A rabbit antiserum detects a VH J558 subgroup marker highly expressed among anti-alprenolol antibodies

A rabbit antiserum raised against anti-alprenolol mAb 14C3 detects common antigenic determinants (ADC3) in 10 out of 14 anti-alprenolol mAb that use different germ-line VH and/or Vk genes. The anti-14C3 antiserum binds only to H chains in immunoblots, therefore suggesting that at least part of the ADC3 determinants may be encoded by H chain V region genes. Analysis of VH gene family usage among the anti-alprenolol mAb reveals that the expression of ADC3 correlates with utilization of VH genes that belong to the J558 gene family, regardless of the JH, Vk, and Jk genes. To determine whether the ADC3 determinants are general V region markers or whether they are unique to anti-alprenolol antibodies, we have extended our analysis to a random panel of antibodies that also use VH genes of the J558 family. Among 23 mAb of various specificities, 14 react with the anti-14C3 antiserum in immunoblot and in ELISA, irrespective of antibody specificity. Adsorption of the antiserum on one of these positive antibodies results in a loss of reactivity toward both anti-alprenolol and unrelated antibodies. Therefore, several but not all antibodies that use a J558 VH gene also express the complete set of epitopes defining ADC3. These results strongly suggest that ADC3 are markers of a subset of J558 VH gene products. The anti-14C3 antiserum may thus constitute a "serologic probe" for identification of a VH gene subgroup from the J558 gene family.     

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.     

The immune response towards beta-adrenergic ligands and their receptors. VI. Idiotypy of monoclonal anti-alprenolol antibodies

Four murine monoclonal antibodies specific for alprenolol, a synthetic beta-adrenergic ligand, with different binding properties towards alprenolol and other beta-adrenergic antagonists and agonists (as described in a previous report) were used to induce anti-idiotypic responses in rabbits and mice. Three of the rabbit anti-idiotypes inhibited, and one increased the binding of tritiated dihydroalprenolol to the Ab1 against which they were induced. The syngeneic mouse anti-idiotypes all had an inhibitory effect on the ligand binding to their corresponding Ab1. Cross-reactivity tests of the xenogeneic and syngeneic anti-idiotypes were positive only for two monoclonal anti-alprenolol antibodies. Cross-reaction could be shown neither on a panel of 15 other monoclonals, nor on polyclonal anti-alprenolol antibodies of the BALB/c and the C57BL/10 mice. These results suggest that the immune response against alprenolol results in antibodies with mostly private idiotypic determinants. Moreover, the properties of the anti-idiotypic response against the same monoclonal antibody seem to be different according to the species used for anti-idiotypic induction.     

Molecular analysis of heavy and light chains used by primary and secondary anti-(T,G)-A--L antibodies produced by normal and xid mice

The primary (1 degree) antibody response to (T,G)-A--L shows limited heterogeneity, consisting mostly of side chain-specific antibodies that bind GT and that express the TGB5 idiotype (Id). The secondary (2 degrees) response is very diverse: antibodies that bind the backbone A--L constitute a third of the response, and a high proportion of the side chain-specific antibodies do not bind GT and are TGB5 Id-. To provide a molecular basis for understanding this difference in repertoire expression, we analyzed the Ig genes used by heavy and light chains of 1 degree and 2 degrees side chain-specific anti-(T,G)-A--L hybridoma antibodies (HP). Southern blot restriction analysis and nucleotide sequence analysis of the expressed genes used by three TGB5 Id+ 2 degrees HP showed usage of three different VH genes in two VH gene families (36-60 and J558), different D segments, and two different Vk1 genes (the Vk1A and Vk1C subgroups). Thus, antibody heterogeneity in the 2 degrees response is contributed by combinatorial diversity of distinct germ-line genes. Nucleotide sequence analysis of the expressed genes used by TGB5 Id+ 1 degree HP showed use of highly homologous VH genes in the J558 VH gene family and highly homologous Vk1A genes. The majority of TGB5 Id+ 1 degree HP from different donors gave similar heavy and similar light chain gene rearrangements by Southern blot restriction analysis, after correction for known or potential J region differences. The combined nucleotide sequence and Southern blot restriction analysis data suggest that most 1 degree B cells use the same or very similar VH and Vk genes, i.e., the 1 degree response is paucigenic. Different D segments were used by the TGB5 Id+ 1 degree and 2 degrees HP that were sequenced, and there was no apparent correlation between TGB5 idiotypy and VH, D gene, or JH gene usage. However, all TGB5 Id+ HP sequenced used highly homologous genes from the Vk1 group. Expression of a Vk1 light chain correlates with, but is not sufficient for, TGB5 idiotypy, because one GT-binding, TGB5 Id- HP was found to use a Vk1C subgroup light chain. By Southern blot and nucleotide sequence analysis, the Vk genes used by two TGB5 Id+ 2 degrees HP from xid mice are highly homologous, if not identical to the Vk1A gene(s) used by 1 degree and 2 degrees Id+ HP from wild-type mice.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.     

The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations

We have determined the nucleotide sequences of the expressed VH and Vk genes from 13 secondary (2 degrees) hemagglutinin (HA) (Sb) specific hybridomas derived from a single mouse. These antibodies share an Id, H37-68 (68Id) that dominates the 2 degrees HA(Sb) response in this mouse, but is rare or absent from 2 degrees responses of other mice. We find that these antibodies derive from five clones. The H chains of these antibodies are encoded by a single VH gene joined to a variety of DH and JH genes. The length of complementarity-determining region (CDR) 3 and sequence of the D-J junction are restricted, suggesting selection on CDR3 of the H chain. The L chains are more diverse. In the presented examples, they are encoded by the Vk21C and Vk21E genes and a Vk9 gene, and are joined to Jk1, 2, or 4. Each antibody is extensively mutated. The nature and distribution of the mutations suggests that 68Id-producing cells have been selected by Ag, although there are differences regarding the domain (VH, Vk, or both) in which mutations were selected. The implications of these findings on the idiosyncratic nature of the 68Id antibody response to HA(Sb) are discussed. There are two unusual characteristics regarding somatic mutation in these hybridomas. Whereas the expressed VH and Vk21 genes appear to have accumulated mutations at a high rate (1 to 1.5 x 10(-3)/base pairs/division, the expressed Vk9 genes appear to have accumulated mutations at a 5 to 15-fold lower rate than the expressed VH genes in the same cells. There is also a surprisingly high number of parallel silent somatic mutations in the VH genes, of which all but one are clustered to a 28-bp region in framework region 2 and CDR 2-encoding segments. The probability that this could have occurred by a random mutational process is essentially zero.     

Immunoglobulin diversity: analysis of the germ-line VH gene repertoire of the murine anti-GAT response.

A cDNA clone was constructed from a mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) BALB/c monoclonal antibody heavy chain. Its sequence, covering codons -5 to 162 and therefore encompassing the complete V-D-J region, was determined. Surprisingly, the sequence of the VH gene-encoded region was almost identical with that of the BALB/c VH anti-HNP (4-hydroxy-3-nitrophenyl) acetyl VH region, suggesting that the same VH germ-line might be used to encode two heavy chains contributing to antibodies of discrete specificities. A specific VH probe was derived and annealed to Eco RI and Bg1 II restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains that differ in their H chain allotypes. Under stringent conditions, only a few bands were identified by Southern blotting. The different patterns observed suggest that the VH anti-GAT repertoire differs between these strains even though their various anti-GAT antibodies express the same public idiotypic specificities.     

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.     

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.     

The variability, arrangement, and rearrangement of immunoglobulin genes

The multiplicity of heavy-chain variable-region (VH) genes in mouse and human DNA has been estimated using a mouse heavy-(H) chain cDNA clone. We found about 10 hybridization components in mouse DNA and about 20 components in human DNA. Cross-hybridization studies of variable region (V) genes indicate that these components represent the numbers of genes within the VH subgroups of each of these species. The arrangement and rearrangement of the H-chain gamma subclasses have been studied in order to assess possible mechanisms of the H-chain switch. Evidence has been found for rearrangement events involving the gamma 2a and gamma 2b constant-region (CH) genes in DNA from cells making IgG2a and IgG2b respectively. In addition we found that cells making IgG2a lack detectable genes for gamma1 and gamma 2b. Both sets of observations are discussed in relation to H-chain diversity and the switch.     

Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species

The lambda x, a new mouse Ig lambda L chain, is produced by rearrangement of the V lambda x, J lambda 2, and C lambda 2 gene segments. The V lambda x amino acid sequence is as divergent to other V lambda as to Vk gene sequences. Additionally, its third hypervariable region (CDR3) is four amino acids longer than those of all other variable gene segments of murine L chain. We have cloned and sequenced the germ-line V lambda x gene and found that the unexpected CDR3 length is encoded by the V lambda x gene. Junctional diversity is prevented by a TAA termination codon localized at the V lambda x 3' extremity. Moreover, we show a striking conservation of the V lambda x sequence in various mammalian species. Portions of the V lambda x sequence display more than 70% of nucleotide sequence identity with rabbit and human variable regions. These results suggest that V lambda x predated the divergence of mammalian species.     

Antibody engineering for the analysis of affinity maturation of an anti-hapten response.

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-bearing anti-NP antibodies which by itself leads to an increase of hapten-binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.     

Germ-line affinity and germ-line variable-region genes in the B cell response

The predominance of germ-line genes in IgM expression was evaluated from the nucleotide sequences of mRNA, derived from 10 hybridoma cell lines, coding for the VH and VL regions of anti-5-dimethylaminonaphthalene-1-sulfonyl (anti-Dns) IgM antibody. At least six germ-line VH gene segments distributed among four families are used in this response. Seven of the 10 independently rear-ranged VH genes were identified as germ line, with the other three possibly germ line. In all of them the D and JH portions retained the germ-line sequences of the D and JH segments from which they were derived. Maximum diversity was found in the D segments and the use of noncoded nucleotides at the VH-D and D-JH junctions. Of the eight cell lines expressing the lambda light chains, all were germ line and involved the three subtypes. Maximum affinity for the homologous ligand was found among the seven cell lines identified as expressing germ-line gene segments. Thus any somatic mutation among the remaining 3 cell lines did not provide enhanced affinity and the observed affinity of each cell line can be described as germ-line affinity. It is further suggested that the anti-Dns selectivity of the IgM antibodies is associated primarily with the CDR3 regions.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Restricted Ig variable region gene expression among Ly-1+ B cell lymphomas

The majority of the characterized Ly-1+ B cell lymphomas of B10.H-2aH-4bp/Wts origin (the CH series) bear surface Ig related by Ag specificity or idiotype or both. To determine the genetic basis for these structural similarities, we have sequenced the VH and VL region genes expressed by 10 CH lymphomas, and have compared their VH and V kappa gene rearrangements by Southern blot analysis to one another and to those of four other CH lymphomas. Sequence analysis identified only five different VH, and seven different VL genes, and indicated that these V genes are essentially unmutated. CH lymphomas which express the identical VH gene share at least one idiotope. Thus, the basis for shared idiotype and specificity is due in most cases to the use of the same V gene. This restriction in V gene expression is not due to the preferential use of V genes of any particular VH family or VL group, as the expressed V genes belong to four different VH families and four V kappa groups, and include V lambda 1 and V lambda 2. We hypothesize that Ag selection accounts for the restriction in V gene usage among CH lymphomas.     

A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin

Approximately 50% of the primary antibody response of BALB/c mice to the A/PR/8/34 influenza virus hemagglutinin is directed to the Cb site, one of the four major antigenic regions of the molecule. To determine the structural basis of the anti-Cb site response, we have examined the paratypic and genetic diversity exhibited by a panel of 24 primary and 4 secondary response mAb specific for this antigenic region. Reactivity pattern analysis demonstrated 20 distinct fine specificities among these antibodies, and V region gene sequence analysis showed that they are encoded by 17 different VH gene segments from 6 VH gene families and 14 different VK gene segments from 6 VK gene groups. Despite this overall diversity, many of the antibodies can be placed in a limited number of sets based on the shared expression of VH and/or VK genes. One set contains antibodies encoded by a single gene of the VK4/5 group in combination with one of two closely related genes from the J558 VH family. This set accounts for half of the Cb site-specific primary response hybridomas, indicating that the representation of the various anti-Cb site B cell specificities during the primary response to A/PR/8/34 influenza virus is not uniform. The preferential participation of B cells expressing this VH/VK combination is largely responsible for the dominance of anti-Cb site antibodies in the primary anti-hemagglutinin response.     

Genetic diversity of murine rheumatoid factors

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.     

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.