V4-34 Gene Research Articles

A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1

A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.

Preferential IgH V4-34 gene segment usage in particular subtypes of B-cell lymphoma detected by antibody 9G4

Immunoglobulin heavy chain locus variable gene segment V4-34 (V H4.21) use in productive heavy chain (IgH) gene rearrangements has been described in a number of human reactive and autoimmune B cell responses, and has been shown to be frequently used in some series of cases of diffuse large cell lymphoma (DLCL). The 9G4 antibody is relatively specific for the V4-34 gene product and can be used to screen for cells that use V4-34 in their productive IgH locus rearrangements. The purposes of this study were to determine the sensitivity of the 9G4 antibody against DLCL cases known to use V4-34, then to screen a variety of human lymphoma types for 9G4 reactivity. Frozen tissue sections were cut from 118 cases of various human lymphomas. Generalized 9G4 membrane reactivity was identified in 78% of DLCL cases known to use V4-34. 9G4 reactivity varied by lymphoma type for the unknown cases, with diffuse large cell lymphoma (30%) and mantle cell lymphoma (28%) showing statistically significant differences ( P < .001) from the expected value of 6% V4-34 positivity in peripheral blood B cells. This nonrandom increased utilization of V4-34 in productive IgH locus rearrangements supports the hypothesis that Ig binding specificity may play a role in lymphomagenesis.

Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.

The V4-34 gene segment is commonly used by human monoclonal IgM alloantibodies against blood group antigens and by cold-reactive red cell autoantibodies with anti-I or anti-i specificity. This study was conducted to determine whether cold agglutinin activity is found among the V4-34-encoded alloantibodies. Fifty-four human IgM monoclonal antibodies (MoAbs) against Rh system antigens were tested for cold agglutinin activity against red cells lacking the relevant Rh system antigen and for reactivity with tissue I and/or i antigens using immunohistochemistry. The findings were correlated with the utilization of the V4-34 segment as determined in an enzyme-linked immunosorbent assay with an antibody (9G4) that is specific for this gene product and were also correlated with other serologic properties. Of the MoAbs, 59 percent were 9G4-positive. Of the 9G4-positive subset, 16 and 44 percent agglutinated native adult (express I) and cord (express i) cells, respectively, at 4 degrees C; these levels rose to 84 and 94 percent, respectively, with the use of papain-treated cells. The red cell antigens recognized at 4 degrees C were cleaved by endo-beta-galactosidase, which is consistent with their being I and i. Of the 9G4-positive subset, 53 percent bound to tissue i antigen. These reactivities were not found among 9G4-negative MoAbs. Endo-beta-galactosidase treatment of red cells enhanced Rh system antibody agglutination by 9G4-negative MoAbs. Anti-I/i reactivity is common among IgM Rh system MoAbs and is shown only by the V4-34-encoded subset. This finding has implications for the use of MoAbs for Rh system typing of blood.

Phage surface expression for analysis of recognition sites of human autoantibodies: Comparison of single chain Fv and Fab

Human autoantibodies can mediate the pathology of autoimmune disease. To plan therapeutic intervention based on inhibition of binding to autoantigen, it is desirable to map the amino acid residues involved in recognition. Bacterial expression provides a simple and rapid system for production and mutagenesis of recombinant antibody fragments, but there are problems with reproducible folding and dispersity of soluble products. Expression at the surface of phage particles avoids both these problems, and allows variable region gene sequences to be expressed as functional monomers. To apply this technology, we have expressed a human anti-DNA monoclonal antibody, derived from a hybridoma, at the surface of the phage. The presence of the phage particle allowed highly sensitive detection of antibody activity by ELISA, so that minimal levels of expression were required. By measuring the amount of expressed antibody protein, it was possible to analyze the antibody activity/microgram. We expressed the antibody as FAB or single chain (sc) Fv, and found binding activities to be closely similar. The parental anti-DNA antibody utilizes the human V4-34 gene and displays the associated conformation-dependent 9G4 idiotope. Both the Fab and scFv expressed the idiotope, confirming similar molecular folding. This technology can be applied rapidly and efficiently to investigations of human autoantibody structure, with the option for expression as either Fab or scFv, and the only requirement being knowledge of VH and VL sequences.

The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells.

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.

Variable region gene segment utilization in Rhesus monkey hybridomas producing human red blood cell-specific antibodies: Predominance of the VH4 family but not VH4-21 (V4-34)

Structural analyses of human immunoglobulin gene segments from monoclonal cell lines provide valuable information regarding the antibody repertoire. This information, in conjunction with a nearly complete knowledge of the human immunoglobulin germline repertoire, now allows further investigation into the underlying molecular basis responsible for some of the observed biases found in the expressed repertoire. One human heavy chain variable region gene segment, V4-34 (VH4-21), is one of the most prevalent gene segments in the expressed repertoire. The overwhelming presence of the V4-34 gene segment suggests that it may play an important role in immune responses. However, there is currently little information regarding its presence and potential importance in nonhuman primates. In order to determine if this gene segment is used by lower primates in a similar manner we determined the molecular structure of the variable region gene segments that are expressed by macaque monoclonal heterohybridomas that are specific for human red blood cell antigens. Eleven of the 12 hybridomas are derived from Rhesus monkeys ( Macaca mulatta) and one is from a cynomologous monkey ( Macaca fascicularis), all of which have been immunized with human red blood cells. The predominance of a VH4-like family and the specific absence of a VH4-21 equivalent led us to further characterize the macaque VH4 gene family at the germline level. Therefore, germline gene segments from the macaque equivalent to the human VH4 gene family are also described.

V H repertoire in human B lymphocytes stimulated by CD40 ligand and IL-4: Evidence for positive and negative selection mechanisms coupled to CD40 activation

In the normal immune system, B cells are thought to be negatively or positively selected at various checkpoints during their maturation; a process that maintains a broad immunoglobulin repertoire while eliminating non-functional or potentially harmful autoreactive antibodies. This study tested the hypothesis that utilization of certain immunoglobulin heavy chain variable region (V H) genes, possibly as a consequence of intrinsic affinity for various ligands, directs positive or negative B cell selection coupled to B cell activation in the periphery during the immune response. The specific prediction that the V H repertoire of CD40-activated B cells would differ from the repertoire of unstimulated cells from the same donor, was tested by assessing V H utilization among human B cell clones grown in vitro, follwing stimulation with CD40 ligand (CD40L) and IL-4. The results showed that, although utilization of the known V H families and of individual V H3 genes was similar to that found in unstimulated B lymphocytes of the same donor, utilization of individual V H4 genes in CD40-activated B cells displayed a pattern that was markedly different from that of the unstimulated B cells. An allele of V4-61, V4-61b, was over-represented among the activated cells and, in contrast, the V4-34 gene (known to encode cold agglutinins with strong autoreactive properties) was modestly represented among the V H4 activated B cells, although V4-34 was overwhelmingly predominant in the repertoire of resting B cells. These results point to the existence of selection mechanisms that operate during B cell activation in the periphery. These mechanisms may favor B cells utilizing certain V H genes and disfavor the cells that utilize other genes, possibly because utilization of the latter confers autoreactivity.

High Frequency of Somatic Mutations in the VH Genes Expressed in Prolymphocytic Leukemia

Prolymphocytic leukemia (PLL) is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed lg heavy chain variable region (VH) genes in eleven cases of de novo PLL Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%), with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed VH genes diverged from their putative germline counterpart, and in eight cases the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and VH4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of lg VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.

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.

Predominant V‐Region Gene Configurations in the Human Antibody Response to Haemophilus influenzae Capsule Polysaccharide

The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V-region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin-conjugated Hib PS vaccine. The VH gene segments that encode for these antibodies belong to the VH3 gene family, of which two are related to the V3-23 gene and two to the VH3b subfamily. Both hybridomas that express a V3-23-related gene use short D-segments (3 bp), the JH6 gene segment and a V kappa gene derived from the A2 germline gene. The two hybridomas that express VH3b genes use D-segments of conventional length (24-33 bp), the JH4 gene segment and a non-A2 V kappa gene. Comparison of our sequences with those reported by others suggests that the above patterns of V-region gene segment association exemplify two V-region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B-1 B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.