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Characterization of idiotype interactions during the immune response to ferredoxin. idiotype and epitope… Weaver, Michael Stanley 1982

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CHARACTERIZATION OF IDIOTYPE INTERACTIONS DURING THE IMMUNE RESPONSE TO FERREDOXIN. IDIOTYPE AND EPITOPE SPECIFIC INTERACTIONS DETERMINE THE OUTCOME OF CHALLENGE WITH ANTIGEN by Michael Stanley Weaver M.Sc, The University of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA (C) September 1982 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 Microbiol a The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 10 A/CA^VWJK^ DE-6 (3/81) i ABSTRACT Anti-idiotype antisera were raised i n rabbits to two monoclonal a n t i -bodies, Fd-1 and Fd - 2 , with s p e c i f i c i t y for each of the two antigenic epitopes found on the ferredoxin (Fd) molecule. The anti-idiotype antisera (anti-Fd - 1 and anti-Fd - 2 ) were used to demonstrate that one of the idiotypes (Fd -1) was expressed at s i g n i f i c a n t levels i n most anti-Fd antisera raised i n BIO.BR mice while the second idiotype (Fd -2) was infrequently expressed. Examination of anti-Fd sera raised i n other mouse strains demonstrated that expression of the Fd -1 idiotype mapped to the IgH gene complex and was found i n the antisera of a l l mouse strains examined with the I g - 1 ^ allotype. When splenocytes from Fd-immune B10.BR mice were treated with anti-Fd - 1 and transferred to irradiated syngeneic recipients, the adoptive secondary response was s i g n i f i c a n t l y higher i n animals receiving treated c e l l s as opposed to control animals which re-ceived normal rabbit serum treated c e l l s . This response produced a net increase i n antibody to both epitopes and the r e l a t i v e amount of Fd-1 idiotope was not s i g n i f i c a n t l y altered. Further studies with separated c e l l populations showed that the overall increase of anti-Fd antibody produced was attributable to the effects of the a n t i - i d i o t y p i c serum on a population(s) of T c e l l s . Treatment of mice with the Fd-1 monoclonal antibody (which should react with a n t i - i d i o t y p i c c e l l s ) had an analogous effect to that of the anti-idiotype. Treated mice produced heightened levels of antibodies directed to both epitopes of Fd. Treatment of mice with second anti-idiotype, anti-Fd - 2 , was found to enhance the anti-Fd response of B10.BR mice and abrogate the non-responder status of DBA/2 mice. Additional evidence indicates that the Fd -2 idiotype could be expressed on a suppressor c e l l population which may be a predominant regulatory element i n both BIO.BR and DBA/2 mice. i i TABLE OF CONTENTS Page No. Abstract i Table of Contents 11 L i s t of Figures . . . .' i v L i s t of Tables v Acknowledgement v i Chapter I. Introduction and l i t e r a t u r e review 1 (a) H i s t o r i c a l outline . 1 (b) Definitions of idiotype 2 (c) Structural correlates of idiotype markers 3 (d) Experimental demonstrations of id i o t y p i c interactions . . . . 8 (e) Need for a simple experimental model 10 Chapter I I . Detection of sp e c i f i c idiotype i n the serum of Fd-immune BIO.BR mice 14 (a) Introduction 14 (b) Materials and Methods 15 ( i ) Animals 15 ( i i ) Antigens and immunization 15 ( i i i ) Assay for s p e c i f i c antibody 16 (iv) C e l l fusion 1 8 (v) Production of anti-idiotype serum . ^ (v i ) A f f i n i t y i s o l a t i o n of anti-idiotype antibody and l OA preparation of an a n t i - i d i o t y p i c immunoadsorbent z , u ( v i i ) Assays for a n t i - i d i o t y p i c antibody z u ( v i i i ) Determination of combining s i t e s p e c i f i c i t y of a n t i -idiotype serum 21 ( i x ) P u r i f i c a t i o n of idiotype bearing antibodies from the serum of immune mice 22 i i i (c) Results 22 ( i ) Binding s p e c i f i c i t y of monoclonal anti-Fd antibodies . . . 26 ( i i ) Raising and immunoadsorption of antisera to monoclonal anti-Fd antibodies 26 ( i i i ) Characterization of anti-idiotype antiserum to Fd-1 . . . 29 (iv) Characterization of anti-idiotype antiserum to Fd-2 . . . 35 (d) Discussion 35 Chapter I I I . C e l l u l a r expression of Fd-1 idiotype 41 (a) Introduction 41 (b) Materials and Methods 44 ( i ) Radioimmune assay for idiotype 44 ( i i ) In vivo administration of anti-idiotype 45 ( i i i ) Adoptive transfer of anti-idiotype treated spleen c e l l s 45 ( i v ) -^Cr release cy t o t o x i c i t y assay 46 (v) Preliminary characterization of idiotype expression . . . 47 ( v i ) Description and standardization of RIA 48 ( v i i ) Results of serum survey for idiotype expression 52 ( v i i i ) In vivo effects of anti-idiotype 52 ( i x ) Studies using antibody feedback 61 (c) Discussion . 66 ( i ) Effects of anti-idiotype on responding c e l l s 68 Chapter IV. Cellular Expression of Fd-2 idiotype . 75 (a) Materials and Methods 75 (b) Results 76 (c) Discussion 83 (d) Summary Discussion 86 Bibliography 88 i v LIST OF FIGURES Page No. Figure 1. Amino acid sequence of ferredoxin 12 2. T i t r a t i o n of anti-Fd antibodies by ELISA 25 3. Blocking ELISA using Fd-1 monoclonal antibody 27 4. Binding of Fd-2 monoclonal antibody to the N- or C-fragment or to whole Fd 28 5. Competitive i n h i b i t i o n of monoclonal antibody Fd-1 by antigen and anti-idiotype 31 6. Lack of i n h i b i t i o n of serum and monoclonal antibodies to KLH by anti-idiotype serum anti-Fd-1 32 7. Competitive i n h i b i t i o n of anti-Fd-1 idiotype by Fd . . . . 33 8. S p e c i f i c i t y of anti-idiotype selected serum antibody . . . 34 9. Inh i b i t i o n of Fd-2 binding to Fd i n the presence of anti-Fd-2 36 10. S p e c i f i c i t y of anti-Fd-2 serum for Fd-2 antibody 37 11. S p e c i f i c i t y of anti-Fd-2 serum for Fd-2 antibody 38 12. Competitive i n h i b i t i o n of anti-Fd-2 by antigen 39 13. In h i b i t i o n of N-determinant directed antibodies by anti-idiotype to Fd-1 49 14. Radioimmunoassay for c i r c u l a t i n g idiotype i n mouse serum 50 15. Treatment of T- or B-enriched c e l l subpopulations with anti-Fd-1 plus C» 59 16. Model for i d i o t y p i c interaction i n the immune response to Fd 74 17. Radioimmunoassay for c i r c u l a t i n g idiotype i n mouse serum . 77 V LIST OF TABLES Page No. Table 1. Characteristic antibody t i t r e s i n BIO.BR mice 23 2. Analysis of anti-Fd sera taken from mice of various haplotypes and allotypes after secondary immunization with Fd 53 3. Administration of anti-Fd-1 antibody i n vivo 55 4. Complement dependent cyto t o x i c i t y of anti-Fd-1 serum on selected c e l l u l a r targets 56 5. Analysis of anti-Fd sera from an adoptive secondary response i n BIO.BR mice 57 6. The influence of anti-Fd-1 on the adoptive primary or secondary response of BIO.BR mice to Fd . 60 7. Analysis of anti-Fd sera i n an adoptive primary response i n BIO.BR mice 62 8. The influence of anti-Fd-1 on the adoptive secondary response of BIO.BR mice to Fd 63 9. The influence of the monoclonal Fd-1 on the immune response to Fd i n BIO.BR mice 65 10. Expression of the Fd-2 idiotype i n BIO.BR mice 78 11. Influence of anti-Fd-2 on the primary anti-Fd response of BIO.BR and DBA/2 mice 79 12. Lack of an anti-Fd response i n nonresponder mice co-immunized with mitogen 81 13. I n a b i l i t y of the enhanced response of anti-Fd-2 treated mice to persist i n adoptive transfer 82 14. Influence of Fd-2 antibody on the primary anti-Fd response of BIO.BR mice 84 v i ACKNOWLEDGEMENT With the completion of a task which at one time appeared to be composed of endless p o s s i b i l i t i e s , I would l i k e , i n reviewing this important phase of my training, to acknowledge the contributions of many individuals. Barbara K e l l y f i r s t provided moral encouragement, technical advice and eventually, friendship to an at times uncertain student. Doug Kilburn and Geof Hoffman provided valued guidance as members of my advisory committee. My fellow student Lydia Sikora shared with me the achievements and p i t f a l l s of a re-warding research project. My family lent their support and expressed con-tinued f a i t h i n my a c t i v i t i e s . J u l i a Levy, my research supervisor, acted as my advisor and counsel through a period of great personal growth. Her lab-oratory was a challenging and rewarding environment i n which to work and myself and other students benefitted greatly by exposure to her o r i g i n a l and perceptive thinking. Lastly, I must acknowledge the assistance and friendship offered by the other members of Dr. Levy's laboratory. A l l of the individuals mentioned above contributed to an experience which proved to be enjoyable and worthwhile. It i s i n memory of these people and of interesting times that I extend my sincere thanks. 1 CHAPTER 1: INTRODUCTION While the physical universe of Immunology occupies a r e l a t i v e l y f i n i t e space, the theoretical boundaries which have been crossed i n an attempt to un-derstand the workings of the immune system are quite remarkable. Many of i t s fron t i e r s have been shared with a l l i e d studies i n genetics and molecular b i o l -ogy. The growth of Immunology represents developments on many different fronts so much so that even research, for instance on the expression of immunoglobulin genes, might be viewed as much a biochemical problem as an immunological one. In the present era, the search for new models inevitably seems to draw the attention of theorists to the problems of Immunology. Much speculation has focused on mechanisms which are involved i n the generation of di v e r s i t y and regulation of immune responses. To date, no contemporary theory has gone as far towards unifying these two apparently incompatible issues as has the con-cept of network theory which was f i r s t proposed i n an address to the Pasteur I n s t i t u t e by Niels Jerne i n 1974 (1). H i s t o r i c a l outline From the i n i t i a l observations of Landsteiner, interactions between a n t i -body and antigen molecules have been defined according to the pr i n c i p l e of com-plementarity i n which an immunoglobulin molecule or ligand combined with a par-t i c u l a r epitope by vir t u e of i t s immunological s p e c i f i c i t y (2). I t has become apparent that this degree of s p e c i f i c i t y necessitates the existence of an ex-tensive repertoire of antibody s p e c i f i c i t i e s . This o r i g i n a l concept was reaf-firmed i n 1957 when Burnet published the Clonal Selection theory which placed d i v e r s i t y i n the context of population genetics by defining a clonal d i s t r i b u -tion of antibody s p e c i f i c i t i e s amongst unique populations of antigen reactive c e l l s (3). Burnet's theory therefore defined c e l l interaction models on the 2 basis of the a b i l i t y of c e l l s to bind the same antigen, or to associate by virt u e of shared s p e c i f i c i t y . A l l forms of immunological interaction were nec-es s a r i l y antigen driven events controlled by contact with the external environ-ment. I t became clear, however, that assumptions made concerning the role of self-tolerance i n this model could not be made i n view of a number of experi-mental findings which demonstrated the existence of autoantibodies which were directed towards host components, including reports that the combining s i t e s of antibody molecules could be immunogenic i n the host immune system (4,5). In 1974 Niels Jerne proposed a theory whereby c e l l s could communicate through the sharing of variable region receptors by l i f t i n g the r e s t r i c t i o n s on s e l f tolerance and permitting c e l l u l a r interactions to occur through the production of complementary receptors (6). This concept can eas i l y be viewed as a r e f l e c t i o n of clonal selection theory back on i t s e l f , transcending the isolated immune system of Burnet into an elaborate network displaying the novel a b i l i t y to communicate with i t s e l f v i a the capacity for s e l f recognition and to interact with i t s environment through contact with external antigen. Quoting from Jerne's simple description: "The immune system i s a network of antibody molecules and lymphocytes that recognize and are recognized .by other antibody molecules and lymphocytes." (6). Definitions of idiotype Interactions within this form of network are based upon a concept of idiotype which has undergone several changes i n d e f i n i t i o n since i t s inception. According to Oudin, an idiotype was defined as the set of unique determinants presented by antibodies induced by a given antigen (7). This d e f i n i t i o n takes into account the existence of ind i v i d u a l antigenic s p e c i f i c i t i e s of immunoglob-u l i n molecules which appeared to be associated with the antigen binding a c t i v -i t y of the antibody molecule. The i d i o t y p i c property of an antibody was 3 therefore related to i t s antigen s p e c i f i c i t y and thereby became correlated with the phenomenon of antibody d i v e r s i t y . In subsequent years, i t was made evident that antibody molecules were more highly d i v e r s i f i e d than the degree envisaged by Oudin, whose d e f i n i t i o n was l a t e r shown to include a sizeable de-gree of molecular heterogeneity. More recent studies u t i l i z i n g monoclonal antibodies of either myeloma or hybridoma o r i g i n , have resulted i n a further refinement of the term. The idiotype of a given monoclonal antibody molecule i s , i n a c t u a l i t y , a composite property composed of a number of ind i v i d u a l a n t i -genic determinants or epitopes. Jerne used a more s p e c i f i c term -idiotope, which was taken to represent an in d i v i d u a l i d i o t y p i c epitope.. An important d i s t i n c t i o n i s therefore made i n Jerne's theory between the notion of idiotope and combining s i t e per se. The only c r i t e r i o n for this d e f i n i t i o n i s that they be confined to the variable portion of the molecule. Since each of these speci-f i c i t i e s are operationally defined by the existence of a complementary a n t i -idiotype antibody, i d i o t y p i c relatedness between antibody molecules can vary i n terms of the number of shared s p e c i f i c i t i e s and hence, number of a n t i - i d i o -topes with which they interact. I t i s also useful to note that the quantum unit i n a l l network interactions i s an idiotope-anti-idiotope pair. In other words, an idiotope i s i n d i r e c t l y defined by the s p e c i f i c i t y of the antibody which rec-ognizes i t . Because i d i o t y p i c complementarity i s the central p r i n c i p l e of net-work theory i t i s t h i s concept which i s alluded to i n a l l subsequent uses of the term idiotype throughout this thesis. Structural correlates of idiotype markers Efforts to define the nature of i d i o t y p i c determinants more precisely have r e l i e d on two avenues of investigation: serological and biochemical analysis of antibody molecules of defined idiotypy. A number of serological approaches have been used i n attempting to define idiotype expression i n vivo. As these 4 have often involved r a i s i n g of anti-idiotype under d i f f e r i n g conditions, i t i s important to consider several q u a l i f i c a t i o n s which are i m p l i c i t i n Jerne's theory yet have sometimes been overlooked. In the following paragraphs several experimental d e f i n i t i o n s of idiotypy are described which reveal that the terms of each are not necessarily equivalent and would hold different consequences within a Jernian network. Jerne states that regulation i n the network occurs through a process of self-recognition. As mentioned previously, exceptions to the rule that s e l f -reactive lymphocytes do not exist have been described i n the l i t e r a t u r e . For example, i n a number of diseases which appear to have an autoimmune basis a n t i -bodies are present which bind to endogenous IgG and DNA (8,9). Experimentally, i t has been found that host idiotypes are not p a r t i c u l a r l y immunogenic i n the sense that large quantities of anti-idiotype cannot readily be obtained. Only through prolonged immunization usually with the i d i o t y p i c antibody conjugated to a foreign c a r r i e r protein has i t been possible to drive the network i n the d i r e c t i o n of producing s i g n i f i c a n t amounts of anti-idiotype (10). In contrast, allogeneic idiotypes do appear to be readily immunogenic, that i s , antibodies which block combining s i t e s of allogeneic antibody can be e a s i l y produced. Functionally, these a l i o - or xenoantisera act analogously to auto-anti-idio-type. The actual i d i o t y p i c determinant recognized by these two reagents could be unrelated, while being indistinguishable on a serological or functional ba-s i s . A problem arises when an attempt i s made to determine i d i o t y p i c related-ness between antibody molecules using these two forms of anti-idiotype. I t i s possible that molecules which are i d i o t y p i c a l l y related i n an autologous net- ... work may not be recognized as such by a foreign immune system. Jerne's d e f i n i -t i o n inherently depends upon the concept of i d i o t y p i c complementarity, an abstract relationship for which i t i s d i f f i c u l t to define a serological 5 equivalent. I t must therefore be acknowledged i n the absence of concrete struc-t u r a l evidence, that present definitions of idiotypy based on di s s i m i l a r genetic systems only constitute formal approximations of the relationship proposed by Jerne. Immunization across species barriers has been p r e f e r e n t i a l l y u t i l i z e d i n serological studies i n favour of both the speed with which this mode of immuni-zation can y i e l d a n t i - i d i o t y p i c s p e c i f i c i t y as w e l l as the quantities of reagent that can be isol a t e d . Some of the e a r l i e s t studies showed that idiotypes were not shared by different animals when immunized with the same antigen (11). Some evidence that cross-reactions might be observed between different i n d i v i d -uals of the same species was discovered although the incidence was found to be very low (12). Interest i n using idiotypes as genetic markers developed only after reports of i d i o t y p i c cross-reactions were reported with defined antigen systems i n mice. For example, the response of Balb/c mice to the phosphoryl-choline hapten i s almost e n t i r e l y dominated by antibodies bearing the T15 idiotype (13). In another hapten system, 4-hydroxy-3-nitro-phenyl-l-acetyl (NP), 85% of primary anti-NP antibodies express a major idiotype (14). In the arsonate system, i t was found that 20-70% of anti-arsonate antibodies bear a common idiotype (15) i n A/J mice. Between 30 and 50% of antibodies to group A streptococcal carbohydrate i n A/J mice share a common idiotype which i s desig-nated A5A (16). In the sense that pooled serum antibodies are composed of the combined products of many different lymphocyte clones, the i r anti-idiotypes are i n r e a l i t y oligoclonal i n s p e c i f i c i t y and define idiotypy which i s biased i n favour of the more predominant idiotypes i n the ex i s t i n g antibody population. I t has only become possible to probe the i d i o t y p i c heterogenity of oli g o -clonal systems with the advent of c e l l fusion technology (17). I t i s now pos-s i b l e to perform i d i o t y p i c studies using monoclonal antibodies obtained by c e l l 6 fusion i n place of pooled serum antibodies. Using a large set of monoclonal antibodies with s p e c i f i c i t y for the arsonate moiety, the idiotype system desig-nated as CRI has been resolved into a large number of public and a small number of private i d i o t y p i c s p e c i f i c i t i e s (18). P a r a l l e l findings were observed i n . the NP hapten system using a panel of monoclonal anti-idiotype antibodies raised to a single NP-binding hybridoma protein i n mice (19). These cases demonstrate s i m i l a r i t i e s which have also been observed i n a number of other systems (20). The predominance of i n d i v i d u a l hybridoma idiotypes was found to be low, suggesting that the t o t a l repertoire of .serum antibodies may be very diverse. These examples aptly i l l u s t r a t e the serological d i s t i n c t i o n between Oudin's c r i t e r i o n and Jerne's concept of idiotype. Accurate biochemical data has only recently been obtained by exploiting the precision afforded by the existence of monoclonal probes and the precise markers which they define. Comparative amino acid sequence analysis of a n t i -body V-regions has shown that for heavy chains there exist four regions of ex-treme v a r i a b i l i t y or hypervariable regions which map to residues 31-37, 51-68, 84-91 and 101-110 (21). I t was subsequently shown that the residues i n three hypervariable regions constituted antigen-contacting residues i n the actual antigen binding s i t e , hypervariable regions have also been referred to as complementarity determining regions (22). This role has been strongly sup-ported by a f f i n i t y l a b e l l i n g and Xeray crystallographic data which showed that residues within hypervariable regions were distributed over the f r o n t a l surface of the immunoglobulin molecule i n positions which were d i r e c t l y exposed to the solvent and for reasons of conformation could conceivably f i l l the role of antigen combining s i t e (23). In taking into account the sequence data of framework domains of V-regions, several groups have shown that the actual alpha-carbon backbone of immunoglobulin 7 V-regions i s almost i d e n t i c a l between different molecules (24). I t would appear that even between di s t a n t l y related species a very high degree of con-formational homology has been retained. This structural s i m i l a r i t y has been used as a basis for model building using an established framework prototype as a foundation for predicting combining s i t e structures based on information from X-ray d i f f r a c t i o n analysis and primary sequence data (25). Sequence analysis of antibody molecules which comprise an i d i o t y p i c family of myeloma and hybridoma proteins with dextran binding a c t i v i t y has revealed several important facts concerning the contribution of heavy and l i g h t chains towards the actual idiotope (26). Light chains of dextran antibodies are a l l of the lambda type and appear to be almost i d e n t i c a l according to their isoelec-t r i c focusing patterns. Preliminary amino acid sequence data on two myeloma proteins M104E and J558 revealed that their V regions are i d e n t i c a l . Complete amino acid sequences of ten i n d i v i d u a l heavy chain variable regions have pro- . . vided primary structural correlates of several V-region idiotype markers. A correlation between an i n d i v i d u a l idiotype (Idl) on protein J558 (defined by a monoclonal anti-idiotype) and the presence of a s p e c i f i c sequence -Arg-Tyr-at positions 100/101 of the J558 heavy chain was found by comparative sequencing (27). This represents the f i r s t demonstration of an i d i o t y p i c marker with the D segment of VJJ. Support for t h i s assignment was demonstrated i n a modifica-tion study which showed direct evidence for the p a r t i c i p a t i o n of the tyrosyl residue at position 101 i n the interaction between J558 and i t s anti-idiotype. I n d i r e c t l y , the presence of a second category of i d i o t y p i c determinant IdX has been correlated to amino acid residues 54 and 55 of J558 confirming the inde-pendence of these markers on the basis of d i f f e r e n t i a l modification. Two dextran-binding myelomas J558 and Hdex24 which possess the same i n d i -vidual idiotype (Idl) were diazotized to low l e v e l s . Both proteins lost the 8 I d l marker under these conditions. When diazotization was performed i n the presence of hapten (1-0-methyl-alpha-D-glucopyranoside) J558 retained the I d l marker whereas Hdex24 did not. I t would appear that the i d i o t y p i c determinant modified i n the case of J558 i s i n the hapten binding s i t e whereas i n the case of Hdex24 i t was not. This finding implies that residues from different loca-tions i n the t e r t i a r y structure may be juxtaposed to form the same i d i o t y p i c determinant. In the case of J558, this determinant i s located within the D seg-ment and i t s expression may r e f l e c t the phenomenon of junctional d i v e r s i t y pro-posed by Early e_t a l or could represent an example of a germline D segment mini-gene (28,29). Genetic mapping of i d i o t y p i c determinants therefore remains a complex i s -sue. Despite the best efforts of investigators to obtain a molecular correlate, the ultimate d e f i n i t i o n may escape detection. For example, for an amino acid correlate ascribed to the D-segment, various mechanisms could account for i t s presence.. Where i t can be shown that the same marker can be correlated with dif f e r e n t s i t e s on different molecules, i t might also be predicted that a given idiotype may.be dependant on conformation and require pa r t i c u l a r sequences i n other portions of the V regions for i t s expression. A given minigene may -thereby produce an I d l i n one context but f a i l to do so i n another. This un-certainty i s reflected i n the following comment: "Indeed, one can never be cer-tain what i s being mapped- a V segment, a D segment, a J segment, the a b i l i t y to produce certain somatic variants, or some combinations of these." (27). Experimental demonstrations of i d i o t y p i c interactions Although idiotypes were o r i g i n a l l y detected on antibody molecules as early as 1953, the existence of such i n d i v i d u a l antigenic s p e c i f i c i t i e s on immuno-globulins remained a serological c u r i o s i t y u n t i l the phenomenon could be placed into the frame of reference of antibody d i v e r s i t y (1). The potential u t i l i t y of 9 this property became apparent as information l i n k i n g idiotypy to antigen speci-f i c i t y became known and was subsequently exploited to probe i d i o t y p i c cross-, r e a c t i v i t y between genetically related individuals. Expression of idiotype markers has since been linked to the Ig-1 locus and i s a subject which has been reviewed (30). In addition, i t has been widely demonstrated that anti-idiotype antisera can be raised between individuals of the same inbred s t r a i n (iso-immunization) as wel l as i n the same in d i v i d u a l from which the immunizing i d i o -type was o r i g i n a l l y obtained (autoimmunization) (31). Evidence demonstrating the existence of auto-anti-idiotypic immunity during the course of the normal immune response i n vivo f u l f i l l s Jerne's postulate on the existence of t h i s form of interactions within the host network (1). Experimental confirmation of other network postulates has been found i n a number of systems. Support for the role of i d i o t y p i c interactions i n regulation of immune responses and the predominance of T lymphocytes i n this mode of regulation was shown by several investigators (32-34). These examples of TH-cell manipulations using antisera raised to antibody variable regions also confirm the existence of shared i d i o -typic repertoires between T and B c e l l s . In a number of instances this finding has been extended to idiotypes on T - c e l l derived regulatory factors (35). Ad-ministration of minute quantities of anti-idiotype i n vivo can therefore result i n profound and long-lasting consequences i n the immune system. Stable a l t e r a -tions i n the balance of regulatory T lymphocytes have been obtained as a result of i d i o t y p i c perturbations during the course of immune responses. This suggests that idiotypes may be one l e v e l at which immune responses can be e f f e c t i v e l y manipulated. While the concept of i d i o t y p i c interaction i s now widely accepted and con-tinues to provide concise experimental data on isolated phenomena, i t has been used mainly to demonstrate temporal interactions, for example, generation of 10 or Tg c e l l s as members of an i d i o t y p i c series. Within a functional network, however, alterations i n the a c t i v i t y of one component must have a compensatory effect on other elements with which they are i n equilibrium. I t i s essential i n order to probe the more comprehensive issues i n network theory to design experiments which allow one to measure not only the behaviour of a single net-work parameter, but to follow the changing relationship between linked i d i o -typic events simultaneously. The lack of clear experimentation along these lines has so far kept network theory from gaining wide acceptance into the f i e l d of c e l l u l a r immunology. This i s largely due to the fact that idiotypes have been investigated i n macromolecular systems i n which the measurable idioTv type: specific, portion constitutes a very small portion of the overall immune response. Antigen-linked (hapten-carrier) interactions form the basis for Ir gene controlled responses but under these conditions interactions between i d i o -t y p i c a l l y d i s t i n c t compartments cannot be measured. This lack of correlation with the known models of I r gene control has led various authors to attempt a theoretical integration of network concept with existing data on I r gene con-t r o l l e d c e l l interactions (36). Need for a simple experimental model The existence of idiotypy among pathways regulating the immune response has been shown i n a number of independent systems. A common approach.of such studies has been i n the method of preparation of anti-idiotype reagent. In general, a f f i n i t y p u r i f i e d antibody has been used to raise heterologous a n t i -idiotype which has been used to define major s t r a i n s p e c i f i c idiotypes (13-15). Subsequent analysis of monoclonal antibodies with s p e c i f i c i t y for haptens, amino acid copolymers, and dextrans have indicated that the previously defined major i n t r a s t r a i n idiotypes can be further resolved into families of closely related but not necessarily i d e n t i c a l molecules (18-20). The degree of 11 heterogeneity amongst antibodies expressing a c o l l e c t i v e idiotype i s , conse-quently, uncertain. Cross-reactivity of an anti-idiotype which i s complement .. tary to such an antibody population can establish i d i o t y p i c relatedness i n func-t i o n a l studies, but cannot present a formal demonstration of the connectedness amongst members of an i d i o t y p i c series as this type of reagent has not been shown to discriminate c l o n a l l y distributed receptors. Recent evidence has been reported to suggest that different forms of the same antigenic determinant can trigger different B c e l l precursors (37-38). Balb/C mice immunized with the T-independent antigen dextran B1355s respond with the expression of two unique idiotyp es i n addition to the normal cross-reactive idiotype. Greater than 90% of these antibodies express lambda l i g h t chains. By contrast, spenic B c e l l s stimulated with a T dependent form of a n t i -gen, dextran coupled to hemocyanin, carried a large proportion of kappa a n t i - , body, less than 25% of which expressed the cross-reactive idiotype. This selec-tion for different i d i o t y p i c B c e l l s may occur by means of T c e l l s which re c r u i t precursor B c e l l s i n an i d i o t y p i c a l l y s p e c i f i c fashion. Association of a given haptenic determinant with a variety of c a r r i e r determinants could thereby i n -fluence the selection of hapten s p e c i f i c B c e l l s . By presenting such a haptenic determinant i n an environment of variable TJJ or Tg, idiotype expression may be modulated. This notion i s germane to the chemistry of synthetic antigens which may vary i n terms of molecular weight and conformation, land to the study of haptenic groups which must be coupled to large molecular weight carriers i n order to become immunogenic. The studies which are described i n this thesis were undertaken with the aim of describing network interactions using a model system whose immunochemical properties were wel l defined. The ferredoxin (Fd) molecule i s a small electron transport protein, isolated from Clostridium pasteurianum (Figure 1). I t has a TRYPSIN H^N-ALA-TYR-LYS-ILE-ALA-ASP-SER-CYS-VAL-SER-CYS-GLY-ALA-CYS-ALA-SER-ALA-CYS-PRO-VAL-ASN 1 21 ALA-ILE-SER-GLN-GLY-ASP-SER-ILE-PHE-VAL-ILE-ASP-ALA-ASP 22 35 THR-CYS-ILE-ASP-CYS-GLY-ASN-CYS-ALA-ASN-VAL-CYS-PRO-VAL-GLY-ALA-PRO-VAL-GLN-GLU-COOH 36 t 55 CARBOXYPEPTIDASE A Figure 1: Amino acid sequence of ferredoxin. Underlined sequences indicate the two major antigenic determinants. Arrows demonstrate the point of trypsin cleavage i n the N-terminal determinant and the point at which carboxypeptidase A a c t i v i t y i s terminated i n the C-terminal determinant. 13 t o t a l amino acid content of f i f t y - f i v e residues a l l of which are present i n a single polypeptide chain (39). In view of i t s molecular weight and antigen-i c i t y i n several species, the molecule has been a convenient subject for immuno-l o g i c a l analysis. E a r l i e r studies using synthetic peptides representing d i f -ferent regions of the molecule showed that the sequences located i n the N--terminal heptapeptide (NH^-ala-tyr-lys-ile-ala-asp-ser) and the COOH-terminal pentapeptide (-ala-pro-val-gln-glu-COOH) constituted the two major antigenic determinants. In equilibrium d i a l y s i s experiments, i t was shown that these peptides bound e s s e n t i a l l y 100% of the antibody synthesized i n rabbits to Fd (40). The s p e c i f i c i t y of these assignments i s also supported by chemical modi-f i c a t i o n studies (41), and by the results of c e l l u l a r studies which indicate that the NH2-terminal determinant appeared to be the stronger of the two i n terms of cell-mediated functions, while the COOH-terminal determinant was pre-dominant i n serological assays (42). These results are representative of c e l l cooperation models i n immunology and have been the basis for genetic i n v e s t i -gations of Fd responses i n congenic strains of mice (43). At the molecular l e v e l , t h i s antigen exhibits properties which lend themselves to immunochemical analysis. Located within each of those regions which have been i d e n t i f i e d as antigenic determinants are s i t e s susceptible to hydrolysis by s p e c i f i c proteo-l y t i c enzymes. Digestion of ferredoxin with selected enzymes results i n a mole-cule retaining 52 or 53 of i t s o r i g i n a l 55 amino acid residues but possessing only a single functional antigenic determinant (44). Through the< use of two selective enzymes trypsin and carboxypeptidase A, monospecific fragments having the i n d i v i d u a l characteristics of either an NH2-terminal or the COOH-terminal determinant can be obtained. 14 CHAPTER 2. DETECTION OF SPECIFIC IDIOTYPE IN THE SERUM OF Fd-IMMUNE BIO.BR SERUM Introduction The ferredoxin molecule (mw 5500) i s among the smaller antigens which have been u t i l i z e d i n immunological studies. Of currently available proteins such as myoglobin (45), lysozyme (46), and staphylococcal nuclease (47), and i n s u l i n , only i n s u l i n (48) has a comparable molecular weight. While Fd i s immunogenic i n mice, peak antibody t i t r e s are moderate i n comparison to the levels of antibody produced i n response to more complex antigens. Analysis of purified antibody by i s o e l e c t r i c focussing suggests that the repertoire of d i f -ferent antibody species i s limited to a small number of detectable spectrotypes (44). An examination of idiotypy might provide a more accurate characteriza-tion of the degree of antibody d i v e r s i t y present i n the anti-Fd repertoire. Customarily, researchers have produced anti-idiotype reagents by immunizing animals against the a f f i n i t y p u r i f i e d antibodies pooled from a number of i n d i -viduals. In view of the demonstrated v a r i a b i l i t y i n expression of inherited idiotypes between related individuals, however, studies based on this approach cannot be used to demonstrate absolute i d e n t i t y i n idiotype repertoire but only overlap i n certain species or between individuals. Studies i n other animal systems had shown that the immune response to Fd was limited to only two determinants of the molecule. Isolation of quantities of p u r i f i e d anti-Fd antibody s u f f i c i e n t to perform controlled i d i o t y p i c studies was precluded due to the fact that after normal immunization protocols, only small amounts of antibody could be obtained. This necessitated either the pool-ing of sera from large numbers of individuals or using serum from hyperimmunized donors. A decision was made instead to attempt to i s o l a t e monoclonal antibodies to ferredoxin. This approach presents the advantage of enabling one to f i r s t 15 determine the epitope s p e c i f i c i t y of the antibody before subsequently attempt-ing to characterize i t s idiotype amongst the population of antibody molecules having similar epitope binding s p e c i f i c i t y i n serum. Studies were therefore undertaken with the aim of producing anti-Fd monoclonal antibodies and deter-mining their binding s p e c i f i c i t y for the two Fd determinants using immunochemi-c a l techniques and second, of r a i s i n g antisera to these molecules i n order to determine the role of these idiotypes i n the immune response. Materials and Methods Animals Female mice 6-8 weeks of age of the following strains, were purchased from the Jackson Laboratories, Bar Harbour, Maine: BlO.BR/OSn DBA/2, C57BL/6, C3H, CBA, SJL, AKR, SM, C58, BlO.D2/OSn, STB, MaMY, CE. Young adult female New Zealand white rabbits were obtained from the University of B r i t i s h Columbia animal care centre breeding unit. Antigens and immunization Keyhole limpet haemocyanin (KLH) was obtained from Calbiochem, La J o l l a , C a l i f o r n i a . Ferredoxin (Fd) was p u r i f i e d i n the laboratory from cultures of Clostridium pasteurianum grown and p u r i f i e d according to the procedures of Martenson (49). B r i e f l y , crude Fd was isolated by acetone extraction of harvested c e l l pastes followed by p r e c i p i t a t i o n with 90% saturated (NH^)2S04 and subsequent elution from DEAE Cellulose (Sigma). Fd was then p u r i f i e d from the crude preparation by molecular exclusion chromatography on Sephadex G-50 (Pharmacia) using the c r i t e r i o n for purity of A390/280 as described. Frac-tions exhibiting ratios of greater than or equal to 0.7 were used i n the experi-ments. Mice were immunized with 50 ug of s p e c i f i c antigen (Fd or KLH) emulsified 16 i n complete Freund's adjuvant (CFA-Gibco) by intraperitoneal i n j e c t i o n on day 0. Primary immune sera were obtained on day 21 by r e t r o - o r b i t a l bleeding. Secondary immunizations were given on day 28 by intraperitoneal i n j e c t i o n of 50 yg of antigen emulsified i n incomplete Freund's adjuvant (IFA Gibco) and secondary immune sera were subsequently taken on day 35. In experiments involv-ing adoptive secondary responses, animals were bled on day 35 as w e l l as on day 49. For c e l l fusion experiments, mice which had received secondary injections of antigen 4 weeks previously were given intraperitoneal injections of either 100 yg of Fd i n CFA three days before using their spleens i n fusion experiments. Rabbits were used i n the preparation of antiserum to normal mouse immuno-globulins (Pentex) and a n t i - i d i o t y p i c antisera to the monoclonal antibodies Fd-1 and Fd-2. Animals were i n i t i a l l y immunized with 1.0 mg of the appropriate mouse immunoglobulin emulsified i n CFA by intramuscular i n j e c t i o n . Subsequent injections of antigen i n IFA were given u n t i l antibody t i t r e s i n serum reached levels of approximately 20 mg/ml. Antisera were obtained 7 and 14 days follow-ing the most recent i n j e c t i o n by bleeding from the marginal ear vein. Assay for s p e c i f i c antibody Antibodies i n immune serum were quantified by using the enzyme linked immunosorbent assay (ELISA) described by Voller et_ al_ 1976 (50). Conjugates of enzyme with anti-immunoglobulin were prepared i n the laboratory using the following procedure. Rabbit anti-mouse immunoglobulin was pu r i f i e d from hyper-immune rabbit serum by adsorption to normal mouse IgG which had been covalently linked to Sepharose 4B-CL (Pharmacia) with cyanogen bromide (51). Specific antibody was eluted from the matrix i n pH 2.3 glycine buffer and immediately neutralized with 3M NaOH. Those fractions containing s i g n i f i c a n t quantities of protein (0.5 mg/ml) were pooled and dialysed against phosphate buffered 17 saline (PBS) solution overnight and concentrated to 5 mg/ml against polyethyl-eneglycol mw 2 0 , 0 0 0 (Fisher). Concentrated antibody was . stored at - 20°C u n t i l required. Five mg of alkaline phosphatase enzyme pu r i f i e d from ca l f intestine (Sigma type v l l - s , Boehringer-Mannheim) was dialyzed against PBS (Sigma enzyme) or used d i r e c t l y without d i a l y s i s (Boehringer-Mannheim enzyme). Two mg of a f f i n i t y isolated rabbit anti-mouse IgG (RaMIg) was combined with 5 mg of enzyme i n a t o t a l volume of less than or equal to 1 . 5 ml i n PBS. Ten micro-l i t r e s of glutaraldehyde solution ( 2 5 % EM grade, JB EM Services, Dorval) were added and the mixture l e f t for 9 0 min at room temperature with slow s t i r r i n g . After reaction with the cross-linking reagent, the conjugated material was dialyzed overnight against 0 . 0 5 M Tris HCL pH 8 . 0 and stored for use as a 5 0 % solution i n glycerol at 4°C i n the dark. Conjugates prepared i n this way were standardized against a fixed amount of normal mouse Ig which had been coated onto the s o l i d phase of polystyrene substrate binding plates (Immulon I-Dyna- . tech) by overnight incubation with 0 . 1 ml/well of carbonate coating buffer con-taining 1 0 0 mg/ml of mouse IgG (Pentex). After washing with PBS-Tween buffer followed by a br i e f incubation ( 2 0 min) with 0 . 2 % bovine serum albumin (BSA) i n PBS solution, coated plates were incubated with 0 . 1 ml/well of a d i l u t i o n series of enzyme conjugate ranging from 1 / 1 0 0 - 1 / 1 0 , 0 0 0 . Working dilutions of conjugate were chosen i n order to y i e l d an O . D . ^ Q ^ ^ of 1 . 0 i n a 3 0 min reac-tion time with substrate. Generally, optimum working strength of conjugate prepared using this procedure was obtained at a d i l u t i o n of 1 / 1 0 0 0 - 1 / 1 5 0 0 . No evidence of any decline i n the working strength of conjugate was noticed over intervals as long as 2 4 months. The s e n s i t i v i t y of this assay for s p e c i f i c anti-Fd antibody has previously been calibrated using a radioimmunoassay ( 4 3 ) . Under the conditions used, the lower detection l i m i t of the ELISA for a n t i -Ferredoxin antibody i s 1 0 ng. 18 C e l l fusion The conditions used i n the production of hybridoma c e l l l ines were essen-t i a l l y those of the protocol described by Oi and Herzenberg (52) with minor modifications. F i r s t as hyperimmunization does not appear to d i r e c t l y increase the l i k e l i h o o d of generating s p e c i f i c hybridomas an alternate immunization regime based upon adoptive transfer was used. Spleen c e l l s were removed from donor mice 7 days following secondary i n j e c t i o n of antigen (Fd or KLH) and adoptively transferred to syngeneic sublethally irradiated (500 rad) recipients, using 5x10^ c e l l s per mouse. These animals were rested for 2 weeks during which time they received an a n t i b i o t i c solution (Pfizer) i n the i r drinking water. The mice then received a t h i r d dose of Fd (100 yg i n IFA) intraperitoneally and t h e i r spleens were taken for fusion 3 days l a t e r . The myeloma c e l l l i n e used, SP2/0 (53), was obtained from the Salk C e l l D i s t r i b u t i o n Center, La J o l l a , Cal-i f o r n i a . Prior to fusion, myeloma c e l l s were cultured i n 100 ml volumes i n 250 ml capacity tissue culture flasks (Falcon 3013) at a s t a r t i n g density of 10^ cells/ml and reaching a f i n a l density l i m i t of l-2xl0~Vml. The culture medium consisted of freshly prepared (not more than 1 week) Dulbecco's Modified Eagle Medium supplemented with 2mM glutamine, 5mM sodium pyruvate, 100 ug/ml penicillin/streptomycin a n t i b i o t i c solution, 2mM sodium bicarbonate (Fisher) and 10% by volume f e t a l c a l f serum (Gibco). C e l l cultures at a l l stages were incubated i n a humidified environment of 10% CO2 at 37°C. Polyethyleneglycol (Serva-mw 4,000 p u r i f i e d for GLC) was prepared on the day of fusion by auto-claving of s o l i d PEG followed by d i l u t i o n with s t e r i l e PBS to a 50% solution (w/v). This solution was t i t r a t e d to pH 7.4-7.8 using s t e r i l e IN NaOH before use. Spleen c e l l s were fused with SP2/0 c e l l s at a r a t i o of 5:1 i n 50% PEG using the procedure of slow d i l u t i o n with warm (37°C) serum-free PBS. After d i l u t i o n and p e l l e t i n g by centrifugation, fused c e l l s were incubated at 37°C 19 for 30 min i n a small volume of tissue culture medium. The c e l l s were then gently resuspended to a concentration of 5x10^ c e l l s total/ml i n medium con-taining 20% FCS and 10^ mouse red blood cells/ml and dispensed into 96-well tissue culture plates (Costar 4596) using 0.1 ml/well of c e l l suspension. The plates were placed into p l a s t i c boxes with loosely f i t t i n g l i d s to retard evaporation and fed on the following day with double strength HAT medium. Hybridoma cultures were screened for antibody production between 7-10 days using the ELISA procedure described e a r l i e r . Subsequent d e t a i l s including cloning by l i m i t i n g d i l u t i o n , growth as ascites i n vivo and p u r i f i c a t i o n on protein A Sepharose are as described i n Oi and Herzenberg (52) with the excep-tion that hybridomas were cloned on feeder layers of mouse red blood c e l l s i n place of mouse thymocytes. Production of Anti-idiotype Serum Anti-idiotype serum was produced i n a New Zealand White rabbit by i n j e c t i o n of monoclonal antibody (Fd-1) isolated from ascites f l u i d on Protein A Sepha-rose. For the primary i n j e c t i o n , 1.0 mg of pu r i f i e d Fd-1 i n complete Freund's adjuvant was given intramuscularly. Subsequent injections of Fd-1 were given as alum precipitate intraperitoneally at intervals of two weeks. Serum used throughout t h i s study was taken two weeks following the fourth i n j e c t i o n . P r i o r to testing serum for anti-idiotype a c t i v i t y , rabbit antibodies reacting with constant region determinants of Fd-1 were removed by adsorption onto a column of mouse immunoglobulins coupled to Sepharose 4B using the CNBr procedure (51). Rabbit anti-Fd-1 serum (8.0 ml) was passed through the column twice. Samples of adsorbed rabbit anti-Fd-1 serum used i n anti-idiotype assays contained 10% by volume of normal B10.BR serum i n order to preclude the p o s s i b i l i t y of detect-ing reactions toward non-idiotypic epitopes on mouse immunoglobulin. 20 A f f i n i t y I s o l a tion of A n t i - i d i o t y p i c Antibody and Preparation of an A n t i - i d i o -type Immunoadsorbent: Anti-idiotype antibody was p u r i f i e d from normal mouse Ig-adsorbed rabbit a n t i - i d i o t y p i c antiserum by using an a f f i n i t y column of the monoclonal antibody Fd-1 coupled to Sepharose 4B. Anti-idiotype antibodies were recovered from the column by e l u t i o n with .05 M glycine HC1 pH 2.2 and immediately neutralized with 1 M NaOH. Pu r i f i e d antibody was then assayed for anti-idiotype a c t i v i t y against Fd-1 antibody as described below. Active material was coupled to CNBr activated Sepharose 4B at a concentration of 3 mg per 1 ml packed beads. Under the conditions used approximately 90% coupling e f f i c i e n c y was obtained. Assays for Anti-idiotype A c t i v i t y Assays for anti-idiotype a c t i v i t y of rabbit antiserum to the monoclonal antibody Fd-1 were performed using the ELISA. A c t i v i t y of i n h i b i t o r s was meas-ured against a constant concentration of a s p e c i f i c antibody which had been previously t i t r a t e d to y i e l d an absorbance at 405 nm of 1.0 after t h i r t y minutes incubation i n a standard system. Specific antibodies used i n these assays con-sisted of serum from Fd immune B10.BR mice, serum from KLH immune B10.BR mice, monoclonal antibody to Fd and a rabbit antiserum to the hybridoma protein Fd-1. In stage 1 of an i n h i b i t i o n assay, doubling dilutions of i n h i b i t o r s were mixed with a constant amount of the s p e c i f i c antiserum and incubated at 4°C overnight i n microtitre trays. Aliquots of 0.1 ml for each sample were then transferred to ELISA plates bearing the appropriate antigen bound to the s o l i d phase. From this stage onwards, processing of the plates was done according to standard ELISA technique. The tests were developed with alkaline phosphatase labeled sheep anti-rabbit immunoglobulin. 21 Determination of Combining Site S p e c i f i c i t y of Anti-idiotype Serum Interaction between rabbit anti-hybridoma protein and the hybridoma protein was measured i n the pre-sence of antigen . as follows: Protein binding ELISA plates (Cooke Dynatech) were f i r s t coated with p u r i f i e d monoclonal antibody to Fd (Fd-1) by incubating them i n the presence of 0.1 ml per w e l l of carbonate coatr-ing buffer containing 20 yg per ml", of protein A p u r i f i e d Fd-1 for 45 min. F o l -lowing this step, Fd-1 coated plates were incubated with 500 yg/ml of bovine serum albumin i n PBS buffer for 15 minutes i n order to saturate unbound protein binding s i t e s on the s o l i d phase. This step was undertaken i n order to reduce background colour development i n the assay due to nonspecific adsorption. After washing the plates with PBS-Tween buffer, monoclonal antibody coated plates were incubated with varying concentrations of antigen, either Fd or KLH, for 1 hour. KLH served i n this case as a non-specific control, to control for non-s p e c i f i c blocking by soluble proteins. The plates were then washed again and incubated with a solution of rabbit anti-idiotype serum at a d i l u t i o n of 1/512. Binding of the rabbit anti-idiotype to the mouse monoclonal antibody coated plates was allowed to proceed for 30 min after which the plates were washed and the wells incubated with 0.1 ml of an alkaline phosphatase conjugate of sheep anti-rabbit Ig at a d i l u t i o n of 1/2000 for an additional 30 min. After a further 1 hour incubation and f i n a l washing with buffer, the enzyme substrate reaction was allowed to continue for 30 minutes. Colour development was then determined spectrophotometrically using a Titertek Multiskan spectrophotometer (Flow Labs Inc). 22 P u r i f i c a t i o n of Idiotype Bearing Antibodies from the Serum of Immune Mice Idiotypic antibody was p u r i f i e d from the serum of BIO.BR mice immunized 4 weeks previously with 50 yg Fd i n CFA. One ml of pooled serum was passed over the anti-idiotype immunoadsorbent column, and bound antibody was recovered i n the acid eluate. Antibody i n the eluted f r a c t i o n was then t i t r a t e d for Fd binding a c t i v i t y by ELISA. Serum anti-Fd antibody eluted from the a n t i - i d i o -type column was tested i n an i n h i b i t i o n assay as described above against measured quantities of whole Fd, i t s N- or C-terminal fragments, as we l l as a f f i n i t y isolated a n t i - i d i o t y p i c antibody. Tests for the s p e c i f i c i t y of this antibody were devised i n collaboration with Lydia Sikora (44). Results Typical antibody t i t r e s i n BIO.BR mice which are high responders to Fd, immunized with ferredoxin are shown i n Table 1. Twenty-one days after a primary immunization with Fd, levels of s p e c i f i c antibody average about 3.0 yg/ml. In the secondary response, measured 7 days after i n j e c t i o n of Fd this average t i t r e r i ses to about 12.0 yg/ml. In comparison to t y p i c a l antibody t i t r e s found after immunization with commonly studied antigens (NP, ARS) these t i t r e s are much lower. Given the molecules' low molecular weight and i t s limited number of antigenic determinants this moderate l e v e l of immunogenicity i s not surprising. Anti-hapten antibody t i t r e s of up to 5.0 mg/ml have been obtained i n mice after immunization with hapten coupled to large molecular weight carriers (17-18). Production of such large amounts of antibody presumably r e f l e c t s the magnitude of T c e l l help provided by the action of c a r r i e r s p e c i f i c T lymphocytes. In comparison, the Fd antigen can be viewed as a molecule which consists of two covalently linked haptenic groups. According to the rules of hapten c a r r i e r interactions there can be only a single c a r r i e r moiety providing help for the alternate determinant and antibody responses to Fd may therefore r e f l e c t this 23 Table 1: Characteristic antibody t i t r e s i n BIO.BR (responder mice). BIO.BR mice were immunized with Fd and bled following a primary (day 0) or secondary (day 28) antigen stimulus. Anti-Fd t i t r e s are ex-pressed as a mean value for 6 animals. Stage Time of Bleed ug/ml.anti-Fd±S.E.M. 1° response day 21 3.4±0.7 2° response day 35 11.9±3.2 24 l i m i t a t i o n . Quantitative i s o l a t i o n of milligram quantities of anti-Fd antibody which would be required for the preparation of an anti-idiotype i n either mice or rabbits was precluded by the low antibody t i t r e s obtained. I t was also f e l t that a more d e f i n i t i v e analysis of t h e l i d i o t y p i c repertoire and the role of in d i v i d u a l idiotypes i n immune regulation could be obtained by i s o l a t i n g mono-clonal anti-Fd antibodies using the c e l l fusion technique of Kohler and M i l s t e i n (17). In a preliminary study i t was found that by using standard pro-tocols, the frequency with which s p e c i f i c anti-Fd secreting hybrid clones could be obtained was considerably lower than was obtained using a more immunogenic antigen such as KLH (raw 3x10 ). In order to increase this frequency, a d i f -ferent immunization schedule was designed. This protocol was based upon an adoptive transfer system i n which mice were given a secondary i n j e c t i o n 5 days before adoptive transfer into sublethally irradiated syngeneic recipient mice. Following spleen c e l l transfer, recipients were rested for 10-14 days. A third i n j e c t i o n of Fd was then given, and the spleens were removed 3 days l a t e r for c e l l fusion. Using the adoptive transfer protocol, a marked increase i n the y i e l d of s p e c i f i c anti-Fd producing clones was obtained. The i s o l a t i o n and characterization of two hybridomas derived from an adoptive transfer as w e l l as an e a r l i e r fusion have been described (44). B r i e f l y , these two hybridomas, Fd-1 and Fd-2, were obtained i n fusions between spleen c e l l s from Fd immune BIO.BR mice and the SP2/0 myeloma c e l l l i n e . Because the parental myeloma does not synthesize a cryptic l i g h t chain, the antibodies produced by the two hybrid-omas contains no heterologous l i g h t chain and are therefore s t r i c t l y monoclonal. An antigen binding t i t r a t i o n comparing the two monoclonal antibodies with a n t i -Fd serum i s presented i n Figure 2. 25 U 8 16 32 6U 128 256 512 1/dilution i Figure 2: T i t r a t i o n of anti-Fd antibodies by ELISA. Samples of B10.BR a n t i -Fd serum (O—O), Fd-1 monoclonal antibody (A—A) and Fd-2 mono-clonal antibody (0~0) were incubated on Fd-coated ELISA plates at the indicated r e l a t i v e d i l u t i o n . The lowest d i l u t i o n repre-sents a concentration of 10% serum (v/v) or 16 ug/ml of pu r i f i e d monoclonal antibody. 26 Binding s p e c i f i c i t y of monoclonal antl-Fd antibodies At the molecular l e v e l , Fd exhibits properties which are amenable to immunochemical analysis. Located within each of those regions which have been i d e n t i f i e d as antigenic determinants are sites susceptible to hydrolysis by sp e c i f i c proteolytic enzymes. As the molecule possesses a single lysine r e s i -due, i t i s susceptible to trypsin cleavage at only one peptide bond, between lys-3 and ala-4. Trypsin cleavage thus e f f e c t i v e l y destroys the NH2-determi-nant while leaving the remaining 52 residues of the molecule i n t a c t . Similar-l y , carboxypeptidase A can remove the two COOH-terminal amino acids, but w i l l not degrade the molecule past val-53 because of the presence of proline at position 52. This digestion process then e f f e c t i v e l y removes a s i g n i f i c a n t part of the COOH-terminal antigenic determinant. Digestion of whole Fd with either of these selective enzymes results i n a molecule which retains 52 of i t s o r i g i -nal 55 amino acid residues, but possess only a single antigenic determinant (66). By using these monospecific fragments of Fd i n an i n h i b i t i o n assay i n the ELISA the Fd-1 antibody was shown to be sp e c i f i c for the N^-determinant and the Fd-2 antibody was found to bind the COOH-determinant Figure 3 and Figure 4. (These figures and the digestion protocol are included with the kind per-mission of Lydia Sikora). Raising and immunoadsorption of antisera to monoclonal anti-Fd antibodies Adult New Zealand white rabbits were immunized with protein A p u r i f i e d monoclonal anti-Fd antibody as described i n Materials and Methods. Following the f i f t h i n j e c t i o n , these animals were bled and tested for r e a c t i v i t y towards mouse immunoglobulins using a standard ring p r e c i p i t i n test against normal BIO.BR serum. High antibody t i t r e s (2 mg/ml) were generally present and the rabbit antisera were f i r s t extensively adsorbed on an immunoadsorbent prepared with mouse IgG as described i n Materials and Methods. The immunoadsorbent bed 27 1001 0.25 0.5 1 2 4 8 Inhibitor, /ig Figure 3: Blocking ELISA carried out with the Fd s p e c i f i c monoclonal a n t i -body, Fd-1, at a concentration of 0.5 ug/ml of a f f i n i t y p urified antibody. The antibody was incubated overnight with dilutions of native Fd (A—A), N-fragment (O—O) or C fragment (Q-O) and tested on ELISA plates coated with native Fd at 4.0 Ug/ml. The test was performed i n quadruplicate. 28 12 10 h o fe a u 6 c o n w O M a < Fd Figure 4: The binding of hybridoma Fd-2 to either the N- or C-fragment, or to whole Fd on ELISA plates. Antigen or fragments were affixed to plates at 4 Ug/ml and monoclonal antibody was reacted for 30 min. at a concentration of 0.5 yg/ml. The test was performed i n quad-ruplicate and demonstrates the i n a b i l i t y of Fd-2 to bind the N-determinant, even at 5.0 Ug/ml which constitutes levels of ten-f o l d molar antibody excess. 29 volume used was s u f f i c i e n t to remove 15 mg of anti-mouse IgG a c t i v i t y from hyperimmune serum. Antisera to the monoclonal antibodies were adsorbed three times on this column and a l l assays to characterize their a n t i - i d i o t y p i c speci-f i c i t y were run i n the presence of 5% v/v of normal BIO.BR mouse serum to pre-clude the p o s s i b i l i t y that the s p e c i f i c i t y of the antisera were directed towards non-idiotypic immunoglobulin determinants. . Under these conditions, no reac-t i v i t y of either adsorbed antiserum towards normal BIO.BR immunoglobulins has been detected. Differences i n amino acid composition of hypervariable regions effect changes i n combining s i t e structure which are responsible for different binding s p e c i f i c i t i e s of antibody molecules. Structural evidence has indicated that amino acid substitutions are found i n both combining s i t e as wel l as areas ad-jacent to th i s portion of the molecule. Jerne recognized that since some of the i d i o t y p i c determinants (idiotopes) could be located within or im-mediately adjacent the antibody combining s i t e , anti-idiotype .anti-bodies (anti-idiotopes) should demonstrate the property of blocking the binding of antigen. A special term, paratope, was reserved for idiotopes which were located within the combining s i t e . Combining s i t e as w e l l as non-combining s i t e related idiotopes have recently been demonstrated using monoclonal a n t i -idiotype reagents (19). In the case of a polyvalent anti-idiotype anti-serum some degree of paratopic s p e c i f i c i t y would be expected as the antiserum should recognize a variety of in d i v i d u a l idiotopes. This factor was therefore taken ... into consideration during the characterization of the a n t i - i d i o t y p i c antisera to the monoclonal anti-Fd antibodies. Characterization of anti-idiotype antiserum to Fd-1: The s p e c i f i c i t y of the anti-idiotype antibody was i n i t i a l l y tested by assessing i t s a b i l i t y to i n h i b i t the binding of the monoclonal Fd-1 to Fd i n 30 the ELISA. Controls were run using soluble Fd with Fd-1 and show that both Fd and the anti-idiotype i n h i b i t binding of the monoclonal antibody Fd-1 to Fd coated plates (Fig. 5). To demonstrate the s p e c i f i c i t y of this i n h i b i t i o n , equivalent amounts of the anti-idiotype were tested with BIO.BR anti-serum d i -rected to KLH. No i n h i b i t i o n by the anti-idiotype was noted i n these studies, indicating that the anti-idiotype was directed s p e c i f i c a l l y towards anti-Fd antibody (Fig. 6). To determine whether the anti-idiotype antibody was directed s p e c i f i c a l l y to the antigen binding s i t e s of the Fd-1 antibody, an ELISA was carried out i n which Fd-1 was bound to the s o l i d phase of ELISA plates and the binding e f f i -ciency of the anti-idiotype was assayed i n the presence of increasing amounts of soluble antigen (Fd). As can be seen i n Fig. 7, the binding capacity of the anti-idiotype was substantially blocked i n the presence of antigen, i n d i c a t i n g that a major portion (approx. 70%) of the a n t i - i d i o t y p i c a c t i v i t y i s directed to regions of the Fd-1 antibody involved i n or adjacent to antigen binding. In order to establish a correlation between the N-determinant binding s p e c i f i c i t y of the monoclonal Fd-1 and i t s anti-idiotype, an anti-idiotype immu^ noadsorbent column was prepared and used to select antibodies from immune serum of BIO.BR mice. The material isolated i n this way was then analysed for both idiotype expression and determinant binding s p e c i f i c i t y . Results of this study are presented i n Fig. 8. This experiment shows that the anti-idiotype column s p e c i f i c i a l l y selects N-determinant binding antibody from immune serum. That a small proportion of this antibody i s inhibited at the highest levels of C-determinant may r e f l e c t the p o s s i b i l i t y either that trypsin digestion of t h i s preparation may not have been carried to completion and therefore would conceiv-ably be contaminated with small quantities of the N-determinant or, that the determinant recognized by Fd-2 extends somewhat beyond the lysine residue such P that 31 Figure 5: Competitive i n h i b i t i o n of monoclonal antibody Fd-1 by antigen and anti-idiotype. Inhibition of binding to Fd coated plates was measured i n the presence of i n h i b i t o r s , either Fd or rabbit a n t i -Fd-1, as described i n Materials and Methods. Percent binding of Fd-1 i n the presence of i n h i b i t o r s i s expressed r e l a t i v e to con-t r o l s incubated in the presence of NRS. Inhibitors: (©—•) rabbit anti-Fd-1 serum. (O---O) Fd 100 yg/ml. 32 1 i i : 512 256 128 64 32 16 8 4 i | ' / d i l u t i o n » | ! I , i > I Figure 6: Lack of i n h i b i t i o n of serum and monoclonal antibodies to KLH by anti-idiotype serum anti-Fd-1. Inhib i t i o n of serum and monoclonal antibody binding to KLH was measured as described i n Materials and ^Methods. Values plotted represent average of samples run i n t r i p -l i c a t e . (A—A) B10.BR anti-KLH serum; (•"•) B10.BR IgG 3 mono-clonal anti-KLH; (• ;A) B10.BR IgM monoclonals anti-KLH. Solid lines show results of the reaction of the various antisera or mono-clonal antibodies i n the presence of anti-Fd-1 i n which no i n h i b i -t i o n i s seen. Dotted lines show results for equivalent antisera i n the presence of increasing amounts of KLH. 33 100 % Binding A g concentrat ion ug / ml Figure 7: Competitive i n h i b i t i o n of anti-Fd-1 idiotype by Fd. Binding of anti-Fd-1 to Fd-1 coated plates was measured i n the presence of Fd as outlined i n Materials and Methods. Percent binding of anti-Fd-1 in the presence of Fd i s expressed r e l a t i v e to a control incubated i n the presence of equivalent concentrations of KLH. 34 Adsorp t ion of B10.BR A n t i - F d to I,- S e p h a r o s e : .125 . 2 5 .5 1 2 4 8 16 Inhibitor c o n c e n t r a t i o n u g / m l > Figure 8: S p e c i f i c i t y of anti-idiotype selected serum antibody. Serum a n t i -bodies to Fd were isolated over an anti-Fd-1 anti-idiotype column and assayed for percent binding to Fd coated plates i n the presence of soluble i n h i b i t o r s . Percent >„• binding of antibody i n the presence of each i n h i b i t o r was calculated according to a s p e c i f i c i t y control consisting of equivalent concentrations of purified normal rabbit IgG. Points shown represent the mean of t r i p l i c a t e samples. Standard deviations between replicates (not shown) were i n a l l cases l e s s than 2% (•--•) Fd; (*--*) N-Fd; (A—A) C-Fd; (o—O) A f f i n i -ty purified rabbit anti-Fd-1. 35 even after trypsin digestion Fd-1 retains a small degree of a f f i n i t y for this portion of the molecule. Characterization of anti-idiotype antiserum to Fd-2: The adsorbed rabbit antiserum raised to Fd-2 was also tested i n an ELISA for i t s a b i l i t y to i n h i b i t the binding of Fd-2 to Fd coated plates (Fig. 9). S p e c i f i c i t y of anti-Fd-2 serum for Fd-2 i s indicated i n Figs. 10 and 11. In Fig. 10, anti-Fd-2 was tested i n an ELISA for i t s a b i l i t y to block Fd-2 binding to Fd coated plates. Strong i n h i b i t i o n was seen i n the presence of a n t i - i d i o -type serum as w e l l as an a f f i n i t y p u r i f i e d f r a c t i o n of anti-Fd-2 antibody. In Fig. 11, anti-Fd-2 was tested i n an ELISA to determine whether i t s p e c i f i c a l l y bound Fd-2 alone, or exhibited cross-reactivity with Fd-1 also. As measured i n both the binding i n h i b i t i o n assay (Fig. 10) or i n the direct binding assay (Fig. 11) no cross-reactivity of anti-Fd-2 with Fd-1 was detected. In order to determine i f this i d i o t y p i c s p e c i f i c i t y of the antiserum recognized a combining s i t e related determinant, the degree of competition between Fd and anti-Fd-2 for the Fd-2 antibody was measured i n an ELISA. The results of this assay are presented i n Fig. 12. Approximately 45% of the anti-idiotype i s prevented from binding to Fd-2 i n the presence of Fd, in d i c a t i n g that a s i g n i f i c a n t proportion of the anti-idiotype detects determinants closely associated with the antibody combining s i t e . Discussion This chapter has presented data concerning the immunochemical characteri-zation of the anti-Fd response i n mice. This antigen has previously been ana-lyzed i n both guinea pigs and rabbits (40). Studies using antigenic peptides synthesized by the s o l i d phase M e r r i f i e l d technique showed that i n d i v i d u a l antigenic determinants could be related to small peptides of defined sequence 36 Figure 9: Inhibition of Fd-2 binding to Fd i n the presence of anti-Fd-2. Dilutions of anti-Fd-2 serum (O—o) or a f f i n i t y purified antibody (O—O) 16 ug/ml, were co-incubated with a standard concentration of Fd-2 antibody (2 ug/ml f i n a l ) prior to loading onto Fd-ELISA plates. Percent binding of Fd-2 i s expressed r e l a t i v e to an assay control. 37 ! I ; - I I 1 I . 8 . 512 256 128 6 1 » 32 16 8 tt 1 / d i l u t i o n , Figure 10: S p e c i f i c i t y of anti-Fd-2 serum for Fd-2 antibody. S p e c i f i c i t y was assessed by direct binding to idiotype coated plates. Dilu t i o n of anti-Fd-1 (o—O) or anti-Fd-2 (O—-O) serum plus normal mouse serum were incubated on an ELISA plate precoated with Fd-2 antibody. Binding of rabbit antibodies was measured with a sheep anti-rabbit Ig enzyme conjugate. 38 I F i g u r e 11: S p e c i f i c i t y o f a n t i - F d - 2 s e r u m f o r Fd-2 a n t i b o d y . B i n d i n g o f Fd-1 a n t i b o d y (2 y g / m l ) t o F d c o a t e d p l a t e s w a s m e a s u r e d i n t h e p r e s e n c e o f a n t i - F d - 1 s e r u m (O—o) o r a n t i - F d - 2 s e r u m ( O - — O ) . 39 Figure 12: Competitive i n h i b i t i o n of anti-Fd-2 by antigen. Binding of a n t i -Fd-2 to Fd-2 coated plates was measured i n the presence of Fd as outlined i n Materials and Methods. Percent binding of anti-Fd-2 i n the presence of varying quantities of Fd i s expressed r e l a t i v e to a non-specific control (KLH). 40 (41). These conclusions were reached using the available techniques of antibody measurement and lymphocyte transformation assays. Monoclonal reagents permit one to design highly s p e c i f i c assay systems. Using an ELISA the binding of p r o t e o l y t i c a l l y degraded fragments of Fd showed that the two Fd monoclonal antibodies described here were directed to separate determinants. The existence of unique N- and C-determinant binding antibodies confirms assignments which were made i n e a r l i e r Fd studies. While the c e l l fusion technique yields antibody products which may not accurately represent the normal repertoire i n serum and would therefore not rule out the existence of additional determinants, no evidence was found i n previous studies to suggest that these might exist i n s i g n i f i c a n t quantities. By using a conventional protocol, rabbit antisera were raised to each of these monoclonal antibodies. After the appropriate absorptions, i t was shown that a portion of the remaining a c t i v i t y was s p e c i f i c for combining s i t e related determinants of the hybridoma protein. These determinants were found to be unique to the hybridoma i n question as no r e a c t i v i t y of anti-idiotype with antibody directed towards a d i s s i m i l a r antigen was detected. The anti-idiotypes thus defined can therefore be used to investigate questions concerning the pre-dominance of a-particular idiotype i n serum, and the presence i f any, of simi-l a r idiotypes on receptors of c e l l s involved i n regulating the immune response to Fd. These issues are .discussed i n two chapters f i r s t from the point of view of the Fd-1 antibody (chapter 3), followed by studies based on the Fd-2 a n t i -body (chapter 4). 41 CHAPTER 3: CELLULAR EXPRESSION OF Fd-1 IDIOTYPE Monoclonal antibodies have proven to be useful as probes i n the study and mapping of individual antigenic determinants on complex molecules. The s p e c i f i c i t y of these reagents has enabled researchers to obtain precise information on the location of s p e c i f i c epitopes on experimental antigens. Assignments of antigenic determinants have been made on the basis of r e a c t i v i t y of antibody with enzymatically cleaved fragments of the native antigen, cross r e a c t i v i t y of antibody for polymorphic forms of the same antigen or deductively, on the basis of cross r e a c t i v i t y of antibody for synthetic analogues (54-56). One factor which these studies f a i l s to account for however, i s the role of t e r t i a r y structure i n the recognition of s p e c i f i c antigenic determinants by antigen reactive lymphocytes. The process by which B c e l l s recognize antigen including the structure and genetics of the immuno-globulin receptor which i t displays has been well characterized for many years. Although a large amount of work has, i n the past decade, been directed towards elucidation of the equally s p e c i f i c receptor on the T c e l l , many questions remain unanswered. The most d e f i n i t i v e work in this area has u t i l i z e d small haptenic molecules as antigens rather than the more complex polypeptides. With use of these haptens i t has been shown quite convincingly that s p e c i f i c T c e l l s bear i d i o t y p i c determinants which react with a n t i -i d i o t y p i c antisera raised against the major idiotypes expressed i n the serum of immunized animals (57,58). I t would appear that when such s t e r i c a l l y stable structures as these haptens are used as antigens, both reactive B and T c e l l s share common idiotypy, presumably by way of s i m i l a r i t i e s i n the structure of their respective antigen receptors. While i t i s generally accepted that V markers can be found on T c e l l s , no V markers have yet been detected. With regard to B and T c e l l recognition of more complex antigenic 42 structures, the information i s not as clear cut. Immunoglobulin receptors on B c e l l s are dependent for their interaction not only on the primary structure of an antigen epitope but also on i t s secondary and t e r t i a r y structure. At the T c e l l l e v e l of recognition, there are a number of examples i n the l i t e r a t u r e indicating that secondary and t e r t i a r y structures may not be of great importance. Thus i t has been found that antibodies raised to native lysozyme do not cross-react with i t s reduced and carboxymethylated derivatives, while cell-mediated immunity was cross-reactive regardless of whether animals were immunized with native or denatured material (59). Similar observations were made by Parish using native and chemically modified f l a g e l l i n (60). More recently, studies on i n s u l i n and i t ' s antigenic peptides have shown essenti a l l y the same results (61). There are at least two explanations for these data: either B c e l l s (or Ig) recognize determinants which are conformation dependent while T c e l l s recognize different determi-nants which are not configuration dependent and are d i s t i n c t from those recognized by B c e l l s , or the same determinants are recognized by both c e l l types but the T c e l l receptor i s not as configuration dependent as i s the B c e l l receptor. Studies on the commonly used hapten systems have provided useful i n f o r -mation regarding the role of i d i o t y p i c interactions between T and B c e l l s recognizing the same determinant. In general, i d i o t y p i c interactions have so far dealt only with responses towards individual epitopes whereas i n c l a s s i c a l immunology, immune responses are primarily based upon a cooperative association between clones of lymphocytes s p e c i f i c for different epitopes of a complex antigen molecule. Where idi o t y p i c regulation can so demonstrably regulate immune responses towards a given epitope, then i t i s conceivable that the effects may also influence the outcome of responses towards other i d i o -t y p i c a l l y unrelated, but s t r u c t u r a l l y associated determinants on the same 43 molecule. Associative recognition models of c e l l cooperation were o r i g i n a l l y based on the premise that T and B c e l l s did not recognize the same determi-nants of an antigen (62). Idiotypic studies have since shown that t h i s i s not the general case, hence the d i s t i n c t i o n between haptenic and ca r r i e r determi-nants may only be a r e l a t i v e one. I t would seem unlikely that i d i o t y p i c recognition could function separately, and i n i s o l a t i o n of, interactions based on hapten-carrier recognition. These two modes of lymphocyte communi-cation might be more closely interrelated. Predictable as t h i s hypothesis may be, i t has yet to be v e r i f i e d experimentally. Chemical haptens have served as useful models of lymphocyte recognition although these are not i n t r i n s i c antigenic epitopes, and must be conjugated to large molecular weight carriers i n order to become immunogenic. I t i s presumed that this provides s u f f i c i e n t T c e l l help for hapten s p e c i f i c B c e l l s . In view of the demonstrated a b i l i t y of T c e l l s to recognize hapten according to i d i o -typic studies, t h i s interpretation may be incorrect i n the sense that the c r u c i a l interaction missing from t h i s example may not l i e i n the absolute s p e c i f i c i t y of individual T and B c e l l s (polarity) but i n the manner i n which they are connected. The emphasis of hapten research has been placed s t r i c t l y on the outcome of i d i o t y p i c manipulation on the response to the hapten i t s e l f . Considering the complexity of the macromolecular immunogen however, responses to the s p e c i f i c hapten represents a small component of a potentially very heterogeneous immune response. Were i t not for t h i s fact, i t might be possible to study the influence of i d i o t y p i c manipulations involving the hapten on the responses towards other determinants of the immunogen. The complexity of most protein antigens precludes fractionation of the entire molecule into defined antigenic portions p a r t i c u l a r l y i f the molecule were large enough for t e r t i a r y structure to play a s i g n i f i c a n t role. Idiotypic probes on the other hand do constitute a highly spe c i f i c marker against which 44 responses to the whole antigen may be monitored. This i s especially relevant to the case of Fd as i t consists esse n t i a l l y of two haptenic groups for which s p e c i f i c monoclonal antibodies have been defined. Materials and Methods Radioimmune assay for idiotype Idiotype expression i n mouse serum was measured using a s o l i d phase radioimmune assay. A f f i n i t y - i s o l a t e d anti-idiotype antibody was used at a concentration of 1 lig/ml i n pH 9.6 carbonate buffer for a period of 1 hour at 37°C. Subsequently, coated plates were washed with PBS-Tween buffer and incubated for 15 minutes with PBS-Tween buffer containing 0.1% by weight of bovine serum albumin (Sigma) and normal B10.BR serum (.25%). Plates were then washed i n PBS-Tween buffer p r i o r to incubation with assay samples. Test samples of immune sera at a d i l u t i o n of 1/40 i n PBS-Tween buffer and control samples of non-immune sera containing added quantities of monoclonal Fd-1 antibody ranging from 1 ng - 1280 ng/ml were dispensed i n t r i p l i c a t e to the wells of prepared plates. After 60 min incubation at room temperature, the plates were washed, and then incubated as before with 0.1 ml/well of a 125 solution of I-labelled Fd-1 monoclonal antibody labelled by the chloramine T procedure (63) using a t o t a l of 150,000 cpm/well. After 60 min incubation, the plates were washed, and the individual wells were cut out using a single edged razor blade and the amount of r a d i o a c t i v i t y bound per well was counted i n a Beckman Biogamma radioisotope counter. A standard i n h i b i t i o n curve was constructed using cold Fd-1 monoclonal antibody as a competitor. Percent i n h i b i t i o n was calculated using cpm/well i n the presence of normal mouse serum as zero, and taking cpm i n the lower plateau portion of the curve as 100% i n h i b i t i o n . Percent i n h i b i t i o n of binding i n the presence of test sera was then read d i r e c t l y from this standard curve. Values obtained were corrected 45 for d i l u t i o n and are expressed as nonogram equivalents of idiotype per ml of serum.. In vivo administration of anti-idiotype B i o l o g i c a l effects of anti-idiotype were assessed i n whole animals by in j e c t i o n of quantities of anti-idiotype intraperitoneally into non-immune mice. In early experiments the b i o l o g i c a l effects of anti-idiotype was assessed i n groups of mice given injections of anti-idiotype or normal mouse Ig i n comparison to mice whic h received no injections p r i o r to antigen. One week following i n j e c t i o n of anti-idiotype, mice were immunized with 50 ug Fd i n CFA intraperitoneally on day 0. Twenty-one days after priming, they were bled and t i t r e s of anti-Fd antibody as w e l l as r e l a t i v e amounts directed to the N- or C-determinant were measured. Specific idiotype was determined by ELISA and RIA. In l a t e r experiments quantities of whole anti-idiotype serum were substituted i n place of a f f i n i t y isolated antibody. Adoptive transfer of anti-idiotype treated spleen c e l l s -Spleen c e l l s from either immune or non-immune BIO.BR mice were resuspended i n anti-idiotype serum at a concentration of 12 ug idiotype binding capacity per ml, or i n an equal d i l u t i o n of normal rabbit serum which had been adsorbed against mouse Ig for a period of 45 min. on ice . Cells were then washed three times with Dulbecco's modified Eagle medium (Gibco) and resuspended i n rabbit complement (Lowtox, Cedarlane) at a f i n a l d i l u t i o n of 1/5 i n medium and incu-bated for 45 min. at 37° C. Cells were then washed three times i n medium, adj-usted to a concentration of 2 x 10 8 cells/ml and injected i n t r a p e r i t o n a l l y (0.1 ml/mouse) into normal B10.BR mice which had received 500 rad whole body i r r a d i a t i o n from a gamma source (Gammacell 200, Atomic Energy of' Canada) 24 hr. previously. Immediately after c e l l transfer, recipient mice were given an intraperitoneal i n j e c t i o n of 50 ug Fd i n CFA. In some experiments animals were also immunized 46 with KLH (100 yg) i n CFA. Antibody t i t r e s of recipient mice were determined from sera taken 21 days post transfer when non-immune c e l l s were used while t i t r e s of transferred immune spleen c e l l s were determined from sera taken 7 and 21 days post-transfer. In some experiments, spleen c e l l s were f i r s t fractionated before treatment with anti-idiotype or normal rabbit serum. T lymphocytes were enriched from suspensions of whole spleen c e l l s using the technique of nylon wool adherence. Nylon wool columns were prepared according 8 to the method of Ju l i u s et_ ail. (64) . A t o t a l of 3 x 10 spleen c e l l s were passed over nylon wool (20g) packed to a volume of 20 ml into a 30 ml syringe ba r r e l . C e l l recovery using this procedure varied between 18-27%. T c e l l depleted spleen c e l l suspensions were prepared using treatment with a mono-clonal anti-Thy 1.2 antibody ( g i f t of Dr. H-S Teh) plus rabbit complement. Spleen c e l l s were resuspended i n 200 yg/ml of anti-Thy 1.2 antibody i n culture g medium at a density of 10 cells/ml and incubated at 4°C for 30 min. The c e l l s were then washed, resuspended i n the equivalent volume of medium containing a source of rabbit complement (Low Tox, Cedarlane) 20% v/v, and incubated for 30 min at 37°C. The c e l l s were then washed three times i n culture medium and counted. Viable c e l l recovery under these conditions ranged from 52-57%. 51„ • • Cr release cytotoxicxty assay Two target c e l l l ines were u t i l i z e d i n an assay designed to measure the complement dependent cytotoxic a c t i v i t y of a n t i - i d i o t y p i c antiserum anti-Fd-1. The l i n e s i n question were both hybridoma c e l l l i n e s producing monoclonal anti-Fd antibody Fd-1 and Fd-2 as described i n the text of the thesis. Target c e l l s were obtained from the ascites f l u i d of mice i n which the particular hybridoma c e l l l i n e was being passaged. These c e l l s were labelled using the following procedure: 2 x 10^ c e l l s were incubated with 250 yCi Na~^CrO, (New 47 England Nuclear) i n a volume of 0.5 ml in 10% DMEM for 1 hr at 37°C with occasional shaking. The c e l l s were then washed twice i n medium and reincubated for a further 30 minutes. Labelled c e l l s were then washed twice more, counted, and resuspended to a concentration of 10 /ml i n culture medium. At the same time, doubling dilutions of normal rabbit serum and anti-Fd-1 serum were prepared i n culture medium in U-bottom 96 well microtitre plates (Dynatech). 4 A t o t a l of ten m i c r o l i t r e s of labelled c e l l suspension containing 10 target c e l l s was then added to each well and the plates were incubated for 45 minutes at 4°C. The c e l l s were then pelleted by centrifugation, washed once in .2 ml/ well of ice-cold culture medium and resuspended i n .15 ml of culture medium containing a source of rabbit complement (Low-Tox, Cedarlane Labs) at a f i n a l concentration of 10% v/v. The plates were incubated i n the presence of comple-ment for 45 minutes at 37°C and then centrifuged. Aliquots of the supernatant f l u i d (.1 ml) were removed for counting of soluble isotope in a radioisotope counter (Beckman Biogamma). Specific cytotoxicity was calculated i n r e l a t i o n to two controls: (a) spontaneous release (complement only) and (b) 100% release (soluble i n Triton buffer) using the following formula: „ „ . e. . t . .. Test - Spontaneous „ ,„„„ % Specxfxc cytotoxxcity = _ X 100% 100% - Spontaneous Results Preliminary characterization of idiotype expression Previous immunogenetic studies have shown that the proportion of antibody i n serum directed towards the N- and C-determinants of Fd varies between different strains of mice (66). Using sera from individual mice of two representative strains which had been t i t r a t e d for N- and C-determinant reactive antibody, an attempt was made to determine the r e l a t i v e amounts of idiotype expressed i n immune serum. Individual sera which had previously been 48 t i t r a t e d and assayed for N-determinant antibody a c t i v i t y were tested with the anti-idiotype to determine the extent to which they could be inhibited. Varying quantities of the anti-idiotype or equivalent concentrations of normal rabbit serum were incubated with constant amounts of previously t i t r a t e d a n t i -sera. These were subsequently tested for their a b i l i t y to react with Fd i n the ELISA. Percent i n h i b i t i o n s of N-determinant r e a c t i v i t y were estimated on previous calculations of N-determinant s p e c i f i c antibody present i n each a n t i -serum sample (44). Results are shown in figure 13. In B10.BR mice, i n which N-determinant reactive antibody i s r e l a t i v e l y low (between 20 and 30% of t o t a l antibody), i n h i b i t i o n by the anti-idiotype serum ranged from 0-72%. In three of the s i x individual antisera tested, at least 50% of the N-determinant sp e c i f i c antibody was i n h i b i t a b l e by the anti-idiotype. In B10.S mice, i n which N-determinant s p e c i f i c antibody i n the anti-Fd population i n whole serum i s approximately 65%, a similar pattern i s observed. This indicates that the rabbit anti-idiotype serum i s reacting with a s i g n i f i c a n t amount of N-deter-minant s p e c i f i c antibody i n both these strains of mice. The results show that the Fd-1 idiotype constitutes a variable, but s i g n i f i c a n t proportion of N-determinant s p e c i f i c antibody i n individual mice and that the idiotype i s conserved between two strains of mice which share id e n t i t y at the Ig-1 locus. While this form of competitive binding assay yields useful q u a l i t a t i v e information on idiotype expression, i t i s not p a r t i c u l a r l y suited to screening large numbers of samples. I t was also f e l t that a more direct measure of idiotype expression could be obtained using a quantitative radioimmune assay. Description and standardization of RIA Anti-Fd-1 anti-idiotype was therefore used i n a s o l i d phase radioimmune assay i n order to survey expression of the s p e c i f i c Fd-1 idiotype amongst individual mice of the BIO.BR as well as i n other related strains. A n t i -49 '00 I A % H Inhibition I 1 I i I • B10.BR Individuals B10-S Individuals - 1/8 - H 1 /16 Figure 13: Inhibition of N-determinant directed antibodies by anti-idiotype to Fd-1. Binding of individual anti-Fd serum samples to Fd coated plates was assayed i n the presence of rabbit a n t i - i d i o -type. Percent i n h i b i t i o n of N-determinant s p e c i f i c antibodies was calculated as described i n Materials and Methods. (A) Individual serum samples from BIO.BR mice. (B) Individual serum samples from B10.S mice. Dotted lines indicate results obtained at serum di l u t i o n s of 1:8 and s o l i d l i n e s , results obtained at 1:16. 50 Figure 14: Radioimmunoassay for c i r c u l a t i n g idiotype i n mouse serum. Varying quantities of unlabelled Fd-1 i n normal B10.BR serum were incu-bated over anti-idiotype coated wells of a v i n y l microtitre plate. After 1 hr. the samples were washed out and replaced with standard 125 amounts of I-labelled Fd-1. Percent i n h i b i t i o n of binding was calculated r e l a t i v e to control samples incubated with normal BIO.BR serum alone. 51 i d i o t y p i c antibody which had been a f f i n i t y isolated by passage of serum over a column of Fd-1 bound to Sepharose-4B-8L (Pharmacia) was used to coat the wells 125 of polyvinyl microtitre plates. I-labelled Fd-1 antibody w i l l bind to anti-idiotype coated wells and this interaction can be quantitated by choosing a concentration of probe which f a l l s within the range of 50-75% saturation of the s o l i d phase, the point at which maximal s e n s i t i v i t y of this form of RIA i s attained. Pre-incubation of the s o l i d phase with set quantitites of unlabelled Fd-1 quantitatively i n h i b i t s the binding of labelled Fd-1. In t h i s way a competition curve can be constructed which can be used as a reference i n comparison with which concentrations of idiotype present i n unknown samples may be determined. A sample competition curve i s shown i n figure 14. The lower l i m i t of s e n s i t i v i t y attainable with this assay depends upon two p r i n c i p a l factors. F i r s t l y , the s e n s i t i v i t y i s d i r e c t l y dependent upon the sp e c i f i c a c t i v i t y of the probe. While high s p e c i f i c a c t i v i t i e s are desireable i n this assay, conditions of radioiodination were choosen i n order to minimize the extent of damage done to the probe. Under the chosen conditions, i t can be seen that concentrations of idiotype down to several nanograms per ml can readily be detected. As i t was necessary to perform simultaneous determinations of antibody t i t r e s , determinant s p e c i f i c i t y as well as idiotype on each of the individual serum samples tested i t was necessary to economize on the quantitites of serum which were consumed i n each assay. Ten m i c r o l i t r e s of serum were diluted to a concentration of 1/40 so that each sample could be run i n t r i p l i c a t e (3 x 0.1 ml). The effective lower l i m i t of s e n s i t i v i t y for this d i l u t i o n then becomes 40 x 1 ng/ml or 40 ng/ml of idiotype. Taking into account the range of v a r i a t i o n observed between normal samples which presumably contain no idiotype, the s t a t i s t i c a l baseline was established at 45 ng/ml. Significance i n this system i s defined as 2 standard deviations about the mean 52 therefore concentrations of idiotype less than or equal to 45 ng/ml are l i s t e d as zero. Results of serum survey for idiotype expression Enzymatic degradation of Fd e f f e c t i v e l y destroys one of the antigenic determinants, generating a molecule which i s immunologically monovalent. The derivatives which are obtained following either trypsin or carboxypeptidase A treatment can be used i n an ELISA to determine the quantities of antibody i n immune serum which bind to either the N- or the C-determinant. The proportion of N/C antibody i s f a i r l y constant within a given s t r a i n so that character-i s t i c s t r a i n s p e c i f i c values can be calculated (66). Strains of different H-2 haplotype have been found to d i f f e r i n this s p e c i f i c r a t i o i suggesting some form of MHC-linked control over the amount of antibody produced to the two determinants. As large numbers of serum samples which had previously been t i t r a t e d for N- and C-antibody were available, these were selected i n order to perform assays for idiotype content. In this way i t i s possible to calculate a figure which expresses idiotype as a percentage of the t o t a l N-specific antibody. Since this calculation takes into account the t i t r e of anti-Fd antibody present, t h i s figure can be used as a basis for comparison of i d i o -type expression between individuals and between strains. The results of a survey for idiotype i n immune serum from several different strains are pre-sented i n table 2. Idiotype expression i s very c l e a r l y limited to strains of mice which share id e n t i t y at the Ig-l° locus. This survey, involving strains of mice congenic at H-2 and representing different allotype linkage groups f a i l e d to reveal any association with other Ig-1 a l l e l e s . The expression of Fd-1 i s apparently not influenced by genes within the H-2 region. In vivo effects of anti-idiotype As administration of anti-idiotype has been shown to have marked effects Table 2. Analysis of anti-Fd sera taken from mice of various haplotypes and allotypes after secondary immunization with Fd. Idiotype Strain Number of Animals Haplotype Allotype Anti-Fd (yg per ml ± SEM) Anti-N (yg per ml) Expression (ng per ml ± SEM) % Idiotype Expression BIO.BR 12 k b 9.0 1.8 ± 1.3 310 ± 37 22.9 BIOS Pool s b 1.2 0.7 88 13.3 C57BL/6 6 b b 10.5 ± 3.8 2.2 237.4 ± 76.8 10.8 SJL 6 s b 1.4 0.7 250 36.8 CE/J 6 k f 14.0 ± 0.03 1.2 ± 0.2 0 0 AKR/J 6 k d 10.8 ± 0.16 1.2 ± 0.2 0 0 ST/J 6 k a 12.3 ± 1.0 2.4 ± 0.2 0 0 RF/J 6 k c 10.7 ± 0.1. 0.5 ± 0.2 0 0 C58/J 6 - k a 13.2 ± 0.04 1.1 ± 0.3 0 0 U> 54 on the course of immune responses i n vivo, a preliminary experiment was carried out to determine i f anti-Fd-1 had any particular effects on the immune response to Fd i n mice. Groups of unprimed BIO.BR and DBA2 mice were given a single dose of p u r i f i e d anti-Fd-1 or normal rabbit Ig by intraperitoneal i n j e c t i o n . One week l a t e r , these animals were immunized with 50 yg Fd i n CFA and bled 21 days l a t e r . Using a single fixed dose of anti-Fd-1 (15 yg/mouse) injected 1 week before Fd, no direct effect on the primary anti-Fd response of BIO.BR or DBA2 mice was observed (table 3). Similar results were obtained i n mice treated with either anti-Fd-1 or NRS and immunized with KLH (data not shown). Because i t has been shown i n other systems that the effects of a n t i -idiotype may depend upon either the dose administered or the subclass (32), rather than attempt to fractionate the limited quantities of anti-idiotype available, i t was f e l t that a functional characterization of the antiserum should f i r s t be undertaken. A "^Cr release assay was therefore designed to test the cytotoxic potential of anti-Fd-1 on the appropriate target c e l l s . Anti-Fd-1 was found to have a measurable cytotoxic t i t r e at a d i l u t i o n of 1/8 (table 4). The cytotoxic property of the anti-idiotype was then used under controlled conditions i n which spleen c e l l s were removed from immune mice and treated i n v i t r o with either normal rabbit serum or anti-Fd-1 plus C and then adoptively transferred into syngeneic irradiated recipient mice along with a 2° challenge of Fd. Control animals were immunized with KLH. These animals were then bled after 7 days and their individual antibody t i t r e s determined by ELISA. Data from this experiment i s shown i n table 5. Anti-Fd-1 plus C treatment of spleen c e l l s resulted i n an enhanced t i t r e of anti-Fd antibody i n comparison to the control group, i n which c e l l s were exposed to NRS plus C. When these sera were analyzed for N-determinant antibody i t was observed that the amount of anti-N-antibody present had increased. This increase was found to be proportionate to the overall increase i n anti-Fd 55 Table 3: Administration of anti-Fd-1 antibody i n vivo. Small groups of BIO.BR and DBA/2 mice were given an intraperitoneal i n j e c t i o n of normal rabbit Ig (nRIg) or a f f i n i t y isolated rabbit anti-Fd-1 anti-body 7 days prior to immunization with Fd or KLH. Experiment No. 1 Strain BIO.BR BIO.BR Treatment nRIg anti-Fd-1 ug/ml anti-Fd±S.E.M. 12.3±3.2 10.8+4.8 DBA/2 DBA/2 BIO.BR BIO.BR nRIg anti-Fd-1 nRIg anti-Fd-1 0.2±0.1 0.4±0.2 ug/ml anti-KLH±S.E.M. 2.0±0.8 1.4±0.3 56 Table 4: Complement dependent cytotoxicity of anti-Fd-1 serum on selected c e l l u l a r targets. % Specific cytotoxicity Target c e l l l i n e treatment d i l u t i o n : 1/4 1/8 1/16 1/32 Fd-1 hybridoma NRS +C 8.1 6.2 6.6 0.0 anti-Fd-1+C 27.3 12.6 5.1 3.3 anti-H-2 d +C 37.4 38.4 15.6 17.1 Fd-2 hybridoma NRS +C 6.3 2.6 0.0 0.0 anti-Fd-1+C 1.3 5.0 0.0 0.0 anti-H-2d+C' 41.9 30.0 13.3 6.8 SP1/0 myeloma NRS +C 2.8 2.6 0.0 0.0 anti-Fd-1+C 6.6 1.0 1.7 0.0 anti-H-2 d +C 21.4 13.2 5.6 9.7 Table 5, Analysis of anti-Fd sera from an adoptive secondary response i n B10.BR mice. Spleen c e l l s from Fd-primed animals were treated with either NRS + C or anti-Fd-1 + C' prior to transfer. Data are averages from 6 individuals per group. Idiotype % Anti-Fd Anti-N Expression Idiotype Treatment Antigen (yg per ml ± SEM) (yg/ml ± SEM) (ng per ml ± SEM) Expression NRS + C Anti-Fd-1 + C NRS + C 1Anti-Fd-1 + C Fd Fd KLH KLH 1.0 ± 0.6 2.9 1.3 0 (4.85 0.70) 0 (4.36 1.92) 0.2 ± .02 0.6 ± .08 87.2 ± 71.0 358.4 ± 297.0 39.8 56.5 + Amount of N-specific antibody as calculated by spec i f i c r e a c t i v i t y on ELISA plates to which degraded fragments of Fd containing only the N- or C-determinant had been attached. Expression of the Fd-1 idiotype i n individual antisera calculated by RIA. % of the N-specific antibody which reacted with anti-Fd-1 in the RIA. +Anti-KLH t i t r e (yg/ml ± S.E.M.). 58 t i t r e , hence the actual percent of N-antibody remained constant. For this to be true there must therefore have been a corresponding increase i n antibody directed towards the C-determinant. When sera were analyzed for idiotype content, i t was found that idiotype expression had increased as w e l l , but by a factor exceeding the general increase i n anti-N antibody, indicating that the actual percentage of idiotype had risen. In a second experiment, the role of complement i n the adoptive transfer was examined. Recipient mice received 2 x 10^ Fd primed spleen c e l l s which had been treated with NRS plus C , anti-Fd-1 plus C or anti-Fd-1. Immediately after adoptive transfer, mice were immunized with both Fd and KLH i n CFA. They were bled 7 and 21 days after treatment. The results are shown in table 6 i n which i t can be seen that the anti-Fd-1 treatment enhanced the adoptive secondary response to Fd whether complement was added to the system or not. Neither treatment had any effect on the response to KLH. Alterations i n the magnitude of antibody responses have been induced i n other studies by the action of anti-idiotype °n regulatory T c e l l s which express idiotype. The effects of anti-Fd-1 treatment are reminiscent of such r e s u l t s , and an experiment was devised i n order to determine whether anti-Fd-1 was exerting an effect at the l e v e l of T c e l l s or of B c e l l s . Spleen c e l l s were f i r s t fractionated using the p r i n c i p l e of nylon wool separation of T c e l l s and treatment with anti-Thy 1.2 plus C for depletion of T c e l l s . These T-enriched and T-depleted c e l l populations were then treated separately with normal rabbit serum or anti-Fd-1 as before and adoptively transferred into i r r a d i a t e d recipients following the combinations outlined i n figure 15. These results demonstrate that anti-Fd-1 i s primarily affecting T c e l l s as an enhanced anti-Fd response i s obtained only i n combinations where T c e l l s were exposed to the action of anti-idiotype independently of whether or not B c e l l s were s i m i l a r l y treated. Treatment of B c e l l s alone with anti-Fd-1 does not 59 Ug ANTIBODY PER ml TREATMENT ANTIGEN I 2. £ 4 T CELLS B CELLS NRS NRS Fd ' 1 • NRS ANTI-Fd-I Fd ' 1 ' ANTI-Fd-I ANTI-Fd-I Fd 1 ANTI-Fd-I NRS Fd | ' Figure 15: Treatment of T-or B-enriched c e l l subpopulations with anti-Fd-1 + C'. Cells were isolated as described i n Materials and Methods and treated with anti-Fd-1 + C' i n v i t r o . Treated c e l l s were adoptively transferred to sublethally irradiated syngeneic recipients which were subsequently challenged with antigen. Animals were bled on day 7 and t i t r e s of anti-Fd antibody were determined by ELISA. Table 6. The influence of anti-Fd-1 on the adoptive primary or secondary response of BIO.BR mice to FD. Spleen c e l l s from Fd-immune or naive BIO.BR were treated with NRS plus complement or anti-Fd-1 ± complement prior to in j e c t i o n into irradiated BIO.BR recipients and bled either 7 or 21 days l a t e r . Each experiment had 6 (expts. 1 and 2) or 10 (expts. 3 and 4) animals per group. Time of Anti-Fd P* Anti-N Idiotype % Idiotype Expt. No. Treatment Bleed (yg/ml ± SEM) Anti-N (yg/ml ± SEM) Expression P* Expression (ng/ml ± SEM) 1** NRS + C primary 21 days 1.82 + 0.95 7.28 + 0.25 60.0 ± i,32 8.3 anti-Fd-1 + C 21 days 3.89 + 1.83 NS 1.49 + 0.75 121.0 ± 57 NS 8.1 2** NRS + C secondary 7 days 1.01 + 0.57 0.24 + 0.21 87.2 ± 41 39,8 anti-Fd-1 + C 7 days 2.94 + 1.34 NS 0.64 + 0.34 358.4 ± 297 NS 56.5 ^ NRS + C secondary 7 days 1.07 + 0.53 0.36 + 0.33 ND anti-Fd-1 + c 7 days 2.12 + 0.90 <0.05 0.56 + 0.29 ND anti-Fd-1 7 days 2.90 + 0.95 <0.05 0.83 + 0.44 ND NRS + C secondary 21 days 3.19 + 1.92 0.97 + 0.70 340 ± 192 anti-Fd-1 + c 21 days 13.27 + 4.38 <0.025 4.24 + 0.14 870 ± 4.76 NS 16.4 anti-Fd-1 21 days 11.34 + 4.77 <0.05 2.28 + 0.85 380 ± 144 NS 14.3 Differences i n t i t r e s between anti-Fd-1 treated mice and NRS treated controls were analyzed by Students' t test to establish the significance of differences between the groups. NS indicates values p => .05. Six animals per experimental group. t^c >V Ten animals per experimental group. *** Percentages are based on values obtained only from sera of mice which had detectable levels of N-epitope s p e c i f i c antibodies and therefore do not r e f l e c t a direct percentage of numbers shown i n columns 6 and 7. ON o 61 s i g n i f i c a n t l y change the response from that of the control group. I t appears, therefore, that at least at the l e v e l of antibody production, that the Fd-1 idiotype i s expressed on a subpopulation of T c e l l s which may be susceptible to the action of anti-idiotype. Owing to the h i s t o r i c a l development of these experiments, the sera from the o r i g i n a l T plus B c e l l adoptive transfer experiment were not available for analysis so that a second experiment was performed u t i l i z i n g only the combinations i n which T c e l l s alone were treated with anti-idiotype and sub-s t i t u t i n g unprimed spleen c e l l s ; - While the antibody t i t r e s resulting from t h i s repeated experiment were not as high as those observed i n the i n i t i a l experiment, p a r a l l e l findings to the results of the whole spleen experiment were seen when the sera were compared for anti-N antibody and i d i o -type content (table 7). As before, the increase i n N-antibody r e f l e c t s the increase i n t o t a l anti-Fd antibody, while the increase i n idiotype exceeded this value. The fact that similar results were observed when these experi-ments were performed on unfractionated spleen c e l l s or using a T-enriched c e l l population suggests that a common idiotype-expressing T c e l l may be the component which i s responsible for these effects. In order to determine whether differences i n t i t r e between anti-Fd-1 treated and NRS treated T populations were maintained, a further experiment was done i n which primed T c e l l s were treated as described above. Recipients were immunized with both Fd and KLH i n CFA, and bled 7 and 21 days l a t e r . Results are shown i n table 8 i n which i t can be seen that anti-Fd-1 treated animals have s i g n i f i c a n t l y higher levels of antibody that do controls after 21 days. KLH responses were not s i g n i f i c a n t l y different between the two groups. Studies using antibody feedback In other systems, i t has been shown that immune responses normally Table 7. Analysis of anti-Fd sera i n an adoptive primary response i n B10.BR mice. Spleen c e l l s of BIO.BR mice were enriched for B and T c e l l populations using nylon wool or anti-thy-1 plus C. T c e l l enriched populations were treated with either a n t i -Fd-1 or NRS before mixing with B c e l l s (NRS treated) and administration to irradiated recipients. Data presented are averages from 8 individuals per group. m Idiotype Treatment . _ „, . „ „ . „ T .. Antx-Fd Anti-N Expression % Idiotype T c e l l s B c e l l s (yg per ml ± SEM) (ng per ml ± SEM) (ng per ml ± SEM) Expression NRS NRS 0.72 ± 0.34 288 ± 35 60 ± 34 20.8 Anti-Fd-1 NRS 1.15 ± 0.34 435 ± 4 2 121 ± 4 6 27.8 Table 8. The influence of anti-Fd-1 on the adoptive secondary response of BIO.BR mice to Fd. Spleen c e l l s from Fd-primed B10.BR mice were separated by nylon wool or anti-thy-1 + complement to y i e l d T or B c e l l enriched populations. These populations were treated with either anti-Fd-1 or NRS pr i o r to transfer into irradiated recipients. In experiment 2, recipients were immunized with both Fd and KLH. Animals were bled 7 and 21 days l a t e r . There were 6 animals per group in experiment 1 and 12 per group i n experiment 2. .v Anti-N- Fd-1 Idiotype Experiment Treatment Time of Anti-Fd P epitope Expression % Idiotype Number T Cells B Cells Bleed (yg/ml ± SEM) (yg/ml ± SEM) (ng/ml ± SEM) Expression 1 NRS NRS 7 days 1.22 + 0.88 ND NRS anti-Fd-1 7 days 1.26 + 0.72 NS ND anti-Fd-1 NRS 7 days 3.05 + 2.83 NS ND anti-Fd-1 anti-Fd-1 7 days 2.25 + 2.20 NS ND T c e l l s only 2 NRS 7 days 1.40 + 0.55 ND anti-Fd-1 7 days 2.80 + 0.80 NS ND NRS 21 days 3.93 + 1.27 1.30 ± 0.80 627 ± 484 37.2 anti-Fd-1 21 days 23.63 + 12.00 <.005 3.32 ± 1.10 555 ± 264 16.7 Differences i n t i t r e s between anti-Fd-1 treated and NRS treated c e l l s were analyzed by Students' t test to establish significance of differences between groups. NS indicated non-significance and p values of >.05. 64 involve the p a r t i c i p a t i o n of anti-idiotype regulatory T c e l l s i n addition to the conventional role played by antigen binding, i d i o t y p i c T c e l l s (67). Idiotype s p e c i f i c T c e l l s have also been generated after immunization with idiotype bearing antibody, idiotype conjugated thymocytes and following administration of anti-idiotype antibody (68-70). This form of immunity to idiotype has been shown to s i g n i f i c a n t l y a l t e r the normal course of immune responses. Induction of a n t i - i d i o t y p i c T c e l l s under these conditions however, i s not equivalent with evidence for their role during the normal response induced by antigen. In order to determine whether a n t i - i d i o t y p i c T c e l l s played any role i n the anti-Fd response, an experiment was designed i n which non-immune mice were injected with the monoclonal Fd-1 antibody prior to immunization with antigen. Under these conditions a n t i - i d i o t y p i c lymphocytes would therefore be placed i n contact with idiotype before antigen binding lymphocytes were f i r s t stimulated by Fd. This experiment i s outlined i n table 9. Mice were given an intraperitoneal in j e c t i o n of 10 yg p u r i f i e d Fd-1 prior to immunization with Fd or a control antigen, KLH 7 days l a t e r . Two weeks after immunization, the mice were bled and their t i t r e s of antibody to Fd and KLH determined by ELISA. As seen i n group 1, administration of Fd-1 alone did not induce synthesis of anti-Fd antibody, nor were appreciable quantities of residual Fd-1 detectable. When Fd-1 was administered followed by immunization with Fd the antibody t i t r e which resulted was at least 3-fold higher than that obtained with Fd alone. In contrast, the s p e c i f i c i t y control groups 4 and 5 were not s i g n i f i c a n t l y affected by this same treatment. Pre-exposure of the mice i n group 2 to idiotype has resulted i n marked increases in their t i t r e of anti-Fd antibody. Sera from groups 2 and 3 were also analyzed for the proportion of N/C s p e c i f i c antibodies present. The ratios between groups 2 and 3 are not s i g n i f i c a n t l y different. Thus, the increase i n Table 9. The influence of the monoclonal Fd-1 on the immune response to Fd i n B10.BR mice. Seven days prior to immunization, animals received 10 yg of Fd-1. Animals were bled 7 and 14 days after immunization, were reimmunized on day 28 and bled again on day 35. In a l l instances, mice treated with Fd-1 alone showed no anti-Fd t i t r e , and mice treated with Fd-1 and Fd had s i g n i f i c a n t l y higher t i t r e s of anti-Fd antibody than did controls. There were 12 animals in each experimental group. Time of Anti-Fd or Idiotype Bleed anti--KLH Anti-N Expression % Idiotype Day -7 Day 0 Day 28 (day) dig/ml + SEM) (yg/ml ± SEM) (ng/ml ± SEM) Expression Fd-1 _ _ 7 ND 182 ± 65 Fd-1 Fd - 7 1.65 + 0.64 <, .005 0.34 ± 0.87 112 ± 53 Fd-1 KLH - 7 0.92 + ** 0.16 ND 133 ± 64 Fd _ 7 0.49 + 0.26 0.15 ± 0.02 104 ± 61 — KLH — 7 1.05 + 0.25 ND Fd-1 - - 14 0 Fd-1 Fd - 14 3.33 + 1.30 <, .005 ND - Fd - 14 0.50 + 0.40 Fd-1 - - 35 0 40 Fd-1 Fd Fd 35 27.18 + 7.98 < .05 10.45 ± 3.78 386 ± 156 3.7 - Fd Fd 35 11.38 + 4.63 5.30 ± 2.20 2450 ± 1644 46.2 Differences between Fd-1 treated immunized mice and untreated controls were analyzed by Students' t-test to establish significant differences between the two groups. Titres were calculated to KLH in these instances. Differences are not si g n i f i c a n t between the two groups. 66 anti-Fd antibody c l e a r l y involves a marked increase i n the amount of anti-C antibody i n treated mice. When mice from groups 2 and 3 were tested after a secondary i n j e c t i o n of Fd the same pattern of results was obtained. Animals which had received Fd-1 before antigen exhibited an increased t i t r e of t o t a l anti-Fd antibody, and a decreased r a t i o of N/C antibody. The effect of passive idiotype i n this experiment appears to be mainly on the production of an t i-C ant ib o dy. Discussion In the mouse, there exists a system of a l l e l i c polymorphisms involving the constant regions of immunoglobulin heavy chains. These antigenic s p e c i f i c i t i e s or allotypes, are inherited i n simple Mendelian fashion. Genes which code for the constant portion of immunglobulin heavy chains are located within a closely linked cluster located on chromosome 12 (30). The largest number of a l l e l e s occurs at the IgH-1 locus (IgG ). Linkage between the individual l o c i i s so strong that few recombinants between l o c i have been documented. Most strains which have the same Ig-1 a l l e l e also share a l l e l e s at each of the other l o c i . IgGH genes therefore tend to segregate as haplo-types, hence linkage groups are generally l i s t e d according to their Ig-1 a l l e l e s only. Unlike the c l e a r l y i d e n t i f i a b l e antigenic s p e c i f i c i t i e s of a l l o t y p i c markers, i d i o t y p i c determinants result from the association of l i g h t and heavy chains and are theore t i c a l l y dependent upon the segregation of at least two genes, In some cases genes linked to the l i g h t chain locus on chromosome 6 have been shown to govern expression of idotypic markers ( i b i d ) . In the majority of cases, however, idiotype markers of induced antibodies have been linked to the genes coding for heavy chain allotype. In the Fd system, rabbit anti-idiotype antiserum was used to define 67 binding s i t e related i d i o t y p i c determinants of the monoclonal Fd-1 antibody. When th i s anti-idiotype was used to determine the presence i n immune sera of molecules sharing idiotypy with Fd-1, two observations were apparent. F i r s t , idiotype bearing molecules were detected only when the sera had been derived from mice sharing identity at the Ig-1 locus. This i s i n accord with similar linkage studies performed using defined idiotypes of either polyclonal (71) or monoclonal antibodies (72). Second, i t was shown that idiotype was present i n 50% of the sera tested and th i s expression represented a variable proportion (between 0 and 100%) of the t o t a l anti-N population present. This l e v e l of v a r i a b i l i t y i n the expression of a r e s t r i c t e d idiotype i s consistent with observations from other monoclonal systems. Analysis of a large number of levan binding myeloma proteins has revealed the existence of two main categories of i d i o t y p i c determinants. Anti-idotypic sera prepared to 10 different levan binding myeloma proteins showed that each possessed i t s own unique individual i d i o t y p i c s p e c i f i c i t y (IDI) which was not shared by any other myeloma. In addition, most of the proteins carried a variable number of idiotypes which were shared by a number of other molecules (27). This second group of shared idiotype was termed a cross-specific idiotype (IdX). Private and public idiotypes are also present i n the antibody response to the arsonate hapten (18). On the basis of defined monoclonal i d i o -type markers t h i s response appears to be quite heterogenous. When sera from immune mice were screened for the presence of a particular idiotype belonging to the I d l category, only 50% of the sera contained detectable levels of idiotype, representing less than 2% of the total.antibody present. Private idiotype determinants therefore appear to be present at very low concentra-tions i n immune sera. Analogous findings were also observed i n a system using monoclonal antibodies to the hapten 4-hydroxy-3-nitro-phenyl-l-acetyl (NP). Hybrid c e l l l i n e s secreting monoclonal antibodies against idiotypes 68 of one selected anti-NP hybridoma protein, Bl-8, were isolated and used to characterize the variable portion of Bl-8 and other anti-NP antibodies a l l belonging to an antibody family which carries a predominant id i o t y p i c marker (19). Monoclonal anti-idiotypes defined private, combining s i t e related idiotopes which were expressed on 1-10% of serum antibodies, as well as public, non-combining s i t e related idiotopes which were more widely expressed on 10-100% of serum antibody. Expression of private idiotypes was also shown to be linked to the Ig-1 locus. In th i s system, private i d i o t y p i c determinants also appear to represent a minority of the serologically detectable idiotypes. On the basis of the r e s t r i c t e d range of expression observed for Fd-1, the idiotype would appear to belong to the I d l category. In the absence of additional idiotype markers for the N-determinant however, t h i s assignment must be considered tentative as the rabbit anti-idiotype would probably also be detecting a public s p e c i f i c i t y which i s present i n low concentration. Effects of anti-idiotype on responding c e l l s When immune spleen c e l l s were treated with anti-idiotype plus C and transferred to irradiated recipient mice, the anti-Fd response i n the recipients was found to be increased i n comparison with control animals. When sera from these animals were examined for idiotype expression i t was noted that while the re l a t i v e proportion of N/C antibody was unchanged, the absolute amount of Fd-1 idiotype increased 2-4-fold. Despite the fact the idiotype bearing B c e l l s may have been exposed to anti-idiotype plus C' they have cl e a r l y not been eliminated from the treated spleen c e l l population. The enhanced idiotype t i t r e argues either i n favour of a simple mechanism of B c e l l stimulation which i s independent of complement or a more indirect mechanism acting upon idiotype bearing T c e l l s which may or may not be 69 dependent upon C'. The observation that enhanced response to Fd was obtained either i n the presence or absence of C1 implies C independence. However, i t does not establish the mechanism of activation which could involve c e l l stimulation by antibody, or the elimination i n vivo of antibody coated c e l l s . The l a t t e r p o s s i b i l i t y appears to be the most favoured one at this time. Because anti-idiotype treatment of adoptively transferred spleen c e l l s did not appear to be eliminating idiotype bearing B c e l l s , since increased idiotype expression was observed with treated c e l l s , i t was considered possible that the treatment was affecting a T c e l l population. When the appropriate experiments were performed i t was found that an increased anti-Fd response was observed only i n the case where the transferred T c e l l s had been treated with anti-idiotype. This increase, again, was reflected both i n l e v e l of anti-Fd antibody and Fd-1 idiotype. In view of the fact that the same treatment of B c e l l s was without effect, i t was concluded that anti-idiotype treatment se l e c t i v e l y affected an idiotype bearing T c e l l population whereas B c e l l s did not appear to be susceptible to i t s mode of action. While the primary outcome of anti-idiotype treatment can be accepted as a s t r i c t l y i d i o t y p i c effect, additional observations can be made. In the majority of hapten systems studied to date, anti-idiotype effects are usually r e s t r i c t e d to changes i n the expression of the corresponding idiotype while t o t a l antibody levels remain unchanged. As mentioned e a r l i e r however, these haptens are administered i n the form of multideterminant derivatives of immunogenic car r i e r s and usually result i n the formation of substantial quantities of anti-hapten antibody. At the idiotype l e v e l t h i s antibody has been found to be very heterogenous. When one considers the fact that hapten i t s e l f constitutes only one species on a large array of different epitopes, then the relationship of any single . anti-hapten idiotype to the overall network of anti-immunogen idiotopes might actually be remote. In the case of 70 a more limited immunogen such as Fd where the immune response i s confined to only two known epitopes, the repertoire of idiotopes may be smaller, hence the antibody network could be less stable with respect to any given idiotype. Idiotypic manipulations might theoretically perturb other members of the repertoire at a measureable l e v e l . In the case of Fd, idi o t y p i c manipulation does measurably affect the l e v e l of t o t a l antibody. After allowing for r e l a t i v e increases i n N-specific antibody i t i s clear that the increase i n idiotype content does not account for the overall increase i n t o t a l anti-Fd antibody. In other words, the observed increment i n idiotype f a l l s short of the actual increment i n anti-N antibody. There i s a s i m i l a r i t y i n th i s observation to the findings of Reth and co-workers who found that regulation by a given anti-idiotype antibody need not be re s t r i c t e d to the expression of the corresponding hapten binding idiotype but could extend to other hapten binding idiotypes as well (72). With Fd however, anti-idiotype treatment also resulted i n an increase i n production of antibody directed towards the unrelated C-determinant. I t i s clear, therefore, that alterations i n the a c t i v i t y of T c e l l s governing idiotype expression can affect not only the production of idiotype but can extend to other members of the repertoire as well . Previous studies on Fd have shown that there are only two immunologically reactive regions present and these two epitopes are active at both the B and T c e l l l e v e l (41). Thus, i n terms of hapten c a r r i e r interactions, i t may be assumed that during the response to Fd, one epitope provides the help necessary to generate a response to the other. The idiot y p i c regulatory T c e l l influenced by anti-idiotype i n these experiments i s specif i c for the N-determinant . Since N-determinant binding T c e l l s also provide the help necessary for the anti-C response, i t may be that the idiotype bearing T c e l l affects the function of other N-binding T helper c e l l s which may 71 or may not be i d i o t y p i c a l l y related. I t i s impossible at t h i s time to define more precisely the nature of the c e l l s involved i n generating the increased help for the C-determinant response since treatment of primed T c e l l s with anti-idiotype may pre f e r e n t i a l l y affect one i d i o t y p i c population over another. Although B c e l l s have been shown to participate i n anti-idiotype regulation (73), the effects which are observed are the same whether the experiment was performed using whole or separated spleen c e l l s and would appear to rule out any direct effects of anti-idiotype caused by bridging of idiotype bearing T and B c e l l s . I t appears then, that the anti-Fd-1 antiserum i s capable of affecting a population of immunoregulatory T c e l l s , possible suppressor c e l l s , which bear the Fd-1 idiotype. This treatment results not only i n an increased l e v e l of antibody directed to the C-determinant (increased help from N-specific T helper c e l l s ) , but also i n an increased expression of the Fd-1 idiotype i n N-specific B c e l l s . In order for both these events to occur, i t i s necessary to postulate that the idiotype T immunoregulatory c e l l s have a dual function: regulation of N-specific help, and regulation of idiotype expression by way of an a n t i - i d i o t y p i c population of, presumably, T c e l l s . A model incorporating these observations i s shown i n figure 16. Prior to contact with antigen the network of potentially reactive c e l l s exists i n a non-immune or ground state i n which idiotype and anti-idiotype elements are i n a state of equilibrium (74). This state of homeostasis can be easily distrubed by the introduction of antigen, idiotype or anti-idiotype. Normally, contact with antigen leads to a s h i f t i n the balance between id i o t y p i c elements resulting i n antibody synthesis. Prior contact with i d i o -type on the other hand, might potentially induce p r o l i f e r a t i o n or elimination of idiotype binding c e l l s ( a n t i - i d i o t y p i c c e l l s ) biasing the existing equilibrium i n a way which would result i n an abnormal response to antigen. 72 This prediction was investigated by Forni e_t a l . (75) who observed stimulation of IgM anti-SRBC plaque forming c e l l s following passive administration of anti-SRBC IgM. The authors postulated a mechanism involving direct stimula-tion of a n t i - i d i o t y p i c T helper c e l l s by the administration of id i o t y p i c antibody. This procedure was applied to the Fd response where a marked enhancement i n the t i t r e of anti-Fd antibody was observed as a result of pre-immunization with monoclonal Fd-1 antibody. Unlike the former case however, the enhanced response was dependent on antigen as no anti-Fd response was detected after administration of Fd-1 alone. When the sera were analyzed for their N/C ra t i o i t was found that the ratios remained unchanged and that the major s h i f t i n antibody production was again attributable to a sign i f i c a n t increase i n C-specific antibody. In order to accomodate these findings the model presented i n f i g . 16 includes linkage between idiotype and anti-idiotype bearing T c e l l s . In other studies, administration of idiotype iji vivo has been shown to stimulate specificT c e l l functions, although the outcome i s somewhat dependent on the mode of immunization (32). Conjugation of idiotype to syngeneic thymocytes has been used to induce a n t i - i d i o t y p i c helper and suppressor T c e l l s depending upon whether they were administered intravenously or subcutan-eously (69). One could l o g i c a l l y extend t h i s q u a l i f i c a t i o n to include the poss-i b i l i t y , that a ;single mode of immunization may induce multiple subpopulations of immunoregulatory c e l l s which could have competing effects. The major i n t e r -actions can only be defined by manipulating c e l l s under highly controlled conditions. Several of the above mentioned factors may be contributing i n some degree to the outcome of the Fd experiments. Also important i s the fact that s p e c i f i c events involving B c e l l s have not been explored i n t h i s study, so that i d i o t y p i c events have been largely interpreted from the point of view of T c e l l a c t i v i t y . Nevertheless, i t has been demonstrated that the anti-Fd response i s sensitive to perturbations at the l e v e l of idiotype and anti - i d i o t y p e s and i t i s anticipated that these effects are most l i k e l y mediated through lymphocytes. 74 \ (o) ANTI-Fd-I (b) 1 1 1 I /A i d -Fd-I binding diotypic regulation i d * N-binding •uppreteor id + N- binding (c) id t \ N-bindlng \ helper idiotyplcally unrelated C-binding Figure l S : Model for i d i o t y p i c interaction i n the immune response to Fd. A major id"1" regulatory T c e l l ( c e l l b) controls the l e v e l of help generated by other N-epitope binding T-cells ( c e l l c) which may or may not be i d i o t y p i c a l l y related but control the le v e l of antibody produced by B c e l l s reactive with the C-epitope. Elimination of this c e l l ( c e l l b) by anti-Fd-1 increases the response to the C-epitope. Since the response to the N-epitope i s also increased i n this process, a second, i d - regulatory T c e l l i s also postulated (this population (a) would be bound by the Fd-1 monoclonal since they are an t i - i d i o t y p i c ) . Elimination of either of these c e l l populations destroys this l e v e l of network regulation. 75 CHAPTER 4: CELLULAR EXPRESSION OF Fd-2 IDIOTYPE Evidence presented i n the previous chapter showed that the Fd-1 idiotype was expressed on a s i g n i f i c a n t portion of antibodies i n serum which bound the N-determinant. Treatment of immune spleen c e l l s with anti-idiotype affected the anti-Fd response by increasing the production of idiotype as well as other members of the anti-Fd repertoire. The second monoclonal antibody, Fd-2, i s s p e c i f i c for the C-determinant of Fd. An a n t i - i d i o t y p i c antiserum was raised to this protein and could therefore be used to investigate questions similar to those which were studied using anti-Fd-1 serum. Many studies have shown that the i d i o t y p i c repertoire can be quite extensive. Immune responses include the appearance of both shared, as well as private idiotypes. The immune system seems capable of mounting a response which while predictable i n the case of more common idiotypes, involves numerous idiotypes which are not shared but nevertheless may constitute a sizable percentage of the expressed repertoire. It i s not known whether the l e v e l of expression of any given idiotype i n the serum r e f l e c t s the s p e c i f i c i t y of s p e c i f i c T helper to T suppressor c e l l s . This question has been i n v e s t i -gated using the two anti-idiotypes which have been characterized i n the Fd system. E a r l i e r , i t was shown that T c e l l s bearing the Fd-1 idiotype exerted a s i g n i f i c a n t influence over the expression of Fd-1 idiotype i n serum a n t i -body. The other idiotype in this system, Fd-2, provides an additional marker which allows for comparison of idiotypy at the serological and c e l l u l a r l e v e l . Materials and Methods The following experimental methods and conditions used i n this chapter are i d e n t i c a l to those used i n the Fd-1 studies which were described i n d e t a i l i n the Materials and Methods section for chapter 3. 1) Radioimmune assay for idiotype. 76 2) In vivo administration of anti-idiotype. 3) Adoptive transfer of anti-idiotype treated spleen c e l l s . The protocol used i n chapter 4 i s i d e n t i c a l except, where indicated, DBA recipients were not irradiated. 4) In vivo passive transfer of idiotype. Results Anti-idiotype serum to Fd-2 was prepared and characterized as described i n chapter 2. This serum was used i n a radioimmunoassay i n order to look for the presence of similar idiotypes among serum antibodies to Fd. A sample standard curve of th i s RIA i s presented i n figure 17. As with the RIA for Fd-1, the assay i s sensitive for idiotype levels of about 45 ng/ml. Unlike e a r l i e r findings with Fd-1 however, Fd-2 does not appear to be expressed as part of the normal anti-Fd repertoire i n BIO.BR mice. A small number of individual mice tested produced detectable levels of the idiotype however, even in those cases this expression constituted only a minor proportion of their anti-C antibody (table 10). A p i l o t experiment using a f f i n i t y p u r i f i e d anti-Fd-2 was undertaken to determine whether i n vivo administration of anti-Fd-2 would affect the response of BIO.BR mice towards Fd. As shown i n table 11, prior administration of 15 yg of anti-Fd-2 to BIO.BR mice markedly enhanced the Fd response i n comparison to the control group which received the same quantity of normal rabbit Ig. When the experiment was repeated using a genetic non-responder s t r a i n DBA/2, anti-Fd-2 was able to induce a strong primary response. The antibody t i t r e s of these animals approached the values which are normally seen i n responder strains. According to genetic analyses, DBA/2 mice f a i l to respond to Fd due to a suppressor effect which appears to be inherited as a codominant t r a i t 77 Figure i.7,: Radioimmunoassay for c i r c u l a t i n g idiotype i n mouse serum. Varying quantities of unlabelled Fd-2 i n normal BIO .BR serum were incu-bated over anti-idiotype coated wells of v i n y l microtitre plates. After 1 hr., the samples were washed out and replaced with " I O C standard amounts of J I - l a b e l l e d Fd-2. Percent i n h i b i t i o n of binding was calculated r e l a t i v e to control samples incubated with normal BIO.BR serum alone. 78 Table 10. Results of radioimmune assay for Fd-2 idiotype i n immune sera of BIO.BR mice. BIO.BR mice were immunized with Fd and bled 21 days lat e r . Sera from these animals were analyzed for idiotype content using a s p e c i f i c RIA for Fd-2 idiotype. Anti-Fd (yg/ml) Fd-2 (ng/ml) 1.6 0 2.4 120 1.8 0 3.2 75 4.4 0 2.2 0 3.0 0 2.7 0 2.1 140 3.1 0 4.5 0 5.3 0 79 Table 11: In vivo administration of anti-Fd-2 antibody. Small groups of BIO.BR and DBA/2 mice were given a single intraperitoneal injec-t i o n of 15 yg of rabbit Ig or a f f i n i t y p u r i f i e d anti-Fd-2 a n t i -body 7 days prior to immunization with Fd or KLH. s t r a i n treatment yg/ml anti-Fd±S.E.M. BIO.BR BIO.BR RIg_; 15 yg anti-Fd-2 15 yg 12.3±3.2 31.5±6.1 DBA/2 DBA/2 Rig 15 yg anti-Fd-2 15 yg 0.2±0.1 15.0+13.7 st r a i n treatment yg/ml anti-KLH±S.E.M. BIO.BR BIO.BR Rig 15 yg anti-Fd-2 15 yg 2.0±0.8 2.4±0.6 80 controlled by the I region (43). Despite the small sample size, the effects of the previous experiment were f e l t to be s u f f i c i e n t l y s i g n i f i c a n t to warrant further investigation. Two experiments u t i l i z i n g the a b i l i t y of anti-Fd-2 to induce responsiveness i n DBA/2 mice were devised. In the f i r s t experiment, DBA/2 mice were primed with Fd i n the presence of two mitogens, LPS plus a n t i -Ig. Assuming that unresponsiveness resulted from a lack of appropriate T c e l l help, then coimmunization with mitogen might provide a missing signal s u f f i c i e n t to induce activation of Fd spe c i f i c B c e l l s . As indicated i n table 12 however, each of these treatments f a i l e d to induce a response. The induc-tive effect of anti-Fd-2 i n nonresponder mice might, alternately, have resulted from the stimulation of a spec i f i c helper population. An experiment designed to test t h i s p o s s i b i l i t y was performed. Spleen c e l l s from DBA/2 mice which had received i n vivo anti-Fd-2 and responded i n the i n i t i a l t r i a l > experiment were adoptively transferred to non-immune syngeneic DBA/2 recipients which were subsequently immunized with Fd. The recipients were bled after 10 days to determine whether t h i s resulted i n the adoptive transfer of an anti-Fd response into DBA recipi t e n t s . As seen i n table 13, the recipient animals did not however, respond. This result implies that the anti-idiotype may have acted by eliminating a population of suppressor c e l l s rather than through the stimulation of helper c e l l s . After anti-idiotype treatment, adoptive transfer of the responding c e l l population into normal unirradiated recipients serves to reconstitute the o r i g i n a l environment i n which suppression i s dominant. Transferred c e l l s would then f a i l to respond as a result of interaction with suppressor c e l l s _in s i t u . This interpretation of events i s based on an assumption that idiotype bearing suppressor c e l l s are eliminated by anti-idiotype In vivo. This prediction was tested i n an adoptive transfer system i n which the donated lymphocytes were obtained from Fd primed DBA/2 mice. Donated spleen c e l l s , the 81 Table 12: Lack of an anti-Fd response i n nonresponder mice (DBA/2) co-immu-nized with mitogen. The indicated quantities of mitogen were administered by intraperitoneal i n j e c t i o n 3 days prior to immuni-zation with Fd. Treatment yg/ml anti-Fd Fd 0 Fd + LPS (15 yg) 0 Fd + RaMIg (15 yg) 0 82 Table 13: I n a b i l i t y of the enhanced response of anti-Fd-2 treated mice to persist following adoptive transfer to normal, nonirradiated hosts. s t r a i n treatment of donor c e l l s ug/ml anti-Fd±S.E.M. BIO.BR Rig 4.16±1.04 BIO.BR anti-Fd-2 3.52±0.48 DBA/2 DBA/2 Rig anti-Fd-2 0.1310.30 0.10±0.05 83 presumed source of suppressor c e l l s , were treated i n v i t r o with anti-Fd-2 plus C' and adoptively transferred to irradiated syngeneic recipients, which were then immunized with Fd. Antibody t i t r e s of the recipients were deter-mined from sera bled 21 days after immunization. The results of this experi-ment are shown i n table 14. As i n the case of the In vivo experiment, exposure of c e l l s to a f f i n i t y p u r i f i e d anti-Fd-2 resulted i n a strong primary response. Following secondary challenge with Fd th i s response i s maintained. Discussion The results of anti-Fd-2 treatment of immune spleen c e l l s d i f f e r i n several ways from the effects which were observed using anti-Fd-1. The i d i o -types defined by these reagents appear to be expressed i n very different amounts during a normal immune response i n B10.BR mice. Whereas the Fd-1 idiotype i s a regularly expressed component of the anti-Fd repertoire, the Fd-2 idiotype i s very infrequently expressed and always i n very low concentra-tion i n immune serum. The two anti-Fd monoclonal antibodies were obtained i n separate fusion experiments. Despite the apparently random nature of c e l l hybridizations, i t cannot be concluded with any certainty that the products obtained v i a c e l l fusion are representative of the antibody repertoire existing at the time of fusion. Metzger and coworkers observed that the idiotypes of hybridomas derived at different times during the anti-lysozyme response differed s i g n i f i c a n t l y from those expressed on serum antibodies which were consistent throughout the response (76). These findings add evidence to e a r l i e r suspicions that c e l l fusion may be selective towards lymphocytes i n a parti c u l a r state of d i f f e r e n t i a t i o n . In contrast to their r e l a t i v e paucity i n serum antibody, the effects of treatment of c e l l s with anti-Fd-2 greatly exceeded those induced by anti-Fd-1. While administration of anti-Fd-1 in vivo had no short term effects, anti-Fd-2 84 Table 14: A b i l i t y of anti-Fd-2 to abrogate the nonresponder status to Fd i n DBA/2 mice. Fd-primed DBA/2 mice served as donors of spleen c e l l s which were treated with anti-Fd-2 serum and adop-t i v e l y transferred to irradiated DBA/2 recipients together with Fd. Recipients were bled at day 21. On day 28 a 2° challenge with Fd was given, and recipients were again bled on day 35. st r a i n treatment yg/ml anti-Fd±S.E.M. 21 days 35 days DBA/2 Rig 0.6010.10 0.15+0.06 DBA/2 anti-Fd-2 3.28+0.53 10.10±2.60 85 administration markedly enhanced the anti-Fd response and was also active i n adoptive transfer. In experiments designed to investigate the mechanism of this e f f e c t , no evidence for the induction of helper c e l l s in anti-Fd-2 treated mice was observed. Rather, the effects of anti-Fd-2 treatment observed i n the nonresponder s t r a i n are more consistent with the existence of an id i o t y p i c suppressor c e l l . Anti-Fd-2 treatment of primed DBA/2 spleen c e l l s resulted i n a response which exhibited both the ki n e t i c s (peaking at 21d) and antibody t i t r e s which are seen i n a normal primary anti-Fd response i n BIO.BR mice. The putative suppressor c e l l would be active i n both the BIO.BR s t r a i n , i n which i t may only be p a r t i a l l y e f f e c t i v e , and the DBA/2 stra i n i n which i t predominates. A potent suppressor c e l l with similar characteristics has been described i n the immune response to lysozyme. Genetic nonresponsiveness of the BIO s t r a i n to lysozyme was found to be due to the activation of suppressor T c e l l s by a r e s t r i c t e d portion of the antigen. Removal of th i s epitope converted BIO mice to responder status (77). A n t i -Fd-2 treatment may be an equally selective means of overiding t h i s type of suppression i n DBA/2 mice. While the expression of FD-1 idiotype was found to be r e s t r i c t e d to a particular allotype linkage group i n BIO.BR mice, t h i s may r e f l e c t differences i n idiotype production which are below the levels of detection afforded by the idiotype assay employed. Using a similar assay for Fd-2, only very low le v e l s of idiotype were detected i n the BIO.BR st r a i n . In contrast, i n vivo and adoptive transfer experiments indicated that the Fd-2 idiotype may be expressed on a subpopulation of lymphocytes i n two strains, B10.BR and DBA/2, which do not share allotype. Although this difference i n allotype r e s t r i c t i o n may seem discordant, other experimental studies have furnished evidence which suggests that these findings may not be inconsistent with network principles. In studying delayed type hypersensitivity reactions with the arsonate hapten, 86 H i r a i and coworkers found that idiotype s p e c i f i c T suppressor c e l l s could be generated by immunization of mice with idiotype conjugated spleen c e l l s (78). Interestingly, these idiotype s p e c i f i c suppressors could be generated i n a number of strains of different allotype linkage groups which did not normally express the idiotype. The potential exists i n these strains of recognizing an allotype r e s t r i c t e d idiotype. In an unrelated study, i t was observed that immunization of Balb/c mice with the T15 myeloma protein induced T c e l l helper a c t i v i t y which was spec i f i c for T15 as well as other PC binding myeloma proteins which were negative for the T15 idiotype (79) . The authors proposed on the basis of this finding that T c e l l recognition of idiotypes was re s t r i c t e d to germ l i n e s p e c i f i c i t e s while B c e l l s , having undergone extensive somatic mutation, would generate a spectrum of variant idiotypes. According to this proposal, T c e l l s would recognize predominantly germ l i n e encoded idiotypes while antibodies to B c e l l i d i o t y p i c variants might not completely cross-react with T c e l l s . Although this speculation might not be applicable to the xenogeneic anti-idiotypes used i n this study, i t does provide a possible explanation for the differences i n allotype r e s t r i c t i o n of idiotype expression which have been observed i n Fd as well as i n other systems. Summary Discussion This thesis has presented experimental descriptions of effects which were induced as a result of the treatment of isolated spleen c e l l s with, or administration to whole animals of, i d i o t y p i c and a n t i - i d i o t y p i c elements of a defined antigen system. Many of these r e s u l t s , while consistent with the logic of i d i o t y p i c interactions, cannot be used as a basis on which to propose a comprehensive model that c l e a r l y states the role of i d i o t y p i c interactions in regulating the immune response to ferredoxin. They do indicate, however, 87 that idiotypy i s a s i g n i f i c a n t mode of communication between lymphocytes as evidenced by the altered immune responses exhibited following the administration of idiotype or anti-idiotype. The c e l l u l a r mechanisms responsible for observed s h i f t s i n antibody and idiotype production i n this system have not however, been f u l l y characterized. One dilemma which must be addressed i n studies of t h i s type i s the difference between the goals of c e l l u l a r immunology and those of network theory. C e l l u l a r immunology seeks to define spe c i f i c temporal interactions between lymphocyte subpopulations of d i s t i n c t functional a c t i v i t y . This notion i s embodied i n the widespread usage of phenotypic markers which are presumed to id e n t i f y functionally mature T c e l l populations (80). In contrast to t h i s c e l l u l a r view, network theory avoids the use of functional terms and r e l i e s instead upon the more s t a t i s t i c a l concepts of homeostasis and symmetry i n order to predict the behaviour of the immune system i n response to a given stimulus (74). I t would not be surprising, considering differences i n their underlying l o g i c , that the design and interpretation of experiments would not be mutually consistent between these two theories. The experiments described i n t h i s thesis represent an attempt to explore the significance of both c e l l u l a r and network theory within the confines of a simple antigen system i n mice. In general, the results have been more amenable to a network interpretation, perhaps i n part because idiotypes afforded a readily tested experimental parameter. Nevertheless, this situation became possible because the interactions described are r e a l . 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