Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.     

Autoantibody-encoding kappa L chain genes frequently rearranged in lambda L chain-expressing chronic lymphocytic leukemia

Patients with kappa L chain expressing chronic lymphocytic leukemia (CLL) frequently have leukemia cells reactive with a murine mAb, designated 17.109. Raised against a monoclonal IgM rheumatoid factor autoantibody, this mAb recognizes a major kappa-L chain-associated cross reactive Id, designated 17.109-CRI. Molecular studies reveal that the 17.109-CRI in CLL is a serologic marker for expression of a conserved kappa L chain V region gene (V Kappa gene) of the V Kappa 3 subgroup, designated Humkv325. We isolated an upstream gene fragment of Humkv325 to examine for Ig gene rearrangements of this and other closely related V Kappa 3 genes by Southern analyses. Consistent with Humkv325 encoding the 17.109-CRI, we find that the genomic DNA from all 17.109-reactive leukemia cell populations have gene rearrangements that are detected using this probe. In addition, we observe V Kappa 3 gene rearrangements frequently in the genomic DNA of lambda L chain-expressing leukemia cells. Of the genomic DNA from 33 lambda-L chain-expressing CLL samples, 8 (24%) had additional nongerm-line bands detected with the Humkv325 probe. Consistent with these bands representing Ig gene rearrangements, the additional band in each but one sample also hybridized with probes specific for the J Kappa region and/or the kappa-deleting element. Using the polymerase chain reaction (PCR), we examined the genomic DNA from all lambda L chain-expressing CLL for V Kappa 3 gene rearrangements to J Kappa and/or Kde. PCR on each DNA sample with V Kappa 3 gene rearrangements detected by Southern analysis generated gene fragments that hybridized specifically with oligonucleotides corresponding to framework or CDR of the Humkv325 gene. Nucleic acid sequence analyses of representative samples confirmed that these DNA contained abortive Humkv325 gene rearrangements. PCR for rearranged V Kappa 3 genes in the DNA of other lambda-L chain-expressing CLL either did not generate any PCR product or produced fragments that failed to hybridize with all Humkv325 oligonucleotide probes. Nucleic acid sequence analyses of the latter demonstrated that these represent abortive V Kappa gene rearrangements involving another conserved V Kappa 3 gene, designated Vg. These studies indicate that Humkv325 and Vg frequently may undergo Ig gene rearrangement independent of their expression. As such, the frequent use of Humkv325 in CLL may be secondary, in part, to an enhanced propensity of this V Kappa 3 gene to undergo genetic rearrangement during B cell ontogeny.     

Structural similarities in the kappa light chains of human rheumatoid factor paraproteins and serum immunoglobulins bearing a cross-reactive idiotype

The monoclonal antibody (MoAb) 17.109 recognizes a cross-reactive idiotype (CRI) associated with the light chains of Waldenstrom's macroglobulins with rheumatoid factor (RF) activity. The MoAb also reacts with a proportion of IgM-RF molecules from the sera of rheumatoid arthritis and primary Sjogren's syndrome patients, and from the sera of seropositive normal human subjects. In the present experiments, we used affinity chromatography to purify the 17.109 CRI-positive immunoglobulin from serum and have analyzed the isolated material by Western blotting. The purified 17.109 CRI-positive material from the sera of rheumatoid arthritis patients, Sjogren's syndrome patients, and normal subjects contained exclusively kappa light chains, and had demonstrated RF activity. In every case the 17.109 CRI-positive isolates reacted with antibodies against synthetic peptides corresponding to both the conserved second and third complementarity-determining regions (CDR) of the monoclonal kappa IgM-RF paraprotein Sie. The binding was inhibited specifically by the free peptides in solution. The antipeptide antibodies did not react appreciably with unfractionated human immunoglobulin. The data establish that the 17.109 CRI-positive immunoglobulin from diverse human sera have similar or identical second and third light chain CDR. These results suggest i) that the MoAb 17.109 identifies the protein product of a single or a very few V kappa genes, ii) that the ability to make kappa light chains with the 17.109-associated variable region is widespread in the human population, and iii) that the 17.109-defined kappa variable region segment is associated with IgM-RF autoantibodies.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.     

Identification of a new V kappa gene family that is highly expressed in hybridomas from an autoimmune mouse strain

We have identified a new murine V kappa family that contains five to seven members, one member of which encodes the L chain V region of an anti-dsDNA antibody produced by a BALB/c hybridoma, C8.5. The cloned C8.5 V kappa gene exhibits highest homology with a human V kappa gene that was cloned from a nonproductive rearrangement but has never been seen in an expressed repertoire. Because this family was first identified in an autoantibody, we studied its expression in an autoimmune mouse strain. This V kappa family is expressed in 20% of hybridomas from NZB mice.     

Convenient plasmid vectors for construction of chimeric mouse/human antibodies

Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and V kappa genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and J kappa probes, respectively, and such fragments can be isolated in lambda-EcoRI and lambda-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG 1 gpt contains human C gamma 1 and Ecogpt genes, and only one EcoRI site upstream of the C gamma 1 gene; pSV2-HC kappa neo contains human C kappa and neo genes, and only one HindIII site upstream of the C kappa gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a V kappa J kappa gene are inserted into pSV2-HC kappa neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Two V kappa germ-line genes related to the GAT idiotypic network (Ab1 and Ab3/Ab1') account for the major subfamilies of the mouse V kappa-1 variability subgroup

V kappa Ig germ-line genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA. Three different clones that strongly hybridized with a V kappa-GAT-specific probe were completely characterized and sequenced. All three genes exhibited common characteristic features in their sequences encompassing the 5' to the 3' noncoding region, with coding sections 95% homologous. A comparison with other V kappa genes shows that the size of the first intron is variability subgroup specific. Moreover, a direct correlation exists between the size of this intron and the entire length of the coding region. Nucleotide sequences of these genes were compared with V kappa chains expressed at the Ab1 and Ab1' levels of the GAT idiotypic network: Ag----Ab1----Ab2----Ab3 (Ab1'). K1A5 and K5.1 genes account for V kappa chains in Ab1 and Ab1' hybridomas, respectively. The high conservation of Ab1' sequences in light chain was also recently reported for the heavy chains, suggesting that immunization with Ab2 (anti-idiotypic) antibodies preferentially stimulates the direct expression of germ-line genes. K5.1 and K1A5 genes belong to the V kappa-1 variability subgroup and encode, without any amino acid substitution, V kappa domain in myeloma TEPC 105 and MOPC 467, which are V kappa-1A and V kappa-1C subgroup prototypes, respectively. These genes are extensively used in different mouse strains and in a number of antibodies of discrete specificities, such as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA.     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.     

Molecular characterization of a supratypic cross-reactive idiotype associated with IgM autoantibodies

Neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphomas (SLL) frequently express surface Ig reactive with the mouse mAb, Lc1. Raised against a human monoclonal IgM with rheumatoid factor activity, Lc1 detects a major cross-reactive Id (CRI) present on the H chain of many monoclonal IgM autoantibodies. In contrast to other major autoantibody-CRI investigated to date, we note that the Lc1-CRI is expressed by subpopulation of cells in the germinal centers, as well as in the mantle zones, of secondary human B cell follicles. To examine the molecular basis for Lc1 expression, we used the polymerase chain reaction to isolate the functionally rearranged Ig VH genes of monoclonal Lc1-reactive B cell populations from six unrelated patients with CLL or SLL. Although the neoplastic B cells from most patients with CLL or SLL express the CD5 surface differentiation Ag, the lymphoma cells from one patient with SLL were CD5-negative. We find that the Lc1-reactive cells from each cell population have Ig rearrangements involving a VH gene of the VH4 subgroup. However, the VH4 genes rearranged in different Lc1-reactive tumor populations may originate from at least two disparate germ-line VH4 genes. Also, in contrast to the CD5-positive tumor populations, we find evidence for intraclonal diversity in the functionally rearranged VH4 genes of the CD5-negative SLL. Collectively, this study discerns a degeneracy in the VH4 genes that can encode the Lc1 CRI, indicating the term "supratypic cross-reactive idiotype" may best describe the specificity of the Lc1 mAb. Also, this study suggests that expression of CD5 may delineate categories of B cell SLL that differ in their relative rates of constitutive Ig V gene somatic mutation.     

Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease

The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells.     

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.     

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.     

Dissociation of expression of two rheumatoid factor cross-reactive kappa L chain idiotopes in rheumatoid arthritis.

mAb 6B6.6 and 17.109 recognize two distinct kappa III L chain cross-reactive idiotopes (CRI) present on approximately 2/3 of IgM kappa rheumatoid factor (RF) paraproteins. To determine the distribution of these two CRI and their relationship to each other among polyclonal RF, sera from 86 RA patients and 49 controls were analyzed for the presence of 6B6.6- and 17.019-bearing RF by using sensitive solid phase ELISA. Levels of CRI(+) RF were estimated by using 6B6.6(+) and 17.019(+) RF standards. Detectable levels (greater than or equal to 195 ng/ml) of CRI(+) RF were rarely present in the control sera (8% for 6B6.6; 0% for 17.109), whereas 59% of RA sera contained measurable CRI(+) RF (48% for 6B6.6; 35% for 17.109; 21% for both). Where detected, CRI(+) RF were present in low concentrations (6B6.6: 1.21 +/- 1.56 micrograms/ml; 17.109: 1.20 +/- 1.15 micrograms/ml) and constituted a small fraction of the total IgM RF in these sera (6B6.6: 0.9 +/- 2.2%; 17.109: 0.8 +/- 0.9%). There was no correlation between either RF CRI and levels of IgM RF (r less than 0.1, p greater than 0.5). Levels of 6B6.6(+) RF did not correlate with 17.109(+) RF (r = -0.11, p = 0.47). In selected sera that contained both RF CRI, it was possible to selectively absorb 6B6.6(+) RF. Taken together, these data indicate the mutual independence of these two RF CRI among polyclonal RF and suggest the presence of distinct regulatory mechanisms governing their expression. Moreover, that these two CRI constitute a small proportion of polyclonal RF, in contrast to their striking predominance among monoclonal RF paraproteins, argues for the importance of other germline VL genes contributing to polyclonal RF production or the presence of extensive somatic mutation among polyclonal RF in RA.     

Complete amino acid sequence of the heavy-chain variable region from an A/J mouse antigen-nonbinding monoclonal antibody bearing the predominant arsonate idiotype

The 1F6 hybridoma protein, exhibiting the predominant cross-reactive idiotype (CRI) associated with the immune response to p-azophenylarsonate in A/J mice but failing to bind the hapten arsonate, was elicited following immunization with rat anti-CRI [Wysocki, L.J., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The dissociation of idiotype and antigen binding in this hybridoma provides an opportunity to determine structural features involved in antigen binding and idiotypic sites. The complete heavy-chain variable region (VH) amino acid sequence was obtained by automated Edman degradation of the intact chain and fragments due to CNBr cleavage, trypsin digestion, mild acid hydrolysis, and carboxypeptidase A digestion of a CNBr fragment. Comparison of the CRI+ arsonate-nonbinding 1F6 sequence with the CRI+ germ-line VH gene sequence reveals that the 1F6 heavy chain differs from the germ-line-encoded amino acid sequence at seven positions within VH [Siekevitz, M., Gefter, M. L., Brodeur, P., Riblet, R., & Marshak-Rothstein, A. (1982) Eur. J. Immunol. 12, 1023-1032]. The 1F6 VH appears to arise from the CRI+ germ-line VH by somatic mutation at at least seven amino acid residues, each of which could be due to a single nucleotide base change. The diversity (D) gene-encoded segment of 1F6 is similar to that of the CRI+ antigen-binding hybridoma 36-65 except for two amino acid substitutions. Further, the idiotype (CRI) is preserved despite use of a JH4 gene segment in 1F6 as compared to JH2 in all CRI+ arsonate-binding hybridomas examined to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-reactive idiotype family observed in the phthalate-specific B cell repertoire of adult BALB/c mice: diversity of IgM compared with IgG monoclonal anti-phthalate antibodies

A highly conserved clonotype has been identified within the repertoire of B cells specific for the negatively charged hapten phthalate. The prototype of this phthalate-specific clonotype is a primary-response hybridoma (2E9) that produces a mu,kappa anti-phthalate antibody. The 2E9 monoclonal antibody was found to share idiotypic determinants with several other independently-derived mu,kappa and gamma 1,kappa anti-phthalate monoclonal antibodies and with a significant proportion of conventional anti-phthalate antibodies derived from all of the BALB/c mice immunized with phthalate-keyhole limpet hemocyanin. Competitive RIA analysis of the 2E9 idiotypic relatedness between primary and secondary response antibodies was consistent with the hypothesis that the primary response mu,kappa antibodies represent a conserved germ-line product, whereas the secondary response to gamma 1,kappa antibodies reflect somatic variants of the 2E9 clonotype. Further analysis with a site-specific anti-idiotype reagent suggests that the idiotypic differences between mu,kappa and gamma 1,kappa monoclonal antibodies occur at positions outside of the combining site. Fine specificity analysis of the monoclonal antibodies expressing the 2E9 cross-reactive idiotype (CRI) also supports this hypothesis. Seven to 35% of the anti-phthalate antibodies after a single immunization with phthalate-KLH and 1 to 10% of the antibodies after a second immunization express the 2E9 CRI. The 2E9 CRI was also found in several other strains of mice, and its expression was associated exclusively with anti-phthalate antibodies.     

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.     

Myeloma with multiple rearranged immunoglobulin kappa genes: only one kappa gene codes for kappa chains

In many myelomas more than one kappa gene is rearranged (2-5). We are reporting here the results of studies undertaken to determine whether all the rearranged genes are expressed. It was found that in the myeloma NS-1 three different rearranged kappa genes exist. In a subline of NS-1 and several hybridomas produced by fusion of mouse spleen cells with NS-1 it was found that production of NS-1 kappa chains was correlated with the presence of one of the three kappa genes. Loss of this "expressed" gene eliminated the synthesis of the NS-1 kappa chains, loss of one of the other two rearranged kappa genes did not. It is hypothesized, that allelic exclusion (20) of kappa genes generally operates by the functional rearrangement of one kappa gene; other rearrangements are relatively frequent, at least in myelomas, but mostly they are nonfunctional and thus scrambled antibody molecules do not arise.