systems). 1 X 10^ c e l l s i n 100 u l DMEN ( b i o t i n free) were treated with a 1/100 d i l u t i o n of a b i o t i n conjugated r a t anti-mouse Thy 1.2 monoclonal antibody or 1/100 d i l u -t i o n of a b i o t i n conjugated r a t anti-mouse Lyt 2 monoclonal antibody, or a 1/20 d i l u t i o n of a b i o t i n conjugated r a t anti-mouse Lyt 1 antibody ( a l l the monoclonal antibodies used.here were obtained from Becton Dickinson). After 30 minutes incubation on i c e , the c e l l s were washed twice and resuspended i n 100 u l DMEM containing 1/100 d i l u t i o n of avidin conjugated FITC. Controls were treated d i r e c t l y with fluorescent reagent (avidin conjugated FITC) without the f i r s t ; s t e p antibodies. The c e l l s were incubated on i c e f o r 30 minutes, washed* t h r i c e , resuspended i n 1.5 ml DMEM plus 5% FCS, and analysed on the FACS. Mr. Tuidor Buican performed the FACS an a l y s i s . XII. Freezing of the Clones The method f o r free z i n g was provided by Dr. D.G. Kilburn, who obtained i t from Dr. H. Mostowski of the National I n s t i t u t e of Health, Bethesda, Maryland. C e l l s were washed twice i n complete medium and at l e a s t 3 X 10 c e l l s were resuspended i n 0 . 5 ml of 10% Dextran (Sigma)-90% FCS solu t i o n 13 The c e l l s were incubated f o r 1/2 to 1 hour at room temperature. Then 0.5 ml of an ice cold mixture of 10% Dextran, 15% DMSO and 75% FCS were added to the c e l l s drop by drop while vortexing at a very low speed. The c e l l mix-ture was transferred to a freezing v i a l . The freezing v i a l was stored i n a styrofoam box, frozen at -70°C for 24 hours and then transferred to l i q u i d To thaw, v i a l s were warmed rapidly i n a 37 C water bath. Cells were washed twice with complete medium ans resuspended i n 2 ml of medium consisting of TCGF-CM, 10 6 B6 irradiated spleen c e l l s and 2.5 X 10 6 D2 irr a d i a t e d spleen c e l l s . The c e l l s were fed with TCGF-CM after 2-3 days. When s u f f i c i e n t growth was detected, the c e l l s were transferred to f l a s k s . XIII.Production of EL4-IL2 EL4 thymoma c e l l s were given by Dr. V. Paetkau of the University of Alberta. The o r i g i n a l l i n e was obtained from the laboratory of Farrar et a l . and the production of IL2 by phorbol rayristic acetate (PMA) stimulation of EL4 thymoma c e l l s had been described previously(53). B r i e f l y , EL4 thymoma c e l l s were washed thoroughly, resuspended to a concentration of 1 X 1oV ml and mixed with 10 ng/ml of PMA (Sigma). At the end of the 40 hours incubation period, the culture supernatant was harvested, s t e r i l i z e d and stored at -20°C. An aliquot was tested f o r i t s a b i l i t y to support the p r o l i f e r a t i o n of low numbers of thymocytes i n the presence of Con A (45). u RESULTS• I. Source of T C e l l Clones The source f o r the establishment of the clones i s shown i n Figure I . Using t h i s method, 2 classes of clones have been derived. One class consisting of different types of c y t o l y t i c clones was presumably of B6 o r i g i n . Although the 11—2 phenotypes of these clones \iere not tested, they are presumed to be of B6 o r i g i n since the r e a c t i v i t y of these clones was against DBA/2 antigens(data to be shown l a t e r ) . The a c t i v i t y of these clones was not stable and had only lasted f o r about 6 months. The second class of clones consists of a c y t o l y t i c clone and a helper clone. These clones were d found to be of H-2 origin(data to be shown l a t e r ) . These clones are stable and can be recovered after freezing. One explanation f o r the derivation of H-2^ clones from a B6 anti-D2 MLR i s that some D2 T c e l l s had survived the i r r a d i a t i o n , thus giving r i s e to T c e l l clones of both H-2^ and H-2^ o r i g i n . I I . Characteristics of Anti-D2 Cytolytic Clones A. P r o l i f e r a t i o n Requirements of Clones 14,23,25,27 As indicated i n Table I, a l l the clones proliferated well i n the presence of TCGF and did not p r o l i f e r a t e at a l l i n the absence of TCGF. None of the clones proliferated well when cultured alone with DBA/2 irr a d i a t e d spleen c e l l s . Maximal p r o l i f e r a t i o n was usually observed i n the presence of both TCGF and stimulator c e l l s , except for clone 27 which proliferated just as well with TCGF alone. B. S p e c i f i c i t y of the Clones The s p e c i f i c i t y of clones 14,23,25 and 27 was assessed by com-paring t h e i r l y t i c a c t i v i t i e s against various LPS blast targets. Table II shows s p e c i f i c i t i e s of these four clones against four di f f e r e n t targets: DBA/2 (Il-2 d), AKR (H-2 k), SJL (H-2S) and C57BL/6 (H-2 b). I t can be seen from th i s table that a l l the clones lysed DBA/2 LPS blasts but not AKR, B6 or SJL. TABLE I. PROLIFERATION REQUIREMENTS OF CLONES 14,23,25,27' CLONE 'STIMULATOR TCGF CPM ( M ± S.E.M. ) 14 - 87 + 32 14 + - 150 + 32 14 + + 16059 ± 1679 14 - + 7826 + 1352 25 - - 54 + 15 25 + - 254 + 47 25 + • + 4775 + 436 25 + 2985 + 174 23 - - 47 +9 23 + - 414 + 33 23 + + 12555 + 1290 23 - +. 5256 + 214 27 - 243 + 17 27 + - 2225 + 163 27 + + 12610 + 83 27 - + 12166 + 277 a. 1 X 10 *• c y t o l y t i c cloned c e l l s were cultured with or without 25f0 r a t TCGF, 50 mM alpha methyl mannoside and 5 x 10 ; DBA/2 i r r a d i a t e d stimulating spleen c e l l s i n round bottomed wells f o r 48 hours i n 37 C. TABLE II. SPECIFICITY OF CLONES 14, 23, 25, 27 FRACTION SPECIFIC LYSIS OF LPS. BLASTS' CLONE AKR B6 SJL D2 14 0.10 0.13 0.12 0.49 25 0.05 0.04 0.04 0.31 .23 -0.03. -0.05 -0.03 0.95 27 0.02 -0.04 -0.01 0.24 a. E f f e c t o r to Target r a t i o was 2:1. 17 LPS b l a s t s suggesting t h a t a l l the clones were s p e c i f i c f o r DBA/2 a l i o a n t i -gens. C. Fine S p e c i f i c i t y o f Clones 14, 23, 25, 27 In order to map the f i n e s p e c i f i c i t y o f the i n d i v i d u a l c l o n e s , they were t e s t e d against LPS b l a s t s d e r i v e d from v a r i o u s H-2 recombinant s t r a i n s . The r e s u l t s are shown i n Table I I I . Clones 14 and 25 showed s i g n i f i c a n t r e a c t i v i t i e s a g ainst b l a s t s expressing the H-2D^ antigens. Clones 23 and 27 gave a more complex r e a c t i v i t y p a t t e r n . Clone 27 d i d not r e a c t appreciab-l y w i t h any of the H-2 recombinants, suggesting t h a t the r e a c t i v i t y of clone 27 i s probably d i r e c t e d toward some antigens other than H-2^. Clone 23 was more, d i f f i c u l t to analyse. I t l y s e d P815 which i s known to be Ia antigen negative. However, i t also k i l l e d a v a r i e t y o f t a r g e t s which share only the H-2 IC r e g i o n w i t h 11-2^. U n f o r t u n a t e l y , f u r t h e r work was not done because. -the a c t i v i t y o f the clones was l o s t before f u r t h e r c h a r a c t e r i z a t i o n could, be made. I I I . C h a r a c t e r i s t i c s o f Clone 13 A. P r o l i f e r a t i o n Requirements o f Clone 13 Table IV shows the p r o l i f e r a t i o n requirements o f Clone 13. The r e s u l t s were s i m i l a r to those seen f o r the c y t o l y t i c clones described above i n t h a t i t was TCGF dependent, d i d not p r o l i f e r a t e i n response to antigen alone and the p r o l i f e r a t i o n was best i n the presence of both TCGF and i r r a d i a t e d B6 spleen c e l l s . B. S p e c i f i c i t y o f Clone 13 The s p e c i f i c i t y o f clone 13 was t e s t e d by using two assays. The f i r s t assay system used was d i r e c t e d a t d e t e c t i n g both suppressor and c y t o l y t i c f u n c t i o n s . In t h i s assay, 2 X 10"+ responder LN or spleen c e l l s , 10^ s t i m u l a -t o r c e l l s and 10^ c e l l s of the clone i n question were c o c u l t u r e d . The combi-n a t i o n o f 2 X 10 f responder c e l l s and 10 s t i m u l a t o r c e l l s always r e s u l t s i n TABLE I I I . FINE SPECIFICITY OF CLONES 14, 23, 25, 27 18 FRACTION SPECIFIC LYSIS BY CL0NEa TARGET K IA IB IJ IE IC D U 23 25- 27 B10.A k k k k k d d 0.68 0.67 0.46 0.03 ' B 1 0 . A ( 3R ) b b b b k d d. 0.83 0.65 0.52 -0.02 B10.A(2R) k k k k k d b 0.15 0.87 0.07. 0.01 B10.D2 d d d d d d d 0.68 0.55 0.45 0.10 DBA/2 d d d d d d d 0.89 0.68 0.53 0.33 P815 d d d d d d d 0.79 0.29 0.74 0.79 a. The effector to target.ratio used was 1:1. TABLE IV. PROLIFERATION REQUIREMENTS OF CLONE 13' CLONE STIMULATOR TCGF CPM ( M + SEM 1 3 _ - 84+27 13 13 13 105 + 4 + 3068 + 251 + 6988 + 358 1 X 10 v clone 13 c e l l s w i t h o r without 2% r a t TCGF, 50 mM alpha methyl mannoside and 5 X 10 B6 i r r a d i a t e d s t i m u l a t i n g spleen c e l l s were c u l t u r e d i n round bottomed w e l l s f o r 48 hours i n 37 C. Cultures were pulsed H Thymidine f o r 6 hours, then harvested and counted. 20 the generation of a reasonably high CTL response a f t e r 5 days of incubation, but i f the added clones are suppressive or c y t o l y t i c towards the responding population, then one expects the CTL response to become abrogated. Table VA shows that clone 13 suppressed only the B6 anti-D2 response and B6 anti-AKR response but not the AKR anti-D2 response, suggesting that clone 13 was spe-c i f i c f o r the B6 responder population. However, these r e s u l t s d i d not i n d i -cate whether clone 13 was eit h e r a c y t o l y t i c clone or a suppressor clone. Clone 13 was tested f o r i t s l y t i c a c t i v i t y against B6 and D2 LPS 51 blasts i n the Cr release assay. Table VB shows that clone 13 lysed B6 blasts and not D2 b l a s t s . This indicated that clone 13 was a c y t o l y t i c clone. C. Fine S p e c i f i c i t y of Clone 13 Table VI shows the f i n e s p e c i f i c i t y of clone 13. Clone 13 suppressed only the responses of responders carrying the H-2D^ antigens, namely B10, 1310.A(2R) and B10.A(4-R). This r e s u l t i n d i c a t e d that clone 13 was H-2D s p e c i f i c . D. Phenotypic Markers of Clone 13 As depicted above, clone 13 was derived from a B6 anti-D2 parental c e l l l i n e whose s p e c i f i c i t y was against H-2D^. The next step was to determine i f clone 13 was of H-2 o r i g i n , that i s , a s e l f k i l l e r , or of H-2 o r i g i n , that i s , a D2 anti-B6 k i l l e r . I t can be seen from Table VII that clone 13 was of 11-2^ o r i g i n and was therefore a conventional -D2 anti-B6 k i l l e r clone. The r e s u l t s of the FACS analysis of clone 13 are shown i n Figures 2A and 2B. Figure 2A shows that clone 13 was high i n Thy 1.2. Figure 2B -f shows that clone 13 was Lyt and very low i n Lyt 1 antigens. E. C h a r a c t e r i s t i c s of Clone 13 Supernatant U8 hour supernatants were obtained from cultures of clone 13 i n complete medium and these were tested f o r suppressive a c t i v i t y i n the gene-ra t i o n of CTL responses. It was found that the supernatant had suppressive 21 TABLE V (a)SPECIFICITY OF CLONE 13 IN TIIE CTL ASSAY 3 X 10^ 1 X 10 5 5 X I O 4 - B6 1 X 10 4 % SPECIFIC RESPONDER STIMULATOR SPLEEN CELLS CLONE 13 LYSIS (2000 R) (2000 R) (2000 R) ' B 6 - _ — - 0.0 + 1.1 B6 D2 • - 46.1 + 19.0 B 6 D2 0.0 + 1.0 B6 — — - 0.0 + 3.5 B6 AKR - 34.4 + 5.0 B6 AKR 5.1 + 6.5 AKR _ — - -0.2 + 0.8 AKR D2 - 31.2 + 14.7 AKR D2 + 56.6 + 18.7 AKR + - • 1.4 + 1.7 AKR D2 + - 58.8 + 18.0 • MR D2 + : + 43.0 + 21.5 a. Cultures with the indicated additions were set up i n V bottom m i c r o t i t e r wells i n a volume of 0.20 ml. The culture s were assayed 3 1^ on day 5 against 2 X 1CT ' Cr l a b e l l e d P815 (H-2d) or 5 1 C r l a b e l l e d RDM-4 (H-2 k) target c e l l s . TABLE V(b)SPECIFICITY OF CLONE 13 IN THE 5 1 C r RELEASE ASSAY' EFFECTOR TO TARGET PERCENT SPECIFIC LYSIS OF TARGET RATIO B6 BLASTS(H-2b) D2 BLASTS(H-2d) 20 to 1 50.1 + 4.5 1.6 + 2.0 10 to 1 48.9 + 7.2 - 0.4 + 0.6 5 to 1 48.4 + 8.2 - 0.8 + 0.9 2.5 to 1 40.9 + 0.3 3.9 + 0.2 1.25 to 1 34.4 +.10.1 1.2 + 1.1 a. The assay time was 3.5 hours TABLE VI. FINE SPECIFICITY OF CLONE 13 23 RESPONDER H-2 STIMULATOR CLONE 13 ' % SPECIFIC I K ABJEC D (2000 R) ( 2000 R) LYSIS B10 b bbbbb b _ 2.8 + 1.3 B10 II + - 3.3 + 4.4 B10 n + + 3.2 + 1 . 5 -B10.A k kkkkd d 3.1 + 1.2 B10.A II + - 6.6 + 2 .5 B10.A it + + . 9.2 + 5.9 B10.A(2R) k kkkkd b _ 3.2 + 0.9 B10.A(2R) ti + - 23 ,5 .+ 6.6 B10.A(2R) II + + 3.2 + 1.1 B10.A(3R) b bbbkd d _ _ 4.2 + 2.5 B10.A(3R) it - 46.9 + 26 .4 B10.A(3R) II + + 62.0 + 20.8 B10.A(4R) k kbbbb b _ 3.3 + 2.1 B10.A(4R) II + - 20.8 + 13.9 B10.A(4R) n • -j. + 2.8 + 1.3 B10.A(5R) b. bbkkd d _ 2.1 + 0.9 B10.A(5R) ti + - 39.8 + 18 .6 B10.A(5R) II + + 33 .7 + 8 .5 B6 b bbbbb b 1.9 + 0.9 B6 • II + — 65 .4 + 16.3 B6 II + + 3 .4 + 1 .6 a. Culture conditions same as Table VA. TABLE VII. H-2 PHENOTYPE OF CLONE 13 C E L L ANTIBODY3, PERCENT DEAD CYTOTOXIC INDEX EL4 (H-2b) - 2.2 — EL4 anti-D d 35.8 0 .34 EL4 anti-K 20 .6 0.20 EL4 anti-K 75.0 0.75 EL4 anti-D 84.0 0.84-P815 (H-2d) _ 3.8 -P815 an t i - D d 84 .5' 0.84 P815 a n t i - K d 63.1 0.62 P815 anti-K 4.4- 0.01 P815 a n t i - D b 2.8 -0.01 Clone 13 _ 13 .3 -Clone 13 ant i - D d 93.2 .0.92 Clone 13 anti-K 86.2 0.84 Clone 13 a n t i - K b 8.8 -0.05 Clone 13 anti-D. 7 .9 -0.06 a. Anti-D d: (B10.AKH X A.SW) anti-ATH Anti-K d: (B10 X A) anti-B10.D2 Anti-D b: (A X B10.D2) anti-B10,A(5R) Anti-K b: (B10.A(5R) X A/SN) anti-BlO.A 25 FIGURE 2A. THY 1.2 Markers ©N CLONE 13 . 1 X 1 0 6 v i a b l e clone 13 c e l l s were incubated with medium (1) or with a b i o t i n conjugated r a t anti-mouse Thy 1.2 monoclonal antibody (2) f o r 30 minutes on i c e . This was followed by s t a i n i n g with a v i d i n conjugated FITC. The FACS IV was used to analyse the fluorescence s t a i n i n g . The units used f o r the fluorescence i n t e n s i t y were a r b i t r a r y . Refer to the Materials and Methods section f o r complete d e t a i l s . 26 F L U O R E S C E N C E 27 FIGURE 2B. LYT MARKERS ON CLONE 13. Clone 13 c e l l s were incubated with medium (1), b i o t i n conjugated anti-Lyt 1 (2) or bi o t i n conjugated anti-Lyt 2 (3), followed by staining with avidin conjugated FITC. 28 F L U O R E S C E N C E 29 a c t i v i t y (Table VIII). The suppressive a c t i v i t y was nonspecific '( Table IX) and could not be reversed by the addition of IL2 ( Table IX ). In order 51 to see i f the supernatant had l y t i c a c t i v i t y , i t was tested i n the Cr 5 release assay. It was found that the supernatant from approximately 2 X 10 51 c e l l s had very low l y t i c a c t i v i t y i n the 3 hour Cr release assay when compared with the clone i t s e l f . A 2:1 Effector/Target r a t i o o f clone 13 to B6 bl a s t s gave 67 % s p e c i f i c l y s i s but a 1/20 d i l u t i o n of supernatant from 4 X 10^ c e l l s only r e s u l t e d i n 12 % s p e c i f i c l y s i s (Table XI). This super-natant not only suppressed CTL generation but also i n h i b i t e d a c t i v a t i o n of spleen c e l l s by Con A and LPS (Table X I I ) . IV. C h a r a c t e r i s t i c s of Clone 9 A. C e l l Surface Markers of Clone 9 Table XIII shows the r e s u l t s of the antibody-dependent complement-mediated c y t o l y s i s of clone 9 by various antibodies with s p e c i f i c i t i e s against \h-2° or II-2 d antigens. The r e s u l t s i n d i c a t e that clone 9 expressed H-2Dd and H-2Kd antigens. FACS analysis showed that clone 9 was Thy 1 + (Figure 3A), Lyt 1~ (Figure 3B), and Lyt 2~ (Figure 3C). B. P r o l i f e r a t i o n C h a r a c t e r i s t i c s of Clone 9 The p r o l i f e r a t i o n c h a r a c t e r i s t i c s of clone 9 were s i m i l a r to those of clone 13. As shown i n Table XIV, clone 9 was TCGF dependent and p r o l i f e -rated most i n the presence of both antigen and TCGF. C. Helper A c t i v i t y of Clone 9 Helpers were detected by adding the clones to cultures containing 3 5 3 X 10 B6 responder spleen or LN c e l l s and 10 i r r a d i a t e d D2 spleen c e l l s . The r e s u l t s are shown i n Table XV. In the absence, of clone 9 , no CTL res-ponse was detected. However,with the addition of 1.25 X 10-^  to 4.2 X 10^ " clone 9 c e l l s , s i g n i f i c a n t cytotoxic responses were observed. The c y t o l y t i c a c t i v i t y did not increase with higher numbers of clone 9 once the plateau TABLE VIII. SUPPRESSIVE ACTIVITY OF CLONE 13 SUPERNATANT3, BD RESPONDER SPLEEN CELLS D2 STIMULATOR SPLEEN CELLS (2000 R) CLONE 13 SN % SPECIFIC LYSIS 2 X 10 4 1 X 10' - 0.0 + 0.4 - 32.8 + 13.1 1/320 0.3 + 0.6 1/640 0.2 + 0.8 1/1280 -0.2 + 0.7 1/2560 -0.2 + 0.7 1/5720 -0.2 + 0.6 1/10240 0.1 + 0.8 a. Culture conditions same as i n Table VA . 31 TABLE IX. SPECIFICITY TESTS OH CLONE 13 SI!'' RESPONDER STIMULATOR (2000 R) B 6 SPLEEN CELLS (2000 R) CLONE 13 SN ! % SPECIFIC LYSIS B 6 _ — — 0.9 + 1.1 BG D2 - - 76.9 + 27.1 B6- D2 - + 19.5 + 11.0 AKR — — - 0.4 + 1.1 AKR D2 - - 6 4 . 4 + 15.1 AKR D2 - + 3.8 + 5.3 B 6 _. — - -0.5 + 3.6 B6 AKR - - 43.3 + 18.4 B 6 AKR - + 7.0 + 12.8 AKR _ 5 X 10 4 — 0.0 + 1.5 AKR D2 II - 74-4 + 8.1 AKR D2 II + 14.6 + 11.9 a. Culture conditions'same as i n Table VA except the d i l u t i o n of clone 13 SN used was 1/5. TABLE X. SUPPRESSION BY CLONE 13 SUPERNATANT'CANNOT BE REVERSED BY IL2 B6 RESPONDER D2 STIMULATOR CLONE 13 SN EL4-!L2 b % SPECIFIC SPLEEN CELLS SPLEEN CELLS ( 1/40 ) LYSIS ( 2000 R ) 3 X 10^ - - - 0.0 + 0.6 II 1 X 10 5 - - 52.6 + 12.0 n + - 9.4 + 3.9 it - 1/80 93.9 + 9.6 n - 1/40 . 87.6 + 6.7 II - 1/20 86.5 + 8.6 II - 1/10 80.6 + 6.1 II » + 1/80 8.6 i 4.3 II » + 1/40 15.7 + 5.6 n » + 1/20 12.6 + 5.3 II I. J . 1/10 6.7 + 1.3 a. Culture conditions same as i n Table VA. b. EL4-IL2 was obtained from supernatants of phorbol myristic acetate-stimulated EL4 variant (53). ii TABLE XI. COMPARISON OF ACTIVITIES OF CLONE 13 CELLS AND CLONE 13 SUPERNATANT IN TIE 5 1 Cr RELEASE ASSAY CLONE 13 : TARGET' RATIO % SPECIFIC LYSIS CLONE 13 SN % SPECIFIC LYSIS 27.6:1 13.8:1 6.9:1 3.45:1 1.72:1 74-3 + 0.9 74.5 + 1.2 71.4 +0.8 65.3 + 1.6 66.9 + 5.4 1/20 1/40 1/80 1/160 1/320 12.2 + 12.0 11.5 + 0.5 0.7 + 3.2 1.8 + 0.7 0.2 + 0.1 a. The target used was B6 ;LPS blasts at .1 X 10 A c e l l s per well. b. The clone 13 SN was secreted from 4 X 10 6 clone 13 c e l l s i n a period of 43 hours. / TADLE XII. SUPPRESSION OF CON A AND LPS ACTIVATION BY CLONE 13 Slf B6 SPLEEN CELLS . MITOGEN CLONE 13 SN CPM ( H+SEH) 5 X.10 5 Con A Con A LPS LPS 1/10 1/10 1525 + 302 .14361 + 697 1799 +118 52101 + 2989 2147 + 169 a. B6 spleen c e l l s were cultured with 2 ug/rnl Con 'A or '20. ug/ral LPS with or without clone 13 SN i n round bottom wells for 72 hours. Cultures were pulsed with H thymidine f o r 6 hours, harvested onto glass f i b e r f i l t e r s and then counted. TABLE XI I I . H-2 PHENOTYPE OF CLONE 9 CELL ANTIBODY* . PERCENT DEAD CYTOTOXIC INDEX EL4 (H-2U) EL4 •EL4 EL4 EL4 anti-D anti-K C anti-K* anti-D d b P815 (H-2 ) P815 P815 P815 P815 d anti-D anti-K^ anti-K anti-D* Clone 9 Clone 9 Clone 9 Clone 9 Clone 9 anti-D anti-K^ anti-K anti-D* 3 2.2 35.8. 20.6 75.0 84.0 0.34. 0.19 0.75 0.84 3.8 84.5 63.1 4-4 2.8 0.S4 0.62 0.01 -0.01 13.8 90.0 61.8 15.8 12.0 1.04 0.66 0.02 -0.03 a. The antibodies used here were similar to the ones described i n Table VII. 36 FIGURE 3A. THY 1.2 MARKERS ON CLONE 9. Clone 9 c e l l s were incubated with medium (1) or a b i o t i n conjugated anti-Thy 1.2 (2) antibody, followed by st a i n i n g with a v i d i n conjugated FITC. 37 F L U O R E S C E N C E 38 FIGURE 3B. LYT 1 MARKERS ON CLONE 9. Clone 9 cells were incubated with • medium (1) or a biotin conjugated rat anti-mouse Lyt 1 antibody (2), followed by staining with avidin conjugated FITC. C E L L N U M B E R 10° 3 5 IO1 3 5 102 3 5 IQ3 41 40 FIGURE 3C. LYT 2 MARKERS ON CLONE 9 . Clone 9 c e l l s were incubated with medium (1) or a b i o t i n conjugated r a t anti-mouse Lyt 2 antibody (2), followed by st a i n i n g with a v i d i n conjugated FITC. o — LO-CO — 64 128 192 256 F L U O R E S C E N C E TABLE XIV. PROLIFERATION REQUIREMENTS OF CLONE 9* • CLONE STIMULATOR TCGF CPM ( M + SEM ) 9 - - 7 2 + 8 9 + - 71 + 5. 9 + + 24922 + 758 9 - + 1219 + 60 a. Culture conditions were s i m i l a r to those described i n Table IV. 43 TABLE X V . HELPER ACTIVITY OF CLONE 9' B6 RESPONDER D2 STIMULATOR CLONE 9 PERCENT SPECIFIC LN CELLS SPLEEN CELLS (2000 R) (2000 R) LYSIS 3 X IO 3 — - 0.0 + 1.2 I I 1 X 10 5 - 2.1 '+ 1.0 I I 1 1.25 X 1 0 3 21.4 + 11.2 I I I I 5.25 X 1 0 3 23.5 + 15.3 1 I I 1.05 X 1 0 4 25.2 + 18.5 I I I I 2.10 X 10^ 30.6 + 15.4 I t I I 4.20 X 10^ 38.9 + 24.6 a. Cultures were set up i n c o n i c a l bottom m i c r o t i t e r t r a y s i i a volume of 0.20 .ml. On day 5, 2 X 10 3 5 1 C r l a b e l l e d P815 uere .added to each w e l l . Assay time =4.5 hours. had been reached. D. S p e c i f i c i t y o f Clone 9 When clone 9 was added to c u l t u r e s c o n t a i n i n g B6 as responders and D2 o r AKR as s t i m u l a t o r s , c y t o t o x i c responses were generated (Table XVI, l i n e s 3,12). However, when clone 9 was added to c u l t u r e s . c o n t a i n i n g AKR as responders, no response was observed (Table XVI, l i n e 6 ) . Even when i r r a -d i a t e d B6 spleen c e l l s were added together w i t h AKR responders, there was s t i l l no response generated (Table XVI, l i n e 9). These r e s u l t s suggested t h a t the s p e c i f i c t a r g e t o f clone 9 was the B6 responder p o p u l a t i o n . The s p e c i f i c i t y o f clone 9 was mapped by using d i f f e r e n t K-2 recom-binant s t r a i n s as responders. Table XVII shows t h a t clone 9 only helped B10, B10.A(2R) and B6, but not B10.A or B10.A(3R). B10.A(2R), B10 and B6 expressed the H-2Db antigens, suggesting t h a t clone 9 was s p e c i f i c f o r H-2Db. The s p e c i f i c i t y produced as a r e s u l t o f the he l p e r a c t i o n o f clone 9 was next determined. The same c u l t u r e system was used except t h a t on day 5, c u l t u r e s were s p l i t and t e s t e d a g a i n s t two t a r g e t s , namely P315 (H-2 ) and B6 (H-2 b) b l a s t s . As shown i n Table X V I I I , o n ly anti-D2 CTL's were a c t i -vated s i n c e o n ly P815 t a r g e t s , and not B6 b l a s t s , were l y s e d . This r e s u l t t h e r e f o r e i n d i c a t e d t h a t clone 9 was not a p o l y c l o n a l a c t i v a t o r . E. K i n e t i c s o f the A c t i o n of Clone 9 In order to determine the time o f a c t i o n o f clone 9 i n the deve-lopment o f CTL responses, clone 9 was added to the assay system a t d i f f e -r e n t times during the 5 day development o f the response. Table XIX shows t h a t clone 9 only helped when i t was added together w i t h the responders and s t i m u l a t o r s a t the i n i t i a t i o n o f the c u l t u r e s . Clone 9 t h e r e f o r e e x h i b i t e d i t s e f f e c t d u r i ng the f i r s t 24 hours on the course o f the development o f the c y t o t o x i c response. 45 TABLE XVI. SPECIFICITY OF CLONE 9 & RESPONDER LN CELLS STIMULATOR SPLEEN CELLS (2000 R) B6 ACCESSORY SPLEEN CELLS (2000 R) CLONE 9 (2000 R) % SPECIFIC LYSIS B6 . _ — — 0.0+0.7 B6 D2 - - 2.3 + 2.3 B6 D2 - 36.4 + 19.7 AKR — — — ' -0.4 + 1.5 AKR D2 - - -1.5 + 1.2 AKR D2 - 10 4 -1.4 +1.2 AKR — 5 X 10^ — -1.6 + 1.4 AKR D2 ti - -1.7+1.9 AKR D2 I! -1.2 +1.8 B6 — — — 0.0 + 5.6 B6 AKR - - -2.3 + 5.3 . B6 AKR — .10 4 30.0 + 16.1 a. Culture conditions were the same as i n Table XV. Cr. l a b e l l e d P815 or RDM-4 v;ere used as targets on the day of the assay. 46 TABLE XVII. FINE SPECIFICITY OF 'CLONE 9 AND CLONE 9 SUPERNATANT51 1ESPOMDER 11-2 D2 STIMULATOR HELPER % SPECIFIC LN CELLS K I-ABJEC D SPLEEN CELLS LYSIS (2000 R) B10 b bbbbb b — — 0.0 + 3.2 B10' II + - 6.2 + 7.5 BIO it -1- 10/l- clone 9 39.3 + 15.9 B10 ii + 1/5 'clone. 9 SN b 2 9 . 7 + 19.8 B10.A k kidded d — — -0.1 + 0 . 7 B10.A II + - -0.5 + 1.6 B10.A ii + 10^ clone 9 0.5 + 2.9 B10.A ii + 1/5 clone 9 SN 1 . 2 + 3.0 B10.A(2R) k kidded b — - -1.3 + 1 . 4 B10.A(2R) II + 1 0 ^ clone 0 . 7 + 3.3 B10.A(2R) n + . 9 23.4 + 12.2 B10.A(2R) ii + 1/5 clone 9 SN 8.0 +8.4 B10 .A(3R) b bbbkd d - - -1.6 + 2.0 B10.A (3R) ii + 10^ clone -0.8 + 1 . 4 B10.A(3R) ii + 9 6.0 + 8.2 B10 .A(3R) II + 1/5 clone 9 SN 2.6 + 3.6 B6 b bbbbb b — — 3.6 + 6.5 B6 II + - 5.5 + 5.1 B6 II 10^ clone 9 56.2 + 18.5 B6 II + 1/5 clone 9 SN 2 4 . 3 + 1 2 . 9 a. Culture conditions viere the same as i n Table XV. b. The clone 9 SN used here was unconcentrated. 47 TABLE XVIII. SPECIFICITY OF CTL ACTIVATION BY CLONE 9 AND CLONE 9 SN a B6' RESPONDER D2 STIMULATOR B6 SPLEEN HELPER % SPECIFIC LYSYS OF: b LN CELLS SPLEEN CELLS CELLS P815(H-2 ) B6 BLAST(H-2 ) (2000 R) (2000 R) 6 X 1 0 ^ - 5 X 10^ - 0.0+1.1 0.0+5.4 i- 10 5 " - 4.1 + 3.7 -2.1 + 9.1 ii " » 10^ c e l l s 20.2 + 10.8. -5.4 + 3.5 » » " 1/10 SN 35.0 + 13.7 -5.6 + 5.6 a. Cultures were set up in. c o n i c a l bottom v e i l s i n a volume of 0.2 ml. 3 51 On day 5, cultures were s p l i t and tested against 2 X 10 . Cr-l a b e l l e d P815 or B6 b l a s t s . Assay time = 4.5 hours, b. B6 i r r a d i a t e d spleen c e l l s were added to the assay to increase the s e n s i t i v i t y of the assay f o r the detection of helper a c t i v i t y i n the clone 9 supernatant. 4-S TABLE XIX. KINETICS OF THE ACTION OF CLONE 9 a B6 RESPONDER D2 STIMULATOR CLONE 9 DAY ADDED % SPECIFIC SPLEEN CELLS SPLEEN CELLS ( 2000 R ) LYSIS (2000 R) 3 x 10 3 - - - 0.0 + 1.2 ii 1 X 10 5 - - - 1 . 0 + 0.6 II II day 0 62.4- + 1 7.6 II II ii day 1 2.2 + 10.2 II II day 2 1.8 + 3.3 II II it day 3 8.9 + 14.5 II II n day 4 1.7 + 2.2 a. Culture conditions were the same as i n Table XV. - . 4 9 F. Requirement of Accessory C e l l s f o r Clone 9 Function When the dose of D2 stimulator c e l l s i n the helper assay uas reduced 5 1 from 10 to 10 ^  and a l l the other conditions were kept the same, there was no CTL response generated (Table XX, compare l i n e 3 with l i n e 10). With the addition of i r r a d i a t e d B6 or AKR spleen c e l l s , higher CTL responses were generated (Table XX). This r e s u l t suggests that there was a requirement of accessory c e l l s f o r the function of clone 9. This requirement d i d not seem to be g e n e t i c a l l y r e s t r i c t e d . I t i s probable that the accessory c e l l s pro-vided signals which act s y n e r g i s t i c a l l y with clone 9 i n the generation of CTL. G. Clone 9 Acts Through Factor(s) Supernatant from clone 9 was obtained by c u l t u r i n g 1 X 10^ to 2 X 10' clone 9 c e l l s i n complete medium f o r 48 hours. This supernatant was harves-ted, concentrated ten times and then tested f o r helper a c t i v i t y . The r e s u l t s show that clone 9 SN exhibited the same properties as clone 9. Clone 9 SN was s p e c i f i c f o r H-2Db antigens (Table XVII). I t only helped i n the a c t i v a -t i o n of anti-D2 clones i n a B6 anti-D2 response • (Table XVIII). I t also acted early i n the development of CTL (Table XXI). The r e s u l t s on the k i n e t i c s of action of clone 9 or clone 9 SN showed that clone 9 SN was required on day 0 but d i d not i n d i c a t e i f i t was required from day 0 to day 5, the day of the assay. Unlike TCGF, when clone 9 SN was tested i n the thymocyte p r o l i f e -r a t i o n assay, i t did not exhibit any potentiating e f f e c t on the thymocyte p r o l i f e r a t i o n (Table XXII). I t also d i d not have any a c t i v i t y i n suppor-t i n g the p r o l i f e r a t i o n of clone 13 (Table XXIII). H. A c t i v a t i o n of CTL i n the Presence of Clone 9 SN and TCGF The r e s u l t s above showed that clone 9 required accessory c e l l s f o r i t s f u n c t i o n . I t i s possible that the accessory c e l l requirement was i n the form of TCGF. To t e s t t h i s , the accessory c e l l requirement was substituted 50 TABLE XX. REQUIREMENT OF ACCESSORY CELLS FOR CLONE 9 FUNCTION* B6' RESPONDER LN CELLS D2 STIMULATOR SPLEEN CELLS ( 2000 R ) SPLENOCYTES ( 2000 R ) CLONE 9 % SPECIFIC (2000 R) LYSI£ 3.2 X 10' 1 X 10' 5 X 10^ B6 5 X 10^ AKR 10 4 10' 10' 0.0 + 1.5 2.2 + 2.4 11.8 + 5.5 6.5 ± 5.0 32.6 + 17.6 8.5 + 10.6 24.2 + 15.1 3.2 X 10 3 1 X 10 4 5 X 104- B6 5 X IO 4 - AKR 10 10' 4 10 4 0.0 +0.8 0.0 + 0.6 -1.0 + 0.7 0.7 ± 1.2 16.0 + 11.7 N.D.b a. Culture conditions the same as i n Table XV. b. N.D. = Not Done. . 51 TABLE XXI. KINETICS OF THE ACTION OF CLONE 9 SN' B6- RESPONDER D2 STIMULATOR B6 SPLEEN CLONE 9 SN DAY % SPECIFIC LN CELLS SPLEEN CELLS CELLS (1/10) ADDED LYSIS (2000 R) (2000 R) 3 X 1 0 3 _ 5 X 10^ - - 0.0 + 2.0 II 1 X 10 5 n - - 0.3 + 0.4 II II II + day 0 51.4 + 18.8 II II II + day 1 11.9 + 12.7 II II it + day .2 1.2 + 6.8 1 II it + day 3 -3.3 + 3.7 II II II + day 4 -6.6 + 0.2 a. Culture conditions were s i m i l a r to those described i n Table XV. 52 TABLE XXII. ACTIVITY OF CLONE 9 SN IN THE THYMOCYTE PROLIFERATION ASSAY* B6 THYMOCYTES CON A (2 UG/ML) FACTOR COUNTS PER MINUTE 5 10 J - - 389 + 81 it + - 978 + 24 II + 1/4 TCGF 22594- + 273 it + 1/8 TCGF 14633 + 1078 it 1/16 TCGF 7334 + 178 M + 1/4 clone 9 SN 856 + 134 it + 1/8 " 924 + 104 II + 1/16 " 758 + 48 a. T r i p l i c a t e cultures were set up i n round bottom m i c r o t i t e r trays and incubated. On day 3, cultures were pulsed with ^H-thymidine f o r 6 hours, then harvested and counted. 53 TABLE XXIII. ACTIVITY OF CLONE 9 SN IN THE CLONAL PROLIFERATION ASSAY a CLONE 13 FACTOR CPM 5 X 10 3 1/4 TCGF 1/8 " 1/16 » 1/4 clone 9 SN 1/8 " 1/16 463 + 55 2665 + 410 1276 + 79 481 + 38 114 + 18 142 + 14 142 + 32 a. Culture conditions were the same as i n Table XXII. 54 TABLE XXIV. ACTIVATION OF CTL IN THE PRESENCE OF CLONE 9 AND TCGF' B6 RESPONDER CLONE 9 D2 STIMULATOR TCGF b DAY TCGF % SPECIFIC LN CELLS SN (1/20) SPLEEN CELLS 10%' ADDED LYSIS (2000 R) 3 X 10 3 - - - - 0.0 + 4.8 . II + - - - -4.8 + 1.9 II 10 4 - - -4.0 + 3.4 II + 10 4 - - -2.2 + 1.8 II - + day 0 -5.9 + 2.1 1! + --_ + II -4.6 + 3.0 II - . 10 4 II 46.6 + 26.0 II 10 4 + II 95.2 + 27.8 II - - + day 1 -0.8 1 3.1 II + + II -6.3 + 2.7 II - 10 4 + II 7.0 + 11.4 II + 10* + II 59.6 + 26.7 a. Culture conditions were the same as i n Table XV. b. 50 mM alpha methyl mannoside was added whenever TCGF was added to the cultures. 55 with 10 % (v/v) TCGF . Table XXIV ( l i n e 4) shows that clone 9 SN d i d not help when the stimulator dose was only 10 4 . When TCGF alone was added , a s i g n i -f i c a n t response (4-6.6%) was observed ( l i n e 7) . However, when clone 9 SN was added together with TCGF, the response observed was much higher (95.2%) than the added responses of the groups with clone 9 SN alone or TCGF alone ( l i n e 8). This higher response therefore appeared to be the r e s u l t of a synergis-t i c e f f e c t between clone 9 SN and TCGF. Furthermore, i t should be noted that the clone 9 SN showed t h i s s y n e r g i s t i c e f f e c t only i f the stimulator c e l l s were added . This r e s u l t suggested that i n order f o r clone 9 to amplify the CTL response, TCGF and antigen are required. 56 DISCUSSION The method used here to generate clones from an allogeneic system i s a s l i g h t modification of the methods used by other workers as previously described (14,15,30). Cells recovered from a B6 anti-D2 MLR were allowed to adapt to an environment consisting of TCGF, stimulator c e l l s and syngeneic accessory c e l l s f o r around 3 months before cloning was performed. The clones were maintained using the same conditions throughout. With t h i s method, 5 c y t o l y t i c clones and 1 helper clone were generated. I t should be noted that the primary aim of the project was to rai s e B6 clones from a B6 anti-D2 parental c e l l l i n e . However, as i t turned out, both B6 and D2 clones were iso l a t e d . An explanation f o r t h i s unexpected occurrence i s that some irradiated D2 stimulator c e l l s had survived the I r r a d i a t i o n . Therefore, i t i s imperative that i f one i s using a method simi-l a r to the one described here, one must always check the H-2 phenotype of the clones before proceeding with t h e i r characterization. In our case, we i n i t i a l l y assumed that the clones were of B6 o r i g i n , and the H-2 phenotype was only checked during the l a t t e r phase of the project. This could lead to wrong conclusions regarding the function of the clones. For example,, clone 13, which was f i r s t thought to be of B6 o r i g i n , was concluded to be a suppressor clone since i t suppressed the B6 CTL response. However, i t was subsequently shown that clone 13 was a H-2a c y t o l y t i c clone which suppressed the B6 CTL response by k i l l i n g the B6 responder c e l l s . The analysis of clone 9 was s i m i l a r l y complicated due to the same reason. One major problem encountered i n t h i s project was the d i f f i c u l t y i n recovering c e l l s after freezing. I n i t i a l l y , the normal freezing procedure applied for tumor c e l l s was used (52). Unfortunately, t h i s method did not work f o r these clones. Some workers had reported success . using si m i l a r procedures except that most of them used a controlled cooling apparatus 57 f o r f r e e z i n g . (37,38). Since we d i d not have such an apparatus a v a i l a b l e f o r use, we had to r e s o r t to another f r e e z i n g p r o t o c o l . This method r e q u i r e d the a d d i t i o n of dextran to the f r e e z i n g medium. Nabel e t . a l . (25) a l s o reported using a .high molecular weight substance (hydroxyethyl starch) i n t h e i r f r e e z i n g p r o t o c o l . I t seemed t h a t having a high molecular weight subs-tance i n the f r e e z i n g medium i s important i n p r o t e c t i n g the c e l l s . The c y t o l y t i c clones i s o l a t e d here e x h i b i t e d s p e c i f i c k i l l i n g a c t i -v i t y a g ainst D2 a l l o a n t i g e n s . Clones 14 and 25 were r e a c t i v e against H-2Dd antigens. Clone 27 was r e a c t i v e against some antigen(s) coded o u t s i d e H-2. Clone 23 had a more complex p a t t e r n o f r e a c t i v i t y which was not c l e a r l y d e f i n e d . Some o f the clones obtained by Glasebrook e t . a l . (18) e x h i b i t e d s i m i l a r patterns o f r e a c t i v i t y . Their clones also r e q u i r e d the presence o f both TCGF and s t i m u l a t o r s / f e e d e r c e l l s f o r maximum p r o l i f e r a t i o n . While t h e i r clones had been claimed to be l o n g l i v e d , the clones d e r i v e d here were only s t a b l e f o r about 6 months and a f t e r t h a t t h e i r a c t i v i t i e s g r a d u a l l y waned. The clones thawed a f t e r f r e e z i n g were f u n c t i o n a l l y i n a c t i v e . The only s t a b l e c y t o l y t i c clone whose a c t i v i t y could be recovered a f t e r f r e e z i n g was clone 13. I t was a H-2 clone w i t h a r e a c t i v i t y against H-2D antigens. S i m i l a r to the other c y t o l y t i c clones analysed f o r c e l l s urface markers (18), clone 13 was Thy 1 , s l i g h t l y L y t 1 and L y t 2 . An a d d i t i o n a l f e a t u r e o f clone 13 was t h a t the SN d e r i v e d from i t suppressed n o n s p e c i f i c a l l y CTL generation and mitogen a c t i v a t i o n . This SN was only 51 weakly a c t i v e i n the Cr r e l e a s e assay. Thus, i t not only k i l l e d c e l l s c a r r y i n g the s p e c i f i c antigen but i t also secreted substances i n the SN which were not antigen s p e c i f i c i n t h e i r suppressive f u n c t i o n s . I t i s probable t h a t the production of these suppressive substances i n the SNri was a subsequent step to the r e c o g n i t i o n of t a r g e t antigen by c y t o t o x i c c e l l s . So f a r there i s no r e p o r t i n the l i t e r a t u r e regarding the a c t i v i t y o f the SN produced by c y t o l y t i c c l ones. I t i s c l e a r t h a t f u r t h e r work needs to be 53 done on the nature of the suppressive substances i n the SN. The p h y s i o l o g i -c a l properties of these molecules remains to be determined. The helper clone, termed clone 9, appeared to be a novel helper c e l l . I t s major c h a r a c t e r i s t i c s d i f f e r e d from those described by other workers (1, 6, 14, 19, 25, 29, 36, 40, 41)• This helper clone was Thy 1 V, Lyt 1" and Lyt 2~. Most of the helper clones reported previously are Lyt 1 + (6, 15). The p r o l i f e r a t i o n c h a r a c t e r i s t i c s of clone 9 were s i m i l a r to the c y t o l y t i c clones. I t did not p r o l i f e r a t e well with stimulators alone. However, i t p r o l i f e r a t e d well In TCGF without stimulators. The maximal p r o l i f e r a t i o n was usually observed when i t was cultured with both TCGF and alloantigens. Thus, clone 9 i s TCGF dependent; i t cannot induce TCGF produc-t i o n or produce TCGF i t s e l f . Clone 9, which i s of D2 o r i g i n , was found to augment the CTL responses of B6 responder c e l l s . I t was s p e c i f i c i n helping responders which carry the H-2D baiitigens. Clone 9 only helped the a c t i v a -t i o n of anti-D2 CTLs i n a B6 anti-D2 response. In order f o r clone 9 to help, i t required the presence o f accessory c e l l s . This i s the f i r s t report of a helper clone which helped another s t r a i n to generate CTL responses against surface antigens i n an a n t i g e n - s p e c i f i c fashion, that i s , not through the action o f TCGF. Similar to other clones reported previously (18, 25, 39, 46), clone 9 mediated i t s action through soluble f a c t o r s . The SN from clone 9 e x h i b i -ted the same c h a r a c t e r i s t i c s as clone 9. I t d i d not contain IL2 as tested i n the thymocyte p r o l i f e r a t i o n assay and the c l o n a l p r o l i f e r a t i o n assay. It was s p e c i f i c i n i t s action and acted early during the development of the CTL response. Furthermore, i t was found to act i n synergy with TCGF i n the augmentation of the CTL response. The current model on CTL a c t i v a t i o n suggests that a minimum of 59 two signals are required i n the generation of CTL (20, 42, 43). Precursor c e l l s are required to i n t e r a c t with antigen (signal 1) i n the f i r s t few hours of the response whereupon they s t a r t to express TCGF receptors. For further expansion of these p a r t i a l l y activated CTLs, TCGF (signal 2) i s required. At t h i s point, i t i s s t i l l not clear where clone 9 f i t s i n t h i s scheme, but i t can be speculated that clone 9 probably p a r t i c i p a t e s during the : period between the a c t i v a t i o n and expansion step. I t i s possible that clone 9 or i t s SN may have some helper r o l e i n making the CTLs more recep-t i v e to TCGF. The picture presented by the two s i g n a l model i s s t i l l i n -complete. Other s t i l l unresolved factors aside from IL2 may also be involved i n CTL generation. A recent f i n d i n g by two independent groups had shown that a f a c t o r (termed CTL d i f f e r e n t i a t i o n factor) which i s d i s t i n c t from IL1 or IL2, i s required f o r the d i f f e r e n t i a t i o n of CTL precursors into mature CTLs (51,52). The factor(s) produced by clone 9 may also be one of the other factors required f o r CTL generation. Further analysis of the SN w a s - d i f f i c u l t because clone 9 does not produce factors i n large q u a n t i t i e s . Work i s currently i n progress to define the nature of the factors secreted by clone 9 and t h e i r mode of action i n CTL generation. The a v a i l a b i l i t y of clones such as the ones described o f f e r s the p o s s i b i l i t y of studying the mechanisms of T c e l l a c t i v a t i o n , the T c e l l antigen receptor r e p e r t o i r e , c e l l u l a r i n t e r a c t i o n s between T c e l l subsets, the mechanisms of c e l l mediated lympholysis, T c e l l d i f f e r e n t i a t i o n -markers and T c e l l f a c t o r s . ABBREVIATIONS B6: C57BL/6 B10: C57BL/10 CTL: Cytotoxic T Lymphocyte CTL-P: Cytotoxic T Lymphocyte Precursor Con A: Concanavalin A D2: DBA/2 DMEM: Dulbecco's Modified Eagles Medium FACS: Fluorescence Activated C e l l Sorter FCS: F e t a l C a l f Serum FITC: Fluorescein isothiocyanate HEPES: N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid IL2: Interleukin 2 LPS: Lipopolysaccharide MLR: Mixed Lymphocyte Reaction SN: Supernatant TCGF: T C e l l Growth Factor TCGF-CM: 25% TCGF, 50 mM alpha methyl mannoside and complete medium 61 BIBLIOGRAPHY 1. Apte, R.N., Lowy, I., De Baetsalier, P., and Mozes, E. (1981) E s t a b l i s h -ment and char a c t e r i z a t i o n of continuous helper T c e l l l i n e s s p e c i f i c to poly (L-tyr,L-glu)-poly ( D L -ala)—poly ( L - l y s ) . J . Immunol. 127, 25-30. 2. Braciale, T.J., Andrew, M.E. and Brac i a l e , V.L. (1981) Heterogeneity and s p e c i f i c i t y of cloned l i n e s of influenza v i r u s - s p e c i f i c cytotoxic T lymphocytes. J . Exp. Med. 153, 910-923. 3. Andrew, M.E. and Brac i a l e , T.J. (1981) Antigen- dependent p r o l i f e r a t i o n o f cloned continuous l i n e s of H-2 r e s t r i c r e d influenza v i r u s - s p e c i f i c cytotoxic T lymphocytes. J . Immunol. 127, 1201-1204. 4. Bianchi, A.T.J., Hooijkaas, H., Benner, R., Tees, R., Nordin, A.A., Schreier, M.H. (1981) Clones of helper T c e l l s mediate antigen-s p e c i f i c , H-2 r e s t r i c t e d DTII. Nature 2_20, 62-63. 5. Dennert, G. (1979) C y t o l y t i c and p r o l i f e r a t i v e a c t i v i t y of a permanent T k i l l e r c e l l l i n e . Nature 277, 476-4-77. 6. Dennert, G., Swain, S., Waterfield, J . , Warner, J . , Dutton, R. (1981) Fine s p e c i f i c i t y mapping of two a l l o s p e c i f i c T c e l l l i n e s : recog-n i t i o n of private s p e c i f i c i t i e s i n the H-2 IA subregion. Eur. J . Immunol. 1_1_, 62-64. 7. Swain. S., Dennert, G., Wormsley, S. and Dutton R. (1981) The-Lyt pheno-.. type of a long-term a l l o s p e c i f i c T c e l l l i n e . Both helper and k i l l e r a c t i v i t i e s to IA are mediated by Ly 1 c e l l s . Eur. J . Immunol. 11, 175-180. 8. Fathman, C G . and Kimoto, M. (1981) Studies u t i l i z i n g murine T c e l l clones: Ir genes, Ia antigens and MLR stimulating determinants. Immunol. Rev. 54., 57-79. 9. Finn, O.J., Bohiver, J . and Kaplan, H.S. (1979) Induction, establishment i n v i t r o , and characterization of f u n c t i o n a l , a n t i g e n - s p e c i f i c , carrier-primed murine T - c e l l lymphomas. Proc. Natl. Acad. S c i . USA 76, 4033-4037. 10. G i l l i s , S. and Smith, K.A. (1977) Long-term culture of tumor s p e c i f i c cytotoxic T c e l l s . Nature 268, 154-156. 11. G i l l i s , S. and Smith, K.A. (1977) In v i t r o generation of tumor s p e c i f i c cytotoxic lymphocytes. Secondary allogeneic mixed tumor lymphocyte culture of normal murine spleen c e l l s . J . Exp.Med. 14-6, 468-482. 12. Baker, P.E., G i l l i s , S. and Smith, K.A. (1979) Monoclonal c y t o l y t i c T-c e l l l i n e s . J . Exp. Med. V£, 273-278. 13. Glasebrook, A., and F i t c h , F. (1980) T - c e l l l i n e s which cooperate i n generation of s p e c i f i c c y t o l y t i c a c t i v i t y . Nature 278, 171-173. 14. Glasebrook, A. and F i t c h , F. (1980) A l l o r e a c t i v e cloned T c e l l l i n e s . I. Interaction between cloned amp l i f i e r and c y t o l y t i c T c e l l l i n e s . J . Exp. Med. 151, 876-895.. 62 14. Glasebrook, A. and F i t c h , F. (1980) A l l o r e a c t i v e cloned T - c e l l l i n e s . I . I n t e r a c t i o n between cloned a m p l i f i e r and c y t o l y t i c T c e l l l i n e s . J . Exp. Med. 151, 876-895. 15. Glasebrook, A., Quintans, J . , Eisenberg, L. and F i t c h , F. (1981) A l l o r e a c t i v e cloned T - c e l l l i n e s . I I . P o l y c l o n a l s t i m u l a t i o n o f B c e l l s by a cloned helper T c e l l l i n e . J . Immunol. 126, 240-244. 16. Lutz, C.T., Glasebrook, A.L. and F i t c h , F. (1981) A l l o r e a c t i v e cloned T - c e l l l i n e s . I I I . Accessory c e l l requirements f o r the growth o f cloned c y t o l y t i c T lymphocytes. J . Immunol. 126, 1404-1408. 17. L u t z , C.T., Glasebx'ook, A.L. and F i t c h , F. (1981) A l l o r e a c t i v e cloned T - c e l l l i n e s . IV. I n t e r a c t i o n o f a l l o a n t i g e n and T c e l l growth f a c t o r s (TCGF) to s t i m u l a t e c y t o l y t i c T lymphocytes. J . Immunol. 127, 391-392. 18. Glasebrook, A.L., Sarmiento, M., Loken, M.R., Dial y n a s , D.P., Quintans J . , Eisenberg, L., L u t z , C.T., Wilde, D. and F i t c h , F.W. (1981) Murine T lymphocyte clones w i t h d i s t i n c t immunological f u n c t i o n s . Immunol. Rev. 54_, 225-265. 19. Hapel, A.J., Lee, J.C., F a r r a r , W.L.+and Ihle_, J.N. (1981) Establishment of continuous c u l t u r e s o f Thy 1.2 , Lyt 1 ,2~ T c e l l s w i t h p u r i f i e d i n t e r l e u k i n 3. C e l l 25, 179-186. 20. Herrmann, S.H., Weinberger, 0., Burakoff, S.J., Mescher, M.F. (1982) A n a l y s i s of the two s i g n a l requirement f o r precursor c y t o l y t i c T lymphocyte a c t i v a t i o n using H-2 i n liposomes. J . Immunol. 128, 1968-1974. 21. Hunig, T. and Bevan, M.J. (1982) Antigen r e c o g n i t i o n by cloned c y t o l y t i c lymphocytes f o l l o w s r u l e s p r e d i c t e d by the a l t e r e d s e l f hypothesis. J . Exp. Med. 155, 111-125. 22. Kappler, J.W., Skidmore, B., White, J . and Marrack, P. (1981) Antigen-i n d u c i b l e , H - 2 - r e s t r i c t e d , i n t e r l e u k i n - 2 - p r o d u c i n g T c e l l hybridomas. Lack o f independent antigen and H-2 r e c o g n i t i o n . J . Exp. Med. 153, 1198-1214. 23. L i n , Yun-Lu and Askonas, B.A. (1981) B i o l o g i c a l p r o p e r t i e s o f an i n f l u e n z a A v i r u s - s p e c i f i c k i l l e r T c e l l clone. I n h i b i t i o n of v i r u s r e p l i c a t i o n i n vivo and i n d u c t i o n o f delayed-type 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 . J . Exp. Med. 154, 225-234. 24. MacDonald, H.R., C e r r o t t i n i , J . C , Ryser, J.E., Maryanski, J.L., Taswel l , C , Widmer, M.B. and Brunner, T. (1980) Q u a n t i t a t i o n and c l o n i n g o f c y t o l y t i c T lymphocytes and t h e i r p recursors. Immunol. Rev. j31_, 93-123. 25. Nabel, G., Fresno, M.., Chessman, A. and Cantor, H. (1981) Use o f cloned populations o f mouse lymphocytes to analyse c e l l u l a r d i f f e r e n t i a t i o n . C e l l 23, 19-28. 26. Narimatsu, II. and S a i t o , K. (1981) Murine c e l l l i n e s t h a t help i n induc-t i o n and generation o f 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 s from thymocytes. J . Immunol. 126, 95-99. 63 27. Rosenberg, S., Schwartz, A. and Spiess, P.J. (1978) In v i t r o growth o f murine T - c e l l s . I I . Growth o f i n v i t r o c e l l s c y t o t o x i c f o r a l l o -antigens. J . Immunol. 121, 1951-1955. 28. R i c c i a r d i C a s t a g n o l i , P., Dor i a , G. and A d o r i n i , L. (1981) Production of a n t i g e n - s p e c i f i c suppressor T c e l l f a c t o r by r a d i a t i o n leukemia virus-transformed suppressor T c e l l s . Proc. N a t l . Acad. S c i . USA 78, 38CK-3813. 29. S r e d n i , B., Tse, H.Y., Ghen, C. and Schwartz, R. (1981) A n t i g e n - s p e c i f i c clones o f p r o l i f e r a t i n g T lymphocytes I . Methodology, s p e c i f i c i t y and MHC r e s t r i c t i o n . J . Immunol. 126, 341-347. 30. Von Boehmer, H. and Haas, W. (1981) H-2 r e s t r i c t e d c y t o l y t i c and non-c y t o l y t i c clones: I s o l a t i o n , s p e c i f i c i t y and f u n c t i o n a l a n a l y s i s . Immunol. Rev. _5_4_, 27-56. 31. Nabholz, M., Conzelmann, A., Acuto, 0., North, M., Haas, W., P o h l i t , H., Von Boehmer, H., Hengartner, H., Mach, J.P., Engers, H. and Johnson, J.P. (1980) E s t a b l i s h e d murine c y t o l y t i c T c e l l , l i n e s as t o o l s f o r somatic c e l l genetic a n a l y s i s o f T - c e l l f u n c t i o n s . Immunol. Rev. 51, 125-156. 32. Engers, H., C o l l a v o , D., North, M., Von Boehmer, H., Haas, W., Hengart-ner, H.and Nabholz, M. (1980) C h a r a c t e r i z a t i o n o f cloned c y t o l y t i c murine T - c e l l l i n e s . J . Immunol. 125, 1481-1486. 33. Nabholz, M., Engers, H.D., C o l l a v o , D., North, M. (1978) Cloned T - c e l l l i n e s w i t h s p e c i f i c c y t o l y t i c a c t i v i t y . Curr. Top. M i c r o b i o l . Immunol. 81, 176-187. 34-. Haas, W., Mathur-Rochat, J . , P o h l i t , H,, Nabholz, M. and Von Boehmer, H. (1980) Cyt o t o x i c T c e l l responses to haptenated c e l l s . I I I . I s o l a t i o n and s p e c i f i c i t y a n a l y s i s o f continuously growing clones. Eur. J . Immunol. 10, 828-834. 35. Schwaller, R., R o l l i n g h o f f , M. and Wagner, H. (1980) Fine s p e c i f i c i t y . ' .. and c y t o l y t i c a c t i v i t y o f continuously growing a l l o r e a c t i v e c y t o t o x i c T lymphocyte clones. Scand. J . Immunol. 11_, 44-9-453. 36. S c h r i e r , M.H., Iscove, N.N., Tees, R., Aarden, L. and Von Boehmer., H. (1930) Growth requirements, s p e c i f i c i t y and r e t e n t i o n o f f u n c t i o n i n long-term c u l t u r e . Immunol. Rev. _51_, 315-336. 37. S c h r i e r , M. and Tees, R. (1981) Long-term c u l t u r e and c l o n i n g o f s p e c i f i c helper T c e l l s . Immunological Methods, v o l I I ( L e f k o v i t s , I . and P e r n i s , B., eds.) Academic Press, New York, p. 263-275.-38. Haas, W. and Von Boehmer, H. (1982) Methods f o r the establishment of continuously growing c y t o l y t i c T c e l l clones. J . Immunol. Meth. 52, 137-148. 39. Taniguchi, M., S a i t o , T. and Tada, T. (1979) A n t i g e n - s p e c i f i c suppressive f a c t o r produced by a t r a n s p l a n t a b l e I - J bearing T - c e l l hybridoma. Nature 273, 555-558. 6 4 40. Watson, J . (1979) Continuous p r o l i f e r a t i o n o f murine a n t i g e n - s p e c i f i c helper T lumphocytes i n c u l t u r e . J . Exp. Med. 150, 1510-1519. 41. Widmer, M.B. and Bach, F.H. (1981) Antigen-driven helper cell-independent cloned c y t o l y t i c T lymphocytes. Mature 294., 750-752. 42. Wagner, II., Hardt, C., Stockinger, H., Pfizenmaier, K., B a r t l e t t , R., and R o l l i n g h o f f , M. Impact o f thymus on the generation o f immuno-competence and d i v e r s i t y o f a n t i g e n - s p e c i f i c MHC-restricted c y t o t o x i c T lymphocyte precursors. Immunol. Rev. 58, 95-129. 4 3 . Larsson, E.-L. (1981) Mechanism of T c e l l a c t i v a t i o n I I . Antigen- and lectin-dependent a c q u i s i t i o n o f responsiveness to TCGF i s a non-mitogenic, a c t i v e response o f r e s t i n g T c e l l s . J . Immunol. 126, 1323-1326. 44. G i l l i s , S., Ferm, M.M., 0u, W. and Smith, K.A. (1978) T c e l l growth f a c t o r : Parameters of production and q u a n t i t a t i v e microassay f o r a c t i v i t y . J . Immunol." 120, 2027-2032. 45. Shaw, J . , Monticone, V. and Paetkau, V. (1978) P a r t i a l p u r i f i c a t i o n and molecular c h a r a c t e r i z a t i o n of a lymphokine(co-stimulator) r e q u i r e d f o r the mitogenic response of mouse thymocytes i n v i t r o . J . Immunol. 120, 1967-1973. 46. Nabel, G., Greenberger, J.S., Sakakeeny, M.A. and Cantor, H. (1981) M u l t i p l e b i o l o g i c a c t i v i t i e s lof a cloned Inducer X c e l l p o p u l a t i o n . Proc. N a t l . Acad. S c i . USA 78, 1157-1161. 47. Altman, A., S f e r r u z z a , A., Weiner, R.G. and Katz, D.H. (1982) C o n s t i t u t i v e and mitogen-induced production of T c e l l growth f a c t o r by s t a b l e T c e l l hybridoma l i n e s . J . Immunol 128, 1365-1371. 48. Morgan, D.A., R u s c e t t i , F.W. and G a l l o , R. (1976) S e l e c t i v e i n v i t r o growth of T-lymphocytes from normal human bone marrow. Science 193, 1007-1008. 49. Smith, K.A. (1980) T - c e l l growth f a c t o r s . Immunol. Rev 51, 337-357. 50. Wagner, H., Hardt, C , Rouse, B.T., R o l l i n g h o f f , H., Scheurich, P. and Pfizenmaier, K. (1982) D i s s e c t i o n of the p r o l i f e r a t i v e and d i f f e r e n -t i a t i v e s i g n a l s c o n t r o l l i n g murine c y t o t o x i c T lymphocyte responses. J . Exp. Med. 155, 1876-1881.. 51. Raulet, D.H. and Bevan, M.J. (1982) A d i f f e r e n t i a t i o n f a c t o r r e q u i r e d f o r the expression o f c y t o t o x i c T c e l l f u n c t i o n . Nature 296, 754-757. -52. 0 i , . V.T. and Herzenberg, L.A. (1980) Immunoglobulin-producing h y b r i d c e l l l i n e s . Selected Methods i n C e l l u l a r Immunology( M i s h e l l , B.B. and S h i i g i , S.M., eds) W.H. Freeman and Company, San F r a n c i s c o , p. 351-372. 53. F a r r a r , J . J . , F u l l e r - F a r r a r , J . , Simon, P.L., H i l f i k a , M.L., S t a d l e r , B.M. and F a r r a r , W.L. (1980) Thymoma production of T c e l l growth f a c t o r ( i n t e r l e u k i n 2 ) . J . Immunol. 125, 2555-2558. 65 54. Rubin, B., G o l s t e i n , P., Nordfang, 0. and He r t e l - W u l f f , B. (1980) Generation of H-2 r e a c t i v e T c e l l l i n e s t h a t bear the 5936 i d i o t y p e s . J . Immunol. 124,., 161-167. 55. Simon, M.M.' and Eichmann, K. (1980) T c e l l subsets p a r t i c i p a t i n g i n the generation o f c y t o t o x i c T c e l l s . Springer Semin. Immunopathol. J3, 39-62. 56. Cantor, H. and Boyse, E.A. (1975) F u n c t i o n a l subclasses o f T lymphocytes bearing d i f f e r e n t Ly antigens. I . The generation of f u n c t i o n a l l y d i s t i n c t T c e l l subclasses i s a d i f f e r e n t i a t i v e process independent of antigen. J . Exp. Med. 1_4J_, 1376-1389. 57. Kohler, G. and M i s t e i n , C. (1976) D e r i v a t i o n o f s p e c i f i c antibody-producing t i s s u e c u l t u r e and tumor l i n e s by c e l l f u s i o n . Eur. J . Immunol. 6, 511-513. 58. Aai-den, L.A. et a l . (1979) Revised Nomenclature f o r a n t i g e n - n o n s p e c i f i c T c e l l p r o l i f e r a t i o n and helper f a c t o r s . . J . Immunol.. 123r 2978-2979. 

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