Heterogeneity of anti-idiotypic antibodies to anti-DNA antibodies in humans.

Serum antibodies in some patients with systemic lupus erythematosus (SLE) were found to have specificity to idiotypes (Id) of 0-81 (human monoclonal anti-single-stranded DNA (ssDNA) antibody) but not to Id of NE-1 (human monoclonal anti-double-stranded DNA (dsDNA) antibody) or pooled human IgM. The interaction of the antibodies and 0-81 was blocked by the co-existence of free ssDNA. Some of SLE sera also showed preferential binding to Id determinants of NE-1, which included the antigen-binding sites of the dsDNA antibody. Some other SLE sera reacted with both Id of 0-81 and NE-13. Thus, there was heterogeneous population among human anti-Id autoantibodies to anti-DNA antibodies. The anti-Id activity was commonly detected in inactive SLE sera, and less frequently in normal controls, suggesting some regulatory role for anti-Id antibodies in the production of autoantibodies.     

Increased presence of common systemic lupus erythematosus (SLE) anti-DNA idiotypes (16/6 Id, 32/15 Id) is induced by procainamide

Sixty-seven patients on treatment with procainamide were examined for the presence of two common idiotypes of anti-DNA antibodies (16/6 Id and 32/15 Id). These idiotypes have been shown previously to have clinical relevance in patients with systemic lupus erythematosus (SLE). An enzyme-linked immunosorbent assay (ELISA) with rabbit anti-Id antibodies revealed increased concentrations of the 16/6 Id and 32/15 Id in 25 (37%) and 16 (24%) patients, respectively. Five of eight patients with drug-induced lupus had elevated titers of both idiotypes. A high correlation (R=0.56,P<0.001 for 16/6 Id) was found between Id levels and anti-single-stranded DNA (ssDNA) antibody titers and between 16/6 Id titers and antihistone antibodies (IgG,R=0.43; IgM,R=0.25). It seems that procainamide, a component known to be associated with drug-induced lupus, may induce an increased production of common anti-DNA idiotypes in apparently normal subjects.     

Incidence of anti-DNA idiotype-positive cells in human peripheral blood

Human anti-DNA idiotype (Id)-bearing cells in peripheral blood were sought using mouse monoclonal anti-Id antibodies to human monoclonal anti-DNA antibodies. Pretreatment with acid pH or pronase P or preincubation in human serum-free medium markedly decreased the number of anti-Id-reactive cells. Pronase P-treated cells were able to bind to anti-Id once more after incubation for 18 hr at 37° C, indicating the resynthesis of internal idiotypic determinants on the cells. Antiidiotype-reactive cells retained the same idiotypes in their cytoplasma. The cells expressing anti-DNA idiotypes, termed 0-81 and NE-1, were detected in the circulation of most patients with active lupus nephritis but not in those of inactive systemic lupus erythematosus (SLE) and healthy subjects. The idiotype-positive cells occurred in up to 5–10% of B cells from some patients with SLE in the active stage but became undetectable in remission. A limited number of anti-DNA Id-positive cells responsible for anti-DNA production might be preferentially expanded during acute episodes of the disease in some patients with SLE.     

Evidence that a putative anti-idiotypic monoclonal antibody may actually be recognizing circulating immune complexes.

Murine monoclonal antibodies (mAb) reacting with affinity-purified antihistone antibodies (AHA) from serum of a patient with systemic lupus erythematosus (SLE) were obtained. One of them, 8B3, was initially considered to recognize idiotypic (Id) determinants in AHA since (a) it reacted with AHA but not with control IgG; (b) this reactivity could be inhibited using affinity-purified AHA, but not with control IgG or whole serum; (c) affinity-isolated 8B3+ antibodies showed antihistone activity and not other activities tested so far; (d) antihistone activity due to 8B3+, but not that of 8B3- from the same serum, could be fully inhibited by the presence of 8B3 mAb in the antihistone assay and (e) serum levels of 8B3 reactivity were higher than normal in SLE patients with AHA (56%), in contrast with SLE patients without AHA (6%). From these results it was deduced that 8B3 defined a cross-reactive Id shared by a subset of AHA in SLE patients. However, the present results suggest that (a) 8B3 mAb did not recognize AHA or Ig, but did recognize a 55 kDa histone-binding protein; (b) this 55 kDa protein was present free at low concentration in all human sera, but also associated with IgG in 8B3+ SLE sera and (c) these complexes are responsible for the false positive results in the antihistone assay as shown for DNA/anti-DNA complexes. Thus, mAbs recognizing the non-Ig moiety of circulating immune complexes may resemble anti-Id antibodies with features of the so-called epibodies. These immune complexes may be responsible for false positive results and caution should be exercised in the interpretation of results.     

Pathogenic anti-DNA idiotype-reactive IgG in intravenous immunoglobulin preparations.

This study was addressed to explore the reactivity of natural anti-idiotypes from commercial lots of immunoglobulins to several idiotypes (Ids), usually expressed by anti-DNA molecules in lupus nephritis. Eleven intravenous immunoglobulin (IVIG) preparations and nine (three polyvalent and six hyper-immune) intramuscular IgG were investigated for specific content of anti-DNA, anti-F(ab')2 and antibodies reacting with several anti-DNA IgG Ids. Two samples (nos 6 and 11) showed high reactivity with allogeneic F(ab')2 and with F(ab')2 of myeloma proteins bearing the anti-DNA Id 3I+ and the 8.12+. Since both 3I and 8.12 Id markers are known to characterize pathogenic anti-DNA IgG in systemic lupus erythematosus (SLE), anti-Id antibodies to these markers were obtained by absorbing the IVIG samples nos 6 and 11 to Sepharose columns coupled with pooled F(ab')2 fragments of 3I(+)-F4(+)-8.12(+)-myeloma proteins. Inhibition experiments showed that anti-8.12 Id-eluted IgG induced a selective suppression of the DNA-reactive antibodies derived from patients with active lupus nephritis to their substrate, suggesting the involvement of 8.12+ molecules in the SLE glomerular damage. Since 8.12+ anti-DNA are nephritogenic antibodies, the occurrence of anti-8.12+ Id in commercial IVIG may be of potential therapeutic relevance in modulating the pathogenic SLE Id network. Previous variable results of IVIG treatment in SLE, such as resolution of proteinuria or worsening nephritis, could be related to variable enrichment of different lots of IVIG in suppressive anti-pathogenic Id antibodies.     

Towards an idiotype vaccine against mammary tumors. Induction of an immune response to breast cancer-associated antigens by anti-idiotypic antibodies

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross-reacting antigens. B11 is a monoclonal IgG generated by the human-human hybridoma technique using axillary lymph node of breast cancer patients. 4.6/6 is a monoclonal IgG established by the mouse-human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti-idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.6/6. Following exhaustive adsorption on unrelated human-Ig, fetal calf serum and purification on B11 or 4.6/6 affinity columns, the anti-Id antibodies were shown to react specifically with their respective Id. The binding of these anti-Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/or HuMTV antigens. Rabbit anti-B11 and rabbit anti-4.6/6 anti-Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV-related antigens could be demonstrated. The sera of the mice contained anti-MMTV antibodies and delayed-type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected. Our results support the notion that anti-Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral-associated antigens.     

A peptide derived from a polyreactive monoclonal anti-DNA natural antibody can modulate lupus development in (NZBxNZW)F1 mice

In lupus-prone (NZBxNZW)F1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti-DNA antibodies. While these D23 Id+ antibodies were able to regulate anti-DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti-DNA antibodies and furthermore could participate in the lupus-like syndrome. To explore further the role of the D23 Id+ antibodies, we injected young B/W mice with a peptide corresponding to the VH CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA-reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti-DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.     

Private idiotypes on human polyclonal IgG anti-DNA antibodies are not expressed on coexisting IgM anti-DNA antibodies in systemic lupus erythematosus

Sharing of private idiotypes (Id) on human polyclonal IgG anti-double-stranded DNA (dsDNA) with coexisting IgM anti-dsDNA was investigated using rabbit (R) anti-Id raised against IgG anti-dsDNA. The R-anti-Id showed specificity to private Id in or near the antigen-binding sites. The R-anti-Id poorly bound to the immobilized enriched IgM anti-dsDNA preparation but significantly bound to IgG anti-dsDNA preparation by a direct-binding ELISA (0.020 OD vs 0.295 OD, respectively). The R-anti-Id poorly inhibited the binding of IgM anti-dsDNA to immobilized dsDNA but significantly inhibited the binding of IgG anti-dsDNA to dsDNA (6% vs 55% inhibition, respectively). This was confirmed by poor inhibition of binding of the R-anti-Id to immobilized IgG anti-dsDNA by the enriched IgM anti-dsDNA preparation (maximum of 26% inhibition at 50 micrograms/ml). Nonsharing of private Id between IgG and coexisting IgM anti-dsDNA may represent the idiotypic diversity of human anti-DNA antibodies secondary to the frequent occurrence of somatic mutation on anti-DNA antibody during class switching.     

Clonal frequency analysis of B cells producing pathogenic anti-DNA antibody-associated idiotypes in systemic lupus erythematosus.

In order to identify the mechanism responsible for autoantibody production in systemic lupus erythematosus (SLE), B cell repertoires associated with anti-DNA idiotypes were explored by a limiting dilution analysis using Epstein-Barr virus (EBV) transformation methods and ELISA spot assays. The frequencies of B cell clones producing antibodies to DNA and to conventional antigens, tetanus toxoid, dinitrophenyl, or keyhole limpet hemocyanin were higher in active SLE compared to those in inactive SLE and in normal subjects. In addition, there was a disproportionate increase in anti-DNA antibody- and anti-DNA idiotype (Id)-producing clones at the precursor cell levels as well as at the mature cell level. On the other hand, numbers of anti-Id clones against anti-DNA-Id, termed 0-81 Id, were markedly increased at inactive stages of the disease but not at active stages. These were confirmed by serial studies in some patients with SLE. These results support a two-step mechanism for autoantibody production, in which initial polyclonal activation is followed by an antigen-driven process, and indicate an alteration of the precursor B cell repertoire in SLE, which may also associate with a preferential expansion of anti-DNA clones.     

A Murine Monoclonal Anti-Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.     

Production and characterization of a human monoclonal anti-idiotype to anti-ribosomal P antibodies

Autoantibodies to ribosomal P protein (anti-P) are a specific hallmark of systemic lupus erythematous (SLE). Several authors found significant associations of anti-P antibodies with neuropsychiatric, hepatic, and renal disease. We now report the isolation by phage display of human anti-idiotype (Id) monoclonal antibody fragments as single-chain Fv fragment (scFv) against anti-P antibodies. The V gene repertoires were derived from the RNA obtained from the B cells of a SLE patient. Affinity-purified anti-P antibodies were used for the selection of bacterial clones producing anti-P-specific scFv antibody fragments and little reactivity with normal IgG and other IgG antibodies. The anti-Id antibody recognizes a public idiotope broadly cross-reactive with polyclonal anti-P antibodies and inhibited binding of anti-P to ribosomal P antigen in immunoassays and on Jurkat cells. The anti-Id scFv antibody fragment may have therapeutic implications in SLE. They may also be used as probes in the study of the structure of the idiotype.     

In vitro production of an anti-DNA idiotype by lymphocytes of normal subjects and patients with systemic lupus erythematosus

We investigated whether normal B cells can synthesize antibodies with an idiotypic marker that occurs with high frequency in anti-DNA antibodies of patients with systemic lupus erythematosus (SLE). This idiotype, Id16/6, has been found in the serum of patients with active SLE and in monoclonal anti-DNA antibodies derived from unrelated patients with the disease. We found that cultured lymphocytes of all normal subjects tested produced Id16/6 when stimulated by pokeweed mitogen (PWM). By contrast, lymphocytes from SLE patients produced Id16/6 even without mitogenic stimulation, whether or not they were obtained from patients in remission or relapse. Relapsed patients' lymphocytes spontaneously produced the highest levels of Id16/6 which was found in IgG and IgA, in addition to IgM. The majority of Id16/6 produced by PWM-stimulated lymphocytes from either normal subjects or patients in remission did not bind to nucleic acid. In relapse, however, the nucleic acid-binding proportion of Id16/6 rose substantially, indicating that the spontaneously activated B cells in active SLE differ from the subset of B cells that produce Id16/6 upon PWM stimulation. The findings suggest that the lupus Id16/6 family is conserved in normal individuals and it consists of two populations of antibodies with different antigenic specificities. The major set is not directed against nucleic acid antigens; its antigenic specificity is unknown and it dominates the Id16/6 family that appears after PWM stimulation. The other, minor set binds to nucleic acids and becomes prominent in clinically active lupus. These two sets of idiotypically related antibodies may be connected by an immunoregulatory network.     

The role of anti-idiotypic antibodies in the induction of experimental systemic lupus erythematosus in mice

We have recently reported the induction of experimental systemic lupus erythematosus (SLE) in mice by a human anti-DNA monoclonal antibody (mAb) that bears a common idiotype, the 16/6 Id. In the present report we investigated the role of the idiotypic network in the induction of experimental SLE by using a murine anti-idiotypic mAb specific for the 16/6 Id. This anti-idiotypic mAb induced experimental SLE similarly to the 16/6 Id. Thus, following immunization, in addition to 16/6 Id+ antibodies, the mice produced antibodies to various nuclear antigens: single-stranded DNA, double-stranded DNA, poly(I), poly(G), Ro, La, Sm and ribonucleoproteins. Similarly to the 16/6 Id-immunized mice, the mice injected with the anti-16/6 Id mAb exhibited elevated erythrocyte sedimentation rate and leukopenia. The murine anti-16/6 Id mAb was found to be more effective than the 16/6 Id, in causing earlier onset of proteinuria and renal damage. These results suggest that the idiotypic network and particularly anti-idiotypic antibodies specific for anti-DNA common idiotypes found in SLE, play an important role in the induction of SLE in mice.     

Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

The Importance of the Pathogenic 16/6 Idiotype in the Induction of SLE in Naive Mice

We have previously demonstrated the pathogenicity of the common anti-DNA idiotype designated 16/6 Id. Immunization of naive mice with the 16/6 Id induced SLE-like disease characterized by serological (e.g. anti-dsDNA and anti-Sm auto-antibodies), clinical (increased ESR, leucopenia and proteinuria), and pathological (16/6 Id deposition in kidneys) parameters. To elucidate further the role of the 16/6 Id in SLE induction the following studies were carried out: BALB/c mice were immunized with SA-1, a human anti-DNA monoclonal antibody carrying the 16/6 Id; TB-68, a mouse monoclonal anti-tuberculosis (TB) glycolipid, which binds dsDNA and carries the 16/6 Id; TB-72, a mouse monoclonal anti-TB glycolipid that binds DNA and does not harbour the 16/6 Id; and 4B4, a human anti-Sm antibody that carries the 16/6 Id. SLE was induced in BALB/c mice only when immunized with SA-1, TB-68, and 4B4, namely antibodies with diverse binding capacities albeit having the 16/6 Id. Our studies further support previous evidence on the pathogenic role attributed to the 16/6 Id in SLE, and suggest that SLE is most probably an idiotype-induced disease.     

Relation between lymphocytotoxic antibodies, anti-DNA antibodies and a common anti-DNA antibody idiotype PR4 in patients with systemic lupus erythematosus, their relatives and spouses.

Forty-two patients with systemic lupus erythematosus (SLE), 65 of their healthy relatives and 20 spouses were studied for the presence of lymphocytotoxic antibodies (LCA), anti-lymphocyte antibodies (ALA), antibodies to DNA and a common idiotype (Id) PR4. Seventy-one per cent of the patients had positive levels of LCA, and in 34% the PR4 Id was detected; normal levels were found in their families. Anti-PR4, an anti-Id, failed to block the lymphocytotoxic activity in those nine patients who both carried the Id and had LCA. This indicates that the Id was not present on LCA. There was no correlation between anti-DNA antibodies and LCA, suggesting that different mechanisms are involved in their expression.     

Ligand-binding and idiotypic cross-reactions between anti-DNA antibodies and antibodies to Klebsiella K30 polysaccharide in patients with systemic lupus erythematosus or rheumatoid arthritis

Antibodies to DNA and K30p were purified by affinity procedures and tested for their ability to cross-react with K30p and DNA, respectively. Anti-ssDNA antibodies were shown to react with solid-phase K30p and be inhibited in a dose-dependent manner by soluble ssDNA and K30p. Conversely, anti-K30p antibodies were found to bind immobilized ssDNA and be inhibited with soluble inhibitor. These results show that certain subpopulations of anti-ssDNA and anti-K30p antibodies overlap in their ligand-binding specificities. Idiotypic (Id) analysis showed that anti-K30p antibodies did not react significantly with an anti-Id reagent directed against a common anti-DNA framework Id (AM Id), thus clearly separating K30p-binding anti-DNA antibodies into an AM Id-negative population. When anti-DNA antibodies were probed with an anti-Id reagent directed against a framework Id present on anti-K30p antibodies (SP Id), substantial reactivity was found. Thus the SP Id identifies a subpopulation of antibodies capable of binding both K30p and DNA. These results show that a subset of anti-ssDNA antibodies cross-react with K30p antigen and are idiotypically related to a subset of antibodies reactive with K30p.     

A murine monoclonal anti-idiotype to anti-ribosomal P antibodies: production, characterization, and use in systemic lupus erythematosus

Overt anti-ribosomal P (anti-P) autoantibodies are restricted to a subset of systemic lupus erythematosus (SLE) patients, and are potentially pathogenic. Covert anti-P are detected in all other individuals. An idiotype (Id) network is nonoperational in those with overt anti-P, whereas it is functional in all others. The aim of this study was to produce a murine monoclonal (mAb) anti-Id to characterize the anti-P Id network in SLE. BALB/c mice were immunized with F(ab')(2) fragments of IgG anti-P from a patient with a broadly cross-reactive Id. One mAb was chosen (mAb41) that reacted preferentially to the immunogen. This IgG1 mAb bound comparably in ELISAs to affinity-purified anti-P from 11 SLE patients with overt anti-P. This binding was partially inhibited with ribosomal P antigen. In contrast, it did not bind to affinity-purified control autoantibodies, nor to normal human IgG. mAb41 inhibited anti-P binding to ribosomal P antigen in immunoassays and on Jurkat cells. No change was detected in patients' anti-P antibodies over time when mAb41 was used in Id-specific ELISAs. We conclude that mAb41 is an anti-Id that recognizes a public idiotope within the antigen-combining site of anti-P antibodies. Thus, it is analogous to its human counterparts, and potentially, would modulate the pathogenicity of anti-P autoantibodies in vivo.     

Age-dependent responsiveness to interleukin-6 in B lymphocytes from a systemic lupus erythematosus-prone (NZB x NZW)F1 hybrid.

The cellular mechanisms for the production of IgG anti-DNA antibodies were studied. Culture of T and B cells from old (NZB x NZW)F1 mice led to the production of IgG anti-DNA antibodies. We found that direct cell contact was partly necessary for the production of IgG anti-DNA antibodies. Fixation of the T cells showed that lymphokines were largely responsible for the antibody synthesis. Antibodies to mouse interleukin-6 (IL-6) inhibited the production of these antibodies in the T-B cell coculture. Human IL-6 could induce small "resting" B cells from the old (NZB x NZW)F1 mice to produce IgG anti-DNA antibodies in a dose-dependent fashion. The response was inhibited by an anti-human IL-6 monoclonal antibody. Large or small B cells from young (B/W)F1 mice or Balb/c mice were not induced by IL-6 to antibody production. Therefore, the capacity of the B/W mice to produce the IgG anti-DNA antibodies correlated with the ability of the B cells to respond to IL-6 and with the age at which the mice begin to have signs of the disease.     

Expression of inactive stage anti-dsDNA idiotypes on anti-ssDNA antibodies in a lupus patient during active stage of lupus cerebritis.

The possibility that idiotypes (Ids) defined on anti-double stranded DNA (dsDNA) antibodies during active and inactive stages of lupus (1/84 Id and 4/90 Id, respectively) were expressed on anti-DNA antibodies during a subsequent active period (9/90) of the disease was investigated in a lupus patient with lupus cerebritis. Using rabbit (R)-anti-Ids specific to 1/84 Id and 4/90 Id in inhibition assays, the 4/90 Id was shown to be expressed on the framework regions of anti-single stranded DNA (ssDNA) but poorly on co-existing anti-dsDNA antibodies of active (9/90) stage. The 1/84 Id was poorly expressed on both types of 9/90 anti-DNA antibodies. While the 9/90 anti-ssDNA significantly bound to immobilized ssDNA and several single-stranded polynucleotides, only ssDNA inhibited the binding of the anti-ssDNA to ssDNA, suggesting its monospecificity toward ssDNA. Western blot analysis following isoelectric focusing showed that a spectrotype pattern of 4/90 Id-positive 9/90 anti-ssDNA IgG was similar to that of the 4/90 anti-dsDNA, suggesting that they are of related clonal origin. The present study suggests the idiotypic heterogeneity of anti-DNA antibodies and the shift of antigen specificity within an idiotypically related anti-DNA population during exacerbation of the disease.