Somatic diversity in CDR3 loops allows single V-genes to encode innate immunological memories for multiple pathogens

The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized L-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.     

The importance of the light chain for the epitope specificity of human anti-U1 small nuclear RNA autoantibodies present in systemic lupus erythematosus patients.

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd approximately 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3-11) and the same lambda (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r lambda light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.     

Analysis of IgE antibodies from a patient with atopic dermatitis: biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3

To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the V(H)6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V(H)6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with V(H)1, V(H)3, and V(H)4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.     

Many human immunoglobulin heavy-chain IGHV gene polymorphisms have been reported in error

The identification of the genes that make up rearranged immunoglobulin genes is critical to many studies. For example, the enumeration of mutations in immunoglobulin genes is important for the prognosis of chronic lymphocytic leukemia, and this requires the accurate identification of the germline genes from which a particular sequence is derived. The immunoglobulin heavy-chain variable (IGHV) gene repertoire is generally considered to be highly polymorphic. In this report, we describe a bioinformatic analysis of germline and rearranged immunoglobulin gene sequences which casts doubt on the existence of a substantial proportion of reported germline polymorphisms. We report a five-level classification system for IGHV genes, which indicates the likelihood that the genes have been reported accurately. The classification scheme also reflects the likelihood that germline genes could be incorrectly identified in mutated VDJ rearrangements, because of similarities to other alleles. Of the 226 IGHV alleles that have previously been reported, our analysis suggests that 104 of these alleles almost certainly include sequence errors, and should be removed from the available repertoire. The analysis also highlights the presence of common mismatches, with respect to the germline, in many rearranged heavy-chain sequences, suggesting the existence of twelve previously unreported alleles. Sequencing of IGHV genes from six individuals in this study confirmed the existence of three of these alleles, which we designate IGHV3-49*04, IGHV3-49*05 and IGHV4-39*07. We therefore present a revised repertoire of expressed IGHV genes, which should substantially improve the accuracy of immunoglobulin gene analysis.     

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBL^hi) or without (MBL^lo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.

Methodology/Principal Findings

For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBL^lo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBL^hi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)⁺ clonal B-cells.

Conclusions/Significance

These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBL^lo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.     

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.     

Latent a1 VH germline genes in an a2a2 rabbit. Evidence for gene conversion at both the germline and somatic levels

We have previously reported the sequences of putative latent a1 cDNA derived from an alpha 2 alpha 2 rabbit. Significant similarity to nominal a1 cDNA sequences was noted, but none of the latent sequences were completely a1-like. We have now probed a genomic library, produced from the same alpha 2 alpha 2 rabbit, for evidence of germline latent a1 VH genes. Four hundred ninety-four VH+ clones were screened with oligonucleotides specific for a1 diagnostic regions of framework region 1 (FR1) and FR3. Twenty-two percent of the VH+ clones hybridized with an a1FR3 oligonucleotide probe. Two a1 FR1 probes yielded weak signals with 6% to 13% of the VH+ clones. Twenty VH genes from clones positive for one or more of the a1-specific oligonucleotide probes were sequenced, revealing 14 unique germline VH genes. All but one of these genes were 85% to 92% identical to the VH1-a1 nominal gene prototype, with sequence identity extending into the leader intron. Most genes displayed extended regions of similarity to a1 in FR1, FR3, or both and expressed 13 to 17 of the 21 allotype-associated residues, consistent with the nominal a1 sequence. The a1-like sequences were variously interspersed with short non-a1 segments, suggestive of germline gene conversion. Although none of the germline a1-like VH genes we have isolated from the alpha 2 alpha 2 rabbits are identical to the known a1 genes or protein sequences from alpha 1 alpha 1 rabbits and 8 of 14 are pseudogenes, most could make significant contributions to the synthesis of a complete nominal a1 sequence by serving as a pool of sequence donors during somatic gene conversion.     

Genomic screening by 454 pyrosequencing identifies a new human IGHV gene and sixteen other new IGHV allelic variants

Complete and accurate knowledge of the genes and allelic variants of the human immunoglobulin gene loci is critical for studies of B cell repertoire development and somatic point mutation, but evidence from studies of VDJ rearrangements suggests that our knowledge of the available immunoglobulin gene repertoire is far from complete. The reported repertoire has changed little over the last 15 years. This is, in part, a consequence of the inefficiencies involved in searching for new members of large, multigenic gene families by cloning and sequencing. The advent of high-throughput sequencing provides a new avenue by which the germline repertoire can be explored. In this report, we describe pyrosequencing studies of the heavy chain IGHV1, IGHV3 and IGHV4 gene subgroups in ten Papua New Guineans. Thousands of 454 reads aligned with complete identity to 51 previously reported functional IGHV genes and allelic variants. A new gene, IGHV3-NL1*01, was identified, which differs from the nearest previously reported gene by 15 nucleotides. Sixteen new IGHV alleles were also identified, 15 of which varied from previously reported functional IGHV genes by between one and four nucleotides, while one sequence appears to be a functional variant of the pseudogene IGHV3-25. BLAST searches suggest that at least six of these new genes are carried within the relatively well-studied populations of North America, Europe or Asia. This study substantially expands the known immunoglobulin gene repertoire and demonstrates that genetic variation of immunoglobulin genes can now be efficiently explored in different human populations using high-throughput pyrosequencing.     

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

Cloning the antibody response in humans with inflammatory central nervous system disease: analysis of the expressed IgG repertoire in subacute sclerosing panencephalitis brain reveals disease-relevant antibodies that recognize specific measles virus antigens.

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands (OGBs) in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV) or Cryptococcus neoformans, respectively. Using SSPE as a model system, we have developed a PCR-based strategy to analyze the repertoire of IgG V region sequences expressed in SSPE brain. We observed abnormal expression of germline V segments, overrepresentation of particular sequences that correspond to the oligoclonal bands, and substantial somatic mutation of most clones from the germline, which, taken together, constitute features of Ag-driven selection in the IgG response. Using the most abundant or most highly mutated gamma H chain and kappa or lambda L chain sequences in various combinations, we constructed functional Abs in IgG mammalian expression vectors. Three Abs specifically stained MV-infected cells. One Ab also stained cells transfected with the MV nucleoprotein, and a second Ab stained cells transfected with the MV-fusion protein. This technique demonstrates that functional Abs produced from putative disease-relevant IgG sequences can be used to recognize their corresponding Ags.     

Antibody repertoire development in fetal and neonatal piglets. XIII. Hybrid VH genes and the preimmune repertoire revisited

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise approximately 80% of the preimmune repertoire. However, so-called "hybrid" VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied > 3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for > 95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.     

Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL

In 10–20% of the cases of chronic lymphocytic leukemia of B-cell phenotype (B-CLL), the IGHV1-69 germline is utilized as VH gene of the B cell receptor (BCR). Mouse G6 (MuG6) is an anti-idiotypic monoclonal antibody discovered in a screen against rheumatoid factors (RFs) that binds with high affinity to an idiotope expressed on the 51p1 alleles of IGHV1-69 germline gene encoded antibodies (G6-id⁺). The finding that unmutated IGHV1-69 encoded BCRs are frequently expressed on B-CLL cells provides an opportunity for anti-idiotype monoclonal antibody immunotherapy. In this study, we first showed that MuG6 can deplete B cells encoding IGHV1-69 BCRs using a novel humanized GTL mouse model. Next, we humanized MuG6 and demonstrated that the humanized antibodies (HuG6s), especially HuG6.3, displayed ～2-fold higher binding affinity for G6-id⁺ antibody compared to the parental MuG6. Additional studies showed that HuG6.3 was able to kill G6-id⁺ BCR expressing cells and patient B-CLL cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, both MuG6 and HuG6.3 mediate in vivo depletion of B-CLL cells in NSG mice. These data suggest that HuG6.3 may provide a new precision medicine to selectively kill IGHV1-69-encoding G6-id⁺ B-CLL cells.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Intraclonal diversity in the VH genes expressed by CD5- chronic lymphocytic leukemia-producing pathologic IgM rheumatoid factor.

The leukemic cells from 95% of patients with B cell chronic lymphocytic leukemia (CLL) coexpress B cell differentiation antigens and the pan-T lymphocyte surface antigen CD5 (Leu 1). As such, CLL generally may be considered a malignancy of the CD5 B cell, a minor B cell subpopulation implicated in the production of autoantibodies. Recent data indicate that CD5+ leukemic cells may express autoantibody-associated V region genes with little or no somatic mutation. We examined the Heavy chain V genes expressed by an unusual CLL that secretes rheumatoid factor (RF) autoantibodies but does not express the CD5 surface Ag. Nucleic acid sequence analyses of the rearranged VH genes of three independent rDNA clones demonstrated intraclonal diversity not apparent in previously studied cases of CD5+ CLL. Comparison of the rearranged VH genes reveals that they belong to the VH4 gene subgroup and share highest homology with a rearranged VH gene (Ab44) that encodes a polyreactive autoantibody. That these productively rearranged VH genes encode the RF generated by this unusual CLL population is demonstrated by immunoblotting of the RF paraprotein using primary sequence dependent antipeptide antisera. These results indicate that CD5- CLL, like their CD5+ counterparts, may produce RF. However, unlike CD5+ CLL examined to date, CD5- CLL may have intraclonal diversity in their expressed Ig genes.     

Molecular characterization of the anti-idiotypic immune response of a relapse-free neuroblastoma patient following antibody therapy: a possible vaccine against tumors of neuroectodermal origin?

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne's network theory, a subset of anti-idiotypic Abs (Ab2beta) carries an "internal image" of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for V(H) and between 93.90 and 99.60% for V(L). An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient's serum did. GK8 alone inhibited >80% of the patient's anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for GD2-positive [corrected] tumors.     

VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions

In this study we investigate the molecular genetic basis for VHa- Ig. Knowing that the expression of VHa allotype Ig is suppressed by neonatal injection of rabbits with anti-VHa allotype antibody, and that the decreased level of VHa allotype Ig, VHa+, in the suppressed rabbits is compensated for by an increase in VHa- Ig, we determined the nucleotide sequences of 41 VDJ genes from a2/a2 rabbits neonatally suppressed for the expression of a2 Ig. We compared these nucleotide sequences to each other and identified two groups of VH sequences. We predict that the molecules of each group are encoded by one germline VH gene. Inasmuch as VHa+ Ig is encoded predominantly by one germline VH gene, VH1, it appears that more than 95% of the VDJ repertoire of rabbits may be encoded by as few as three germline VH genes. A genomic VDJ gene whose VH sequence was similar to those of group I molecules was expressed in vitro and was shown by ELISA to encode molecules of the VHa- allotype, y33. Analysis of the D regions in the VDJ gene indicated that germline D2b and D3 gene segments were preferentially used in the VDJ gene rearrangement. A comparison of sequences of D regions of the 41 VDJ gene rearrangements in 3-, 6-, and 9-wk-old rabbits to sequences of germline D gene segments showed an accumulation of mutations in the D region. Inasmuch as we have previously shown that V regions of rabbit VDJ genes are diversified, in part, by somatic gene conversion, it appears now that rabbit VDJ genes diversify by a combination of somatic mutation and somatic gene conversion.     

Partial molecular genetic map of the rabbit VH chromosomal region

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.     

Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.