IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

Shared N-terminal sequences in monclonal IgMkappa and IgGkappa proteins from a patient with a complex multiple paraprotein disorder.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgM kappa and IgG kappa paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgC, and IgA proteins.     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

Preferential use of lambda L chain in lamin B autoantibodies

The generation of lamin B autoantibodies was examined in a patient with SLE. Lamin B autoantibodies in this individual's serum were almost entirely IgG1 lambda at the onset of the autoimmune response. There was no evidence of IgM to IgG class switching, and no apparent diversification of the autoantibody response over the next 2 yr. The kappa/lambda L chain ratio of the lamin B autoantibodies was 0.13 by ELISA, with a kappa/lambda ratio of 0.70 for the patient's total IgG. In contrast, anti-histone autoantibodies in the patient's serum contained primarily kappa L chain. The kappa/lambda L chain ratios of lamin B autoantibodies from sera of four other patients with SLE were also consistent with restricted heterogeneity of lamin autoantibodies. In two, lambda L chain predominated, and in two others, the lamin B autoantibodies contained predominantly kappa L chains. The restricted heterogeneity of lamin B autoantibodies could be related to targeting of a single immunodominant autoepitope, to the existence of a particular L chain gene or genes that preferentially undergo somatic mutation, leading to the production of lamin B reactive clones, or to a combination of both mechanisms.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

B cell maturation in the chicken Harderian gland

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.     

Immunoglobulin light and heavy chain isotypes in skin diseases: restricted distribution in bullous pemphigoid and linear IgA bullous dermatosis

To assess the heterogeneity of immunoglobulins involved in various skin diseases, direct and indirect immunofluorescence studies of skin biopsies and sera, respectively, for kappa and lambda light chains, were performed. The anti-basement membrane zone (anti-BMZ) antibodies of patients with bullous pemphigoid showed a predominance of kappa light chains, and patients with linear IgA bullous dermatosis showed a predominance of one light chain that was sometimes kappa and sometimes lambda. The bullous pemphigoid autoantibodies were then studied for IgG subclass distribution; a predominance of IgG4 was found. Although other explanations are possible, the light chain restriction in bullous pemphigoid most likely reflects heavy chain restriction and preferential association of heavy and light chain isotypes. The basis of the heavy chain restriction is not apparent. The light chain restriction in linear IgA bullous dermatosis may represent a restricted idiotypic repertoire.     

Surface mu heavy chain expressed on pre-B lymphomas transduces Ca2+ signals but fails to cause growth arrest of pre-B lymphomas.

Little is known about the role of signals transduced by cell surface IgM (sIgM) expressed during early B cell development. A subclone (1.6) of the late pre-B cell lymphoma 70Z/3.12 was used to study signal transduction by surface mu heavy (H) chain before and after transition to the early immature B cell stage, and the functional consequences thereof. Although kappa L chain expression can be induced on 1.6 cells by LPS or cytokines, immunoprecipitations indicated that the non-induced 1.6 cells expressed mu H chain with an alternative protein(s) which may be a surrogate light chain(s). Consistent with this, anti-mu but not anti-kappa or anti-lambda antibodies caused transient Ca2+ mobilization in noninduced 1.6 cells. The Ca2+ signal was derived from both intracellular stores and Ca2+ influx in either noninduced cells or in cells that had been preinduced to express kappa L chain. Thus, the ability of mu H chain to mobilize Ca2+ as a second messenger does not depend upon the expression of mature L chains. The immature B lymphomas, WEHI-231 and CH1, express mature forms of IgM and undergo growth arrest when stimulated by anti-mu antibody. In contrast, signals generated by mu H chain on either noninduced or preinduced 1.6 cells or in the sIgM+ pre-B cell transfectant 300-19 mu lambda 36/8 did not cause growth arrest. These results suggest that mu H chain expressed on pre-B cells is capable of mobilizing Ca2+, but that this signal alone is insufficient to induce growth arrest in the pre-B cell.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Immunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by replication-competent Rauscher murine leukemia virus: transformation of B cells at various stages of differentiation

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.     

Duplicated variable region genes account for double isotype expression in a human leukemic B-cell line that gives rise to single isotype-expressing cells.

The SSK41 cell is an immunoglobulin IgM+ human neoplastic B-cell line that switches to IgG+ cells (SSK gamma) spontaneously. We isolated a derivative of SSK41 (SSKWB) that expressed both IgM and IgG. Studies on the Ig heavy chain gene organization have shown that the SSKWB cell had two identical VDJ genes on the same chromosome; one linked to the C mu gene and the other to the C gamma 1 gene, both of which are transcribed to produce mu- and gamma-mRNAs with the same VDJ sequence. We also isolated the two switch variants derived from the SSKWB cell by cell sorter: 1G (IgG+) and 11M (IgM+). The 1G cells contained two populations; one had a similar genetic organization as SSK gamma and expressed only IgG1, and the other carried the same genetic organization as the SSKWB cell but produced aberrantly spliced mu-chain mRNA, in which the hydrophobic signal sequence exon is directly joined to the C mu exon. Te 11M cell deleted the VDJ-C gamma 1 segment of the SSKWB cell and expressed IgM. These results raise the interesting possibility of another mechanism for class switching.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

人源噬菌体抗体库的构建及抗VEGF抗体的初步筛选分析

应用噬菌体表面呈递技术构建人抗体组合文库 .筛选获得了结合血管内皮细胞生长因子( VEGF)的人噬菌体 Fab抗体 ,并对所获抗体的多样性进行了进一步分析 .从不同人群外周血淋巴细胞提取总 RNA,经反转录后采用家族特异性免疫球蛋白可变区基因引物与免疫球蛋白信肽序列引物 ,通过改变 PCR条件或半套式扩增分别获得全部亚型的轻、重链抗体 Fab段 ,并重组到噬粒载体 p Comb3H中 ,经电转化大肠杆菌 XL- 1 Blue,构建了 1 .5× 1 0 8完整组合抗体库 .利用 VEGF12 1对该库经过 4轮固相筛选后 ,获得 1 2个可与 VEGF特异结合的阳性克隆 .酶谱分析表明了所获抗体克隆的多样性 .为通过基因工程改造 ,进一步获得可用于临床的人源 VEGF抗体奠定了基础 .     

Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.     

Light chain determines the binding property of human anti-dsDNA IgG autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies.