Functional analogues of the VKOx1 gene in different strains of mice: evolutionary conservation but diversity based on V-J joining

Monoclonal antibodies to the hapten phenyloxazolone were raised 7 days after immunization in mice of six strains (BALB/c, C57BL-Igha, DBA2, RF, A/J, and CE). Hybridomas were selected that produced 260 idiotype-positive antibodies, and their light chain mRNA were partially sequenced. (RF is an idiotype-negative strain, and sequencing was done without this selection.) All newly sequenced BALB/c, C57BL-Igha, DBA/2, A/J, or CE VK segments had a 100% nucleotide homology with the VKOx1 (H3) germline gene. This gene codes for one third of early BALB/c phenyloxazolone antibodies, and according to our results the same gene has a significant role in the early response of at least five strains of mice. Four RF hybridomas had identical nucleotide sequences, suggesting that they express a non-mutated nucleotide sequence of a new VK germ-line gene (VKOx2). This gene codes for a CDR1 which is two amino acids longer than the CDR1 coded by the VKOx1 gene, but otherwise the two genes are related (94.5% sequence homology). All but one of the 16 kappa chains studied had the J5 segment; this segment had the same sequence in all six strains. One RF antibody had the J4 segment the nucleotide sequence of which differs from the BALB/c J4 segment in two places. Three of the kappa chains had an extra long CDR3. Long and "normal" kappa chains were probably coded by the same pair of germ-line genes (VKOx1 and J5, or VKOx2 and J5). The length heterogeneity was probably caused by a lack of precision in VK-JK joining.     

Generation of heavy chain-loss mutants in a B cell hybrid mediated by syngeneic idiotype-specific spleen cells

Idiotype-specific spleen cells from appropriately primed BALB/c mice cause a marked and irreversible suppression of the membrane and secreted forms of idiotype-positive immunoglobulin (Ig) of an antigen-specific B cell hybrid clone (2C3E1). The suppression of this BALB/c B cell line has been observed in vitro and in vivo, and appears to require intimate contact between effector spleen cells and target 2C3E1 cells. The observed suppression in the 2C3E1 cell line is due to an induced mutation or a selection of pre-existing mutants within the 2C3E1 cell population, because the resultant light and heavy chain-loss variants are phenotypically stable in vitro and in vivo in the absence of any further active suppression. Biochemical analysis of the 2C3E1 cells after this suppression indicates that all of the variants are negative for the production of idiotype-positive Ig. Heavy chain synthesis by the variants is almost totally eliminated, and light chain synthesis is decreased by 10 to 90%. Spleen cells from identically primed nude mice do not induce any alteration in the 2C3E1 cell line, suggesting that induction or selection of the heavy and light chain-loss mutants requires the presence of mature T lymphocytes. The generation of idiotype-negative 2C3E1 variants during the period of tumor growth in the spleen (but not elsewhere) may represent one mechanism by which this tumor escapes the host's immune recognition.     

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.     

Monoclonal antibodies from mice bearing polyoma virus-induced tumor

Summary Monoclonal antibodies were produced by fusing NS1/1 myeloma cells with splenocytes from A. BY mice bearing syngeneic polyoma virus-induced SEYF-a tumors.From six separate fusion experiments 514 hybridomas were obtained, 45 of which were found to secrete SEYF-a-binding antibodies. The binding patterns of antibodies secreted by eight hybridomas to a panel of tumor cells and to normal mouse fibroblasts were analyzed by means of an indirect radioimmunoassay. Seven hybridomas were found to secrete antibodies that bound to all cell lines tested. This indicated that certain SEYF-a-associated antigens are widely distributed on a variety of seemingly nonrelated tumor cells.One hybridoma secreted antibodies that exhibited a high binding activity to SEYF-a cells, a low binding activity to two members of the tumor panel, and none at all against most of its constituents, including normal fibroblasts. The results of the binding experiments were further supported by absorption experiments.A subclass analysis of the immunoglobulins secreted by the various hybridomas revealed that three clones secreted IgG1; one clone secreted IgM; and three clones secreted IgG2a. Polyacrylamide gel electrophoresis of two of the secreted antibodies indicated a high degree of homogeneity of the heavy and the light chain of the corresponding antibodies, as would be expected from monoclonal products.The results of this study demonstrate the feasibility of obtaining anti-tumor monoclonal antibodies from tumor bearers, representing the immune response of the tumor bearer against antigens associated with his syngeneic tumor.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Idiotype-specific T lymphocytes responsible for the selection of somatic variants of a B cell hybrid

A B cell hybrid (2C3E1), which when cultivated in vitro stably expresses a serologically defined private idiotype on the cell surface, undergoes a somatic variation which culminates in the generation of idiotype-negative variants when propagated as a tumor in syngeneic BALB/c mice. Lyt-1+ and Lyt-2- BALB/c T cells derived from appropriately primed spleen cells when co-cultured with 2C3E1 tumor cells in vitro are responsible for the generation or selection of the idiotype-negative tumor variants. The idiotype-specific effector T cells responsible for the variant production in vitro and in vivo are triggered either by viable or irradiated 2C3E1 tumor cells, but not by soluble idiotype-positive monoclonal antibody secreted by the tumor. The idiotype-positive antibody did stimulate the production of the effector T cells when the soluble protein was covalently linked to the plasma membrane of BALB/c spleen cells, but not when the idiotype-positive protein was presented on allogeneic C57BL/6 spleen cells. A single exposure of spleen cells to 2C3E1 cells in vivo was sufficient to prime the idiotype-specific T cells, but the frequency and reproducibility of variant selection increased when the in vivo primed effector T cells were restimulated by irradiated 2C3E1 cells in vitro prior to co-cultivation with the wild-type 2C3E1 cells. The initial variant B cell lines could be segregated into several different phenotypes. However, after extensive cultivation either in vitro or after repeated transfer in vivo, all of the variant cell lines acquired a single stable phenotype that was characterized by the loss of both the surface and secreted idiotype marker and antigen-binding activity associated with the wild-type 2C3E1 cells.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.     

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.     

Serologic and structural characterization of immunoglobulin chains secreted by rabbit-mouse hybridomas

Serologic and primary structural analyses of Ig chains secreted by several rabbit-mouse hybridomas have shown that these hybrid cells produce heavy (H) or light (L) chains identical to those isolated from rabbit sera. Two of the cell lines (7D2, 7D6) secreted rabbit H chains with a m.w. of 55,000 each of which expressed a full complement of variable and constant region allotypes (a3, d11, e15). These apparently normal rabbit H chains were secreted in a complex with a m.w. about 130,000, and serologic studies indicated that this complex contained a covalently linked mouse kappa L chain. Two other cell lines (4C1, 12F2) produced allotype b4 L chains with m.w. of 23,000 and 25,000, and a third (1D4P5) produced an allotype b5 L chain with a m.w. of 23,000. Serologic analyses indicated that the allotypes on these chains are equivalent to those expressed by normal rabbit Ig molecules. Partial amino acid sequence data obtained for the L chain products showed them to be typical of rabbit L chains, and to be significantly different from mouse L chains.     

Immunologic memory to phosphocholine. IV. Hybridomas representative of Group I (T15-like) and Group II (non-T15-like) antibodies utilize distinct VH genes

The anti-phosphocholine (PC) memory response elicited in BALB/c mice by phosphocholine-keyhole limpet hemocyanin (PC-KLH) contains two groups of antibodies distinguished by their fine specificity for PC and p-nitrophenylphosphocholine (NPPC). Group I antibodies are inhibited by both PC and NPPC, while Group II antibodies are inhibited appreciably only by NPPC; only Group I antibodies are dominated by the T15 idiotype. Anti-PC hybridomas representative of the memory response to PC-KLH were produced to examine the variable region genes expressed by memory B cells. Two IgM hybridomas were of the Group I type, because they were inhibited by both PC and NPPC and they bound to the pneumococcus R36A. However, only one of these antibodies (PCM-2) expressed a T15 idiotope, while the other (PCM-1) did not express any of three T15 idiotopes. Despite its negative T15 idiotype profile, N-terminal amino acid sequencing of PCM-1 purified heavy chain and Southern blots of the hybridoma DNA indicated that it utilizes the T15 VH and JH1 genes. Three hybridomas, IgG1, IgM, and IgE, typical of Group II antibodies, were examined; these were negative for three T15 idiotopes and displayed measurable avidity only for NPPC in a PC-protein binding inhibition assay. These three hybridoma antibodies, like serum Group II IgG1, did not measurably bind to the bacterium R36A. The heavy chain amino termini of all three of these antibodies were inaccessible for Edman degradation. Southern blots of DNA from the IgG1 hybridoma revealed the T15 VH gene to be in the germ line configuration only and unassociated with any JH segment, indicating that this Group II antibody utilizes a VH gene different from the T15 family. These results signify that, whereas some diversity of the (anti-PC) memory response may be generated by somatic diversification of variable regions important in the primary response, a significant contribution to the overall heterogeneity of memory antibodies originates in the expression of additional variable region genes.     

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing.

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.     

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.     

Regulation of immunoglobulin biosynthesis in the murine plasmacytoma MOPC 315.

We have examined certain aspects of IgG biosynthesis by constructing hybrids between MPC11 (gamma2b, kappa) and MOPC 315 (alpha,lambda2) that have lost the ability to synthesize one or the other heavy chain. Cells express the three chains in a stable fashion, and both autologous (parental) and heterologous (nonparental) H and L chain pairs form and are secreted. The alpha H chain was found in polymeric form when associated with the heterologous kappa L chain. The lambda2 L chain covalently assembled to the heterologous gamma2b H chain. Surprisingly, autologous pairing was always favored over heterologous pairing in vivo by 5 to 10:1 in terms of rate of assembly. Similar ratios were maintained in the secreted protein. These results suggest that co-expression of particular H and L chain pairs is predetermined. Evolution presumably operates to improve antigen recognition as well as rate of assembly of active molecules.     

Diversity in the available repertoire of murine antibodies reactive with bromelain-treated isologous erythrocytes

Antibodies specific for bromelain-treated mouse RBC (BrMRBC) are of interest as models of "natural autoantibodies" and because of their primary source is Ly-1+ (CD5+) B cells. In earlier work by others, anti-BrMRBC hybridomas prepared by using CBA or NZB "spontaneously activating" peritoneal B cells were all found to produce mAb with a single common H chain V region sequence, by using a novel gene (VH11p), and a single common L chain V region sequence, a member of the Vk9 group (VkBrMp). We prepared anti-BrMRBC hybridomas by using LPS-activated B10.A splenic B cells in order to reveal the maximum available diversity in this repertoire. Data based on binding studies, Northern blot analyses with V region-specific probes, and mRNA nucleotide sequence analysis indicated that there is combining-site diversity in the repertoire of anti-BrMRBC hybridomas. There was considerable variation in trimethylammonium (a constituent of phosphatidyl choline) binding efficiency, and one of the anti-BrMRBC mAb showed no detectable binding. Northern blot analyses indicated 6 of 11 mAb to be of the VH11p/VkBrMp type, including one dual reactive anti-[BrMRBC + SRBC] mAb. Sequence analyses of the H chain V regions of four of the non-VH11p mAb revealed utilization of four distinct VH, three of which are very similar to the VH expressed by Ly-1+ B cell clones or lymphomas, as reported by others. However, because the VH11p/VkBrMp-type mAb were all relatively efficient at lysing BrMRBC and binding trimethylammonium, we suggest that affinity considerations may determine the selective predominance of B cells with this V region configuration from an available repertoire of considerable diversity.     

β淀粉样肽单克隆抗体可变区基因的5′RACE扩增及序列分析

目的:克隆并分析抗β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构,以及对该抗体进行人源化改造奠定了基础。     

Nucleotide sequence of the constant region of a chicken mu heavy chain immunoglobulin mRNA.

We have recently reported the sequence of a chicken Ig lambda light chain cDNA clone, isolated from a spleen partial cDNA library (1). In this paper, we describe the characterization of a cDNA clone coding for the chicken constant (C) region of the secreted mu chain. This is the first report on the nucleotide and amino acid sequence of a chicken Ig heavy chain constant region. It contains the 3' untranslated region of the mu mRNA up to the poly(A) tail, and, in comparison with the mouse Cmu sequence, displays the overall domain size and organization of a secreted mu chain, i.e.: a characteristic COOH-terminal region, a Cmu4, a Cmu3, a Cmu2, and part of a Cmu1 domain. The sequence homology between these two species ranges from 45% for the Cmu4 to 18% for the Cmu2. Thus, the Cmu sequence appears much less conserved between chicken and mouse than their respective lambda light chain constant regions (1). These results, together with some distinctive features of the Cmu2 domain, may be of evolutionary relevance and will be further discussed.     

Somatic mutation and clonal expansion of B cells in an antigen-driven immune response.

The variable (V) regions of three closely related monoclonal antibodies produced by hybridomas which had been isolated from a single mouse were sequenced at the level of the mRNA. The sequences and the restriction analysis of the immunoglobulin loci carried by the hybridoma cells indicate that the antibodies are derived from cells belonging to a single B cell clone. The sequence data imply a high frequency and stepwise occurrence of somatic point mutations in the expressed V region genes and substantial clonal expansion of B cells in the mouse. The mutations appear to be randomly introduced into heavy and light chain V region genes. Mutations are also seen in the complementarity determining regions which may thus have been involved in the selection of the cells producing the three antibodies.     

Dissecting the anti-desmoglein autoreactive B cell repertoire in pemphigus vulgaris patients

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. V(H) gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two V(H) genes. V(L) gene segment use was diverse even among IgG hybridomas suggesting that the V(L) is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in V(H) use, is somatically mutated, and is Ag selected.