Idiotypic Specificities Research Articles

Molecular analysis of the anti-GAT repertoire in three strains of mice

GAT-specific antibodies which express the CGAT (or pGAT) public idiotypic specificities are encoded by a paucigene system. Antibodies expressing discrete specificities, i.e. anti-GAT and anti-NP, appeared to use the same VH germ-line genes, an observation which directly supports the importance of random H-L pairing to generate a large number of distinct antibody molecules.

Interrelationships among distinct idiotypic specificities

The fine idiotypic properties of a hybridoma (H51.85.2) anti-GAT antibody which lacks both CGAT and GA-1 idiotypes are described. We identified another idiotype, termed GA-2 idiotype, on H51.85.2 hybridoma antibody. The GA-2 idiotype is present in all mouse strains tested and is induced by GA-related antigenic determinants. An interesting relationship between GA-1 and GA-2 idiotype on hybridoma anti-GA antibodies was observed. All GA-1+ hybridomas also express GA-2 idiotype. In contrast, GA-2+ hybridomas can express a full set, a fraction, or none of the GA-1 idiotypic determinants. The data together with previous amino acid sequence studies strongly suggest that H51.85.2 hybridoma antibody was derived from the same VH and VL genes encoding the GA-1+ hybridoma antibodies.

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: Localization and characterization of idiotypic determinants

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) ( K a values ranging from 5 × 10 6 to 10 10 M −) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (γ, κ) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15–20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained γl heavy (H) chains and 4-6-10 contained both γl and γ2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which denned binding mechanism diversity. The location of determinants recognized by antiidiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotye by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (~25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the V H- and V L-chain interaction must exist for the expression of these idiotypes.

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.

Idiotypic studies of monoclonal anti-tobacco mosaic virus antibodies from one mouse

Monoclonal antibodies have been prepared from one BALB/c mouse immunized with tobacco mosaic virus. The monoclonal antibodies are distributed into three subgroups recognizing different epitopes on tobacco mosaic virus subunits. The idiotypic specificities of these monoclonal antibodies have been studied using syngeneic antiidiotypic sera. A sharing of idiotypic specificities has been observed between members of each subset. These idiotypes are not recurrent in BALB/c mice immunized with tobacco mosaic virus.

Variable-region sequences of five human λ-chain proteins reacting with an idiotypic antibody to the MCG bence-jones protein

An antibody to the V-region of BJ protein MCG has been used to detect other human λ-chains with similar idiotypic specificities. Five such proteins have been found and sequenced. They show relatively strong sequence homologies to some segments of the MCG V-region. The results of this study indicate that L-chains which contain short segments of their V-regions that are identical can be readily detected by the use of appropriate antisera.

Interstrain conservation of the murine GAT-specific antibody V kappa repertoire as analyzed at the germline gene level.

A cDNA library was constructed in pBR322 from mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) monoclonal antibody kappa chain. Two cDNA clones were extensively characterized. One, L XI 62, was derived from an aberrant V kappa-J kappa rearrangement which resulted in a frame-shift at position 96, leading to a stop codon at the very beginning of the constant region. The second, L XIX 27, 1150 bp long, was unequivocally assigned to a GAT-specific kappa chain, by comparison of its nucleotide sequence with the previously determined NH2-terminal amino acid sequence of the isolated kappa chain. A specific probe, containing the leader and most of the V kappa gene-encoded region, was prepared from this clone and hybridized to EcoRI and BamHI restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains. Under stringent conditions, similar patterns were observed for all three strains, and consisted of a small number of bands (3-5). Under nonstringent conditions, patterns were again very similar when the different strains were compared, although 15-20 bands could be identified. These observations support the hypothesis that the GAT-specific kappa chains found in antibodies expressing the public CGAT idiotypes are encoded by a very small number of germline genes. This V kappa repertoire seems extremely conserved between the three strains that were analyzed, an observation which correlates with the interstrain conservation of these public idiotypic specificities.

A bi-directional immune network mechanism: Simultaneous induction of idiotype and anti-anti-idiotype in rabbits immunized with antibody to a common idiotypic specificity of anti-VHa2-allotype antibodies

Recently, we identified a common idiotypic specificity ( IdC) present on all of the allo and auto anti-VHa2-allotype antibodies tested. In this report, we immunized allotype-matched rabbits with anti- IdC Ab. When an a 1 a 1 homozygote was immunized with anti- IdC Ab (also from an a 1 a 1 rabbit), two distinct Ab populations were induced which were isolated through the use of immunoadsorbent column chromatography. One of these Ab populations, adsorbed to and eluted from an insolubilized a2 IgG column, was anti-VHa2-allotype Ab (Ab 1), which reacted with a2 Ig and inhibited the reaction between the a2 allotype and anti-VHa2-allotype Ab. The other Ab population, adsorbed to and eluted from an insolubilized immunogen anti- IdC column, was anti-anti- IdC Ab (Ab 3), which was specific for the immunogen anti- IdC Ab. In another experiment, when an a 1 a 1 heterozygous rabbit was immunized with anti- IdC Ab, only the Ab 3 was induced, which appeared to be identical to the Ab 3 induced in the a 1 a 1 rabbit. However, when the expression of the a2 allotype in an a 1 a 2 heterozygous rabbit was suppressed and then this rabbit was immunized with anti- IdC Ab, both Ab 1 and Ab 3 were induced. These data indicate the occurrence of a bi-directional immune response within our immune network system. According to Jerne's network theory, Ab 3 appeared to be induced through a stimulation by the idiotope of the immunogen Ab 2, whereas Ab 1 appeared to be induced through a reverse stimulation by the paratope of the immunogen Ab 2. Furthermore, the production of Ab 1 in these rabbits exhibiting the bi-directional immune response was at least 3–5 times greater than that of Ab 3 production indicating that the response through the reverse stimulation appeared to be dominant. The importance of this bi-directional immune response is discussed in this paper.

Specificity and idiotype analysis of a monoclonal anti-DNA antibody

The binding properties and idiotypes of a monoclonal anti-DNA antibody were studied to investigate the control of specificity in this autoimmune response. This antibody, A9, was derived from the fusion of spleen cells of an MRL- lpr lpr mouse and the cell line X63-Ag8.653. In inhibition binding studies, A9 showed preference for single-stranded DNA and bound both ribo- and deoxyribohomopolymers. An antiserum prepared in a rabbit against A9 showed anti-idiotypic specificity after absorption with BALB c immunoglobulins and the BALB c myeloma MOPC 195. At least two distinct idiotypic specificities could be demonstrated by this serum, one apparently exclusive to A9 and one commonly expressed in sera of MRL- lpr lpr mice. Serum levels of the commonly expressed A9 idiotype were also elevated in sera of C 3H mice expressing the lpr gene suggesting association with B-cell activation induced by this genetic mechanism. The demonstration of a commonly expressed A9 idiotype indicates that some autoantibody variable region determinants may be genetically controlled.

Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.

The common idiotypic specificity of anti-a2 allotype antibodies: contribution of H and L chains to idiotype expression

Previously an idiotypic specificity common to anti-a2 allotype Ab from 15 rabbits was detected and characterized. The contribution of H and L chains to the expression of this common idiotypic specificity was determined. Two types of recombinant IgG were prepared: (1) H chains from anti-a2 Ab with L chains from normal a3b4 IgG. and (2) L chains from anti-a2 Ab with H chains from normal a3b4 IgG. By inhibition of binding radioimmunoassay, recombinant IgG having either H or L chains from anti-a2 did not inhibit the binding between anti-a2 Ab and anti-idiotype Ab. By direct binding radioimmunoassay, recombinant IgG of H chains from anti-a2 Ab with L chains from a3b4 IgG reacted with anti-idiotype Ab whereas recombinant IgG with H chains from normal a3b4 IgG and L chains from anti-a2 Ab did not react. These results indicate that the common idiotypic specificity of anti-a2 Ab is determined primarily by the H chain; however, the full expression of the common idiotypic specificity requires the interaction of both H and L chains from anti-a2 Ab.

Analysis of the repertoire of anti-idiotypic B-cell responses to self-I-Ak- or -I-Ek-reactive monoclonal antibodies in A.TL mice.

Twenty anti-idiotypic antisera (anti-Ids) were produced in A.TL mice to self-I-Ak or -I-Ek-reactive monoclonal antibodies (mAbs), constructed in the A.TH anti-A.TL combination. The reactivity of these anti-Ids was examined in a panel of 31 anti-Iak A.TH mAbs, using direct idiotype binding, cross-competitive inhibition of idiotype binding, and isoelectrofocusing (IEF) assays. Among 13 anti-Ids produced against anti-I-Ak mAbs, one only recognized individual idiotypic specificities (IdIs) on its corresponding mAb, while the 12 others identified homologous IdIs and recurrent idiotypic specificities also expressed on heterologous anti-I-Ak and/or I-Ek mAbs. Two sets of major cross-reactive idiotypes (IdXs) were characterized on two groups of mAbs recognizing public Ia.1, I-Ak,f,u and r) or private (Ia.2, I-Ak) determinants clustered in two spatially distinct epitope regions of the I-Ak molecule, respectively. By contrast, most (5/7) of the anti-Ids raised against mAbs recognizing polymorphic or monomorphic (Ia.7-like) I-Ek determinants displayed specificity apparently restricted to their corresponding mAb IdIs. This finding contrasted with the previous characterization, using xenogeneic anti-idiotypic reagents, of an interstrain IdX expressed on all mAbs defining Ia.7-like determinants in the IEk epitope group I. These data indicate that A.TL mice can readily develop anti-idiotypic responses towards self Ia-reactive mAb minor idiotypes (IdIs) and that recognition of anti-Iak mAb IdXs in such mice is preferentially observed when anti-I-Ak mAbs are used as immunogens.

Idiotypes on B lymphocytes: association with immunoglobulins.

Several idiotypic (Id) specificities have been identified by immunofluorescence on the membranes of B cells in the spleens of nonimmune mice. These determinants are displayed at frequencies varying from 0.22 to 1.83% of B cells and are entirely immunoglobulin (Ig) in nature. No Id determinants were observed on the membranes of four Slg- Abelson virus-transformed pre-B cell lines that are sensitive to LPS. Under our experimental conditions, we did not observe a significant increase in 3H-thymidine incorporation subsequent to the in vitro incubation of splenic lymphocytes from normal mice with various amounts of anti-Id antibodies specific for several cross-reactive (IdX) or individual Id. Similarly, in utero exposure to anti-Id antibodies against myeloma proteins specific for T-independent antigens displaying B cell mitogenic properties did not alter the proliferation of lymphocytes induced by these mitogens. In contrast, exposure to anti-Id antibodies in vitro as well as in utero had a profound and specific effect on the corresponding antibody responses and maturation of small lymphocytes into plasma cells. When normal B cells were cultured with the B cell mitogen LPS in the presence of anti-Id antibodies directed against the J558IdX, specific suppression of the maturation of IdX+ plasma cells was observed. In mice exposed to maternal anti-Id in utero, we observed a severe inability to mount an immune response to any compound that elicited antibody molecules bearing the Id that was complementary to the maternal anti-Id. These same maternally Id-suppressed mice, however, gave a normal response when the same compound was presented as a mitogen. Our results reinforce the concept that Id on membranes of B lymphocytes are associated only with Ig receptors and do not support the possibility of mitogen-like poly-clonal stimulatory properties of anti-Id antibodies.

Structural analysis of monoclonal anti-arsonate antibodies: Idiotypic specificities are determined by the heavy chain

Hybrid immunoglobulin molecules were constructed from the isolated heavy and light chains of monoclonal anti-arsonate antibodies which differed in their expression of a cross-reactive idiotype. The reconstructed molecules were tested for public and private idiotypic determinants associated with the A-strain response to the hapten p-azophenylarsonate and it was found that both an appropriate heavy chain and an appropriate light chain were required for expression of idiotypic determinants. While appropriate heavy chains could be derived only from idiotype-positive antibodies, appropriate light chains could be derived not only from idiotype positive antibodies, but also from certain idiotype-negative molecules. Similarly, recombinant (hybrid) molecules constructed from two idiotype-positive immunoglobulins differing quantitatively in the degree of idiotypic character, expressed the character at a level which correlated with that of the heavy chain donor. Thus, while the serological expression of idiotypic determinants occurs only in the context of the appropriate V HV L combination, these data demonstrate that the determinants comprising the major cross-reactive idiotype of the anti-arsonate response of A/J mice have their bases in heavy chain structures.

Immunoglobulin diversity: analysis of the germ-line VH gene repertoire of the murine anti-GAT response.

A cDNA clone was constructed from a mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) BALB/c monoclonal antibody heavy chain. Its sequence, covering codons -5 to 162 and therefore encompassing the complete V-D-J region, was determined. Surprisingly, the sequence of the VH gene-encoded region was almost identical with that of the BALB/c VH anti-HNP (4-hydroxy-3-nitrophenyl) acetyl VH region, suggesting that the same VH germ-line might be used to encode two heavy chains contributing to antibodies of discrete specificities. A specific VH probe was derived and annealed to Eco RI and Bg1 II restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains that differ in their H chain allotypes. Under stringent conditions, only a few bands were identified by Southern blotting. The different patterns observed suggest that the VH anti-GAT repertoire differs between these strains even though their various anti-GAT antibodies express the same public idiotypic specificities.

Analysis of a major rat idiotype associated with anti-gat antibodies

An anti-idiotypic antiserum was raised in a rabbit against a pool of purified F.334 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.334 anti-GAT sera from primary and secondary anti-GAT responses. Anti-GAT sera of 13 inbred rat strains, with different RT1 haplotypes and with different heavy- and light-chain allotypes, all express idiotypic determinants cross-reacting with GAT-13. Thus, like in mice anti-GAT antibodies from rats express public idiotypic determinants. The anti-idiotypic serum also recognizes a highly conserved idiotypic specificity present on mouse and guinea-pig anti-GAT antibodies. The mouse, rat and guinea-pig express a similar highly conserved idiotypic specificity after immunization with GAT. All anti-GAT antibodies from the mouse and guinea-pig bear this idiotypic specificity. These results confirm the existence in the anti-GAT response of interspecies cross-reactive idiotypic determinants.

Idiotypic specificities of rabbit antibodies to immunoglobulin allotypes.

Idiotypic specificities of rabbit antibodies to immunoglobulin allotypes.

Monoclonal anti-gat antibodies with different fine specificities express the same public idiotype

We have studied the idiotypic specificities expressed by 22 anti-GAT hybridoma products (HP). These antibodies, although derived from cells of mice with three distinct heavy-chain linkage groups (BALB/c, Igh-l a, DBA 2 , Igh-l c and C57BL 6 , Igh-l b) all express the same public idiotypic specificity, p. GAT, defined by the heterologous binding of anti-idiotypic serum 715 to C57BL 6 anti-GAT antibodies. None of these antibodies expressed the strain-restricted idiotypic specificity, s. r. GAT- l, defined by the binding of anti-idiotypic serum JL 122 to BALB/c anti-GAT antibodies. BALB/c anti-GAT HP could be shown to fall into three subsets with respect to their fine antigenic specificity for GAT, GT and GA. An individual idiotypic specificity, i 1- GAT (defined by syngeneic anti-idiotypic sera raised against one of the BALB/c HP), was also found on a group of BALB/c HP which all shared a similar fine antigenic specificity pattern. Taken together, these observations suggest that the expressed mouse anti-GAT repertoire derives from a very few V-germ-line genes (V H-GAT and V K-GAT) which are highly conserved in the species, and which determine the structure resulting in the p. GAT idiotypic specificity. The variations in fine specificity and individual idiotype are likely therefore to reflect somatic variations affecting these germ-line genes.

Contribution of the H- and L-chains and of the binding site to the idiotypic specificities of mouse anti-GAT antibodies

The contribution of the H- and L-chains to the structure of the main idiotype of anti-poly (Glu 60-Ala 30 Tyr 10) (GAT) antibodies has been studied. This idiotype has been previously divided into four types of specificity: (1) the highly conserved idiotypic specificity ( h. c. GAT) is expressed by anti-GAT antibodies from the guinea-pig, rat and mice; (2) the public specificity ( p. GAT) is expressed in an identical form by all anti-GAT antibodies from all strains of mice tested and by all hybridoma products (HP) with anti-GAT activity; (3) the strain-restricted specificity ( s.r.GAT-1) is only expressed by anti-GAT antibodies from strains with Ig-1 a, Ig-I c and Ig-I e allotypic markers; and finally (4) the individual specificity i j ,- GAT defined on HP G5 is also expressed by most of the hybridoma protein with anti-poly (Glu 50 Tyr 50) (GT) activity. In this paper we-show that h. c. GAT, p. GAT and i j - GAT require the interaction of H- and L-chains to be expressed: (1) isolated H- and L-chains from HP G5 did not express these specificities; and (2) recombinant molecules composed of H- and L-chains from HP with anti-GAT activity and an irrelevant myeloma protein (MOPC21) never expressed h. c. GAT, p. GAT and i j - GAT, We next investigated the relationship between the GAT binding site and the p. GAT. h. c. GAT and s. r. GAT-1 idiotypic specificities. GAT and GT were not able to inhibit the binding to s. r. GAT-1 while they inhibit the idiotypic binding to p. GAT and h. c. GAT. A GAT fragment of mol. wt 3000 was also shown to inhibit the binding of p. GAT and h. c. GAT to the appropriate sera. Thus p. GAT and h. c. GAT are very close to the GAT combining site while s. r. GAT-1 represents an idiotypic specificity located outside the GAT binding site.

Specificity and idiotypic analysis of monoclonal antibodies directed against the MOPC460 idiotype

Eight syngeneic anti-idiotypic hybridomas (IDMs) have been obtained against the BALB/c myeloma protein MOPC460 which displays anti-TNP activity. The study of their anti-idiotypic specificity allowed us to distinguish them into two groups which define the presence of at least two idiotypic determinants or idiotopes in the MOPC460 idiotype. The biochemical analysis of the monoclonal antibodies is consistent with this dichotomy. This analysis, in fact, showed a striking correlation between anti-idiotypic specificity and biochemical characteristics of the monoclonal antibodies. Consequently. the idiotypic specificities of three of these hybridomas were studied. In accordance with what is expected. our results clearly indicate a strong idiotypic similarity for hybridomas belonging to the same group and a lack of idiotypic cross-reactivity between the two groups.