Comparison between hybridoma and Fab/phage anti-RhD: their V gene usage and pairings

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 V kappa families (V kappa 1 and V kappa 3). However, the 13 lambda chains were not as restricted, using 10 V lambda germline genes from 4 families (V lambda 1, V lambda 2, V lambda 3 and V lambda 8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from V kappa 1 (97%) and the 24 lambda chains used 10 V lambda germline genes from 5 families (V lambda 1, V lambda 2, V lambda 3, V lambda 4 and V lambda 7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 V kappa germline genes used by the IgG (DPK9). However, the repertoire of the V lambda germline genes used in these two systems is different, with analysis showing greater diversity in V lambda gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% of the IgG hybridoma anti-RhD.     

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.     

Molecular and serological diversity of anti-DNA autoantibodies from NZB and (NZB X NZW) F1 mice

We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of the total murine Ig repertoire is represented in the anti-DNA autoantibodies present in these strains.     

Perspectives on Antigenicity and Idiotypy

HIV-1 infection affects many of the cellular components vital for the maintenance of immune homeostasis. Similar to the T cell superantigen effect on T cell expansion and depletion in AIDS, HIV components with B cell superantigenic properties could be responsible for the observed B cell activation and skewing of VH family usage. Current data on possible B cell superantigen properties of HIV proteins (gp120) are mostly based on studies describing the clonality and VH family usage of immunoglobulins in HIV infection. Various laboratories reported independently an unusual skewing of the VH-repertoire of antibodies that appears not to be random. According to these observations, an enrichment of VH 1 and VH4 family - paralleled a depletion of VH3 family - utilizing anti-HIV-1 gp120 and p24 antibodies in HIV-1 infected individuals and a loss of total VH3⁺ Ig in patients with late stages of AIDS. Polyclonal and monoclonal (VH1, VH4, and VH5) anti-p24 and gp120 antibodies share a crossreactive idiotype (IF7). IF7 like antibodies were found in the serum of HIV-1 infected individuals, persisting in the course of infection, perhaps contributing to the depletion of VH3⁺ Ig. Furthermore, a restriction of clonal heterogeneity of anti-p24 and anti-gp120 antibodies was detected by isoelectric focusing and indicated by skewed kappa/lambda light chain isotype ratios, indicating clonal dominance of certain sets of anti-HIV-1 antibodies during infection. Taken these findings together, a strong case for the involvement of a B cell superantigen can be made, although the mechanism of B cell depletion is not fully understood.     

V genes of the primary antibody response of C57BL/10 mice to the hapten phenyloxazolone

The mRNA of ten monoclonal phenyloxazolone (phOx) antibodies originating from the primary (day 7) response of C57BL/10 mice were partially sequenced. The sequences were analyzed together with those of two previously published antibodies. The C57BL response does not have a predominant subset of antibodies like the BALB/c response has (VH-Ox1/V kappa-Ox1 JK5). Probably, C57BL mice lack the VH-Ox1 gene and, as a consequence, their V kappa-Ox1 gene does not have a main role in the anti-phOx response. Five V kappa and six VH genes were found to participate. All five V kappa genes or their "alleles" had previously been found from the BALB/c response to 2-phenyloxazolone (phOx). On the other hand, the two strains use different VH genes for the anti-phOx response. Most C57BL antibodies were coded by VH genes of group 1 which has only minor role in the BALB/c response. The remaining VH genes were from group 7. Our data show that one V kappa segment (e.g. V kappa-Ox1) can code for anti-phOx antibodies with several, even widely different, VH genes. On the other hand, they emphasize the role of certain VH/VL gene combinations for the anti-phOx specificity. Thus, VH genes of group 7 were found to code for anti-phOx antibodies only together with the V kappa 45.1 gene.     

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

Cationic residues in pathogenic anti-DNA autoantibodies arise by mutations of a germ-line gene that belongs to a large VH gene subfamily.

The F1 progeny of autoimmune NZB and normal SWR mice uniformly develop severe and accelerated lupus nephritis. The (SWR x NZB)F1 mice produce an oligoclonally expanded population of nephritogenic anti-DNA autoantibodies that share a recurrent cross-reactive idiotype (Id564), use highly homologous VH genes and surprisingly have the CH region allotype of the normal SWR parent. From extensive library analyses, we isolated 15 SWR germ-line genes which are most closely related to the pathogenic autoantibody VH564 gene and which also belong to the NPb subfamily of J558 VH genes. We found that the pathogenic VH genes are probably somatic variants of a SWR germ-line VH gene, SW6-3, and they have several basic amino acid substitutions, in addition to those already present in the SW6-3 germ-line gene. Since these pathogenic autoantibodies are not detectable in the sera of the normal SWR mice despite the presence of the SW6-3 gene, strong selection and expansion of B cells expressing the SW6-3 VH gene is probably occurring in (SWR x NZB)F1 lupus-prone mice. We also isolated eight germ-line genes from the NZB mouse which are homologous to SW6-3. The autoimmune NZB parent that rarely develops nephritis lacks the SW6-3 gene, but has several highly homologous germ-line VH genes that would encode less cationic antibodies. These results establish a correlation between structure and pathogenic potential of VH genes.     

Diversity and somatic hypermutation of the Ig VHDJH,V kappa J kappa,and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone

Burkitt's lymphoma (BL) is a malignancy of B cells characterized by chromosomal translocations involving the immunoglobulin (Ig) and c-MYC gene loci. To address the putative role of antigen in the clonal expansion of these neoplastic B cells, we analyzed the VHDJH and VLJL gene segments expressed by the established cell lines derived from six endemic BL and six sporadic BL. Eight BL cell lines used genes of the VH3 family, two of the VH4, and two of the VH1. Eight VL chains were kappa (four members of the V kappa3, two of the V kappa1, and two of the V kappa2 subgroups) and four lambda (three members of the V lambda1 and one of the Vl ambda3 subgroup). The VH gene utilization was stochastic (i.e., it reflected the relative representation of the different VH gene family members in the human haploid genome). In contrast, the VL gene utilization was skewed toward the overutilization of the V kappa3 and V lambda1 gene subgroups. When compared with those of the respective germline genes, the sequences of the expressed Ig VDJ genes displayed nucleotide differences that resulted from somatic hypermutation. In three endemic and three sporadic BL cells, nucleotide changes yielding amino acid substitutions segregated within the complementarity determining region, indicating the application of a positive pressure for replacement mutations and suggesting that these neoplastic lymphocytes underwent a process of clonal selection driven by antigen, perhaps emerging from or transitioning through germinal centers.     

Human antibody responses to bacterial antigens: studies of a model conventional antigen and a proposed model B cell superantigen.

We have investigated the human antibody repertoires that bind to two different classes of bacterial antigens. Immunization with the conventional antigen, type b capsular polysaccharide of Haemophilus influenzae Hib PS, uniformly induces IgA and IgG responses dominated by clones that use heavy chains structurally related to two subsets of VH3 genes, while in a minority of subjects antibodies from the VH1 or VH4 families are co-induced. In contrast, the "alternative binding site" of Staphylococcal Protein A (SPA) represents an unconventional determinant, because; (i) SPA is bound by a large proportion of non-immune IgM, IgA and IgG F(ab')2, (ii) SPA is bound only to Fab from the VH3 family, which can be encoded by at least four different germline genes, (iii) SPA binding is independent of VL usage, (iv) by flow cytometry SPA is bound by > 15% of tonsilar B cells, but not to T cells. (v) In vitro stimulation with an SPA containing mitogen induces the preferentially production of Ig bearing a VH3 marker. Taken together, these studies characterize a VH family restricted binding interaction that is distinct from the properties associated with conventional antigens such as Hib PS. Based on these data we propose that SPA represents a prototype for a B cell superantigen.     

A new germline VH36-60 gene is used in the neonatal primary and adult memory response to (T,G)-A--L.

Our previous studies of the neonatal primary response to (T,G)-A--L showed that the majority of anti-(T,G)-A--L antibodies bind the copolymer L-Glu:L-Tyr (GT), share idiotypy (Id), and use the H10 germline VH gene from the VHJ558 family and a V kappa 1 gene. We also identified two hybridomas from different neonatal donors that produced GT+, Id+ antibodies using a V kappa 1 gene with a VH gene from the VH36-60 family. In the study reported here, we show that both neonatal hybridomas use the same germline VH gene from the VH36-60 gene family. However, the VH gene sequence is different from previously identified germline genes of the VH36-60 gene family. To determine whether the expressed heavy chain gene had undergone somatic mutation, we isolated the corresponding germline gene from kidney DNA. Sequence analysis of this gene shows that it is a new member of the VH36-60 family which is not mutated in the neonatal antibodies. Furthermore, the deduced amino acid sequences of the two neonatal antibodies are identical not only in the VH region but also in the VH-D-JH joins, suggesting that there is a strong selection for CDRIII among neonatal anti-(T,G)-A--L antibodies using this germline gene (designated here as VH3A1) with a V kappa 1 gene. Also, the VH gene from the VH36-60 family that we showed previously was used by an adult memory B cell clone specific for (T,G)-A--L, can now be identified as a rearrangement of the VH3A1 germline gene. Elucidation of the germline variable region genes that are used in the antigen-specific neonatal response will help us understand the mechanisms that shape the preimmune B cell repertoire during B cell development.     

Molecular and genetic characterizations of five pathogenic and two non-pathogenic monoclonal antiphospholipid antibodies

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis, recurrent fetal loss and thrombocytopenia. Antiphospholipid antibodies, detected by enzyme-linked immunoabsorbent assays (aCL) and/or in vitro blood clotting assays (LAC) are strongly associated with APS. Both the molecular structures used by pathogenic antiphospholipid antibodies and the genetic mechanisms leading to their production are unknown. We describe here the variable region genes of seven IgG antiphospholipid antibodies derived from two APS patients. Of these, five are pathogenic as defined in a mouse model of thrombosis and two are not. Analyses of the expressed variable region genes show no preferential V gene usage. However, similar to anti-DNA antibodies, pathogenic antiphospholipid antibodies contain an increased number of arginine residues in the third complimentarity-determining region (CDR3) of their H chains. The increased accumulation of arginine residues in the V(H) CDR3 may act to enhance antigen binding, promote disease and point to the importance of the H chain in the pathogenic potential of certain antiphospholipid antibodies.     

Molecular analysis of human immunoglobulin heavy chain variable region associated determinants recognized by anti-VH3 antibodies 7B4, B6 and D12

7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3-30, V3-30.3 and V3-33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3-11 and V3-7 genes; D12 also reacts with some V3-43 gene Ig. We show here, by site-directed mutagensis, that the lysine at position 57 in the complementarity-determining region 2 (CDR-2) of the V3-30 gene product is crucial for epitope recognition by all three anti-VH3 MoAbs. Further analysis of the amino-acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR-2. Comparing the efficiency of 7B4 reactivity with VH3 gene-encoded human Ig indicates that amino-acid position 4 in the frame region 1 (FR-1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3-30, V3-30.3 and V3-33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)-2, SIV and HTLV-4. We also report that other tetrapeptide sequences found on the 56-59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV-1 isolates. The possible significance of these observations is discussed.     

V genes of oxazolone antibodies in 10 strains of mice

One pair of V genes (V kappa 45.1 and V11) code for a great portion of phenyloxazolone (anti-phOx) antibodies in 10 strains of mice. This combination replaces the first-known major combination VHOx1-V kappa Ox1 in some strains, and is important in most strains. C57BL/10 and SJL mice have an additional subset of antibodies encoded by genes V kappa 45.1 and V13 (a relative of V11). All three genes involved (V kappa 45.1, V11 and V13) have "allelic" variation. Four alleles of V11 were found, one in Igh haplotypes a, c and g, the second in haplotypes d, j and n, the third in b, and the fourth in f. The most distant alleles d, j, n and f had 10 nucleotide differences out of 429 determined (97.7% homology). Only one allele of the V13 gene was found from anti-phOx hybridomas but two others have been published. Three alleles of the V kappa 45.1 gene were found; one in NZB mice (Ig kappa haplotype b) another in CE (haplotype f), and the third in eight strains including representatives of three Ig kappa haplotypes (a, c and e). The three alleles had greater than 99.0% homology. The V11 and V13 genes that code for anti-phOx antibodies in C57BL/10 and SJL mice were different from the related genes found from the C57BL/10 germ line. C57BL/10 mice must have a chromosome bearing two V11 and two V13 genes. RF mice were found to have two V11 genes, and both code for anti-phOx antibodies. Our data show that the majority of antibodies in the anti-phOx response are encoded by the same restricted collection of V genes in most mouse strains. Antibody responses appear to be no less heritable than other functions of the body.     

Variable region structure and staphylococcal protein A binding specificity of a mouse monoclonal IgM anti-laminin-receptor antibody.

Staphylococcal protein A is a cell wall-attached polypeptide that acts as a B-lymphocyte superantigen. This activation correlates with specific VH gene segment usage in the B-cell receptor. B-cell receptor assembled from members of the VH3 family in humans, or S107 family in mice, has an intrinsic affinity for protein A. Human VH3-derived antibodies bind to domain D of protein A. We have characterized a mouse IgM monoclonal antibody that binds protein A. The sequencing of the variable region suggests an almost germline-encoded VH derived from S107 family and a V kappa 8-derived VL. The binding specificity of the monoclonal antibody was tested with various recombinant constructions derived from protein A. We show that, unlike human VH3-derived antibody, mouse S107-derived immunoglobulin binds to the B domain of the bacterial superantigen.     

Identification of conformation-dependent epitopes and V gene selection in the B cell response to type II collagen in the DA rat.

Collagen-induced arthritis (CIA) is a widely used model for rheumatoid arthritis. Induction of CIA in rats using rat type II collagen (CII) results in a chronic arthritis in which anti-CII antibodies are believed to play a pathogenic role. In this study, we analyzed the epitope selection and V gene usage in the anti-CII response in the DA rat. A panel of CII-reactive B cell hybridomas was established from the draining lymph nodes 11 days after immunization. All of the CII-specific antibodies bound cartilage in vivo, showing that these are true autoantibodies. These antibodies were all IgG and specific for several distinct triple helical epitopes on CII. Interestingly, the major epitope, recognized by four different antibodies, was identical with the major B cell epitope in the mouse CII located at position 359--369 (denoted as C1(III)). The Q52 and PC7183 V(H) gene families encoded 12 out of 14 sequenced heavy chains. There was a relatively more heterogeneous usage of V(L) genes as the antibodies were encoded by four different V(kappa) families (V(kappa)1, V(kappa)2, V(kappa)12/13 and V(kappa)RF). As in the mouse, some of the V genes used showed germline characteristics. We conclude that the immune response in the rat shares epitope specificity and a constrained V gene repertoire with the mouse. However, the V genes used for recognition of the closely related collagen structures differed considerably between mouse and rat, indicating an influence of the species-specific variation in the V gene repertoire.     

Endogenous VH gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.     

Selective and polyclonal induction of high levels of lambda light chain-bearing immunoglobulins in BALB/c and SJL mice

Polyclonal B cell activation is V gene independent and consequently results in the unselected expression of both V lambda and V kappa genes. In an attempt to selectively and polyclonally trigger lambda light chain-bearing B cells, we immunized mice with low doses of lipopolysaccharide (LPS) coupled to antibodies specific for the constant region of lambda 1 chains. Analyses of the sera of treated mice revealed that LPS anti-lambda 1 selectively triggers lambda-but not kappa-bearing B cells to high rate Ig secretion. Surprisingly, immunized SJL mice (rlo lambda 1 phenotype) mounted a lambda + response comparable to that detected in mice with the r + lambda 1 phenotype. Because we could definitively establish that the majority of the induced antibodies belong the lambda 1 subtype, our results have strong implications both for the study of the repertoire of lambda 1-bearing B cells and for our understanding of the functional mechanism(s) by which the r lambda 1 locus acts.     

The efficiency of B cell receptor (BCR) editing is dependent on BCR light chain rearrangement status

Anti-DNA knock-in mice serve as models for studying B cell tolerance mechanisms to a ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6xBALB/c)F1 mice, each expressing an unmutated or somatically mutated anti-DNA heavy (H) chain, combined with one of three different light (L) chains, namely V(kappa)1-J(kappa)1, V(kappa)4-J(kappa)4 and V(kappa)8-J(kappa)5. In vitro analysis of the various Ig H/L chain combinations showed that all had a similar specificity for single-stranded DNA and double-stranded DNA, but that antibodies encoded by the mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains exhibited significant levels of serum anti-DNA activity. However, while B cells from mice carrying the V(kappa)1-J(kappa)1 transgenic L chains were tolerized almost exclusively by L chain receptor editing in an affinity-independent manner, the mice expressing V(kappa)8-J(kappa)5 L chains have utilized affinity-dependent clonal anergy as their sole mechanism of B cell tolerance. V(kappa)4-J(kappa)4 targeted mice exhibited an intermediate phenotype with respect to these two mechanisms of B cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell tolerance and that the efficiency of L chain editing is directly related to the number of available J(kappa) segments on the expressed V(kappa) allele.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Repertoire of murine lambda-positive variable domains: polyclonal induction of lambda isotypes and their associated pattern of antibody specificities

The diversity of lambda variable (V lambda) domains is extremely restricted when compared to those of VH and V kappa. In addition, each V lambda domain is determined by the C lambda domain. For these reasons, the lambda system is an excellent model for the study of the associated VH region repertoire. The study of V lambda domain diversity has been limited by the small contribution (approximately 5%) of lambda-bearing proteins to the total Ig pool. We now show that treatment of BALB/c mice with rabbit anti-lambda 1 antibodies coupled to lipopolysaccharide induces a production of polyclonal lambda 1 light chain-bearing Ig whereas, conversely, treatment with rabbit anti-lambda 2 antibodies induces a production of polyclonal lambda 2 + lambda 3 light chain-bearing Ig. The antigenic specificities of these two distinct lambda populations were then studied using B1355 dextran, (4-hydroxy-3-nitrophenyl)acetyl (NP) and 2,4-dinitrophenyl (DNP) as antigens. The anti-alpha(1-3)dextran antibody specificity was found to be mediated exclusively by antibodies bearing the lambda 1 isotype whereas the anti-NP and anti-DNP antibody specificities are mediated by both the lambda 1 and lambda 2 + lambda 3 isotypes. In addition, various mouse strains with the VHa or VHb allotypic haplotype and the rlo lambda 1 or r+ lambda 1 phenotype were treated with rabbit anti-lambda 1 antibodies. The lambda 1 anti-NP and anti-DNP antibody specificities were similar in all strains whereas the lambda 1 anti-alpha(1-3)dextran specificity was linked to the presence of the VHa allotypic haplotype. The mouse strains with the rlo lambda 1 or r+ lambda 1 phenotype did not differ in terms of their patterns of lambda 1 antibody specificities.