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Idiotypic networks and immunoregulation Singhai, Rakesh 1985

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IDIOTYPIC NETWORKS AND IMMUNOREGULATION By RAKESH SINGHAI B.Sc, University of British Columbia, 1981 M.Sc., University of British Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standards University of British Columbia December, 1985 © Rakesh Singhai, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^V^iQIJLZO(^0 The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date IQ ftA* /?Q& DE-6 (3/81) i i Abstract The role of an idiotype borne on a monoclonal antibody (Pd-B2) against the C-determinant of the protein ferredoxin (Fd) was investigated in mice of the B10.D2 (H-2d) non-responsive strain. It had previously been shown that when anti-idiotype (anti-Id) plus complement (C) treated T cells and non-immune untreated B cells were adoptively transferred into irradiated syngeneic B10.D2 mice, the mice response to Fd by making anti-Fd antibodies, suggesting a role for Id-bearing T cells in Fd-specific non-responsiveness. In this series of experiments, i t was shown that when T cells were treated with Id+C and adoptively transferred to B10.D2 in an analogous manner, such mice also became responsive to Fd, suggesting that Id-mediated network at the T cell level may control non-responsiveness in animals. This possibility was confirmed when it was shown that adoptive co-transfer of both of the T cell populations (each in itself confering responsiveness) restored the network as manifested by re-establishment of non-responsiveness in recipient animals. Panning studies demonstrated that anti-idiotypic (Id ) T cells are Lytl +2~3 whereas Fd-B2 Id + T cells are Lyt l~2 +3. T cell lines and clones were derived from B10.D2 mice which had been converted to responsiveness to Fd by the Id or anti-Id as above. One T cell line, and a clone derived from i t , was shown to secrete interleukin-2 (IL-2) not only in the presence of anti-Fd-B2 idiotype antibodies but also in the presence of Fd. Both of these reactivities were found to be i i i MHC-restricted. This shows that non-responder status of H-2 mice is not due to a defect at the level of antigen presentation. Fine specificity analysis of one Id + T cell cloned line showed that i t was reactive only to the anti-idiotypes or the fragment of Fd containing the C-determinant but not to the peptide fragment of Fd devoid of this determinant. Furthermore, i t was found that presentation of both the antigen and the anti-Id to the specific clone could be blocked by the Fd-B2 monoclonal antibody, indicating that the anti-id represents an internal image of the Fd molecule at the level of the C-determinant. Idiotype-mediated network interactions were also found to regulate the anti-P815 mastocytoma immune response. It was shown that when the idiotype-bearing P815-specific DBA/2 T cell hybridoma product, A10, is injected into DBA/2 mice on days -14 and 0 prior to tumour cell challenge, the animals, upon subsequent challenge, show a significant tumour regression over the controls. The activity of the AlO-defined idiotype was demonstrated to be both tumour and strain specific. Using A10- and rabbit-(anti-AlO)-antibody-coated dishes as panning surfaces, i t was shown that the removal of A10+ (Id+) - but not anti-AlO (Id-) T cells leads to a significant regression of tumour. When both Id + and Id T cell populations are adoptively co-transferred to irradiated syngeneic recipients, the tumour growth of specifically treated animals resembles that of the controls. This suggests that a strong similarity but not identity exists between anti-Fd and anti-P815 immunoregulatory mechanisms. iv TABLE OF CONTENTS PAGE Abstract i i List of Tables v i i i List of Figures ix List of Abbreviations xi Acknowledgements xiv Chapter 1 1.1 Introduction 1 1.2 Historical Review 3 1.2.1 p-azobenzenearsonate system 3 1.2.2 Phosphorylcholine 11 1.2.3 4-hydroxy-3-nitrophenyl acetyl (NP) 13 1.2.4 (Glutamic acid 6 0 alanine3° tyrosine 1 0) random terpolymer (GAT) 16 1.2.5 Lysozyme 21 1.2.6 Staphylococcal nuclease 23 1.3 Review of the ferredoxin system 26 Chapter 2 2.0 Materials and Methods 31 2.1 Experimental animals 31 2.2 Purification of antigens 31 2.3 Immunization and collection of sera 32 2.4 Standard ELISA 32 2.5 Generation of the idiotype-bearing anti-Fd antibody Fd-B2 and anti-idiotypic antiserum 33 V PAGE 2.6 Adoptive transfer in the Fd system 34 2.6.1 Preparation of B and T cell enriched population and reconstitution 34 2.6.2 Lyt determination by panning 35 2.7 Idiotype or anti-idiotype bearing T cell lines and clones 37 2.7.1 Idiotype-bearing T cell lines 37 2.7.2 Anti-idiotype bearing T cell lines 39 2.8 IL-2 release assay as a measure of antigen-specific T cell proliferation 39 2.8.1 Antigen-specific IL-2 release 39 2.8.2 Preparation of Con-A induced blasts 40 Chapter 3 3.0 Evidence for network-mediated immunoregulation in B10.D2 Fd-non-responder mice 44 3.1 Introduction 44 3.2 Results 45 3.2.1 Effect of the Fd-B2 idiotype on T cells from Fd-non-responding mice 45 3.2.2 Evidence for an idiotypic network controlling the Fd response 48 3.2.3 Titration of the Id + and anti-Id T cells against each other 52 3.3 Discussion 55 Chapter 4 4.0 Lyt phenotype of the idiotypic and anti-idiotypic suppressor T cells 62 4.1 Introduction 62 vi PAGE 4.2 Results 64 4.2.1 Lyt phenotype of anti-idiotype-positive T cells 65 4.2.2 Lyt phenotype of idiotype-positive T cells 70 4.3 Discussion 75 Chapter 5 5.0 Evidence for Fd-B2 idiotype-anti-idiotype mediated network at the level of T cell clones 78 5.1 Introduction 78 5.2 Results 79 5.2.1 Generation of B10.D2 derived idiotype-reactive (anti-idiotypic) T cell lines 79 5.2.2 Characterization of Fd- and anti-idiotype-reactive T cell lines 85 5.2.3 Cloning of Fd and anti-idiotype reactive T cell lines.... 90 5.2.4 Fine specificity of Fd- and anti-idiotype-reactive clone 2 93 5.2.5 MHC-specificity of heterogenous T cell subcultures 100 5.3 Discussion 100 Chapter 6 6.0 Network-mediated immunoregulation of anti-P815 tumour response 107 6.1 Introduction 107 6.2 Materials and Methods 110 6.2.1 Preparation of affinity-purified A10 material 110 6.2.2 Intravenous administration of A10 molecule I l l 6.2.3 Cellular immunization 112 v i i PAGE 6.2.4 Preparation of rabbit anti-AlO antiserum 112 6.2.5 Adoptive transfer of panned splenocytes 113 6.3 Results 113 6.4 Discussion 140 Chapter 7 7.0 Summary 148 References 151 v i i i LIST OF TABLES PAGE Table No. 6.1 Mean survival times of treated or control mice 120 6.2 Effect of A10 or equivalent BW ascites material on tumour growth 123 6.3 Survival time of mice actively immunized with irradiated tumour cells 135 6.4 Effect of A10 on the syngeneic P815-non-related tumour L1210 145 ix LIST OF FIGURES PAGE Figure No. 2.1 Titration of IL-2 secreted by the EL4 cell line 42 3.1 Depletion of an Id + (Fd-B2) recognizing T cell population changes a non-responder strain for Fd into a responder 46 3.2 Reconstitution of unresponsiveness with Id +-depleted and anti-Id-depleted T cells 49 3.3 Titration of Id + and anti-Id (Id~) plus complement treated T cells against each other 53 3.4 A model depicting a minimal set of cells relevant to the discussion of the response of B10.D2 mice to Fd 58 4.1 The anti-Id (Id -) T cells do not express the Lyt 2 antigen 66 4.2 The Id - T cells expressed the Lyt 1 antigen 68 4.3 The Id + T cells do not express Lyt 1 71 4.4 The Id + T cells express the Lyt 2 antigen 73 5.1 Initial screen of a B10.D2-derived idiotype-reactive T cell line 80 5.2 MHC restriction of Fd-dependent T cell lines 83 5.3 Initial screen of Fd-dependent cell line from B10-D2 mice 86 5.4 IL-2 release in culture supernatants of Fd or anti-id-dependent T cell lines 88 5.5 Specificity of cloned T cells 91 5.6 IL-2 release in culture supernatants of anti-idiotype-reactive cloned T cell line C2 94 5.7 Ability of the Fd-B2 idiotype to block antigen presentation and subsequent IL-2 release by the C2 clone 96 X PAGE 5.8 Fine specificity of clone C2 98 5.9 MHC restriction of the Fd-dependent T cell lines 101 6.1 Effect of soluble A10 molecule on P815 tumour growth 116 6.2 Survival curve of mice shown in figure 6.1 118 6.3 Effect of A10 equivalent BW5147 ascites material on tumour growth 121 6.4 Tumour growth in irradiated mice with panned splenocytes 125 6.5 Survival curve of mice shown in Figure 6.4 127 6.6 Effect of removal of A10+ or A10- cells on tumour growth 129 6.7 Effect on tumour growth following cellular immunization 133 6.8 Immunization with other cell lines 136 6.9 Survival of mice immunized with various tumours 138 6.10 Lack of allogeneic activity of A10 suppressor molecule 141 6.11 In vivo specificity of A10 molecule 143 xi LIST OF ABBREVIATIONS ABA p-azobenzenearsonate A10 suppressor T cell hybridoma or its secretory product directed to the tumour P815 APC antigen presenting cells B16G a monoclonal antibody against a suppressor factor derived from the heterogeneous suppressor T cell population C complement CAMAL-1 a monoclonal antibody used as an irrelevant control, directed to a human leukemia-associated antigen C-determinant trypsin-digest of ferredoxin devoid of N-determinant activity CPA Complete Freund's Adjuvant c H constant region of immunoglobulin heavy chain genes Con-A concanavalin-A CPM counts per minute CRI a major cross-reactive idiotype CTL cytotoxic T lymphocyte DH diversity region of the heavy chain DME Dulbecco's modified Eagle1s medium DTH delayed type hypersensitivity ELISA enzyme-linked immunosorbent assay FCS foetal calf serum, heated at 56°C, 30 min. Fd the protein ferredoxin derived from Clostridium pasteurianum Fd-B2 a monoclonal antibody directed to the C-determinant of Fd x i i GAT H-2 Id anti-Id (Id") Ig Igh Igl IL-2 JH kDa KLH 2-ME M-fragment MHC yCi Vg Ml mm mM MLR M m.w. ng N-determinant random terpolymer of (glutamic3°alanine3°tyrosine10) MHC of mice the idiotype Fd-B2 antibodies/antiserum to Fd-B2 immunog1obu1in immunoglobulin heavy chain (gene) immunoglobulin light chains (gene) interleukin-2 joining region of the heavy chain kilodalton Keyhole limpet haemocyanin 2-mercaptoethanol ferredoxin digested with trypsin and carboxypeptidase and devoid of both C- and N-determinants Major Histocompatibility Complex micro Curie micro gram micro litre millimetre millimolar mixed leukocyte reaction Molar molecular weight nanogram ferredoxin digested with carboxypeptidase-A NHS normal human serum, heated at 56°C for 30 min NP (4-hydroxy-3-nitrophenyl)acetyl NIP (4-hydroxy-5-iodo-3-nitrophenyl)acetyl NNP (4-hydroxy-3,5-dinitrophenyl)acetyl non-sp MAb (Mab) the irrelevant control monoclonal antibody CAMAL-1 NRIg normal rabbit immunoglobulin PBS phosphate buffered saline PC - phosphorylcholine PPC plaque forming cells PMA phorbol myristic acetate RaMIg rabbit anti-mouse immunoglobulin rpm revolutions per minute SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM standard error of mean ThC helper T cells 3[H]TdR 3H-labelled thymidine TsC suppressor T cells TsF a factor secreted by TsC and capable of replacing TsC activity Vu heavy chain variable-region genes xiv ACKNOWLEDGEMENTS I would like to express my appreciation to my Supervisory Committee, Drs. H.-S. Teh, D. Kilburn, and D. Waterfield, and to my research supervisor, Dr. Julia G. Levy for multi-level support and constant interest and enthusiasm during my research. I would also like to thank al l the past and present members of the Levy laboratory particularly, Randy Chu, and especially Dr. Pat M. Logan for her friendship and advice at both professional and personal levels. I am indebted to Dr. Kilburn for making available to me the cell lines and reagents cited in the thesis, to Dr. Teh for the same reasons and to Dr. Waterfield for the IL-2 assay. I thank Ms. Rosario Bauzon for typing this thesis and Randy Chu for a critical read of i t . Finally, I thank my supervisor, Dr. Julia G. Levy for her friendship, guidance and most of a l l , for believing in me. To her, I shall always be grateful. 1 Chapter 1 1.1 Introduction In 1974 Niels Jerne proposed the network theory which provided a foundation based on idiotypic interactions to explain the regulation of the immune system (1). The central proposition of the theory is that the immune system uses an immune response directed against determinants borne on antigen-specific receptors to regulate itself (2). That is, every member of this immune system can, at least potentially, recognize an antigen and be recognized as an antigen. Such interactions would have either enhancing or suppressive effects with respect to a particular antigen within the same host animal. The components of an immunoglobulin which confer immunogenic properties to i t , with respect to the same animal or another member of the same inbred strain, reside in the variable regions of the heavy and light chains and are encoded by the V, D and J region genes. Such unique individual specific determinants are called idiotopes and a family of idiotopes is called the idiotype, a term originally coined by Oudin (3) and independently also described by Kunkel (4). Anti-idiotypic antibodies or anti-idiotypes, therefore, are antibodies which recognize idiotypes. When an animal encounters an antigen i t responds with the proliferation of antigen-specific lymphocytes. This leads to an increase in the concentration of antigen-specific molecules. In the case of B cells such 2 molecules are immunoglobulin. According to Jerne (1) a sufficiently high concentration of antigen-specific antibodies makes them immunogenic to the extent that a response against the idiotypes of the first set of antibodies is triggered and anti-idiotypic antibodies are synthesized. Anti-idiotypes bear their own determinants and stimulate the generation of anti-anti-idiotypic antibodies and in this way an entire network of interconnected cells and molecules is perturbed. The theory has two important features. First, is the assumption that the perturbation includes the steady state interactions of idiotypes and anti-idiotypes before the introduction of the antigen. Secondly, i t also includes the idiotype-anti-idiotype antigen interactions which arise only due to the antigen stimulation. If one allows for stimulatory and inhibitory idiotype-anti-idiotype reactions then the system eventually achieves a new equilibrium after the original perturbation. Yet another property of these idiotype-anti-idiotype reactions is that some induced antibodies bearing a particular idiotype are regulated independently of their ability to bind the antigen which initially stimulated their production. Although the network concepts and assumptions have been discussed in terms of antibodies (thus, in terms of B cells), the same concepts also apply to T cells. Furthermore, i f T and B cells have common idiotypes, then both may be active partners in the network. The theory has provided a significant stimulus and framework for the design and interpretation of a vast number of experiments during the past decade. However, as yet, the network theory has neither been proven nor disproven inspite of the volumes of literature i t has instigated. Some of 3 this literature is reviewed below. This is followed by a description of the ferredoxin molecule as an antigenic probe and its use in experiments which provide clear evidence for an idiotype-anti-idiotype mediated T cell network. Finally, some experiments are reported which form the basis of a continuing study on idiotype-mediated network interactions in a murine anti-tumour response. This latter study was undertaken in an effort to develop a tumour analogue of the ferredoxin model discussed in this thesis. 1.2 Historical Review 1.2.1 p-azobenzenearsonate system Among the variety of synthetic and natural immunologic probes available, the p-azobenzenearsonate (ABA) hapten represents one of the most thoroughly studied systems. When strain A mice are immunized with Keyhole limpet haemocyanin (KLH) conjugated to ABA (KLH-ABA) they respond by producing anti-ABA antibodies bearing a major cross-reactive idiotype (CRI) which is borne on 20-70% of the anti-ABA antibodies (5,6). The CRI bearing antibodies were initially characterized with the use of xenogeneic anti-idiotype (anti-id) antisera raised in rabbits (5). Early studies clearly established the immunoregulatory role of CRI with the discovery that injections of rabbit anti-id antibodies two weeks prior to challenge with the antigen KLH-ABA led to a complete suppression of CRI bearing antibodies without altering the total expression of anti-ABA antibodies in 4 A/J mice (7,8). This observation served as a basis for subsequent studies in which the suppression of CRI-bearing antibodies was attributed to B cell clonal dominance (9,10). A more complex and multistage mechanism emerged from experiments which elucidated the role of T cells in regulation of CRI-positive antibodies. These studies exploited the fact that A/J mice could be suppressed for CRI expression with injections of anti-idiotype antibodies, and be subsequently hyperimmunized with KLH-ABA. In a series of elegant experiments, using hyperimmunized mice as above, i t was shown that suppression with respect to CRI could be adoptively transferred to naive A/J mice with the nylon wool column enriched, anti-Thyl.2 plus complement sensitive fraction of splenocytes (11,12). The suppressive cells were shown to form rosettes with A/J red blood cells (RBC) coated with CRI positive Fab fragments. No T cell rosettes were seen when non-specific Fab coated RBC's were used. The formation of such rosettes was inhibited by A/J anti-ABA serum but not by normal A/J serum or by serum from suppressed, hyperimmunized mice. As expected, F(ab')2 fragments of rabbit anti-id, but not normal rabbit F(ab*)2, inhibited rosette formation. Lastly, ABA analogues which bind anti-ABA antibodies, were also shown to inhibit rosette formation. All these results convincingly demonstrated that in these studies the transfer of suppression could be attributed not only to T cells but to T cells bearing anti-idiotype receptors (11,12). Another important observation made was that the receptors of anti-idiotypic cells were synthesized endogenously and could be destroyed by incubation with trypsin as seen by the lack of 5 rosette formation after such treatment. Rosette forming ability was, however, restored after 24 hours in tissue culture. In contrast to the above results, when suppressor T cells were adoptively transferred to irradiated A/J animals which had previously been primed with ABA, typical CRI levels were observed, suggesting that a secondary (and subsequent) response is not susceptible to the influence of suppressor T cells (13). This effect could be overcome with a co-transfer of purified secondary B cells along with the suppressor T cells and was observed regardless of the carrier protein used. The possibility was considered that the target for suppression by anti-id TsC might be idiotype-positive suppressor cells which, upon stimulation by the anti-id TsC, could be the immediate effectors. The issue then arises regarding the manner in which anti-id receptor-bearing TsC are stimulated. Since the immune system was perturbed with the antigen and anti-id antibodies, both of which presumably react with the idiotype receptors and not the anti-idiotype receptors, one would expect that at first idiotypic T cells would be stimulated which in turn would stimulate the production and/or activation of anti-idiotypic TsC as proposed by Jerne (1). This could also explain the observation that usually eight weeks were needed after hyperimmunization for the maximal induction of anti-id TsC (11,12). In support of this hypothesis, Lewis and Goodman showed the existence of idiotypic receptor-bearing TsC which had the capacity to suppress antibody response (14). Due to the fact that in a l l these studies T cells were manipulated with the idiotype (or anti-idiotype), i t was assumed that the 6 T cell repertoire shares topographic configurations with idiotypes cross-reactive with B cells. Subsequent work demonstrated that suppressor T cells could be induced in adoptive transfer experiments upon injection of idiotype-conjugated thymocyte cells into syngeneic mice (15). However, i t was further shown that there was no H-2 restriction for the formation of TsC resulting from injection of idiotype conjugated to thymocytes since the source of cells used seemed to be irrelevant (15). Also, work by Hirai and colleagues showed that these suppressor cells could function across an allotype barrier (16). In another series of experiments, the question of regulation was addressed using a panel of CRI-positive monoclonal antibodies (17). Two approaches were employed. In the first, monoclonal antibody was coupled to thymocytes and injected into mice which were subsequently immunized with KLH-ABA. In this type of approach, each of the monoclonal antibodies caused the generation of CRI-specific TsC but the anti-ABA response was normal. In the second approach, rabbit anti-id prepared against a CRI-positive monoclonal antibody was injected into mice before immunization with KLH-ABA. This technique also led to a dramatic suppression of CRI without altering the anti-ABA response. The results were taken to indicate that the synthesis of CRI-bearing antibody or the suppression of its synthesis activates a network of idiotypic interactions which influence almost a l l of the idiotypic response. These data also support the previous and ongoing biochemical studies which suggest the close relationship between CRI-bearing antibodies (18-27). 7 The cellular studies described above culminated in the definition of ABA-specific suppressive factors which had the same immunoregulatory properties as the cells which presumably secreted them (28). The source of suppressor factors was culture supernates of splenocytes from mice which were anti-idiotypically suppressed for CRI and hyperimmunized with KLH-ABA. It was found that when normal A/J splenocytes were incubated for 4 hours with the dialyzed culture supernate and adoptively transferred into irradiated naive A/J mice which were subsequently challenged with KLH-ABA, the recipients made normal levels of anti-ABA antibodies. However, splenocytes incubated in culture supernate of nonsuppressed mouse cells did not result in transfer of CRI-specific suppression. Transfer of suppressive activity was abrogated when splenocytes from hyperimmunized mice were treated with anti-thy antiserum and complement. In addition, no suppressive activity was secreted by cells which adhered to anti-Fab coated polystyrene dishes. These observations support the origin of suppressive factors as being from T cells. Using classic affinity chromatographic and biochemical techniques, the suppressive factors were resolved into two distinct entities. One molecule was shown to carry CRI-determinants, binding to the hapten ABA as well as to anti-idiotype antibodies. It was also found to express I-J determinants as shown by the binding of suppressive activity to anti-I-J immunoadsorbent columns. The factor which expressed anti-id determinants bound to CRI-positive but not to CRI-lacking antibodies, and i t too expressed I-J determinants. Both 8 factors were in the 50-100 kilodalton range as determined by gel filtration. The suppressive activity of both factors was sensitive to trypsin but not to RNase or DNase. These observations were used to conclude that the idiotypic and anti-idiotypic suppressive factors represented two different protein molecules which coexist in solution separately rather than as a complex and the idiotype-factor recognizes the anti-idiotype factor and vice versa (28,29). Further information on idiotype-anit-idiotype network interactions, and the possible role of T suppressor factors (TsF) was obtained in experiments involving suppression of ABA-specific delayed type hypersensitivity (DTH). Numerous studies using this approach were conducted which resulted in the characterization of two distinct subpopulations of suppressor cells. One family of suppressor T cells, called Tsl, was generated upon immunization with ABA-coupled thymocytes of syngeneic recepients and was demonstrated to carry CRI determinants on cell surface (30). These cells were produced when mice were immunized with 30-100 x 10^ ABA-coupled splenocytes and were found to suppress ABA-specific T cells mediating DTH (Tdth), and cytotoxic T lymphocytes (CTL) (30,31). The Tsl were shown to be susceptible to lysis by anti-CRI and complement treatment and were Lyt l +23 (32,33). In studies carried out to assess the genetics of Tsl function, i t was concluded that Tsl cells function in an allotype-restricted fashion (32,33) and therefore, in addition to idiotype-anti-idiotype interactions, allotype linked genes may be responsible for cellular interaction signals for TsC. It was also possible to generate Tsl with intravenous (i.v.) injections of 9 anti-id antibodies (31,34) which were similar to ABA-coupled thymocyte-induced Tsl cells according to a l l the parameters used to characterize the hapten induced cells. These Tsl were shown, upon culturing, to secrete suppressor factors (designated as TsFl) into the supernatant . The suppressive activity was shown to be between 50 and 70 kilodaltons (31,35-37). The properties of this TsF were similar to the suppressor factor described by Hirai and Nisonoff (28). The factor bore V and I-J encoded determinants, was heat and low pH labile and was H present in either cell culture supernatants or in a preparation of mechanically disrupted cells. TsFl had similar immunosuppressive properties to Tsl. Further studies with TsFl showed that i t acted by inducing a second-order suppressor T cell population, designated as Ts2 (30,35-40). Ts2 did not bear CRI determinants as they could not be killed by anti-CRI plus complement treatment. These cells, however, did bind to, and could be eluted from, CRI-coated dishes but not ABA-coated plates. These and other results indicated that Ts2 were anti-idiotypic and were active in the afferent and efferent mode (35,40) in contrast to Tsl which were idiotypic and only functioned in the afferent mode (30,31). Anti-idiotypic receptor-bearing Ts2 could also be induced solely by the administration of TsFl (29). It was found that Ts2, as was the case with Tsl, also produced a suppressive factor, called TsF2 which could be obtained by mechanical disruption of Ts2 cells (41,42). TsF2 was found to be suppressive in an antigen-specific manner and like the cells from which i t was derived, was 10 able to suppress immune responses in both the efferent and afferent mode (41,42). Studies showed that TsF2 carried determinants encoded by the H-2 complex, and could bind to CRI but not to immobilized ABA. Therefore, TsF2 was anti-idiotypic in nature and. furthermore, was both H-2 and Igh-1 restricted (42). These studies eventually led to the generation of T cell hybridomas resulting from fusions between ABA-induced TS1 and BW5147 thymoma cells (43,44). The factors secreted by these hybrid cells were biochemically and immunologically similar to the equivalent cell culture supernatant or mechanically disrupted cell preparation-derived TsFl. However, since then, an ABA binding, CRI-bearing non-immunoglobulin, originally thought to be a T cell suppressor factor, has been described (45,46) and found to be ubiquitous in nearly a l l tissues tested. In addition, several reports have further complicated the situation in the ABA system. These include reports that: (a) some CRI-positive immunoglobulins are unable to bind ABA (47); (b) CRI appears to be rarely present at the B cell precursor level (48) but is magnified by two orders of magnitude following immunization with ABA-carrier protein conjugates (this increase is probably not due to antibody affinity-dependent clonal expansion since CRI bearing anti-ABA antibodies do not have especially high affinity for ABA (49,50); (c) B cell repertoire is not representative in the hyperimmune anti-KLH-ABA response, and regulatory genes may play a major role in influencing idiotype expression (51); and (d) alternative immunization protocols can lead to CRI expansion in CRI-negative strains (52). However, new research in this system should serve to clarify some of these issues. 11 1.2.2 Phosphorylcholine Phosphorylcholine (PC) represents another antigenic system in which immunoregulation has been investigated. Studies with this antigen began with the discovery that plasmacytomas produced by mineral o i l treated BALB/c mice secreted immunoglobulins which reacted with polysaccharides of bacterial cell walls (54). Further analysis of the antigen demonstrated i t to be phosphorylcholine (55). One of the several BALB/c derived myeloma proteins studied was TEPC 15 (T15). When BALB/c mice are challenged with Pneumococcus pneumoniae (R36A vaccine) they make a very strong anti-PC respone which is up to 98% dominated by the idiotype represented by T15 (56). Use of a variety of mouse strains established that T15 was a dominant idiotype in the anti-PC humoral response of BALB/c, C57BL/6, C56, ST and 129 strains of mice but not of other strains. These studies concluded that T15 idiotype was linked to the IgVR locus (57). Immunoregulatory work has shown that when adult mice are pre-treated with anti-T15, the subsequent anti-PC response is transiently devoid of T15 bearing antibodies for a period of two to three weeks at the end of which the levels of T15 become normal (58). However, a very different observation is made when neonatal animals are pretreated with the anti-idiotype. In this case the expression of T15 idiotype is suppressed for the l i f e span of the treated animal (59) even though the anti-PC response becomes normal. Further studies have shown that this persistent suppression could be adoptively transferred by the manipulation 12 of anti-T15 induced Lyt 2+ TsC (60). A recent study has shown that when monoclonal anti-idiotypes directed to various T15 idiotopes are administered to neonates a suppression of T15-positive response is observed upon challenge with PC although expression of the particular idiotype may s t i l l be detectable (61). Investigations undertaken to resolve the role of T cells in regulation of T15-positive anti-PC response have revealed that at least two helper T cells (ThC) are involved. These studies demonstrated that one ThC is required for the recognition of an MHC product (62) while a second ThC is involved in T15 recognition (13). A subsequent report indicated that both cells are necessary to synergistically cooperate for the production of T15 dominated anti-PC antibodies (64). Recently, experiments were conducted in order to investigate the fine specificity of anti-PC suppressor T cell activity in antibody production in vitro using PC-coupled syngeneic spleen cells as the immunogen (65). These studies indicated that PC-specific TsC needed for suppressing antibody response bear very homogenous T15 idiotypes on their functional receptors (65). AT cell hybridoma has been generated which binds PC as well as fluorescent anti-T15 antibodies (66). This hybridoma secretes a factor possessing a PC binding site as well as T15 determinants, but i t does not express the T15 germline gene segment (67). In a recent report concerning the behaviour of Th cells in the anti-PC response, i t was indicated that there exists a Thl-Th2 induction loop in which the former cell carries a T15-like receptors and the latter possesses anti-T15-like receptors (68). This work also demonstrated that 13 idiotope-specific priming induced non-idiotypic-specific T cells. It was concluded that the idiotypic T cell network in this system was based on a different selection of idiotope determinants than the selection of the B cell idiotype network. In another report which examined the regulation of T15 idiotype dominance, i t was found that whereas the anti-PC memory response bearing T15 idiotype is controlled by the B cell genotype and not the genotype of T cells, the T cell genotype controls the magnitude of the secondary adoptive transfer reponse (69). In any case, to date the manner in which T15 idiotype specific suppressor cells are induced, what their specificity is and how the different immunocompetent cells interact remain questions that await further research. 1.2.3 14-hydroxy-3-nitrophenyl acetyl (NP) In addition to the previous haptens, immunoregulation has also been examined in the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten system. In this system, C57BL/6, 101, or LP mice immunized with NP on a protein carrier (for example, bovine serum albumin or chicken serum globulin) produce anti-NP antibodies which bear a major cross-reactive idiotype designated as NP-b, in the primary response (70,71). In addition, these antibodies are heteroclitic in that anti-NP antibodies show greater affinity for related haptens such as (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) or (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP) than for NP itself (72). 14 Idiotypic analysis of the primary anti-NP response of C57BL/6 mice showed that expression of idiotype NP-b was linked to the Igh-b allotype (71). NP-a, a second idiotype, was shown to be controlled by the Igh-a allotype (73) and in fine specificity analysis i t was suggested that NP-b and NP-a idiotypes represent allelic forms as both are linked to \ light chain bearing antibodies detected most frequently in the primary response, are heteroclitic (74) and are mutually exclusive in a l l strains studied to date (73). Anti-NP monoclonal antibodies bearing either NP-a or NP-b idiotype have been raised and comparative studies between these two types of monoclonal antibodies show that some NP-b positive monoclonal antibodies share idiotypic determinants with those which are positive for NP-a (75). Of the many monoclonal antibodies bearing NP-b idiotype, Bl-8, IgM subclass, represents an especially well characterized one. Bl-8 expresses germ line V and V genes (76,77), and bears at least two H \ idiotypes which can be detected by two monoclonal anti-idiotypes, Ac3, and Acl46 (78). Analysis at the molecular level has shown that both V„ and D J J genes contribute to idiotype expression (79) and minor changes in the primary sequence can dramatically affect idiotype specificity as demonstrated with a mutant of Bl-8 (79). Other studies have been carried out to demonstrate that the idiotypic repertoire is a result of V region polymorphism and that the antigen binding site and idiotype are structurally separate (80,81). In parallel with serologic and molecular studies on idiotypes, the role of T cells in idiotype regulation was investigated. It was 15 demonstrated that TsC which suppress the DTH response in Igh-V mice can be induced by immunizing C57BL/6 mice with NP-conjugated syngeneic splenocytes (82). The suppressor T cells were shown to be restricted by Igh-V and MHC genes (83), and i t was found that DTH suppression involved two distinct T cell subpopulations (84). One of these subpopulations bound NP and carried NP-b idiotypic determinants (84) whereas the other population expressed an anti-idiotypic receptor, as was the case in ABA and PC haptenic systems (85). A 1977 study demonstrated that NP binding, NP-b idiotype positive, Ig serologic marker negative molecules could be isolated from primed T cells by antigen binding (85). Recently, a cell hybridoma has been generated in corroboration of the earlier studies (86). The BIO.BR derived hybridoma binds to NP, expresses NP-b idiotype and framework Ig V determinants, and secretes a T suppressor factor H which is specific to the NP-6 positive anti-NP response, I-J positive and is distinguishable from the cell surface receptor (86). Several in vivo studies using monoclonal anti-idiotype antibodies have been performed. In one of these studies i t was demonstrated that monoclonal anti-NP-b idiotype antibodies suppress the response to NP-coupled sheep red blood cells and that this suppression could be mediated by IgG but not by monoclonal IgM, IgD, or by F(ab)2 fragments (87). As well, administration of small amounts of monoclonal NP-b bearing antibody Bl-8 caused an increase in Bl-8 levels in the anti-NP response (79). In another study (88) using monoclonal anti-Bl-8 idiotype to induce idiotype specific suppression in neonate C57BL/6 mice, it was concluded that idiotype suppression by regulatory T cells may be 16 perturbed by antigen interacting with idiotypic antibodies on the B cell surface and may therefore play a role in establishing tolerance not only for the expressed antibody repertoire, but for self antigens in general. In a more recent report (89) i t was found that NP coupled to isologous gamma globulin can replace anti-idiotypic antibody in the induction of neonatal chronic idiotype suppression. From this study the authors inferred that an autoantigen in mice is able to produce a T cell dependent suppressive mechanism that controls expression of antigen-specific antibodies via the recognition of antibody idiotype. Therefore, the authors concluded that an idiotype network is involved in the control of tolerance and the available antibody repertoire (89). To date, however, no clear regulatory mechanism has been proposed in the NP system. And to make matters more difficult, i t was shown that nearly a l l of the NP-b idiotype of T cell preparations is due to conventional NP-b bearing anti-NP anibody strongly bound to T cell surfaces (90). These findings certainly introduce a level of complexity in the interpretation of the data for T cell idiotype responses and idiotype expression control. However, active research is currently underway to clarify these problems. 60 30 10 1.2.4 (Glutamic acid alanine tyrosine random terpolymer (GAT) GAT is a synthetic polypeptide consisting of various proportions of glutamic acid, alanine and tyrosine. In a study of immune responses to GAT and related copolymers the linkage of the anti-GAT 17 response to H-2 was demonstrated (91). However, another study reported non-MHC linked gene effects on the anti-GAT response (92). The use of GAT and related copolymers as immunogenic probes has been detailed elsewhere (93) and the present discussion will confine itself to the major observations concerning idiotypes and T cells, and their role in immuno regu1at ion. Idiotypic analysis of anti-GAT antibodies began in the late 1970's with the observation that when various inbred strans of mice are immunized with GAT they respond by making antibodies bearing an interstrain cross-reactive idiotype. Different xenogeneic anti-idiotypic reagents were used to define the idiotypes: those idiotypes characterized with a guinea pig anti-idiotype are designated as CGAT (95) while those defined with the use of rabbit anti-idiotype are called pGAT (97). A recent study demonstrated that both anti-idiotypic reagents may define similar determinants and that CGAT and pGAT have probably identical specificities (96). Analysis of rat and guinea pig anti-GAT antisera showed that guinea pig anti-GAT sera contain both CGAT and pGAT idiotypes (94,97). Further, immunization of mice with poly (glutamic acid, tyrosine) (GT) (98), but not with poly (glutamic acid, alanine) (GA) (99) causes the expression of CGAT. Biochemical (100) and immunological analysis further indicated that a l l inbred murine strains examined express CGAT and pGAT (94,95) and i t was inferred that this reflects the highly conserved nature of common anti-GAT V and V germline genes, as both H K GAT-responder and non-responder mouse strains make CGAT/pGAT bearing antibodies. 18 CGAT bearing monoclonal antibodies have been produced (100) some of which also express idiotypic determinants (GA-1 idiotype) associated with anti-GA antibodies (101). In studies comparing intrastrain and allotype associated idiotypic specificities, i t was found £1 C © that mice of Igh-1 , Igh-1 , and Igh-1 allotypes express a common idiotype (srGAT-1) (102) whereas mice of Igh-b allotype share an idiotype designated as Gte (103). In studies using conventional and recombinant strains of mice, i t was shown that the Gte idiotype maps to the V H region in proximity to Igh-NP, Igh-Np/b and Igh-Bgl markers, but i t does not appear to be linked to the Lyt 3 (IgK) loci (104). In addition, some antibodies which only recognize GAT also express a cross-reactive idiotype in common with antibodies against poly(GT) or poly (glutamic acid, lysine) (GL) (105). Along with the idiotype bearing anti-GAT antibody response studies, structural analysis of pGAT/CGAT idiotypes was undertaken which demonstrated that in order for these idiotypes to be expressed light chain-heavy chain interactions are needed (106). Furthermore, idiotype-anti-idiotype reactions could be blocked with the antigen (106) indicating that the pGAT/CGAT specificities were associated with the antigen binding site (106). Amino acid sequence analysis of CGAT positive anti-GAT monoclonal antibodies shows that the anti-GAT repertoire is highly limited as the amino acid sequence of these antibodies is identical at the kappa light chain level and that CGAT idiotype defines a new V K locus (107). An extensive study of CGAT-bearing heavy chains demonstrated that: (1) pGAT/CGAT determinants are linked with heavy chains of limited 19 heterogeneity, (2) CGAT-lacking GA-1 positive heavy chains, inspite of being associated with a different light chain, appear to be similar to CGAT and, (3) CGAT-positive monoclonal antibody amino acid sequences are expressed in polyclonal anti-GAT serum (96). These and other findings led to the hypothesis that CGAT and GA-1 specificities are markers for germline V and V genes (96). H K Molecular genetic studies of genes encoding anti-GAT antibodies have been conducted. In sequence analysis of VDJ mRNA of four BALB/c pGAT positive anti-GAT monoclonal antibodies i t was demonstrated that two monoclonals were identical and the other two differed in 3 and 8 positions only (107). A cDNA probe from the V region of one of the H homologous monoclonal antibodies was developed. The Southern blot pattern of germline DNA from 3 strains of mice differing in Igh allotype was developed with various restriction endonuclease digests, using stringent hybridization conditions. Resulting data showed 3-7 non-identical gene fragments (108). This suggests that there are only a small number [3-7] of germline genes involved and that the anti-GAT repertoire at the V H level is dissimilar between at least the three strains examined, even though their anti-GAT antibodies are pGAT positive (108). In another recent report, as an example of heavy and light chain association defining antibody specificity, comparisons between the V gene sequences of H antibody to GAT and NP were made, showing that some heavy chains were derived from the same germline V gene but they are associated with H different light chain isotype gene products (109,110). VR genes were also analyzed and the Southern blot analysis of restriction enzyme digests 20 showed that these genes are also limited in an anti-GAT repertoire (111). It was observed that three germline genes are present in a l l strains examined and a l l these V regions are very similar at least in primary K structure. However, any one strain may not use a l l of these Vv genes in its anti-GAT response (112). Studies investigating the involvement of pGAT/CGAT in the anti-GAT T cell response have also been conducted. Early work showed that when genetically non-responding mice are immunized with GAT they respond by generating T suppressor cells from which immunosuppressive factor(s) may be extracted (115). The factor has affinity for GAT, expresses I-J determinants but not Igh or Igl constant region markers (114) and can induce the in vitro production of Ts2 cells in responder strains (115). It has been shown that this TsF bears CGAT idiotypic determinants (116) and recently T cell hybridomas capable of making factors have become available (117). Another T cell hybridoma, derived from a responder strain, has been produced and shown to produce a TsF similar in characteristics to the non-responder TsF (118,119). None of the factors have yet being analyzed in terms of their role in idiotype-mediated regulation. In a definitive report analyzing the molecular genetics of ten GAT-specific suppressor T cell hybridomas, of which six secrete CGAT positive factors, three GAT-specific helper T cell lines and hybridomas, it was observed tht T and B cells that recognize the same antigen do not transcribe similar heavy chain V region gene segments even though extremely sensitive detection methods were employed (120, 121). In 21 another recent study examining the public idiotopes associated with anti-GAT response i t was found that GAT-specific T cells could be primed by monoclonal anti-idiotypic antibodies (122). It was concluded that the monoclonal anti-id antibodies may be influencing T cell activity in an indirect but unknown manner (122). Further work on regulation in the GAT system is continuing and i t is thought that regulatory mechanisms in this system are generally similar to those in the ABA hapten system. 1.2.5 Lysozyme Due to their availability, small size, number of variants in nature and the ease of purification, lysozymes have served as useful probes for immunoregulation. In a study of the antigenic structure of native hen egg-white lysozyme (HEL) using rabbit and goat primary anti-HEL antibodies, i t was shown that HEL contained at least three antigenic conformational regions (123). Soon, however, i t was shown that HEL was comprised of many other, mostly, conformational determinants when heterologous (124,125) and monoclonal antibodies (126) were used. Studies initiated to investigate the nature of regulation involved in the anti-lysozyme response determined that while the H-2 linked genes control the responsiveness to lysozyme, non H-2 genes regulate the magnitude of the hyperimmune response (127). Investigations of T and B cell interactions in the HEL specific response show that both T and B cells recognize many distinct epitopes and that antigen bridging is required for the regulation of anti-HEL immune response (128). An 22 extensive series of experiments showed that the same HEL fragment could contain several different T and B cell determinants (128-132). Furthermore, studies in genetic non-responder animals suggested that helper and suppressor cells recognize different epitopes (130). An analysis of the structural relationship between carrier determinants and antigenic determinants has recently confirmed the requirement for antigen bridging between B and T cells (133). Analyses of idiotypes in the HEL response have also been undertaken in which data from responder animals reveal the presence of a predominant cross-reactive idiotype (IdXL) which does not appear to be linked to either the H-2 or Igh allotype (134). Curiously, however, polyclonal antibodies only against the NC-HEL fragment bear IdXL (134), whereas anti-HEL monoclonal antibodies specific for non NC-HEL determinants also bear IdXL (135,136). Evidence for a selective mechanism for idiotype expression was obtained when i t was shown that IdXL-positive antibodies appear with a corresponding decrease of IdXL negative antibodies during the maturation of anti-HEL antibody response (136). The generation of B cell hybridomas using HEL immune splenocytes from mice in various stages of anti-HEL response has verified the above observation (133,137). In addition to IdXL expression at the B cell level, evidence has been obtained for its expression at the T cell level as well. In an extensive study i t was shown, using BIO recombinant strain mice that administration of anti-idiotype reagents could induce T suppressor cells in HEL-non-responders. In addition, the anti-IdXL plus complement had a 23 cytotoxic effect upon the anti-HEL T suppressor cells (131). Recently, a T cell line which produces a factor with suppressor activity toward in vivo primary and secondary anti-HEL responses has been described (138). The factor exhibits exquisitely fine epitope specificity in that i t has suppressive activity neither toward the closely related ringneck pheasant lysozyme nor to the L l l fragment of HEL (138). In further characterization of the factor specificity, i t was demonstrated that the factor is directed toward the phenylalanine residue at the third position of HEL (139). These studies also showed that this factor displayed both V and H-2 restriction (139). To date, however, the idiotypic profile H of this factor has not been reported. In another recent study examining the presence of IdXL expression and recognition by HEL-primed T cells (Th, Ts inducers, Ts effector), it was found that among the three cell subpopulations only the TsC possesses IdXL determinants whereas Tsi is anti-idiotypic in that i t can be depleted after adsorption to IdXL-coated plastic dishes (140). This complementary specificity of the Tse and Tsi was taken as evidence for communication between these cells based on idiotypic recognition (140). Studies are currently in progress to investigate these interactions. 1.2.6 Staphylococcal nuclease The enzyme staphylococcal nuclease (nuclease) from Staphylococcus aureus is another protein which has served as a useful immunological probe. The biochemical and biophysical properties of this enzyme are well understood (141). Initial studies involved the analysis 24 of antigenic determinants located on the enzyme (142) and measurements of anti-nuclease antibody activity consisted of nuclease enzyme activity assay (143). Further studies showed that B cell antigenic determinants are highly dependent upon the native tertiary configuration of the molecule (144) which hindered further progress in the determination of epitopic structures at the antibody level. T cells, however, were shown not to be so dependent upon the native conformation and in fact they responded very strongly to peptide fragments of the enzyme (145-147). The primary murine anti-nuclease response is linked to an H-2 associated gene (148). However, secondary and subsequent responses do not seem to be linked to either the H-2 or Igh genes (149). Further investigations demonstrated that whereas the H-2 genes control the epitope specificity of the anti-nuclease response (150), non-H-2 genes regulate the response magnitude (149). Studies have been undertaken to define the possible significance of idiotypes in the anti-nuclease response. In an examination of genetic linkage and strain distribution of anti-nuclease idiotypes using congenic recombinants i t was shown that, in the case of A/J anti-nuclease idiotypes, no H-2 associated gene linkage was apparent (151) whereas such linkage was necessary for the epitope response to nuclease (150). This study further revealed that anti-nuclease antibodies of A/J and SJL mice each expressed predominant intrastrain cross-reactive idiotypes, although the two idiotypes did not have common determinants (151). The A/J and SJL idiotype expressions were each linked to their respective Igh loci (151). BALB/c mice, however, were found to express 25 both of these idiotypes in their anti-nuclease antibodies. A study of antigenic regions of nuclease demonstrated the presence of distinguishable idiotypes on anti-nuclease antibodies each one of which were found to map to different V genes and were further used in analysis of V - C H H H recombination events in BAB.14 strain mice (152). Regulation studies of anti-nuclease antibodies show that when porcine anti-BALB/c idiotype is administered to BALB/c mice they respond, upon immunization with nuclease, by making idiotype bearing antibodies which do not bind nuclease (153). Such treatment of BALB/c mice induced the production of antigen-specific helper T cells which were sensitive to anti-idiotype and complement mediated lysis (153). Analogous results were obtained when animals were primed with the antigen alone (154) and these findings also held true in the A/J strain and its anti-nuclease idiotype bearing helper T cells (155). In a series of experiments designed to further exploit idiotypic interactions i t was demonstrated that porcine (anti-BALB/c idiotype) antisera and complement lyse only helper T cells from primed BALB/c mice but not from B10.D2 (156). Consistent with this result, B10.D2 mice did not make anti-nuclease antibodies of BALB/c idiotype. However, when B10.D2 mice are treated with porcine anti-(BALB/c idiotype) antibodies, they produce BALB/c idiotype bearing antibodies which do not bind nuclease as well as idiotype positive, antigen-specific helper T cells (156). These results allow a distinction between antigen-combining sites and idiotypes at the B cell level but not at the level of T cells. Clearly, whatever idiotype mediated immunoregulatory events occur in the nuclease system they appear to be very complex and await further clarification. 26 Some of the complexities of the various antigenic systems do not appear to be present in the ferredoxin system since ferredoxin (Fd), as an antigen, has many significant advantages over others. These include: (a) Fd is a small, naturally occurring, biochemically well defined, and stable molecule such that anti-Fd antibodies recognize both native and denatured forms equally well (94), (b) Fd is a bacterial protein with no mammalian autologous counterparts, thereby, reducing the possibility of autosensitization, (c) only two determinants exist at the B cell level and fragments bearing either one of the determinants to the exclusion of the other can be prepared relatively easily, (d) monoclonal antibodies prepared against Fd so far only react with either one of the two determinants and not any hypothetical third one, and finally, (e) Fd has no in vivo or in vitro toxic properties. 1.3 Review of the ferredoxin system Fd, an electron transport molecule produced by the anaerobe, Clostridium pasteurianum. has served as an antigenic probe for the dissection of various aspects of the immune response in Dr. J.G. Levy's laboratory over the past several years. Fd consists of a single, cysteine rich, fifty-five amino polypeptide of pi 1.9 (157,168). The eight cysteinyl residues, arranged roughly evenly in the middle part of the protein, do not participate in disulfide bonding, nor do they appear to be needed in eliciting an immune response (159). Rather, crystallographic work indicates that the native Fd molecule assumes a hairpin-like 27 structure (in which the amino- and carboxy termini are in close proximity to each other) such that the cysteinyl residues of each Fd molecule bind two f"e^S2 molecules via the sulfhydryl moieties (160). Due to its reasonably well characterized immunochemistry, relative structural simplicity and small size, Fd constitutes a well suited immunological probe. In view of the structural simplicity of the peptide, i t was possible to determine the epitopes of Fd. To that end a variety of small peptides, constituting various portions of Fd, were synthesized and used in competition assays to define their similarity or dissimilarity to epitopes of native Fd. Using the inhibition of complement fixation between specific rabbit antisera and oxidized Fd, and the synthesized peptide fragments as competitors, i t was found that the amino terminal heptapeptide and the carboxy-terminal pentapeptide formed two dominant determinants of oxidized Fd since they bound different populations of antibodies (161,162). Subsequent studies, in which rabbit antisera to 14 oxidized Fd was analyzed using C-acetylated peptides in equilibrium dialysis, determined that essentially a l l antibodies formed in response to Fd were directed toward the two determinants defined by these two peptides (162). Later studies performed with inbred mice confirmed and extended these findings (163). This work formed the basis of experiments which established that the magnitude as well as determinant selection (164) in the anti-Fd response of inbred mice were controlled by I-A region gene products of the murine MHC such that H-2 animals are high responders. "b s d H-2 and H-2 are intermediate and H-2 animals are effectively 28 non-responders (>10, 5, and 0 v»g Ab/ml, respectively) (164). In more recent years, attempts have been made to characterize the idiotypes of the Fd system. Using rabbit antisera raised against whole anti-Fd antibodies from various strains of mice, evidence was obtained for the presence of at least three major cross-reactive idiotypes in the anti-Fd response of three mouse strains (165). The same study showed that eight strains of mice differing at Igh or Igl loci can express each of these idiotypes and therefore these cross-reactive idiotypes are apparently unrelated to heavy or light chain allotype alleles. However, H-2 linkage to the idiotypes was observed but with no predominant pattern. These serological data from heterologous sera were confirmed with monoclonal antibodies. Antibody heterogeneity of each idiotype family was demonstrated with biochemical data (165). In a slightly different approach, monoclonal antibodies to Fd were raised in BIO.BR high responder mice and analyzed for the presence upon them of immunoregulatory idiotypes with the use of anti-idiotypic antisera raised in rabbits. This approach demonstrated that one of the antibodies (Fd-1), specific for the amino-determinant represented an idiotype that is dominant on anti-amino-determinant antibodies and is present in the majority of BIO.BR mice tested (166). Further work showed that this idiotype was found in anti-Fd sera of mice with the Igh-b allotype (167). Fractionated cell population studies carried out in adoptively transferred irradiated syngeneic mice showed that both the idiotype and anti-idiotype treatment of T cells, but not B cells, resulted in a significant increase in the anti-Fd secondary response to both determinants. The relative 29 amount of Fd-1 idiotype, however, was constant (167). A second monoclonal antibody, directed toward the carboxy determinant (designated as Fd-B2 and generated by a fusion of Fd hyperimmune spleen cells from a BIO.BR mouse with SP/2 myeloma cells) was also analysed in a manner similar to Fd-1. Preliminary observations indicated that the idiotype represented by the monoclonal antibody Fd-B2 was rarely expressed in the anti-Fd sera of BIO.BR mice (168). Using a very limited number of animals, the same study suggested the intriguing possibility that in vivo treatment of mice with anti-Fd-B2 (anti-idiotype) resulted in enhancement of anti-Fd response of BIO.BR mice and abrogation of the non-responder status of DBA/2 (H-2d) mice. It was postulated that the Fd-B2 idiotype could be a predominant regulatory element in BIO.BR and DBA/2 mice by acting on a suppressor cell population (168). In an expanded and carefully controlled study, using a sensitive inhibition assay, i t became clear that the Fd-B2 idiotype was rarely present, and then only at very low levels (<30 ng/ml) in BIO.BR anti-Fd antisera (168). In genetic linkage studies, the Fd-B2 idiotype was not found in anti-Fd antisera of other strains with Igh-b allotype, nor in anti-Fd antisera from strains of Igh allotype other than b. Thus, although a B cell product, Fd-B2 does not represent a common idiotype expressed at the B cell level. When either the idiotype (Fd-B2) or anti-idiotype was administered to Fd-immune or non-immune BIO.BR mice, the subsequent anti-Fd response was significantly enhanced, while expression of the Fd-B2 idiotype was s t i l l not detectable in the resulting antisera. Similar observations were made in H-2 mice of allotype other than Igh-b 30 indicating that this effect was not linked to the Igh genes. Adoptive transfer studies were performed in which T cells from a variety of mice were treated with either, Fd-B2 or anti-Fd-B2 (anti-idiotype) plus complement prior to adoptive transfer into irradiated syngeneic mice as well as unprimed syngeneic B cells. Invariably, the responses to Fd in mice reconstituted with idiotype or anti-idiotype-treated T cells were significantly increased compared to appropriately treated controls (168). Perhaps the most important effect observed was that idiotype-bearing T cells existed in B10.D2 non-responder (Igh-b, H-2d) mice since the manipulation of such cells with the anti-idiotype resulted in the conversion of non-responder mice into responder animals (170). In order to more clearly define the role of the Fd-B2 idiotype in immunoregulatory interactions of B10.D2 mice with regard to Fd, as well as to characterize the implicated suppressor T cells, further studies were undertaken. In view of the rare expression of Fd-B2 idiotype, attempts were made to generate T cell lines bearing this idiotype in order to carry out in vitro network studies usually performed with more common idiotypic systems. Finally, some network interactions were elucidated in the P815 mastocytoma tumour system in order to study the commonality of network interactions between tumour and soluble antigen. 31 Chapter 2 2.0 Materials and Methods 2.1 Experimental animals. BIO.BR, B10.D2, DBA/2 CBA, and C57BL/6 female mice (used at the age of 5-12 weeks of age) were either purchased from Jackson Laboratories (Bar Harbor, Maine) or were bred from mating pairs (Jackson Labs) and maintained in our animal breeding facility. New Zealand white female rabbits (8-10 weeks of age) were obtained from the University of B.C. Animal Care Unit and were housed in our animal facility. 2.1 Purification of antigens. Ferredoxin was either purchased from Sigma (F7629) or isolated and purified from Clostridium pasteurianum using the method of Mortenson (171) and Tanaka et al. (1974) as modified by Sikora (94). Briefly, the cells were grown, stored and lysed as described in detail by Sikora (94). The Fd containing supernatants of cell lysates were applied to 1.0 M phosphate buffer (pH 6.5) equilibrated DEAE-Sephadex A-25 resin (Pharmacia, Sweden) and washed with 0.15 M NaCl. Fd was eluted in a one-step procedure with the same buffer containing 1.0 M NaCl. Unideterminant fragments of Fd were generated by enzyme digestion of iron-sulfide free Fd, also described by Sikora (94). 32 -Keyhole limpet haemocyanin (KLH) was purchased (Calbiochem #37H805) as a salt slurry in (NH^ JgSO^ , dialysed extensively in phosphate buffered saline (PBS) and was stored in 1 mg/ml aliquots at 4°C or -20°C. 2.3 Immunization and collection of sera. Immediately following adoptive transfer of variously treated enriched T cell populations (see below) B10.D2 mice were immunized subcutaneously (s.c.) in the right flank with 50 yg Fd in PBS emulsified 50:50 in complete Freund's adjuvant (CFA) H37Ra (Difco Laboratories, Detroit, Michigan). As an internal control, mice received 50 yg KLH in 50% CFA s.c. in the other flank. Control mice were immunized with KLH only. Mice were individually bled from the t a i l vein on day 21 for primary immune sera. The animals were boosted on day 28 as above and bled on day 35 for secondary immune sera. The anti-Fd and anti-KLH titres of these antisera were measured by using a standard ELISA. The antisera were stored at -20°C. 2.4 Standard ELISA. Anti-Fd and anti-KLH antibodies were quantified using an enzyme-linked immunosorbent assay (ELISA) as described previously (173). Briefly, 96 well polystyrene ELISA plates (Dynatech Immunolon I) were coated with 0.1 ml of 1.0 yg/ml Fd or KLH in carbonate coating buffer 33 (pH 9.6). The plates were incubated overnight at 37 C in a humidified incubator. After washing three times with PBS-Tween washing buffer, 0.1 ml of anti-Fd or anti-KLH antisera in various dilutions (ranging from 1:100 to 1:800) were applied to plates in quadruplicate. The plates were incubated for 1 h at room temperature and then washed three times with PBS-Tween. Next, 0.1 ml of previously titrated rabbit anti-mouse Ig (Ramlg) conjugated by glutaraldehyde (173) to the enzyme alkaline phosphate (Boehringer Mannheim) was added to each well and incubated for 1 h at room temprature. The plate was washed three times with PBS-Tween again and 0.1 ml of substrate solution (1 tablet of p-nitrophenyl phosphate, Sigma #104-105, per 5 ml of diethanolamine buffer, pH 9.8) was added to each well. Colour development was allowed for 1, 1.5, 2 and 2.5 hr intervals at room temperature and quantitated at 405 nm with a Titertek Multiscan (Flow Laboratories) spectrophotometer. Absorbance readings were compared to a standard curve which was generated by using control anti-Fd or anti-KLH antiserum containing known amounts of specific antibodies as described (208). 2.5 Generation of the idiotype bearing anti-Fd antibody Fd-B2 and  anti-idiotypic antiserum. The monoclonal antibody Fd-B2 was generated by Dr. M.S. Weaver (168) in Dr. J.G. Levy's laboratory through a fusion of Fd hyperimmune spleen cells from B10.BR mice with SP/2 cells using standard techniques (174). The resulting hybridoma (Fd-B2) produced a monoclonal antibody which binds the C-determinant of the Fd molecule. 34 The production and characterization of anti-Fd-B2 (anti-idiotype) serum has been described previously (166,169). Briefly, anti-idiotypic antiserum was produced in a New Zealand white rabbit by the intramuscular injection of the monoclonal antibody Fd-B2 isolated from ascites fluid on protein A-sepharose. Anti-mouse Ig constant region activity of the antisera was removed by extensive adsorption onto Sepharose 4B (Pharmacia) beads to which mouse Ig had been coupled by the CNBr activation procedure (209). Such adsorbed anti-idiotypic antibodies were further affinity purified using a Sepharose 4B column to which the monoclonal Fd-B2 had been coupled (209). 2.6 Adoptive transfer in the Fd system. 2.6.1 Preparation of B and T cell enriched populations and  reconstitution. Non-immune spleen cells from B10.D2 mice were enriched for T cells by passage over nylon wool columns as described by Julius et al. (175). Cell recovery was routinely in the range of 30%, approximately 95% of which were T cells as determined by anti-Thy-1 plus complement treatment as described previously (169). Purified T cells were maintained in Dulbecco's Modified Eagle's Medium (DME) (Gibco Laboratories, Grand Island Biological Co., Grand Island, N.Y.). B cell enriched populations from non-immune B10.D2 splenocytes were prepared by double treatment of cells with anti-Thy-1 (1:20) on ice for 40 min followed by 2 washes in DME 35 after which the cells were treated with rabbit complement (1:10) at 37°C for 40 min (Cedarlane Labs. Ltd., Hornby, Ont.) as described (169). Purified non-immune B10.D2 splenic T cells were treated in a variety of ways (see below) prior to their use in reconstitution of irradiated syngeneic animals. Reconstitution was routinely carried out as follows. Non-immune B10.D2 mice were irradiated sublethally with 600 rads from a gamma source (Gammcell 200; Atomic Energy of Canada, Ottawa). Each 7 mouse was given a total intravenous (i.v.) injection of 10 treated or 7 control T cells and 10 non-immune B cells. The mice were immediately immunized as described above. 2.6.2 Lyt determination by panning. Anti-Lyt-1 and anti-Lyt-2 monoclonal antibodies (both directed to common allelic determinants) were prepared from the cell lines T1B104 and T1B105 respectively. The cell lines were the kind gift of Dr. D.G. Kilburn (Department of Microbiology, U.B.C.) and were originally obtained from the ATCC (Rockville, MD., U.S.A.). Both cell lines were allowed to overgrow in DME containing 10% foetal calf serum (FCS) until cell death and lysis occurred (as confirmed with Eosin Y dye exclusion). Culture supernatants were harvested, centrifuged to remove debris and stored at 4°C until used. Panning was carried out according to procedures described by Wysocki and Sato (176) with minor modifications. Plastic tissue culture plates (Beckton Dickinson and Co., Cockeysville, MD., U.S.A; 36 100x20) were coated overnight at 4 C. The plates were washed three times with PBS, after which 5.0 ml of B10.D2 nylon wool enriched T cells 7 in DME at a concentration of 1x10 cells/ml were added. Incubation was carried out for 40 min at 4°C after which the plates were gently rotated several times. Plates were further incubated at 4°C for another 30 min. Controls consisted of T cells incubated on plates which had previously been coated with 1% FCS in DME. After incubation, individual plates were gently rotated manually to resuspend non-adherent cells which were pipetted off. Three gentle washes with cold PBS were carried out and pooled with the non-adherent populations. Adherent cells were removed by pipetting 5 ml of 5% FCS in PBS at 37°C into the plates and gentle scraping with a rubber policeman. The plates were then flushed with Pasteur pipettes and the eluted cells were harvested by centrifugation. Separated cell populations were washed three times with DME, resuspended, and counted using Eosin Y exclusion for a viability determination. Subsequently, the non-adherent T cell populations were treated either with the idiotype Fd-B2 or anti-idiotype (10 yg protein 7 per 10 cells/ml) plus complement, or with the appropriate controls (irrelevant monoclonal antibody of the same subclass as Fd-B2 or normal rabbit Ig plus complement). Experimental groups of 12 B10.D2 mice 7 received 10 T cells consisting of reconstituted adherent and 7 non-adherent populations, and 10 unprimed B cells. Reconstituted animals were immunized as described. 37 2.7 Idiotype or anti-idiotype bearing T cell lines and clones. T cell lines were generated essentially as described by Kimoto and Fathman (77) with the modifications noted below. 2.7.1 Idiotype-bearing T cell lines. Eight B10.D2 irradiated (600 rads) females (6 weeks of 7 age) were adoptively reconstituted with i.v. injections of 4x10 naive syngeneic nylon wool-enriched T cells (per mouse) which had been treated 7 with the idiotype Fd-B2 (10 yg/10 cells on ice) plus complement (as before) (thereby, presumably, eliminating anti-idiotypic T cells). Each 7 animal was also given 4x10 naive syngeneic B cells prepared as described above. Following a 7 day rest period, the animals were challenged with 25 yg Fd in CFA per hind foot pad and then 10 days later popliteal and inguinal lymph nodes were removed. Lymphocyte cultures 5 6 (total volume 15 ml) containing 10 cells/ml, 50 yg Fd/ml, 2.5x10 irradiated (3600 rads) B10.D2 spleen cells in RPMI-1640 with lOmM Hepes, _5 5x10 M 2-mercaptoethanol (2-ME) and 10% heat inactivated normal human serum (NHS) (Canadian Red Cross Blood Bank, Vancouver, Canada), were produced in tissue culture flasks (Falcon) and incubated for 9 days at 37°C, in a humidified incubator with 10% C0^. Live cells were separated by centrifugation on Lympholyte-M (Cedarlane). Viable cells from the interface were recultured into medium (RPMI-1640 with Hepes, 3 glutamine at 2x10 M, 2-ME, 100 yg/ml streptomycin, and 100 U/ml 38 penicillin) with 10% NHS following two washes in the culture medium with 3% FCS. A fraction of viable cells were saved and entered into antigen-specific interleukin-2 (IL-2) release assays (see below). T cells were cultured in the presence of Fd (100 yg/ml) and irradiated B10.D2 splenocytes (2.5x10^ cells/ml) for another 9 days. This cycle of separating live cells with the addition of fresh irradiated antigen presenting cells (APC's) and antigen was repeated every 9 days at the end of which the cells were washed and recultured. After every wash some cells were saved and tested for IL-2 release under various conditions (see Chapter 5). After the third nine-day cycle these T cells were subdivided into two cultures. Both cultures were identical except that one culture contained Fd whereas the other contained anti-idiotype antibodies as the antigen. Cells from T cell lines were cloned by limiting dilution (96-well tissue culture plate, Becton Dickinson) in 200 yl culture medium plus 10% NHS, 50 jag/ml antigen and a feeder layer of 5 irradiated splenocytes (10 cells/well) following a four-day pulse of 1% IL-2-containing EL4 cell line supernatant derived from EL4 cells stimulated with phorbol myristic acetate (222). Only wells showing single colonies were scaled up to 10 ml cultures and maintained exactly as described by Kimoto and Fathman (177). 39 2.7.2 Anti-idiotype bearing T cell lines. Six female B10.D2 mice were treated with i.v. injections of the anti-idiotype (10 yg/mouse) and rested for 1 week. Mice were then challenged in both hind foot pads with 25 yg Fd-B2 monoclonal antibody in CFA per mouse. Ten days later the popliteal lymph node cells were excised and treated exactly as above, the exception being that the Fd-B2 monoclonal protein was used as the antigen in culture. 2.8 IL-2 release assay as a measure of antigen-specific T cell  proliferation 2.8.1 Antigen-specific IL-2 release. T cells from lines or clones were tested for IL-2 release in the presence or absence of the test antigen. In each assay (carried 4 5 6 out in duplicate) 10 or 10 T cells were cultured with 10 irradiated splenocytes. The culture volume was 200 yl and each culture contained 35 yg/ml antigen, 10% NHS and 5xl0~5 M 2-ME in RPMI-1640. Incubation (37°C, 10% CO^) was continued for 48 h. Culture supernatants were harvested, centrifuged twice with a change of centrifuge tube each time to remove contaminating cells and tested on concanavalin-A (Con-A) induced blasts as described below. Control cultures either contained no antigen or an irrelevant antigen. 40 2.8.2 Preparation of Con-A induced blasts. The presence of IL-2 was detected according to a modification of a previously described method (178). Single cell splenocyte suspensions from (BALB/C X CBA)F1 males (6-8 weeks old) were prepared at 15xl06 cells in 5 ml RPMI-1640 containing 5% FCS and _5 5x10 M 2-ME in 16 mm plastic dishes. Con-A (Sigma, Type IV, No C-2010) was added at 2 yg/ml and the cultures were incubated for 48 h following which the cells were washed and centrifuged gently (800 rpm, 10 min). The blasts were distributed into 96 well flat bottom tissue culture 4 plates (Flow Laboratories) at 2x10 cells per 0.1 ml RPMI-1640 and 2.5% FCS. To each well 0.1 ml of putative IL-2 containing supernatant ranging in doubling dilution from 1:2 to 1:16 were added. Each dilution was 3 tested in duplicate. After two days of incubation, [H]thymidine 3 ( [H]TdR) (New England Nuclear) was added at 1 jiCi/jil per culture 18 hrs before harvest. Cells were harvested on glass microfibre filters (Whatman) and radioactivity was counted.on a scintillation counter (TRI-CARB 4550, Packard) (for 1 min per sample) using standard scintillation cocktail of PP0/BIS MSB (Syndel Labs) in toluene (4g/litre). Results are expressed as counts per minute (CPM) which never differed from each other by 12% for each duplicate pair. Controls consisted of culture supernatants from non-specific antigen or no-antigen containing T cell proliferative cultures. Every experiment also contained a standard growth curve in which IL-2 from the EL4 cell line was used in 4 log dilutions ranging from neat to 1:10 supernatant. Such a curve is 41 shown i n f i g u r e 2 . 1 . The methods employed f o r network s t u d i e s i n t h e P815 mastocytoma tumour system a r e d e s c r i b e d i n Chapter 6 . 42 Figure 2.1. Titration of IL-2 secreted by the EL4 cell line. EL4 cells were suspended at 10^ cells/ml in RPMI-1640 supplemented with 5% FCS and incubated with 10 ng/ml of PMA (phorbol myristic acetate) in ethanol at 1 mg/ml for 18-24 h. Supernatants were harvested by centrifugation and stored frozen at -70°C in 0.5 ml aliquots. The presence of IL-2 activity was measured by using the proliferation of 48 h Con-A-induced 5 splenocyte blasts (2x10 cells/ml) in the presence of various dilutions 3 of PMA-stimulated EL4 cell supernates and H-thymidine (1 yCi/10 yl per culture). Culture volumes were 200 yl. Con-A-induced blasts were harvested on to fibreglass f i l t e r discs using a cell MASH harvester and 3 H-thymidine uptake was measured using a scintillation counter. Results are indicated by counts per minute (cpm). Each point on the graph is an average of duplicate which were consistently within 10% of each other. 1/ S E R I A L DILUTION I 44 Chapter 3 3.0 Evidence for Network-Mediated Immunoregulatlon in B10.D2 Fd-Non-Responder. 3.1 Introduction Previous experiments using the monoclonal anti-Fd antibody Fd-B2 have established the possibility of its recognition in an idiotypic T cell network in a variety of mouse strains with unrelated allotype or haplotype. The role of id-bearing T cells had also been investigated in non-responder mice of the B10.D2 strain. Studies involving either intravenous treatment of mice with the anti-id antiserum or adoptive transfer of nylon wool-enriched T cells treated with the anti-id antiserum into irradiated syngeneic animals showed that such treatments abrogated the non-responder status of B10.D2 animals whereas control animals remained non-responsive (169). In order to further elucidate the role of the idiotype Fd-B2 in a possible antigen-specific immunoregulatory network, a series of adoptive transfer experiments were performed. These are described below. 45 3.2 Results 3.2.1 Bffect of the Fd-B2 idiotype on T c e l l 3 from  Fd-non-responding mice. Nylon wool-enriched T cells from 6-8 week old Fd non-immune mice were treated with 10 yg of the monoclonal Fd-B2 per 7 10 cells/ml DME for 60 min on ice followed by treatment with rabbit complement as indicated in Methods. Control cells were treated with an irrelevant monoclonal antibody of the same subclass as Fd-B2 (IgG2b). Groups of 12 B10.D2 females which had 24 h previously been irradiated with 600 rads were given 10 treated or control T cells along with an equal number of Fd non-immune anti-thy-1 antiserum treated syngeneic splenocytes. Immediately following adoptive transfer the mice were challenged with 50 yg Fd and KLH (an internal control) or with KLH alone in a 1:1 mix of CFA. Mice were rested and bled on day 21, boosted on day 28 and re-bled on day 35 as described in Materials and Methods. Individual sera were analyzed for anti-Fd and anti-KLH antibodies as well as N:C-determinant specific antibody ratios in standard ELISA test wells established in this laboratory. A very sensitive competitive inhibition ELISA was developed to measure the level of idiotype present in anti-Fd antisera (169). The results of such an experiment shown in Figure 3-1 indicate that while the anti-KLH response was the same in a l l groups, the anti-Fd response of specifically treated mice approximated that of normal high-responders whereas control B10.D2 mice maintained their non-responder 46 Figure 3.1. Depletion of an Id + (Fd-B2) recognizing T cell population changes a nonresponder strain for Fd into a responder. Non-immune B10.D2 nylon wool-purified T cells were treated with the monoclonal antibody g Fd-B2 (IgG2fe; 10 yg/ml/10 cells) or an equal amount of an irrelevant monoclonal antibody (Nonsp-mAb) in phosphate-buffered saline, pH 7.2, or in DME plus rabbit complement (C). B cell suspensions were prepared by treatment of nonimmune B10.D2 splenocytes with a monoclonal anti-Thy-1.1 plus C. Groups of twelve previously irradiated (500 rads) 7 B10.D2 mice received a 50:50 mixture of 2x10 treated T cells and B cells i.v. and were immediately immunized with 50 yg each of Fd and KLH in 50% complete Freund's adjuvant. The 21-day primary response is shown. 47 T R E A T M E N T O F T C E L L S A N T I G E N N O N - s p - m a b * C ug A N T I B O D Y P E R ml £ & l£_ K L H F d - B 2 » C F d K L H P B S I C F d K L H 48 status. These results together with those described in an earlier study (169), showed clearly that non-responder B10.D2 mice have T cell populations which bear both the Fd-B2 idiotype and its respective anti-idiotype, and that the removal of either population resulted in T cells capable of helping to generate a substantial anti-Fd antibody response under the conditions described here. 3.2.2 Evidence for an idiotypic network controlling the Fd  response. The results reported above and those of a previous study strongly suggested the existence of a T-T cell idiotype-anti-idiotype connected network which operates in regulating the immune response to Fd. The disruption of this communication by uncoupling the T-T cell interaction by removal of either the idiotype-bearing or anti-idiotypic T cells results in a population capable of helping in the antibody response to Fd. It was, therefore, argued that i f this were true, the non-responder status could be re-established i f T cells treated with either the idiotype or anti-idiotype (each population In itself being a "responder" population) were mixed together and used in syngeneic reconstitution. To show this, nylon-wool purified non-immune B10.D2 T cells which had previously been treated with the idiotype or anti-idiotype plus complement were adoptively transferred into irradiated syngeneic recipients, either one population at a time or mixed together. One group of mice received cells which were sequentially treated with both the 49 Figure 3.2. Reconstitution of unresponsiveness with Id+-depleted and anti-Id-depleted T cells. Id and anti-Id treated B10.D2 T cells and nonimmune B cells were prepared and adoptively transferred to groups of 12 mice as described in Figure 3.1. Mice were immunized with Fd and KLH as an internal control. The 21-day anti-Fd and anti-KLH antibody response is shown, a and b refer to T cell populations which were first treated with anti-Id plus complement, washed and then treated with the idiotype Fd-B2 plus complement and washed again prior to adoptive transfer into irradiated mice. C E L L S O F T R E A T M E N T  A N T I - l d ' C I d l C ug A N T I - F d A N T I B O D I E S P E R ml 1,0 2 0 30 40 50 ug ANT IK L H ANTIBODIES P E R ml 2 0 AO 6 0 g o 190 10 7 10 7 10 + +° 51 idiotype and the anti-idiotype, each treatment being followed by lysis 7 with complement. All mice received a total of 10 T cells with an equal number of naive B cells. The results in Figure 3.2 show that mice which received idiotype (Id) or anti-idiotype (anti-Id) treated T cells mounted a very substantial anti-Fd response, whereas those reconstituted with an equal mixture of both cell populations made a marginal response (26 and 31 as opposed to 4 yg anti-Fd antibody/ml). Interestingly, mice which were reconstituted with the doubly treated T cells make a significantly higher anti-Fd response than those reconstituted with the id or anti-id treated T cells (see discussion below). The anti-KLH response of a l l four groups is similar. The slight anti-Fd response seen in the mice reconstituted with both T cell populations together is not a surprising observation since imbalance of various known and unknown physiological interactions between the Id + and anti-Id populations could have resulted from in vitro manipulations of the cell populations, as well as possible modifications of auxiliary mechanisms of unknown character. (Also, see further results below in this chapter). In any case, these results provide clear evidence that an id- anti-id network between T suppressor cells exists and that i t can be uncoupled and, perhaps more importantly, re-established by the methods described here. 52 3.2.3 Titration of the Id + and anti-Id T c e l l 3 against  each other. The experiments reported above, although providing evidence for the existence of an id-anti-id network as well as its precise "tight coupling", do not provide clues as to the stoichiometry of the network reactions between the two cell populations involved in the non-responding mice. The question was asked as to whether or not one Id + T cell is needed to be in communication with an anti-Id T cell to maintain a non-responding situation to Fd. Groups of previously irradiated non-immune B10.D2 mice were reconstituted with various numbers of either id or anti-id plus complement treated nylon-wool enriched T 7 cells for a total 10 T cells. All mice also received non-immune ahti-thy-1 plus complement treated syngeneic splenocytes. The results in. Figure 3.3 show that the maintenance of the non-responder status occurred, only when the Id and anti-Id treated T cells approach a 1:1 ratio whereas an excess of either population (Id treated vs. anti-Id treated; 9:1, 7:3, 5:5, 3:7, 1:9) leads to a sizeable anti-Fd response. A l l groups showed an unaltered anti-KLH response. These results, although they are not conclusive, do argue for a very tight one-to-one communication channel between the Id and anti-Id bearing T cell populations. The results could also imply (see Discussion) i f any Id or anti-Id T cells are not coupled then the presence of free (uncoupled) cells leads to anti-Fd response. 53 Figure 3.3. Titration of Id + and anti-Id (Id ) plus complement treated T cells against each other. Experiment was carried out as in Figure 3.1 using groups of 12 animals. The 21-day anti-Fd and anti-KLH response is shown. N U M B E R OF T C E L L S T R A N S F E R R E D 9 /10 3< 10 J i t S l O A N T I G E N gg A N T I B O D Y P E R ml 10 2 0 3 0 4 0 7 X 1 0 6 9 X 1 0 K L H Fd K L H Fd K L H Fd K L H F d K L H 3-55 3.3 Discussion The main aim of this study was to establish the existence of and characterize an idiotypically mediated Fd-specific network potentially responsible for the non-responsiveness state of B10.D2 (H-2d) mice. Previously, studies had shown that either intact mice treated with anti-Id or irradiated mice reconstituted with anti-Id plus complement treated nylon-wool purified naive (Id +) T cells make a measurable anti-Fd response upon challenge with the antigen. This finding suggests that elimination of an id-bearing T cell population disrupts immune regulation in such a way as to make possible an anti-Fd response. It had previously been shown that, at least in Fd-high responder mice an anti-idiotypic T cell population also existed and that the treatment of this population with the idiotype also led to an enhanced anti-Fd antibody response upon challenge with Fd (169). As shown in Figure 3.1, an analogous T cell population also exists in non-responder B10.D2 mice. These results, therefore, strongly argue for the existence of Id-bearing and anti-idiotype bearing T suppressor cells (Tsl and Ts2, respectively) in B10.D2 animals. Several network models have been proposed to account for the results obtained in various antigen systems (see Introduction) which account for the role of T cells in immunoregulation. However, the earliest network model to specifically include T cells described the suppressed state as involving both id-bearing (antigen-recognizing) and anti-idiotypic T cells (179). Subsequently, the work of Nisonoff (28) and Benacerraf (28,30,180) and colleagues clearly showed the role of idiotypic 56 and anti-idiotypic T cells in suppression within the arsonate system in which the id and anti-id T cells were demonstrated after the introduction of antigen into the system. In the present system, on the other hand, the presence of both id and anti-id-bearing T cells is demonstrated in the unperturbed, immunologically naive, T cell population. In addition, Hoffmann (182) has suggested in the context of the symmetrical network that T cells either suppress or help immune responses depending on their network connectivity, specifically on whether they are strongly or weakly connected to rest of the network via idiotype-anti-idiotype interactions. The suppressed (non-responsive) state in that model is considered to be of high connectivity for the antigen-specific and anti-idiotypic T cells; the immune state is said to reflect a condition of the lowest connectivity, whereas the virgin state is interpreted to be of intermediate connectivity. The results described in this chapter are consistent with that concept. According to the above idea, the Id and anti-idiotypic cells specific for Fd are viewed as having relatively high network connectivity in the unperturbed non-responder mouse, as compared with their counterpart cells in a high-responder mouse. Treatment of T cells with Id plus complement k i l l s the anti-Id cells (181), leaving the i d + T cells without their complementary receptor-bearing counterparts, and thus with a low connectivity. Such cells would then theoretically be able to function as helpers, since the essential difference, according to Hoffmann's model (182), between helper and suppressor T cells resides in their network connectivity. Analogously, exposure to anti-id antibody plus complement 57 kills the i d + cells. An immune response can then be mounted. Accordingly, recombination of the two subpopulations increases the connectivity of both i d + and anti-id T cells to the high level resembling the situation in the naive animal, and therefore, the non-responder state is restored. Figure 3.4 shows a model, which has been previously described (181), that represents an attempt to define anti-Fd regulation in B10.D2 mice. Seven of the twelve conceivable sets of cells are shown. Three classes of relevant idiotypes are depicted in the model. Id represents the idiotype on the C-determinant specific monoclonal antibody Fd-B2, and anti-Id represents its anti-idiotypic partner. Id' are the remaining idiotypes which recognize the C-determinant while Id" represents N-determinant recognizing idiotypes. Assuming linked recognition ("cognate" help), as is to be expected for Fd as an antigen, and given the relationship of idiotype regulation to conventional hapten-carrier effects (167), the Id" set of T cells would function as the helper cell for antibody responses to the C-determinant. Although direct evidence is available only for T . B T j t and B_.„ in B10.D2 animals, the model Id Id' Id" includes B and T cells in each antigen-specific set in order to maintain symmetry, and analogy with other investigations. As for the six anti-idiotypic cells (B anti-Id, T anti-Id', T anti-Id", etc.) not shown in Figure 3.4, only the effects of T anti-Id were evident in this study. Figure 3.2 also shows that when B10.D2 mice are reconstituted with T cells which have been doubly treated (that is, with both the idiotype and the anti-idiotype plus complement), a very high anti-Fd 58 Figure 3.4. A model depicting a minimal set of cells relevant to the discussion of the response of B10.D2 mice to Fd. The antigen has two + +' antigenic determinants, C and N. Id and Id are Id that recognize + +" C, and Id is cross-reactive with Fd-B2. Id is the set of Id that recognize N. A dominating role is played by the Id + and Id - T cells, that are capable of suppressing the response only i f they are both present. Anti-Id cells for the other populations presumably exist, but at sufficiently low levels that they have not made themselves manifest in our studies. 0 0 ^ ^ -N. 60 antibody response is made. This observation argues for the presence of Pd-specific helper T cells that are outside the Fd-B2 idiotype-anti-idiotype system which in the context of the proposed model, would be T cells of low connectivity or helper T cells of the Id" set. Assuming that once the antigen has bound to the antigen presenting cell (AP cell), with specificity being conferred upon the normally non-specific AP cells by virtue of antigen-specific T cell factor being cytophylic for AP cells (182), such AP cells become activated. Upon activation, the AP cells secrete non-specific second signal factors that allow the differentiation of B cells (which have previously bound the antigen via cell surface immunoglobulin). As is the case with most signal acceptor sites (for example, hormone receptor), i t is assumed that AP cells bear a finite number of acceptor molecules to which antigen-specific T cell factors can bind in a non-specific manner. As a result of this non-specific binding of antigen-specific factors, the AP cells specifically become charged with those antigen-specific factors (182). Because of the simultaneous presence of both the idiotypic and anti-idiotypic T cells in the environment, the AP cell surface can be said to be "jammed". This jamming occurs due to the mutual stimulation of Id and anti-Id T cells which results in the release of respective factors (with specificities of cells of origin) competing against the Id" factors for the factor acceptor sites on the surface of AP cells. This would, therefore, prevent the AP cells from presenting the antigen to Id" T cells for an anti-Fd response. However, once both the Id and anti-Id T cells have been eliminated the AP cells are released from jamming and an immune 61 response can occur (181). Since the Id" T cells are acting as helpers, the above interpretation of events is compatible with Id" T cells having low connectivity. Although i t is difficult to quantitate the "amount" of connectance required between the id and anti-idiotypic cells for non-responsiveness to be maintained, Figure 3.3 may suggest that a one-to-one contact is needed between the Id and anti-Id cells for them to jam up the AP cells. Each mouse was reconstituted with various amounts of 7 Id or anti-Id plus complement treated T cells (for a total of 10 cells), the actual number of Id and anti-idiotypic T cells which participate in the network is obviously far less. Such crude estimates indicate that the cells involved in the network are extremely potent and in a precisely coordinated state with each other. When such a precise coordination is disrupted an immune response becomes possible (as seen in Figure 3-3 in the case of T cell ratios other than 1:1). The slight anti-Fd response seen in doubly reconstituted mice (Figure 3.2) could be due to experimental inability to recreate the exact conditions required for this one-to-one coordinated state between the Id and anti-idiotypic T cells. The scheme presented in Figure 3.4 shows the Id and anti-Id T cells without Lyt phenotype markers assigned to them. In order to further clarify the cell-cell interactions and possibly shed more light upon the nature of cells, the suppressor T cell populations were characterized in terms of their Lyt cell surface markers. This work is discussed in the next chapter. 62 Chapter 4 4.0 Lyt Phenotype of the Idiotypic and anti-idiotypic suppressor T Cells. 4.1 Introduction Over the past several years monoclonal and polyclonal serologic reagents have been used to identify cell-surface structures and to correlate the presence of these structures with functionally distinct subpopulations of T cells. Since the original work by Harvey and Cantor and others (183-185), the Lyt family of T cell surface molecules have become the most extensively studied differentiation markers in the mouse. These studies involved the analysis of the effect of anti-Lyt alloantisera and monoclonal antibodies plus complement on T cells that were involved in various immune responses. In these studies i t was found that T cells involved in delayed type hypersensitivity and T-dependent antibody responses were Lyt l +,2 ,3 , whereas cytotoxic T cells and suppressor T cells were Lyt 1 ,2+,3+. This is a generality, and there is now ample evidence that Lyt subgroups do not precisely distinguish functional subpopulations. Using the conclusions of Cantor and Boyse, numerous studies were undertaken in which functions were assigned to T cells based upon the Lyt phenotype of T cells in question. Subsequently, the assigned T cell functions were used to derive a variety of mechanisms of action to explain 63 the particular observations in a given antigen system (see Introduction, Chapter 1). Recently, however, i t has been shown that the correlation between Lyt phenotype and function of T cells is not absolute (186). Studies by Nakayama, Herzenberg, Asofsky and their colleagues (185,187,188), using flow microfluorometric techniques, indicate that Lyt 1 antigen is present on most T cells with different degrees of expression by different T cell subpopulatiorts. According to these studies Lyt 1 + cells in complement-mediated cytotoxicity assays are Lyt 1 + bright in flow microfluorometry whereas Lyt l - cells are Lyt dull. Therefore, Lyt 2,3 is a more reliable and absolute marker for T cell characterization. In her analysis of cytotoxic and suppressor T cell activities, Swain (189) has suggested that the Lyt phenotype correlated with the class of MHC molecules recognized by a T cell rather than the cell's effector function. According to Swain's hypothesis, Lyt l +,2 3 phenotype correlates with specificity for class II MHC determinants, while Lyt 2,3 would correlate with specificity for class I MHC products or for unrestricted interactions. In support of these suggestions, Vidovic et al. and Pierres et al. (190,191) have reported on class I specific alloimmune helper T cells which express Lyt 2 whereas heterogenous or cloned CTL specific for class II bear virtually no Lyt 2,3 antigen. The assignment of restriction elements to Lyt 1 + or 2+3 T cells appears to be generally true, and this kind of grouping also pertains to the human situation in which T4+ cells are class II restricted and T8 + are class I restricted. 64 With the above contentious interpretation of the significance of the Lyt phenotype in mind, an attempt was made to characterize the Id and anti-Id bearing T cell populations in B10.D2 mice in terms of the Lyt cell surface markers. It was hoped that such phenotypic assignment, together with the results in Chapter 2, may provide clues regarding relationship between and possible mechanism of action of Id and anti-Id bearing T suppressor cells in the present system. 4.2 Results Initially, for a mass scale characterization of T cell populations according to Lyt phenotype, the panning technique described by Wysocki and Sato (176) was used, with slight modifications (see Materials and Methods). Lymphocytes adhering to tissue culture plates previously coated with either the anti-Lyt 1 or anti-Lyt 2 monoclonal reagents were eluted from the plates after the plates were washed free of non-adherent cells. It was found consistently that the recovery of adhered cells per 5 plate was 1% (10 cells) or less compared to the total number of nylon 7 wool-enriched cells applied to the plates (3-5x10 cells). Therefore, the reverse approach was used in which non-adherent cell populations were carefully removed free of the adherent cell populations and prepared for subsequent treatments before adoptive transfer into syngeneic mice. 65 4-2.1 Lyt phenotype of anti-idiotype-positive T cells In the first of this extensive series of experiments nylon wool enriched T cells from non-immune mice were panned on plastic plates coated with monoclonal anti-Lyt 2 and separated into adherent and non-adherent populations. Non-adherent cells (i.e., Lyt l +,2~) were treated with either the idiotype or an irrelevant monoclonal antibody (MAb) plus complement. They were mixed with equal numbers of recovered 7 7 adherent cells and used at a concentrations of 10 T cells plus 10 non-immune B cells per mouse in adoptive transfer experiments. The results are shown in Figure 4.1 in which i t is evident that treatment of the Lyt l +,2 populations with idiotype plus complement converted the recipient mice into responders, indicating that the anti-idiotypic T cells involved in the non-responsive status was of the Lyt l +,2 phenotype. Specificity of the response was indicated by the fact that the anti-KLH response of a l l animals was essentially the same. The reciprocal experiment was performed with T cells panned over monoclonal anti-Lyt-l + antibody coated plates. The non-adherent population (i.e., Lyt 1 2+) was treated with either the idiotype or an irrelevant MAb plus complement as described. In these adoptive transfer experiments, none of the mice responded to Fd whereas al l responded equally well to KLH (Figure 4.2). These data show that the anti-Id T cell in this system is Lyt-l +2~ and apparently belongs to the helper subpopulation according to the classical understanding of phenotypic markers. 66 Figure 4.1. The anti-Id (Id ) T cells do not express the Lyt 2 antigen. Nylon wool enriched B10.D2 splenic T cells were incubated on plastic plates coated wih a monoclonal anti-Lyt 2 antibody and separated into adherent and non-adherent populations (see Materials and Methods). Non-adherent cells (i.e. , Lyt 2 ) were treated with either the Id, or an irrelevant monoclonal antibody (Non-sp-mab) or PBS plus C and mixed with equal numbers of adherent T cells and non-immune B cells and adoptively transferred to groups of previously irradiated 12 B10.D2 mice. All animals were immunized with Fd and KLH (see Figure 3-1). 35-day secondary response is shown. T R E A T M E N T u « ANTIBODY PER m I OF T C E L L S ANTIGEN g 2 4 6 8 10 PBS + C Fd KLH Non-ip-maln-C Fd KLH Fd-B2<-C Fd KLH 68 Figure 4.2. The Id T cells expressed the Lyt 1 antigen. Non-immune B10.D2 T cells were prepared, panned on anti-Lyt 1 coated plates, and treated as described in Figure 4.1 and Materials and Methods. 35-day secondary anti-Fd response is shown. 69 T R E A T M E N T ANTIGEN ug ANTIBODY PER ml 4 6 8 10 P B S + C Fd KLH 1 1 ' Non-»p-mab + C Fd KLH Fd-B2 + C F d KLH • 1 ' 70 4.2.2 Lyt phenotype of idlotype-posltive T cells The lyt phenotype of the idiotype-bearing T cells was investigated in an analogous manner. B10.D2 T cells were panned over anti-Lyt-1 coated plates and treated with either anti-idiotypic antiserum or normal rabbit immunoglobulin (NRIg) plus complement before adoptive transfer. The non-adherent cells (Lyt-1~2+) after treatment with anti-id were capable of converting B10.D2 recipients to the responder status (Figure 4.3). On the other hand, when Lyt l + 2 ~ cells (non adherent to anti-Lyt 2 plates) were treated with anti-Id plus complement none of the recipients responded to Fd (Figure 4.4), whereas the KLH response in a l l groups was equivalent. It would therefore appear that the idiotype-bearing (thus antigen specific) T cell in this system is of the Lyt 1 ,2+,3+ phenotype whereas the anti-idiotypic population is Lyt l +,2 ,3 -All these experiments were performed at least twice with 12 animals per group per experiment. Each experiment included KLH as an internal control. As indicated in Figures 4.1-4.4, no ambiguous results were obtained. As an additional control, B10.D2 T cells were panned over plates coated with 10% FCS only and after separation into adherent and nonadherent fractions the latter population was treated with either NRIg or the irrelevant MAb and adoptively transferred to irradiated B10.D2 as before. Upon subsequent challenge with Fd, a l l animals maintained anti-Fd non-responsive status, whereas the anti-KLH response in a l l these animals was normal. 71 Figure 4.3. The Id + T cells do not express Lyt 1. This experiment shows the effect of id on anti-Lyt 1 non-adherent B10.D2 nylon wool purified T cells in adoptive transfer to B10.D2 mice. See Figure 4.1 and Materials and Methods. Controls consisted of T cells treated with normal rabbit immunoglobulin (NRIg) or PBS plus C. 72 ufl ANTIBODY PER ni TREATMENT OF T CELLS KLH PBS * C Fd KLH 73 Figure 4.4. The Id + T cells express the Lyt 2 antigen. Non-immune B10.D2 T cells were panned on anti-Lyt 2 coated plates, and treated described in Figure 4.1. 35-day secondary response is shown. 74 ug ANTIBODY P E R ml T R E A T M E N T OF T C E L L S A N T k F d - B 2 + C 3-75 4-3 Discussion The work described in this chapter and reported previously (194) was undertaken in order to further characterize the Id + and anti-idiotypic suppressor T cell populations according to their Lyt cell surface antigens. Such information, i t was reasoned, might shed further light upon possible immune response mechanisms utilized by the two T cell populations. When these studies were being carried out i t was expected, perhaps naively, that the Lyt phenotype of cells would conform to the general conclusions of Cantor and Boyse which have been corroborated by numerous other investigators (see Introduction of this chapter). The results actually obtained in terms of the Lyt cell surface markers of Id + and anti-idiotypic T cells were quite different from the expected findings. Using the panning technique described by Wysocki and Sato, as modified according to Materials and Methods, i t was demonstrated that the Id + T cells were Lyt 1,2 +, the anti-Id T cells were Lyt l +,2~. However, a more popular observation has been, for example in the arsonate system (29), that the Id + (antigen binding) suppressor T cells (Tsl) is Lyt l +,2~ and anti-idiotypic T cell (Ts2) is Lyt 1",2+. Since many authors have argued against a rigid correlation between cell surface antigen and cellular function, the results reported here are neither surprising nor unusual. Nevertheless, these results do constitute, as far as is known, the first evidence of lack of correlation between function and Lyt phenotype at the whole cell population level 76 rather than at the level of cloned cells or cell lines. More recently, in studies based on human T-lymphocyte clones, several workers have reported a lack of correspondence between T4+ cells as "helper/inducer" and T8+ cells as "suppressor/cytotoxic" cells (195) as has been widely accepted. Meuer et al . (194) have suggested, rather reminiscent of the suggestion made by Swain in the murine system (189), that T4+ CTL clones are directed against MHC class-II antigens, whilst T8+ clones only recognize MHC class-I molecules, and hence that T8 and T4 markers predict the MHC restriction elements of cells rather than their biological function. However, even this assertion has been disputed as several carefully designed studies demonstrated that same T4+ clones are MHC class-I antigen specific (195) whereas T8+ clones maybe class-II specific (196). Therefore, in view of every possible cell-surface phenotype on T cell clones of different function, one could conclude that such flexibility also extends to in vivo cell subpopulations and that the presence or absence of Lyt 1 and Lyt 2 in mouse or T4 and T8 in man is not necessarily indicative of the function of cells of interest. The question that arises then is: "Are the Lyt phenotype findings presented here consistent with the idea of connectivity proposed in the model depicted in Figure 3.4?" As MHC class-I antigens are widely distributed on a variety of cell types i t is reasonable to assume that class-I recognizing cells would perceive a constant signal and, therefore, be persistently stimulated. Within the context of a network such persistent stimulation would cause the stimulation and selection for the opposite partner, namely anti-idiotypic cells for Lyt 2 + cells. The 77 consequences of such preferential stimulation and selection become clear in that the Lyt 2+ have high network connectivity with their anti-idiotypic opposites, and therefore Lyt 2+ cells tend to function as suppressor cells. However, in light of the poor in correlation between cell surface phenotype and function as discussed above, this sentence should not be misconstrued as an inviolable statement. Nor does this imply that other cell surface markers which correlate well with the functional status of cells do not exist. The results reported so far concern whole cell populations in an in vivo system. It was believed that both the idiotype-bearing and anti-idiotypic reagents described here could be used as tools in the generation of T cell lines and clones which could subsequently be used in in vitro experiments to confirm and extend the in vivo results. The following chapter constitutes a description of this work. 78 Chapter 5 5.0 Evidence for Fd-B2 idiotype-anti-idiotype mediated network at the  level of T cell clones 5.1 Introduction In view of the results discussed in Chapters 3 and 4, i t was thought that the Fd-B2 idiotype and the anti-idiotype reagents might offer a unique opportunity to definitively test some network concepts in an in vitro situation. To that end, attempts were directed to the development of T cell lines and clones bearing on their surfaces idiotype or anti-idiotype reactive molecules. In addition, basic questions regarding the requirement for MHC identity between antigen-reactive T cells and antigen-presenting cells (APC's), the relationship between the antigen and the anti-idiotype, as well as the epitopic fine specificity of T cells which might participate in network pathways remained unaddressed in the Fd system. T cell lines and clones were generated from appropriately manipulated B10.D2 mice (see below). These cells were characterized with respect to their idiotype-anti-idiotype interactions, MHC-specificity and their antigenic fine specificity. 79 5.2 Results 5.2.1 Generation of B10.D2 derived idlotype-reactive  (anti-idiotypic) T cell lines Previously, i t had been reported that the i.v. administration of anti-idiotype antibodies into B10.D2 mice converted them to responders (169,170). Mice so treated were immunized with the monoclonal antibody Fd-B2 in CFA (1:1) in both hind foot pads as described in Materials and Methods. T cells derived from popliteal lymph nodes of these mice were cultured in complete RPMI medium supplemented with 10% normal human serum (NHS) and 100 yg/ml of the antibody Fd-B2 as well as irradiated syngeneic splenocytes. The cells were cultured and maintained according to the modifications of Fathman's procedure (177) described in Materials and Methods. In a typical test for antigen (in this case, Fd-B2) 5 specificity, 10 T cells were incubated for two days with 35 yg Fd-B2/ml. Control antigens were CAMAL-1, an isologous monoclonal antibody directed toward a human protein, and partially purified protein derivative (PPD) from Mycobacterium tuberculosis (Connaught Laboratories, Willowdale, Ont., Canada). Sham cultures containing no antigen were also generated. Culture supernatants were tested two days later for the presence of IL-2 as an indicator of antigen-specific T cell proliferation in an assay using 3 two day Con-A-induced blasts in the presence of [H]TdR (see Materials and Methods). The results of such screen are shown in Figure 5.1. The 80 Figure 5.1. Initial screen of a B10.D2-derived idiotype-reactive T cell line. T cells from females treated with anti-idiotype antibodies prior to challenge with the idiotype Fd-B2 were cultured in complete RPMI-1640 supplemented with 10% NHS. The cells were tested for IL-2 release in the 5 6 presence of indicated antigens (35 yg/ml) at 10 T cells/ml and 10 irradiated splenocytes in duplicate. Harvested culture supernatants were tested in duplicate for the presence of IL-2 activity using 48 h Con A induced splenocyte blasts (as in Figure 2.1) in the presence of 3 H-thymidine (1 yCi/10 yl per culture) by doubling dilution titration starting at 1:2 (1:4 final dilution) to 1:16. The results of 1:2 (1:4 final) dilution are shown and are expressed as CPM. 81 A N T 16 E N 0 NO A g PPD C A M A L - I F d - B 2 C P M X I O 4 6 10 82 results indicateed that the T cells thus selected reacted only with the specific idiotype of Fd-B2 and not with the control monoclonal or with PPD. These observations supported the contention that these T cells bear anti-idiotypic receptors on their surfaces. Following preliminary testing, the cells were twice harvested and maintained in antigen-containing cultures as described. The reactivity of these cells with the Fd-B2 idiotype in the context of APC's from mice of H-2b, H-2d and H-2k haplotypes was investigated. The results in Figure 5.2 showed that the anti-idiotypic T cells from B10.D2 (H-2d) mice proliferated only when Fd-B2 idiotypic determinants are presented in association with self-MHC gene products, since no proliferation occurred when the Fd-B2 antibody was presented the context of non-self MHC antigen-bearing splenocytes. These findings indicate that these idiotype-anti-idiotype interactions, at least at the T cell level may not be a simple consequence of ligand-receptor binding between T cells. However, one cannot rule out the possibility that activation could be due to the binding of the idiotype by the anti-idiotype since this experiment was not done in the absence of APC's. That idiotype-mediated activation does not occur in the presence of APC's from mice of non-self haplotype, however, does support the contention that MHC may play a role in idiotype-anti-idiotype recognition (see Discussion below). Comparisons between Figure 5.1 and 5.2, furthermore, show the time-dependent enrichment of T cells specific for the Fd-B2 idiotype. 83 Figure 5.2. MHC restriction of Id-dependent T cell lines. APC's were derived from BIO.BR (H-2k), C57BL/6 (H-2b), DBA/2 (H-2d), and B10.D2 d (H-2 ) irradiated splenocytes. Assay conditions were the same as in Figure 5.1. Cells were incubated with the antigen listed above and culture supernatants (final dilution 1:4) were tested for IL-2 as shown as 3 cpm H-thymidine uptake by Con-A blasts. oo s I s B I 1-I 8 l l n n n n n n n n f l n n r i n n n n n Li i •• l j 11 * j . 3 ~ h •» Q „ 7, « < o- s a « « * 1 f i h i i 85 5.2.2 Characterization of Fd- and anti-idiotype-reactive T cell lines Irradiated (Id + T cells) B10.D2 females were adoptively 7 reconstituted with i.v. injections of 4x10 naive syngeneic nylon wool-enriched T cells which were treated with Fd-B2 plus complement (as described in Materials and Methods). Animals were also given an equal number of naive syngeneic B cells prepared as described. This procedure rendered recipients depleted of anti-idiotypic T cells and converted them to the Fd-responder status by this process. After a 7-day period the mice were challenged with 25 yg Fd in CFA in each hind foot pad and 10 days later animals were sacrificed and popliteal and inguinal lymph nodes were excised. The cells were cultured in complete RPMI medium supplemented with NHS in the presence of 50 yg Fd/ml and irradiated syngeneic naive splenocytes. Figure 5.3 shows that the cell line established from such cultures was specifically reactive toward Fd. In addition, unexpectedly, reactivity was also seen to anti-idiotype antibodies. The cells did not produce significant levels of IL-2 in the presence of any other test materials (NRIg, PPD). In further attempts to demonstrate that some individual T cells may recognize both Fd and anti-idiotype determinants, two subcultures of the T cell line containing either Fd or anti-idiotype antibodies were initiated. If the anti-idiotype did express an equivalent internal image as the antigen, then T cells reactive with one might be reactive with the other. That this was indeed the case is shown in Figure 5.4. It is evident that Fd-primed T cells were not only reactive to Fd 86 F i g u r e 5 . 3 . I n i t i a l s c r e e n o f Fd-dependent c e l l l i n e f r o m B10.D2 m i c e . A s s a y s were c a r r i e d i n t h e p r e s e n c e o f i n d i c a t e d a n t i g e n s u s i n g t h e c o n d i t i o n s d e s c r i b e d i n F i g u r e 5 . 1 . 87 C PM X 10" 3 A N T I G E N 0 g * 6 | 10 NO A g P PD NR Ig ANTI- ld 88 Figure 5.4. IL-2 release in culture supernatants of Fd- or anti-id-dependent T cell lines. Assays were carried out as in figure 5.1. in the presence of: 0 0, Fd; • • , anti-idiotype antibodies to the Fd-B2 ; • • , anti-Id; • • , Fd; k A, NRIg; • • , PPD; 0 G, no antigen. • • and • • refer to cell lines maintained in the presence of Fd, and 0 0 and • O refer to cell lines maintained in the presence of anti-idiotype. 36 ' 1 4 S 16 l /SERIAL DILUTION OF C U L T U R E SUPERNATANTS 90 but also toward the anti-idiotype. Reciprocally, subcultured anti-idiotype primed T cells were specific for the anti-idiotype. However, they also recognized Fd. While these results further supported the contention that some clonotypic cells recognize both Fd and the anti-idiotype, they do not prove i t . 5.2.3 Cloning of Fd and anti-idiotype reactive T cell lines T cells from the T cell line maintained either with Fd or anti-idiotype were subcloned by limiting dilution in the presence of Fd or anti-idiotype, and were given pulses of IL-2 (177) containing supernates of EL4 cell line cultures which had been induced with phorbol myristic acetate (PMA) (222). Cells were allowed to grow until colonies occupied 20-30% of the well-bottom surface area. A total of 12 T cell clones in 0.2 ml cultures were identified and expanded up to 10 ml cultures with their respective antigens. Of these, 7 were daughter clones from the Fd stimulated T cell lines and 5 were from anti-idiotype stimulated cell lines. Of the former subclones, one died within 5 days of scaling up to 10 ml and two subclones of the latter type died during the same period. Subsequently, a l l Fd-incubated subcultures were shown to test negatively. Clones of T cells isolated from the anti-idiotype selected cell line were tested for the release of IL-2 in the presence of anti-idiotypic antibodies (Figure 5.5). It can be seen that only clones 2 and 4 show a substantially greater reactivity toward anti-idiotype antibodies over NRIg controls. Clone 2 was selected for further study. 91 Figure 5.5. Specificity of cloned T cells. Cloned T cells from the Fd-dependent T cell lines were tested for IL-2 production at a 4 concentration of 5x10 cells per ml in the presence of Fd, anti-Id, or NRIg. Representative results from various clones are shown, clones Al and A2 were maintained in Fd but were shown not to be reactive to Fd. Therefore, they were not further tested. C P M XIO" 3 C L O N E AN TIG E N ° J !2 !£ Al F d A2 F d Cl ANTI - ld N R i s C2 A N T H d NRIg C3 A N T I - l d NRIg ANTI-ld NRIg 93 With-the availability of cloned T cells, i t was possible to test directly the network concept of anti-idiotype being an internal image of the antigen (1,205). Figure 5.6 shows that when T cells from clone 2 were incubated with anti-idiotype antibodies, they responded positively. When the same cloned cells were incubated with Fd (Figure 5.5), or with a combination of Fd and anti-idiotype (Figure 5.7), they also respond positively. Thus, the same cloned T cells responded to both the antigen and the anti-idiotype. When clone 2 cells were incubated with a mixture of Fd and anti-idiotype in combination with an excess of the idiotype bearing monoclonal antibody Fd-B2, no proliferation was seen, indicating that Fd-B2 bound to and then prevented the recognition of both the antigen and the anti-idiotype (Figure 5.7). In the control for this experiment, the presence of an excess amount of CAMAL-1 did not cause inhibition of reactivity due to the presence of Fd and anti-idiotype. In cultures containing PPD and NRIg with CAMAL-1 idiotype control antibody, only slight reactivity presumably due to the presence of NRIg is observed. These studies strongly suggest that at least in this system anti-Fd-B2 is an internal image of Fd. 5.2.4 Fine specificity of Fd- and anti-idiotype-reactive Clone 2 In an examination of epitopic fine specificity, clone C2 cells were incubated with Fd, the N fragment of Fd, the C fragment or the M fragment (devoid of both N- and C-termini) (163). Figure 5.8 shows that clone 2 cells respond to Fd and to the C fragment but not to the N or M fragments. These observations demonstrated that these T cells recognized 94 Figure 5.6. IL-2 release in culture supernatants of an anti-idiotype-reactive cloned T cell line (clone C2) in the presence of 0 0, anti-Id; • 0, Fd; k i , NRIg; • • , PPD; 0 0, no antigen. Antigen concentrations were at 35 yg/ml. 96 Figure 5.7. Ability of the Fd-B2 idiotype to block antigen presentation and subsequent IL-2 release by the C2 clone. Cells were incubated in the presence of anti-Id and Fd (17.5 yg/ml each for a total of 35 yg antigen per ml) or NRIg and PPD and the Fd-B2 idiotype or a control monoclonal CAMAL-1 (100 yg/ml). 97 ANTIGEN 0 NO Ag A NT|-l<J*Fel ANTI'ld* Fd •Fd-B2 ANThld«Fd •CAMAL-1 PPD+NRIg *Fd-B2 PPD»NRIg • CAMAL-1 C P M X IO_i S° j o 98 Figure 5.8. Fine specificity of clone C2. Cells were grown in the presence of the reagents (35 yg/ml) shown and culture supernatants tested at a final dilution of 1:4 for the presence of IL-2. 99 ANTIGEN 0 NO AO P P D C P M X 10 10 20 100 the C-determinant of Fd molecule and indicate that the anti-idiotype carried an image of the C epitope, as expected. 5.2.5 MHC-specificity of heterogenous T cell subcultures The MHC-specificity of subcultures primed with either Fd or anti-idiotype is shown in Figure 5.9. T cells from the Fd-stimulated subcultures recognize Fd only when APC's of H-2d MHC are used (B10.D2 and DBA/2) but not when APC's of H-2k (BIO.BR) or H-2b (C57BL/b) are used. Figure 5.9 also shows that the anti-idiotype is also recognized within the context of self MHC, further supporting the idea that idiotype-anti-idiotype interactions involve a level of complexity imposed by MHC molecule. However, for the reasons outlined in section 5.2.1, this cannot be stated with absolute certainty. 5.3 Discussion The preceding experiments were undertaken in order to further test network concepts. T cell lines were raised from genetic non-responders (B10.D2) which were manipulated extensively prior to their use as lymphocyte donors. Idiotype-bearing T cell lines were generated from mice which were irradiated and then reconstituted with syngeneic non-immune B cells and idiotype plus complement treated T cells (see Chapter 2 and references 169, 181). After 7 days the mice were challenged in both hind foot pads with Fd in CFA. Popliteal and inguinal lymph nodes were 101 Figure 5.9. MHC restriction of the Fd-dependent T cell lines. APC's were derived from the strains of mice shown in Figure 5.2. Assay conditions were the same as in Figure 5.1. 102 SOURCE OP APC'S eio-eft {H-2") ANTIGEN 0 NO Ag ~ PPO NRIg CPM KIO " —35 4J) CB7BL/6 <H-2b) NO Ag PPD NRIg Fd DBA/2 (H-2") PPO NRIg BlO-DZ lH-24) NO Ag PPD NRIg Fd I 103 used as sources of T cells which were cultured with Fd as the antigen as described in Chapter 2 using the protocol developed by Kimoto and Fathman (177). These T cells were shown to be reactive to anti-idiotype antibodies, indicating that at least cells in Fd-maintained cultures bore the Fd-B2 idiotypic determinants on their surfaces and suggesting that perhaps the same cells may be responding to both the antigen and the anti-idiotype. Subcultures of these cells, incubated with either Fd or anti-idiotype showed reactivity not only toward the antigen with which they were incubated but also toward the other antigen (Figure 5-4), further supporting the contention that Fd-reactive T cells could be anti-idiotype-reactive and vice versa. This suggestion supports the hypothesis that anti-idiotype, at least in some cases, is an internal image of the antigenic determinants, as originally proposed as part of the Network Theory (1). Support for this concept was provided when i t was shown that anti-idiotype antibodies directed toward a variety of hormone-specific antibodies were able to mimic hormone specific effects (206,207), suggesting that internal images may exist within the immune system which reflect configurations from the molecules outside i t . Further examples of anti-idiotype antibodies as internal images of antigens have been provided (223,224). It is well documented that T cells bear idiotype determinants, recognizable by anti-idiotype antibodies directed to idiotypes expressed on immunoglobulins. The now classic experiments of Eichmann and colleagues with the A-CHO system (A5A-id) showed that antigen (A-CHO) primed T and B cells could produce A5A i d + 104 PFC in vitro after stimulation with anti-idiotypic antibodies (225). In addition, recent reports show that monoclonal anti-idiotype antibodies stimulate antibody production to phosphorylcholine (227), S. pneumoniae (228) and E. coli (229). As well, preliminary experiments have been initiated to investigate the possibility of anti-idiotypes as surrogate antigens for use in vaccines where the monoclonal anti-idiotype antibodies mimic hepatitis B surface antigen (230). Proof at the clonal level of T cell^-recognition of the anti-idiotype as the internal image of antigen remains to be established. This concept was tested directly, using single cell-derived clones from a T cell line incubated with anti-idiotype antibodies (Fd-incubated subcultures did not survive the expansion procedure). It was shown that cells from one clone, desginated as Clone 2, were not only reactive toward the anti-idiotype but also toward Fd (Figure 5.6 and 5-7), but not with PPD nor with NRIg, confirming that the same cell can recognize both the antigen and the anti-idiotype. In addition, clone 2 cells were also reactive to a mixture of Fd and anti-idiotype in combination with a control monoclonal antibody CAMAL-1 but not in combination with the idiotype-bearing antibody Fd-B2. These two observations together definitively prove that, at least in the Fd-B2 idiotype system, the anti-idiotype is an internal image of Fd, probably the C-determinant since Fd-B2 monoclonal antibody recognizes that part of Fd (168). That this is indeed the case was shown (Figure 5.8), confirming the previous suggestion of these cells' fine specificity. However, as pointed out recently by Roitt (231), the anti-Id may not resemble the antigen (Fd) in shape but 105 may have the same or similar contact residues interacting with the idiotype as does the idiotype with the antigen. It is generally accepted that the restriction elements involved in antigen presentation to T cells involve a rigorous requirement of identity at the MHC (class II) level between antigen-reactive T cells and APC's (199,200). This MHC restriction has been narrowed down to the I-A subregion of the MHC (201) unless responder status is I-E based (221). Therefore, i t was of interest to determine whether or not similar MHC-restriction could be demonstrated with B10.D2 Fd-immune T cell lines. Using irradiated splenotypes as APC's from a limited variety of haplotype mice, i t was shown that Fd-reactive H-2d T cells responded only when the antigen was presented within the context of the H-2d MHC (Figure 5.9). It was surprising to find that, at least in the case of the present idiotype-reactive (anti-idiotypic) T cell lines, the cell lines described responded to the antibody Fd-B2 within the context of self H-2 gene products (Figure 5.9). That this could be the case is indicated by the observation that the anti-idiotype was not recognized by Id + T cells in the context non-self MHC genes. However, uncertainty exists in their interpretation since the activity of these T cells was not tested with respect to the anti-idiotype in the absence of APC's. It is not known whether this additional indicated MHC-restriction at the idiotype-anti-idiotype level is a phenomenon unique to the present T cell lines or i f this is a more general occurrence. Questions regarding idiotype-anti-idiotype interactions may be tested more directly using immortalized T cell produced from the cell lines described here. Such an 106 undertaking was considered beyond the scope of this thesis. Among the issues that could be further addressed with T cell hybridomas are: (a) the similarity or dissimilarity between idiotype-anti-idiotype interactions and simple receptor-ligand binding reactions, (b) a more direct test of the hypothesis that idiotype structures on immunoglobulins as well as MHC-encoded products might represent restriction elements for T cells (202-204), (c) testing the observation made by others that some T cell hybridomas from antigen-MHC-restricted T cell lines may be more flexible in their restriction than the parental T cells (201), and (d) assessment of individual idiotype- and anti-idiotype-bearing T cell hybridomas as representatives of corresponding T cell subpopulations. 107 Chapter 6 6.0 Network-mediated immune-regulation of antl-P815 tumour response 6.1 Introduction Network mediated regulation of the immune response to soluble antigens has occupied the attention of immunologists for some time (see Chapter I for a partial review of this work). However, investigations into the immunoregulation of anti-tumour responses have been more limited. One of the better studied tumour systems with respect to the regulation of immune response against i t is the methylcholanthrene-induced mastocytoma P815. This DBA/2 strain tumour has been the subject of active research in this laboratory with the i n i t i a l findng that when live tumour cells are introduced subcutaneously into mice they respond by producing splenic cytotoxic T lymphocytes (CTL) which appear to be active for 3-5 days (210). Furthermore, i t was shown that the CTL activity was abrogated with the appearance of tumour specific TsC and that the presence of TsC coincided with an accelerated growth of the tumour. The CTL thus described could be characterized in vitro (211). Further research on the tumour-specific TsC showed that a tumour-specific suppressor factor (TsF) from heterogenous TsC could be purified on an affinity column containing immobilized P815 membrane material. TsF thus isolated was antigen specific, could be inactivated by 108 antisera directed to the Ir region of H-2 , and had a molecular weight (m.w.) in the region of 70,000 (212,213). Affinity-purified P815 TsP was used to raise antisera in both allogeneic (C57BL/6) and syngeneic (DBA/2) mice. Theantisera could inactivate TsF as well as eliminate TsC in the presence of complement in vitro (214). In vivo, the syngeneic antiserum significantly prolonged the li f e of tumour-bearing mice (215). A monoclonal antibody (B16G) against the TsF derived from the heterogenous TsC populations was raised by the fusion of TsF-immune BALB/c splenocytes and the NS-1 myeloma cell line (215). The monoclonal antibody B16G was shown to recognize TsC and a TsF from the spleens of DBA/2 mice. It was further apparent that B16G recognized a public specificity of DBA/2 TsF and therefore inhibited the regulation of a variety of predictable immunological reactions (216). Studies undertaken to characterize the Bl6G-reactive material revealed that when TsC extracts were absorbed and eluted from a B16G immunoadsorbent column, specifically purified material reacted with B16G in an ELISA, was biologically active in that i t was suppressive in a standard mixed leukocyte reaction (MLR) and its activity could be eliminated by the addition of B16G to the MLR cultures (217). Biochemical studies resolved that this heterogenous TsC derived TsF was apparently a doublet of 40 and 45 kDa under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the native material appeared to be of 80-90 kDa (217). Subsequently, the monoclonal antibody B16G was used to screen T cell hybridomas derived from the fusion of the thymoma BW5147 and 109 thymocytes of DBA/2 mice immunized with P815 membrane extracts 5 days prior to the fusion (218). A T cell hybridoma was isolated and cloned from such a fusion and screen and designated as A10. Material secreted by A10 in either ascites or in cultures with or without serum specifically bound both to the B16G and P815 membrane extract immunoadsorbent columns but not to appropriate irrelevant control columns (218). Control materials from BW5147 ascites purified over the specific immunoadsorbent columns were also prepared. Analysis of AlO-material specifically purified in such a manner showed that i t had an in vivo biologic activity in that the A10 material injected at the same time as the tumour cells into DBA/2 mice significantly enhanced the growth of P815 tumour cells, but not of other DBA/2 syngeneic tumours such as L1210 or M-l. Also, unlike the Bl6G-purified whole spleen derived DBA/2 TsF, A10 material did not inhibit in vitro MLR thus demonstrating its specificity under in vitro conditions. Biochemical studies showed that the native A10 material purified over B16G or P815 membrane extracts was of 80-90 kDa and in SDS-PAGE i t appeared as a doublet of 45 and 43 kDa (218). Preliminary unpublished results (J.K. Steele) indicated that when affinity-purified A10 preparations were injected (20 pg per animal) intravenously and immediately challenged with 10 P815 cells subcutaneously, an i n i t i a l accelerated tumour growth was observed followed by a regression period 14-21 days after the tumour challenge such that 10-15% of A10 treated mice survived significantly longer than did the controls. Assuming, based on previous related observations (219), that the A10 substance is a soluble product of an Ly t l + , i d + Tsl of the 110 type described by Benacerraf (220), i t was postulated that the A10 molecule's target might represent a population of anti-idiotypic Ts2 effectors which may activate idiotype-positive contrasuppressor cells. Thus, a direct or indirect inducement of tumour regression may be envisioned by the prevalence of immunity to the tumour. In order to investigate the validity of this hypothesis and the assumptions implied, studies were designed to dissect some of the potentially underlying network interactions. 6.2 Materials and Methods All studies were conducted with freshly purified A10 and equivalent BW5147-derived control preparations. 6.2.1. Preparation of affinity-purified A10 extracts. The purification of A10 material for experiments has been described in detail (218). Briefly, the CNBr linking procedure was used to couple the monoclonal antibody B16G or P815 membrane extracts (218) to Sepharose 4B resin by the usual method (209). Control columns consisted of similarly immobilized isologous monoclonal antibodies directed to the protein ferredoxin or to a protein associated with the membrane of the human lung cancer cell line A-549. A B16G column that had lost its binding capacity was also used as a negative control. I l l To purify A10 TsF from A10 ascites fluid applied to B16G columns, 0.1N HC1 was used and acid fractions were immediately neutralized with 1.5 M Tris-HCl, pH 7.0. Equivalent material from BW5147 ascites fluid was isolated similarly from B16G or control columns. Such materials were prepared and stored at 4°C for not more than 24 h and brought up to room temperature prior to intravenous use. Both the A10 and BW materials were provided to me by J.K. Steele using the methodology defined here. His generous assistance is gratefully acknowledged. 6.2.2 Intravenous administration of A10 molecule. Groups of DBA/2 female mice were treated intravenously with 20 yg/0.1 ml per mouse of freshly purified A10 preparations on day -14 and day -7 or day -7 and day 0 or only on day 0. All mice on day 0 3 received 2x10 live P815 tumour cells (subcutaneously) which had been freshly harvested from ascites passaged in DBA/2 mice. The controls received PBS or BW5147 thymoma material treated in the same manner as the A10 material. All groups were rested for 7 days following tumour implantation and then were monitored daily for tumour growth. Tumours were measured in terms of their cross-sectional areas with the aid of an engineer's precision caliper and the size was recorded in square millimeter units. Survival times of test animals were also recorded. 112 6.2.3. Cellular immunization. The cell lines EL4, BW5147 (obtained from ATCC), P815, L1210 and A10 T cell hybridoma were maintained in DBA/2 mice as ascites and removed from mice approximately 2 h prior to use. For cellular immunization, cells of each line were washed and resuspended in sterile PBS, irradiated (10,000 rads) emulsified in 50% CFA and injected 7 subcutaneously into the left flank of DBA/2 or C57BL/6 mice at 2x10 cells per 0.1 ml per animal. The mice were rested for two weeks and boosted in exactly the same way as above. A week later, identical injections of irradiated appropriate cells were administered and the animals were rested one week further. Subsequently, the DBA/2 mice were 3 challenged with live 2x10 P815 cells or with the syngeneic control 3 tumour L1210. Immunized C57B1/6 mice were challenged with 5x10 EL4 cells. Tumours were measured as above. 6.2.4. Preparation of rabbit anti-AlO antiserum. Rabbit anti-AlO (R anti-AlO) was prepared by Agnes Chan. New Zealand white female rabbits were immunized with two intramuscular injections, two weeks apart, of A10 material, purified on immunoadsorbent columns as above. Eluted material was emulsified with 50% CFA prior to immunization. Anti-AlO activity was tested with the ELISA. 113 6.2.5. Adoptive transfer of panned splenocytes. Tissue culture plates (100x20mm, Becton Dickinson and Co.) were coated with sterile, freshly prepared A10 suppressor molecule, equivalent BW5147 ascites material, normal rabbit immunoglobulins (NRIg) or R anti-AlO antibodies at a concentration of 16 yg/ml in a total volume of 5 ml PBS at 4°C overnight. The plates were washed and cells from naive DBA/2 mice were panned for non-adherent cell populations in exactly the same manner as described in section 2.6.2. Subsequently, the 7 recovered non-adherent cells (2x10 cells/mouse) were injected intravenously into irradiated (600 rads) DBA/2 recipients which were then 3 rested for 4 weeks. The mice were then challenged with 10 viable P815 cells. The tumour dose was reduced compared to other experiments in these studies because i t was believed that, given the vigor of the tumour, a greater dose might have overwhelmed the irradiated adoptively reconstituted hosts before any possible network mechanisms manifested themselves. In a l l cases tumour measurements were discontinued when 50% of the animals were dead in any one group of a multi-group experiment. However, animal survival was recorded until a l l tumour-bearing animals died. 6.3 Results The original observations which instigated this series of experiments were that when groups of DBA/2 mice were injected intravenously with either 20 yg of A10 material (prepared as described in Section 6.2.1 of 114 this Chapter) or sterile PBS and immediately challenged with 2x10 viable P815 tumour cells subcutaneously and subsequently monitored for tumour growth, i t was found that some of the mice treated with A10 showed an accelerated tumour growth followed by a significant regression of the tumour compared to PBS controls (226). This effect was reproducible in a number of trials. It was proposed that due to the injection of (presumed) Tsl factor A10, the anti-P815 T cell network is perturbed such that the antigenic activation and clonal activation of Tsl which would normally occur is bypassed. Instead, a direct stimulation of anti-idiotypic Ts2 effectors takes place which may then recruit contrasuppressors that counteract the suppressor T cells and allow anti-tumour immunity to dominate. If this is the case, then i t should be possible to trigger contrasuppression of TsC earlier. In order to test the contention that anti-P815 immunity could be manipulated using the principles of Network Theory, groups of 8 DBA/2 female mice were injected intravenously on day -14 with 20 pg of A10 protein preparations per animal. On day -7 the same group and another group of previously naive animals received A10 in the same manner. On day 0, the latter group of animals received an additional injection of A10. In total, a l l experimental groups received 2 doses of A10, one on day -14 and day -7, and another on day -7 and day 0. Controls were treated with sterile PBS on the same schedule as day -14, -7 animals. On day 0, freshly harvested and washed viable P815 cells were implanted subcutaneously into a l l mice. One week later, two dimensional measurements of tumours were taken daily and recorded as the surface area 115 of tumour. Typical results of such an experiment are shown in Figure 6.1. It is evident that in the day -14, -7 and day -7, 0 groups there was an i n i t i a l acceleration of tumour growth compared to the PBS controls. However, commencing on day 14, a period of regression occurred which lasts to day 21 following which tumour growth resumes. In control groups, however, 50% of the animals were already dead by day 19 (Figure 6.2 and Table 6.1) with the last death occurring on day 21. On the other hand, 50% of deaths in AlO-treated mice occurred on or after day 30 with a l l animals dying by day 49. These two results indicate that tumour-protective mechanisms can be manipulated and brought into play upon the introduction of A10 TsF, presumably by a direct or indirect abrogation of anti-tumour specific CTL activities. As a further control, this experiment was repeated using the day -14, -7 A10 injection protocol and BW5147 ascites preparations, "purified" in the same manner as A10 (see Section 6.2.1 this chapter), were used as controls along with PBS. This experiment was undertaken to ascertain whether or not the BW5147 (BW) component of the A10 hybridoma had conferred this tumour suppressive activity upon A10 material. As Figure 6.3 shows, regression of tumour was seen only in mice treated with A10 whereas the tumour growth of the BW-material treated group is similar to the PBS control group. Significantly, prolonged survival was seen only in the A10 treated group whereas BW-derived material had no effect on survival (Table 6.2). From these results i t was postulated that the introduction of A10 into mice may have elicited the production of Ts2 which may be the effector population. It was reasoned that reconstitution of mice lacking 116 Figure 6.1. Effect of soluble A10 molecule on P815 tumour growth. A10 material was affinity purified as in Materials and Methods and given (20 yg per mouse) on days -14 and -7 (• • ) or days -7 and 0 ( 0 0) to groups of 8 DBA/2 mice. Controls consisted of animals treated with sterile PBS ( A A ). On day 0 a l l animals were challenged wth 3 2x10 P815 subcutaneously (s.c.) in the hind flank and tumour growth was measured by using an engineer's precision caliper in terms of surface area 2 (mm ). Significance was derived by comparing the results to PBS-controls by using Student's one-tailed t-test. A = p<0.005; • = <0.025. 117 9 10 12 H 16 16 20 DAYS AFTER TUMOUR IMPLANTATION 118 Figure 6.2. Survival curve of mice shown in Figure 6.1. A A, PBS control; • §, A10 on days -14, -7; 0 0, A10 on days -7, 0. 119 MICE 6 < SURVIVING 1 IB zo 23 S o -DAYS T3 « J * B — ^ " ~ » 0 Table 6.1. Mean Survival times of treated or control mice. Animals were treated intravenously prior to tumour cell challenge with affinity purified A10 or PBS. Each experimental group consisted of 8 females. Treatment Mean survival time p15 (day + S.E.M.)a 50 yl PBS on day -7 and day 0 18.5 ± 0.76 20 yg A10 on day -7 and day 0 35.3 ± 3.52 <0.005 20 yg A10 on day -14 and day -7 35.9 ± 3.61 <0.005 aS.E.M. = Standard error of mean. ^Significance was derived by comparing treatment results with PBS controls using a one-tailed Student's t-test. 121 Figure 6.3. Effect of A10 equivalent BW5197 ascites material on tumour growth. Groups of mice were treated with 20 pg of A10 material ( 0 0 ) or an equivalent BW5147 ascites material ( • • ) or PBS ( A •& ) according the day -14, day -7 schedule as in Figure 6.1. Tumour growth was monitored as in Figure 6.1. k - p<0.005, * = p<0.025, calculated as before. 122 2 0 0 10 12 14 16 IB 20 22 DAYS AFTER TUMOUR IMPLANTATION I 123 Table 6.2. Effect of A10 or equivalent BW ascites material on tumour growth. Conditions were same as in Table 6.1. Treatment Mean survival time p a (day + S.E.M.) 50 yl PBS on day -7 and day 0 30.1 ± 2.58 20 yl BW ascites material on 34.5 ± 3.18 N.S.a day -14 and day -7 20 yl A10 on day -14 and day -7 39.8 ± 3.67 0.03 aN.S. = not significant. 124 either of these cell populations (A10+-Tsl and its complementary anti-idiotypic Ts2 partner) or both of them would be manifested by tumour regression. The latter experiment was performed since i f no Tsl-Ts2 type of network existed then such an experiment would clearly show this. Irradiated DBA/2 mice were reconstituted with syngeneic splenocytes which were panned fi r s t over AlO-coated plates to remove anti-AlO (anti-idiotypic) T cells, washed, and then panned over anti-AlO coated plates (to remove AlO-positive cells). Control cell suspensions were sequentially panned over BW5147 ascites preparations and NRIg-coated plates, (see Section 6.2.5). The reconstituted mice were rested for 4 weeks and subsequently challenged with 1000 live P815 cells and tumour growth was monitored as before. Figure 6.4 suggested that removal of both A10+-Tsl and/or its coresponding anti-idiotypic partner leads to the regression of tumour over a period of several days. Results in Figure 6.5 dramatically emphasize the survivability of mice, compared to the controls, whose tumours underwent regression. These observations were reproducible. The findings of the above experiments were taken as evidence that the P815 anti-tumour response is mediated by a T cell network defined by the A10 idiotype. In order to reveal the various components of the putative network, a series of panning studies were carried out to selectively remove one population or, the other, or both or finally neither, using plastic dishes coated with either the A10 material, R-anti-AlO antibody, BW ascites material or NRIg. The results of such an experiment are shown in Figure 6.6 in which the measurements of appropriate control groups (see 125 Figure 6.4. Tumour growth in irradiated mice (600 rads) reconstituted with splenocytes panned over A10-, followed by anti-AlO-antibody-coated plates ( 0 0 ) or over BW ascites-material- or NRIg-coated plates ( O • ). Mechanics and conditions were the same as in Materials 7 and Methods. Each mouse (8 per group) received 2x10 cells intravenously and were rested for 4 weeks prior to tumour challenge with 1000 cells. A = p<0.005; • = p<0.025. 126 O A Y S A F T E R T U M O U R IMPLANTATION 127 Figure 6.5. Survival curve of mice shown in Figure 6.4- 0 • , mice reconstituted with cells panned over AlO-followed by anti-AlO coated dishes, or with 0 0, BW ascites material-followed by NRIg-coated dishes. 1001 % OF MICE SO SURVIVING 0 129 Figure 6.6. Effect of removal of A10+ or A10 T cells on tumour growth. Irradiated DBA/2 mice (8 per group) were reconstituted with: cells panned over (a) anti-AlO-antibody-coated dishes ( 0 0 ) (control: NRIg coated dishes); (b) AlO-coated dishes ( A A) (control: A10 equivalent BW ascites material); (c) anti-AlO-coated dishes, followed by panning over A10 coated dishes ( • • ) (controls as in figure 6.4); (d) mice reconstituted with cells as in (a) and another suspension of cells as in (b) ( • • ) (controls as in (a) and (b). All the control curves overlapped each other and therefore were averaged ( • • ). A, indicates p<0.005; *, p<0.01; f, p<0.05. 160 DAYS AFTER TUMOUR IMPLANTATION 131 legend to Figure 6.6) have been averaged since the growth curves of a l l control animals were virtually identical. It is clear from this figure that when the mice are reconstituted with cells lacking the A10+ Tsl populations (due to adherence to anti-AlO coated dishes) a regression of tumour is observed by day 14 and is persistent until day 18. When, in the reciprocal experiment, mice were reconstituted with cells panned over AlO-coated dishes, thus transferring anti-idiotypic Ts2 cells, a similar tumour regression was not observed. These two observations together indicate that whereas A10+ (id +) cell appears to be important regulator of the anti-P815 tumour response anti-idiotype bearing Ts2 does not (see discussion below). In a double subtraction experiment mice were reconstituted with cell populations sequentially panned over A10 followed by anti-AlO-coated dishes. In this case as well, usual tumour regression is seen (Figure 6.6), confirming the results shown in Figure 6.4-When both cell populations, those panned over either A10 or anti-AlO coated dishes were co-transferred into the same animal, the network seems to re-establish itself as evidenced by the observation that the tumour growth curve of these mice paralleled that of the control groups. Taken together, these results strongly suggested the existence of an A10 idiotype mediated network against the mastocytoma P815. All these results were shown to be reproducible. In parallel studies, the effect of immunization, using whole cells, on tumour growth was investigated. Groups of 8-10 mice were primed and boosted with either A10 or BW cells or PBS in CFA (1:1) according to the 3 protocol described. Subsequently, the mice were challenged with 2x10 132 viable P815 cells and monitored as before. Results in Figure 6.7 showed that tumour growth was significantly lower in A10 immunized mice than PBS controls. Unexpectedly, however, mice immunized with BW5147 also showed tumour regression, suggesting that, perhaps, this cell line may exhibit suppressor-like epitopes against which mice can be immunized. The ascites products, however, were clearly not suppressive (Fig. 6.3, 6.4 and 6.6). The BW5147-induced tumour regression did not seem to translate into better survival of tumour-bearing mice as both PBS and BW5147 immunized mice died within two days of each other whereas AlO-immunized mice live approximately 60% longer (Table 6.3). These results indicate that the A10 cellular immunization procedure results in a similar recruitment of putative Ts2 as does the intravenously administration of A10 soluble product. These observations are reiterated in Figure 6.8 which shows, again, that immunization with A10 influenced tumour growth as well as survivability of tumour-bearing mice (Figure 6.9) whereas BW5147 affected tumour growth but not survival. Other cell lines, EL4 or P815, confer no immunity against the tumour. In a test of possible allogeneic effects of A10, groups of 10 C57BL/6 (H-2b) mice were immunized with EL4 (syngeneic to C57BL/6), BW5147, A10 or PBS. Mice were challenged with EL4 tumour cells (5000 cells/mouse). Data in Figure 6.10 show that A10 immunization had no influence upon tumour growth since the tumour progression of these mice paralleld that of PBS-immunized animals. Therefore, A10 appeared to be specific for P815. The survival of various groups of mice did not significantly differ from each other. 133 Figure 6.7. Effect on tumour growth following cellular immunization. 7 Groups of 8-10 DBA/2 mice were immunized with 2x10 irradiated (10,000 rads) A10 or BW5147 cells or PBS in CFA (50:50), subcutaneously, 3 times over a period of 4 weeks. Cells for immunization were obtained freshly as ascites from carrier animals. One week after the last boost the animals 3 were challenged with 2x10 tumour cells. A10 immunization, 0 0; BW5147, • O; PBS, A. Tumours were measured as before. A, indicates p<0.005; •, indicates p<0.025. 135 Table 6.3. Survival time of mice actively immunized with irradiated tumour cells. Groups of 10 mice were immunized three times with 10? irradiated (10,000 rads) BW5147 or A10 cells in CFA (50:50) over a period of 4 weeks. One week after the last boost the mice were challenged with 2x10^ P815 tumour cells. Immunization Mean survival time p a (day ± S.E.M.) PBS/CFA 21.8 ± 3.16 BW5147 24.6 ± 3.55 N.S. A10 40.3 ± 4.97 <0.025 aBW5147 and A10 values were compared to the PBS/CFA controls. 136 Figure 6.8. Immunization with other tumour cell lines. Mice were immunized as in Figure 6.7 with various tumour cell lines. Mice (8-10 per group) were challenged as before. Immunization with: P815, A -A; EM, • B ; • •, PBS; • • , BW5147; 0 -O, A10. A = p<0.01; f = p<0.05. 137 D A Y S A F T E R TUMOUR IMPLANTATION 138 Figure 6.9. Survival of mice immunized with various tumours. P815; • • , EL4; * i , BW; • •, PBS; 0 0, A10. 139 % OF MICE SURVIVING 140 The specificity of A10 was also shown in in vivo experiments using DBA/2 mice immunized with A10 cells and challenged with P815 and the P815-unrelated tumour L1210. Figure 6.11 shows that whereas L1210 tumour growth was unaffected by A10 immunization, P815 tumour growth underwent the usual regression compared to PBS conrols. A10 had no effect on the survival of mice challenged with L1210 whereas i t did significantly prolong the lives of P815-bearing mice (Table 6.4). 6.4 Discussion It had been speculated that, due to the properties of AlO-Tsf described previously (see Section 6.1 of this Chapter and references 213, 218 and 218), this suppressor molecule may represent a central regulatory element in the immune response to the tumour P815. The experiments reported here were undertaken to elucidate the role of A10 in a possible immunoregulatory circuit. Intravenous administrations of affinity-purified A10 materials were shown to cause tumour regression and enhanced survival of tumour-bearing mice. These effects could be induced to occur earlier, depending on the A10 injection schedule, implying the role for A10 molecule and or its parent or analogous cell in a presumed immunoregulatory pathway. Given the ability of the A10 molecule to bind both a suppressor factor-specific antibody and the antigen (P815 membrane extract) (218), i t was further assumed that the A10 product represented an idiotype-bearing structure and, therefore, its possible counterpart in an immunoregulatory 141 Figure 6.10. Lack of allogeneic activity of A10 suppressor molecule. Groups of 10 C57BL/6 (H-2b) were immunized with: EL4, • •; BW, A A; PBS, O • ; or A10, 0 0, as in Figure 6.7. A, indicates no significance. 142 10 12 14 16 18 20 OAYS A F T E R TUMOUR IMPLANTATION 143 Figure 6.11. In vivo specificity of A10 molecule. Groups of 8 DBA/2 mice were treated with A10 material or PBS on days -14, -7, schedule. On day 0, 8 mice from each of the group were challenged P815 or the tumour L1210 3 (2x10 cells/animal) and tumours were measured as before. AlO-treated, challenged with L1210, • • ; PBS-treated, challenged with L1210, A A, AlO-treated, challenged with P815, 0 0; PBS-treated, challenged with P815, • • . A = p<0.025; • = p<0.05. 144 DAYS A F T E R TUMOUR IMPLANTATION 145 Table 6.4. Effect of A10 on the syngeneic P815-non-related tumour L1210. Groups of 16 animals were immunized as in Table 6.3 with A10 or PBS in CFA. Subsequently, subgroups of 8 animals received either 2x103 P815 cells or an equal number of L1210 animals. Immunization Challenge Mean survival time (day ± S.E.M.) PBS/CFA PBS/CFA L1210 P815 19.7 ± 1.05 25.0 ± 1.15 A10 A10 L1210 P815 18.5 ± 0.76 37.9 ± 3.25 N.S. <0.025 aA10 values of each group were compared to their PBS/CFA counterparts. N.S. = not significant, when compared to PBS controls. 146 pathway would be an anti-idiotype bearing Ts2 population analogous to the Ts2 populations well characterized by Benacerraf (220) and described in this thesis in the Fd system. Evidence was obtained for the idiotype-bearing (A10+) T cell in immunoregulation but not for the anti-idiotype Ts2 counterpart. This could be due to the possibility that either A10 may not be a good panning reagent or our methods may not be appropriate to detect the activity of Ts2 cells. From these observations and by analogy with the Fd-specific Fd-B2 idiotype mediated pathway, one would predict that intravenous administration of anti-AlO (anti-idiotype) antibodies into mice prior to tumour implantation would lead to tumour regression upon subsequent challenge. Previous data support this prediction (216). The analogy to Fd does not seem to be strict, however, since the removal of anti-AlO-bearing cells did not result in as dramatic a regression of the tumour as seen when A10+ cells were removed (Figure 6.6). This observation was consistent over a number of trials. Nonetheless, A10+ cells are important in maintaining suppression as evidenced by the lack of tumour regression in doubly reconstituted mice. This dissimilarity with the Fd-specific network could be a reflection of a number of factors: (a) P815 is a highly complex tumour with probably thousands of determinants on its surface, some of which may induce suppression while others help, (b) some T cell populations in an immunoregulatory pathway may have different levels of connectivity with different partners of the regulatory pathway, (c) P815 as a tumour may undergo spontaneous mutation in vivo, further complicating the regulation 147 of immune response against i t , and (d) A10 may not be a good panning reagent. In view of these levels of difficulties, one can s t i l l argue that A10 must represent an important regulatory idiotype since i t does induce tumour regression. Furthermore, given the complexities of P815, regulatory pathways involved in anti-Fd and anti-P815 responses while not identical, were surprisingly similar. 148 Chapter 7 7.0 Summary Originally, a role for the rarely expressed idiotype Pd-B2, borne on a monoclonal antibody (Fd-B2) directed against the C-determinant of the peptide ferredoxin (Fd), had been implicated in the regulation of the anti-Fd immune response in a number of mouse strains. Evidence had been gathered to support the hypothesis that the idiotype Fd-B2 played a central role through network interactions in the regulation of the immune response to Fd in genetic non-responder mice of the B10.D2 (H-2d) strain. This research represents a detailed investigation of the presumed idiotypically mediated network interactions which occur in the B10.D2 strain. Furthermore, the universality of network principles was investigated by applying these principles to the regulation of anti-tumour responses. It had previously been found that when anti-Fd-B2 (anti-idiotype) antibodies were injected intravenously into B10.D2 mice, or when irradiated mice were reconstituted with syngeneic anti-idiotype plus complement-treated T cells and naive B cells, these mice respond by making antibodies to Fd-B2 upon subsequent challenge with the antigen. This was taken to indicate that anti-idiotype antibodies recognized idiotype-bearing Tsl (since previously no evidence had been found for a direct role of B cells in this network). In the present investigations, 149 B10.D2 mice were also shown to respond to Fd when Fd-B2 antibody plus complement treated T cells were transferred into irradiated syngeneic mice. This was assumed to indicate that the antibody Fd-B2 recognized anti-idiotypic determinants on a second population of T cells (Ts2) whose elimination leads to an anti-Fd response. These results support the hypothesis that a pre-existing network of T cells exists in a state of high connectivity to suppress anti-Fd antibody response. The perturbation of this network with either the idiotype or the anti-idiotype leads to an abrogation of this connectivity which subsequently results in the anti-Fd response. It was possible to demonstrate conclusively the existence of this network by reconstitution into mice of both T cell populations (each of which by itself confers responsiveness) which re-established the high-connectivity network such that the non-responsive state was restored in recipient animals. Additionally, using panning techniques, idiotype-positive T cells were shown to be Lytl~,2 +3, and anti-idiotypic T cells were Lytl +2 3-It was possible to produce T cell lines and clones from appropriately manipulated B10.D2 mice which were reactive to either the idiotype-bearing antibody Fd-B2 or to the anti-idiotype antibodies, or to the antigen Fd itself. It was further demonstrated at the clonal level that a T cell clone which was reactive to anti-idiotype antibodies was also reactive to the antigen Fd. Responsiveness to both of these reagents was shown to be inhibitable by the antigen-binding, idiotype-positive monoclonal antibody Fd-B2. These results constitute an evidence for the idea that in this case the anti-idiotype represents an internal image of the antigen Fd. 150 The internal image represents the C-determinant of Fd since the same T cell clone was reactive to the C-determinant-bearing but not the N-determinant fragment nor the fragment devoid of both determinants of Fd. Antigen-reactive T cells were shown to be MHC-restricted in their reactivity of Fd and evidence was also provided to suggest that idiotypes could possibly represent additional restriction elements by a possible interaction with MHC-encoded products. 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Regulation of the immune response to ferredoxin by T c e l l idiotypic network. J. C e l l . Biochem. Supplement 6A. 3. Slnghai, R., Weaver, K., and Levy, J.G. 1984. Demonstration of a T c e l l Idiotypic network responsible for maintaining the non-responder status ferredoxin In B10.D2 mice. Regulation of the Immune System: (H. Cantor, L. Chess, B. Sercarz, eds.) p. 847-853. Alan R. Liss, Inc., New York. 5. Slnghai, R., Hoffmann, G.W., and Levy, J.G. 1985. Abrogation and reconstitution of non-responsiveness: A correlation with high network connectivity. Bur. J. Immunol. 15:526. 6 . Slnghai, R., Sikora, L.K., and Levy, J.G. 1986. Complexity of the non-responder status- to ferredoxin, In: Immunogenlcity of protein antigens: repertoire and regulation. (B. Sercarz and J. Berzofsky, eds. In press). 7. Slnghai, R., and Levy, J.G. 1986. Isolation of a T c e l l clone which reacts with both antigen and anti-idiotype: evidence for anti-idiotype as internal image for antigen at the T c e l l level. Proc. Natl. Acad. S c i . USA. (submitted for publication). 8. Slnghai, R., Steele, J.K., Stammers, A.T., and Levy, J.G. 1986. Suppressor c e l l circuitry controlling the immune response to syngeneic tumour: Manipulation of the response to the P815 mastocytomas with T c e l l suppressor factors. Submitted for publication. 9. Steele, J.K., Chan, A., Stammers, A.T., Slnghai, R., and Levy, J.G. 1986. Immune regulation in neoplasia: Dominance of suppressor systems. In: Induction and Recognition of the Transformed C e l l , (eds. M.I. Greene, and T. Hamaoka, Plenum Publishing Corporation, New York). 


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