Genetic control of the immune response to the terpolymer l-glutamic acid-l-alanine-l-tyrosine (GAT)—IV: Heterogeneity of idiotype GAT-715

A major idiotype GAT-715 was defined in mice by a rabbit anti-idiotypic antiserum raised against BALB/c anti-poly (Glu⁶⁰-A!a³⁰-Tyr¹⁰) (GAT) antibodies. Corresponding idiotypic specificities were found to be present on anti-GAT antibodies from responder and non responder mice regardless of their allotypic markers. Some of the idiotypic specificities that we have termed highly conserved idiotypic specificities are expressed by rat and guinea pig after immunization with GAT. In this paper we report experiments that show that idiotype GAT-715 can be further analysed and subdivided into public and private idiotypic specificities. All mice tested express the public specificities that represent 80% of idiotype GAT-715. Some strains also express private specificities; no genetic linkage is observed between the inheritance of heavy chain allotypic markers and private specificities. In addition, public and highly conserved specificities have been compared. A structural model for GAT-715 idiotype is discussed.     

Genetic control of the immune response to the L-Glu60-L-Ala30-L-Tyr10 (GAT) terpolymer. V. Three types of idiotypic specificities on BALB/c anti-GAT antibodies

Three types of idiotypic specificities compose the major idiotype of anti-poly (L-Glu60-L-Ala30-L-Tyr10) (GAT) antibodies from BALB/c mice (idiotype termed GAT-715). Assays have been designed to analyzed and study the distribution of these specificities. The highly conserved idiotypic specificity (h.c.GAT) has been assayed by the binding of serum 715-7A4 to radiolabeled rat anti-GAT antibodies. Guinea pig and mouse anti-GAT antisera all express the same h.c.GAT specificity. The public specificity (p.GAT) has been shown to be present in an identical form in all anti-GAT antisera from all strains of mice studied. The assay used for p.GAT was the binding of serum 715-7A4 to C57BL/6 anti-GAT antibodies that express only p.GAT. Finally, the strain-restricted specificity s.r.GAT has also been investigated by radioimmunoassay; this specificity is expressed only by strains BALB/c, BALB/b, BUB/J, DBA/2, DBA/1 and ATL. This expression is independent of known allotypic markers. However, the expression of the s.r.GAT specificity of BALB/c mice follows the genetic distribution of VH genes of BALB/c origin indicating that s.r.GAT can be considered as a genetic marker of some VH gene(s) involved in the specific immune response to the GAT terpolymer.     

Genetic control of the immune response to the terpolymer L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10)(GAT). II. Characterization of a cross-reactive idiotype associated with anti-GAT antibodies from responder and nonresponder mice.

An anti-idiotypic antiserum specific for the antibodies directed against the terpolymer poly(Glu⁶⁰, Ala³⁰, Tyr¹⁰), referred to as GAT, has been prepared in rabbit immunized with anti-GAT antibodies purified from ascites fluid of BALB/c GAT responder mice. The specificity of this serum has been investigated. The interaction between ¹²⁵I-labeled anti-GAT antibodies and the anti-idiotypic antiserum is specifically inhibited by anti-GAT antiserum or anti-GAT ascites fluid. GAT and the copolymer of L-glutamic acid⁵⁰-L-tyrosine⁵⁰, but not the closely related copolymer of L-glutamic acid⁶⁰-L-alanine⁴⁰, inhibit the idiotype-anti-idiotype binding indicating a close association of idiotypic determinants with the antibody combining site. This GAT idiotype was found in the anti-GAT antibodies of all individuals of all inbred strains of mice tested regardless of their allotypic markers. This GAT idiotype has also been found in serum of nonresponder mice immunized with GAT bound to methylated bovine serum albumin. Therefore, the existence of a GAT cross-reactive idiotype represented in a very large number of inbred strains of mice has been identified. These results are in contrast to results obtained in other experimental systems where linkage was always observed between allotypic and idiotypic specificities.     

Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.     

Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera.     

Idiotypic cross-reactivity of anti-GAT and anti-alprenolol antibodies: an approach to the structural correlates of the pGAT idiotypic specificity

Most anti-GAT antibodies in the BALB/c strain express a public idiotypic specificity (pGAT), which is encoded by specific germline genes (VH10, VK5.1 and VK1A5). One or both of these germline genes, referred to as "GAT-specific genes", are also used by four anti-alprenolol antibodies. Anti-Alp and anti-GAT antibodies show no cross-reactivity for the antigens. The light chain of one anti-Alp antibody, 22C4, is encoded as the anti-GAT antibodies by a VK5.1-J2 combination and expresses part of the pGAT idiotopes, whereas the heavy chain is not "GAT"-related. Two anti-Alp using the VH10-VK5.1-J1 association do not express any of the pGAT idiotopes. Sequence comparison of the various CDR sequences points to the predominant role of the VH-CDR2 and VL-CDR3 for the constitution of the pGAT specificity. Regarding VL-CDR3, a drastic change in idiotypic determinants appears to be linked to V-J junctional diversity.     

Isoelectric focusing spectra of rabbit antibodies to Salmonella abortus-equi detected by anti-idiotypic sera

Serum antibodies from rabbits hyperimmunized with Salmonella abortus-equi (S.a-e) were examined by isoelectric focusing in polyacrylamide gel, followed by the coating of the gel with iodinated polysaccharide extracted from bacterial cell walls. The antibody populations in these sera were highly heterogeneous. Anti-idiotypic sera, prepared in allotypically matched rabbits, recognized only a fraction of these antibody populations (generally from one to three clonal antibodies per serum). Different rabbits subjected to the same anti-idiotypic immunization can recognize different antibodies in the same serum to S.a-e.

Homologous and heterologous idiotypes recognized by the same anti-idiotypic serum have shown very similar but never identical pI spectra. It appears that the same group of idiotypic determinants can be markers for several clonal antibodies and are not unique to a single clone.

Two rabbits were studied throughout an immunization course lasting 2 years; we did not observe any change in the clonal product of antibody-producing cells carrying the idiotypic determinants.

     

Human immune response against timothy grass pollen allergens. I. Shared idiotypes on human and murine antigen-B-specific lymphocyte receptors

Enzyme-linked immunoassays were used to examine the binding specificity of affinity-purified anti-idiotypic antibody [against the idiotypic determinant on timothy grass pollen antigen B (AgB)-specific IgE] with murine AgB-specific IgG and IgE antibodies, and the serum from timothy-sensitive patients. In addition, the proliferative response of peripheral blood lymphocytes from timothy-sensitive patients with either antigen or anti-idiotypic antibody was examined. These studies suggest that the idiotypic determinant expressed on murine AgB-specific IgE is highly conserved on human AgB-specific IgE and T cells of timothy-sensitive patients.     

Immune response against poly(Glu60,Ala30,Tyr10) (GAT): immunization with monoclonal anti-idiotypic antibodies leads to the predominant stimulation of idiotypically similar immunoglobulins with anti-GAT activity

Two monoclonal anti-idiotypic antibodies (HP-Id20 and HP-Id22) recognizing two different public idiotopes expressed in the anti-poly(Glu60,Ala30,Tyr10) (GAT) response were used to immunize BALB/c and C57BL/6 mice. From these animals hybridomas were isolated. From BALB/c and C57BL/6 mice eight and seven monoclonal antibodies were characterized, respectively. The reagents were classified according to the expression of the public idiotypic specificity p.GAT (recognized by a rabbit antiserum). The anti-GAT activity and the expression of the various idiotopes characterized on anti-GAT polyclonal and monoclonal antibodies were also studied. Most of the reagents are Ab1'-type of antibody resembling anti-GAT antibodies. One anti-anti-idiotypic monoclonal antibody (Ab3) was also isolated from BALB/c mice. This suggests that in this experimental model the repertoire induced after HP-Id immunization and antigen stimulation is comparable. The idiotypic analysis of a large number of anti-GAT and of Ab1' monoclonal antibodies suggests that only two public idiotopes are involved in the anti-GAT response.     

A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

Idiotypes of rabbit anti-Salmonella abortus-equi antibodies (recent results)

Anti-idiotypic sera which precipitate the anti-Salmonella abortus-equi (Sae) serum used for their preparation (homologous reactions) sometimes precipitate anti-Sae sera other than the one used for their preparation (heterologous reactions). The frequency of these heterologous reactions is about 3 % for 5036 studied reactions. We have detected heterologous idiotypes in several different individuals, which apparently were not distinguished from homologous ones in the reaction in gel medium with the same anti-idiotypic serum. However, the quantity of heterologous anti-Sae serum necessary for inhibiting the homologous reaction is always larger than that necessary for inhibiting the heterologous reaction observed with the same anti-idiotypic serum. Homologous and heterologous reactions indicate that antibodies produced by rabbits submitted to the same anti-idiotypic immunization were sometimes directed against different idiotypes and even against different idiotypic determinants of the same idiotype. We have separated two different IgG idiotypes by the isoelectrofocusing technique. These two idiotypes seem to have different idiotypic specificities.     

Idiotypic Determinants in Alloantigen Receptors I. Role in a Leucocyte Migration Inhibition Reaction

An anti-idiotypic serum was prepared by injecting BALB anti-AKR serum into (BALB X AKR)F1 mice. Pretreatment of BALB anti-AKR immune cells with this anti-idiotypic serum plus complement abrogated a leucocyte migration inhibition reaction (LMIR) against AKR extracts but not against purified protein derivative By itself, the serum induced LMIR in immune cells but not in normal cells. The reaction was strain-specific and anti-Thy 1,2 plus complement sensitive. These results indicate that alloantigen receptors, able to trigger in LMIR in immune cells, have idiotypic determinants similar to those of circulating antibodies of the same specificity. Thus the LMIR is a good model for the study of receptors cell-mediated immune reactions of primed cells.     

A cross-reacting human idiotype (B17) associated with antibodies to N-acetyl-D-glucosamine. Specificity, immunoglobulin class association, and distribution in the population

This report describes the study of the expression of an idiotype in the human population which is associated with antibodies to N-acetyl-D-glucosamine (GlcNAc) present in most human sera presumably due to streptococcal infections. The idiotype is identified with antisera and monoclonal antibodies prepared against the IgM (kappa) antibody secreted by the Epstein-Barr virus-transformed human B cell line B17. At least 90% of 207 individuals tested had immunoglobulin with B17 idiotypic determinants in their sera, as demonstrated with conventional and one monoclonal anti-idiotypic antibody. Another monoclonal anti-idiotypic antibody reacted with antibodies in only a few of the sera. No correlation was found between the level of expression of different idiotopes in individual human sera, suggesting molecular heterogeneity of the B17-positive antibody population. B17-positive immunoglobulins are to a large extent specific for GlcNAc but represent only a minor population of all GlcNAc-specific antibodies in human sera. B17 determinants are on IgM (kappa) in all human sera and on IgG and IgA in some. In addition, some lambda-bearing Ig was found to react with anti-B17 antisera, suggesting the detection of VH-associated idiotypic determinants in this experimental system.     

Studies of Monoclonal Antibodies Specific for Major Histocompatibility Complex Products of the Rat

Polyclonal syngeneic, allogeneic, and xenogeneic and monoclonal syngeneic anti-anti-idiotypic antibodies have been produced against previously described monoclonal anti-idiotypic antibodies with specificity for monoclonal RT1 alloantigen-specific antibodies. The anti-anti-idiotypes could again be shown to be highly specific for the monoclonal anti-idiotype used for the induction of the anti-anti-idiotypic antibodies and to carry the same, or a very similar, idiotype as the original monoclonal idiotypic antibody used to induce the monoclonal anti-idiotypic. Among the 30 syngeneic and allogeneic and the five xenogeneic polyclonal anti-anti-idiotypic antisera and the three monoclonal anti-anti-idiotypes, only one polyclonal antiserum showed binding capacity to the corresponding RT1-encoded antigenic determinants on spleen cells. All the other antibodies were idiotypic but not antigen binding.     

A monoclonal anti-idiotypic antibody against anti-receptor antibodies from myasthenic sera

A monoclonal anti-idiotypic antibody has been developed against anti-acetylcholine receptor antibodies purified from the serum of one myasthenic patient. The idiotype is present on a subpopulation of antibodies directed against the toxin-binding region of the receptor. The monoclonal antibody cross-reacts with antibodies from other myasthenic sera, suggesting shared idiotypic specificities.     

Induction and characterization of isogeneic anti-idiotypic antibodies to BALB/c myeloma S117: lack of reactivity with major idiotypic determinants.

Isogeneic anti-idiotypic antibodies were induced by immunization of BALB/c mice with the BALB/c-derived myeloma protein S117 which binds N-acetylglucosamine-containing antigens including Group A streptococcal carbohydrate (A-CHO). Most BALB/c mice produced anti-S 117 idiotypic antibodies, as shown by various different immunization protocols. The antibodies of individual mice were of intermediate to high affinity (2.8 x 10(6) M-1 to 1.4 x 10(8) M-1). In isoelectric focusing, most individual antibodies were shown to consist of a small number of clonotypes, but each mouse produced its own unique set of clones so that the potential clonal repertoire of strain BALB/c is rather large. Most importantly, all isogeneic anti-idiotypic antibodies were directed against idiotypic determinants of S117 that are not shared with induced anti-A-CHO antibodies, whereas it has been shown previously that allogeneic and xenogeneic anti-S 117 idiotypic antibodies react with idiotypic determinants unique to S 117 as well as with those that are shared with anti-A-CHO antibodies. The data suggest that the expression of S 117 idiotypic determinants in strain BALB/c is not under antibody-mediated, anti-idiotypic feedback control.     

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

The expression and functional involvement of nuclease- specific idiotype on nuclease-primed helper T cells

The expression and functional significance of idiotypic determinants on antigen-specific helper T (Th) cell populations for responses to Staphylococcal nuclease (Nase) were evaluated in an in vitro antibody response system. Trinitrophenyl (TNP)- specific plaque-forming cell responses to TNP-conjugates of Nase (TNP-Nase) were shown to require the cooperation of Nase-primed Th cells as well as unprimed B and accessory cells. The expression on these antigen-primed Th cells of idiotypic determinants cross-reactive with those on anti-Nase antibodies was demonstrated by the specific elimination of Th cells for TNP-Nase by treatment with affinity-purified anti-idiotypic antibodies plus complement. The susceptibility of Nase-primed Th cells to elimination by such treatment was specific in that anti-idiotypic antibodies affected Th cells only from strains normally expressing the same (or a cross-reactive) idiotype on anti-Nase antibodies. A functional role of the idiotypes expressed on Nase-primed Th cells was suggested by the fact that anti-idiotypic antibody present throughout the period of culture, in the absence of complement, suppressed responses to TNP-Nase in an antigen- and strain-specific manner. It was further shown, by cell mixing experiments, that this inhibition appeared to occur at the level of the Th cells and was not dependent on the strain of origin of the B cells. Thus, antigen-specific Nase-primed Th cells express strain-specific idiotypic determinants cross-reactive with, or identical to, those of anti-Nase antibodies. These cell surface idiotypic determinants appear to be functionally involved in the activity of Th cells for the induction of antibody responses to TNP-Nase in vitro.     

Public and individual idiotopes in the anti-poly(Glu60, Ala30, Tyr10) response: analysis by monoclonal antibodies

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.     

Pathogenic Anti-DNA Antibodies in SLE: Idiotypic Families and Genetic Origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 31, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 31 autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.