Restricted utilization of germ-line VH3 genes and short diverse third complementarity-determining regions (CDR3) in human fetal B lymphocyte immunoglobulin heavy chain rearrangements.

Twenty-one independent immunoglobulin heavy chain VH3DJH rearrangements were cloned and sequenced from livers of human fetuses at 7, 13 and 18 weeks of gestation. The VH elements expressed were not somatically mutated. Eight out of the estimated 30 VH3 elements were utilized with a preference for five of them. One of these VH3 sequences, designated FL13-28, represented a thus-far unknown VH3 gene segment. From the six functional JH elements the JH3 and JH4 segments were utilized preferentially and from the estimated 30 D segments the DQ52 element and the Dxp family were found to rearrange frequently. D elements were utilized both in normal and inverted orientation, as single copies or in D to D fusions. Addition of N nucleotides, removal of nucleotides from the coding sequences and utilization of DIR elements (D genes with irregular recombination signals) further expanded the third complementarity-determining region (CDR3) diversity. One fourth of the fetal CDR3 regions lacked N regions. Due to utilization of DQ52, the relative absence of N regions and extensive exonuclease activity operating on the D elements, the fetal CDR3 regions were significantly shorter than those found in adult B lymphocytes.     

Variable region genes of human monoclonal autoantibodies to histones H2A and H2B from a systemic lupus erythematosus patient

An antibody phage library obtained from peripheral blood lymphocytes of a systemic lupus erythematosus (SLE) patient was used to isolate four monoclonal autoantibodies against histones H2A and H2B. Analysis of the variable region sequences revealed that the anti-histone monoclonal antibodies were not clonally related; they used VH genes from three different VH gene families (VH3, VH4, and VH5) and distant members of the Vkappa group (L25, L6, A27, and O8) in conjunction with different D and J gene segments. These observations suggest that certain gene families or segments are not critical in producing anti-histone autoantibodies in SLE. Most of the utilized VH and Vkappa sequences were highly mutated and the complementarity-determining regions (CDRs) varied greatly in length. The VH region of the antibody SLEhis18 had an isoelectric point of 6.1, and 29% of the mutations were changes to acidic amino acid residues. The second CDR (CDR2) of SLEhis18 VH contained one basic and three acidic residues. Acidic residues were observed in the CDR3 regions of VH, but not VL, in all isolated clones; this is unusual, as most autoantibodies are comprised predominantly of non-acidic residues. This is the first report of a systematic sequence analysis of human anti-histone monoclonal antibodies. Our results suggest that certain V genes are not important for autoreactive specificity to histones in SLE; instead, other mechanisms such as an existence of acidic residues and somatic mutations in CDRs are required for specific binding to histones, which might play a role as a stimulatory autoantigen for the activation of autoantibody-producing B-cells and the selection of high affinity antibody.     

Two V(H)5 family genes expressed by human peripheral B cells display differential mutational frequencies in the V(H) region

The heavy chain variable segment gene (V(H))5 family, one of the seven immunoglobulin (Ig) V(H) families, contains two functional genes, VH251 and VH32. To investigate functional differences between these V(H)5 family genes, V(H) segments expressed by human peripheral B cells were sequenced and analyzed. One hundred fifty-three sequences with unique V(H)DJ(H) recombinations were obtained from 17 adults. The mutational frequency of VH32 derived sequences (6.4%) was higher than that of VH251 derived sequences (4.4%), resulting in a significant difference (P<0.01). Significant differences in mutational frequencies between VH251 and VH32 derived sequences were observed in CDRs and FRs. No significant differences were found in CDR3 length distribution, D segment usage, or J(H) segment usage between VH251 and VH32 derived sequences. These results suggest that mutational frequency is affected, in part, by V(H) gene structure. The difference may occur after recombinational events in B cell development.     

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.     

Structural basis of stimulatory anti-idiotypic antibodies

In order to design and produce effective vaccines based upon the idiotype network hypothesis of Jerne, a thorough understanding of the biological and structural aspects underlying the stimulating activities of anti-idiotypic antibodies is needed. Here we determined the nucleotide sequence of the variable heavy and light chain regions of two monoclonal anti-idiotypic antibodies which induce different anti-phosphorylcholine responses. The nucleotide sequences of the variable domains of two monoclonal anti-TEPC 15 (T15) antibodies (F6-3 and 4C11) were determined by the primer extension and Maxam-Gilbert techniques. The nucleotide sequence data show that 4C11 and F6-3 have homologous VH segments and JH segments, but different D regions. The VH segments of both clones belongs to the J558 VH family. Most of the differences among the VH segments are located in CDR2. The VK segments of 4C11 and F6-3 are homologous to the VK gene group 4 and group 8, respectively. Comparison of the sequences of 4C11 and F6-3 with other published anti-idiotype antibodies shows that there is no preferential utilization of immunoglobulin genes. An analysis of the distribution of charged residues and hydropathic comparison studies were used to interpret the sequence of 4C11 in terms of the biological mimicry of antigenic stimulation.     

Immunoglobulin Gene Mutations and Frequent Use of VH1-69 and VH4-34 Segments in Hepatitis C Virus-Positive and Hepatitis C Virus-Negative Nodal Marginal Zone B-Cell Lymphoma

Nodal marginal zone B-cell lymphoma (NMZL) is actually considered as a distinct entity that must be distinguished from extra-nodal and splenic marginal zone lymphomas. To define the cell origin and the role of antigen stimulation we determined the nucleotide sequence of the tumor-related immunoglobulin heavy chain variable genes in 10 cases of NMZL. The results were also evaluated on the basis of the presence of chronic hepatitis C virus (HCV) infection. All 10 cases harbored VH somatic mutations with a sequence homology compared to the closest germline gene, ranging from 83.33 to 98.28%. Interestingly, different VH segments were preferentially used in HCV-positive and HCV-negative patients: three of five HCV-negative NMZLs used a VH4-34 segment joined with different D and JH segments whereas three of five HCV-positive NMZLs used a VH1-69 gene joined with a D3-22 and a JH4 segment, with very strong similarities in the CDR3s among the three different cases. These data indicate: 1) NMZL is derived from B cells that have experienced the germinal center reaction; 2) the preferential usage of a VH1-69 segment in the majority of the HCV-positive NMZL cases with similar CDR3s suggests the presence of a common antigen, probably a HCV antigen epitope, involved in the B-cell selection; and 3) the use of a VH4-34 segment suggests a role of yet unknown B-cell superantigen(s) in the selection of tumor B-cell precursors in HCV-negative NMZL.     

Improvement of anti-Burkholderia mouse monoclonal antibody from various phage-displayed single-chain antibody libraries

To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respectively. We collected high scFvs binders through panning experiment over the large (size ~1 × 10?) random and domain libraries. The quality of the libraries was validated by successful selection of high-affinity clones. Random mutagenesis generated many mutant scFv clones having more than one amino acid changes around framework regions, but not many in CDRs. Surprisingly, the resulting eight higher scFv binders were selected from CDR3 mutations, but not from random mutations. Six of them resulted from CDR3 mutations of light chain, except for two scFvs from heavy chain, showing both Burkholderia pseudomallei and Burkholderia mallei had preferentially influenced the VL CDR3. Furthermore, all eight higher scFvs converted to full format human IgG1 antibodies were expressed transiently in 293T cell line. Five chimeric MAbs showed improved higher binding activity, as much as 0.2-0.3 at O.D. 405 nm, than positive control MAbs. These libraries could be valuable sources for selection of anti-Burkholderia antibodies and discovery of the relevant epitope(s) for developing effective vaccines or therapeutics.     

Diversity of immunoglobulin heavy chain gene segment rearrangement in B lymphoblastoid cell lines from X-linked agammaglobulinemia patients

X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.     

Oligonucleotide Probes to the 5'End of the Framework 3 (FR3) Gene Segment detect Polymorphisms of VH Gene Sequences encoding Biologically Important Amino Acid Residues

In this study, we demonstrate the use of oligonucleotide probes to the most VH family-speeific framework, the Eramework 3 (ER3) gene segment, in the analysis of germline DNA. Compared with the hybridization of restriction enzyme-digested genomic DNA to large, cloned human VH family probes, hybridization with oligonueleotides derived from nucleotides 199 to 258 of the ER3 gene segments both yields less complex patterns and highlights polymorphic variations between individuals. The potentially important role played by amino acid residues encoded by the FR3 gene segment in influencing conformation, antigen binding and affinity of the physically proximal complementarity determining regions (CDRs) suggests FR3 sequence conservation to be biologically significant. Genomic studies combining information from both CDR and framework restriction fragment polymorphisms can prove useful in detecting specific genes and in identifying haplotypes of interest.     

Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl.

As a first step toward defining the molecular interactions between ligands and the IgE antigen-combining site, we report here the cDNA cloning and variable (V) region nucleic acid sequences of the heavy (H) and light (L) chains of 2 monoclonal mouse IgE antibodies to trinitrophenyl (ATCC-TIB142 = IGELa2 and ATCC-TIB141 = IGELb4). In all instances, full-length cDNA clones were obtained to facilitate future expression studies. The H chains were encoded by VH genes from the VH3660 and J558 gene families in context with DQ52 and DSP2.2 diversity (D) mini genes, and JH3 and JH4 joining (J) gene segments, respectively. Vk8/Jk2 and Vk1/Jk5 rearrangements encoded the respective L chain V-regions. Both antibodies exhibited considerable conservation of complementarity determining region (CDR) sequences, which will facilitate template-based computer modeling of the three-dimensional structures of complexes formed between various ligands and these antibodies. From sequence comparison between the dinitrophenyl (DNP)-binding myeloma protein MOPC-315 and these IgE antibodies likely candidates for hapten-contact residues within the binding sites of IGELa2 and IGELb4 have been suggested.     

Organization of the murine immunoglobulin VH complex: placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation

During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183 ). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.     

The primary structure and specificity determining residues displayed by recombinant salmon antibody domains

Previously, single chain fragments of salmon (Salmo salar L.) immunoglobulin variable regions (scFv) were isolated by reactivity towards trinitrophenyl (TNP) or fluorescein (FITC) using phage display technology. The fine specificity of six scFv clones were analysed by ELISA, while the primary structure was determined by DNA sequencing. In addition, preliminary models of one anti-TNP and one anti-FITC clone were built. Here, a follow-up analysis of the primary and tertiary structure of all six clones is focused on the structural basis for hapten specificity. Tertiary structure was analysed by molecular modelling of the antigen combining site. The analysis shows that reactivity to each hapten is maintained by a number of different combinations of VH, D, JH and VL sequences. Accordingly, various sizes of CDR3 on both the heavy and light chain and CDR2 of IgH may support TNP binding. Due to variability of the antigen combining site each clone probably has a distinct binding affinity. However, a feature common among the four scFv antibodies that recognise TNP is a positively charged Arg in CDR2 of either the heavy or light chain. In the majority of the anti-TNP clones localisation of this side-chain is stabilised by a negatively charged Asp in LCDR1. In addition, a Trp in LCDR3 is conserved in all the anti-TNP clones. Also, the anti-FITC clones display a Trp in the LCDR3, suggesting its participation in binding of FITC as well. In combination with a large aromatic amino acid near the N-terminus of HCDR2 and a positively charged Arg in CDR1, these residues probably determine both specificity and affinity towards the FITC moiety.     

Extensive CDR3H length heterogeneity exists in bovine foetal VDJ rearrangements

Analysis of seven variable-diversity-joining (VDJ) gene rearrangements in B splenocytes from a 125-day-old bovine foetus revealed an extensive heavy-chain complementarity-determining region 3 (CDR3H) length variation (9-56 codons). Indeed, the global CDR3H size spectratyping of foetal VDJ rearrangements substantiated such an extensive heterogeneity and was comparable with that noted in peripheral B lymphocytes of adult cattle. These observations are in contrast to species such as humans with extensive germline combinatorial capability where shorter CDR3H length is noted early during B-cell development. Exceptionally long CDR3H (as in adult cattle) was noted in two foetal VDJ rearrangements encoded by a single germline VH gene. Further, two VH genes (gl.110.20 and BF2B5) were preferentially expressed in the foetal VDJ rearrangements. The DH gene-encoded CDR3H region of foetal VDJ rearrangements is remarkable for repetitive GGT (glycine) and TAT (tyrosine) codons that favour the recruitment of somatic hypermutations. It appears that closely related germline DH genes, preferentially used in the hydrophilic reading frame, encode varying CDR3H lengths early during B-cell ontogeny in cattle. A comparison of germline and expressed VH genes, especially in the CDR1 and CDR2, confirms that somatic hypermutations contribute to immunoglobulin (Ig)M antibody diversification in cattle. The biased nucleotide base use and high occurrence of 'hot-spot' triplet (AGPy; AG pyrimidine base) in the CDRs predisposes to somatic hypermutations. Overall, these observations suggest that extensive CDR3H length heterogeneity, including the generation of exceptionally long CDR3H (up to 56 amino acids), and somatic hypermutations contribute to IgM antibody diversification in cattle. The extensive CDR3H length heterogeneity early during the B-cell development may compensate for constraints imposed on antibody diversification owing to the limited germline sequence diversity of genetic elements in cattle.     

Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice

Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation.     

Immunoglobulin heavy chain gene rearrangements in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) appears to involve a defect in human B lymphocyte differentiation which is manifested at the pre-B cell stage. The defect segregates as an X-linked recessive trait but is not a single genetic entity. IgM-producing B cell clones were established by Epstein-Barr virus transformation of peripheral blood mononuclear cells of patients with the XLA defect linked to the DXS3 and DXS17 chromosomal loci. Individual XLA B cell clones were demonstrated to have rearrangements of the JH regions of both immunoglobulin VH region loci. The rearranged JH regions of the B cell clone ALA 19 were molecularly cloned and their nucleotide sequence was determined. Both JH-associated rearrangements (designated 191 and 192) resulted from the juxtaposition of variable (VH), diversity (D) and joining (JH) segments (VHDJH rearrangements). The 191 rearrangement employed a VH segment belonging to VH subgroup III and a JH4 segment. The 192 rearrangement employed a VHII and a JH6 segment. The D191 and D192 segments encompassed 21 and 28 nucleotides, respectively, and showed little homology to each other or to previously reported human D sequences. Surprisingly, both VHDJH complexes had open reading frames. However, in accord with principles of allelic exclusion, only the 191 allele was detectably expressed in the total RNA of the cell. A possible mechanism for the lack of expression of the 192 allele is discussed. We conclude that the DXS3-DXS17-linked XLA defect does not preclude VH to DJH rearrangements or the expression of VH containing heavy chain molecules.     

A VH gene is located within 95 Kb of the human immunoglobulin heavy chain constant region genes

Using cosmids covering about 117 Kb upstream of the human immunoglobulin chain C mu gene, we have identified a potentially functional VH gene, belonging to the VHVI subgroup. This VHVI gene is only about 95 Kb from the C mu gene and is probably the first functional VH segment of the Igh locus. These results illustrate the proximity of the human VH, DH and JH segments involved in creation of the complete heavy chain genes.