Epstein-Barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs.

Peripheral blood lymphocytes from normal individuals and patients with autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis were infected with Epstein-Barr virus, and the culture supernatants were tested for autoantibodies that reacted with normal tissues. Between 58 and 86% of Epstein-Barr virus-transformed cultures produced immunoglobulin M antibodies, and between 9 and 24% of the transformed cultures produced immunoglobulin G antibodies that reacted with normal tissues. Ten Epstein-Barr virus-transformed clones secreting human immunoglobulin M monoclonal autoantibodies were isolated. Four of these monoclonal autoantibodies were studied in depth and found to react with antigens in multiple organs, including thyroid, pancreas, stomach, smooth muscle, and nerves. It is concluded that Epstein-Barr virus can trigger the production of autoantibodies without infecting the target cells to which the autoantibodies are directed.     

EBV-transformation of surface IgA-positive human lymphocytes

Surface IgA-positive human lymphocytes were selected, separated, and immortalized with Epstein-Barr virus (EBV). Selection was carried out by rosetting the cells with ox erythrocytes, coated with rabbit anti-human IgA immunoglobulin, followed by separation on Ficoll-Isopaque. Stable monoclonal lines were established by cloning in agarose. This shows that EBV-transformation is not restricted to the IgM-positive B cell category but can be extended to minority classes of B lymphocytes. The method provides the means to establish other minority categories as continuous lines, depending on the efficiency of the preselection procedure.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

In vitro growth of B lymphocytes infiltrating human melanoma tissue by transformation with EBV: evidence for secretion of anti-melanoma antibodies by some transformed cells

Epstein Barr virus was used to transform the B lymphocytes infiltrating metastatic tumor tissue from seven patients with melanoma. In this way it was possible to establish continuously growing B lymphoblastoid cell lines (LCL) derived from the tumor-infiltrating B lymphocytes from each of the seven patients. Antibody production of up to 50 micrograms/ml could be achieved by such cultures, and the lymphoblastoid cells could be cloned readily by limit dilution on a feeder layer of irradiated fetal fibroblasts. Preliminary analysis of the antibodies produced by lymphoblastoid cell lines established from tumors from two of the patients indicated that most were of IgM type and bound to a panel of melanoma cell target cells, as well as to some nonmelanoma tumor cell lines. Cloned LCL were produced from the tumor-infiltrating B cells from one of the patients, and of 100 such clones tested, 9% secreted antibody that bound to autologous tumor cells, and one of these clones produced antibody that appeared to be melanoma specific.     

Production of human monoclonal antibodies against various antigens of erythrocyte stages of Plasmodium falciparum

Peripheral blood lymphocytes from 20 individuals living in a malaria endemic area (Burkina Faso) were transformed with Epstein-Barr virus. No antigen specific selection, nor stimulation of B-cells were performed prior to transformation. 20 cell lines were established, 14 secreted polyclonal antibodies directed against erythrocytic stages of Plasmodium falciparum. 4 lines were cloned and the supernatant analysed and characterised against Plasmodium falciparum antigens.     

Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro.

Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.     

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.     

A Monoclonal Antibody Defining Human B Cell Differentiation Antigen (HLB-1 Antigen)

A new monoclonal antibody specific for human B cell differentiation antigen (HLB-1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein-Barr virus (EBV)-transformed peripheral B cell line, RPMI-8057. This monoclonal, antibody designated anti-HLB-1 monoclonal antibody (anti-HLB-1), reacted with surface immunoglobulin (sIg)-positive B cells of normal peripheral blood and lymphoid tissues and sIg-positive leukemic cells. The cells of T cell leukemia, non-T non-B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB-1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti-HLB-1 reacted with B cell lines derived from pre-B, Burkitt's lymphoma, B cell type ALL and EBV-transformed peripheral B cells. Anti-HLB-1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non-T non-B ALL cell lines. This newly defined HLB-1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.     

Monoclonal antibodies to alpha DNA polymerase as a marker of cell proliferative activity

A hybridoma cell line (5F) secreting monoclonal antibodies directed to alpha DNA polymerase has been developed. Kinetic studies on peripheral blood lymphocytes stimulated with mitogen and human colon cancer cell lines established in vitro were made by the two autoradiographic techniques of Thymidine Labelling Index and Primer-dependent alpha DNA polymerase Labelling Index and the immunoperoxidase assay (PAP) with monoclonal antibody to alpha DNA polymerase. We demonstrated the exclusively intranuclear presence of alpha DNA polymerase in lymphocytes induced to proliferate and actively growing colon cancer cells in contrast with the cytoplasmic distribution of the enzyme in resting stage populations. The feasibility of using monoclonal antibodies to alpha DNA polymerase to determine cell growth fraction was evaluated.     

Helper factors derived from autologous mixed lymphocyte cultures

Human tonsillar lymphocytes infected with Epstein-Barr virus (EBV) were cultured at a cell concentration of 2 × 10⁶/ml in a 0.01-ml volume in microtest plates and cell lines composed of relatively small numbers of clones (oligoclonal) were established. Culture supernatants of 1020 cell lines thus established were screened for anti-phosphorylcholine (PC) antibody production by passive hemagglutination (PHA) assay and 10 cell lines with high anti-PC PHA titers were obtained. Plaque-forming cell (PFC) assay of these cell lines revealed that in seven cell lines more than 1% of the total cells and especially in two cell lines more than 10% of the total cells were anti-PC antibody-producing cells. Free PC concentrations required to inhibit PHA (8 PHA titer) and PFC (50% inhibition) correlated well in the same cell lines, but among the different cell lines considerable differences (10- to 1000-fold) of the PC concentration were obtained, indicating that anti-PC antibody-producing cells with different binding affinities to PC were transformed in these cell lines. PHA inhibition by monospecific antisera against immunoglobulin heavy-chain classes revealed that anti-PC antibodies produced in these cell lines were of the IgM class. Cloning experiments were performed and one clone stably producing anti-PC antibodies was obtained.     

Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interferon production potentials of various human lymphoblastoid cell lines

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus-induced interferon (IFN). It was found that, in addition to B cells, various T and nonT-nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN-alpha and might become an alternative source of IFN production. Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein-Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN-alpha. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.     

Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus

Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein-Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen.     

Establishment and characterization of a new Epstein-Barr virus transformed cell line from a human B cell lymphoma

We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/microliters) were infected in vitro with Epstein-Barr virus (EBV), and CD20+ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, out-growing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Studies on human porin. V. The expression of "porin31HL" in the plasmalemma is not by cell transformation]

In recent papers we proved "Porin 31HL" to be located on the surface of human, EBV-transformed B lymphocytes. Here we present proof of "Porin 31HL" in the plasmalemma of normal human blood lymphocytes. For this purpose B and T lymphocytes were isolated from human heparinized blood and examined by indirect immunofluorescence techniques using different monoclonal antibodies against purified "Porin 31HL" and some B and T cell markers, respectively. For comparison a number of established cell lines of different origin were employed. Hence it followed that normal B and T cells as well as transformed and leukemic cells express "Porin 31HL" in their membrane. No significant quantitative differences could be seen. Consequently, the location of "Porin 31HL" in the plasmalemma is not a product of transformation.     

Sequence complexity of circular Epstein-Bar virus DNA in transformed cells

A simplified procedure, based on several methods previously used to isolate circular DNA molecules from bacteria, was derived for the preparation of covalently closed circular viral DNA molecules from large quantities of lymphocytes transformed by Epstein-Barr virus. The protocol can be applied both to virus nonproducer lines and to lines containing cells activated to virus production. Sufficient amounts o highly purified viral DNA of intracellular origin were obtained from B95-8 and Raji cells to allow direct visual analysis of their sequence complexities after cleavage with EcoRI and separation of fragments by gel electrophoresis. No major differences in complexity were observed between circular DNA and linear virion DNA from B95-8 cells. The fragment patterns observed in this fashion agree well with those detected by conventional blotting and hybridization methods. The procedure can also be used as an analytical method to assay for small amounts of circular Epstein-Barr virus DNA molecules in other transformed cells. In this connection, no circular Epstein-Barr virus DNA was detected in Namalva cells.