Evidence for shared idiotypy expressed by the IgM, IgG, and IgA serum proteins of a patient with a complex multiple paraprotein disorder.

The results of a comparative idiotypic analysis of multiple Ig paraproteins isolated from the serum of an individual patient, Ca, with Sj?gren's syndrome and Waldenstr?m's macroglobulinemia are reported. At initial presentation, Ca serum was found to contain two major paraproteins, an IgMkappa and an IgGkappa, together with a small elevation in the level of IgA protein. The patient's clinical course was characterized by dramatic and opposing changes in the respective serum levels of the IgMkappa and IgGkappa paraproteins over an extended time period that coincided in part with received chemotherapy. Idiotypic antigenic analysis of the IgMkappa and IgGkappa paraproteins revealed that the two monotypic proteins shared identical idiotypic determinants. The Ca IgA serum fraction, specifically isolated by an immunoabsorbent and free of any IgG and IgM, was shown to possess idiotypic determinants identical to the IgG and IgM proteins. In extensive tests of specificity, the idiotypic determinants shared by Ca IgM, IgG, and IgA proteins were not present in large excesses of heterologous IgM and IgG, nor on Ig molecules contained in a large number of normal and myeloma sera.     

Genetic mechanisms for dominant VH gene expression. The VHB512 gene.

A total of 37 mAb with reactivity for dextran B512 have been studied; 30 of them were products of independent rearrangements and 21 made use of the same VH gene, the VHB512 gene. These results unambiguously established that the immune response to dextran in the high responder mouse strain C57BL/6 was restricted. Idiotypic determinants are located all over the Ig V region. Many but not all Id described so far can be ascribed to protein structures encoded by VH or VL gene segments. The expression of the major Id, 17-9 Id, in C57BL/6 was not absolutely correlated with the expression of the dominant VHB512 gene in the same mouse strain. Inspection of amino acid sequences of the CDR3 of idiotypic positive and negative clones suggested that idiotypic structures may be associated with the expression of Tyr at position 95 and Phe or Leu at position 96 in the H and L chains, respectively. Therefore the indiscriminate use of idiotypic markers to characterize VH genes and the relevance of idiotypic regulation in VH gene expression are questioned. Id-positive and Id-negative clones displayed similar affinity values for dextran, indicating that idiotypic and binding structures were probably separated. The exchange of Asp65 for Gly65 in one of the clones reduced affinity for dextran, suggesting the involvement of CDR2 in dextran binding. The dominant expression of VH genes can be explained by somatic and/or genetic mechanisms. Because somatic mechanisms such as idiotypic regulation or selection based on affinity for dextran did not seem to influence the expression of the VHB512 gene we favor a genetic alternative. We discuss a model based on the distance between VH genes and D and JH elements. This model is compatible with somatic and genetic regulation in other systems and provides a new theoretical approach to the understanding of immune VH dominance and low responsiveness.     

Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

The limited diversity of the mouse gamma-chains anti-GAT repertoire does not seem to be noticeably amplified upon class switch

NH2-terminal sections of H and L chains isolated from five monoclonal anti-GAT antibodies derived from BALB/c mice have been sequenced upon to residue 43. Four among these five antibodies, sharing similar public idiotypic determinants, possess extremely conserved sequences, both for the H, which is apparented to the VH II type, and the L chains, which belong to the V kappa I subgroup. VH sequences are identical up to residue 43 and contain the common sequences (residues 1 to 32) defined for the H chains derived from the DBA/2 IgM anti-GAT monoclonal antibodies. Light chains are also remarkably conserved, a rather unusual situation for kappa-chains. The fifth antibody that expresses only part of the public idiotypic determinants contains very distinctive H and L chains. Its heavy chains are close to the VH I subgroup, whereas its kappa-chains permit definition of a new V kappa subgroup. The repertoire appears to be highly conserved between BALB/c and DBA/2 mice, and does not seem larger in IgG than in IgM antibodies. This latter observation does not speak in favor of a switch-linked amplification of diversity.     

Noncovalent association of heavy and light chains of human immunoglobulins. IV. The roles of the CH1 and CL domains in idiotypic expression

A rabbit anti-idiotypic antiserum was raised against a monoclonal human IgM kappa(Me) in order to analyze the possible modulation of idiotypic expression by Fab constant domains. IgM(Me) fragments, subunits, and domains were prepared by chemical and enzymatic cleavages. All molecular species were shown to have a well-defined secondary and tertiary structure by circular dichroism. Full recombination between domains and subunits was ascertained by difference spectroscopy. The expression of the idiotype on native and recombined fragments, domains, and subunits was quantitated in a competitive enzyme-linked immunosorbent assay (ELISA). Reduced and alkylated Fab, isolated H and L chains, purified Fv(Me), intact VH and VL domains and H-L, VH-L, VL-H, and VH-VL recombinants were compared on a molar basis to native Fab(Me) for idiotypic expression. VH-specific determinants were found, whereas the L chains were virtually devoid of idiotypic activity. Both the peptic FV(Me) fragment, which is composed of intact VH and VL domains, and the recombined VH-VL heterodimer were found to be fourfold less active for idiotype expression than native Fab(Me). However, full inhibition was achieved at high molar concentrations, suggesting that all the idiotopes present on Fab(Me) were expressed on FV(Me) but with a reduced antigenicity. Comparison of VH-L and VL-H hybrid molecules revealed that the presence of the C mu 1 domain was sufficient to restore full idiotypic expression as compared with native Fab(Me). These data support the hypothesis that the first constant domain of the mu heavy chain alters the quaternary interaction between the variable domains, and therefore modulates the expression of the idiotype through longitudinal interactions that are not affected by reduction of the inter-H-L chain disulfide bond.     

Idiotypical identity of IgG myeloma protein with monoclonal IgM, Bence Jones protein, and Fv derived from one patient.

Serum from a patient (KK) with IgG2-lambda myeloma was shown to contain multiple paraproteins corresponding to an IgM-lambda monoclonal protein (MMP), a lambda-type Bence Jones protein (BJP), and a 30 kDa component in addition to the IgG2 myeloma protein (GMP). These proteins possessed common idiotypic determinants, as judged by their monoclonal reactivity with rabbit anti-GMP idiotype antibody (aId) in the immunofixation electrophoresis. Analysis with aId absorbed with either H or L chain of GMP revealed that the 30 kDa component shared both VH and VL with GMP and MMP, while BJP carried only the VL idiotype. The 30 kDa component, however, failed to react with antibody to either the mu, gamma, alpha, kappa, or lambda isotype, indicating that it had an Fv-like molecular composition. These results suggest that myeloma cells of KK had diverged from the same precursor B cell clone to produce MPs of different isotypes and altered molecular constructions.     

Idiotypes on galactan binding myeloma proteins and anti-galactan antibodies in mice.

Antibodies with specificity for beta1,6 linked D-galactoses were induced in mice by immunization with gum ghatti. Idiotypic antisera were prepared in rabbits and mice by immunization with 8 BALB/c IgA(k), beta1,6D-galactan binding myeloma proteins (beta6GALBMP). Two kinds of idiotypic sera were obtained: cross-specific sera that reacted with two or more beta6GALBMP but not other BALB/c myeloma proteins, and individual idiotypic sera that reacted with only the beta6GALBMP used in the immunization. Antibodies with specificity for beta1,6 linked D-galactans shared cross-specific idiotypes with beta6GALBMP. Only one of seven individual idiotypes associated with beta6GALBMP was found on galactan antibodies. Since all beta6GALBMP thus far have the same Vk and VH isotope composition the results indicate an extensive heterogeneity among galactan-binding immunoglobulins in BALB/c mice. It is speculated that some of this diversity may arise from somatic rather than germ line gene mutations.     

Antigenic studies of a VH fragment: demonstration of three sets of antigens, idiotypic, VH subgroup, and VH framework-specific antigens.

The antigenic properties of the VH region of immunoglobulin heavy chains were studied by means of a fragment corresponding to the variable part of the heavy chain of an IgG3 myeloma protein (KUP) and an antiserum made against this fragment. By hemagglutination, hemagglutination inhibition, and immunofluorescence techniques, it was shown that the anti-VH antiserum detected three sets of antigens in the VH region, namely idiotypic antigens, VH subgroup-specific antigens, and VH domain-(framework) specific antigens. The VH fragment inhibited in a VHII subgroup-specific hemagglutination inhibition test system. The VH fragment was thus antigenically similar to the tvh region found in the intact molecules and the light chains were not needed to express the VH subgroup antigens or the VH framework antigens.     

A cross-reactive idiotype on anti-DNA antibodies defines a H chain determinant present almost exclusively on IgG antibodies

We have previously reported two anti-idiotypic antibodies, 3I and 8.12, that recognize L chain determinants on anti-DNA antibodies. We have generated a new anti-idiotypic antibody, F4, that recognizes a H chain determinant on cationic anti-DNA antibodies. F4 reactivity is present in high titer in serum of approximately 60% of SLE patients and on 84 of 706 myeloma proteins. It is preferentially associated with 3I reactive L chains. Furthermore, antibodies bearing both the F4 and 3I idiotypic determinants preferentially bind DNA. Amino acid sequencing of H chains isolated from four F4-reactive myeloma proteins suggests that they derive from two currently identified VH gene families. F4 reactivity is restricted almost exclusively to Ig of the IgG isotype suggesting that F4 may recognize either a somatically mutated hypervariable region or a variable region used late in the immune response. F4, therefore, represents a new idiotypic family preferentially associated with auto-Ag specificity and having features of an Ag-driven immune response.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.     

Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype.

Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag.     

VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes

The utilization of germline genes for the synthesis of autoantibodies has been suspected for many years based on the presence of cross-reactive idiotypes among patients as well as in some healthy first-degree relatives of patients with several autoimmune diseases including SLE. One such system of idiotypes involves anti-Sm antibodies, which are highly specific for SLE. To definitively establish the utilization of germline genes in the Sm system, we produced human-human B cell hybridomas from a patient with SLE who had circulating anti-Sm antibodies. One stable hybridoma designated 4B4 secretes an IgM-kappa mAb that binds Sm and shares idiotypic determinants with other anti-Sm antibodies. A second anti-Sm antibody (3C3), isolated from the same patient was also studied. Oligo(dT) priming was used to produce cDNA corresponding to full length IgM. Sequence analysis revealed that the VH gene segment (1-96) of 4B4 is identical to a VH sequence previously detected in a fetal liver cDNA library by Schroeder and his co-workers as well as a germline VH recently described by Berman and his associates. The identity of a lupus mAb and sequences derived from unrelated individuals provides strong evidence that this autoantibody is a direct copy of a germline gene.     

Characterization of molecular heterogeneity and multispecificity in homologous idiotypic antisera.

The molecular heterogeneity of homologous anti-idiotypic reagents was characterized by a novel isoelectric focusing procedure. Idiotypic antisera directed against the PC-binding plasmacytoma protein T15 were raised in CE and and A/J mice. These antisera were shown to be highly specific by hemagglutination with myeloma protein-derivatized sheep erythrocytes and by radioimmunoassay. Competition experiments performed with affinity-labeled T15 revealed that about 40% of the pooled CE antibody activity was directed against binding site-associated determinants. Further analysis of anti-idiotypic sera from individual animals with the use of isoelectric focusing disclosed heterogeneous populations of antibody molecules distinguishable by isoelectric point and by subspecificity. Each animal expressed a unique spectrotypic profile. In addition, clones reactive with binding site and non-binding-site determinants as well as some clones with specificity for other PC-binding mouse myeloma proteins were detected. These results emphasize the importance of careful selection and thorough absorption of idiotypic antisera.     

Idiotypic and anti-idiotypic determinants on lymphocytes during anti-Rh immunization

Evolution of idiotypic determinants on lymphocytes membrane and presence of other lymphocytes carrying anti-idiotypic determinants, were studied in Rh negative human volunteer blood donors during immunization towards Rh factor. For this purpose E-Rh Rosettes, direct immunofluorescence, inhibition of E-Rh Rosettes by anti-idiotypic sera as well as EA-Rh Rosettes--induced with Fab'2 fragments from anti-Rh antibodies and lymphocytes from the same subject--were examined and compared to the evolution of circulating antibodies. E-Rh Rosettes preceded or accompanied production of anti-Rh antibodies; their frequency decreased after the fifth month following immunization, meanwhile EA-Rh Rosettes increased in parallel with antibody decrease. The direct immunofluorescence and the inhibition of E-Rh Rosettes, by anti-idiotypic sera, show the presence of idiotypic determinants on lymphocyte membranes; the presence of EA-Rh Rosettes, coinciding with the decrease in antibodies demonstrate the existence of lymphocytes bearing auto-anti-idiotypic determinants.     

High idiotypic connectivity of the VH7183-encoded antibodies directed to a murine embryonic carbohydrate antigen, Lewis Y, as ascertained by syngenic anti-idiotype monoclonal antibodies

The Lewis Y Ag is a carbohydrate Ag which is closely related to a well-known murine embryonic Ag, the stage-specific embryonic Ag-1 (SSEA-1), in its biochemical structure. It is expressed at the surface of murine embryonic cells as well as many murine cancer cells. For the analysis of idiotopes carried by the anti-Lewis Y antibodies, we generated two syngenic anti-idiotypic mAb, Id-A1 and Id-B4 (both BALB/c IgG1), which are directed to the idiotypic determinants carried by the anti-Lewis Y mAb, AH-6 (BALB/c IgM). Both Id-A1 and Id-B4 (Ab2) recognized paratope-related idiotopes carried by the AH-6 antibody (Ab1); they specifically inhibited the binding of AH-6 to the Lewis Y Ag. The high idiotypic connectivity of anti-Lewis Y antibodies was noted; the polyclonal anti-idiotype antibody, produced in the sera of BALB/c mice by immunizing AH-6 antibody, cross-reacted with several anti-Lewis Y mAb which has been established in different laboratories. Id-B4 and Id-A1 seem to represent such cross-reactive anti-idiotypic antibodies. Id-A1 recognized an idiotope carried by two out of six panel Ab1 mAb directed to the Lewis Y Ag. Id-B4 reacted with four out of the six panel antibodies, and was considered to recognize a recurrent idiotope of anti-Lewis Y antibodies which occurs more commonly than the idiotope recognized by Id-A1. All of the anti-Lewis Y antibodies which carry idiotopes that react with Id-A1 or Id-B4 were encoded by the VH genes of the VH7183 family; the most D-J proximal VH gene family in BALB/c mice, which is known to be preferentially expressed in embryonic B cells. Immunization of BALB/c mice with keyhole limpet hemocyanin-conjugated Id-B4 and/or Id-A1 induced a significant titer of anti-Lewis Y antibodies (Ab1-like Ab3) in the sera.     

Representation of heavy but not light chain Ig idiotypes on T cell receptors for alloantigens.

We have analyzed idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants by the use of different anti-idiotypic antibodies. Such antisera were produced in (Lewis X DA) F1 rats against Lewis anti-DA alloantibodies (= B cell product) and Lewis T lymphocyte receptors with the same specificity. We found that B lymphocytes bear unique idiotypic determinants which are not present on the corresponding T lymphocytes. T cell unique (not shared by B lymphocytes) idiotypes were so far not detected. T cells idiotypic determinants which are present on heavy but not light chains of the corresponding alloantibodies.     

Genetic control of the immune response to nuclease. II. Detection of idiotypic determinants by the inhibition of antibody-mediated nuclease inactivation.

The humoral response of mice to staphylococcal nuclease has previously been shown to be controlled genetically by H-2-linked Ir gene(s). In order to examine the possible contributions of variable region immunoglobulin genes to this genetic control, we have developed a system for the detection of idiotypic determinants on anti-nuclease immunoglobulin molecules. Antisera to nuclease were raised in two high responder strains, A/J and SJL. The corresponding antibodies were purified by affinity chromotography on Sepharose-nuclease columns, and were used to immunize groups of Lewis rats. An assay system was developed to assess the inhibition of antibody-mediated inactivation of nuclease activity by the rat antisera thus produced. Despite the presence of many species-specific anti-mouse immunoglobulin antibodies in these sera, inhibition of antibody-mediated enzyme inactivation was found to be specific for anti-nuclease antibodies of the immunizing strain. The inhibition could not be removed by extensive absorption with normal serum proteins from the antibody-producing strain, and was shown to require antibodies directed toward binding sites of the anti-nuclease antibodies. This inhibition thus defines idiotypic determinants of anti-nuclease antibodies.     

Characterization of a T-cell clone recognizing idiotypes as tumor-associated antigens

Although heterogeneous T cells recognizing idiotypic determinants have been demonstrated to occur spontaneously in vitro or to be expanded by immunization with antigen or idiotype, their in vitro propagation and cloning was not successful. These previous studies have relied extensively on soluble immunoglobulin to induce proliferation of idiotype-specific T cells. This report describes a unique approach to obtain a stable T-cell clone specific for a monoclonal beta 2-6 fructosan binding myeloma ABPC48 (BALB/c origin), bearing well-defined A48 regulatory idiotopes. Following repeated immunizations with ABPC48 myeloma protein of C.B/R3 mice (H-2d, VHb, CHa), which differ only in the VH locus from BALB/c mice (H-2d, VHa CHa), several stable T-cell clones were obtained after stimulation in vitro with ABPC48 myeloma cells. The proliferation of a T-cell clone A48.B2 was observed with irradiated myeloma cells or hybridomas producing antibodies bearing A48 idiotype encoded by genes deriving from the VH 441-4 family. Proliferation of the clone did not occur with soluble ABPC48 myeloma protein or with Sepharose 4B-bound ABPC48 myeloma protein. Both anti-A48Id and anti-Iad monoclonal antibodies can specifically inhibit the proliferation of this clone when stimulated with ABPC48 myeloma cells. These results demonstrate recognition of idiotypes on B-cell tumours by T cells and implicate the role of class II major histocompatibility complex determinants in this cellular interaction.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.