VH Sequences Research Articles

Use of phage surface expression to analyze regions of human V4-34(VH4-21)-encoded IgG autoantibody required for recognition of DNA: no involvement of the 9G4 idiotope.

The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.

Heavy-chain variable regions in carcharhine sharks: development of a comprehensive model for the evolution of VH domains among the gnathanstomes.

We determined the sequence of 18 DNA clones encoding VH regions of sandbar shark and bull shark. All of these sequences exhibit key structural coding features characteristic of known VH genes of higher vertebrates. These VH sequences disclosed considerable diversity, and can be divided into six families according to the criterion of 80% DNA sequence identity. The overlapping of some VH gene clones to two or more families is a particular feature found in carcharhine sharks, which suggests that VH diversification is a continuing process. The basic sequence patterns of heavy-chain V regions found in all representative gnathanstomes and in VH of the shark heavy immunoglobulin IgW provides evidence for selection of canonical residues in all VH structures. Elasmobranch VH sequences can be divided into two classes or clans, one comprising the 'classical' VH set and the other comprising VHS related to those of IgW (V omega). Phylogenetic analyses place the VH cluster as the root of all the classic VHS and indicates that the V omega set is most probably that of the primordial heavy chain.

The generation of antibody diversity in the turtle.

The Ab response in reptiles has been studied at the protein level, and in turtles some aspects resemble those of cold-blooded vertebrates from other classes. The genetic bases for these features are not clear. The present study is the first on the IgH organization and complexity of a reptilian Ig gene system. The approach to cloning turtle (Pseudemys scripta) sequences is entirely PCR based, and its efficacy is demonstrated by obtaining extensive information on a heretofore unexplored Ig gene system. A number of genomic VH sequences, representing possibly four families, were isolated, as was a genomic C mu 4 clone. These sequences, used as probes, provided proof that in the turtle there is a single IgH locus with multiple VH genes and one C mu gene. In Northern hybridizations, the C mu 4 probe detected two transcripts; of the four VH groups, only one was expressed, and multiple bands indicated the presence of at least two non-mu transcripts. Using reverse transcription-PCR on spleen or liver RNA, an IgM heavy chain sequence was obtained, as were a number of VDJ rearrangements. Among 32 unique VDJ rearrangements from one animal, there were 22 sequence variants at framework 4, suggesting either a very large number of J segments or somatic modification in the variable region. The latter interpretation is supported by point mutations found in framework 3 and CDR3. The number of changes is considerably greater than the deduced Taq misincorporation rate (0.05%).

VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4- 34) gene

The immunoglobin heavy chain variable region (VH) gene usage in multiple myeloma (MM) has not been reported, although a few studies have incidentally identified the VH gene rearranged in small cohorts of MM patients. We used a reverse transcriptase-polymerase chain reaction based technique to analyze the VH gene usage in MM. The VH sequences were obtained after amplification of bone marrow cDNA using the seven VH family-specific and constant region primers. The VH sequences of 72 patients were successfully identified. The frequency of VH family usage in decreasing order was VH3>VH4>VH1>VH5>VH2>VH6>VH7 and corresponded to the functional germline complexity of the VH families. Individual VH genes (VH1-69, VH3-9, VH3-23, and VH3-30) were overrepresented in our cohort of MM patients; some VH genes [VH3-49, VH3-53, and VH4.21 (VH4-34)], which are rearranged with increased frequency in normal circulating B cells, autoimmune diseases, and other B-cell malignancies, were not detected in any MM patient. Compared with germline sequences, an average of 8.8% (range, 2.7% to 16.5%) of the nucleotides had evidence of mutation within each VH sequence. Based on these results, we conclude that (1) the VH gene usage in MM is unique compared with other malignant and nonmalignant B-cell populations, (2) the physiologic process of clonal deletion functions to remove clones that have rearranged VH genes (VH4.21) capable of expressing antibodies, which recognize self-antigens, and (3) the complete lack of VH4.21 gene rearrangement may help to partially explain the paucity of autoimmune phenomena in MM.

Ig VH Gene Mutational Patterns Indicate Different Tumor Cell Status in Human Myeloma and Monoclonal Gammopathy of Undetermined Significance

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig-secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.

Light chain shuffling of a high affinity antibody results in a drift in epitope recognition

Human polyclonal and monoclonal antibodies against pathogens and toxins are potentially useful in the treatment of various diseases. A number of human monoclonal antibodies with protective capacity in vitro have been established by conventional hybridoma technology. However, with the development of phage-display technology, the possibility of specifically tailoring antigen-binding properties has improved substantially. We show here that the reactivity of a high affinity, virus-neutralizing human antibody against the AD-2 epitope of cytomegalovirus gB can be modified by introducing other Vκ sequences together with the original VH sequence. The fine specificity, as determined by the requirement of particular amino acid residues in the epitope, is shifted in these new antibody fragments. It was also evident that the VH Vκ pairing was not promiscuous, since antibody fragments selected by phage display retained light chain sequences very similar to the original hybridoma-derived light chain, proving that a high affinity interaction was very dependent on a co-operativity between both variable domains. These findings show that phage display technology might modify the binding properties of pre-existing, high affinity antibodies.

Genetic control of the humoral immune response to xenografts. II. Monoclonal antibodies that cause rejection of heart xenografts are encoded by germline immunoglobulin genes.

The early phases of the rejection of xenografts exchanged between closely related species are dominated by a vigorous humoral immune response. We have recently used a linker-mediated polymerase chain reaction (LM-PCR) to generate Ig heavy and light chain-specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR-1 VH with the germline equivalent of this gene isolated from newborn LEW liver (provisionally designated VHHAR-1) showed that the two VH sequences share a nucleic acid identity of 99.3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98.2% identical with the JH1 nucleic acid sequence available in the GeneBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the result of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that is responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

Analysis of VH Gene Expression in CD5+ and CD5− B-Cell Chronic Lymphocytic Leukemia

In contrast to highly mutated follicular lymphomas and multiple myelomas, chronic lymphocytic leukemias (CLLs) frequently express VH genes in germline configuration. It is currently unclear whether this difference is related to the expression of CD5 or to the differentiation stage of the B cell when malignant transformation occurs. We have studied the VH sequence of 11 cases of CD5- B-CLL to address the question whether CD5- B-CLL are derived from naive pregerminal B cells (low mutation pattern) or from germinal center-derived memory B cells (high mutation pattern). Among the 12 detected rearrangements (2 distinct rearrangements in 1 case) VH1 family was found in 2, VH2 in 2, VH3 in 4, and VH4 in 4. Nine different VH genes were detected among the 12 rearrangements, including 2 cases expressing V1-69 (51p1) and 1 case expressing V4-39 (VH4.18), previously reported to be overexpressed in CD5+ B-CLL. A higher mutation pattern, following a random distribution, was observed when compared with classical CD5+ B-CLL. However, as reported in normal B cells, these results appeared to be related to membrane Ig phenotype (less mutations in membrane mu delta-expressing forms in leukemias expressing exclusively membrane mu). Overall, the differences found when comparing the mutational profile with classical CD5+ B-CLL were not clearcut and might be explained more by the membrane isotype (mu v mu delta) than by CD5 expression.

VH3-21 B Cells Escape from a State of Tolerance in Rheumatoid Arthritis and Secrete Rheumatoid Factor

Rheumatoid factor (RF) is a characteristic but not pathognomic feature in patients with rheumatoid arthritis (RA). It is unknown whether the repertoire of immunoglobulin genes utilized by RF+ B cells of RA patients is unique and whether RF+ B cells in normal individuals are silenced or deleted. Clonal B cell populations were established from the peripheral blood of normal donors (127 B cell clones), RA patients (113 RF- and 60 RF+ B cell clones) and patients with primary Sjögren's syndrome (82 RF- and 47 RF+ B cell clones) by coculturing with anti-CD3-stimulated T helper cell clones. The cross-reactivity pattern of antibodies secreted by the B cell clones was determined by ELISA on a panel of antigens. The molecular structure of the IgM heavy chains was characterized by VH family-specific RT-PCR and sequencing. VH elements which correlated with RF specificity were identified. The responsiveness of B cells expressing these VH elements to T helper cell signals was compared in normal individuals and RA patients. The majority of RF+ B cells were monospecific when specificity was tested on five antigens. RF+ B cells expressed a significantly different repertoire of VH gene segments than RF- B cells. In particular, the VH3 gene segment V3-21 was not detected in B cell clones from normals but was the most frequent VH element in RF+ B cell clones from RA patients. Most of the V3-21 sequences were in germline configuration. The correlation between RF specificity and V3-21 gene segment usage was maintained in patients with Sjögren's syndrome. V3-21 transcripts were present in peripheral blood B cells from normal individuals. VH3-21+ B cells from RA patients but not from normal donors were responsive to preactivated T helper cells. Stimulation with a bacterial superantigen could overcome the nonresponsiveness of V3-21+ B cells in normal donors and induce the secretion of RF. RF production is correlated with the usage of the V3-21 gene segment in two distinct RF+ diseases. In patients with these diseases, V3-21+ B cells secrete antibodies with RF activity in response to activated T helper cells. V3-21+ B cells remain in a state of nonresponsiveness in normal individuals that can be broken by superantigen stimulation. The germline configuration of VH3-21+ RF+ immunoglobulins in RA patients suggests that the loss of tolerance is not an antigen-driven process.

Analysis of anti-GM1 ganglioside IgM antibodies cloned from motor neuropathy patients demonstrates diverse V region gene usage with extensive somatic mutation.

Serum anti-GM1 ganglioside Abs are abnormally elevated in patients with chronic motor neuropathies and Guillain-Barré syndrome. We have cloned six anti-GM1 IgM Abs from PBMCs of five affected patients. VH and VL gene analysis demonstrated substantial somatic mutation, predominantly clustered around VH CDR2 and FR3 in all six Abs. The Abs SM1, DO1, and WO1, isolated from three different patients, are encoded by closely related VH gene segments with restricted canonical structures. The corresponding L chains are encoded by V lambda II and V lambda VIII genes and also share the same canonical structures, containing nucleotide deletions at the VL/JL boundary to form a VLCDR3 of 10 amino acids. DO1 and WO1 VH segments show nucleotide identities of 95.6% and 88.8%, respectively, to the VH3 member 1.9III, and SM1 shows 87.8% identity to the closely related VH3 member, Hv3005. The Abs BO1 and BO3, derived from a single patient and containing the same VH-D-JH and VL-JL rearrangements, are encoded by the VH3 gene, HHg19, with nucleotide identities of 92.2 and 91.2%, respectively, and have both common and unique somatic mutations. The VH sequence of BR1 has 91.3% identity to the VH4 member, V71-2. These data indicate that neuropathy-associated anti-GM1 IgM Abs can be encoded by both diverse and closely related V genes in association with restricted canonical structures, and that their V genes are somatically mutated, consistent with an origin through an Ag-driven immune response.

Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin.

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

Analysis of Ig VH region genes encoding IgE antibodies in splenic B lymphocytes of a patient with asthma.

An atopic patient with hypersensitivity against house dust mite died as a result of an asthmatic attack. A portion of the spleen was obtained and was used to analyze the spectrum of Ig heavy chain V regions involved in encoding IgE Abs. A nested PCR technique generated 14 cloned VH sequences that had distinct CDR3 regions; 5 of 14 were derived from the minor VH5 family, and the remainder derived from the larger families, VH3 (6 of 14) and VH4 (3 of 14). One of the VH3-derived sequences was present as a repeated sequence in three clones. A control PCR with the same VH primers in combination with JH primers yielded only 1 of 13 sequences from VH5, indicating preferential VH5 usage only for IgE. Analysis of VH5-C epsilon sequences revealed usage of a single gene, DP73, with extensive mutations and several "hot spots" containing common replacement amino acids. However, there was no concentration of replacement mutations in the CDRs, which conventionally would indicate a role for Ag selection. The VH3 and VH4 genes in combination with C epsilon also harbored extensive somatic mutations. From these findings in splenic B lymphocytes, and those of a previous study of blood lymphocytes, it seems that preferential usage of VH5 genes and extensive somatic hypermutation are characteristic of B cells synthesizing IgE in patients with allergic disease.

Sequence analysis and antigen binding characteristics of Ig SCID Ig+ mice.

SCID mice as they age may develop a limited number of functional B and T cell clones. VH and VL gene sequences of hybridoma antibodies derived from the spleens of five adult SCID Ig+ mice revealed nine clonally unrelated, i.e. derived from different precursor lymphocytes, groups of Ig. Of these, four used the VH7183 family, one Q52, three J558 and one VH3609. VH7183 was seen in excess over J558 despite a 5-fold larger number of J558 family members in the mouse genome. Preferential use of D proximal VH genes along with reduced N region addition in the heavy chain VDJ joins of these antibodies suggest derivation from a population of lymphocytes with characteristics of the perinatal repertoire. All of the hybridomas produced kappa light chains of several different VL family groups. Sequences of 23 VH and 22 VL genes of hybridomas derived from one mouse (no. 45) showed that they were all clonally related, differing only in somatic mutations. Autoreactivity of these antibodies to cellular antigens [including (U1)RNP-specific A protein and SmB] was analyzed, and shown to correlate with the geneology and specific mutations within members of the clone. The geneological tree derived from sequence data permitted a study of the microevolution of a clone of B cells and showed that as it developed there was a tendency toward increased strength of self-reactive binding as well as alterations in specificity toward these autoantigens. Analysis of replacement:silent mutation ratios within members of this clone reflected limited affinity maturation in mouse 45 and none for another mouse, no. 58. These data are consistent with the progressive differentiation of B cells in an immunodeficient state in the absence of clonal competition with B cells of similar antigen specificities as they populate the spleen.

The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition.

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.

A new phage display system to construct multicombinatorial libraries of very large antibody repertoires

We present an easy and efficient technique for the construction of large phage-displayed antibody (Ab) repertoires through the recombination of two separate heavy (VH) and light (VL) chain gene libraries. Here, the system has been applied to the display of a chimpanzee anti-HIV gp160 Ab. The process, which makes use of λ phage att recombination sites, leads to the irreversible physical association between plasmid and phagemid vectors carrying, respectively, VL and VH sequences. The heat-inducible expression of the Int recombinase allows perfect control of recombination. Selection of the recombinant phagemid is made possible by the assembly, in vivo, of a genetic marker (chloramphenicol resistance) created only after the correct recombination event. Theoretically, all possible associations between the VL and VH sequences should be obtained, and it should be possible to generate multicombinatorial libraries of close to 1012 clones.

Three new cross-reacting idiotopes as markers for the products of two distinct human VH3 genes expressed in the early repertoire.

This article describes characterization of three new cross-reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing VH3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) VH3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti-idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fc mu, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti-idiotypic MoAbs were shown to be VH-specific reagents by the independence of their reactivity upon the L-chain type, or the antigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for VH3 gene-products was demonstrated by its sole cross-reactivity with VH3 proteins. Each anti-Id had a different reactivity pattern with a panel of MoAbs utilizing different VH3 genes. By relating the VH sequences of the tested VH3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47-encoded proteins, although 3H1 had weak cross-reactivities with a few other VH3 gene-products. 7B4 appeared to be specific for antibodies utilizing DP46-related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize VH3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46-related gene products, B6 and D12 additionally recognized non DP46-related proteins and were thus different to 7B4.

Evolution of T cell receptor genes. Extensive diversity of V beta families in the Mexican axolotl.

We have cloned 36 different rearranged variable regions (V beta) genes encoding the beta-chain of the T cell receptor in an amphibian species, Ambystoma mexicanum (the Mexican axolotl). Eleven different V beta segments were identified, which can be classified into 9 families on the basis of a minimum of 75% nucleotide identity. All the cloned V beta segments have the canonical features of known mammalian and avian V beta, including conserved residues Cys23, Trp34, Arg69, Tyr90, and Cys92. There seems to be a greater genetic distance between the axolotl V beta families than between the different V beta families of any mammalian species examined to date: most of the axolotl V beta s have fewer than 35% identical nucleotides and the less related families (V beta 4 and V beta 8) have no more than 23.2% identity (13.5% at the amino acid level). Despite their great mutual divergence, several axolotl V beta are sequence-related to some mammalian V beta genes, like the human V beta 13 and V beta 20 segments and their murine V beta 8 and V beta 14 homologues. However, the axolotl V beta 8 and V beta 9 families are not significantly related to any other V beta sequence at the nucleotide level and show limited amino acid similarity to mammalian V alpha, V kappa III, or VH sequences. The detection of nine V beta families among 35 randomly cloned V beta segments suggests that the V beta gene repertoire in the axolotl is probably larger than presently estimated.

Isolated VH4 heavy chain variable regions bind DNA characterization of a recombinant antibody heavy chain library derived from patient(s) with active SLE.

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with VH specific oligonucleotide primers. The VH fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to 32P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding VH fragments revealed that they utilized the VH gene family previously described to be associated with autoimmune responses, with a JH6 segment. On VH sequence analysis only one residue substitution in the consensus sequence is needed to form a VH4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of VH regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline VH4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.

Structure-function studies of anti-3-fucosyllactosamine (Le(x)) and galactosylgloboside antibodies.

We are studying murine mAbs against two carbohydrate epitopes, 3-fucosyllactosamine (Le(x), CD15) and galactosylgloboside. The VH domains of both panels of Ab are encoded by VH441, a member of the X24 family of Ig genes. To evaluate the contribution of the heavy chain CDR3 to the affinity of the anti-3-fucosyllactosamine Ab, CDR3-H of PMN6, a low affinity Ab, was replaced by the CDR3 of PM81, a higher affinity Ab. The affinity of the chimeric 6/81 Ab was increased when the heavy chain was paired with the PM81 light chain, but not when paired with another light chain (M5), which differs from PM81 light chain by three amino acids. To evaluate the contribution of somatic mutations to the binding of GalGb4, the 3A9 VH sequence, which contains three amino acid substitutions, was replaced by a germ-line sequence encoded by either VH441 or VHX24. The chimeric Ab, 441/3A9 and X24/3A9, bound Ag as well as the wild-type 3A9 Ab. Computer models of the Fv fragments of PM81 and 3A9 were compared with the crystal structure of the Fv fragment of J539, a galactan-binding myeloma protein that is encoded by the same VH and VK genes as 3A9. The surfaces of 3A9 and J539 have shallow pockets that are potential Ag-binding sites. Replacement of CDR3-H Tyr99, which is a prominent component of the pocket, by Ala abolished the binding of Ag. In contrast, the Fv surface of PM81 contains a large cleft rather than a pocket. These models indicate how the same VH gene segment can be used to encode Abs that exhibit different specificities.

Mantle Cell (Previously Centrocytic) Lymphomas Express VH Genes With No or Very Little Somatic Mutations Like the Physiologic Cells of the Follicle Mantle

To clarify whether mantle cell lymphomas (MCLs) are related to naive pre-germinal center B cells (expressing nonmutated rearranged VH genes) or to germinal center-derived memory B cells (expressing mutated rearranged VH genes), clonal IgH gene rearrangements using DNA from six MCLs were polymerase chain reaction (PCR)-amplified and analyzed for the presence of somatic mutations. For comparison, six cases of B-chronic lymphocytic leukemia (B-CLL) known to display nonmutated rearranged VH genes and six cases of follicle center lymphomas (FCLs) known to display mutated rearranged VH genes were included in our study. The VH region sequences of the six MCLs showed, like those of the six B-CLL cases, no or very little somatic mutations, in contrast to the VH sequences of the six FCL cases, all of which were highly mutated. This is indicative of MCL, like B-CLL, being derived from naive pre-germinal center B cells and furthermore underscores the postulated relationship of MCLs with the B cells of the normal follicular mantle zone because it has recently been shown that this mantle zone is solely or at least largely composed of naive (nonmutated) B cells.