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Ferredoxin T cell idiotype : linkage to immunoglobulin heavy chain alleles or the major histocompatibility… Singhai, Rakesh 1983

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FERREDOXIN T CELL IDIOTYPE. LINKAGE TO IMMUNOGLOBULIN HEAVY CHAIN ALLELES OR THE MAJOR HISTOCOMPATIBILITY COMPLEX RAKESH SINGHAI B.Sc. (Hon.), University of British Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1983 © Rakesh Singhai, 1983 i I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f f^lir./^Q f\i n/n The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date £ Jr/n £ DE-6 (3/81) i i ABSTRACT An anti-idiotypic antiserum was raised in rabbits to a monoclonal antibody with specificity for one of the two antigenic determinants on the ferredoxin (Fd) molecule. The monoclonal antibody (Fd-B2) was derived k b from fusion of spleen cells from Fd-immune BIO.BR (H-2 , Igh ). Examination of an extensive number of samples of Fd-immune serum from BIO.BR and other mouse strains established that the Fd-B2 idiotype is essentially never present in such sera in detectable concentrations (> 30 ng/ml). Administration of the anti-idiotypic antibody (anti-Fd-B2) i.v. to BIO.BR mice, or treatment of BIO.BR T-cell enriched populations with anti-Fd-B2 + complement prior to adoptive transfer to irradiated BIO.BR recipients followed by challenge with Fd resulted in a significant increase in the production of anti-Fd antibodies. This effect was specific and was not accompanied by a change in expression of the Fd-B2 idiotype in the antibody produced. Similarly, injection of 10 yg of Fd-B2 into BIO.BR mice resulted in an enhanced anti-Fd response. When similar experiments were carried out using B10.D2 mice (H-2**, Igh**), which are genetic non-responders to Fd, i t was observed that treatment with anti-Fd-B2 followed by challenge with Fd resulted in production in treated animals of significant levels of antibody to Fd. Again, the antisera thus produced did not contain detectable levels of the Fd-B2 It idiotype. Further experiments using high responder (H-2 ) mice with Igh a allotypes differing from the BIO strains (C57/BR, Igh , and RF/J, i i i Igh ), showed that treatment of these animals with a n t i - F d - B 2 also r e s u l t e d in a highly s i g n i f i c a n t enhancement of the anti-Fd response. The implications of these findings are discussed. iv TABLE OF CONTENTS Page Abstract i i Table of Contents iv List of Tables v Li s t of Figures v i List of Abbreviations v i i Acknowledgements v i i i Introduction 1 Materials and Methods 5 Results 11 Discussion 35 References 40 LIST OF TABLES V Page I. Influence of Fd-B2 or anti-Fd-B2 on primary or secondary response of BIO.BR mice to Fd 15 I I . Influence of anti-Fd-B2 on primary or secondary response of B10.D2 mice to Fd 25 I I I . Influence of anti-Fd-B2 across heavy chain allotype 31 v i LIST OF FIGURES Page 1 Standard curve f o r the determination of Fd-B2 idiotype 7 2 21 day anti-Fd response of B10.BR mice pretreated with anti-Fd-B2 antiserum 13 3 Adoptive anti-Fd response of BIO.BR mice 18 A E f f e c t of Fd-B2 idiotype on anti-Fd response in BIO.BR mice 20 5 Anti-Fd response of B10.D2 animals 23 6 35 day secondary adoptive anti-Fd response i n B10.D2 mice 27 7 21 day primary anti-Fd response of a n t i - i d treated C57/BR mice 29 8 21 day primary anti-Fd response of a n t i - i d treated RF/J animals 32 List of abbreviations: anti-id, absorbed anti-Fd-B2 antiserum; C, rabbit or guinea pig complement; C-determinant, trypsin digested-fragment of Fd; d, day DME, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; Fd, ferredoxin; Fd-B2, a monoclonal antibody directed the C-determinant of Fd Igh, immunoglobulin heavy chain gene allotype; JsV,.. immunoglobulin heavy chain gene of the variable region KLH, key hole limpet hemocyanin; MHC, major histocompatibility complex; N-determinant, carboxypeptidase A digested-fragment of Fd; NRIg, normal rabbit immunoglobulins; v i i i ACKNOWLEDGEMENTS I should like to acknowledge the invaluable guidance and assistance from Dr. Juli a G. Levy during the course of this project. I am also indebted to a l l my colleagues and friends in the lab, in particular, L.K. Sikora and D. Demetrick for their helpful comments and suggestions. 1 INTRODUCTION In the past few years a great deal of intellectual and experimental effort has been devoted to the elucidation of the antigen-specific T c e l l surface receptor. To that end, a number of strategies have been employed. However, a unified concept has not been arrived at, as many studies are contradictory. The most common assumption, based on evidence demonstrating immunoglobulin heavy chain gene (Igh) linkage of both T and B c e l l idiotypes, has been that the variable genes of the immunoglobulin heavy chain (V ) encode the information for the antigen-binding site of H the T c e l l receptor; in a number of instances, anti-idiotypic antibodies were found to bind T-cells or T-cell products (1-7). The implicit hypothesis in these studies is that T-cells use the same gentic information and mechanisms as do B-cells, to generate a diverse antigen-specific receptor repertoire. This hypothesis gained strength when i t was shown that idiotypes characteristic of IgV were apparently H common to T cells and antibodies specific for the same antigens (1,2,5). This, however, does not prove a common genetic origin for T-cell idiotypes and B c e l l products, as recently exemplified by Sege and Peterson, when they reported that anti-idiotypic antibodies directed against anti-insulin antibodies react with the insulin receptor on fat cells (8). The above hypothesis was later restricted somewhat by the discovery of von Boehmer, Hurme, and others (7,9-11) that T-cells which are dependent on recognition of molecules encoded by the major 2 histocompatibility complex (MHO as well as foreign antigens, such as proliferative or cytolytic T lymphocytes, do not seem to express idiotypes which predominate in antibody responded. Prior to these studies, Binz and Wigzell (12-14) had found that idiotype expression on T-cells was linked to the Igh locus but not to the MHC, using anti-idiotypic antisera raised against allospecific rat T-cells. However, in a similar study, Krammer and Eichman (15) reported that both the Igh and MHC contribute to the murine T c e l l idiotype expression, thus implicating both genetic l o c i in expression of the T-cell receptor. More recent reports provided further evidence that the T-cell receptor was linked to the Igh locus by demonstrating that the conventional (16) and monoclonal alloantibodies (17-18) generated react with the putative T-cell receptor since the antibodies compete in a reaction between T-cells and antibody idiotypes (16) or react with factors secreted by T suppressor cells (18). These studies employed cytotoxicity analysis for assaying the T-cell receptor and are subject to the criticism that such assays may contain numerous artifacts as well as possibly being subject to over interpretation of data (19). In direct contrast to previous studies, several investigators support the idea that the genes coding for the antigen-specific T-cell receptor need not be linked to those of antibodies (19). For example, Bellgrau and Wilson (20) showed that in the rat the allospecific T-cell receptors lack polymorphism and, therefore, found no evidence for linkage to polymorphic lo c i such as Igh or MHC. More recently, Clark and Capra (21) have reported the discovery of hapten-specific, idiotype-bearing molecules 3 which are ubiquitous and are found not only in many lymphoid c e l l lines but also in normal liver and spleen c e l l s . The relationship of these molecules to the T c e l l receptor is at this time unproven. We sought to gain insight regarding the nature of the T-cell receptor within the ferredoxin (Fd) system. Fd has been used as an antigen to study aspects of immune regulation for over a decade in this laboratory. It is a bideterminant, single-chain polypeptide that consists of 55 amino acids. One of the antigenic determinants resides within a seven amino acid portion of the NH2-terminal of the molecule (N-determinant), and the other is defined by the five amino acids located at the COOH-terminal (C-determinant) (22). The existence of these two determinants has more recently been confirmed through the generation of monoclonal antibodies to Fd which were found to have specificities exclusively directed to these two determinants (23-24). The murine immune response to Fd has been shown to be under s t r i c t control of the MHC and maps to K/I-A (25): i t was found that in high responding mice (H-2 ) 10-20% of the anti-Fd response is directed toward b s the N-determinant, in intermediate responders (H-2 and H-2 ) the anti-Fd response is directed equally towards both determinants, and H-2** mice are non-responders (24). Recently, we have described an idiotype (Fd-1) which is representative of a large proportion of serum anti-Fd IgG directed toward the N-determinant (26). The expression of this idiotype b in antibody was restricted to mice with the Igh all e l e . Although Fd-1 represents a minor fraction of the total anti-Fd antibody, i t was shown to be involved in idiotypic interactions which not only regulate Fd-1 + expression but also exert significant influence over the response of Fd-1 antibodies with specificities for the other determinant. The present thesis describes a monoclonal antibody (IgG^) directed toward the C-determinant. This monoclonal antibody represents an idiotype (designated as Fd-B2) which rarely occurs in anti-Fd sera and then, only in BIO.BR mice. However, this idiotype plays a significant role in immune regulation of the over-all anti-Fd response as seen by the abil i t y of anti-idiotype-treated T-cells to influence anti-Fd antibody production regardless of the MHC and the Igh allotypes of the treated mice. Our conclusions are consistent with those of Bellgrau and Wilson ( 2 0 ) . 5 MATERIALS AND METHODS The purification of Fd, generation of the monoclonal anti-Fd antibody, Fd-B2, enzyme-linked immunosorbent assay (ELISA) for anti-Fd antibody in immune serum, and the preparation and characterization of anti-idiotypic serum and normal rabbit Ig have been described in detail elsewhere (23). Preparation of anti-idiotypic antibodies Anti-idiotypic serum was produced in a New England white rabbit by injection of monoclonal antibody (Fd-B2) isolated from ascites f l u i d on protein A-Sepharose. Anti-mouse Ig constant region activities were removed by adsorption onto Sepharose 4B beads to which mouse Igs had been coupled. Such adsorbed anti-idiotypic antibodies were further a f f i n i t y purified using a Sepharose 4B column to which the monoclonal Fd-B2 has been coupled. Animals Four to six week old female mice of the following strains were obtained from the Jackson Laboratory (Bar Harbor, Me) and used between the ages of six to eight weeks of age: B10.BR/0Sn, B10.D2/OSnj, C57/BR, and RF/J. 6 Determinant Specificity and Quantification of Antisera The total amounts of anti-Fd antibodies in individual immune sera were quantified by an ELISA procedure described in detail previously (25). The N- or C-determinant specificity of antibodies in antisera was also measured according to previously published methods (24). Briefly, purified Fd was selectively digested with either carboxypeptidase A (which inactivates the C-determinant) or trypsin (which effectively removes the N-determinant). Antisera were assayed in ELISA with whole Fd and both N-sepcific and C-specific fragments. Antibodies directed to either the N-or C- determinant were quantified as a function of relative color development with either Fd or the N- or C-fragments used in the solid phase of the ELISA. Detection of Fd-B2 Idiotype The amount of Fd-B2 idiotype present in anti-Fd antisera was measured using a competition ELISA described by Chang e_t a l . (27). Briefly, microtiter plates (Dynatech Laboratories, Inc., Dynatech Corp., Alexandria, Va) were coated with purified anti-Fd-B2 at 1 ug/ml. Plates were washed and varying amounts of the monoclonal Fd-B2 (used as a standard) or varying dilutions of immune sera (1:5 - 1:250) were incubated together with 15 ng/ml alkaline phosphatase-conjugated Fd-B2 for 2 h at room temperature. The plates were washed and the substrate p-nitrophenyl phosphate (Sigma 104, Sigma Chemical Co., St. Louis, Mo; 1 mg/ml) was added. Absorbance was measured at 405 nm using a Titertek Multiskan 7 FIGURE LEGENDS Figure 1. Standard curve for the determination of Fd-B2 idiotype. Microtiter plates were coated with purified anti-Fd-B2 at (1 vg/ml in carbonate buffer, (pH 9.6) overnight and blocked with 10% normal mouse serum for 30 min. Various amounts of unconjugated Fd-B2 were incubated with 15 ng/ml of alkaline phosphatase-conjugated Fd-B2 for 1.5 h at room temperature. Absorbance at 405 nm was measured after the substrate p-nitrophenyl phosphate was added to the washed plates. % INHIBITION ( A 4 0 5 spectrophotometer (Flow Laboratories, Inc., Rockville, Md). The lower limit of the assay was 30 ng/ml of idiotype. A typical standard curve is shown in Fig. 1. Antigens and Immunization Mice were treated intravenously (i.v) with either 10 ug of af f i n i t y - purified anti-Fd-B2 raised in the rabbit or equal amounts of normal rabbit immunoglobulins (NRIg) and rested for a week. On day 0 the animals were immunized subcutaneously (s.c.) with Fd and KLH (50 ug each) in 50% complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, Mi) or with KLH (Calbiochem-Behring Corp., La Jolla, Ca) alone in CFA. Primary immune sera were collected on day 14 and 21 by bleeding individual mice from the t a i l vein. Secondary s.c. immunizations were given with 50 yg Fd or KLH in CFA on day 28 and sera were taken on days 35 and 42. In another series of experiments, animals were treated with 10 yg of the monoclonal Fd-B2 or an irrelevant monoclonal antibody on day -7. On day 0 and subsequently the animals were immunized with Fd and KLH and bled as described above. Adoptive Transfer of Anti-Idiotype-Treated T-Cells Non-immune spleen cells from B10.BR or B10.D2 mice were fractionated by the nylon wool procedure (28) with a c e l l recovery of about 30%. The collected cells were shown to be mainly T-cells in that greater than 95% of the cells were k i l l e d by treatment with a monoclonal anti-Thy-1.2 10 antibody (a kind g i f t of Dr. H.-S. Teh) plus rabbit complement (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada). The purified T-cells were resuspended in 2 ml of Dulbecco's modified Eagle's medium (DME) (Gibco Laboratories, Grand Island Biological Co., Grand Island, N.Y.) containing 10 ug/ml of anti-Fd-B2, or equal amounts NRIg and incubated on ice for 45 min. The cells were then washed three times with DME and incubated with 1:10 of guinea pig complement (Cedarlane Labs. Ltd.) in 1.0 ml of DME for 45 min on ice. The cells were washed as previously and resuspended at 10 /ml in DME. B-cell enriched suspensions were prepared by treatment of non-immune spleen cells with monoclonal anti-Thy 1.2 antibody plus rabbit complement. Cell recovery after treatment was 48% and the cells 8 were resuspended in DME at 10 /ml. Non-immune B10.BR or B10.D2 mice, which had 24 h previously received 500 rad whole body irradiation from a gamma source (Gammacell 200; Atomic Energy of Canada), were injected intravenously with a mixture of 10^ treated T-cells and 10^ B-cells. Immediately after c e l l transfer, recipient animals were immunized with Fd and KLH (50 pg each) in CFA s.c. and bled 14 and 21 days later. The animals were boosted with Fd (50 yg in CFA) on day 28 and bled again on days 35 and 42. 11 RESULTS Characterization of the Fd-B2 idiotype The monoclonal antibody Fd-B2, was generated by a fusion of Fd hyper-immune spleen cells from BIO.BR mice with SP/2 c e l l s . The resulting hybridoma (Fd-B2) produced monoclonal antibody which binds the C-determinant of the Fd molecule. Anti-idiotypic antisera to this monoclonal antibody were raised in rabbits by methods previously described (23). Examination of a large number of individual hyperimmune sera from Fd-immune BIO.BR mice and other strains, for this idiotype was carried out, and i t was found that this idiotype is rarely expressed in Fd-immune B10.BR mice and not at a l l in other strains. It therefore does not represent a major or conserved idiotype in Fd-immune serum. It was therefore somewhat surprising to observe that this anti-idiotypic antiserum (anti-Fd-B2) had a significant effect on the immune response in mice to Fd (see below). Effects of anti-Fd-B2 idiotype on the Fd response and on the expression of  the Fd-B2 idiotype In order to investigate the possible effect of anti-idiotype raised against Fd-B2, BIO.BR mice (high responders) (H-2^, Igh'3) were treated with anti-idiotype and challenged seven days later with Fd (and KLH as an internal control). Control mice consisted of animals treated with NRIg and challenged with Fd and KLH as well as animals treated with anti-idiotype and challenged with KLH alone. The mice were bled according 12 to protocol and individual sera were titrated for specific response to Fd, KLH, the N- and C-determinants, and the expression of the Fd-B2 idiotype. Results of such an experiment are shown in Fig. 2 and Table I (experiment 1). It can be seen that mice treated with anti-idiotypic antiserum gave a significantly enhanced anti-Fd response compared to control animals. Specificity of the observed effect is indicated by the fact that the anti-KLH response is unchanged in a l l three groups of animals. When these sera were analyzed for determinant specific response, i t was found that the characteristic ratio of N:C-determinant specific antibodies normally observed in high responders is conserved in treated animals (Table I). These sera were further analyzed for the expression of Fd-B2 idiotype in an ELISA assay which detected the idiotype in levels as low as 30 ng/ml (Fig. 1). No idiotype was detected even when sera were diluted only 1:5. Therefore, treatment with this anti-idiotype, while affecting the total Fd response, had no significant influence on expression of the idiotype i t s e l f , or on the ratio of N:C-determinant specific antibody synthesized. This suggested the possibility that the anti-idiotype may be acting at the T-cell rather than the B-cell level. Idiotype expression on T-cell populations In order to investigate the possibility that the anti-idiotypic antiserum was influencing the response to Fd by acting on a T-cell population, a series of adoptive transfer experiments were performed. Naive BIO.BR nylon wool purified splenic T-cells were treated with anti-idiotype plus complement, NRIg plus complement or anti-idiotype alone 13 Figure 2. 21 day anti-Fd response of BIO.BR mice pretreated with anti-Fd-B2 antiserum. Groups of 12 animals were treated with the anti-idiotype (anti-id) or normal rabbit Ig (NRIg) (10 yg, i.v.) and 7 days later were challenged with 50 yg Fd and KLH (as an internal control) or with KLH alone and bled according to protocol. Microtiter plates were coated with Fd (1 yg/ml) or 100 yg/ml of KLH in carbonate buffer overnight. Individual sera were titrated alongside a standard curve with known amounts of anti-Fd antibody. Absorbance was determined at 405 nm; results are described as yg/ml antibody ± S.E. 14 TREATMENT NRIg ANTI-Fd-B2 ANTI-Fd-B2 ANTIGEN Fd KLH KLH Fd KLH -I t p g A N T I B O D Y P E R ml 2 E 14 16 . 1 Table I. Influence of Fd-B2 or Anti-Fd-B2 on Primary or Secondary Response of BIO.BR Mice to Fd. Experiment 3 Treatment Time of bleed Anti-Fd p b Percent 0»' number ug/ml Anti-N ± S.E. ± S.E. NRIg Anti-Fd-B2 21d 21d 7.41 ± 2.20 13.60 ± 1.10 0.05 9.9 ± 2.0 10.3 ± 1.8 NRIg + C Anti-Fd-B2 Anti-Fd-B2 + C 21d 2 Id 21d 1.46 ± 0.52 4.28 ± 1.58 7.90 ± 1.92 0.025 0.001 8.5 ± 2.2 10.5 ± 1.8 9.6 ± 2.6 NRIg + C Anti-Fd-B2 Anti-Fd-B2 + C 35d 35d 35d 5.12 11.48 31.16 1.04 4.69 t 12.55 0.05 0.02 11.8 ± 2.6 9.3 + 1.7 9.8 ± 2.3 Fd-B2 PBS Irrelevant monoclonal 21d 2 Id 21d 10.60 ± 4.15 2.16 ± 0.90 3.21 ± 1.18 0.025 11.3 + 1.7 10.9 ± 1.2 10.2 :'• 2.3 16 Table I - continued Experiment 1 - Groups of 12 mice were given 10 yg protein (i.v.) on day -7, challenged with Fd and KLH on day 0 and bled on days IA, 21 and 35 after a secondary boost on day 28. Experiment 2 - Groups of 12 non-immune mice (2A h previously given 500 rads whole body irradiation) were reconstituted with appropriately treated syngeneic T- and B-cells (2 x 107 cells/mouse). Experiment 3 - Groups of 6 mice were given the indicated treatment (10 yg protein/mouse) Fd-B2, irrelevant monoclonal antibody; or, phosphate buffered saline. Differences in titers between anti-Fd-B2 (with or without complement)-treated mice and NRIg-treated controls (experiments 1 and 2) or, between Fd-B2-treated mice and irrelevant monoclonal antibody-treated (experiment 3) mice were analyzed by Student's t-test to establish the significance of differences between the groups. Percentage of antibody directed to the N-determinant versus the C-determinant as calculated in the ELISA. ^The Fd-B2 idiotype expression in immune sera was below the lower sensitivity limit (30 ng/ml) of the competitive ELISA. 17 as described above. Syngeneic animals, previously irradiated with 500 rads, were reconstituted with 10 7 treated T-cells and an equal number of purified naive B-cells. The mice were challenged with Fd and KLH in CFA and bled according to the schedule described. The sera were once again tested for reactivity to Fd, KLH, the N- and C-determinants and for the presence of the idiotype Fd-B2. Results of the adoptive transfer experiment are shown in Fig. 3 and Table I (experiment 2). It can be seen that mice receiving anti-idiotype4 plus complement treated T-cells.gave a three-fold higher anti-Fd response compared to those mice which received T-cells treated with anti-idiotype or NRIg. This indicates that the complement treatment k i l l s a population of T-cells which bear the Fd-B2 idiotype and appear to regulate the response to Fd. As previously seen, the N- and C-determinant specific antibody ratio was conserved and the idiotype Fd-B2 was not detected (Table I), supporting the contention that the effect of this anti-idiotype is directed only toward T-cells. These results indicate the absence of a T-cell-B-cell network with the Fd-B2 idiotype but do not exclude the possibility of a T-cell-T-cell network. To investigate this possibility, BIO.BR mice were given the Fd-B2 monoclonal (10 ug/mouse) i.v. and challenged seven days later with Fd and KLH (as internal control). Control mice were treated with an irrelevant monoclonal antibody of the same subclass. Fig. 4 and Table I (experiment 3) show that mice treated with the monoclonal Fd-B2 gave a five-fold greater anti-Fd response compared to control animals. The ratio of antibodies specific for N- and C-determinants was found to be conserved and no Fd-B2 idiotype expression 18 Figure 3. Adoptive anti-Fd response of BIO.BR mice. Groups of 12 previously irradiated (500 rads) BIO.BR animals received nylon-wool purified T-cells treated with either NRIg, anti-id or anti-id plus complement (C). Each animal received 10 7 syngeneic T-cells and an equal number of B cells obtained with anti-Thy.1.2 plus C treatment of spleen c e l l s . Animals were immunized and bled as described in the protocol and anti-Fd response was quantitated as in Fig. 2. 19 TREATMENT ANTIGEN TIME OF BLEED 0 NRIgtc ANTI-F(J-B2*C Fd KLH ANTI-Fd-B2 Fd KLH Fd KLH 21 d 21 d 21 d U9 ANTIBODY PER ml _j! 3 6 g If IZ 14 16 10 20 30 NRIgt-C ANTI-Fd-B2 Fd KLH Fd KLH ANTI-Fd-B2+C Fd KLH 35 d 35 d 35 d 3—< 20 Figure A. Effect on Fd-B2 idiotype on the anti-Fd response in BIO.BR mice. Groups of six animals were given either the Fd-B2 monoclonal antibody or an irrelevant monoclonal antibody (NON-SP) (10 ug, i.v.) or given phosphate buffered saline (indicated by a dash) they were challenged with Fd as described in Fig. 2. 21d immune response is shown. 21 TREATMENT ANTIGEN F «KB2 — Fd-B2 F d-B 2 NON-SP Fd KLH Fd KLH Fd K LH Ml ANTIBODY PER ml 4 6 g IC) l£ j£ I6_ 22 could be detected in the 35 day secondary bleed of these mice. These experiments establish the probability that the Fd-B2 idiotype is involved in a T-cell-T-cell network but that this idiotype is rarely expressed as a B-cell product in the sera of responder strains. Is there H-2 linkage of the idiotype? It was of interest to investigate the H-2 linkage of the idiotype present on BIO.BR T-cells. We chose to investigate this in the d to Fd-nonresponding strain B10.D2 (H-2 , Igh ). The experiments were designed to investigate two aspects of the Fd-B2 idiotype: the presence or absence of linkage to H-2, and whether the idiotype is expressed on a major regulatory T-cell population which might play a role in the non-responder status of the B10.D2 mouse. Accordingly, B10.D2 mice were treated with anti-idiotype (10 ug/mouse) i.v. and challenged with Fd and KLH as described previously for BIO.BR mice. Controls consisted of anti-idiotype treated mice challenged only with KLH as well as NRIg treated mice challenged with Fd and KLH. The results are shown in Fig. 5 and Table II (experiment 1). It can be seen that on day 35 anti-idiotype treated B10.D2 animals do indeed respond to Fd whereas the controls do not, indicating that treatment with this anti-idiotype can alter the responder-non-responder status These results also show that this idiotype is not linked to the H-2 complex. In these animals the ratio of N:C-determinant specific antibodies was characteristic of the intermediate anti-Fd responders (24). As predicted, no Fd-B2 idiotype could be detected in immune serum (Table II). 23 Figure 5. Anti-Fd response of B10.D2 animals. Each group contained 12 animals. Animals were treated and challenged with Fd and KLH as described previously. Animals were bled and antibody responses were measured as in Fig. 2. 35d secondary response is shown. 24 TREATMENT NRIg ANTI-Fd-B2 ANTI-Fd-B2 ANTIGEN 0 Fd K L H Fd K L H K L H (jg ANTIBODY PER ml 2 ^ 4 ^ 6 7 8 9 ip 1,1 12 Table I I . Influence of Anti-Fd-B2 on Primary or Secondary Response of B10.D2 Mice to Fd Experiment 3 number Treatment Time of bleed Anti-Fd lig/ml £ S.E Percent' anti-N ± S.E. NRIg Anti-Fd-B2 21d 2 Id 0 0 NRIg Anti-Fd-B2 35d 35d 3.38 ± 0.74 0.0005 30.32 :! 6.51 NRIg + C Anti-Fd-B2 Anti-Fd-B2 + C 21d 21d 2 Id 0 0 0 NRIg + C Anti-Fd-B2 Anti-Fd-B2 + C 35d 35d 35d 0 3.46 ± 0.29 6.09 + 0.68 0.0005 43.4 :i 4.8 0.001 41.1 ± 4.0 12 animals per group i n each experiment. no idiotype detected i n the competitive ELISA 26 These findings were confirmed in an adoptive transfer experiment in which irradiated B10.D2 animals received: NRIg plus complement treated or anti-Fd-B2 plus complement treated T-cells or anti-Fd-B2 only treated T-cells from non-immune B10.D2 animals and challenged and bled as described previously. Mice reconstituted with anti-Fd-B2 plus complement treated T-cells gave a higher anti-Fd response compared to mice which received only anti-Fd-B2 treated T-cells (Fig. 6, Table II). Both groups of these mice demonstrated a significantly higher response than the controls which made no detectable anti-Fd antibody. In responding B10.D2 animals, the Fd-B2 idiotype expression was negative. When B10.D2 animals were treated with the idiotype and subsequently challenged with Fd and KLH, no anti-Fd response was seen, although the anti-KLH response was normal. The implications of this finding are discussed later. Effect of anti-Fd-B2 across heavy chain allotypes The possibility of Fd-B2 idiotype linkage to Igh was investigated k a using two H-2 strains with unrelated Igh allotypes: C57/BR (Igh ) c and RF/J (Igh ). Non-immune mice of both strains were treated with anti-Fd-B2 (10 ug/mouse) (i.v.) or with an equal amount of NRIg. Seven days later the animals were challenged with Fd and/or KLH and bled as before. Results presented in Table III show that both C57/BR (Fig. 7) and RF/J (Fig. 8) animals which were treated with the anti-idiotype gave a significantly higher response as compared to the control animals. In both strains the N:C-specific antibody ratios were unchanged as compared to NRIg treated controls and in agreement with previously obtained results. 27 Figure 6. 35d secondary adoptive anti-Fd response in B10.D2 mice. Ir r a d i a t e d r e c i p i e n t s received enriched syngeneic B c e l l s and treated T - c e l l s as described in F i g . 3. Each group contained 12 mice. 28 T R E A T M E N T A N T I G E N ANTI -Fd - B 2 +C A N T I - F d - B 2 N R I g + C F d K L H F d K L H F d K L H p g A N T I B O D Y P E R ml 4 6 8 10 12 J 1 29 Figure 7. 21d primary anti-Fd response of a n t i - i d treted C57/BR mice. Experiment c a r r i e d out as in F i g . 2 with 6 animals per group. TREATMENT NRIg ANTI-Fd-B5 ANTI-F0-B2 ANTIGEN o Fd KLH Fd KLH KLH H 9 ANTIBODY P E R ml -4 § 9 UL_ Table I I I . Influence of Anti-Fd-B2 Across Heavy Chain Allotype S t r a i n 3 Haplotype Igh Allotype Treatment Anti-Fd V;g/ml ± S.E. P Ant i - N b ± S.E. C57/BR H-2k Igh 3 NRIg 3.31 ± 1.98 10.5 1.89 Anti-Fd-B2 13.64 ± 4.50 0.025 12.4 ± 3.39 RF/J H-2k Igh c NRIg 4.46 ± 2.45 9.8 :.! 1.0 Anti-Fd-B2 20.87 ± 1.05 0.0005 8.5 ± 2.7 36 animals per group i n each experiment. no idiotype detected i n the competitive ELISA. 32 Figure 8. 21d primary anti-Fd response of anti-id treated RF/J animals. Experiment carried out as described in Fig. 2 with 6 animals per group. 33 TREATMENT ANTIGEN NRIg Fd KLH onti - Fd - B2 Fd KLH anti-Fd-B2 KLH ji.q antibody per ml -S § !2 1 2 I-4 W & 2 0 34 As previously seen, sera from neither strains were positive for the presence of the Fd -B2 idiotype. The anti-KLH response in both strains was normal. These results indicate that the effect of anti-Fd -B2 is neither limited by H-2 linked control nor is i t allotype restricted. The possibility, therefore, exists that anti-Fd -B2 serum recognizes some part of an antigen specific T-cell receptor which is neither controlled by H-2 nor linked to heavy chain allotype. 35 DISCUSSION The present work describes the immunoregulatory properties of a ferredoxin specific monoclonal antibody which expresses an idiotype k b designated as Fd-B2. When naive BIO.BR (H-2 , Igh ) (Fd-high responders) mice were treated with anti-Fd-B2 antiserum and subsequently challenged with Fd, the anti-Fd response of treated mice was significantly higher compared to that of controls (Table I) yet the determinant-specificity of the response was unaltered. The idiotype, Fd-B2, could not be detected in the immune sera, using a very sensitive competitive ELISA, supporting the hypothesis proposed by Haas and others that T-cells appear not to express idiotypes which are a common part of the B-cell repertoire (9-11). The above observation suggested that the anti-id may be acting at the level of T-cell rather than the B-cells. When T-cells were treated with the anti-id followed by treatment with or without C and transferred to irradiated syngeneic animals, the adoptive anti-Fd response was significantly higher in treated animals. Complement treatment of T-cells exposed to anti-id resulted in the highest response. However, the ratio of N:C-determinant specific response in a l l groups of mice was unchanged and there was a continued absence of detectable levels of the idiotype. These results support the idea that the anti-id might be directed only toward T-cells. The observation that T-cell k i l l i n g by complement treatment resulted in a higher anti-Fd response than cells not so treated suggested that the idiotype positive cells need to be eliminated before the enhanced effect on the immune response can be seen. 36 Employing a similar experimental approach as above, the H-2 linkage of the Fd-B2 idiotype was investigated. When anti-id-treated B10.D2 (H-2**, Igh*3) (non-responders) animals were challenged with Fd, a secondary anti-Fd response was observed on day 35 (Table II) whereas control animals retained their non-responder status. The ratio of N:C-determinant specific response was typical of intermediate responders (14) but no idiotype could be detected with our assay. These findings were confirmed when the adoptive anti-Fd response of irradiated B10.D2 animals was undertaken: one group received syngeneic anti-id plus C treabed T cells and its response was observed to be significantly higher compared to mice in the group which had received anti-id coated T-cells without C treatment (Table II). The N:C-determinant specific response was similar to that of the intermediate responders, and as seen previously, no idiotype could be detected in the immune sera. The controls did not respond. These results are supportive of the idea that the anti-Fd response in normally non-responder animals is probably due to the elimination of an idiotype bearing T-cell population which confers the d non-responder status through suppression upon H-2 haplotype animals. That the Fd-B2 idiotype is not linked to the H-2 is supported by the k d observations that T-cells of both BIO.BR (H-2 ) and B10.D2 (H-2 ) are influenced by the anti-id. Taken together, the observations in BIO.BR and B10.D2 animals indicate that Fd-B2 idiotype is not involved in a conventional T-cell-B-cell network but may play a role in a T-cell-T-cell network. This possibility is supported by the observation that Fd-B2 treated BIO.BR mice give a significantly higher anti-Fd response than the controls 37 (Table I). This finding could not be repeated in the B10.D2 system. In any case, the results suggest that a population(s) of Fd-B2 idiotype bearing T-suppressor cells (Fd-B2+-Tsc) exists which interact with anti-id bearing T-cells. The latter cells function to regulate anti-Fd response in responding strains but they are unable to overcome the suppressive effects of Fd-B2+-Tsc in non-responding animals. In investigation of the idiotype Fd-B2 linkage to heavy chain k a allotype, the high responding (H-2 ) strains, C57/BR and RF/J (Igh and Igh C, respectively, were treated with the anti-id and subsequently challenged with Fd. Treated mice gave unequivically higher responses than did the controls (Table III). As seen earlier, the N:C-determinant specific response was conserved and no idiotype was detected in the immune sera. These experiments show that this idiotype is not linked to the Igh genes and that perhaps the T-cell "idiotype" is not a product of the genes coding for the variable regions of heavy chain (V ). n In a recent publication, Kubagawa et al (29) report that their monoclonal anti-V„ antibodies seemed to recognize a "hidden" V H n determinant which is not accessible on intact, native Ig and possibly differs from conventional V subgroup determinants. While their H anti-V antibodies seem to bind surface Ig of fixed B lymphocytes, they H recognize neither viable B-cells nor T-cells. In our own system the anti-id does not seem to influence viable B-cells, however, i t clearly does influence T lymphocytes. 3 8 The results reported here suggest a number of interesting possibilities regarding T-cell-T-cell interactions, and tend to preclude a number of possibilities regarding the nature of the receptor on regulatory T-cells. The anti-idiotypic antisera used here was raised to a monoclonal antibody specific for the Fd molecule (Fd-B2). This antibody, however does not represent a common or major idiotype of Fd-immune serum; in fact, i t appears to be rarely i f ever represented in such sera at levels detectable by a sensitive assay. However, the anti-idiotype antiserum (anti-Fd-B2) was found to have a highly significant enhancing effect on the antibody response of BIO.BR mice to Fd, whether administered directly to recipient animals or used to treat T-cells in adoptive transfers. These effects were shown to be specific. These findings showed that common T-cell idiotypes do not necessarily resemble dominant idiotypes represented as B-cell products. It was also shown that the idiotype i t s e l f had a similar effect iri vivo as did the anti-idiotype in enhancing the response to Fd in BIO.BR mice. Increased antibody titers in both instances were not related to an increase in idiotype expression. These results imply the presence of T-cell networks in which B-cells are not directly involved. The finding that anti-Fd-B2 antiserum, when administered to B10.D2 mice followed by priming with Fd resulted in the production of detectable anti-Fd antibody indicated that the T-cells bearing this idiotype play a major regulatory role in maintaining the non-responder status of this mouse strain, since their removal permits a measurable response. Also, these findings demonstrated that the expression of this idiotype on T-cells was not dependent on the H-2 39 haplotype. Finally, our observation that anti-Fd-B2 had an identical effect on the Fd response of H-2 mice with Igh allotypes differing from the BIO.BR strain demonstrated that the expression of this idiotype on T-cells is not linked to the Igh locus. These results suggest that the topographic region on these regulatory T-cells, while structurally resembling the Fd-B2 idiotype, is not a product of the V gene region. n Further work in this lab is being directed toward analysis of the T lymphocytes involved in the idiotype system described here in terms of their phenotype as well as the mechanisms of their interactions. 40 REFERENCES 1. Binz, H., and H. Wigzell. 1976. Antigen-binding, idiotypic receptors from T lymphocytes: An analysis of their biochemistry, genetics, and use as immunogens to produce specific immune tolerance. Cold Spring Harbour Symp. Quant. Biol. 41:275. 2. Rajewsky, K., and K. Eichman. 1977. Antigen receptors of T helper ce l l s . Contemp. Topics in Immunology 7_:69. 3. Binz, H., and H. Wigzell. 1977. Antigen-binding, idiotypic T-lymphocyte receptors. Contem. Top. Immunobiol. 7_:113. 4. Eichmann, K. 1978. Expression and function of idiotypes on lymphocytes. Adv. Immunol. 26:195. 5. Cramer, M., U. Krawinkel, I. Melcher, T. Imanishi-Kari, Y. Ben-Neriah, D. Givol, and K. Rajewsky. 1979. Isolated hapten-binding receptors of lymphocytes. IV. Expression of immunoglobulin variable regions in (4-hydroxy-3-nitrophenyl) acetyl (NP)-specific receptors isolated from murine B and T lymphocytes. Eur. J. Immunol. 9:332. 6. Sy, M.-S., A. Brown, B.A. Bach, B. Benacerraf, P.D. Gottlieb, A. Nisonoff, and M. Greene. 1981. Genetic and serological analysis of the expression of cross-reactive idiotypic determinants on anti-p-azobenzenearsonate antibodies and p-azobenzenearsonate-specific suppressor T-cell factors. Proc. Natl. Acad. Sci. U.S.A. 78:1143. 7. Krammer, P.H. 1981. The T-cell receptor problem. Current Topics in Microbiology and Immunology. Springer Verlag, New York 91:179. 8. Sege, K., and P.A. Peterson. 1978. Anti-idiotypic antibodies against anti-vitamin A transporting proteins react with prealbumin. Nature (London) 271:167. 9. Sherman, L.A., S.J. Burakoff, and B. Benacerraf. 1978. The induction of cytolytic T lymphocytes with specificity for p-azophenyl-arsonate-coupled syngeneic c e l l s . J. Immunol. 121:1432. 10. Hurme, M., K. Karjalainen, and 0. Makela. 1980. Failure to demonstrate public idiotypes on cytolytic cells with specificity for NP-coupled syngeneic cel l s . Scand. J. Immunol. 11:241. 11. von Boehmer, H., and W. Haas. 1981. H-2-restricted cytolytic and non-cytolytic T-cell clones: isolation, specificity and functional analysis. Immunol. Rev. 54:27. 12. Binz, H., and H. Wigzell. 1975. Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants.' IV. Isolation of two groups of naturally occurring idiotypic molecules with specific antigen-binding activity in the serum and urine of normal rats. Scand. J. Immun. 4:591. 13. Binz, H., and H. Wigzell. 1976. Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. V. Biochemical and serological characteristics of naturally occurring soluble antigen-binding T lymphocyte-derived molecules. Scan. J. Immunon. 5:559. 42 14. Binz, H., H. Wigzell, and H. Bazin. 1977. T-cell idiotypes are linked to immunoglobulin heavy chain genes. Nature (Lond.) 264:639. 15. Krammer, P., and K. Eichmann. 1977. T-cell receptor idiotypes are controlled by genes in the heavy chain linkage group and the major histocompatibility complex. Nature (Lond.) 270:733. 16. Owen, F.L., A. Finnegan., E.R. Gates, and P.D. Gottlieb. 1979. A mature T lymphocyte subpopulation marker closely linked to the Ig-1 allotype C locus. Eur. J. Immun. 9:948. H 17. Owen, F.L. 1982. Products of the Ig T-C region of chromosome 12 are maturational markers for T cell s . Sequence of appearance in immunocompetent T cells parallels ontogenetic appearance of T Thy**, d d T ind , and Tsu . J. Exp. Med. 156:703. 18. Tokuhisa, T., Y. Komatsu., Y. Ochida, and M. Taniguichi. 1982. Monoclonal alloantibodies specific for the constant region of T c e l l antigen receptors. J. Exp. Med. 156:888. 19. Jensenius, J.C., and A.F. Williams. 1982. The T lymphocyte antigen receptor-paradigm lost. Nature (London) 300:583. 20. Bellgrau, D., and D.B. Wilson. 1979. Immunological studies of T-cell receptors. II. Limited polymorphism of idiotypic determinants on T-cell receptors specific for major histocompatibility complex alloantigens. J. Exp. Med. 149:234. 21. Clark, A.F., and J.D. Capra. 1982. Ubiquitous nonimmunoglobulin p-azpbenzenearsonate-binding molecules from lymphoid c e l l s . J. Exp. Med. 155:611. 22. Kelly, B., J.G. Levy, and D. Hull. 1973. Cellular and humoral immune responses in guinea pigs and rabbits to chemically defined synthetic peptides. Eur. J. Immunol. 3:574. 23. Weaver, M.S., L. Sikora, and J.G. Levy. 1982. The immune response to ferredoxin: characterization of a major idiotype in serum using monoclonal antibody derived by c e l l fusion. Molec. Immunol. 19:105. 24. Sikora, L., M. Weaver, and J.G. Levy. 1982. The use of unideterminant fragments of ferredoxin in the genetic mapping of determinant specificity of the immune response. Molec. Immunol. 19:693. 25. Sikora, L.K.J., and J.G. Levy. 1980. Genetic control of the immune response to ferredoxin: linkage and mapping of T-cell proliferation and antibody production genes to the MHC of mice. J. Immunol. 124:2615 26. Weaver, M., R. Singhai, L. Sikora, and J.G. Levy. 1983. Identification of an idiotypic marker of a major regulatory T-cell of the immune response in BIO.BR mice to ferredoxin. The relationship of idiotypic regulation to conventional hapten-carrier effects. J. Exp. Med. 157:285. 27. Chang, S.P., M. Brown, and M.B. Rittenberg. 1982. Immunologic memory to phosphorylcholine. III. IgM includes a fine specificity population distinct from TEPC 15. J. Immunol. 129:1559. 44 28. Julius, M.H., E. Simpson, and L.A. Herzenberg. 1978. A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 8:645. 29. Kubagawa, H., M. Mayumi, J.F. Kearney, and M.D. Cooper. 1982. Immunoglobulin V determinants defined by monoclonal antibodies. H J. Exp. Med. 156:1010. 

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