Detection of a regulatory idiotype on a spontaneous neonatal self-reactive hybridoma antibody

To investigate the physiologic relevance of an idiotype-driven regulation of the immune system, we began a search for spontaneous self-reactive hybridomas in neonatal BALB/c mice. We sought hybridoma antibodies reactive with thyroglobulin (Tg) and expressing Id62, a recurrent idiotype with regulatory properties borne on induced adult autoantibodies to Tg. We describe herein such a neonatal Tg binding/Id62-positive monoclonal antibody (mAb) B10H2, and compare it with prototype mAb 62, an adult Tg-binding/Id62-positive mAb. Both mAb react with the same Tg epitope, as demonstrated by their abilities to totally inhibit the binding of the other to Tg. Moreover, as assessed by a double-reciprocal plot of their binding to Tg, their relative affinities for Tg are comparable. Likewise, mAb B10H2 appears idiotypically similar to mAb 62 because it totally inhibits the binding of mAb 62 to homologous anti-idiotype. Finally, as previously shown for mAb 62, mAb B10H2 expresses Id62 independently on both heavy (H) and light (L) chains, as evidenced by the immunoblot binding of a specific anti-Id62 probe to separated H and L chains. By molecular genetic analysis both antibodies appear to have made use of a member of the same VH 7183 gene family. Altogether, these findings suggest that neonatal mAb B10H2 and adult mAb 62 are very similar if not identical, and regulatory idiotype Id62 may be germline encoded. Furthermore this observation supports, in general, the concept of an idiotype-driven regulation for autoimmune responses.     

Expression of idiotype on the surface of human B cells producing anti-DNA antibody

We analyzed the idiotype (Id) expression on the surface of human anti-DNA antibody-producing cells. Murine monoclonal anti-Id antibodies with a specificity for determinants associated with the antigen-binding sites of human monoclonal anti-DNA autoantibodies were prepared. One anti-Id antibody reacted only with surface Id on anti-ssDNA-producing cells, but not with those on anti-dsDNA-producing B cell clones. Another anti-Id antibody did bind the surface Id on anti-dsDNA clones, but not those on anti-ssDNA clones. The interaction between anti-Id and surface Id was inhibited by pretreatment of the clones with DNA or appropriate polynucleotide antigens, or by preabsorption of anti-Id antibodies with free anti-DNA antibodies. Surface IgM and IgD expressed the same Id as the antibody secreted from the clones. The treatment of Id-positive clones by anti-Id antibody induced the redistribution of surface Id on the cells, indicating that these cells serve as targets for the regulatory action of anti-Id antibody.     

A public idiotypic determinant is present on spontaneous cationic IgG antibodies to DNA from mice of unrelated lupus-prone strains

A monoclonal anti-idiotypic antibody (anti-Id) to a public idiotype (Id) present on spontaneous IgG antibodies to DNA from NZB/NZW F1 mice recognized similar determinants on polyclonal and monoclonal IgG anti-DNA antibodies from mice of the unrelated MRL/lpr and BXSB strains. Incubation of the anti-Id with four of five monoclonal Id in solid phase inhibited their ability to bind DNA; however, different Id+ antibodies recognized different epitopes within the DNA molecule. Therefore, the public Id was located close to the antigen-binding regions but did not comprise all of those regions. Analysis of multiple polyclonal and monoclonal antibodies to DNA showed the Id on all subclasses of IgG. However, antibodies bearing the Id carried a neutral or cationic charge (10 of 10 monoclonals with pI greater than 7 were Id+); the presence of the Id on anionic IgG (pI less than or equal to 7) was infrequent (one of 21 serums, one of eight monoclonal antibodies). Therefore, IgG autoantibodies to DNA are constructed from closely related public idiotypes in several mouse strains that spontaneously develop lupus, and that Id is restricted to antibodies with a pI of 7 or greater.     

Mouse monoclonal antibodies to chicken VH idiotypic determinants reactivity with B and T cells

We have prepared mouse monoclonal antibodies against idiotypic (Id) determinants on chicken antibodies to N-acetylglucosamine (NAGA) and p-aminobenzoic acid (PABA) made by inbred line EL 6(3) birds. The monoclonal anti-NAGA Id antibody, termed CId-1, reacted with affinity purified antibodies to NAGA, but not with antibodies specific for PABA, arsanilic acid (Ars), phosphorylcholine (PC), or with normal chicken IgG and IgM. The monoclonal anti-PABA ID antibody, termed CId-2, reacted with anti-PABA antibodies and to a lesser extent with anti-Ars antibodies, but not with anti-NAGA, anti-PC, and normal IgG and IgM. The Id determinants were found among antibodies to NAGA and PABA made by outbred and inbred lines of White Leghorn chickens. The binding of the CId-1 and CId-2 antibodies to intact homologous anti-NAGA and anti-PABA antibodies, respectively, was not hapten-inhibitable in either case. Both anti-Id antibodies reacted specifically with isolated homologous heavy chains, suggesting VH Id specificities. The monoclonal CId-1 and CId-2 antibodies were reactive by immunofluorescence with approximately 0.9 and 0.2%, respectively, of the circulating lymphocytes and with approximately 0.4 and 0.15 of plasma cells. CId-1+ and CId-2+ bursal cells were first detected on the 16th and 14th days of incubation, respectively; both reached maximal frequencies by the 17th day of incubation. The CId-2 antibody reacted exclusively with immunoglobulin-positive cells. The CId-1 antibody also reacted with a subpopulation (0.4%) of immunoglobulin-negative lymphocytes from normal and agammaglobulinemic chickens, and thus would appear to recognize an idiotypic determinant expressed by certain clones of B and T cells.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Anti-idiotypic antibodies recognizing stable epitopes limit the emergence of idiotype variants in a murine B cell lymphoma.

The emergence of Id variants is a major escape mechanism from anti-Id therapy of human B cell malignancies and of the murine B cell lymphoma 38C13. To determine what impact the epitope specificity of anti-Id antibodies has on the prevention of emergence of such Id variants in the 38C13 lymphoma, anti-Id mAb of varying epitope specificity for the Id of 38C13 tumor cells were produced and studied. Some antibodies, produced by immunizing mice with both the wild-type 38C13 IgM and variant IgM, cross-reacted with wild-type 38C13 IgM and with all four members of a panel of variant IgM. These anti-Id did not react with separated 38C13 IgM H or L chains by Western blot, but did react with the cytoplasmic H chain of the surface Ig- variant cell line T2D that expresses the same H chain as wild-type 38C13 in its cytoplasm but does not express any associated L chain. In contrast, anti-Id of narrower specificity did not react with this H chain. This indicated that the broadly cross-reactive antibodies recognized a stable epitope on 38C13 H chain. When a broadly cross-reactive antibody MS11G6 was compared to S1C5, an antibody of narrower specificity, MS11G6, was superior at preventing tumor growth in mice inoculated with 38C13 cells. Moreover, no surface Ig+ variants emerged in escaping tumors in the MS11G6-treated group, whereas such variants were common in the S1C5 treated group. Both anti-Id were of equal efficacy in eliminating wild-type 38C13 cells by using 38C13 cells in tumor inoculums that had just been cloned in vitro, but MS11G6 was also capable of preventing the growth of several surface Ig+ variant cell lines in vivo. We conclude that anti-Id recognizing more stable Id determinants can limit the emergence of Id variants and therefore be more effective therapeutic agents. This finding is of additional importance as additional in vivo and immunophenotypic studies demonstrated that the generation of Id variants was an ongoing process both in cloned parental 38C13 cells and its variants.     

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.     

Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

Autoimmunity induced by HgCl2 in Brown-Norway rats. II. Monoclonal antibodies sharing specificities and idiotypes with mouse natural monoclonal antibodies

Spleen cells derived from BN rats receiving HgCl2 were fused with the nonsecreting rat myeloma cell line IR983F. We screened 59 supernatants from immunoglobulin-secreting hybrids for antibody activity against actin, tubulin, autologous and heterologous myosin, myoglobin, dsDNA, peroxidase, and the haptens TNP, NIP, NNP, and NBrP. Six monoclonal antibodies (mAb) were found to react with antigen(s) of the panel. At least three groups of antibody specificities were identified: clones reacting with TNP (1 IgM, 1 IgE); clones reacting with horseradish peroxidase (1 IgM); and clones possessing widespread reactivity for several antigens as found for mouse natural autoantibodies (2 IgM, 1 IgE). We also analyzed the idiotypic (Id) determinants of the 59 mAb by using anti-Id antibodies described elsewhere prepared in rabbits against the BALB/c D23 natural monoclonal autoantibody and recognizing a BALB/c recurrent Id (Id D23) of natural polyspecific autoantibodies. We found that all rat mAb that possessed widespread reactivities bore this Id. We performed similar studies in sera from normal and mercury-stimulated rats. The results indicate a role for HgCl2 in the stimulation of natural antibodies producing cells and the existence of interspecies cross-reactive Id among mouse and rat natural antibodies.     

Evidence that anti-idiotype induced immunity to experimental African trypanosomiasis is genetically restricted and requires recognition of combining site-related idiotopes

The ability of anti-idiotypic (anti-Id) antibodies to immunize mice against African trypanosomiasis independent of antigen has been confirmed. Of three allogeneic anti-Id antibodies raised against three protective monoclonal antibodies, each with specificity for the variant surface antigen of a clone of Trypanosoma rhodesiense, only one (anti-7H11 Id) was effective in immunizing BALB/c mice against homologous challenge. The immunity was associated with the more rapid and enhanced expression of the corresponding Id after infection. The immunity was restricted to mice bearing genes linked to Igh-Ca, which appeared to control expression of this Id both in response to infection and anti-Id treatment. Another Id, 11D5, appeared to be under similar genetic control. Anti-11D5 Id, however, was ineffective in immunizing mice against infection despite inducing high levels of Id bearing molecules before challenge. The immunizing potential of the respective anti-Id antibodies appeared to be related to the relative concentrations of antibodies reactive with idiotopes near to or within the antigen-combining site, which, in turn, determined the relative proportion of Id-bearing clones activated that had antigen binding activity.     

Properties of anti-idiotypic T cell lines propagated with syngeneic B lymphocytes. I. T cells bind intact idiotypes and discriminate between the somatic idiotypic variants in a manner similar to the anti-idiotopic antibodies

We describe the development of T cell lines possessing a binding specificity for syngeneic T15 idiotopes (Id) expressed on phosphorylcholine (PC)-reactive Ig. The lines were obtained by cultivation of BALB/c splenic T cells with T15 Id+ stimulator cells BCg3R-1d, a BALB/c lymphoma transfected with genomic sequences mu and kappa with S107 (T15) variable regions. Resulting Thyl-2+, L3T4+ cell lines depend on the T15 Id+ BCg3R-1d cells for growth and demonstrate the ability to bind TEPC15, a S107 germline-encoded, PC-specific Ig alpha. The specificity of the 125I-TEPC15 binding was studied in a competitive RIA with various unlabeled Ig. The isolated H and L chains of TEPC15 failed to inhibit the 125I-TEPC15 binding, and the T15-, PC-binding proteins M603 (alpha) and M511 (alpha) inhibited the binding either poorly or not at all. Moreover, the T cell lines had a discriminatory binding specificity for various T15+ Ig that are somatic variants of TEPC15 and that differ from each other in discrete, conformational Id (epitopes) detectable with specific monoclonal anti-Id. The T cell lines could be grouped according to their binding patterns, which were comparable to the recognition patterns of certain monoclonal anti-Id. These data suggest the existence of T cells with specificity for serologically-defined determinants of syngeneic idiotypes.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Monoclonal anti-idiotypic antibodies induce humoral immune responses specific for simian virus 40 large tumor antigen in mice.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.     

Spontaneous shift of anti-DNA antibody idiotypes in systemic lupus erythematosus

Spontaneous shift in Id expression of polyclonal anti-DNA antibodies in a patient, BS, with SLE was investigated. BS had active lupus nephritis in 1982 and developed central nervous system lupus in 1986 without evidence of active nephritis. Two rabbit polyclonal anti-Id (BS-82 and BS-86 R-anti-Id) were raised against affinity-purified anti-DNA antibodies prepared from 1982 serum (BS-82) and 1986 serum (BS-86), respectively. In addition, murine monoclonal anti-Id was prepared against BS-82 Id. Direct binding assays showed that all three anti-Id had preferential binding to the immunizing anti-DNA antibodies (the homologous Id) and poor binding to anti-DNA antibodies prepared from the different dated sample of BS. This was confirmed by inhibition assays of binding of anti-Id to the homologous Id by various Id. Moreover, inhibition assays of binding of various Id to DNA by the R-anti-Id showed that the R-anti-Id was the most effective inhibitor for the homologous Id. Testing for Id expression in serial (1982 to 1986) serum samples of BS with the R-anti-Id as probes showed that BS-82 Id declined and was undetectable after October, 1984, whereas BS-86 Id was first detectable in July, 1985, and increased by June, 1986. These results clearly demonstrate spontaneous shifts in Id expression of human anti-DNA antibodies. The phenomenon of Id shift should be considered in any future strategy for the diagnosis and therapy of human autoimmune disease by anti-Id.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

Preferential association of kappa IIIb light chains with monoclonal human IgM kappa autoantibodies

The predominance of the relatively uncommon V region subgroup isotype kappa III among the light chains of human monoclonal (IgM kappa) anti-IgG antibodies, (i.e., rheumatoid factors), was further documented through sequence analyses of ten such autoantibodies isolated from IgM-anti-IgG cold-insoluble immune complexes (mixed cryoglobulins). The amino-terminal sequence of all ten kappa-chains was characteristic for kappa III proteins and virtually identical to that of a prototype kappa III light chain. Similar sequence identity was found for kappa-chains isolated from three IgM kappa autoantibodies that formed cold-insoluble immune complexes with low-density lipoprotein (LDL). The thirteen light chains were found to be virtually identical in sequence for the first framework region (FR); ten of these proteins sequenced through the first complementarity-determining region (CDR) and into the second FR were markedly similar. The second CDR of five proteins was almost identical in sequence to that of the prototype kappa III-chain. Concordance was also demonstrated between the structural classification of the light chains as kappa III and their immunochemical classification as members of this V region subgroup. Serologic analyses of light chains isolated from seven IgM kappa autoantibodies (six anti-IgG, one anti-LDL) and of one intact IgM kappa anti-LDL antibody showed that each had antigenic determinants common to kappa II proteins. These light chains also expressed the antigenic determinant(s) of a V-region sub-subgroup of kappa III proteins designated kappa IIIb. Our studies confirm the preferential association of kappa III (and kappa IIIb) light chains with IgM kappa anti-IgG antibodies and demonstrate a similar association for IgM kappa anti-LDL antibodies. The finding that these and other types of IgM kappa autoantibodies, e.g., cold agglutinins, have remarkably similar light chains suggests an inherent restriction in the immune response to self-antigens.     

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.     

Detection of in ovo derived idiotypic antibodies. I. A model for maternal-neonatal idiotype network studies.

Idiotypic vaccines appear to have many advantages over conventional vaccines. Maternal Id or anti-Id that are passively transferred to the fetus or neonate could provide another avenue for vaccination. Based on this premise we have investigated the transmission of idiotypic antibodies from dam to embryo by inoculating laying hens and analyzing their egg yolks for the presence of Id, anti-Id, and anti-anti-Id. The Ag chosen for these studies was BSA. After isolation and concentration BSA Id titers were approximately 256,000. The presence of anti-Id antibody in yolk samples is characterized by the ability of anti-Id to inhibit BSA binding to Id. The anti-Id extracted from yolks inhibited BSA binding to Id by 7 to 53%. Not all of the anti-Id present in samples was binding to a paratope-associated Id because 11 to 16% of the fluoresceinated anti-Id bound to Id-coated beads in the presence of excess BSA. Because a portion of the anti-Id antibodies were internal images of the BSA Id, they should be able to mimic Ag. This idea was confirmed when anti-Id inoculated in hens caused the synthesis of antibodies that would bind BSA and could be detected in an ELISA. Binding of anti-anti-Id to BSA-coated wells could be inhibited by preincubation of anti-anti-Id with BSA in solution. The chicken model provides a novel system to investigate maternal-fetal and maternal-neonatal interactions in the idiotypic network and the cellular mechanisms involved in the ontogeny of the Id network in neonates.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Idiotype-specific T helper clones recognize a variable H chain determinant.

Previously we described a Th clone specific for a regulatory idiotype on 3A4, an anti-Id mAb that mimics a murine L1210/GZL tumor-associated Ag. In our studies, we determined the molecular target on the stimulating anti-Id antibody that is recognized by the Th clone. The Th clone responds with proliferation to the H chain of 3A4 but not to the L chain. Furthermore, the 3A4 chain stimulates this Th clone more efficiently than either the intact 3A4 or the Fab fragments, and the presentation by APC of the H chain is more resistant to chloroquine treatment than the presentation of the intact 3A4 molecule. These results suggest that regulatory T cells "see" their target idiotopes as linear sequence determinants present on isolated Ig chains, and show that this might have biologic advantages with respect to the mechanism of Ag presentation.