Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.     

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.     

Molecular analysis of cross-reactive anti-myosin/anti-streptococcal mouse monoclonal antibodies

Nucleotide sequences of VH- and VL-genes of anti-myosin/anti-streptococcal monoclonal antibodies (mAbs) were analyzed and compared with their highly detailed antigen binding reactivities. Antigen-specificities of the cross-reactive mAbs included myosin, streptococcal M-protein, actin, keratin, N-acetyl-beta-D-glucosamine, vimentin, DNA, tropomyosin, troponin, and laminin as previously described. After nucleotide sequence analysis, homology indicated that some of the V gene sequences aligned with antibodies recognizing gangliosides and blood group antigens glycophorin M and N. Therefore, mAb reactivity with gangliosides and glycophorin M and N was identified. The cross-reactive mAbs utilized a heterogeneous group of germline V-heavy genes comprised of nine J558-, four 7183- and two Q52-family VH-genes. Germline V-light genes utilized by the mAbs included six Vkappa4/5-, three Vkappa8-, two Vkappa10-, three Vkappa19- and one Vkappa23-family VL-genes. No preferential VH/VL-chains correlated with any of the 12 different antigen reactivities, even for mAbs with nearly identical cross-reactivities. However, we did find that the cross-reactive mAb germline genes within a V gene family shared more homology among themselves than with other germline genes within their V gene families, suggesting convergent mutation. Cross-reactive mAbs with the highest relative avidity for myosin were found in the VH7183 family which contained two cytotoxic mAbs. Antibodies with V gene sequences most homologous to those of our cross-reactive anti-myosin/anti-streptococcal mAbs had specificities for laminin, DNA, carbohydrates, or blood group antigens and were reported to cause autoimmune disease in mice.     

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.     

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.     

Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.     

Nucleotide sequence analysis of rheumatoid factors and polyreactive antibodies derived from patients with rheumatoid arthritis reveals diverse use of VH and VL gene segments and extensive variability in CDR-3.

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.     

Natural polyreactive immunoglobulin A antibodies produced in mouse Peyer’s patches

To understand the biological function of natural immunoglobulin A (IgA) antibodies in Peyer’s patches (PP), we generated IgA monoclonal antibody (mAb) clones from the PP of normal, unimmunized, specific pathogen-free BALB/c mice and examined their reactivities by enzyme-linked immunosorbent assay (ELISA). Many of these antibodies reacted with more than one antigen examined, suggesting that they were polyreactive Abs. Two mAbs agglutinated several different strains of commensal bacteria isolated from mice. To examine the genetic features of these polyreactive mAbs, the V_H genes of seven different IgA mAbs were sequenced. The V_H genes from the VGAM, J558 and 7183 families were compared with sequence from the mAbs with distinct VDJ rearrangements. One of the mAbs that agglutinated bacteria was encoded by a germline V_H gene, but the V_H region of the other polyreactive mAbs contained between seven and 11 mutated sites. No indication of antigenic selection was observed in the pattern of these mutated sites. Our results show that polyreactive IgA Abs are present in PP as a part of the normal B-cell repertoire. These polyreactive Abs may establish a natural immune homeostasis, and function as a polyreactive sensor to detect pathogenic invasion and to control immune response in the gut.     

Characterisation of a mouse monoclonal anti-idiotype reactive with a V region sequence commonly used by human immunoglobulins.

BACKGROUND: A mouse monoclonal antibody (2C7/IgG2b kappa) has been described recently, which is directed against the major house dust mite allergen Der p 1, and whose epitope specificity is representative of a major component of the human IgE anti-Der p 1 response. AIMS: To characterise an anti-idiotypic antibody (2G10/IgG1 kappa) raised against monoclonal antibody 2C7 as surrogate human IgE anti-Der p 1. METHODS: The specificity of the anti-idiotype antibody 2G10 was determined by competitive inhibition experiments using human and mouse immunoglobulins of known VH gene families. The epitope recognised by monoclonal antibody 2G10 was located on the molecular model of the Fv (fragment variable) region of monoclonal antibody 2C7. RESULTS: The data suggest that monoclonal antibody 2G10 is directed against a crossreactive idiotype on human IgE that is shared by polyclonal IgG. Competitive inhibition studies against human immunoglobulins, representative of VH2, VH3, and VH4 gene families, showed that monoclonal antibody 2G10 is mostly likely to be directed against sequences encoded by either VH3 or VH4 genes. The fact that monoclonal antibody 2G10 binds to the humanized (complementarity determining region (CDR) grafted) CAMPATH-1H antibody, but not to the original rat CAMPATH-1 YTH34.5.6 antibody, indicates that it is directed against a framework region rather than the CDRs. Analysis of amino acids in the VH region for charge, hydrophobicity, and accessibility suggests that reactivity with monoclonal antibody 2G10 is defined by a hexapeptide spanning residues 74-79 within framework region 3. CONCLUSION: The anti-idiotype monoclonal antibody 2G10 could potentially be used as a probe for determining the contribution of the VH3 and VH4 gene segments to antigenic specificity.     

Polyreactive IgM antibodies generated from autoimmune mice and selected for histone-binding activity.

A panel of histone-reactive IgM mAbs was obtained from mice belonging to various spontaneously autoimmune strains. Most of these antibodies were polyreactive, i.e. they showed binding to other cationic antigens (poly-L-lysine, lysozyme, cytochrome c) or to cytoskeletal proteins (actin, myosin, vimentin). The variable regions of these antibodies were encoded by V genes and gene segments belonging to various families. Their H chain third hypervariable regions were unusual in that the D segments were read in all three possible reading frames in contrast to most conventional antibodies and other polyreactive antibodies obtained from normal mice.     

Structure and evolution of mammalian VH families

Antibodies are encoded by a limited number of germline gene segments that undergo somatic diversification through rearrangement and mutation. Because these mutation processes are efficient, it is widely believed that there is little environmental selection pressure for the maintenance of specific antibody gene sequences. We have performed pairwise comparisons of known germline (as opposed to somatically generated) antibody VH elements with the hope of identifying conserved structural features common to sets of VH gene segments. These studies reveal that VH families arose prior to the mammalian radiation and have since been conserved, that this conservation appears to reflect selection at the level of protein sequence, and that the conserved regions are discretely localized on a solvent-exposed face of the heavy chain, at some distance from the antibody combining site. A family-specific region was also identified within the recombinase recognition sequences. Our results provide a context for theories that address the physiological significance of variations in VH family utilization during the development of the immune repertoire.     

Limited repertoire of utilized VH gene segments in a VHa3-allotype-suppressed rabbit.

Between 70 and 90% of serum Ig molecules of normal laboratory rabbits bear one of the serologically defined VHa allotypic specificities, a1, a2, or a3, and are termed VHa+ (a-positive) molecules; the remaining 10-30% of Ig molecules that do not have VHa allotypic specificities are designated VHa- (a-negative). The repertoire of utilized VHa(+)-encoding gene segments has been examined extensively, but only limited studies of the repertoire of utilized VHa(-)-encoding gene segments in normal rabbits have been done. To examine the repertoire of utilized VHa- gene segments, we analyzed VH-encoding cDNA clones from mRNA of a VHa-allotype-suppressed rabbit whose serum Ig was primarily VHa-. For VHa suppression a newborn a3/a3 rabbit was injected periodically with anti-VHa3 antiserum; when it was 2 months of age and its serum Ig was greater than 94% VHa-, cDNA clones were generated from splenic RNA. The nucleotide sequences of eight putative VHa(-)-encoding cDNA clones were compared to those of eight cDNA clones generated from RNA of non-suppressed a3/a3 rabbits. The presumed VHa3-encoding cDNA clones from the non-suppressed rabbits appeared to derive from VH1-a3, the 3'-most germline VH gene segment. In contrast, the VHa(-)-encoding cDNA clones from the suppressed rabbit were distinctly different from VH1 and were most probably derived from germline VH segments other than VH1. Because the expressed VHa- gene repertoire was highly restricted, we propose that VHa- molecules in a3/a3 rabbits may be derived from as few as three germline VH gene segments.     

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.     

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.     

Effect of amino acid substitutions in the heavy chain CDR3 of an autoantibody on its reactivity

In this study, we applied site-directed mutagenesis to the Fab fragment of a mouse IgM (IE12) that was previously shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. Its native structure was very similar to that of a polyreactive natural IgM (ppc15-30). Indeed, they both use the same light chain and the same VH, D and JH segments. However, the N regions differ and the D is translated in two different reading frames, giving different amino acid compositions of the heavy chain CDR3 (HCDR3). Site-directed mutagenesis modified the HCDR3 of IE12 compared to that of the natural antibody and the resulting effects on its reactivity were analyzed. Because the HCDR3 of IE12 is very rich in aliphatic residues, which are hydrophobic, we replaced them with the more hydrophilic residues of the HCDR3 of the polyreactive IgM. In addition, we evaluated the impact of the proline residues in the HCDR3 of IE12 on its activity, because they are known to restrict backbone flexibility. We found that a more hydrophilic HCDR3 conferred to the IE12 Fab a polyreactive profile. Prolines seem to play an important role in this context, because when they were replaced by glycines, the resulting Fab fragments were highly polyreactive. Our results suggest that, for polyreactivity, hydrophilicity and a certain plasticity of the HCDR3 seem to be necessary. Greater flexibility of the CDR, particularly the HCDR3, might be an important characteristic for polyreactive antibodies.      

Human Polyreactive IgM Monoclonal Antibodies with Blocking Activity Against Self-Reactive IgG

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')₂ fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.