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.344 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.344 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.     

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.     

A common idiotype expressed on a murine anti-Sm monoclonal antibody and antibodies in SLE sera.

A rabbit anti-idiotypic antiserum made against a murine monoclonal anti-Sm autoantibody (Y2) was used in a solid-phase radioimmunoassay to investigate idiotypic cross-reactivity among anti-Sm antibodies present in sera from patients with systemic lupus erythematosus. Sera from 25 of 51 SLE patients (49%) containing anti-Sm antibodies were positive for this Y2 idiotype compared to only one of 22 normal human sera. Nine of 28 SLE patients (32%) whose sera were anti-Sm negative were also positive for the Y2 idiotype in low titre. Binding was not due to rheumatoid factor-like activity but was specific for the Y2 determinant and could be eliminated by absorption with Y2 monoclonal antibodies. The anti-idiotypic antibody blocked the ability of 12 of 25 anti-Sm positive lupus sera to bind Sm. Conversely, Sm antigen inhibited the binding of anti-idiotypic antibody in nine of 12 lupus sera.     

A common anti-cardiolipin antibody idiotype in autoimmune disease: identification using a mouse monoclonal antibody directed against a naturally-occurring anti-phospholipid antibody

We have recently produced a series of human monoclonal antibodies reacting with cardiolipin. One of these, H3, a polyspecific IgM/k derived from a normal individual, was used to raise mouse monoclonal antibody to its idiotype. Two anti-idiotypic antibodies, S2.9 (IgG2b) and S2.10 (IgM) were selected for their specific reaction with H3.S2.9 did not react with five other human monoclonal antibodies of IgM/k class despite the fact that these shared some antigen-binding characteristics with H3.S2.9 was able to block the binding of H3 to all of its cross-reactive antigens including cardiolipin, while S2.10 was not. S2.9 was equally efficient in blocking the binding of H3 to three of its cross-reactive antigens, cardiolipin, diphtheria and tetanus toxoids; greater than 90% inhibition could be achieved at an equimolar ratio of H3 to S2.9. The anti-idiotype S2.9 was used to demonstrate the presence of the H3 idiotype in serum. This idiotype was found in amounts greater than that seen in 42 normal individuals, in 30 of 36 patients with systemic lupus erythematosus (SLE), eight of 20 patients with rheumatoid arthritis (RA), 8 of 20 patients with Felty's syndrome as well as 10 of 23 patients with syphilis. Not one of nine patients with drug-induced lupus syndrome had abnormal levels. In patients with SLE and Felty's syndrome there was a good correlation between the amount of anti-cardiolipin antibodies and the amount of H3 idiotype (rs = 0.70 and 0.69 respectively). No such correlation was found in syphilitics or in patients with RA. In patients with SLE the H3 idiotype was present on IgM and IgG anti-cardiolipin antibodies. In 15 of 16 SLE sera with high levels of cardiolipin antibody, S2.9 blocked binding of serum antibodies to cardiolipin by 13-72%, with a mean value of 49%. One patient had a high level of anti-cardiolipin antibody which could not be blocked by S2.9. These results indicate that a mouse monoclonal antibody which reacts with an idiotope in the antigen-binding region of a naturally-occurring phospholipid antibody also defines a common idiotype of anti-cardiolipin antibodies in patients with autoimmune disease.     

The H3 anti-phospholipid idiotype is found in patients with systemic lupus erythematosus (SLE) but not in patients with syphilis.

The human H3 idiotype, defined by a mouse monoclonal antibody S2.9, is commonly found in patients with SLE where it is correlated with the amount of anti-cardiolipin antibodies. No correlation between the amount of anti-cardiolipin antibody and the H3 idiotype is found in patients with syphilis. Using the S2.9 antibody, serum from each of 10 patients with SLE and eight patients with syphilis was separated into H3-bearing and H3-negative fractions. Comparison of the partition of anti-cardiolipin antibody in these two groups of patients revealed that much of the anti-cardiolipin antibody (44-91%) was found in the H3+ fraction in patients with SLE; in patients with syphilis, virtually none of the anti-cardiolipin antibody was H3+. In patients with SLE, the H3+ fraction contained both IgG and IgM and antibodies of both kappa and lambda light chains. The H3+ fraction was polyspecific and frequently reacted with dsDNA.     

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 VHVL 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.     

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.     

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.     

Idiotypic expression of antibodies to retinal S-antigen in experimental autoimmune uveoretinitis.

Retinal S-antigen (S-ag), found in the rod photoreceptors of the eye, is a potent autoantigen that is commonly involved in inflammatory eye disease leading to blindness in man. Antibodies, induced in the experimental model by immunizing rats with S-ag purified from porcine retina, were used to prepare heterologous rabbit anti-idiotypic antibodies. The binding of the four rabbit anti-idiotypes to S-ag antibodies was partially inhibitable by porcine S-ag but not by ovalbumin. The idiotypic determinants were localized to the heavy chains by Western blotting with the anti-idiotypes. The presence of the idiotype recognized by the rabbit anti-idiotype was assessed in antisera from various species containing antibodies to S-ag. All rat sera from animals undergoing experimental autoimmune uveoretinitis by immunization with S-ag from porcine or bovine retina contained antibodies that react to varying degrees with the rabbit anti-idiotype. The intraspecies nature of the idiotypic determinants recognized was demonstrated by the fact that none of the anti-idiotypes showed any reactivity with rabbit or murine antisera to S-ag from porcine, bovine or human retina or to human autoantibodies to S-ag from patients with inflammatory eye disease. Thus, all private and recurrent idiotypic determinants induced in rats by immunization with S-ag appear to be restricted to that species.     

Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals.

The cross-reaction between anti-DNA and anti-cardiolipin IgG antibodies and its relation to the standard test for syphilis was studied with sera and monoclonal antibodies derived from human patients and mice with systemic lupus erythematosus (SLE). Syphilitic sera of humans and rabbits infected with the spirochete Treponema pallidum were also tested in this study. In addition, rabbits were immunized with ssDNA and cardiolipin and the cross-reactions of the induced antibodies were studied in two different assay systems. The results of these experiments suggest: that the anti-DNA and anti-cardiolipin IgG autoantibodies in SLE sera constitute separate antibody populations and, therefore, cardiolipin cannot play a role in the induction of immune response to DNA in SLE; that in immunized experimental animals there is a significant level of cross-reaction between anti-DNA and anti-cardiolipin-the detection of this cross-reaction depends on highly amplified solid phase assay systems which measure low affinity antibodies and that there is no correlation between the activity of syphilitic sera in the serologic test for syphilis and their binding to pure cardiolipin-this implies that cardiolipin may not be the dominant ingredient in this test as previously proposed.     

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.     

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-beta2-glycoprotein I (beta2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to 'pure' phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific 'pure' anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein-Barr virus infection. However, anti-beta2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.     

Characterization of variable-region genes and shared crossreactive idiotypes of antibodies specific for antigens of various influenza viruses

Several syngeneic monoclonal anti-idiotypic antibodies were obtained against PY206, a monoclonal antibody specific for X-31 (H3N2) influenza virus hemagglutinin. This idiotype was found in the sera of BALB/c mice immunized with various influenza viruses. Adsorption experiments indicated that the PY206 Id was borne by antibodies specific for viral hemagglutinin (HA) and/or neuraminidase (NA). This idiotype was identified on other monoclonal antibodies specific for various influenza HAs (H3 and H1). Study of the variable-region (V) genes of these monoclonal antibodies showed that its expression is independent of variable kappa (VK)21 light-chains and that the heavy-chains of the strongly idiotype-positive hybridomas derive from either the variable heavy (VH) J558 or VH 7183 family. Finally, Western blot analysis demonstrated that PY206 idiotypic determinants are located exclusively on the heavy chain.     

Detection of a circulating gastrointestinal cancer antigen in sera of patients with gastrointestinal malignancies by a double determinant immunoassay with monoclonal antibodies against human blood group determinants.

Monoclonal antibodies (MoAbs) produced against determinants A and B of the human ABO blood group system and against the Lea and Leb determinants of the Lewis (Le) blood group system detected these determinants on molecules released by cultured cells of human colorectal, gastric and/or pancreatic carcinoma (Ca) but not by a variety of other cells maintained in culture. Circulating Le antigen could be demonstrated in sera of patients by inhibiting the binding of MoAbs to a target preparation. A double determinant radioimmunoassay (DDIA) was then developed to detect the association of blood group determinants with a previously defined gastrointestinal cancer antigen (GICA). The DDIA with the anti-blood group and anti-GICA antibody was in some cases more sensitive in detecting GICA in sera than using the anti-GICA MoAb alone. Of 55 sera from patients with primary and early recurrent colorectal carcinoma (CRC), 10 (18%) were scored positive in the DDIA using only anti-GICA MoAb. When MoAb binding to a determinant on Leb and on H, type I, was used as first antibody in DDIA followed by anti-GICA MoAb 11 additional sera were reactive, increasing the percentage of positive sera to 38. Using the same combinations of MoAbs, the sensitivity of detection of GICA was only slightly improved from 63 to 66% in sera of patients with advanced CRC. The number of false positive sera from patients with non-malignant gastrointestinal diseases or from healthy donors remained at low levels when anti-blood group determinant antibodies were used together with anti-GICA MoAb. The results indicate that DDIAs with MoAbs against different blood group determinants and tumour associated antigens can improve the detection of circulating antigens in patients with early stage cancer.     

Anti-mitochondrial type 5 antibodies and anti-cardiolipin antibodies in systemic lupus erythematosus and auto-immune diseases.

Twenty sera from patients with systemic lupus erythematosus (SLE) and high titre of IgG anti-cardiolipin antibodies (ACA) were studied in order to evaluate the prevalence of anti-mitochondrial type 5 antibodies (AMA 5). None of these sera were found to be AMA 5 positive but five of 18 were positive for VDRL. Twenty sera from patients with AMA 5 were studied in order to evaluate the prevalence of ACA: only six of 20 were positive for ACA. In contrast to this finding, 15 of the 20 sera positive for AMA 5 were also positive for VDRL (P less than 0.001). The six sera positive for ACA and AMA 5 were absorbed with cardiolipin micelles. This absorption eliminated the ACA activity but not the AMA 5 activity. Despite the clinical similarities between the two groups of patients with AMA 5 or ACA, these data suggest that patients with AMA 5 and patients with ACA belong to two different subsets of SLE or SLE-like syndromes and that AMA 5 antigen is different from cardiolipin.     

Anti-mitochondrial type M5 and anti-cardiolipin antibodies in autoimmune disorders: studies on their association and cross-reactivity.

In a series of 42 positive sera, anti-mitochondrial type M5 antibodies (AMA-M5) were found most frequently in patients with SLE (24) and SLE-like syndromes. Patients with AMA-M5 displayed a higher prevalence of thrombocytopenia, thrombosis, biological false positive seroreactions for syphilis, lupus-like anticoagulant activity and anti-cardiolipin antibodies in comparison with a group of 43 SLE AMA-M5 negative patients. The strong association between anti-phospholipid and AMA-M5 antibodies cannot be explained entirely by cross-reactivity between these two groups of antibodies, as indicated by absorption experiments and studies using affinity purified antibody preparations. However, cardiolipin liposomes were able to reduce partially the titres of AMA-M5 sera, suggesting that a small population of AMA-M5 antibodies exists that cross-reacts with cardiolipin. The existence of this population was further substantiated by our demonstration that an IgM monoclonal antibody, from a patient with Waldenström''s macroglobulinaemia, displayed both anti-cardiolipin and AMA-M5 activity, and AMA-M5 activity was completely inhibited by cardiolipin.     

Lupus anti-DNA antibodies bearing the 8.12 idiotype appear to be somatically mutated

Anti-DNA antibodies in systemic lupus erythematosus (SLE) sera were analyzed using an antiidiotype designated 8.12 which recognizes a determinant on lambda light chains highly expressed in SLE sera. Eight of ten normal individuals had peripheral blood lymphocytes which produced high-titered 8.12-positive antibodies, following transformation with Epstein Barr virus, implying that the 8.12-reactive sequence originates in the germline gene (GLG). Of 58 SLE sera, 32 contained elevated titers of 8.12-reactive antibodies. Twenty-three of these sera had 8.12-reactive anti-DNA antibodies, suggesting a strong correlation between 8.12 idiotype and DNA binding. Moreover, 20 of 26 8.12-reactive IgG antibodies and only 4 of 10 8.12-reactive IgM antibodies bound DNA (P<0.05). These observations strengthen our previous findings in myeloma sera that DNA binding is associated with IgG isotype in the 8.12 idiotype system and suggest that the acquisition of anti-DNA reactivity in antibodies bearing the GLG idiotype 8.12 is achieved by somatic mutation, a feature of an antigen-driven response.     

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.     

Nature of anti-hapten antibodies arising after immune suppression of a set of cross-raactive idiotypic specificities

Anti-p-azophenylarsonate (anti-Ar) antibodies arising in strain A mice immunized with keyhole limpet hemocyanin-Ar have previously been shown to exhibit strong idiotypic cross-reactions. The appearance of the cross-reacting idiotype is suppressed by prior administration of antiidiotypic antibody, but a high titer of anti-Ar antibodies lacking the idiotype is produced in response to the antigen. The present studies indicate that these antibodies do not share idiotype with anti-Ar antibodies arising in other suppressed or nonsuppressed mice of the same strain. Strain A mice therefore produce at least 2 populations of anti-Ar antibodies, one showing intrastrain cross-reactions and the other being unique to each mouse. The results emphasize the great diversity of anti-hapten antibodies produced within an inbred strain. Suppression persisted when 9 weeks were permitted to elapse between administration of antiidiotypic antibody and the first challenge with antigen. This gives further evidence that suppression takes place at a central level and not through simple neutralization by antiidiotypic antibody of anti-Ar antibodies bearing the cross-reactive idiotype. Suppression of idiotype is maintained for a total of at least 5 months.     

Monoclonal anti-cardiolipin antibodies bind to DNA

BALB/c mice immunized with the phospholipid, cardiolipin, produced anti-cardiolipin and anti-DNA antibodies. Seven hybridomas derived from spleen cells of the cardiolipin-immunized mice produced cardiolipin-binding monoclonal antibodies that also bound to the polynucleotides DNA, poly(dT), and poly(I). The seven cardiolipin-induced monoclonal antibodies shared idiotypic determinants with a high frequency idiotypic marker of spontaneously expressed anti-DNA autoantibodies of lupus-prone MRL-lpr/lpr mice. The monoclonal antibodies presumably bound to phosphodiester phosphate groups that occur in both polynucleotides and phospholipids. The results imply that production of anti-DNA autoantibodies does not require immunization by DNA.