Epstein Barr virus transformation of peripheral blood B cells secreting antibodies reactive with cell surface antigens

EBV transformable peripheral blood B cells secreting antibodies reactive with cell surface antigens present on two indicator human leukemia cell lines, NALM1 and U937, were studied. Oligoclonal EBV transformants from patients with a variety of diseases were frequently found to produce cell surface reactive antibodies. Antibody secreting transformants could also, although less frequently, be readily cultured from the PBM of normal volunteers, and represented, by limiting dilution, 1 out of 113 transformable B cells. CD8 antibody had no effect on the frequency of antibody producing B cells, but depletion of CD8+ cells by immunomagnetic methods prior to transformation significantly (P less than 0.05) increased the recovery of antibody secreting B cells to 1/33. Readdition of magnetically depleted cells did not significantly inhibit the transformation of these B cells. During the acute and recovery phases of some infections increasing numbers of these transformable antibody producing B cells appear in the circulation. The majority of antibodies produced were of the IgM class, although IgG antibodies were also detected. IgM antibody producing transformants were tested and some were found to react with autologous and allogeneic normal lymphocytes. These results lend support to the notion that B cells capable of secreting cell surface reactive antibodies, a proportion of which are autoreactive, are present in the normal repertoire of healthy adults, and that these cells are under active regulation by CD8+ cells.     

Generation of human monoclonal antibodies to cancer-associated antigens using limited numbers of patient lymphocytes

A limiting dilution method for the efficient transformation by Epstein-Barr virus (EBV) of human B lymphocytes has been applied to the production of human monoclonal antibodies to ovarian cancer-associated antigens. Limited numbers (e.g., 2 X 10(5)) of EBV-infected B lymphocytes from ovarian cancer patient spleen, lymph node, tumor, ascites and blood were successfully transformed using this method. An immunofiltration assay system was employed to identify EBV transformants secreting IgM antibody which reacted selectively with ovarian cancer patient ascites tumor cells, but not with a mixture of normal cell types. A miniature Western blot assay was utilized to screen for IgG reactivity to protein species in detergent extracts of ovarian cancer tumor cells. EBV-transformed cells selected after screening were then fused with heteromyeloma fusion partner SHM-D33 resulting in efficient recovery of hybridomas secreting MAb of the desired specificity. Human MAbs which selectively react with antigens associated with ovarian cancer tumor cells were obtained.     

The construction and use of a human-mouse myeloma analogue suitable for the routine production of hybridomas secreting human monoclonal antibodies

A human-mouse myeloma analogue termed HMMA2.11TG/O was constructed by fusion of the mouse myeloma cell line P3x63Ag8.653, a mutant derivative of MOPC21, with bone marrow mononuclear cells from a patients with IgA myeloma. The HMMA2.11TG/O cell line is resistant to 6-thioguanine and ouabain and sensitive to HAT. The cell line secretes no detectable immunoglobulin and has a hybrid karyotype and cell surface phenotype. An average fusion efficiency for growth of hybridomas of 1/17,000 fused cells was obtained in fusions with human peripheral blood mononuclear cells (PBM), Pokeweed Mitogen (PWM) stimulated PBM, and Epstein-Barr Virus (EBV) transformed polyclonal B cell lines. Over 75% of hybrids secrete detectable immunoglobulin and the cloning efficiency of the hybrids at 1 cell/well averages 25%. Antibody secreting cloned hybridoma cell lines were obtained by fusion directly with PBM from an immunized volunteer and by fusion with in vitro, secondarily immunized, EBV transformed polyclonal cell lines. Five hybridomas secreting human monoclonal IgM anti-tetanus antibodies and 2 secreting human monoclonal IgG anti-tetanus antibodies were selected and cloned from 6 fusions performed specifically for anti-tetanus antibody. Immunoglobulin and antibody secretion by cloned hybrids has been stable for 5-10 months at present. Immunoglobulin and antibody secretion in routine cultures passaged every 3-4 days has been 8-42 micrograms/ml. This human-mouse myeloma analogue should prove useful for the routine production of human monoclonal antibodies.     

In vitro production of anti-HLA-DR antibodies by human B-cell lines

Human B-cell lines secreting antibodies which react preferentially with the HLA Class II antigen DR5 have been produced. Supernatants from these cell lines reacted with lymphocytes from all DR5 positive donors and a minority of DR6 positive donors but were negative on lymphocytes of other phenotypes. The cytotoxic activities of the supernatants apparently depend on IgM antibody. These results demonstrate the potential of B-cell lines for the in vitro production of antibodies to HLA-DR antigens.     

Immunization of normal human splenocytes in vitro to produce human monoclonal antibodies to cellular antigens

We have obtained human monoclonal antibodies to polymorphic cell surface determinants by immunizing normal human splenecytomy in vitro to allogeneic cells. Splenocytes from young patients undergoing splenectomy secondary to traumatic injury are separated into T and B lymphocyte populations. The T lymphocytes are irradiated with 1500 rad to selectively inactivate T suppressors. Responder T and B cells are then recombined at a 1:1 ratio. Maximal IgM and IgG production is obtained when pokeweed mitogen and irradiated stimulator cells are added to the cultures. Stimulator cell specific antibody levels peak at day 7 of in vitro immunization, and fall thereafter. Fusion of the immunized splenocytes to a human fusion partner as early as day 4 results in hybridomas secreting antibodies to cellular antigens. Transformation by EBV, expansion and fusion of the immunized cell line also yield hybridomas secreting stimulator cell specific antibody.     

Human monoclonal antibodies to cytokeratins associated with squamous cell carcinoma

Human lymphocytes from a lymph node draining the tumor-bearing area of a patient with a large primary squamous cell carcinoma of the oral mucosa were fused with the nonproducer mouse myeloma, NS-1, to produce interspecies hybridomas. Of 95 hybridoma culture supernatants tested, 23 contained from 0.5 to 50 micrograms/ml of human IgM or IgG. Six supernatant fluids containing greater than 15 micrograms/ml of Ig were tested by indirect immunoperoxidase and immunofluorescence against sections of the autologous carcinoma. Five IgM (lambda) monoclonal antibodies stained the cytoplasm of autologous and allogeneic squamous carcinoma cells. All five monoclonal antibodies stained all layers of normal epidermis but each antibody stained the superficial keratin layer most intensely. Two of the five hybridoma antibodies were further tested. Both antibodies stained all types of normal epithelium; a network of fibers characteristic of intermediate filaments in cultured squamous carcinoma cells and cultured fibroblasts; Z lines in skeletal muscle; and axons in peripheral nerve fibers. We conclude that all five IgM monoclonal antibodies recognize cytokeratins associated with the autologous squamous cell carcinoma. Two of the five hybridoma antibodies recognize an antigenic determinant common to all types of intermediate filament proteins. These data indicate that cytokeratins released by squamous carcinoma cells induced an antibody response in this patient.     

Generation and characterisation of bovine antigen-specific T cell lines.

15 antigen-specific T cell lines have been generated from eight individual cattle immunised with ovalbumin. Several sources of interleukin-2 (IL-2) were used, including a supernatant from a gibbon cell line (MLA-Sup), human recombinant IL-2 (hrIL-2) and bovine recombinant IL-2 (brIL-2). These IL-2 sources were used alternately with autologous peripheral blood mononuclear cells (PBM) together with ovalbumin to generate the lines. They grew least well in MLA-Sup and best in brIL-2. FACS analysis indicated that the lines generated with the recombinant IL-2s were extremely homogeneous in that the majority of cells were BoCD4+ (bovine CD4 equivalent) and therefore of TH phenotype. The lines were antigen specific and responded to antigen only in the presence of autologous PBM and not allogeneic (MHC class I nonidentical) PBM. However, allogeneic PBM did support their proliferation to ConA. No MLR response was observed by the cell lines to allogeneic PBM. The response to antigen was inhibited by anti bovine class II mAbs but not an anti bovine class I mAb. The subpopulation of PBM which acted as antigen presenting cells for these bovine TH cell lines had typical macrophage characteristics.     

Cross-reactivity of Epstein-Barr virus-specific immunoglobulin M antibodies with cytomegalovirus antigens containing glycine homopolymers.

Timely and reliable detection of acute primary human cytomegalovirus (HCMV) infection is important in prenatal screening programs and for differential diagnosis of infectious mononucleosis-like disease. Enzyme-linked immunosorbent assays (ELISAs) based on HCMV proteins enable the sensitive detection of immunoglobulin M (IgM) antibodies during primary infection. However, concerns have been raised about possible cross-reactivities of the HCMV antigens used for the design of such ELISAs with IgM antibodies induced by Epstein-Barr Virus (EBV). In this study we investigated whether IgM antibodies generated during acute EBV infection reacted with recombinant HCMV antigens. Serum samples from patients with primary EBV infection frequently scored positive when tested in different HCMV IgM ELISAs, irrespective of whether conventional or recombinant antigens were used for the design of the HCMV IgM assays. Such cross-reactive IgM antibodies were found to be directed against short glycine-rich motifs contained within the nonstructural HCMV proteins pUL44 and pUL57. Further analyses revealed that these glycine-rich motifs were major antigenic domains for IgM antibodies induced during HCMV infection. Their deletion from recombinant proteins abrogated reactivity with IgM synthesized during HCMV infection. EBV-induced IgM antibodies that reacted with HCMV antigens showed similar kinetics of reactivity in HCMV- or EBV-specific assays in the course of primary EBV infection, indicating that the two populations of antibodies were highly overlapping. The results demonstrate that primary EBV infection leads to the induction of IgM antibodies that specifically bind to widely used diagnostic antigens of HCMV. This has to be considered in the interpretation of HCMV-specific IgM assays.     

Characterization of Antibodies Reacting with Husband''s Lymphocytes in Sera from Patients with Trophoblastic Malignancies

Lymphocytotoxic antibodies reacting with husband's lymphocytes were demonstrated in 24 sera from women with trophoblastic neoplasia studied before any chemotherapy. These antibodies exhibited a maximal cytotoxic activity at +4 degrees C. They reacted mainly with B lymphocytes as assessed by microlymphocytotoxicity assays using B- or T-cell enriched subpopulations and T- or B-cell lines as targets. They could not be absorbed out with platelets or erythrocytes, and did not react with autologous lymphocytes. In one double-labelling immunofluorescence experiment, these antibodies could be stained by rhodamine-conjugated Fab'2 anti-mu fragments and thus appeared to belong to the IgM class. They were shown to react mainly with surface Ig (SIg) bearing lymphocytes, plus a minor SIg negative subset. Studies with panels of allogeneic normal B cells and of HLA homozygous B-cell lines showed that the target antigen(s) recognized by these antibodies is clearly distinct from HLA-D antigens. Corresponding antigens seem to be expressed on placenta, since the lymphocytotoxic antibodies could be absorbed out with trophoblastic homogenates and one serum reacted with JAR cells (cultured choriocarcinoma line). An enhancing role of such antibodies in the growth of trophoblastic malignancies may be suggested.     

Production of human monoclonal IgG and IgM antibodies with anti-D (rhesus) specificity using heterohybridomas

Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh-negative or DB cells. Concentrations of both antibodies reached between 25 micrograms/ml and 50 micrograms/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody.     

Production of human monoclonal antibodies to myeloperoxidase.

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported.     

Monoclonal antibodies defining shared human macrophage-endothelial antigens.

We have selected several monoclonal antibodies (mAbs) producing using human rheumatoid arthritis (RA) synovial macrophages (m phi s) as immunogen. Of these, mAbs 8H2, 10G7 and 10G9 showed cross reactivity with endothelium, suggesting common antigens between these cell types. We have determined the spectrum of reactivity of these mAbs on hematopoietic cell lines, peripheral blood cells, and inflammatory and non-inflammatory tissues by immunohistochemistry. MAb 8H2 does not react with the myeloid cell lines HL60 (myelocytic), U937 (histiocytic lymphoma), and K562 (erythroleukemia), or with peripheral blood cells. In normal and inflamed tissue sections, mAb 8H2 reacts with m phi s and endothelial cells. In contrast, mAb 10G7 does not react with peripheral blood cells, but reacts with HL60, U937, and K562 cell lines, as well as with m phi s and endothelial cells in inflamed and noninflamed tissues. MAb 10G9 does not react with myeloid cell lines, but reacts with monocytes and platelets in peripheral blood. In both normal and inflamed tissues, mAb 10G9 reacts with m phi s and endothelial cells. The antigens identified by these three mAbs were characterized biochemically, by enzymatic digestion of RA synovial tissue m phi s followed by a cellular ELISA, as well as by reactivity of the mAbs with NIH-3T3 cells genetically engineered to express known myeloid antigens. These mAbs reacted with protein or glycoprotein antigens distinct from the known myeloid antigens CD13, CD14, CD33, CD34, CD36, and c-fms. These mAbs should prove to be a valuable tool for studying m phi s and endothelial cells and their shared antigenic determinants.     

Human--human hybridomas secreting lipid A reactive monoclonal antibodies

Five human-human hybridoma cell lines secreting monoclonal antibodies (mAbs) against lipid A (LA) were produced by cell fusion of Epstein-Barr virus (EBV) transformed human peripheral blood lymphocytes and a human lymphoblastoid cell line KR-4. All these mAbs were isotyped as IgM(kappa) and reacted with lipopolysaccharides (LPS) and LA of various gram-negative bacteria. Whereas the binding of only four of the five mAbs to solid-phase LA was blocked by polymyxin-B sulphate, the mitogenic effect of LPS and LA on murine B lymphocytes was inhibited by all five mAbs. These results demonstrate that the human immune system recognizes at least two common epitopes in lipid A of various gram-negative bacteria.     

Enrichment and depletion of thyroglobulin autoantibody synthesizing lymphocytes.

Lymphocyte populations enriched for (or depleted of) a receptor for thyroglobulin (Tg) have been prepared from Hashimoto peripheral blood mononuclear cells (PBM) by rosetting with Tg coated erythrocytes. Removal of Tg binding cells from PBM or B cell preparations resulted in greater than 85% reduction in their ability to synthesize Tg antibody when stimulated with pokeweed mitogen (PWM) or EB virus (EBV); the depletion was specific since the ability of Tg receptor negative cells to secrete microsomal antibody and total IgG was unimpaired. Hashimoto lymphocytes (PBM or B cells) enriched for Tg binding cells produced only small amounts of Tg antibody when cultured with PWM even in the presence of irradiated T cells and monocytes; exposure to autoantigen followed by mitogen appeared to be inhibitory. However, the Tg receptor positive fraction was readily activated by EBV to synthesize Tg antibody with a specific activity 4-10 times higher than that secreted by unfractionated lymphocytes. The ability to isolate Tg specific B cells from peripheral blood will facilitate the development of EBV transformed cell lines secreting monoclonal Tg antibody and such antibodies will provide invaluable probes in the investigation of autoimmune thyroid disease.     

Characterization of cold reactive lymphocytotoxic antibodies in malaria.

Characterization of cold reactive lymphocytotoxic antibodies present in sera from Thai adults with malaria revealed that the antibodies are predominantly 19S (IgM), directed against both autologous and allogeneic mononuclear cells, complement-dependent, present in titres ranging from 1:2 to 1:16, and exhibit greater lymphocytotoxic activity during the acute stage of malarial infection than during the convalescent stage. The lymphocytotoxic antibodies were primarily directed against B cell targets or both B as well as T cell targets. In addition some sera were reactive with enriched monocyte/macrophage indicator cells at 15 degrees C, but not 37 degrees C. Antibodies directed against B cell targets were lymphocytotoxic both at 15 degrees C as well as 37 degrees C. The results indicate that IgM lymphocytotoxic antibodies in the sera of patients with malaria are directed primarily against B cells with reactivity to a lesser extent against T cells and macrophages and thus may play an immunoregulatory function in the humoral immune response to malaria infection.     

Human mast cells detected by monoclonal antibodies.

We report the establishment of seven mouse-mouse hybridoma cell lines secreting monoclonal antibodies with specificity for granule components of all human mast cells. Reactivity is directed against a molecule which is also found intracytoplasmically in human mature small intestinal enterocytes, liver parenchymal cells, and kidney proximal tubule epithelial cells. No reactivity of these antibodies was found with any other human or animal cell type examined. In particular, the antibodies did not react with basophils or other haemopoietic cell types. This study shows the potential of specific monoclonal antibodies as a tool for identifying and enumerating infiltrating mast cells in tissues. Such antibodies should be of value in investigations into the role of the mast cell in immunological reactions and hypersensitivity diseases.     

Detection of tube agglutination 37 degrees C-only antibodies by solid-phase red cell adherence

There are no published data on the detection of tube agglutination (TA) 37 degrees C-only antibodies by solid-phase (SP) red cell adherence assays using anti-IgG-coated indicator red cells. Thirteen examples of TA 37 degrees C-only antibodies were tested by conventional SP methods. Four TA 37 degrees C-only antibodies failed to react by SP. Three were anti- Lea, considered clinically insignificant, and one was anti-E, an antibody of potential clinical significance. The remaining nine TA 37 degrees C-only antibodies reacted by SP, including three anti-c, two anti- D, two anti-E, one anti-N, and one anti-M. The anti-M reacted with indicator red cells that lacked the red cell antigen and failed to react with IgG-coated indicator red cells whose anti-IgG component had been neutralized, indicating the antibody contained an IgG component. Two anti-D and one anti-c continued to react in an SP test using neutralized anti-IgG antigen-positive indicator red cells, i.e., indicator binding independent of antiglobulin, suggesting an IgM nature to these antibodies. Therefore, many TA 37 degrees C-only antibodies can be detected by SP either through detection of an IgG component by the anti-IgG of the indicator red cells, or through IgM crosslinking of antigen-positive indicator red cells to antigen-positive SP reagent red cell membranes.     

Production and Characterization of Human-Human and Human-Mouse Hybridomas Secreting Rh(D)-Specific Monoclonal Antibodies

Human monoclonal antibodies specific for the Rh(D) antigen were produced by cell lines generated by the fusion of pooled Epstein-Barr virus (EBV)-transformed B-cell lines secreting Rh(D) antibodies with the murine myeloma cell line NS.1 or with the human lymphoblastoid cell line HOA.1. The selection of hybrids was achieved in RPMI 1640 medium containing HAT and ouabain. Higher fusion efficiency was obtained with the NS.1 cell line; however, the hybrids with HOA.1 exhibited a greater clonal stability. The products of four clones (three human-human and one human-mouse) that consistently secreted antibodies for over 11 months were tested for specificity with a panel of red cells of various Rh phenotypes. The supernatants of all four clones showed anti-Rh(D) specificity but failed to react with the red cell Du phenotypes categorized as DV(Dw+) and DVI. Two of the three human-human clones secreted IgM(lambda) and the third IgG(kappa). The human-mouse clone produced IgG(kappa) antibody.     

Cytotoxic T cell recognition of Epstein-Barr virus-infected B cells. III. Establishment of HLA-restricted cytotoxic T cell lines using interleukin 2

Epstein-Barr virus (EBV)-specific cytotoxic T cell precursors, present in the circulation of previously infected (seropositive) individuals, have been reactivated in vitro by challenging with autologous EBV-transformed cells, and the reactivated populations subsequently expanded as interleukin 2 (IL2)-dependent cell lines. These lines were dominated by T cells possessing the cytotoxic/suppressor cell surface phenotype and, when tested for effector function in chromium-release assays, demonstrated potent EBV-specific, HLA-A and -B antigen-restricted cytotoxicity even when derived from seropositive donors whose initial cytotoxic response to in vitro reactivation was relatively weak. With all the lines tested from 10 seropositive donors, strong killing of autologous EBV-transformed cells was observed in the absence of any significant lysis of autologous mitogen-stimulated lymphoblasts or of a panel of EBV genome-negative cell lines sensitive to natural killing. Furthermore, the availability of IL2-expanded effectors cell populations allowed their being tested upon a wide panel of allogeneic EBV-transformed targets such that the dominant HLA-restricted reactivities within these populations could be identified. Monoclonal antibody blocking experiments confirmed that lysis of the autologous EBV-transformed cell line by IL2-expanded effectors could be specifically inhibited (a) by pretreatment of the target cells with antibodies binding to the HLA/beta 2-microglobulin complex, and (b) by pretreatment of the effector cells with the cytotoxic/suppressor T cell-specific antibody Leu 2a.     

Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.