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In vivo effects of T suppressor molecules : specificity and dose effects Deal, Heather Elizabeth 1988

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IN VIVO EFFECTS OF T SUPPRESSOR MOLECULES: SPECIFICITY AND DOSE EFFECTS by HEATHER ELIZABETH DEAL B.A., Oberlin College, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1988 © Heather Elizabeth Deal, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date DE-6 (2/88) A B S T R A C T The suppressor circuit and it's components have been studied extensively in this laboratory and others for the past ten years . This laboratory has produced factor-secret ing hybr idoma cel ls which are ana logous to first-order T suppressor ce l ls d i rected against the tumor P 8 1 5 . A monoclonal antibody has been raised which recognizes a common portion of suppressor cel ls and factors. T h e s e tools are used in this study. It had been seen that when 20 ng A 1 0 F (factor secre ted by A 1 0 , the Ts1 analogue) was injected into a mouse concurrent ly with P 8 1 5 , the suppress ion of the mouse 's immune response was boosted. This led to increased tumor growth and accelerated death. However, when A 1 0 F was injected ten days prior to the mouse receiving P 8 1 5 , the opposi te effect was seen . M ice had smal ler tumors and longer survival t imes. This was not the contrasuppressive effect descr ibed by other laboratories, as the effect seen was not merely an abrogat ion of suppress ion , but rather enhancement of the immune response . The specificity and dose response of the effect was examined. This immune enhancement effect was not speci f ic within the context of syngeneic tumor sys tems. It was found that the same effects were seen when P815 was replaced with L1210 or M-1, both a lso being H - 2 d tumors. In fact, L1210 w a s more sensi t ive to A 1 0 F than P 8 1 5 . There was some level of specif ici ty to the enhancemen t effect. W h e n A 1 0 F was rep laced with F d 1 1 F , a suppressor factor ra ised to ferredoxin, no effect was seen . Converse ly , A 1 0 F did not produce the same effects as Fd11F in the ferredoxin system. Suppressor deletion therapy was used in both of these systems to confirm that suppressor cel ls were responsible for the effects seen . Dose response studies showed that the enhancement effect was dose dependent. Doses of A10F below 20 ug did not produce ii enhancement in the P815 system. Enhancement was seen with lower doses of A10F in the L1210 system, but the effect did decrease at the lower doses. A model is proposed for the data presented. iii T A B L E O F C O N T E N T S P A G E Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations x Acknowledgements xiii I. Introduction 1 A . Suppressor History 1 B. Network and Suppression 5 1. Network Review 5 2. Network in the Suppression System 6 C . History of the P815 Project 8 D. Thes is Proposal 11 II. Materials and Methods 12 A . Purification of T Suppressor Molecules 12 1. B16G-Sepharose Column 12 a. B 1 6 G Production 12 b. Column Preparation 12 iv / 2. Generation of A10 and Fd11 Molecules 13 B. Quantification of T Suppressor Molecules 14 1. Spectrophotometry 14 2. B IO-RAD 14 3. Lowry 14 C . Biochemistry of the T Suppressor Molecule 14 1. EL ISA Analysis 14 2. Ge l Electrophoresis 15 D. In Vivo Assays 16 1. Pre-injection of T s F 16 a . Dose Effects 16 i. Experimental Animals 16 ii. Cel l Lines 16 iii. Treatment Course 16 iv. Analysis of Results 16 b. Specificity 17 i. Experimental Animals 17 ii. Cel l Lines 17 iii. Treatment Course 17 iv. Analysis of Results 18 2. Suppressor-Delet ion Therapy v a. Abrogation of Non-Responder Status 18 i. Experimental Animals 18 ii. Antigen 18 iii. B16G/Hematoporphyr in Preparation 18 iv. Treatment Course 19 v. EL ISA 19 vi . Analysis of Results 19 b. Enhancement of Tumor Resistance 20 i. Experimental Animals 20 ii. Cel l Lines 20 iii. Monoclonal Antibody/Hematoporphyrin Preparation 20 iv. Treatment Course 20 v. Analys is of Results 21 E. Serum Antibody Assay 21 1. Experimental Animals 21 2. Treatment Course 21 3. EL ISA 21 III. Results 23 A . Purification of T Suppressor Molecules 23 B. Quantification of T Suppressor Molecules 23 1. Spectrophotometry 23 vi 2. B I O - R A D 24 3. Lowry 24 C . Biochemistry of the T s F 24 1. Ge l Electrophoresis 24 2. EL ISA Analys is 26 a . T s F Analysis 26 b. Antibody Analysis 26 D. In Vivo A s s a y s 26 1. Pre-injection of T Suppressor Molecule 30 a. Dose Effects 30 b. Specificity 32 2. Suppressor Deletion Therapy 43 a. Abrogation of Non-Responder Status 43 b. Tumor Protection 44 E. In Vitro Antibody Assay 46 IV. Discussion 50 V . References 57 vii LIST O F T A B L E S T A B L E P A G E I Experiment #3. Survival times of mice treated with low doses of A1 OF prior to receiving P815 34 II Experiment #4. Survival times of mice treated with low or high doses of A1 OF prior to receiving P 8 1 5 35 III Experiment #5. Survival times of mice treated with A1 OF prior to receiving P 8 1 5 or M-1 36 IV Experiments #6 and #7. Survival times of mice treated with low or high doses of A1 OF prior to receiving L1210 40 V Experiment #8 . Serum antibody levels in mice which had undergone suppressor deletion therapy , then received ferredoxin 45 VI Experiments #10 and #11. Serum antibody levels in mice treated with A1 OF or Fd11F prior to receiving ferredoxin 49 viii LIST O F F I G U R E S FIGURE P A G E 1 Lowry protein quantification assay standard curve 25 2 S D S - P A G E of A1 OF and D M E M / 1 0 % F C S control material 27 3 EL ISA results of B 1 6 G binding to A1 OF 28 4 EL ISA results of B 1 6 G or E A 2 binding to A1 OF eluted from B 1 6 G or E A 2 coated affinity columns 29 5 Experiment #1. Tumor growth in mice treated with A1 OF prior to receiving P815 31 6 Experiment #2. Survival rate of mice treated with A1 OF prior to receiving P815 33 7 Experiment #5. Tumor growth in mice treated with A1 OF prior to receiving L1210 38 8 Experiment #5. Tumor growth in mice treated with A1 OF prior to receiving M-1 39 9 Experiment #6. Tumor growth in mice treated with low or high doses of A1 OF prior to receiving L1210 41 10 Experiment #7. Tumor growth in mice treated with low or high doses of A1 OF prior to receiving L1210 42 11 Experiment #9. Survival rate of mice receiving L1210, then undergoing suppressor deletion therapy 47 12 Experiment #9. Tumor growth of mice receiving L1210, then undergoing suppressor deletion therapy 48 13 Model for cellular interactions involved in suppression 54 ix LIST O F A B B R E V I A T I O N S ABA - azobenzenzarsonate aid - anti-idiotypic A10 - suppressor T cell hybridoma directed to P815 A1 OF - molecule secreted by A10 B B S - borate buffered saline B 1 6 G - monoclonal antibody against common epitope of suppressor factors BSA - bovine serum albumin C. - celsius C A M A L - 1 - monoclonal antibody against putative leukemia-associated antigen CFA - complete Freund's adjuvent cm - centimeter CTL - cytotoxic T lymphocyte DM E M - Dulbecco's modified Eagle's medium EA2 - monoclonal antibody against common epitope of suppressor factors ELISA - enzyme-l inked immunosorbent assay FCS - fetal calf serum Fd - ferredoxin Fd11 - suppressor T cell hybridoma directed to ferredoxin Fd11F - molecule secreted by Fd11 GAT - L-glutamic acid-L-alanine-L-tyrosine GT - L-glutamic acid-L-tyrosine x Abbreviations (continued) Hp - hematoporphorin id - idiotype I FA - incomplete Freund's adjuvent IL-1 - interleukin-1 IL-2 - interleukin-2 i.v. - intravenous kD - kilodalton KLH - keyhole limpet haemocyanin MAb - monoclonal antibody ug - microgram u.l - microliter mg - milligram MHC - major histocompatability complex ml - milliliter mm - millimeter nm - nanometers NP - 4-hydroxy-3-nitrophenyl acetyl PAGE - polyacrylamide gel electrophoresis PBS - phosphate buffered saline RexMlg - rabbit anti-mouse immunoglobulin rpm - revolutions per minute xi Abbreviations (continued) SDS-PAGE - sodium dodecyl sulfate-polyacrylamide gel electrophoresis 16A - monoclonal antibody against the C terminus of the ferredoxin molecule SRBC - sheep red blood cells ThC - T helper cells TsC - T suppressor cells Ts1 - first-order T suppressor cells Ts2 - second-order T suppressor cells Ts3 - third-order T suppressor cells TsF - T suppressor factor TsF 1 - factor secreted by Ts1 T s F 2 - factor secreted by Ts2 T s F 3 - factor secreted by Ts3 xii A C K N O W L E D G E M E N T S I would like to thank everyone in the Levy laboratory for their ass i s tance , and for their humour, without which I might never have f inished. In particular, I would like to thank Anthea Tench S tammers and Randy C h u for their contributions to this project. Spec ia l thanks go to Dr. Ju l ia Levy for her support and seemingly l imitless pat ience. Th is thesis is dedicated to Bruce Rennie. xiii I. I N T R O D U C T I O N A . Suppresso r History. In 1798 Edward Jenner publ ished a paper descr ib ing a smal lpox vacc ine made from cowpox v i rus, and the f ield of Immunology was born (1). It was nearly 100 years later that ce l ls were first impl icated in the immune p rocess . In 1883, when El ie Metchnikoff observed large cel ls clustering around a rose thorn stuck in a starf ish, cel ls suddenly became the subject of intense study (reviewed in (1)). Even at that, it was many years before the funct ions of differing populations of cel ls began to be understood. In 1942, Landsteiner and C h a s e conducted a se r i es of exper imen ts in wh ich they i nduced de layed- t ype hypersens i t iv i ty or contact sensit iv i ty in mice (reviewed in (1)). They then transferred sensi t ive ce l ls from these mice into naive mice and demonstrated that the recipient mice d isp layed the same sensitivit ies as the donor mice. Th is was the first exper imental ev idence of a populat ion of cel ls which cause sensit ivi ty. In 1970, Gershon and Kondo studied the product ion of ant ibodies in response to sheep red b lood cel ls (SRBC)(2 ,3 ) . They d iscovered that mice which had been irradiated, then repopulated with T and B cel ls did not produce antibodies to S R B C . However, when mice were repopulated with B cel ls a lone, a normal antibody response was mounted. They then showed that this tolerance to S R B C could be adoptively transferred to naive mice with the transfer of T cel ls from a tolerant donor. Thus , a population of T cel ls which serve to down-regulate the immune 1 response, or suppressor cel ls, was postulated. The existence of T suppressor cel ls , or T s C , was proved in 1977 by Cantor and Boyse (4), who isolated a population of L y - 1 " 2 3 + T cel ls which were capab le of distinct suppressive activity and which had no helper effects. The last ten years have seen the number of researchers studying suppression increase steadi ly! The result ing data have often proved difficult to ana lyse . Severa l models have been descr ibed as a result of these data. In 1977 and 1978, Benacerraf publ ished severa l papers examin ing supp ress ion in the L-g lu tamic ac i d -L -a l an ine -L - t y ros i ne (GAT) and L - g l u t a m i c ac id-L- tyros ine (GT) sys tems (5,6). In these papers, it was suggested that more than one cell might be involved in suppress ion, and that a soluble factor, or T s F , was produced by these cel ls. Th is model w a s further def ined by Ge rshon in 1980 in a paper p resen ted to the Fourth International Cong ress of Immunology (7). He proposed that more than one type of suppressor cel l w a s neccessary to explain differences between T s C populations regarding drug sensitivit ies, irradiation sensit iv i ty and levels of ant igen speci f ic i ty. B a s e d on his work with Cantor (8), G e r s h o n proposed that three separate cel ls were present in the suppressor circuit which were ac t iva ted in a l inear manner . S e v e r a l other laborator ies have p roposed s imi lar mode ls , consist ing of two or three suppressor cel ls , and containing factors produced by each cel l which would activate the next cel l in the cascade (9 - 31). In 1984, Benacerraf reviewed his work in the G A T , G T , azobenzenearsonate (ABA) and 4-hydroxy-3-ni t rophenyl acetyl (NP) sys tems and presented his model (11). W h e n an antigen is introduced into the sys tem, it is seen in its so luble form by first-order suppressor cel ls (Ts-i). T h e s e ce l ls d isplay the surface molecules L y - 1 + 2 " , Q a - 1 + , l - J + , are referred to as the 2 inducer suppressors and are antigen speci f ic . T s 1 's secrete a factor, T s F 1 , which act ivates a second ce l l , T s 2 . The T s 2 , or t ransducer cel ls have the phenotype L y - 1 + 2 + , Q a - 1 + , l - J + , and secre te a factor, T s F 2 . Th is factor act ivates T s 3 > or effector supp resso rs with a sur face phenotype of L y - 1 " 2 + , l - J + . T s 3 ' s a lso secrete a soluble factor, T s F 3 , which then acts on a target ce l l . The action of the T s F 3 is antigen requiring, but not antigen speci f ic. The target cel ls identif ied by Benacer ra f are B ce l l s . Interactions between these ce l ls and factors is major histocompatabi l i ty complex (MHC) restr icted. The ce l ls at each level of the c a s c a d e can be replaced functionally by the factors they produce. This model is general ly accepted , although there are several detai ls which still aren't c lear. Al though T s ^ s are generally bel ieved to recognize soluble ant igen, Dorf has found in the N P system that accessory cel ls are neccessary at all s teps in the suppression cascade , including its induction. In all c a s e s , he found that antigen or factor must be presented to T s C ' s by another cel l (12,13). There are also conflicting theories on the reason for the induction of suppress ion rather than help. O n e theory is that the concentrat ion of interleukin-1 (IL-1) or interleukin-2 (IL-2) present when ant igen is s e e n determines whether supp ress ion or help dominates (14,15). Another theory is that antigens possess separate determinants which are recognized by T helper cel ls (ThC's) or T s C ' s (16). T suppressor factors have been character ized as two chain molecu les , one chain of which is antigen binding and one chain of which bears an l-J determinant (14,28). Factors have also been reported which appear to be composed of a single chain (17,18). The mechanisms of 3 interactions between TsC's and TsF's are not understood. There are some who feel that, as TsF's have been shown to directly bind antigen, antigen bridges may play a part in these interactions (19). Most results indicate that these interactions are MHC restricted, but there are some laboratories reporting that some levels of the suppressor cascade are not MHC restricted (20,21,22). Antigen specificity is also not thoroughly understood. Many laboratories have reported that they can demonstrate antigen specificity at all levels of suppression (19,21,22), yet others demonstrate that at least the effector level of suppression is not antigen specific (15,23,24). Both B cells (21,25) and ThC (11,26) have been implicated as the target cells of suppression. TsC's have also been shown to down-regulate cytotoxic T lymphocytes (CTL's) in vitro (27). Benacerraf, Gershon, and others describe a three cell suppression cascade, but other labs have identified two major cells in the cascade, inducers and effectors (28,29,30). Finally, there have been reports of a special population of cells which exist soley to control suppression (31,32,33,34). These reports have generally been viewed with scepticism. There are data supporting the theory that TsC's and TsF's can provide feedback control for themselves (35) . All in all, there is a good deal more known about suppression now than there was ten years ago. Several problems keep the suppressor circuit from being completely understood. First, the MHC restriction often observed is related to the l-J gene product, which maps to the MHC region. However, when the MHC region was cloned and characterized, it was found that there was no sequence long enough to contain the l-J gene outside of the genes already defined (36) . Second, the TsF has yet to be sequenced. These days, cloning is believing. Hopefully this problem will be resolved soon. Lastly, the phenomenon of suppression is being studied by many 4 different groups in many different antigen sys tems. This leads to results which are extremely difficult to compare to each other. The very complex models proposed by some laboratories with suppressor ce l ls , accessory ce l ls , present ing ce l ls , cont rasuppressor ce l ls , etc. make collating these resul ts extremely difficult. S imp le mode ls with the min imum number of parameters neccessa ry to explain the data will make interpretation more straightforward. However , until more b iochemica l detai ls of suppress ion are def ined, the immunologica l da ta produced will cont inue to lend itself to a variety of interpretations. B. Network and Suppress ion . 1. Network Rev iew. In 1974, N ie ls Je rne p roposed a sys tem of cel lu lar interact ions which he termed "network" (37). The sys tem differed from the c lonal select ion theory of Burnet (37) in that it a l l owed for ce l l -ce l l in teract ions and cel lu lar act ivat ion without the s t imulus of external ant igen. Jerne proposed that the variable regions of antibody molecules can themselves be seen as ant igens. The unique portion of the var iable region which is recogn ized in this manner is referred to as the idiotype. Hoffmann e laborated on this theory (39,40) with the stipulation that the interactions between idiotypes are symmetr ical . This means that any given idiotype can both see and be seen by a complementary idiotype. These idiotypes can be general ized as "+" or "-" , or as idiotype (id) or anti-idiotype (aid) . Ids and a i d s are essent ia l ly st icky ends , and will b ind to anything complementary to themse lves . Thus , an idiotypic cel l can stimulate an anti- idiotypic ce l l , which can in turn st imulate another idiotypic cel l as wel l as stimulate the original idiotypic ce l l . The interactions between id and a i d cel ls can be inhibitory as well as 5 stimulatory. Ke lsoe has shown that there is a concurrent rise of a id cel ls and fall of id cel ls, and v i c e - v e r s a (41). T h e interact ions between id and a i d ce l ls probably occur as a result of cross- l inking of receptors. Another mode of interaction between these cel ls could be mediated by id present on a monomeric factor, which would then bind to the receptors on an a id cell and block them from cross- l inkage (40). There has been exper imental ev idence for years that impl icate T cel ls in idiotypic networks (42 ,43 ,44 ,45) . The sugges t i on that T ce l l s carry id iotypic de terminants was conf i rmed when it was shown that the T cel l receptor p-gene is capab le of rearrangements similar to those seen in variable region genes (46). It has also been shown that T and B cel ls can interact through direct receptor-receptor binding (47). T h e id iotypic network is thought to normal ly exist in a state of equ i l ib r ium. Introduction of a foreign antigen forces the system out of equil ibrium. After the antigen has been c l ea red , the sys tem returns to an equi l ibr ium which can be different from that at which it s tar ted (40). 2. Network in the Suppress ion Sys tem. Je rne (37), Hof fmann (39) and G e r s h o n (6) p roposed that id iotypes may play an important part in the cel lu lar interactions of the suppressor c a s c a d e . Idiotypic sur face and factor determinants influence the interactions between these T s C ' s and between T s C ' s and their target cel ls (9,10,30,48,49). When an antigen enters the sys tem, it st imulates T s 1 cel ls which recognize the ant igen. These cel ls a lso display idiotype on their surface. The T s i ' s secrete T s F 1 which also bind ant igen and express idiotype. T h e s e factors then st imulate a i d T s 2 ' s , which 6 secrete aid, non-antigen binding TsF 2. This factor then either recognizes an idiotypic target cell, or stimulates a third branch in the suppressor cascade. The putative idiotypic target cell could be either a ThC or a B cell. Hoffmann's model supports the theory of a need for factors and antigen to be presented by accessory cells (40). This model also addresses the issue of immunity versus suppression. Hoffmann proposes that very low or very high amounts of antigen lead to increased amounts of both id and aid cells, a prerequisite for suppression. Moderate amounts of antigen lead to activation of accessory cells which would then secrete a non-specific factor such as IL-1. This factor would activate those cells which induce immunity. The observation that higher levels of IL-1 are related to the activation of help versus suppression could be used as an argument for this model (14,15). Antigen specificity in suppression is also addressed by the network model. As Ts^s are activated directly by antigen, it could be supposed that the receptor which recognizes the antigen would be specific. The Ts2's, however, are activated by idiotypic determinants and therefore recognize idiotype rather than antigen. Target cells of Ts2's would need only bear idiotype to be recognized. Another possible application of network theory to antigen specificity involves the nature of the TsF's. If Ts 1 is a monomeric factor, then it might bind to the aid receptors of the Ts 2 's without cross-linking and activating them. The presence of the specific antigen to which TsF 1 binds could then provide the cross-linkage. Experiments which show that antigen is necessary for induction of Ts 2 's support this theory (6,50,51,52). There are other results 7 which show that ant igen is not necessary for T s 2 induct ion (53,54). T h e apparent loss of antigen specifity in the efferent branch of suppress ion could also be expla ined in the context of network. The simplest explanat ion is that T s F 2 is a bivalent molecule which can cross-l ink any idiotypic receptors. One laboratory has reported that TsF-| consists of one cha in , and T s F 2 of two cha ins (52). Another possibil i ty is that, if T s F 2 ' s are monomers , they can be cross- l inked by any antigen which contains determinants which resemble self, or id. Th is would explain those results suggest ing that the final stage of suppress ion is not ant igen speci f ic , but is antigen requiring (11). Alternately, if T s F 2 ' s are monomers, they may act by blocking the receptors of their target ce l l s . The importance of idiotypic networks in suppression has been demonstrated by the use of anti-idiotypic antibody vacc ines . The use of these vacc ines has been successsful ly tested in the tetanus toxiod (55), hepatit is B (56), Streptococcus pneumoniae (57), t rypanosomias is (58) and a number of tumor ant igen sys tems (59,60,61,62) . T h e ' internal image ' nature of anti- idiotypic mo lecu les (63,64) a l lows them to act as a vacc ine by either neutral iz ing the idiotypic T s l ' s (60), or by activating idiotypic T h C (65). A vacc ine has been deve loped for a B cel l l ymphoma using secreted id directly from the lymphomic cel ls (66). Idiotypic networks in immunology are rapidly becoming further understood and more general ly accep ted . Carefu l manipulation of id-aid interactions in suppressor circuits may shed light on some of the inconsistancies seen in data involving these circuits. 8 C . History of the P815 Project. Suppress ion in the P815 tumor system was first examined by Takai in the late 1970's (27,67,68). Subcu taneous injections of P 8 1 5 tumor into syngene ic mice led to initial rapid growth of the tumor, a brief slowing of tumor growth, then a resurgence of rapid growth leading to death after 20 to 25 days . The brief s lowing of tumor growth and following growth increase were shown to be c a u s e d by C T L ' s and T s C inhibition of those C T L ' s , respect ively. The T s C ' s produced by these mice produced a T s F speci f ic for P 8 1 5 which could functionally replace the T s C ' s in an in vitro C T L assay (69). This factor was shown to bind to columns coated with P815 membrane extracts, but not to those coated with extracts from L1210 (a syngene ic tumor). The T s C was not M H C restricted, although it expressed surface la . The surface phenotype of the T s C w a s Ly-1 + 2 - (70). Ant isera was raised against T s F . The antisera were found to react with both T s F and T s C (71). In 1983, a monoclonal antibody, B 1 6 G , was produced which bound both T s F and T s C (72). Th is monoclonal antibody was later found to bind T s F ' s raised in a number of other sys tems, inc lud ing T s F ' s f rom human tonsi l lar t i ssue (73). T h u s , B 1 6 G r e c o g n i z e s a c o m m o n determinant on T s F ' s . Pre l iminary s tud ies were run on T s F sepa ra ted from sp leen cel l supernatants from D B A / 2 mice (74). In 1985 , a hybr idoma, A 1 0 , was ra ised which secre ted P815-spec i f i c T s F , A 1 0 F (75,76). Th is factor was character ized as a potential dimer with two units of 43 kD and 45 kD. More recent data (77) suggest that there could be subunits of 32 kD, 50 kD, 80 kD, and a 140 kD unit which could represent a combination of the other subunits. A 1 0 F binds to both P815 and B 1 6 G co lumns, and binds to B 1 6 G in enzyme- l inked immunosorbent assays (ELISA 's ) . Injection 9 of A10 into mice concurrent with administration of P815 results in a significant increase in tumor growth rate and decrease in the length of time of survival. A10F does not have these effects on the syngeneic tumors L1210 and M-1. A10F significantly inhibits production of CTL's specific for P815 in vitro (78). As this hybridoma and factor displayed the characteristics of Ts 1 and TsF 1, attempts were made to raise a Ts 2 to A10 (9). The resulting hybridoma, A29, displayed the characteristics of an anti-idiotypic second-order TsC. A29 secretes a factor, A29F, which binds B16G but not P815. Calcium flux studies showed that A29 cells responded to A10F in a significant and specific fashion. Mice were immunized with A10F in an attempt to increase their resistance to P815 (79). The results showed that A10F provided significant protection when injected at 20 u.g/mouse 10 days prior to tumor administration. These results were not comparable to those referred to as contrasuppression (23-26), as there was not merely an abrogation of suppressive effects, but a significant enhancement of immunity. Other projects in this laboratory have produced related data. It has been found that suppressor cells can be deleted in vivo by linking B16G to a photosensitive molecule, hematoporphyrin (Hp)(80,81). This conjugate is then injected into a mouse which is then exposed to strong light to activate the Hp, which then kills any cells which it bound to. Mice receiving this treatment have been shown to be significantly protected against P815. Another suppression circuit is being examined. A Ts 1 hybridoma (Fd11) has been raised against the 1 0 ant igen molecu le ferredoxin (Fd)(64,82). Extens ive work is being done on the cel l and it's sec re ted factor, F d 1 1 F . The nonresponder status of D B A / 2 mice to fer redoxin and the relat ionship of that status to suppress ion is a lso being studied (83,84). It has been found that el imination of either the id or a id cel l populat ions leads to abrogat ion of the non-responder status. D. Thes is Proposa l . In this study, the phenomenon of enhanced immunity following injection of A 1 0 F ten days prior to tumor cel l injection is examined. The specificity and dose response of this effect is s tud ied . T h e invo lvement of s u p p r e s s o r ce l l s in the rate of tumor growth and in the non- responder status of D B A / 2 mice to ferredoxin is conf i rmed through suppressor deletion therapy. A model is proposed which would explain the data. 1 1 II. M A T E R I A L S A N D M E T H O D S A . Purif ication of T Suppressor Molecu les . 1. B 1 6 G - S e p h a r o s e Co lumn. a . B 1 6 G production. The B 1 6 G monoclonal antibody (MAb) w a s produced by, and has been studied for seve ra l yea rs in this laboratory (72). It was ra ised against T supp resso r mo lecu les with activity for the P815 tumor sys tem, and appears to bind to all of the T suppressor molecules produced by this laboratory (76). For the purposes of these exper iments, the hybr idoma B 1 6 G was grown as an asci tes tumor in Ba lb /c mice which had been bred in the U B C Department of Microbiology's animal breeding facility. The mice had been injected intraperitoneally 10 days to 2 months ear l ier with 0.5 milli l itres (ml) of pr is tane. Asc i tes was co l lec ted from the mice, pooled and stored at 4 ° Ce lc ius (C.) for no more than 2 weeks . M A b was precipitated from the poo led asc i tes with 5 0 % saturated ( N H 4 ) 2 S 0 4 , and centr i fuged at 15,000 rpm in a Dupont R C 5 B centrifuge for 30 minutes. The resulting precipitate was d isso lved in phosphate buffered sa l ine ( P B S ) and d ia lysed in P B S for 18 to 24 hours. B 1 6 G was either re-dia lysed in borate buffered sal ine (BBS) for immediate use or lyophi lysed and stored at 4 ° C . for use in future experiments. Control MAb 's C A M A L and 16A were obtained and stored in the same manner. b. Co lumn preparat ion. 1 2 Protein content of B 1 6 G samples was determined using spectrophotometric readings at 280 and 260 nanometers (nm). Immunoadsorbent co lumns were prepared by combining B 1 6 G at 5.1 mil l igrams (mg)/ml in B B S to cyanogen bromide activated Sepharose 4 B beads using the method of Axen et. al.(85). 10 ml of coated beads were packed into a 16 X 130 centimeter (cm) co lumn, and stored in P B S / 0 . 0 2 % sodium azide at 4 ° C . 2. Generat ion of A10 and Fd11 Molecules. A 1 0 and Fd11 T-suppressor molecu les , or factors (TsF) , were produced by growing the hybr idomas in Du lbecco 's Modi f ied E ag l es medium ( D M E M ) with 1 0 % fetal calf serum ( F C S ) by vo lume, in 1 0 % C 0 2 with 100% humidity at 3 7 ° C . Spent supernatant was harvested in one to two liter batches and centrifuged to eliminate cel ls and cellular debris. The supernatant was then passed over an immunoabsorbent column of B 1 6 G M A b linked to Sepharose 4 B at 4 ° C . Separa te co lumns were used for col lect ion of A1 OF, Fd11F and control material. Flow rates of 50-75 ml/hour were used , and material was passed over the co lumns once only. Co lumns were w a s h e d extensively with co ld P B S until spectrophotometr ic ana lys is showed no apprec iable protein in the exudate. Suppressor molecules were eluted from the co lumns with cold 0.1 M HCI and col lected in 16 1ml fractions. Eluted fractions were read for absorbance at 280 nm and those fractions containing protein were pooled. The pooled material was then neutral ized with 3 M Tr is(hydroxymethyl)- aminomethane (pH 8.9) and d ia lysed overnight in P B S . Amounts of protein obtained ranged from 100 to 200 mg/ml of eluted material as measured by the method of Lowry (86). Puri ty w a s moni tored by us ing po lyacry l im ide ge l e lec t rophores is ( P A G E ) . Control material was obtained in the same manner from equivalent vo lumes of D M E M / 1 0 % F C S . Al l procedures were carr ied out at 4 ° C . Resul t ing material was stored in al iquots at -70° C . 1 3 until used . B. Quantif ication of T Suppressor Molecu les. 1- Spectrophotometry, Protein content of samples was calculated using absorbance at 280 and 260 nm on a Var ian D M S 90 spect rophotometer us ing the formula (OD 280).(1.5) - (OD 260)(0.76) = mg/ml pro te in . 2. B I O - R A D . Protein content of samples was a lso calculated using the Bradford ( ) commass ie blue protein ana lys is method ( B I O - R A D #500-0006). 3. L o w r y . The Lowry method (86) of protein quantification was used . Suppressor molecules were quantif ied by taking three different dilutions of the sample and taking the average protein/ml by extrapolat ion from a s tandard curve genera ted using bovine serum albumin ( B S A ) . A new standard curve was run for each assay . C . Biochemistry of the T Suppressor Molecule. 1. E L I S A Ana lys is . E n z y m e l inked immunosorbent a s s a y s (EL ISA) were u s e d to test the binding capabi l i t ies of A 1 0 and Fd11 T s F ' s . T suppressor molecule samp les were tested for protein content by the Lowry method and diluted to 20 i ig /ml . These samples were appl ied in 100 nl al iquots to Dynatech Immulon I plates in doubl ing di lut ions. The plates were then incubated 1 4 overnight at room temperature. P la tes were washed three t imes with P B S / 0 . 0 5 % Tween-20 . B 1 6 G MAb was appl ied to the plates at either 500 u.g/ml or 100 ng/ml in 100 u.l al iquots. The plates were then incubated for one hour at room temperature and again washed three times with P B S / T w e e n . The plates were incubated with alkaline phosphatase conjugated rabbit anti-mouse Ig (RocMlg) for one hour at room temperature and once aga in w a s h e d three t imes with P B S / T w e e n . The p lates were then deve loped with S i g m a phospha tase subst rate (S igma #104-105) . P la tes were read on a F low Ti tertech spect rophotometer at 405 nm at time intervals from 15 minutes to 2 hours. Contro ls for non-speci f ic binding at all levels were run on each plate. The Titertech was zeroed on wells containing buffer a lone. E L I S A s were also used to analyse B 1 6 G and E A 2 MAb ' s . Factor eluted from either a B 1 6 G or an E A 2 affinity column was plated at 20 ug/ml in 100 uJ/well and incubated at room temperature for one hour. Plates were washed and B 1 6 G or E A 2 MAb ' s were added in limiting dilutions starting with 1 mg/ml. Both MAb 's were tested against A 1 0 F from both co lumns. Plates were developed as described above. 2. G e l Elect rophoresis . Sod ium dodecy l sulphate polyachry lamide reducing gel e lect rophores is ( S D S - P A G E ) was used to confirm that samples contained T s F and to monitor levels of contaminating protein. The Laemell i (87) method was used . Protein samp les were mixed with a reducing solution (10mM dithiothreitol, 2 % S D S , 1 0 % g lycero l , pH 6.8), and heated for 3 minutes at 100° C . G e l s were 1 mil l imeter (mm) thick. A 1 0 % separat ing gel and a 4 % stacking gel were used . G e l s were run at a constant current of 25 mi l l iamps, and with protein samples at 3 doses to check for artifacts. Ge l s were 1 5 then f ixed in 5 0 % methanol and stained with silver nitrate using the method of Wray (88). D. In Vivo Assays . 1. Pre- in ject ion of T s F . a. Pose Effects, i. Exper imenta l An ima ls . D B A / 2 J mice between 8-12 w e e k s of age were used in this a s s a y . Any g iven exper iment used all males or all females . Al l an imals were ra ised in the U B C Department of Mic rob io logy an imal facil i ty. i i. Ce l l L ings . The P815 mastocytoma of D B A / 2 J mice was used as the tumor model in this study. This cel l l ine has been maintained in this laboratory for the past d e c a d e . It is maintained in the peritoneal cavit ies of D B A / 2 J mice. On the day of use , cel ls were removed from asci tes tumors carr ied in D B A / 2 mice, washed once in P B S , counted and resuspended at 1 0 5 cel ls /ml . When not in use , the cel ls were suspended in 10% dimethylsulphoxide and stored in liquid nitrogen. iii. Treatment C o u r s e . On day -10 of each experiment, mice were injected in the tail vein with A 1 0 T s F in a vo lume of 100 ul P B S . Amount of the T s F var ied from 0.08 ug to 40 ug . O n day 0, 1 0 4 tumor cel ls were injected subcutaneously in the right flank. iv. Ana lys is of Resul ts . Eight mice were used in each exper imental group. Tumor s ize was measured with cal ipers on a daily bas is , and average tumor s ize per group of mice calculated. Once more than 4 1 6 mice had died in a given group, tumor s ize was no longer read. Each death was recorded and the average survival time recorded at the end of a 50 day per iod. M ice which had no detectable tumors after 50 days were cons idered cured . Students t-test was used to calculate statistical s igni f icance. E a c h group was compared with a control group which had not received any T s F , but had rece i ved a tumor ce l l in ject ion. Resu l t s were c o n s i d e r e d s ign i f icant at p< 0 .05. b. Specificity, i. Exper imenta l An ima ls . D B A / 2 J mice between 8-12 weeks of age were u s e d in this a s s a y . Any given exper iment used all males or all females . Al l an imals were ra ised in the U B C Department of M ic rob io logy an imal facil i ty. ii. Ce l l L ines . The P 8 1 5 mastocy toma, the L1210 leukemia, and the M-1 rhabdomyosarcoma, all tumors of D B A / 2 J mice, were used as the tumor models in this study. Al l three cel l l ines have been grown in this laboratory for the past decade . They are mainta ined and harvested as descr ibed above. For some experiments, L1210 was resuspended at 1 0 4 / m l . iii. Treatment C o u r s e . On day -10 of each experiment, mice were injected in the tail vein with either A10 or Fd11 T s F in a volume of 100 u.l P B S . Concentrat ion of the T s F ' s var ied experimentally. On day 0, tumor cel ls were injected subcutaneously in the right flank. P815 tumor cel ls were injected at a concentrat ion of 1 0 4 cel ls in 100 u.1 of P B S . M-1 tumor cel ls were administered in the same manner . L1210 tumor ce l ls were injected at a concentrat ion of 1 0 3 or 1 0 4 ce l ls in 100 u.l of P B S . 1 7 iv. Ana lys is of Resul ts . Eight mice were used in each exper imental group. Tumor s ize was measured with cal ipers on a daily bas is , and average tumor s ize per group of mice calculated. E a c h death was recorded and the average survival time recorded at the end of a 50 day period. Mice which had no detectable tumors after 50 days were cons idered cured. Students t-test was used to calculate statistical s igni f icance. Each group was compared with a control group which had not received any T s F , but had received tumor cel ls. Resul ts were considered significant at p< 0.05. 2. Suppressor -de le t ion Therapy . a . Abrogat ion of non-responder status. i. Exper imenta l An ima ls . D B A / 2 J mice were obtained from the U B C Department of Microbiology animal breeding facility. They were between the ages of 8 and 12 weeks when used. ii. A n t i g e n . T h e fer redox in mo lecu le is a 55 amino ac id po lypep t ide f rom Clostridium pasteurianum. It has been used extensively as an antigen model in this laboratory (64, 82-84) . A hybr idoma has been ra ised, F d 1 1 F , which produces an ant igen-speci f ic suppressor molecule for ferredoxin. Th is T s F has been shown to be capab le of convert ing non-responder mice to responders (83,84). iii. B 1 6 G / H e m a t o p o r p h y r i n P repa ra t i on . H e m a t o p o r p h y r i n d ihyd roch lo r i de (75%) ( S i g m a H1875) w a s u s e d in these exper iments. Hematoporphyr in (Hp) is a l ight-sensit ive molecule which has been shown to kill target cel ls when assoc ia ted with a M A b speci f ic for those ce l ls , then exposed to light (80,81). 1 8 Hp was conjugated to B16G and CAMAL by Dr. Daniel Liu of QuadraLogic Technologies. iv. Treatment Course. On day -1, mice were injected in the tail vein with PBS, B16G/Hp (87 ug/33 ug) or CAMAL(an irrelevent antibody)/Hp (240 ug/36 ug), all in a volume of 100 ul. Mice were kept in the dark for 4 hours to allow the MAb to find it's target cells. The mice were then placed under a strong light for 4 hours to activate the Hp. Following this treatment, mice were returned to their normal quarters. On day 0, mice were injected subcutaneously with 15 ug ferredoxin in complete Freund's adjuvent (CFA). Mice were bled on day 20, and reinjected with ferredoxin on day 21. On day 28, mice were again bled. Each experimental group contained 6 mice. v. ELISA. An ELISA was used to measure relative antibody levels in the day 20 and day 28 serum samples. Immulon Dynatech II plates were coated overnight at room temperature with 0.2 ug/well ferredoxin in 100 ul carbonate coating buffer. Plates were incubated for one hour at room temperature, then washed 3 times with PBS/Tween. Normal mouse serum and a known positive serum were diluted 1/200 and used as controls. Experimental sera were diluted 1/100 and 1/200. Serum samples were added in 100 ul aliquots and plates were incubated for one hour at room temperature. Plates were then washed again and incubated for one hour at room temperature with RtxMlg-alkaline phosphatase. Finally, after another washing, plates were developed with Sigma phosphatase substrate. Plates were read on a Flow Titretech at 405 nm after one hour, two hours and twenty-four hours. vi. Analysis of Results, The average absorbance of the serum samples from each group was calculated for a 1 9 given dilution and time of development. The B 1 6 G / H p and C A M A L / H p groups were each compared to the P B S control group. The student's t-test was used to determine di f ferences, with p-values of less than p=0.05 cons idered significant, b. Enhancement of Tumor Resistance. i. Exper imenta l An ima ls . D B A / 2 J mice from the U B C Department of Microbiology animal breeding facility were used . Mice were received at 6 to 8 weeks of age and used within a month. ii. Ce l l L ine. The D B A / 2 J leukemia L1210 was used in this experiment. iii. Monoc lona l Ant ibody/Hematoporphor in Preparat ion . The M A b E A 2 was raised against the T s F A 1 0 in this laboratory two years ago. It has been shown to bind to the suppressor cel ls and molecules in common use in this laboratory (unpublished data). It is grown and maintained in the same manner that the M A b B 1 6 G is. Hp and E A 2 were comb ined in a ratio of 50 u.g Hp/1mg E A 2 in 1ml P B S , and incubated overnight at room temperature. iv. Treatment Cou rse . On day 0, mice were injected with 5 X 1 0 3 L1210 cel ls subcutaneously in the flank. On day 5 , mice received P B S , 10 ng Hp alone, 200 up, 16A (an irrelevent MAb) /10 ng Hp, or 200 ng EA2 /10 n9 H P a " in a volume of 200 in the tail ve in . Mice were then kept in the dark for 4 hours to allow the E A 2 to find its target cel ls . Next, mice were exposed to f luorescent light for 4 hours to act ivate the Hp. Fol lowing this course of treatment, mice were returned to their normal quarters. 20 v. Ana lys is of Resu l ts . S ix mice were used in each experimental group. Tumors were measured on a daily bas is , and average tumor s ize calculated per group per day. T imes of death were recorded and used for survival statist ics. Mice showing no ev idence of tumor after 50 days were considered cured. Each group was compared with mice receiving tumor only. Resul ts were calculated using the student's t-test, and cons idered significant when p<0.05. E. Serum Antibody Assay 1. Exper imenta l An ima ls . D B A / 2 J mice were obtained from the U B C Department of Microbiology animal breeding facility. Mice were 6 to 8 weeks old when received, and used within a month. 2. Treatment Cou rse . On day -21 , mice were injected in the tail vein with P B S , 1.0 ug A 1 0 or 1.0 ug Fd11 . M ice were then hyper immunized on the fol lowing schedu le : day 0, 50 ug Fd/50 ug keyhole limpet haemocyan in (KLH) (as an internal control) in C F A , injected subcutaneously in separate s i tes; day 21 , b leed ; day 28, 50 ug Fd/50 ug K L H in incomplete Freund 's adjuvent (IFA); day 35, b leed; day 43 , C-fragment of the Fd molecule; day 50, b leed. 3. EL ISA . E L I S A s were used to test anti-ferredoxin and ant i -KLH antibody levels in supernatants and serum samples . Dynatech Immulon I plates were coated with 100 ul Fd or K L H at 1 ug/ml in carbonate coat ing buffer (pH 9.6) overnight at room temperature. P la tes were then washed three t imes with P B S / T w e e n . Ant ibody-containing and control serum samp les were added to 21 plates coated with both antigens in var ious dilutions. Plates were incubated for 1 hour at room temperature, and again washed three t imes with P B S / T w e e n . A lka l ine-phosphatase conjugated R a M l g was then added to each wel l , and plates were incubated for 1 hour at room temperature. P la tes were then deve loped with S i g m a phosphate substrate at room temperature at read at regular time intervals on a Flow Titretech at 405 nm. Media-only and posit ive serum controls were run on each plate. All incubations were carried out in the dark. 22 III. R E S U L T S A. Purif ication of T Suppressor Molecu les . Vary ing amounts of suppressor molecu le , or factor, were obtained from hybr idoma supernatants . Y ie lds ranged from 500 ug/litre of supernatant to "lmg/1. Most ba tches y ie lded 600-800 ug/l. C o l u m n s were run very slowly and large batches (1.5-3 I) of supernatant were run at a t ime to min imize l osses that might be incurred during the purif ication p rocess . In genera l , Fd11 produced lower amounts of T s F than A 1 0 . Y ie lds of protein from D M E M / 1 0 % F C S were quite low - on the order of 75 ug/l of medium. Th is w a s prepared for potential use as control mater ia l . It a lso showed that the levels of non-speci f ic binding to the B 1 6 G affinity co lumn were minimal . B. Quantif ication of T Suppressor Molecules. 1. Spec t rophotomet ry . This laboratory has used spectrophotometry to estimate protein content in samples for some years . Absorbance is read at 260 and 280 nm, and the formula [1.5(absorbance 280 nm) - 0 .76(absorbance 260 nm) = mg/ml protein] has been used for ana lys is . Recent ly , however, it has been found that results using this method are unrel iable. Attempts at confirming protein est imates using other quantification methods have been fruitless. Protein content est imates have been off by as much as two-fold. Repeat ing experiments using these est imates proved difficult. It was dec ided not to rely on this method any further. 2. B I O - R A D . The B I O - R A D commass ie blue protein quantification method was tested on these protein s a m p l e s . S a m p l e s were a l lowed to equil ibrate with the dye for at least 30 minutes before reading. When samples were read, absorbance read-out c l imbed steadily as long as the samples were in the machine. This seemed to be due to the dye staining the cuvettes. Consistant readings were unattainable. W h e n standard curves were generated eventual ly, they var ied considerably from assay to assay . This was true even when using bovine serum albumin (BSA) from the same batch for the standard protein. 3. L o w r y . The B I O - R A D and absorbance methods of quantif ication were rep laced by the Lowry assay . This assay took a little longer to make reagents and to execute, but produced by far the best resul ts. A b s o r b a n c e readings remained constant whi le reading. S tandard curves were virtual ly ident ical from a s s a y to a s s a y (fig.1), and s h o w e d very little d i f ference between batches of B S A . Al l samp les used in these a s s a y s were ana lysed , or re-ana lysed, for protein content using the Lowry method. C . Biochemistry of the T s F . 1. Ge l E lect rophores is . S D S - P A G E gels were run for each batch of T s F to ensure that it had the banding pattern typical of a T s F (fig. 2). Th is pattern usually includes bands around 140kD, 80kD, 50kD, and 30kD (77). Three different d o s e s of each samp le were tested to ensure that bands were 24 Figure 1. Lowry protein quantif ication assay standard curve. Protein samples were tested in 50 ul , 100 u l , and 200 ul vo lumes, and the ug protein/ml ca lcu la ted from extrapolated X-ax is coordinates. decreas ing in intensity with the samp le di lut ions, and not merely art i facts. A s a one step pur i f icat ion p r o c e s s w a s all that w a s u s e d (i.e. affinity b ind ing) , there w a s a lways contaminat ing protein from culture medium containing 10% F C S . These ge ls were also used to check the levels of the contaminat ing protein. Banding patterns which were watched for were those indicating large amounts of contaminating F C S or indicating that ant ibody was leaching off of the affinity co lumn. The sample shown in figure 2 (lane 1) shows a typical banding pattern for T s F . Lane 2 shows what D M E M / 1 0 % F C S control material from a B 1 6 G column looks like. 2. ELJSA Analysis, E L I S A s were run on each new M A b and T s F sample to test their in vitro binding ability, a. T s F analysis. T s F was bound to plates in concentrations from 20 ng/ml down to 0 ng/ml . B 1 6 G M A b was then added at 500 ng/ml or 100 ng/ml, and the plate deve loped. Resul ts were used to ensure that the T s F was capable of being bound by B 1 6 G , and to make sure that this binding titrated out (fig. 3) . b. Ant ibody analys is . Limiting dilutions of B 1 6 G or E A 2 were added to a f ixed amount of T s F (2 ng /we l l ) . E L I S A ' s were used to look for contaminating material, which wouldn't bind efficiently to B 1 6 G or E A 2 . Resul ts were a lso used to compare the ability of T s F eluted from a B 1 6 G column to that eluted from a E A 2 column to bind to B 1 6 G or E A 2 (fig. 4). It was found that material eluted from an E A 2 column bound more efficiently to both E A 2 and B 1 6 G . D. In Vivo A s s a y s . 26 Lane 1 Lane 2 Figure 2. S D S - P A G E analys is of A10F and DMEM/10% F C S . Lane 1 shows a typical banding pattern for A10F, and lane 2 shows a typical banding pattern for DMEM/10% F C S . Figure 3 . E L I S A results. A10 plated in limiting dilutions and bound with either 500 ug/ml or 100 ug/ml B 1 6 G . Plate was read after 1 hour. 28 0.600 -0.000 -| 1 1 1 1 • 1 1 1 • r 1000 500 250 125 62 31 15 7.5 3 0 M-g/ml B16G or EA2 Figure 4. E L I S A results. Compar ison of binding abil it ies of A10 T s F from B 1 6 G (B16GF) and E A 2 (EA2F) co lumns to E A 2 and B 1 6 G ant ibodies. P la tes were coated with 2 u.g/ml A 1 0 F . B 1 6 G or E A 2 was added in limiting dilutions. Plate was read after 30 minutes. 29 1. Pre- iniect ion of T Suppressor Molecu le . a . Dose Effects. A ser ies of exper iments were performed to answer the quest ion of dose dependence when T s F is pre-injected. A s ment ioned, it had been seen that 20 u.g A10 T s F injected into a mouse i.v. 10 days prior to a subcutaneous tumor injection led to an increased resistance to the tumor relative to a P B S control. D M E M / 1 0 % F C S has been run over a B 1 6 G affinity co lumn, and the eluted mater ial used as a control in similar exper iments (76). Th is control was found to have no effect on tumor growth different from that of P B S (unpubl ished data). For these exper iments, it was dec ided to use P B S rather than try to generate the large amounts of control protein that would be needed. Unfortunately, the large amounts of T s F needed for each of these exper iments prevented the use of the same batch of T s F for all of them. Incubator space limited the amount of supernatant that could be grown at one t ime. Ba tches were tested for typical banding patterns on gels and tested for binding capability by EL ISA . This was done to ensure that the same protein was being used from experiment to experiment. Exper iment #1 w a s a repeat of an an experiment done by Dr. Kev in Stee le in this laboratory us ing 20 u.g of A 1 0 T s F . A 1 0 T s F was administered 10 days prior to P 8 1 5 , and subsequen t tumors measu red dai ly. M ice receiv ing the T s F had signif icantly s lower tumor growth (fig. 5). This was the same effect that Steele had seen . M ice in exper iment #2 rece ived the s a m e treatment. In ana lys ing the data from exper iment #2 and subsequent exper iments, it was dec ided to use survival t ime rather than tumor growth as the pr imary cri teria for protect ion. It is felt that this is a more stringent measure , as the rate of tumor growth isn't a lways directly related to the mouse 's eventual state 30 150 100 -CM E E 0) N CO L. o E r- 50 --B- P815/PBS H>- P815/A10F 3 0 Figure 5 . Exper iment #1. Mice were injected with 20 ug A 1 0 F ten days prior to receiving P815 tumor subcutaneously. Tumor growth was measured with cal ipers daily, and the average tumor s ize calculated for each group of 8 mice. Data shown are tumor s ize versus days post tumor injection. Those data points marked with an aster ix indicate when the tumor a rea dif fered signi f icant ly from that of the control group (p<0.05 to p<0.02). 31 of health. In experiment #2, the 20 u.g dose of A10 again led to protective effects against P815 (fig. 6, table II.). Experiment #3 asked whether lower doses of A10 would produce the same effect. The following doses were tested; 10 u.g, 2 \ig, 0.4 ng, and 0.08 u.g. Survival times for these mice were not different from the control mice (table I). It was concluded from these data that the effects seen after pre-injecting A10 TsF were dose dependent, with high doses being neccessary to see the effects. In experiment #4, a higher dose of A10 TsF was tested to see if the protective effects could be amplified. Mice received 43.5 \ig, p BS or 0.06 ug as a low dose control. Those mice receiving 43.5 u.g A10 TsF had significantly longer survival times than the PBS controls (table II). The degree of significance of this difference is higher than that of data from mice receiving 20 u.g A10 TsF (p<0.005 versus p<0.05). The mice receiving the higher dose also showed 4 out of 8 mice cured, versus 1 out of 8 for those receiving 20 ng. The mice receiving a low dose of A10 TsF showed no difference in survival times relative to the PBS controls (table II). Thus 43.5 ug of A10 TsF produced a higher degree of protective effects than 20 u.g- The cut-off point for effective protection is between 10 and 20 ug-b. Specificity. The specificity of effects seen by pre-injecting A10 TsF was tested in two different directions. First, specificity relative to the P815 tumor was tested. Two other H-2d tumors were tested, L1210 and M-1. Experiment #5 tested 20 ug A10 TsF against both of these tumors as well as P815. Mice receiving any of the three tumors were significantly effected by this treatment. Mice treated with A10F before receiving either P815 or M-1 had significantly longer survival times than untreated mice (table III). Mice treated with A10F, then receiving 120 100 Figure 6. Exper iment #2. Mice were injected with 20 ug A 1 0 F ten days prior to receiving P815 tumor subcutaneously . Dates of death were noted, and average surv iva l t imes ca lcu la ted for each group of 8 m ice . Surv iva l t ime for mice rece iv ing A 1 0 F (27.2 +/- 2.1 days) di f fered signi f icant ly from that of mice receiving P B S (22.2 +/- 1.3 days) (p<0.05). There was one cured mouse in the group receiving A 1 0 F . 33 Treatment P B S 0.08 ug A 1 0 F 0.4 ug A 1 0 F 2.0 ug A 1 0 F 10.0 ug A 1 0 F Exper iment # 3 Surv iva l (davs) 25 .5 +/- 2.9 20.9 +/- 1.7 24.2 +/- 3.1 24.1 +/- 2.6 27.7 +/- 2.4 S ign i f i cance NS N S NS N S Table I. Experiment #3. Survival rates of mice receiving low doses of A 1 0 F . Mice were injected with low doses of A 1 0 F ten days prior to receiving P 8 1 5 tumor subcu taneous ly . Da tes of death were no ted , and ave rage surv iva l t imes ca lcu lated for each group of 8 mice. Signif icant di f ferences were ca lcu lated for each exper imental group relative to the P B S control group using the student 's t - t es t . 34 Treatment Exper imen t P B S #2 20 ug A 1 0 F Surv ival (davs) 22.2 +/- 1.3 27.2 +/- 2.1 S ign i f i cance p < 0.05 Cu res 0 1 P B S #4 23.9 +/- 2.5 0 0.06 ug A 1 0 F 25.4 +/- 2.8 N S 0 43.5 ug A 1 0 F 35.5 +/- 1.8 p < 0.005 4 Table II. Experiment #4. Survival rates of mice receiving var ious doses of A 1 0 F . Mice were injected with different doses of A 1 0 F ten days prior to receiving P815 tumor subcutaneously . Dates of death were noted, and the average survival time and rate was calcu lated for each group of 8 mice. Signif icant di f ferences were ca lcu lated for each experimental group relative to the P B S control groups using the student 's t-test. 35 Treatment Exper iment Survival (davs) S ign i f i cance C u r e s P B S / P 8 1 5 # 5 22 .2 +/- 1.3 0 20 u.g A 1 0 F / P 8 1 5 27 .2 +/- 2.1 p < 0.05 1 P B S / M - 1 33.1 +/- 0.9 0 20 ng A 1 0 F / M - 1 36.6 +/- 1.5 p < 0.05 0 Tab le III. Exper iment #5. Survival rates of mice receiving P B S or A 1 0 F prior to P 8 1 5 or M-1 . M ice were injected with 20 u.g A 1 0 F ten days prior to receiving P 8 1 5 or M-1 tumor subcutaneously . Dates of deaths were noted, and average survival t imes and rates were ca lcu lated for each group of 8 mice. Signi f icant d i f ferences were ca lcu la ted for each exper imenta l group relative to the P B S control groups using the student's t-test. 36 L 1 2 1 0 d ied l oo qu ick ly to show a d i f ference in surv iva l t imes , but their tumors were s igni f icant ly sma l le r than control mice (fig. 7). T h e M-1 tumors were a lso signi f icant ly smal le r in untreated mice than when mice were treated with A 1 0 F (fig. 8). In subsequent exper iments , the dose of L1210 adminis tered was reduced from 1 0 4 ce l ls to 1 0 3 ce l ls per mouse so that survival t imes would be longer. Experiment #6 studied the dose effects of A10 T s F in the L1210 system. Mice received 43.5 ug A 1 0 F , 0.06 ug A 1 0 F or P B S . A s expected, the high dose of A 1 0 F produced a protective ef fect , with su rv iva l t imes s igni f icant ly h igher and tumors sma l l e r ( table IV, f ig . 9). Surpr is ingly, the low dose also produced a significant difference in tumor s ize , although none in surv iva l t imes (fig. 10). Exper iment #7 conf i rmed the results seen in experiment #6. The d o s e s in this case were 40 ug A 1 0 F and 0.04 ug A 1 0 F . T h o s e mice receiv ing 40 ug A 1 0 F had signif icant di f ferences from the control mice in both survival t imes and tumor growth rates, as had those mice receiv ing 43 .5 ug in experiment #6 (table IV, f ig. 10). Those mice receiving 0.04 ug A 1 0 F s h o w e d s imi lar resul ts to those receiv ing 0.06 ug A 1 0 F in that their tumors were significantly smal ler, but their survival t imes were the same as those of the control mice. There were 4 out of 8 cures in mice receiving 40 ug A1 OF, and 3 out of 8 cures in mice receiving 0.04 ug A 1 0 F . It was concluded from these experiments that A10 T s F showed protective effects against L1210 and M-1 , as well as P 8 1 5 . The effective dose of A 1 0 F was far lower in the L1210 sys tem than the P815 system. It was also concluded that the effect of A 1 0 F in the L1210 system was not artifactual, as it did titre out. The second test for specificity looked at the question of whether or not another 37 500 0 -J 1 1 1 1 1 0 1 0 2 0 3 0 Day Figure 7. Exper iment #5. M ice were injected with 20 ug A 1 0 F ten days prior to receiv ing L1210 tumor subcu taneous ly . Tumor s i z e s were m e a s u r e d dai ly with cal ipers, and average tumor area was calculated for each group of 8 mice. Data points marked with an asterix indicate when tumor area differed signif icantly from that of the control group (p<0.05). 38 600 Figure 8. Exper iment #5. M ice were injected with 20 u g A 1 0 F ten days prior to receiving M-1 tumor subcutaneously. Tumor s ize was measured daily, and the average tumor a rea calcu lated for each group of 8 mice. Data points marked with an asterix indicate when tumor a rea dif fered signi f icant ly f rom that of the contro l g roup (p<0.05 to p<0.005). 39 Treatment Experiment Survival (davs) Significance Cures PBS/L1210 #6 16.2 +/- 0.5 0 0.06 ug A10F/L1210 17.7 +/- 0.7 NS 0 43.5 ug A10F/L1210 19.3 +/- 0.8 p < 0.01 0 PBS/L1210 # 7 23.0 +/- 1.8 0 0.04 ug A10F/L1210 31.6 +/- 1.5 NS 3 40 ug A10F/L1210 36.9 +/- 0.5 p < 0.025 4 0.04 ug Fd11F/L1210 25.4 +/-4.1 NS N/D 40 ug Fd11F/L1210 21.5 +/- 2.1 NS N/D Table IV. Experiments #6 and #7. Survival rates of mice receiving L1210 and A10F, Fd11F or PBS. Mice were injected with various doses of A10F or Fd11F ten days prior to receiving L1210 tumor subcutaneously. Dates of death were noted, and the average survival times and rates calculated for each group of 8 mice. Significant differences were calculated for each experimental group relative to the PBS control groups using the student's t-test. 40 Figure 9. Exper iment #6. Mice were injected with 0.06 u.g A 1 0 F (LA10F) , 43.5 n g A 1 0 F ( H A 1 0 F ) , or P B S ten d a y s pr ior to rece iv ing L 1 2 1 0 tumor subcutaneously . Tumors were measured daily, and average tumor area calculated for each group of 8 mice. Data points marked with an asterix indicate when tumor area differed significantly from that of the control group (p<0.05 to p<0.005). 41 200 10 12 14 16 18 20 22 Days Figure 10. Exper iment #7. Mice rece ived 0.04 ug A 1 0 F (LA10F) , 40 ug A 1 0 F (HA10F) or P B S ten days prior to receiving L1210 tumor subcutaneously . Tumor s ize was read daily, and average tumor area calculated for each group of 8 mice. Da ta po in ts ma rked with an aster ix ind icate w h e n tumor a r e a di f fered signif icantly from that of the control group (p<0.05). 42 suppressor factor could produce the same effects. The other T s F used was Fd11 T s F , which was ra ised in the ferredoxin sys tem. These two suppressor factors are known to be very similar, but thought not to be identical (89). L1210 was chosen as the tumor system due to it's sensitivity. In this experiment, mice received P B S , 0.04 ug Fd11F or 40 ug Fd11 T s F on day -10 and 1 0 3 L1210 ce l ls on day 0. Neither dose altered the survival t imes of these mice (table IV). This supported the belief that Fd11 T s F and A10 T s F are two different molecules. 2. Suppressor Deletion Therapy. The suppressor deleting process is being studied extensively as a potential method of treating a variety of d i seases , including severa l cancers (81). A monoclonal antibody speci f ic for the target cel ls of choice is l inked to a toxin, hematoporphorin in this c a s e . Hematoporphorin (Hp) is a l ight-sensit ive molecule which is lethal to the cel l it is assoc ia ted with upon being exposed to light. Immediatly after receiving the M A b / H p conjugate, mice were put in the dark for four hours to allow the M A b to find its target cel ls . After this per iod, mice were p laced under a f luorescent light for at least four hours to allow the Hp to become activated and kill the target ce l l s . M ice were then returned to their normal quarters. a. Abrogation of Non-Responder Status. Exper imen t #8 a s k e d whether or not de le t ion of s u p p r e s s o r ce l l s from the non-responding mouse 's repertoire would alter it's ability to produce ant ibodies to a particular ant igen. D B A / 2 J mice do not normally produce significant amounts of antibody to the antigen ferredoxin (82). In this experiment, mice were given B 1 6 G / H p one day before they received ferredoxin. An irrelevent antibody, C A M A L - 1 , was conjugated to Hp and used as a control along with a P B S control. The amount of Hp was kept consistant in all injections, despite the fact that B 1 6 G and C A M A L - 1 had bound to Hp in different amounts. This was because Hp is a toxin by itself, and cou ld effect the survival rates of the mice if not contro l led. M ice rece ived more C A M A L - 1 than B 1 6 G . Mice were bled after the initial injections. Three weeks later mice were boos ted with fer redoxin, then b led aga in . Leve ls of serum ant ibodies to ferredoxin were compared between the two b leeds. The results showed that the B 1 6 G / H p treatment had produced mice capab le of producing significantly more antibodies to ferredoxin than those receiving P B S or C A M A L - 1 / H p (table V) . Therefore, in this sys tem, it can be sa id that suppressor cel ls are involved in the non-responder status, as deleting them leads to abrogation of this status, b. Tumor Protect ion. S u p p r e s s o r delet ion is becoming a potential ly powerful tool in the f ield of tumor therapy (81). In experiment #9, suppressor deletion was used in the L1210 tumor sys tem. The M A b used was E A 2 . E A 2 , like B 1 6 G , is a M A b raised in this lab which recognizes a common port ion of supp resso r mo lecu les (unpubl ished data). A n irrelevent M A b , 16A , w a s used in addition to a P B S control. Hp alone was also used as a control due to its toxicity. MAb 's were not conjugated to Hp as had been done in previous experiments. The conjugation process had been found to decrease the binding ability of MAb 's (unpublished data). The MAb 's and Hp were mixed together and incubated overnight at room temperature. This method also a l lowed for controlling quantities of both Hp and the M A b as opposed to determining the amount of Hp to be injected, then injecting whatever amount of M A b had bound to the Hp along with the Hp. M i c e rece ived 5 X 1 0 3 L1210 ce l ls subcu taneous ly , which is enough to kill an untreated mouse in 14 to 28 days. Five days later, they received E A 2 / H p or one of the controls 44 Q D Reading Treatment Devt. T ime Pre-b leed Pos t -b leed Signi f icance P B S 2 Hours 0.289+/-0.029 0.311+/-0.033 NS B 1 6 G / H p 0.291+/-0.040 0 . 3 6 0 + / - 0 . 0 2 6 p < 0.01 C A M A L / H p 0.329+/-0.046 0.362+/-0.053 NS P B S 24 Hours 0.722+/-0.160 0.826+/-0.184 NS B 1 6 G / H p 0.700+/-0.241 1.049+/-0.066 p < 0.01 C A M A L / H p 0.953+/-0.226 1.110+/-0.250 NS Table V. Experiment #8. Antibody levels in mice treated with B 1 6 G / H p , C A M A L - 1 / H p or P B S . M i ce were injected with B 1 6 G / H p , C A M A L / H p or P B S one day before receiving ferredoxin. Three weeks later mice were b led, then boosted with ferredoxin aga in . A week later mice were b led aga in , and their relat ive se rum levels of anti-ferredoxin ant ibodies pre- and post-ferredoxin boost measured by E L I S A . S e r a were di luted 1/100 for E L I S A ana lys is . Signi f icant d i f ferences were ca lcu la ted for each set of exper imental data relative to the P B S control data using the student 's t - t es t . 45 i.v.. Mice which had their suppressor cells deleted with EA2/Hp all survived long-term, and in fact had rejected their tumors completely within 25 days (fig. 11). Control groups had an average survival rate of 33%. These survivors represent a phenomenon referred to as spontaneous cures, which are often seen in in vivo assays. Tumor growth rates were significantly lower than those of mice receiving 16A/Hp or Hp alone (fig. 12). In this system, suppressor deletion proved to be a very effective tool against L1210 tumors. E. In Vitro Antibody Assay. In experiments #10 and #11, mice were injected with 1 ug Fd11F or 1 u.g A10F ten days prior to starting an immunization regime of ferredoxin and KLH (as an internal control). Serum titre of anti-ferredoxin and anti- KLH antibodies were measured at the end of the immunization schedule. Those mice which had received Fd11F had higher titres of anti-ferredoxin antibodies than those receiving A10F in each of two experiments (table VI). Anti-KLH antibody levels did not differ between the two groups in either experiment. 46 Figure 11 . Exper iment #9. Surv iva l curves of mice receiving E A 2 / H p , 16A /Hp , Hp or P B S . Mice were injected subcutaneously with 5 x 1 0 3 L1210 cel ls. Five days later they rece ived E A 2 / H p supp resso r delet ion therapy, or 1 6 A / H p , Hp or P B S as controls. Dates of deaths were noted, and average survival t imes and rates calculated for each group of 6 mice. 47 200 Figure 12. Exper iment #9. Tumor growth in mice receiving E A 2 / H p , 16A/Hp or Hp a lone. M ice were injected subcutaneous ly with L1210 five days prior to receiving E A 2 / H p , 16A/Hp or Hp alone. Tumors were measured dai ly, and average tumor s ize calculated for each group of 6 mice. Data points marked with an asterix indicate where tumor a rea differed significantly from that of the control group (p<0.02 to p<0.005). 48 Treatment Exper iment 1 u g F d 1 1 F #10 1 ug A 1 0 F O D (405) rx-Ferredoxin ra-KLH 0.988+/-0.071 0 . 3 2 6 + / - 0 . 0 4 2 0.741+/-0.065 0 . 2 8 7 + / - 0 . 0 1 6 1 ug Fd11F 1 ug A10F #11 0.907+/-0.037 0.853+/-0.052 0 . 3 0 7 + / - 0 . 0 1 3 0 . 3 1 9 + / - 0 . 0 3 2 Tab le VI. Exper iments #10 and #11. E L I S A ' s were performed on se ra of mice treated with F d 1 1 F or A 1 0 F prior to being immunized with ferredoxin and K L H . P la tes were coated with either ferredoxin or K L H . S e r a were di luted 1/2000 for testing against ferredoxin, and 1/500 for testing against K L H . P la tes were read after 30 minutes. 49 IV. D I S C U S S I O N The T suppressor factor, A 1 0 F , has been used extensively in this laboratory for the past four years . It is bound by the monoclonal antibody B 1 6 G , which binds non-idiotypically to suppressor molecu les (76). A 1 0 F has been shown to induce measurab le suppress ion both in vivo and in vitro (74,78). However , as this factor is p roduced by a hybr idoma and the purif ication p rocess is not stringent, there w a s cons iderab le batch to batch variabil i ty. This variabil ity made duplication of prior results very difficult. A regime was deve loped to minimize the effects of the batch variability. This regime was also fol lowed when producing F d 1 1 F . E a c h batch was treated in the fol lowing manner : Three to four l itres of spent supernatant were produced at a time. This resulted in batches of factor which were large enough to permit at least one complete experiment, and often more, being carr ied out using material from the same batch. The supernatant was then passed over a B 1 6 G coated affinity column at 4 ° C . Supernatant w a s only p a s s e d over the co lumn once , and at a relatively s low rate of 50 ml/hour. The co lumn was then washed with cold P B S until the fall through contained no more than trace amounts of protein as measured by absorbance. Often, this meant washing for up to 24 hours. The material bound to the column was eluted using one column-volume of 0.1 M HCI, and col lected in 1 ml al iquots. This material was tested for protein content using spectrophotometric absorbance readings. Those fractions which contained protein were d ia lysed overnight in P B S . 50 Protein quantif ication of this material was then carr ied out using the method of Lowry. Using this method for producing and quantifying A 1 0 F led to quite consistant y ields of 100 to 150 ug T s F / l of supernatant. Once a known quantity of putative T s F had been obta ined, the banding pattern on polyacry lamide ge ls and the capabil i ty to bind to B 1 6 G in an E L I S A were used to est imate the quality of the samp le ; that is, the portion of the protein in the sample which was T s F rather than non-speci f ic protein. With the hybr idomas having been grown in 1 0 % F C S , there was a lways some level of contaminating protein. Those samples which contained a typical amount of total protein but either showed a non-typical ge l banding pattern or didn't bind efficiently to B 1 6 G in E L I S A were not used for exper iments. Thus , all batches of T s F used in these experiments were chosen for use because of their similarity to each other. It has been well establ ished that when A 1 0 F is administered to a mouse concurrent with P815 tumor ce l ls , the immune system of the mouse is suppressed , resulting in enhanced tumor growth and reduced survival time (74). This effect was achieved when the quantity of A 1 0 F used was between 10 and 20 ug/mouse. The suppress ive effect of A 1 0 F was antigen specif ic in that the tumor growth of L1210 tumors was not affected by A 1 0 F administration (74). The effects of speci f ic immune suppress ion on the rate of tumor growth has been further conf i rmed in this study and others by the use of suppressor delet ion therapy (81). In these exper iments, mice were injected with B 1 6 G M A b conjugated to hematoporphyrin (Hp), a l ight-sensitive molecule. The mice were kept in the dark for four hours to allow the M A b to find its target cel ls (suppressor cel ls in this case) . Mice were then kept under a strong light for four hour to activate the Hp, which then kil led any cel l with which it w a s assoc ia ted . It has been shown that delet ion of suppressor cel ls and factors in vivo by means of photodynamic therapy 51 leads to enhanced immunity. Mice which were treated in this manner prior to receiving tumor cells had smaller tumors and longer survival times than did untreated mice. The L1210 tumor system was used for this experiment. Mice which received the deletion therapy all survived long-term, as opposed to a spontaneous cure rate of 33% in the control animals. Those tumors which did grow in the experimental animals were significantly smaller than those growing in the control animals. The effects of suppressor deletion has also been examined in the ferredoxin system. The DBA/2 mice used in this experiment are non-responders to ferredoxin, and normally do not produce antibodies to ferredoxin (82). Results show that non-responder mice receiving suppressor deletion therapy become capable of producing antibodies to ferredoxin. Together, these experiments demonstrate that suppressor cells and factors are powerful down-regulators in the systems studied here. Suppression decreases immunity to tumors, and is involved in the non-responder status to ferredoxin. Boosting levels of TsF 1 in a mouse leads to increased suppression of immunity. Deletion of suppressor cells and factors in vivo removes these effects, resulting in enhanced immunity and abrogation of the non-responder status. This study also describes a phenomenon which is quite different than that seen in the experiments described above. Preadministration of A10F by ten days leads to decreased tumor growth and increased survival times (79). This presumptive enhancement of the immune response is not seen when the dose of A10F used is below 10 ug/mouse. The specificity of this immune enhancement was examined. First, the antigen specificity of the effect of preinjected A10F was examined. It was found that this phenomenon is non-specific within the H-2d system. Tumor growth was significantly reduced in both the L1210 and M-1 tumor systems, as well as 52 in the P815 system when A 1 0 F was preinjected. S e c o n d , the effect of a T s F other than A 1 0 F was examined . It was found that the immune enhancement effect is A 1 0 F speci f ic in this respect. F d 1 1 F , a suppressor factor raised in a non-related antigen sys tem, did not increase resistance to L1210 when pre-administered. L1210 was chosen for this experiment due to its sensitivity to pretreatment by A 1 0 F . Converse ly , A 1 0 F does not cause the same effects as Fd11F in the ferredoxin sys tem. Administration of Fd11F to a non-responder mouse leads to abrogation of the n o n - r e s p o n d e r s ta tus and resu l ts in ant ibody product ion fo l lowing immun iza t ion with ferredoxin. M ice which had received A 1 0 F produced a lower antibody titre than those receiving F d 1 1 F . Th is result has been conf i rmed by Randa l l C h u in this laboratory (unpubl ished obse rva t i on ) . The apparent ability of a population of cel ls to act in either a suppress ive or enhancing manner has been reported previously (14,90). The activity of those ce l ls was inf luenced by such factors as presence of IL-1 and antigen, and B cell activation. Green et. a l . have reported that the dual s ignals of both antigen and IL-1 are neccessary for T h C activation (14). Without these two s ignals suppress ion is s e e n . Kotani et. a l . have reported that recognition of a M H C product on the surface of unactivated B cel ls leads to suppress ion , and that recognit ion of the M H C product on activated B cel ls leads to help (90). In the system descr ibed here, the difference between suppress ion and enhancement of immunity appears to be l inked to the presence or absence of antigen. A model for these results is suggested by examination of the idiotypic network involved, (fig. 13) Administrat ion of tumor cel ls to a mouse act ivates a suppress ive response . Those suppressor cel ls which respond are idiotypic and recognize an antigenic determinant on the 53 Figure 13. Mode l for the cel lular interactions involved in the suppressor circuit. W h e n an antigen such a tumor cel l enters the mouse, idiotypic Ts1 cel ls proliferate in response. Idiotypic T h C and B cel ls also proliferate. Al l of these ce l ls recognize some epitope on the tumor ce l l , although not necessar i ly the same one . T h e s e cel ls are recognized in turn by anti-idiotypic T s 2 ce l ls , which then proliferate in response . The Ts2 ce l ls can then interact with any populat ion of ce l ls bearing the complementary idiotype on their receptors. 54 tumor ce l l . The tumor cel ls a lso activate T h C and B cel ls which are idiotypic and also recognize an ant igenic determinant on the tumor ce l l . Th is antigenic determinant could be the same or different from that recogn ized by the suppressor ce l l s . The expand ing i d + T s 1 populat ion stimulates expansion of a population of a id T s 2 cel ls. These cel ls are then capable of recognizing and down-regulat ing the act ivated i d + T h C and B cel ls . When a large amount of i d + T s F 1 is injected without ant igen, there is no concurrent activation of i d + T h C and B cel ls . The i d + TsF ' s st imulate a id T s 2 as usual . The expanded a id T s 2 cel ls and factor do not, however, have their normal target cel ls to act upon. The M H C determinants on the surface of unstimulated B cel ls might not be recognized by these T cel ls. Lack of antigen might also deprive T h C ' s of a necessary second signal for activation. By the time antigen is introduced ten days later, the activated a i d T s 2 ' s have back stimulated i d + T s 1 's in the absence of appropriate target ce l ls . The excess ive amounts of i d + Tsi's and a id T s 2 ' s present concurrently para lyze this network of interactions, render ing the mouse incapable of suppress ing the immunologica l response to the ant igen. R e s p o n s e s to L1210 and M-1 tumor ce l ls are a lso affected by this para lys is due to their s imi lar i ty to P 8 1 5 tumor ce l l s . The idiotypic network cou ld lose s o m e level of its fine specif ic i ty as it expands , which would lead to less speci f ic responses to ant igen. It is a lso poss ib le that a similar epitope on each of these syngeneic tumor cel l l ines is recognized by T s 1 cel ls . If so , then the same population of i d + Ts cel ls would be activated by all three tumors. Thus, paralys is of the suppressor circuit induced by T S F H , which has been raised in response to P815 , 55 would render mice incapable of mounting a suppress ive response to L1210 and M-1 as well as P 8 1 5 . The F d 1 1 F , having been raised to a thoroughly different ant igen, is too dissimi lar from the A 1 0 F to affect the response to these tumor ce l ls . The i d + T s ^ a i d T s 2 circuit responsive to ferredoxin should be affected, but not that responsive to P 8 1 5 , L1210 and M-1 . In conc lus ion , these experiments and others predict a situation in which a given cell 's activity is strongly inf luenced by idiotypic networks and by the environment in which the cell is act ivated. Further studies are certainly necessary to confirm the model presented here or any other mode l . S u c h exper iments could examine the F d 1 1 F response more c lose ly to see if it mirrors that of the A 1 0 F response. The M H C specificity of this system has yet to be addressed. A detai led examinat ion of the cel l populat ions present in the sp leens of mice injected with large quantit ies of T s F could be undertaken using cel l surface labell ing. Binding capabi l i t ies between activated versus unactivated ThC ' s and B cel ls and T s 2 ' s could be examined. These experiments shou ld shed more light on the cel lular interactions occurr ing in an imals when their immune sys tems are manipulated, either by antigen or by idiotypic elements. 56 V. R E F E R E N C E S 1. K le in , J . 1982. Immunology: The sc ience of se l f -nonsel f d iscr iminat ion. John Wi ley & Sons , Inc. Toronto. 2. Ge rshon , R. and K. Kondo. 1970. Cel l interactions in the induction of tolerance: The role of thymic lymphocytes. Immunology 18:723. 3. Gershon , R. and K. Kondo. 1971. Infectious immunological tolerance. Immunology 21: 903. 4. Cantor , H. and E. B o y s e . 1977. Regulat ion of cel lular and humoral immunity by T-cel l subc lasses . Cold Spring Harbor Symp. Quant. 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