Chimaeric monoclonal antibodies encoded by the human VH26 gene from naïve transgenic mice display a wide range of antigen-binding specificities.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.     

Isolation and sequence of a cDNA coding for the immunoglobulin mu chain of the sheep.

A sheep cDNA library was screened with a human C mu probe, and the complete nucleotide sequence of a 1923 nt cDNA was determined. It contains sequences corresponding to all the exons (VH, DH, JH, CH1, CH2, CH3 and CH4) characteristic of the immunoglobulin mu heavy chain regions. The deduced amino acid sequence shows a percentage of identical residues in the range 65-45% when compared with the mu chains of various species. The VH region of this clone is clearly related to a group of genes that includes mouse VH36-60 and VHQ52, human VH2, VH4 and VH6 gene families and Xenopus VHII gene families. The constant region shows an unusual repartition of cystein and proline residues at the beginning of the CH2 domain, that may result in a molecule with enhanced stability and reduced flexibility.     

建立Ig基因富集的DNA库进行2F7单克隆抗体基因克隆

用小鼠型抗人小细胞肺癌单克隆抗体的2F7杂交瘤细胞DNA,经限制性内切酶EcoRI或BamHI完全酶解,通过Southern blot,用小鼠Ig基因Cr_(2a)、V_(H)、J_(H)和E_(H)作探针,富集阳性DNA,同时用pSV_2neo质粒为载体与这些DNA重组,经筛选获得了一批杂交阳性的克隆。     

Production of heavy-chain class-switch variants of human monoclonal antibody by recombinant DNA technology

We have previously established a human-mouse heterohybridoma (H6-3C4), which produced a human sperm-immobilizing antibody (mu, lambda of human type). The human rearranged immunoglobulin mu-chain and lambda-chain genes were cloned from the hybridoma H6-3C4. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglobulin gamma 1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switch from mu-chain to gamma 1-chain of H6-3C4 antibody. The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation. The stable transformants produced the human IgG monoclonal antibody, which fully retained specificity to human sperm cells and sperm-immobilizing activity.     

Endogenous immunoglobulin expression in mu transgenic mice

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.     

Construction of representative immunoglobulin variable region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation

We have developed a sensitive and specific method for preparation of representative IgM and IgG cDNA libraries of peripheral blood lymphocytes without in vitro stimulation of B cells and without primer-based bias. The procedure involves preparation of double-stranded cDNA from initial C mu and C gamma constant region primers. Linkers are attached to the cDNA and nonselective polymerase chain reaction (PCR) amplification is performed with the linkers as primers. This product is ligated to M13 RF DNA, and selective PCR amplification is achieved with a nested constant region primer and a vector primer. The product is cloned into M13 phage. More than 85% of the white plaques are positive with a JH probe. Diversity of the library is confirmed by hybridization with VH gene probes and by DNA sequencing. A sample of 7-30 ml of blood from a normal human adult can yield both mu and gamma cDNA libraries. Analysis of the libraries yields a picture of Ig variable gene usage at the time of sampling.     

Suppression of VH, C gamma and C mu gene products in rabbits by maternal immunization against IgM allotype and detection of somatic recombinant molecules in these animals

Suppression of the mu-chain allotype Ms16 was obtained in young heterozygous Ms16/Ms17 rabbits by immunizing the Ms17 mother against the paternal Ms16 allotype. Examination of the concentration of the VH and CH allotypes of paternal origin (a2,Ms16 and e14) in the offspring, revealed not only Ms16 suppression but also VH and C gamma allotypic suppression. The degree of suppression for the C gamma markers (4-25-fold decrease) was much less than that for the C mu or VH markers (50-150-fold decrease). The excess C gamma marker (e14) was found to be present for the major part on molecules possessing the VH allotype derived from the homologous allelic chromosome. The pattern of persistence of molecules with the suppressed C gamma marker in the serum is consistent with the idea that these molecules arise by somatic recombination and that the gene order is VH, C mu and C gamma.     

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.     

VHDJH to JH joining is not blocked in mu-chain-producing pre-B cells, implying the breakdown of allelic exclusion

Intracytoplasmic mu-positive pre-B cells (mu+VH315- cells) transformed with Abelson virus continuously produced cells (mu+VH315+ cells) that were double-stained by anti-mu and anti-VH315 antibodies which specifically recognized the immunoglobulin heavy chain variable region identical or closely related to that of MOPC315 myeloma protein. Southern blot analysis indicated that the mu+VH315+ cells were generated from the mu+VH315- cells by the VH315.D.JH2 to a germline JH3 joining on the non-productive VH315.D.JH2 allele, which resulted in the production of mu-chains with variable region detectable by anti-VH315 antibodies. Therefore, it is strongly indicated that VHDJH to JH joining is not blocked in mu-chain-producing pre-B cells, and thus is free from allelic exclusion machinery.     

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.     

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.     

Crosslinking of the cell surface immunoglobulin (mu-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin kappa locus.

We constructed an expression vector encoding a truncated Ig mu chain that lacks both VH and CH1 domains (mu delta m chain) and introduced the mu delta m vector into the Ig negative Abelson pre-B cell line P17-27. The transfectants expressed a large amount of the mu delta m chain on their surface, which was not complexed with the lambda 5 and VpreB surrogate light chain molecules. While P17-27 transfected with a vector for the intact micron chain (P17-27 micron) shows V kappa rearrangements in culture, V kappa rearrangements were not detected in P17-27 mu delta m cells. When the mu delta m chains on the cell surface were crosslinked by anti-mu antibodies, V kappa gene rearrangements were induced in P17-27 mu delta m. These results strongly suggest that crosslinking of the micron-lambda 5-VpreB complex on the pre-B cell surface generates a signal that activates V kappa gene rearrangement, and that the lambda 5 and VpreB molecules are necessary for the spontaneous crosslinking of surface Ig on pre-B cells.