The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.     

A cross-reactive idiotype on anti-collagen antibodies in collagen-induced arthritis: identification and relevance to disease

Immunization of mice with type II collagen (CII) leads to the production of anti-CII antibodies and, in susceptible strains, to the induction of arthritis. Specifically purified anti-CII antibodies from arthritic DBA/1 mice were used to prepare a rabbit anti-idiotypic antiserum. This antiserum recognizes a cross-reactive idiotype (CRI) present on 20-25% of anti-CII antibodies from DBA/1 mice immunized with bovine CII. The CRI is not present on DBA/1 anti-trinitrophenyl, undetectable in normal Ig and not Igh allotype linked. The presence of this CRI was examined after antigen specific suppression of the anti-CII antibody response by intravenous administration of chick or bovine CII. While intravenous injection of bovine CII, prior to immunization with chick CII, greatly reduces both the incidence of arthritis and the anti-CII response, the fraction of anti-bovine CII which expresses the CRI is increased by this treatment. These findings suggest that the CRI characterizes a disease-unrelated fraction of anti-CII which recognizes bovine and chick CII, but probably not mouse CII. In addition, attempts at idiotypic regulation of arthritis incidence and antibody response by in vivo administration of anti-idiotypic serum also indicate that the CRI-bearing antibody is not important for the induction of arthritis.     

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.     

Detection and characterization of idiotype-specific enhancing cells generated in mice immunized with idiotype

Cellular interaction between MOPC-104E (M104E) cross-reactive idiotypic (CRI) antibody-producing B lymphocytes and lymphocytes generated by immunization with the relevant idiotype, M104E, was investigated. Adoptive transfer of M104E idiotype-primed and normal spleen cells into 600R x-irradiated syngeneic recipient mice resulted in striking enhancement of the M104E-CRI positive antibody response upon simultaneous immunization of recipients with dextran B1355S. The enhancement was not attributable to a simple additive effect but was due to synergistic cooperation between the two lymphocyte populations. This synergistic enhancement of the anti-idiotype immune cells producing CRI antibody was specific for MOPC-104E CRI, and was reproducible in an in vitro culture system. Because of the cellular characteristics of the enhancing cells, they were assumed to be B lymphocytes specific for the corresponding idiotype, since the activity was not abrogated by treatment with anti-Thy-1, anti-Lyt-1, anti-Lyt-2, or anti-brain-associated theta antisera plus complement, but was eliminated by means of a planning method using a rabbit-anti-mouse immunoglobulin-coated or idiotype-coated dish. The mechanisms of interaction between the CRI-positive B cells and anti-idiotypic B cells in response to the thymus-independent antigen dextran B1355S are discussed.     

Expression of three cross-reactive idiotypes on rheumatoid factor autoantibodies from patients with autoimmune diseases and seropositive adults

Approximately one-half of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors (RF] from unrelated individuals with cryoglobulinemia coordinately express three cross-reactive idiotypic antigens (CRI). The CRI are detected with: 1) monoclonal antibody 17.109, which recognizes a conformation-dependent CRI on K-light chains; and 2) two rabbit anti-peptide antibodies that react with primary sequence-dependent CRI (PSL2 and PSL3) corresponding to the conserved second and third K-chain complementarity-determining regions, respectively. In the present experiments, the structural features of polyclonal RF autoantibodies from diverse patients with rheumatoid arthritis and from those with primary Sj?gren's syndrome, and from seropositive elderly subjects without overt autoimmune diseases, were investigated with these three defined anti-CRI reagents. The pattern of expression of the CRI differed among patient groups. Only the RF autoantibodies from Sj?gren's syndrome patients frequently displayed all three CRI. However, the RF from nearly every subject tested, including patients with rheumatoid arthritis, were enriched in the primary sequence-dependent PSL2-CRI as compared to RF-depleted Ig from the same subjects. Amino acid sequence analysis of monoclonal IgM-RF indicates that PSL2-CRI-positive light chains probably represent the products of a single Vk gene. Therefore, a proportion of the polyclonal RF from different autoimmune states may represent somatic variants of this germ-line RF Vk gene which retain the PSL2 sequence as a common element.     

Immunoglobulin chain recombination among antidigoxin antibodies by hybridoma-hybridoma fusion

Conditions necessary for in vitro chain recombination of high affinity (10(9) to 10(12) M-1) antidigoxin monoclonal antibodies resulted in decreased affinity for both intact "native" and chain recombinant molecules. Chain recombination by somatic cell fusion was used instead to study the effects on antigen specificity and idiotypy of recombinants in which an homologous light (L) chain substituted for the parental L chain. The antidigoxin antibody 26-10 utilizes a VL sequence highly homologous to that of antibody 40-20, an antidigoxin antibody which uses a different VH gene than does 26-10 and lacks significant reactivity with an anti-26-10 idiotypic serum. The drug-marked antidigoxin cell line 26-10 (gamma 2a, kappa) and a drug-marked light chain producing variant of antidigoxin hybridoma 45-20 (lambda 1) which lacks both digoxin binding and idiotypy were fused. The fusion progeny (gamma 2a, kappa, lambda 1) which binds digoxin and is idiotype-positive, was selected for kappa loss (resulting in loss of digoxin and idiotype binding) and then fused with a heavy (H) chain loss variant of antidigoxin hybridoma 40-20 (kappa, digoxin nonbinding, idiotype negative). The resultant cell line CR-57 (gamma 2a, kappa, lambda) secretes antibodies which assemble the 26-10 H chain with both the 40-20 kappa-chain and the 45-20 lambda 1-chain. The affinity purified recombinant species consisting of 26-10 H chain and 40-20 kappa-chain expresses complete 26-10 idiotypic determinants. However, this recombinant antibody binds digoxin with decreased affinity and altered specificity relative to native 26-10. The binding specificity pattern nonetheless is most similar to the H chain donor. Amino acid and nucleotide sequence analyses of the respective light chains demonstrate six variable region differences between them, two of which are in complementarity-determining regions and the remainder in the framework. Hybridoma-hybridoma fusion provides an alternative to in vitro chain recombination for studying the contribution of chain combinational diversity to antibody diversity, antigen binding, and idiotypy.     

The NZB X SWR model of lupus nephritis. I. Cross-reactive idiotypes of monoclonal anti-DNA antibodies in relation to antigenic specificity, charge, and allotype. Identification of interconnected idiotype families inherited from the normal SWR and the autoimmune NZB parents

The incidence of lupus nephritis is low in autoimmune NZB mice, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. In a previous study we showed that anti-DNA auto-antibodies produced by the SNF1 mice were qualitatively different from those made by the NZB parents with respect to their isotype, charge, and antigenic specificity patterns. Here we studied idiotypic cross-reactions among the 65 monoclonal anti-DNA antibodies that were derived from four NZB and seven SNF1 mice. A library of 15 anti-idiotypic antibodies were prepared by immunizing rabbits with 15 monoclonal anti-DNA antibodies selected from the panel of 65. We identified 10 cross-reactive idiotype (CRI) families among this large collection of autoantibodies. Five of these CRI families were restricted to cationic anti-DNA antibodies that were exclusively of SNF1 origin, and the strongly cross-reacting members were predominantly IgG2b auto-antibodies with the allotype of the normal SWR parent. The cationic anti-DNA CRI families could be grouped into an interrelated cluster called the Id564 cluster. The other five anti-DNA CRI families were not restricted to any particular parental allotype or charge, although two of these CRI were shared exclusively by SNF1-derived autoantibodies and four of these CRI families could also be grouped into an idiotypically interrelated cluster called the Id512 cluster. In the case of seven out of the 10 CRI families, the idiotypic determinants detected were close to the antigen-binding site of the anti-DNA antibodies. The results indicate that the idiotypic repertoire of anti-DNA autoantibodies produced by the SNF1 mice is different from the NZB parents, and potentially pathogenic (cationic) antibodies produced by the SNF1 mice that are encoded by genes from the normal SWR parent can be identified as distinct CRI families. In the accompanying paper we demonstrate the role of these anti-DNA CRI families in the development of lupus nephritis.     

Immunoglobulin idiotype and anti-anti-idiotype utilize the same variable region genes irrespective of antigen specificity

Idiotypic determinants characterizing certain antibody specificities have been proven valuable structural and genetic markers in studies of antibody diversity and regulation. The heritable predominant idiotype associated with the response to p-azophenylarsonate in A/J mice consists of a set of highly homologous (greater than 95%) heavy and light chain variable region amino acid sequences probably arising by somatic mutation from one or a few V region genes. We examined a peculiar set of monoclonal antibodies that have been defined as CRI by serologic analysis, but that have no affinity for the hapten Ars. These antibodies were elicited by immunization with anti-CRI rather than by the conventional immunization with antigen. The amino acid sequences of the amino terminal half of the V regions of these anti-(anti-CRI) antibodies are indistinguishable from those of conventional Ars-binding CRI antibodies. Thus, Ars-binding CRI and Ars-nonbinding anti-(anti-CRI) are derived from similar or identical VH and VL genes.     

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.     

Immune responses during human Schistosoma mansoni. XVII. Recognition by monoclonal anti-idiotypic antibodies of several idiotopes on a monoclonal anti-soluble schistosomal egg antigen antibody and anti-soluble schistosomal egg antigen antibodies from patients with different clinical forms of infection

A monoclonal human anti-soluble schistosomal egg Ag(SEA) antibody (E5) that stimulates anti-Id T cells and is idiotypically represented in pools of immunoaffinity-purified human anti-SEA antibodies from chronic, generally asymptomatic, intestinal (INT) patients (AM1 and AM5) was used to raise several monoclonal anti-Id: 1C2, 1C6, 4A8, 4F9, and 2A7. Cross-inhibition between these anti-Id identified distinct idiotopes on E5. Anti-SEA preparations from schistosomiasis patients (AM1, AM5, and others) were tested for their inhibition of the E5/monoclonal anti-Id reactions, in competitive ELISA. In either the E5/4A8 or E5/1C6 ELISA system, anti-SEA from INT (AM1 or AM5) or hepatointestinal (HI) (AM7) patients were able to inhibit these reactions. However, anti-SEA antibodies from acute (AM9) or hepatosplenic (HS) (AM3 or AM8) patients did not express Id that were inhibitory in these systems. These results suggest that a relatively high proportion of INT and HI anti-SEA antibodies express a dominant cross-reactive idiotope (CRI) recognized by 1C6/4A8. This CRI is also easily detected in plasmas from individual INT patients. Anti-Id 1C2 reacted strongly with an Id in AM1, AM5, or AM7, but one which also occurred, to a lesser extent, in AM3, AM8, and AM9. Monoclonal anti-Id 4F9 and 2A7 reacted weakly with idiotopes expressed by antibodies from all patients, regardless of the clinical form of their infection. These observations indicate that anti-SEA antibodies from INT and HI, but not acute or HS patients express dominant, CRI that are identified by 1C6, 4A8, or 1C2 and are also expressed on the INT-derived anti-SEA mAb E5.     

Alteration of the B cell surface phenotype, immune response to phosphocholine and the B cell repertoire in M167 mu plus kappa transgenic mice

M167, mu plus kappa, transgenic mice have been analyzed for the expression of the transgene product as a cell surface, Ag-specific receptor and for their ability to respond to Ag. The vast majority of B cells in these H + L transgenics (97 to 99%) express large amounts of the transgene product on their surface and are capable of binding phosphocholine. A total of 4 to 30% of the B cells also express endogenous IgM and IgD H chain products. After immunization with phosphocholine (PC)-conjugated keyhole limpet hemocyanin, more than 1000 micrograms/ml of anti-PC antibody bearing the transgene IgMa allotype marker are produced. Surprisingly, significant amounts of anti-PC antibodies that express the endogenous, IgMb allotype, are also produced; however, these antibodies lack the T15-idiotype which dominates the anti-PC response in their nontransgenic littermate controls. The B cells producing these endogenous anti-PC antibodies also fail to switch to IgG anti-PC synthesis, whereas B cells producing anti-keyhole limpet hemocyanin antibodies readily undergo class switching. These last two observations may be due to the fact that the endogenous anti-PC antibody actually results from mixed mu a + mu b molecules in which the transgene encoded H and L chains are most likely responsible for the binding of PC. Thus, a switch of the endogenous isotype from mu b to IgG would result in a loss of specificity for PC in the IgG molecules produced using the endogenous VH-gene product(s), and mu a + gamma b hybrid molecules are not likely to be formed. This hypothesis is supported by the fact that the majority of (mu a + mu b) hybridomas have the mu b-allotype joined with a VH region other than the VH1 gene which is required for PC-binding and T15 idiotype expression.     

Comparison of variable region primary structures within an anti-fluorescein idiotype family

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.     

A new cross-reactive idiotype-defined family in the phthalate humoral immune response of mice. I. Linkage of VH-Xmp to IgCH allotype locus and mapping with respect to other known VH genes

A cross-reactive idiotype family was previously identified from a very large library of phthalate-specific hybridoma clones. The prototype of this idiotype family is the hybridoma, 2E9, secreting an IgM antibody with phthalate specificity. A portion of both primary and secondary anti-phthalate antibodies elicited in all BALB/c mice tested expresses the 2E9 cross-reactive idiotype. This idiotype has now been found in the anti-phthalate antibodies of several other inbred strains of mice (A/HeHa, DBA/2, and C3Hf/HeHa) tested but not in C57BL/6 mice. Anti-phthalate antibodies elicited from congenic mice BC.8, which express the same IgCH allotype as BALB/c mice but possess C57BL/6 genetic background, contain the 2E9 cross-reactive idiotype, whereas this idiotype is not expressed on the anti-phthalate antibodies derived from another congenic mouse CB.20, which expresses a C57BL/6 IgCH allotype and a genetic background of the BALB/c strain. These results indicate that the gene controlling the 2E9 idiotype is closely linked to the IgCH allotype locus. The 2E9 cross-reactive idiotype was also found in all of the F1 mice (BALB/c X C57BL/6) tested, and the level of expression of this idiotype in the F1 mice was quantitatively equivalent to the allotype/idiotype homozygous mice. The expression of the 2E9 idiotype in the phthalate repertoire has been followed in 12 different wild mouse populations. As expected, the 2E9 idiotype was observed in a large proportion of the wild mouse strains. Surprisingly, several examples of nonconcordance in the expression of idiotype and allotype were observed in these mice. One likely explanation for the linkage breakdown is a crossing over of the heavy chain constant and variable region gene complexes. In the SM/J inbred strain of mice, where such a crossover has occurred, nonconcordance between allotype and 2E9 idiotype expression was demonstrated. By using the recombinant inbred BXD strains of mice, the VH gene encoding the 2E9 idiotype has been mapped with respect to other known VH gene families. Relative to other VH genes the VH-Xmp is situated very close to the IgCH gene region.     

The majority of human monoclonal IgM rheumatoid factors express a "primary structure-dependent" cross-reactive idiotype

Genetic studies of human immunoglobulin variable regions have been hampered by the lack of anti-idiotypic antibodies that recognize specific heavy and light chain variable region sequences. Sixty percent of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors [RF]) from unrelated individuals share a cross-reactive idiotype (CRI) termed Wa. In previous experiments in which we used an enzyme-linked immunosorbent assay, we reported that a synthetic peptide (PSL2), corresponding to the second hypervariable region in the kappa light chain of a monoclonal IgM-RF (Sie), induced rabbit antibodies reactive with several RF paraproteins. In the present experiments, to avoid interference due to the human IgM-RF binding toward rabbit IgG, the reactivity of the anti-PSL2 antibody to the separated heavy and light chains of multiple IgM proteins and Bence-Jones proteins was assessed by the Western blot technique. The PSL2-induced anti-CRI reacted well with the separated kappa chains from 10 out of 12 IgM-RF, zero out of four light chains from IgM proteins lacking anti-IgG activity, and one out of six kappa Bence-Jones proteins. The results show that the PSL2-CRI is associated with RF and is not a kappa subgroup marker. Furthermore, a comparison of the reported light chain sequences of the PSL2-CRI-positive IgM-RF suggests that the majority of human IgM-RF light chains derive from a single germ-line VK gene or from a family of closely related VK genes that is highly conserved in the human population. Synthetic peptide-induced anti-CRI provide a potent tool for analyzing the genetic basis of CRI and abnormal autoantibody production in humans.     

Anti-angiotensin II anti-idiotypic antibodies bind to angiotensin II receptor

An anti-idiotypic serum from a rabbit immunized with one anti-angiotensin II (AII) monoclonal antibody (A25) was shown to identify a cross-reactive idiotope (CRI) shared by six anti-AII monoclonal antibodies, in addition to a binding site-associated private idiotope. This anti-idiotypic reagent bound to rat liver membranes bearing AII receptors; binding was abolished after pretreatment of the membranes with AII. In immunoblotting experiments with rat liver membranes, as well as with rat pituitary homogenates, a 63,000 +/- 2,000 dalton protein was revealed that co-migrated with the AII receptor. After purification by affinity chromatography on an immobilized CRI+-antibody (A41), anti-CRI antibodies could immunoprecipitate the hormone binding activity from detergent-treated rat liver membranes and still recognize the 63,000 dalton protein. In contrast, anti-idiotypic antibodies specific for the private idiotope failed to interact with the AII receptor. Similar results were obtained with a second anti-idiotypic serum produced by immunization with another CRI+ anti-AII monoclonal antibody (A22). The sharing of the CRI determinant between the AII receptor and anti-AII antibodies might account for the reactivity of anti-idiotypic antibodies towards the AII receptor.     

Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries

To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes.     

An unusual case of Waldenstr?m macroglobulinemia with half molecules of IgG in serum and urine

Immunochemical studies are described in an unusual case of Waldenstr?m's macroglobulinemia. Two monoclonal Igs (whole IgG1/kappa and IgG1/kappa half molecules) occurred in the serum in addition to the IgM monoclonal protein. Protein electrophoresis of the serum showed a monoclonal component in the gamma region, and the immunoelectrophoresis allowed detection of a monoclonal IgM/kappa and another abnormality represented by a double precipitin line in serum and urine, observed when antiserum anti IgG was used. The abnormal proteins were purified and further analyzed. The IgG-related proteins were whole four chains IgG monoclonal molecules, 1/2 IgG monoclonal molecules, composed of one heavy and one light chain, and residual polyclonal IgG. The half molecules were antigenically deficient with respect to normal IgG. The idiotypic analysis showed that the three monoclonal proteins shared idiotypic determinants. This patient had clinical and morphological findings of Waldenstr?m's macroglobulinemia and, as observed in other cases, the formation of half molecules was not associated with a distinct clinical syndrome.     

Activation of a functional idiotype network response by monoclonal antibody specific for a virus (M-MuLV)-induced tumor antigen

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.     

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

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

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.