Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

Host defence against C. albicans infections in IgH transgenic mice with V(H) derived from a natural anti-keratin antibody

Fungal infections have been increasing and life-threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans-induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self-antigen keratin. To further study the generation and anti-C. albicans activities of natural antibodies in vivo, we constructed a mu chain transgenic mouse (TgV(H)3B4) using the V(H) gene from 3B4. TgV(H)3B4 had elevated serum anti-keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgV(H)3B4, B cells secreting the anti-keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin-reactive natural antibodies in anti-C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections.     

Pathogenic autoantibodies in systemic lupus erythematosus are derived from both self-reactive and non-self-reactive B cells

Previous studies have shown that both murine and human anti-double-stranded DNA (anti-dsDNA) antibodies can develop from non-DNA-reactive B cells and suggest a crucial role for somatic mutation in dsDNA binding. However, since only a limited number of human anti-dsDNA antibodies have been analyzed previously, we could not exclude other mechanisms for the generation of anti-dsDNA antibodies in patients with systemic lupus erythematosus (SLE). Therefore, we isolated IgM anti-dsDNA antibodies from peripheral blood B cells of a patient with SLE. Three somatically mutated IgM anti-DNA antibodies with pathogenic potential (glomerular binding) were reverted to their germline configuration. Although all three IgM anti-dsDNA antibodies came from the same lupus patient, they displayed different profiles. Reversion to the germline sequence of autoantibodies A9 and B5 resulted in decreased dsDNA binding. In contrast, the germline form of G3-recognized dsDNA as well as the mutated counterpart. These results suggest that mutated IgM anti-dsDNA antibodies may develop from both DNA- and non-DNA-reactive B cells. The implications are that B cell activation occurs in response to self and non-self antigens, while selection after activation may be mediated by self antigen in SLE. Moreover, ineffective tolerance checkpoints may exist before and after antigen activation in SLE.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell.

By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55IU and HmAb rCH8 had 1.09IU/100 micro g of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 micro g IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94IU/100 micro g IgG.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.     

Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens

Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin. Two of these hybrids could be propagated and further characterized. On the basis of inhibition experiments, one was found to be directed against dsDNA; the other was directed mainly against myosin but at the same time reacted significantly with actin, tubulin, spectrin, and dsDNA. Reactivity with myosin seemed to be concentrated in the light meromyosin subfragment, known to be rich in alpha-helical structure. These results indicate: 1) There are reactive B cell clones directed against self antigens. 2) The antibody specificities found for these antibodies are very similar to those found for natural antibodies in normal human serum and for human monoclonal Ig. 3) The widespread reactivity found for the clone mainly reacting with myosin raises the possibility that the determinant recognized by this antibody is a conformational structure that possibly is associated with alpha-helical structures.     

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.     

Monoclonal antibodies against amyloid fibril protein AA. Production, specificity, and use for immunohistochemical localization and classification of AA-type amyloidosis

Monoclonal antibodies against amyloid fibril protein AA were produced by cell fusion of murine P3 X 63-Ag8.653 myeloma cells with spleen cells of immunized Balb/c mice. To increase immunogenicity, protein AA was coupled to horseradish peroxidase (HRP) or human high molecular weight kininogen (HMWK). Using micro-ELISA (enzyme-linked immunosorbent essay) seven hybridoma cell lines secreting antibodies that specifically bind to protein AA have been selected and cloned. When applied to formalin-fixed paraffin sections of a variety of different amyloid types using immunoperoxidase methods, five monoclonal antibodies bound specifically and strongly to amyloid only of the AA type. Since a series of different AA-amyloids could be stained, these reagents may be used to routinely diagnose AA-amyloidosis in tissue sections. A monoclonal antibody against HRP has also been produced that has been utilized to develop a monoclonal peroxidase-antiperoxidases (PAP) complex. When three immunoperoxidase methods were compared, the sensitivity of a conventional rat PAP was comparable to the monoclonal PAP complex, but the latter was easier to handle. Both methods were more sensitive than the indirect immunoperoxidase technique.     

Influence of antibody isotype on passive serotherapy of lymphoma

We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.     

Differences in the repertoire expressed by peritoneal and splenic Ly-1 (CD5)+ B cells.

Purified populations of B cells expressing the Ly-1 and/or Mac-1 surface Ag were isolated from normal unmanipulated mice by cell sorting. The number of lymphocytes in each population secreting antibodies reactive with DNA, bromelain-treated mouse RBC, phosphorylcholine and TNP-keyhole limpet hemocyanin was quantitated by ELISA spot assay. The proportion of B cells secreting Ig in vivo and the repertoire of antibodies they produced varied as a function of B cell phenotype and location. Among peritoneal lymphocytes, those that were Ly-1+ or Ly-1- Mac-1+ secreted Ig 10 times more frequently that Mac-1- Ly-1- B cells from the same location. In addition, the former populations expressed repertoires that were significantly skewed toward the production of antibodies reactive with bromelain-treated mouse RBC (p less than 0.001). In contrast, splenic B cells expressing the Ly-1 surface Ag did not differ significantly from splenic Ly-1- B cells in their expressed repertoire or frequency of Ig production. B cells isolated from the spleen and peritoneum tended to differ in antibody specificity from bone marrow and lymph node-derived lymphocytes. For example, B cells from the spleen secreted anti-DNA antibodies two to four times more frequently than B cells from other organs. These results demonstrate that phenotype and microenvironment influence the repertoire of antibodies expressed by B cells in vivo.     

20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.     

The recombinant chimeric antibody chHAb18 against hepatocellular carcinoma can be produced in milk of transgenic mice

Biologically active recombinant monoclonal antibodies (mAbs) and their derivatives are in demand as therapeutic agents against a variety of cancers. The antibodies are generally produced by mammalian cell culture, but their production in the milk of transgenic animals would help meet the increasing demand. The mouse-human chimeric antibody chHAb18 has been proven to inhibit the invasion and metastasis of human hepatocellular carcinoma (HCC) cells by recognizing the HAb18G/CD147 molecule that is highly expressed on the surface of HCC tissue. Here, we report that transgenic mice generated by co-microinjection of two cassettes encoding the heavy and light chain genes of chHAb18 could highly express functional chHAb18 in their mammary glands. The expression level range of 1.1–7.4 mg ml⁻¹ was independent of transgenic copy number. Immunoassays demonstrated the ability and specificity of chHAb18 to bind purified antigen (i.e., HAb18G) or HCC cells. Recombinant chHAb18 from transgenic milk exhibited affinity almost equal to chHAb18 derived from CHO cells, and was 68% of that of the parental murine antibody, HAb18. In light of successful clinical application of HAb18, the chHAb18 expressed in mammary glands of transgenic mice constitutes an important step towards high-yield and scaled-up production of this antibody.     

Plasmodium yoelii: antibody response in resistant and susceptible mouse strains

The numbers of antigen-reactive antibody-secreting cells, levels of parasite antigen-specific serum antibodies and numbers of red blood cells staining positive for surface immunoglobulin were determined for susceptible and resistant mouse strains following infection with Plasmodium yoelii 17x. As a control, these parameters also were measured using antigen prepared from normal red blood cells. The relatively susceptible C57BL/6 mice produced more antigen-specific antibody-secreting cells and had higher levels of immunoglobulin positive red blood cells than did DBA/2 mice, but the DBA/2 mice had more antigen-specific IgG in their sera. Both mouse strains possessed cells secreting antibody reactive with soluble normal red blood cell antigen; however, C57BL/6 mice had more IgG positive unparasitized RBC than did DBA/2 mice. Despite possessing fewer antibody positive normal RBC, DBA/2 mice had significantly higher levels of serum antibodies that reacted with soluble red blood cell antigen. These data indicate that levels of serum antibody may not reflect the amounts of antibody produced and that use of any single assay to assess the magnitude of the antibody response may give rise to misleading results.     

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     

Identification,isolation, and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.     

Use of monoclonal antibody to immunochemically characterize variant-specific surface coat glycoprotein from Trypanosoma rhodesiense

Monoclonal antibodies were employed to study the molecular basis for charge heterogeneity in variant-specific surface coat glycoprotein prepared from clone CP3B4 of the Wellcome strain of Trypanosoma rhodesiense. Thirteen hybridomas secreting monoclonal antibodies specific for CP3B4 were obtained by fusing murine plasmacytoma cells to spleen cells from mice immunized with purified surface coat glycoprotein. The clone population of CP3B4 trypanosomes was shown to be homogeneous by means of immunofluorescent assays using culture supernatants from each of the 13 hybridomas. No cross-reactivity was found with other variant antigenic types of the same serodeme. Ascitic fluids were generated from 4 of the hybridomas and th molecular and epitopic specificities of the fluids or their IgG fractions were determined isoelectrofocusing of immunoprecipitates of radioiodinated glycoprotein antigen followed by autoradiography revealed that all 3 major components of the charge heterogeneous CP3B4 surface-coat glycoprotein were immunoprecipitated by each of the 4 monoclonal IgG fractions. Immunofluorescent staining of live trypanosomes was obtained with only one of the 4 ascitic fluids. The results show that charge heterogeneity does not derive from a heterogeneous population of parasites. Furthermore, the data indicate that there are at least 2 different epitopic specificities exhibited by the monoclonal antibodies tested and that each of the 3 charge heterogeneous components of the surface coat glycoprotein contains these epitopes. Charge heterogeneity of CP3B4 surface coat glycoprotein may be attributed to post-translational modification or to limited proteolysis.