Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Membrane IgM, IgD, and IgG act as signal transmission molecules in a series of B lymphomas

Increases in intracellular free calcium concentration ((Ca2+)i) were observed in response to anti-immunoglobulin (Ig) antibodies in each of six B cell tumors or B cell hybridomas bearing mu or delta chains on their cell surface. The BAL17 cell line, bearing mu and delta chains on its surface, behaved similarly to mature B cells in the following respects. Anti-IgM and anti-IgD antibodies caused increases in (Ca2+)i and inositol phospholipid metabolism; the initial increases in (Ca2+)i were derived partly from an intracellular Ca2+ pool; lipopolysaccharide, phorbol myristate acetate (PMA), B cell stimulatory factor-1, and antibodies to class I and class II major histocompatibility molecules and to the Fc gamma receptor failed to cause increases in (Ca2+)i or in inositol phospholipid metabolism; and increases in (Ca2+)i and inositol phospholipid metabolism in response to anti-Ig were inhibited by pretreatment with PMA. Furthermore A20, an IgG2a bearing lymphoma, showed increases in (Ca2+)i in response to anti-IgG2a, and a lymphoma cell line (6G8-2E10) expressing membrane IgG2b as a result of DNA-mediated transfer of the gamma 2b H chain gene, showed increases in (Ca2+)i in response to anti-IgG2b. These results indicate that Ig-bearing lymphomas display early events in B cell activation after receptor cross-linkage and can be used for detailed studies of the activation process.     

双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备

双峰驼IgG亚型包含IgG1、IgG2和IgG3,其中IgG2和IgG3为重链抗体,在结构上与IgG1存在显著差异。为获取双峰驼血清中的IgG1、IgG2和IgG3,并分析其抗原特异性和抗体特异性,本文交替使用Protein A和Protein G亲和层析柱,对其分离纯化,并通过聚丙烯酰胺凝胶电泳进行鉴定;之后分别制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体,通过ELISA对制备的多克隆抗体的效价进行测定;最后应用Western blot评估这三个亚型多克隆抗体的特异性,进而对双峰驼血清中IgG1、IgG2和IgG3的抗原特异性进行分析。结果表明,应用Protein A和Protein G亲和层析柱成功分离纯化出双峰驼血清中的IgG1、IgG2和IgG3;并制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体效价均在1∶10000以上,并且所获得的多克隆抗体分别与IgG1、IgG2和IgG3之间均存在交叉反应,但兔抗双峰驼IgG1多克隆抗体较其它两个亚型多克隆抗体特异性低。结果证明,双峰驼IgG1、IgG2和IgG3均具有良好的免疫原性,三者结构虽存在显著差异,但其抗原特性类似。     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Anti-immunoglobulins in multiple myeloma and benign monoclonal hyperglobulinemia]

Sera from 50 healthy adults, from 21 patients with multiple myeloma (MM) and from 11 patients with benign monoclonal hyperglobulinaemia (BMH) and from 28 patients with sarcoidosis were examined for the presence of anti-IgG-activity by passive haemagglutination technique. 62% of healthy adults (titre less than 2) and all of the sera from patients with MM and BMH (titres 32-512) as well as 42% of the sera from sarcoidosis patients were found to be anti-IgG positive. The anti-IgG positive sera showed also anti-(Fab)2-activity (with the exeption of 2 sera from sarcoidosis patients). No significant differences could be found between anti-Ig activity in sera from MM patients with BMH. There was also seen no correlation of anti-Ig with the clinical course of the disease. After column chromatography we could detect the partially simultaneous presence of anti-Ig with anti-Fc-specificity (MW ca. 900,000) and with (Fab)2-specificity (MW 150,000 or less than 90,000). Antibody dependent cytotoxicity (using melanoma cell lines as targets) was significantly inhibited by the isolated anti-Ig-fractions with low molecular sizes (MW less than 90,000). From these results it seems possible that anti-immunoglobulins may play a role in the clinical course of the disease.     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Subclasses of human IgG. I. Production of antisera to IgG subclasses]

Guinea pigs were used for preparing antisera to human IgG subclasses for anti-IgG1, and rabbits--for anti-IgG2, anti-IgG3, and anti-IgG4. Schemes of laboratory animals immunization with myeloma paraproteins of four IgG subclasses were determined. Methods of antisera absorption for bringing them up to strict monospecificity were worked out. Antisera specificity were determined by the precipitation test after Ouchterlony with standard myeloma proteins in the concentration of 1 mg/ml, and in the passive hemagglutination test with erythrocytic antigenic diagnostic agents. Precipitating antisera to four human IgG subclasses were obtained.     

Antifertility effects of immunoglobulins from uterine fluids of semen-immunized rabbits

The effects on fertility of the immunoglobulins, IgG (similar to serum IgG) and secretoy IgA (the predominant IgA in uterine fluid, different from serum IgA) and the fluids from the uteri of semen-immunized rabbits were studied. Female rabbits were immunized either by transvaginal (TV) injections (every 4 or 5 days for 3 weeks) with adjusted semen added to buffered saline containing .5 mg each polyadenylic acid )Poly-A) and polyuridylic acid or a combination of transvaginal injections and systemic injections with rabbit semen mixed with adjuvant (each once weekly for 3 weeks). 2 weeks following the last injection of semen each rabbit received a TV injection followed in 1 week by an instillation of semen and Poly A and U into uterine horns ligated at both ends. 2 weeks following intrauterine (IU) instillation uterine contents were withdrawn. Uteri were reinstilled. Passive hemagglutinating (PHA) and sperm-immobilizing antibody titers were determined on both serum and adjusted uterine fluid (UF) samples. Sperm immobilizin tests and PHA were positive in all serum samples from the contained immunization group, and PHA were positive in 4 out of 7 UF samples of this group. 2 out of 10 and 3 out of 10 samples from the TV group were positive for SI and PHA, respectively, and 4 out of 10 UF samples were PHA positive. Treatment of rabbit semen with UF samples prior to insemination of estrous rabbits resulted in an implantation rate of 2.8% for the combined immunization group and 24.2% for the TV group compared to 79.2% for controls. Some IUF samples without detectable antibodies appeared to inhibit fertility. The implantation rate of sperm treated with immune UF from the TV group absorbed with goat normal serum, anti-rabbit gamma-globulin serum, anti-rabbit-secretory IgA serum, anti-rabbit IgG serum, or sperm, were 3.4%, 88.9%, 42.9%, 64%, and 73. 3% respectively. SIgA and IgG antibodies were seperated by gel filtration. 2 of 4 pooled IgA samples and 3 of 4 pooled IgG samples depressed fertility, with 24.3% and 22.6% implantation and 25.8% 5, and 0% implantation, respectively. To assure that the antifertility effects observed were due to sperm antibodies of each immunoglobin class, SIgA fractions of 2 IUF pools were subjected to anion-exchange chromatography, and both SIgA and IgG fraction were absorbed with goat NS, anti-SIgA, and anti-IgG. IgG fractions absorbed with anti-SIgA or anti-IgG resulted in implantation rates of 0% and 56.8%, respectively, compared to a control of 89.5%. SIgA fractions absorbed with anti-SIgA or anti-IgG resulted in implantation rates of 81.8% and 19.4%. Thus IgG and secretory IgA of the uterine fluids are active in inhibition of fertility.     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Neutralizing activities of early and late IgG fragments from rabbits immunized with herpes simplex virus.

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab')2 showed a low neutralizing activity only after addition of anti-IgG. F(ab')2 of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab' and Fab-II, though the activity of Fab-I was relatively low. Three three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab')2 was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab' did combine with the virus and that the late Fab' exerted a higher blocking effect than the early Fab'.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas

A panel of 10 hybridomas was assembled to assess the influence of various genetic and biological factors upon glycosylation of secreted monoclonal IgG. After exhaustive Pronase digestion of IgG, glycopeptides were characterized chromatographically by apparent size, charge, and concanavalin A (Con A)-Sepharose and Lens culinaris (LcH)-agarose affinity. Six glycosylation phenotypes were found to be common among all clones studied. Despite this phenotypic heterogeneity in glycosylation of IgG, considerable similarity exists between different clones. In particular, virtually all IgG glycopeptides bear a core fucose residue. Second, the majority of the glycosylation repertoire is comprised of two phenotypes, characterized by glycopeptides which differ in affinity for Con A-Sepharose. Of these, the predominantly expressed phenotype is the same for all clones. The carbohydrate structure derived from this phenotype, elucidated by 500-MHz 1H NMR spectroscopy, is (Formula: see text). Significant variability between different hybridomas exists in the relative expression of the two major phenotypes. Other differences between clones may reflect the expression of an additional site which is glycosylated differently. However, there is no apparent correlation of phenotype with either the hybridoma's parentage or the serologically defined polypeptide structure of the IgG which it secretes. In addition to clonal variability, other sources of variability in phenotypic expression were identified. A generational variability is apparent upon continuous culturing of the same hybridoma. Also, differences in culture medium pH or proliferative state of the cells may have a modest influence upon the glycosylation phenotype.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

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.     

Anti-immunoglobulin autoantibodies are not preferentially induced in polyclonal activation of human and mouse lymphocytes, and more anti-DNA and anti-erythrocyte autoantibodies are induced in polyclonal activation of mouse than human lymphocytes

Using a plaque assay with immunoglobulin (Ig)-coated SRBC, we and others have previously reported that the majority of polyclonally activated mouse lymphocytes secreted antibodies that appeared to be IgM anti-IgG autoantibodies. Careful reexamination of this assay, with application of several highly purified mouse serum and myeloma IgG and IgM preparations, revealed that IgM, which was a minor contaminant of Ig preparations, rather than IgG, was responsible for the formation of these plaques. High numbers of plaques could also be detected in assays with polyclonally activated human lymphocytes, Ig-coated SRBC, and anti-Ig developing sera. Of all IgG-, IgM- or IgA-secreting cells, 40 to 100% were detected with SRBC coated with gamma-globulin or Ig of the same isotype as the isotype to which the developing serum was specific; in general, low proportions of all PFC were detected with SRBC coated with Ig of a different isotype. Studies on the sequence of events leading to the formation of plaques with Ig-sensitized SRBC (both in humans and mice) revealed that antibodies detected in these assays were not able to bind to the Ig-coated SRBC (without the presence of developing serum), and therefore were not anti-Ig autoantibodies. It is our conclusion that the plaque assays with Ig-coated SRBC represent another type of a reverse hemolytic PFC assay that detects cells secreting antibodies regardless of their specificity, and these plaques are formed due to the cross-linking by the anti-Ig developing serum of the Ig coated on SRBC and the Ig secreted by lymphocytes. Our results confirmed preferential induction of anti-DNA antibody secreting cells in mice by showing that these antibodies indeed bind to DNA coated on SRBC. In cultures of polyclonally activated human lymphocytes, anti-DNA and anti-erythrocyte autoantibody-secreting cells were over 10 to 100 times less frequent than in mice. These results, therefore, disprove the concept of preferential induction of anti-Ig autoantibodies in the polyclonal activation of mouse and human lymphocytes, and show that anti-DNA and anti-erythrocyte autoantibodies are easily induced in the polyclonal activation of mouse, but not human, lymphocytes.     

Rheumatoid factors induce signaling from B cells, leading to Epstein-Barr virus and B-cell activation

B-cell antigen receptor signaling is initiated upon binding of the antigen to membrane-bound immunoblobulin (Ig), and the anti-Ig antibody (Ab) mimics this signaling. In B cells latently infected with Epstein-Barr virus (EBV), the same signals induce virus activation. We examine here whether rheumatoid factors (RFs), autoantibodies directed against the Fc portion of IgG, induce EBV and B-cell activation. As a source of RFs, RF-producing lymphoblastoid cell line (LCL) clones were isolated from peripheral blood mononuclear cells (PBMC) and synovial cells from patients with rheumatoid arthritis (RA) by EBV transformation. Burkitt's lymphoma-derived Akata cells, which are highly responsive to EBV activation by anti-Ig Abs, were used for the assay of EBV activation. Akata cells expressed IgG3 as membrane-bound Ig. RFs from a synovium-derived LCL were directed to IgG3 and induced EBV activation in 16 to 18% of Akata cells, whereas RFs from another synovium-derived LCL were directed to IgG1 and did not induce EBV activation. Pretreatment of RFs with the purified Fc fragment of human IgG completely abolished EBV activation. Furthermore, B-cell activation was assessed by incorporation of [3H]thymidine. RFs from synovium-derived LCLs efficiently induced B-cell activation, and the addition of CD40 ligand had a synergistic effect. On the other hand, RFs from PBMC-derived LCLs were polyreactive, had a lower affinity to IgG, and did not induce EBV and B-cell activation. The present findings imply a possible role for RFs as EBV and B-cell activators.     

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.     

Antigen-induced rheumatoid factors. Protein and carbohydrate antigens induce different rheumatoid factor responses

Rheumatoid factors, autologous IgM anti-IgG, were produced after immunization with protein or carbohydrate antigens. After immunization with either type of antigen, the kinetics of the rheumatoid factor response reflected the kinetics of the dominant IgG isotype in the anti-antigen response. Secondary immunization with protein antigens induced an IgM rheumatoid factor response which was consistently greater than that seen after carbohydrate immunization, and almost exclusively specific for the IgG1 isotype. In contrast, primary or hyperimmunization with carbohydrate antigens gave rise to a more heterogeneous response dominated by IgM anti-IgG3, with lesser amounts of IgM anti-IgG2b and anti-IgG1. Direct immunization with immune complexes gave similar results, as complexes composed of IgG1 induced exclusively IgM anti-IgG1, whereas those complexes made up of IgG3 gave rise to IgM rheumatoid factors binding IgG3 and IgG2b. Rheumatoid factor production, with isotypic specificity defined by the immunizing antigen, appears to be a natural consequence of immunization with a variety of protein and carbohydrate antigens.     

Human T-helper clones induce IgG production in a subclass-specific fashion.

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 approximately 60%, IgG2 approximately 30%, IgG3 approximately 5-10%, IgG4 less than or equal to 5%) and unlike that observed in resting B cells (which is IgG1 approximately 40% and IgG2 approximately 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG1-specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-gamma secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.