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7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3-30, V3-30.3 and V3-33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3-11 and V3-7 genes; D12 also reacts with some V3-43 gene Ig. We show here, by site-directed mutagensis, that the lysine at position 57 in the complementarity-determining region 2 (CDR-2) of the V3-30 gene product is crucial for epitope recognition by all three anti-VH3 MoAbs. Further analysis of the amino-acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR-2. Comparing the efficiency of 7B4 reactivity with VH3 gene-encoded human Ig indicates that amino-acid position 4 in the frame region 1 (FR-1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3-30, V3-30.3 and V3-33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)-2, SIV and HTLV-4. We also report that other tetrapeptide sequences found on the 56-59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV-1 isolates. The possible significance of these observations is discussed.

Mutational analysis was used to determine the structural basis for the binding of the murine anti-idiotypic (anti-Id) monoclonal antibodies (MoAb) G6 and G8 to VH1-encoded antibodies. The MoAb G6 binds a cross-reactive idiotope present on the heavy (H) chains of immunoglobulins (Ig) encoded by 51p1-related gene segments, but not those encoded by hv1263-related gene segments. Gene segments 51p1 (DP-10) and hv1263 differ by only four amino acids; three in complementarity-determining region 2 (CDR2), and one in framework region 3 (FR3). The MoAb G8 also binds 51p1-related sequences, although it is undetermined whether G8 can bind hv1263-related sequences. In order to localise the Ids recognized by MoAbs G6 and G8 on 51p1-encoded antibodies, recombinant antibodies containing H-chain mutants were expressed in insect cells. Idiotypic analysis on the expressed recombinant proteins definitively localised the reactivity in each molecule. These studies should be important in the structural appreciation for critical serological reagents.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecipitation and immunoblotting, the IgM antibodies 103-7E and 106-10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31-9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103-7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to participate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non-protein surface antigens also contribute to the pathogenesis of the disease.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.

The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9- and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgG1 and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss- or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None of the eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5- B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five of the eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) of the IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.

We have sought to determine whether rheumatoid factors (RF) produced in rheumatoid arthritis (RA) were different from physiological RF produced in normal, healthy adults. RF-secreting clones were established following Epstein-Barr virus (EBV) stimulation of peripheral blood lymphocytes. Ten RF-secreting clones were established from seven RA patients and 16 from six healthy controls. All monoclonal RF (MRF), except two in each group, were monoreactive and ten of these were shown to have low to medium affinity for IgG,Fc, irrespective of their origin. A majority (74%) of the MRF bound to protein A, indicating that genes of the VHIII family were preferentially used for synthesizing these autoantibodies. The expression of cross-reactive idiotypes (CRI) by the MRF did not allow distinction between those derived from RA patients and controls. The VHI-associated CRI G8 and VHIII-associated CRID12 were expressed at low frequency in both panels of RF. These CRI have been shown to be expressed at high frequency in RF paraproteins. However, the idRQ idiotype was expressed within both panels of RF. A possible distinction between polyreactive and monoreactive MRF appeared to be light chain usage since all (four) polyreactive RF used lambda chains while the normal kappa/lambda ratio was observed for monoreactive RF. The frequency of EBV-activated cells secreting IgM bearing CRI or secreting RF was determined and showed that CRI expression occurred with a higher frequency than did RF, suggesting a dissociation between CRI expression and RF activity.

A human hybridoma stably secreting IgA rheumatoid factor (RF) was produced by cell hybridization with peripheral blood lymphocytes of a patient with rheumatoid arthritis. The RF was of the IgA1 isotype with kappa-light chains and was useful for standardization or specificity controls in class-specific RF assays. RF activity was detected only when the IgA molecular were in a polymeric state, and could be measured by enzyme linked immunosorbent assay as well as in conventional agglutination based tests. The RF had the modified Ga fine specificity described previously for several RFs and for protein A. The immunoglobulin V genes used were isolated and sequenced. The light chain was encoded by the VkIV gene rearranged to Jk2; compared to the published VkIV germ line gene there was 90% nucleotide homology. The heavy chain gene used belonged to the VHI family and was rearranged to JH4. Comparisons with published sequences revealed 90% homology with the recently characterized VH gene expressed by RF-TS3, a rheumatoid synovia RF hybridoma.

Human IgM rheumatoid factors have been classified into major (Wa) and minor (Po) idiotypically cross-reactive families on the basis of reactivity with polyclonal anti-idiotypic antisera. Extensive structural studies have revealed that RFs from the Wa CRI family have homologous light chains. The structural basis of this CRI however is not known. In this study we have defined a conformational idiotope requiring the association of the VH and VL of a number of RF from the Wa CRI family for its expression and which thus comprise part of the Wa CRI. A murine monoclonal antibody, G8 specific for the idiotope reacted with 67% of RFs from the Wa CRI family. G8 also reacted with one IgG2K paraprotein with unknown specificity suggesting that the idiotope per se does not confer RF reactivity. Comparative studies of G8 expression relative to previously described sequence-dependent heavy and light chain associated idiotopes suggest that G8 recognises a separate determinant. Furthermore, the G8 idiotope is found on RFs expressing the VHI subgroup of heavy chain. Study of G8 expression in polyclonal IgM in normal sera demonstrated that the idiotope is widely expressed in the population. However, significantly higher levels of IgM bearing G8 were detected in the sera of patients with rheumatoid arthritis.

Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated "sequence-dependent" reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity.

Monoclonal rheumatoid factors (MRF) of the Wa idiotypic family have been studied for Fc gamma epitope specificity and the expression of idiotopes recognized by murine monoclonal antibodies. The majority of MRF recognized the Ga antigen or a closely similar iso-allotypic epitope expressed on IgG1, 2, 4 and IgG3.G3m(s.t.) proteins. MRF having the iso-allotypic specificity expressed a similar profile of V-region sub-group and idiotypic serological markers. One protein, Kas, whilst recognizing a distinct Fc gamma epitope also expressed these V-region markers and was highly homologous, from primary sequence analysis, with a MRF of Ga specificity. We conclude that these MRF recognize overlapping non-identical epitopes possibly including the same immuno-dominant residue and consequently demonstrate restricted V-gene usage.

Two mouse hybridoma antibodies, G6 and H1, with specificity for idiotypic determinants on the heavy chain of human IgM-RF have been produced. Idiotopes recognized by G6 and H1 were expressed on 5/12 and 2/12 RF paraproteins from the Wa idiotype group respectively, but not on paraproteins lacking rheumatoid factor (RF) activity. Inhibition experiments demonstrated that G6 and H1-specific epitopes were located on (or close to) the antibody binding site of RFs, and measurable on the heavy chain but not their light chains. However, it was concluded that the quaternary structure of the antibody-binding site was important for the optimal expression of both idiotopes. The idiotope recognized by G6 was also detected on polyclonal RFs in unfractionated sera from 8/14 patients with rheumatoid arthritis (RA). The results suggest that the G6 idiotope is a highly conserved determinant on the antibody-binding site of RF. These monoclonal antibodies will allow characterization of RF clonality in RA.

A new method has been developed to determine the specificities of polyclonal rheumatoid factors (naturally occurring antibodies which react with human Fc gamma) (RF) found in sera from patients with rheumatoid arthritis. In this method, monoclonal anti-Rh(D) antibodies of known IgG isotype and allotype are bound to erythrocytes and then act as the target IgG antigen for RF in a direct haemagglutination test. Using two monoclonal anti-D antibodies of the IgG3 isotype and G3m(21) allotype, which were cloned from different donors, we found that a large number of rheumatoid sera reacted with both these G3m(21) proteins. In contrast reactivity of rheumatoid sera with polyclonal anti-D of the G3m(21) allotype in the direct haemagglutination test was rare. A strong correlation was found between reactivities to both G3m(21) monoclonal anti-D antibodies but not with a monoclonal anti-D antibody carrying the alternative allele, namely G3m(5). Haemagglutination inhibition experiments using human paraproteins of known IgG isotype and allotype provided some additional evidence that this method can detect RF with specificity for the G3m(21) allotypic determinant or a related allotypic determinant in polyclonal rheumatoid sera. When each patient's autoantibody response was related to their Gm phenotype, we found that the frequency of reactivity for G3m(21) monoclonal anti-D antibodies was significantly greater in patients negative for G3m(21) than in patients positive for the G3m(21) allotype. IgM preparations from patients' sera were dissociated at acid pH but no 'hidden' antibodies were found. We suggest trans-placental sensitization as one of several possible interpretations of this finding.