Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.     

Analysis of the anti-alpha 1 leads to 3 dextran response with monoclonal anti-idiotype antibodies

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.     

Amino acid substitutions in VH CDR2 change the idiotype but not the antigen-binding of monoclonal antibodies to alpha(1----6)dextrans

An idiotype defined by mAb and polyclonal antibodies to 10.16.1, an anti-alpha(1----6) dextran was previously reported to be expressed on most BALB/c anti-alpha(1----6)dextrans with groove-type sites and to involved CDR3 and probably CDR2. By comparing amino acid sequences of VH and VL derived from cDNA of idiotype+ and idiotype- anti-alpha(1----6)dextran hybridoma proteins, an idiotope was assigned to VH CDR2. Substitution of phenylalanine for leucine at residue 52 in CDR2 coupled with amino acid changes at either residue 58 or residues 57 and 60 abolished expression of this idiotype without affecting Ag binding.     

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.     

Anti-immunoglobulin antibodies. II. Expression of individual and cross-reactive idiotypes on syngeneic and homologous anti-idiotype antibodies

Syngeneic anti-(anti-Id) antibodies were prepared against BALB/c anti-A48Id antibodies, BALB/c anti-460Id monoclonal antibodies, and A/J anti-J558 IdI monoclonal antibodies. With these anti-(anti-Id) antibodies we identified cross-reactive idiotypes on syngeneic and homologous anti-A48Id and anti-460Id antibodies. By contrast, tbe idiotypic determinants of A/J anti-J558 IdI monoclonal antibodies were not shared by other syngeneic, homologous, or xenogenic anti-J558 IdI or IdX antibodies. These results suggest that idiotype-antiidiotype reactions that serve as regulatory controls within the immune system are characteristic for each particular antigen system, strain, or species and that such interactions make the system self-limited with respect to the whole antild repertoire.     

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.     

Two families of monoclonal antibodies to alpha(1----6)dextran, VH19.1.2 and VH9.14.7, show distinct patterns of J kappa and JH minigene usage and amino acid substitutions in CDR3.

Nine groove-type mAb to alpha(1----6)dextran were cloned and sequenced. Together with previous reports from this laboratory, the VH and VL of 34 mAb have been sequenced, in which 10 VH19.1.2 and 11 VH9.14.7 combined with the V kappa-Ox1 gene to form two major families of anti-alpha(1----6)dextrans. The same D minigene (DFL16) was used by all VH19.1.2 and VH9.14.7 mAb; however, the patterns of JH and J kappa usage are quite different. VH19.1.2 mAb used only JH3 and J kappa 2, whereas VH9.14.7 mAb used three JH (JH1, JH2, and JH3) and all four active J kappa (J kappa 1, J kappa 2, J kappa 4, and J kappa 5). Relative uniformity in the lengths of VH CDR3 and the junctional sequences is seen in both families. Some mAb from different mouse strains share common structural features. The differences in idiotypic specificities and in the amino acid sequences suggest that VH19.1.2 and VH9.14.7 may differ in the conformation of CDR1 and CDR2. Combining with V kappa-Ox1 gene to generate groove-type combining sites to the single site-filling epitope of alpha(1----6)dextran, the two VH chains may require certain conformations of CDR3. Whether such conformational requirements influence the choice of J minigenes, the selection of the length of VH CDR3 and the sequences at junctions, are discussed.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Generation of helper T cells that recognize a cross-reactive idiotype through a network mechanism.

T cells that recognize the cross-reactive idiotype expressed on the heavy (H) chain of M104E (IgM, lambda 1) were induced in BALB/c mice by immunization with Dextran B-1355. T cells derived from mice immunized with 1 mg of Dextran B-1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or lesser amounts of Dextran B-1355 did not. BCL1Id, which had an immunoglobulin isotype identical to M104E, did not induce proliferation of the T cells. These T cells also proliferated against J558 (IgA, lambda 1) which shared the cross-reactive idiotype of the anti-alpha (1----3) glucosidic linkage antibody with M104E on the H chain. The T cells proliferated more efficiently against F(ab')2-104E, Fab-104E and H104E, the H chain of M104E, than against intact M104E. The T cell proliferative response against the idiotype on M104E or even H104E required macrophages as antigen-presenting cells (APC) and the response was inhibited when APC were treated with NH4Cl or chloroquine, inhibitors of antigen processing. Moreover, anti-CD4 antibody or anti-Ia antibody inhibited the proliferative response. These results indicated that anti-idiotypic T cells of the helper type, which recognized a cross-reactive idiotype associated with Ia molecules in processed form, could be induced physiologically through a network mechanism.     

V region carbohydrate and antibody expression

N-Linked carbohydrates are frequently found in the V region of Ig H chains and can have a positive or negative effect on Ag binding affinity. We have studied a murine anti-alpha(1-->6) dextran V(H) that contains a carbohydrate in complementarity-determining region 2 (CDR2). This carbohydrate remains high mannose rather than being processed to a complex form, as would be expected for glycans on exposed protein loops. We have shown that the glycan remained high mannose when murine-human chimeric Abs were produced in a variety of cell types. Also, when another carbohydrate was present in CDR1, CDR2, or CDR3 of the L chain, the V(H) CDR2 glycan remained high mannose. Importantly, we found that when the anti-dextran V(H) CDR2 replaced CDR2 of an anti-dansyl V(H), the glycosylation site was used, but H chains were withheld in the endoplasmic reticulum and did not traffic to the Golgi apparatus. These results suggest that inappropriate V region glycosylation could contribute to ineffective Ab production from expressed Ig genes. In some cases, a carbohydrate addition sequence generated by either V region rearrangement or somatic hypermutation may result in an Ab that cannot be properly folded and secreted.     

Hypervariable region peptides variably induce specific anti-idiotypic antibodies: an approach to determining antigenic dominance

Rats and rabbits were immunized with synthetic peptides corresponding to the VH hypervariable regions of several alpha (1----3) dextran-specific antibodies from mice to study the efficacy of synthetic peptides in the generation of site-specific anti-idiotypic reagents. Synthetic peptides were made which corresponded to the HV1, HV2, and HV3 hypervariable regions of the heavy chain of M104 (IdX+, IdI-(M104)+), HV2 of HDex 14 (IdX-), and HV3 of J558 (IdX+, IdI(J558)+). The HV1(M104) peptide sequence is found in all dextran-specific immunoglobulins examined and the HV2 and the HV3 peptides span the regions implicated in IdX and IdI expression, respectively. Sera from many rabbits and rats indicate that all five peptides are immunogenic. Antisera to HV3 peptides show excellent binding to the appropriate myeloma proteins, with antisera to the HV3(M104) peptide demonstrating little binding to proteins that differ in HV3 sequence. Antisera generated against HV1(M104) and both HV2 peptides show weak cross-reaction to the appropriate proteins; however, these sera are not idiotypic because they cross-react with immunoglobulins with very limited sequence homology. Thus, it appears that some, but not all synthetic peptides from hypervariable regions will be capable of generating antisera with useful anti-idiotypic specificities. This may reflect differences in the intrinsic antigenicity of various parts of the VH region.     

Effects of V lambda gene diversity on generation of antigen-specific lymphocytes

Previous studies have shown that dextran B1355 (DEX)- and (4-hydroxy-3-nitrophenyl) acetyl (NP)-coupled antigens triggered, respectively, BALB/c and C57BL/6 (B6) lymphocytes in which the V lambda 1 gene and a specific VH gene (VHDEX and VHNPb) have functionally rearranged. In this paper, we studied whether the closely-related V lambda 2 gene can be utilized in association with these VH genes to generate antigen-specific lymphocytes. We found that the VHDEX gene was restrictedly utilized by the V1 lambda 1 gene to generate anti-DEX lymphocytes, and in contrast, both the V lambda 1 and V lambda 2 genes were utilized together with a VHNPb germline gene to form anti-NP lymphocytes. Southern blot and DNA sequencing of an anti-NP hybridoma confirmed that the germline form of the (186-2) VHNPb gene can be used in association with either the V lambda 1 or V lambda 2 genes.     

A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.     

Expression of a cross-reactive idiotope on naturally occurring murine antibodies against 3-fucosyllactosamine, levan, and dextran

We recently identified a cross-reactive Id (6C4) that is expressed on the H chain of many BALB/c mAb against the 3-fucosyllactosamine (3-FL) determinant, Gal(beta 1-4) (Fuc(alpha 1-3] GlcNAc-R. The VH segments of seven mAb that we recently sequenced are encoded by VH441, which also encodes VH segments of antibodies against galactan, levan, and dextran. To analyze the expression of the 6C4 Id on naturally occurring anti-carbohydrate antibodies, we isolated 6C4+ antibodies by affinity chromatography from pools of normal BALB/c serum. Approximately 20 to 30% of antibodies against 3-FL and levan, and all antibodies against dextran, were removed from the sera by passage over a column containing mAb 6C4. Absorption of the eluate with 3-FL beads removed anti-3-FL antibodies but not anti-dextran or anti-levan. The expression of a cross-reactive Id on naturally occurring antibodies against several carbohydrate Ag suggests that these antibodies may participate in an Id network. We also reported previously that BALB/c mice have naturally occurring anti-3-FL antibodies and respond well to immunization against this determinant, whereas C57BL/6 mice do neither. To examine the role of the Igh-C allotype in the regulation of the anti-3-FL response, we studied congenic strains of BALB/c and C57BL/6 mice. Both congenic strains produced anti-3-FL antibodies in response to immunization, but only C.B-20 mice exhibited naturally occurring antibodies. These data suggest that the naturally occurring and elicited antibody responses against 3-FL are differentially regulated.     

Genetic mechanisms for dominant VH gene expression. The VHB512 gene.

A total of 37 mAb with reactivity for dextran B512 have been studied; 30 of them were products of independent rearrangements and 21 made use of the same VH gene, the VHB512 gene. These results unambiguously established that the immune response to dextran in the high responder mouse strain C57BL/6 was restricted. Idiotypic determinants are located all over the Ig V region. Many but not all Id described so far can be ascribed to protein structures encoded by VH or VL gene segments. The expression of the major Id, 17-9 Id, in C57BL/6 was not absolutely correlated with the expression of the dominant VHB512 gene in the same mouse strain. Inspection of amino acid sequences of the CDR3 of idiotypic positive and negative clones suggested that idiotypic structures may be associated with the expression of Tyr at position 95 and Phe or Leu at position 96 in the H and L chains, respectively. Therefore the indiscriminate use of idiotypic markers to characterize VH genes and the relevance of idiotypic regulation in VH gene expression are questioned. Id-positive and Id-negative clones displayed similar affinity values for dextran, indicating that idiotypic and binding structures were probably separated. The exchange of Asp65 for Gly65 in one of the clones reduced affinity for dextran, suggesting the involvement of CDR2 in dextran binding. The dominant expression of VH genes can be explained by somatic and/or genetic mechanisms. Because somatic mechanisms such as idiotypic regulation or selection based on affinity for dextran did not seem to influence the expression of the VHB512 gene we favor a genetic alternative. We discuss a model based on the distance between VH genes and D and JH elements. This model is compatible with somatic and genetic regulation in other systems and provides a new theoretical approach to the understanding of immune VH dominance and low responsiveness.     

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.     

IgA anti-dextran B1355 responses.

Injection of mice bearing the Ig-1a allotype with dextran B1355 results in an IgM antibody response that is generally regarded as thymus independent. Moreover, the antibody is directed to alpha[1,3] determinants on dextran B1355 and shares cross-reacting idiotypic determinants with a lambda 1 IgA (J558) myeloma protein as well as a lambda 1 IgM (MOPC 104E) myeloma protein. In this study, we show that BALB/c (Ig-1a) mice injected with dextran B1355 produced highly significant IgA anti-dextran responses with specificity directed to the alpha[1,3] epitope. Kinetics of the IgA anti-dextran response in BALB/c mice paralleled kinetics of the IgM response. However, the magnitude of the IgA response was markedly T cell dependent and age dependent.     

Igh restriction of the anti-alpha (1-3) dextran response: polyclonal B cell activators induce the synthesis of anti-alpha (1-3) dextran antibodies in lymphocytes from Igha mice only

The only strains of mice which are able to synthesize lambda 1-bearing antibodies in response to alpha (1-3) Dextran are those expressing the Igha allotypic haplotype or those having an Igh V region identical to Igha mice. The experiments reported here were designed to investigate whether the nonresponsiveness of mice which do not express the Igha haplotype is a consequence of an absence of a polyclonal B cell receptor for the alpha (1-3) Dextran TI-antigen. B cells of several mouse strains were stimulated with polyclonal B cell activators (PBA) known to either stimulate non-overlapping B cell subsets or to stimulate B cells at different stages of maturation, i.e., lipopolysaccharide, Nocardia delipidated cell mitogens and alloreactive T helper cells. Whereas all three PBA induced B cells from Igha mice to secrete lambda 1-bearing anti-alpha (1-3) antibodies, the PBA were incapable of inducing B cells from non-Igha mice to mount an anti-alpha (1-3) Dextran response. The data suggest that non-Igha mice lack a functional VH Dex gene for the lambda 1-bearing anti-alpha (1-3) Dextran response.     

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.     

V kappa gene usage, idiotype expression, and antigen binding among clones expressing the VHX24 gene family derived from naive and anti-idiotype immune BALB/c mice

The BALB/c myeloma protein ABPC48 binds beta(2-6)-linked fructosans and expresses genes derived from the VHX24 and V kappa 10 gene families. We have selected 30 hybridomas expressing the VHX24 gene family derived from mitogen-stimulated spleen cells of naive BALB/c mice and mice injected at birth with the syngeneic monoclonal anti-ABPC48Id, IDA10. The majority of mAb with kappa L chains uses V kappa 1. Antibodies reacting with IDA10 use both V kappa 10 and V kappa 1. Most of these VHX24+ mAb reacted with one or more members of a limited panel of predominantly polysaccharide Ag that have been previously observed to interact with antibodies expressing the VHX24 gene family. Nucleotide sequencing of selected VH and V kappa genes shows a very low frequency of somatic mutation. The effect of neonatal anti-Id injection on VHX24-V kappa pairing and Id expression is discussed.