Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence,binding properties,and pathogenicity

When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody–nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5′-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Enhanced antitumor activity of a combination treatment with a mouse/human chimeric anti-MK-1 antibody and lymphokine-activated killer cells in vitro and in a severe combined immunodeficient mouse xenograft model

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers. In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 V_H and Vκ genes to the human Cγ1 and Cκ genes, respectively. In the present study, we tested combination immunotherapy of Ch FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID) bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1 was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1 may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors. Received: 27 November 1998 / Accepted: 23 February 1999     

Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient than infection of the parental CHO cell line, indicating that N-sulfation is important. In contrast, infection of a cell line deficient in 2-O-sulfation was as efficient as infection of the parental cell line, indicating that 2-O-sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack their N-, C6-O-, or C2-O-sulfate groups. Only heparin chains lacking the N-sulfate group lost the ability to neutralize infection, confirming that N-sulfation, but not C6-O- or C2-O-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes N-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264-275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.     

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.     

抗A型产气荚膜梭菌α毒素单链抗体基因的克隆和表达

应用RT PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌α毒素单克隆抗体的杂交瘤细胞中 ,扩增出抗体V_H 和V_L 基因 ,连接成ScFv基因 ,并将其克隆至pGEM T载体中构建了重组质粒pXScFv 2E3。经序列分析证实 ,V_H 和V_L 基因及linker基因拼接正确 ,基因全长为 726bp ,编码 242个氨基酸。随后将其定向克隆于表达载体pHOG21,转化至大肠杆菌XL1 BLUE筛选出表达菌株XL1 BLUE(pHOG 2E3)。经ELISA和SDS PAGE分析表明 ,在20℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 25 %。并且ScFv基因表达产物能够中和α毒素的磷酯酶C活性     

Different pattern of immunoglobulin gene usage by HIV-1 compared to non-HIV-1 antibodies derived from the same infected subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.     

抗炭疽保护性抗原单克隆抗体可变区基因的克隆及序列分析

目的:克隆并分析抗重组炭疽芽孢杆菌保护性抗原(rPA)单克隆抗体4B2、5E1和2A8轻、重链可变区基因。方法:从分泌抗rPA特异性单抗的杂交瘤细胞株4B2、5E1和2A8中提取总RNA;利用RT-PCR技术克隆抗rPA单抗4B2、5E1和2A8的VL、VH基因,并将其连入pMD18-T载体中进行测序分析。结果:4B2VL基因全长378bp,VH基因全长414bp;5E1VL基因全长420bp,VH基因全长426bp;2A8VL基因全长384bp,VH基因全长342bp。通过分析,这些基因均符合小鼠IgGV区基因的特征,含有4个框架区、3个抗原互补决定区,而且抗体特征性的半胱氨酸残基的数量和位置也正确。结论:克隆了抗rPA单抗4B2、5E1和2A8的VL、VH基因,为下一步构建多种形式的基因工程抗体奠定了良好的基础。     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.     

利用B细胞培养和RT-PCR技术从我国HIV-1感染者中筛选膜蛋白特异性单克隆抗体的初步研究

本研究通过采集1型人类免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)感染者抗凝全血,分离出外周血单个核细胞,然后用磁珠分选纯化记忆性B细胞和体外活化记忆性B细胞,促使其分泌抗体,用ELISA法识别阳性B细胞克隆,并提取阳性B细胞的RNA,从中扩增抗体重链和轻链基因并克隆到表达载体中,再用携带重链基因的质粒和携带轻链基因的质粒共转染293T细胞,获得HIV-1特异性人单克隆抗体,进行抗体特性的鉴定。结果从1例HIV-1感染者的记忆性B细胞中筛选出了4株HIV-1包膜糖蛋白(Envelope glycoprotein,Env)特异性人单克隆抗体,其中2株具有较好的抗体依赖细胞介导的细胞毒作用活性,另有1株对HIV-1假病毒有较弱的中和活性。说明我们成功地引进了利用B细胞培养和RT-PCR技术从人体淋巴细胞中筛选特异性抗体基因的人单克隆抗体技术平台。用该技术可以成功获得HIV-1Env特异性单克隆抗体,为将来从能产生高滴度广谱中和抗体的感染者体内筛选广谱中和抗体打下了基础。     

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta- related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation.     

Active antitumor immunotherapy, with or without B7-mediated costimulation, increases tumor progression in an immunogenic murine T cell lymphoma model

 BW-Sp3 is a BW-5147-derived T cell lymphoma with limited immunogenicity since, despite regression of the majority of subcutaneous tumors, an important fraction of the animals will die from metastases. In the present study, the BW-Sp3 cells were transfected with genes encoding B7-1 or B7-2, known to be involved in the induction of T cell responses. The resulting transfectants exhibited a reduced tumorigenicity and did not cause mortality in the syngeneic recipients. Furthermore, immunization with the B7-1 or B7-2 transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTL) that lysed both the transfectants and the wild-type BW-Sp3 cells. Since the B7 transfectants were completely rejected in syngeneic recipients and induced potent CTL recognizing the wild-type BW-Sp3 cells, these engineered cells were considered as candidates for immunotherapy. Vaccinations with the B7-1 or B7-2 transfectants could completely protect the animals from metastatic disease when subsequently challenged with wild-type BW-Sp3 cells. Furthermore, immunization with the B7 transfectants could prolong the survival time of mice that had been challenged intravenously with BW-Sp3 cells. Surprisingly, however, when these transfectants, as well as the wild-type BW-Sp3 cells, were used for vaccination of tumor-bearing animals, the presence of the subcutaneous BW-Sp3 tumors clearly interfered with the outcome of immunotherapy, resulting in increased malignancy, as reflected by a higher incidence of progressing tumors and a reduced survival rate. Possible implications for immunotherapy in humans are discussed. Received: 5 August 1997 / Accepted: 15 August 1997     

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.     

Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells

Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E. coli--were prepared, and designated as HT-1, and 104-5-f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E. coli but not recombinant human IFN-alpha 5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN-alpha 5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The developmental patterns of B cell precursors distinguishing between environmental and nonenvironmental forms of phosphocholine.

We have analyzed the developmental patterns of two groups of B cell precursors in nonimmunized BALB/c mice with respect to their relative proportions, absolute frequencies, V gene usage, fine specificity, and avidity for antigen. One group of B cells (group I) secretes antibodies specific for PC and PC-containing bacteria, whereas the other group (group II) produces antibodies recognizing only nonenvironmental PC-protein conjugates. A marked shift in the proportions of group I and group II occurs during ontogeny: while the group I B cells dominate (greater than 85%) the adult antibody repertoire, the group II B cells have equal representation in neonatal mice from Days 1 to 7, and remain as a significant portion until 2 weeks of age. Examination of the absolute frequencies of group I and group II B cells revealed that the frequency of group II B cells remained relatively stable throughout ontogeny, whereas group I B cells expanded rapidly after 7 days of age to predominate in the adult. Genetic analysis indicated that early group I antibodies were encoded by VH and VL genes different from adult group I antibodies which are mostly encoded by a single VH (S107) and VL (V kappa 22) gene combination (the T15 idiotype). On the other hand, early group II antibodies used VH genes comparable to their adult counterparts. The majority of early group I antibodies have lower avidity for PC than adult T15+ antibodies, whereas the avidity of neonatal group II antibodies varies considerably and is comparable with that of the adult group II antibodies. Our results suggest that the ontogeny of phosphocholine-specific B cells may be regulated according to their fine specificity rather than to their avidity or V gene usage.