Neutral proteinases induce rheumatoid factor production in mouse spleen cell cultures.

Mouse spleen cells were cultured for 4 days in RPMI 1640 medium with 5% fetal calf serum. The neutral proteinases trypsin and plasmin, and bacterial lipopolysaccharide LPS, all polyclonal B lymphocyte activators, stimulated the development of immunoglobulin producing cells as detected by the protein A plaque assay. At the same time, direct plaque forming cells reacting with mouse, human and rabbit IgG and the Fc fragment of human IgG were induced by the stimulants. The plaques could be inhibited by free IgG or Fc fragment. In the culture supernatants, IgM and IgM anti-IgG antibodies were detected by enzyme linked immunosorbent assays. Both general IgM and IgM anti-IgG antibodies increased under the influence of the proteinases and of LPS. The results are discussed in relation to rheumatoid factor production during inflammatory diseases.     

Spontaneous Reverse Haemolytic Plaque Formation

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94%; this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10-1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1, this significantly reduced or abolished spontaneous IgG plaques, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for monocytes, OKM1 and FMC17, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors.     

Production of monoclonal antibodies against prostatic acid phosphatase by in vitro immunization of human spleen cells

Monoclonal antibodies against human prostatic acid phosphatase (PAPase) were produced by immunization of human primary spleen cell cultures. Dissociated spleen cells were cultured for 5-8 days in the presence of 100 ng/ml of PAPase and pokeweed mitogen (1:5000). Following immunization, B cells were isolated and infected with Epstein-Barr virus (EBV). Two weeks after EBV-transformation, cells were fused with either mouse myeloma cells (SP2/OAg14) or human/mouse heteromyeloma cells (SHM-D33). Hybrid clones were screened for anti-PAPase production. In 7 independent immunizations, the average fusion frequency was 3.6 per 10(6) lymphocytes. 18-32% of the hybridomas produced anti-PAPase; approximately 75% of these secreted IgM and 25% secreted IgG. Antibody specificity was determined by immunoassay and immunohistological studies. The procedures described here may be suitable for the production of human monoclonal antibody of a useful specificity.     

Regulation of an anti-self response: lack of influence of exogenous DNA on the in vitro anti-DNA response.

Anti-DNA antibodies occur in outwardly normal individuals as well as in various forms of autoimmune disease. A number of publications have reported on the ability of added DNA to either induce or inhibit the in vitro production of anti-DNA antibody. In this study, the in vitro production of IgM anti-single stranded DNA (alpha ssDNA) antibody by spleen cells from normal or autoimmune mice neither depends upon, nor is inhibited by, the addition of high molecular weight DNA to the culture. The decrease in antibody forming cell plaques, reported previously, is due solely to the artifactual carryover of inhibitory material into the assay system, where it interferes with the expression of plaques by preventing anti-DNA antibody from reaching the DNA-coated erythrocytes. Similarly, plaque forming cell (PFC) methods have not detected alpha ssDNA antibody producing cells in murine spleen cells without culturing, but various other systems for measuring antibody normally detect anti-DNA antibodies in vivo. This discrepancy is also due to inadequate washing of freshly harvested cells to rid them of inhibitory substances which prevent them from registering as PFC. While S1 nuclease was able to prevent PFC interference by purified DNA, it did not remove the inhibitory substances from the culture supernatants; therefore substances other than ssDNA are able to interfere with alpha ssDNA PFC, suggesting that the alpha ssDNA PFC detected are polyspecific. Levels of alpha ssDNA PFC in spleen cells from non-autoimmune mice begin at one-quarter of the peak in vitro response, decrease to one-tenth in the first day and then reach peak values after 3 to 5 days of culture, suggesting that spleen cells are actively producing alpha ssDNA antibodies an in vivo and that then in vitro response is observed. Despite this evidence for an in vitro alpha ssDNA response, this response was not inhibited markedly by 1000 rad gamma-irradiation, while the response to sheep erythrocytes (SRBC) was profoundly suppressed. These findings suggest that anti-self B lymphocytes are resistant to interphase, possibly apoptotic, lymphocyte death due to gamma-irradiation, while anti-nonself B lymphocytes remain sensitive.     

驱虫斑鸠菊对正常小鼠免疫功能的影响

目的探讨驱虫斑鸠菊对小鼠免疫功能的影响,揭示其免疫作用机理.方法利用[3H]-TdR掺入法分别测定了驱虫斑鸠菊对环胞菌素A(Con A)诱导的小鼠脾脏T淋巴细胞和细菌脂多糖(LPS)诱导的B淋巴细胞增殖活性以及脾脏T淋巴细胞分泌白细胞介素-2(IL-2)活性的影响,运用酶联免疫吸附试验测定了驱虫斑鸠菊对小鼠血清中抗体活性的影响;采用流式细胞仪测定脾脏B淋巴细胞表面抗原CD19的表达水平.结果驱虫斑鸠菊的低、中、高3个剂量体内对脾脏T、B淋巴细胞的增殖活性、血清总抗体和针对人A375黑素瘤细胞抗原特异性抗体含量、B细胞表面抗原CD19的表达以及对脾脏T淋巴细胞分泌IL-2活性都具有明显抑制作用.结论驱虫斑鸠菊对机体体液免疫和细胞免疫功能都具有明显抑制作用.     

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.     

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.     

Monoclonal opsonic mouse antibodies specific for streptococcal IgG Fc-receptor

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.     

Monoclonal antibodies to human malignant mesothelioma

Murine monoclonal antibodies were used to identify tumor-cell membrane antigens on a new human mesothelioma cell line. Hybridomas were constructed by fusing SP2/0 mouse myeloma cells with spleen cells from Balb/C mice immunized by the human mesothelioma cell line MT-1. Hybridoma antibody was detected in 55/672 microculture wells that reacted to these MT-1 tumor cells by an indirect¹²⁵I-protein A binding assay. Six cultures produced antibody binding selectively to the MT-1 tumor cells but not to a human lymphoblastoid cell line. These six hybridomas were cloned: three were IgG and three were IgM antibodies. One monoclonal, MAb 45, reacted with 4 of 7 human mesothelioma cell lines but with only 1 of 11 carcinomas, 1 of 3 sarcomas, 4 of 11 melanomas, and 0 of 5 lymphoid lines. The other five monoclonals had a much broader cross-reactivity. Using an immunoperoxidase technique, MAb 45 bound to mixed-type malignant mesotheliomas but not to normal lung and pleura. The specificity of MAb 45 for diffuse mesotheliomas and the low cross-reactivity with carcinomas and normal adjacent tissues suggest that this monoclonal may be clinically useful.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Use of a murine T-cell hybridoma expressing human T-cell receptor alpha- and beta-gene products as a tool for the production of human T-cell receptor-specific monoclonal antibodies.

We describe the production of mouse monoclonal antibodies specific for the human TcR using as the immunogen transfected murine T-cell hybridoma cells coexpressing mouse CD3 with human Jurkat TcR alpha and beta chains. The shortage of monoclonal antibodies (mAbs) specific for the human TcR-V alpha and V beta families reflects the difficulties in their production by conventional methods using whole human T cells or purified soluble receptors as immunogens. As an alternative strategy to circumvent these difficulties, we have generated a transfected mouse T-cell line expressing a human (Jurkat) TcR alpha beta dimer in a complex with mouse CD3. The parental mouse T-cell line, TG40, is a cell surface TcR-negative, cytoplasmic CD3-positive variant of the mouse T-cell hybridoma 2B4. The human-TcR alpha beta expressing mouse transfectant was used to immunize mice with the same genetic background as the parent mouse T-cell line, and a human TcR-specific response was successfully achieved. MAb-producing hybridomas were generated by fusing spleen cells from the immunized mice with the mouse myeloma cell line NSO. Of 124 hybridoma supernatants screens, 72 showed reactivity to the human T-cell line Jurkat. Twenty-four of the hybridomas producing human (Jurkat) TcR-specific antibodies were cloned and screened for reactivity to Jurkat TcR. Several IgG2b and IgM mAbs specific for the Jurkat T cell line were selected on the basis of their ability to modulate surface CD3 expression on Jurkat cells. Most of the antibodies do not stain other TcR-expressing human T cell leukemia cell lines, implying specificity for the variable domains of the Jurkat TcR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of estradiol on antibody secretion of murine hybridomas

The need for increased antibody production by hybridomas has been approached by the addition to cell cultures of different growth factors; in vitro addition of estradiol-17beta (E2) to human blood lymphocytes increases the accumulation of plasma-blasts and Ig-secreting cells. Four different murine-murine hybridomas secreting different monoclonal antibodies (MAbs) were treated with E2. Specific antibody concentration was measured by enzyme-linked immunoadsorbent assay (ELISA) in culture supernatants whereas expression of E2-receptor in the hybridoma cells was determined by polymerase chain reaction (PCR). When E2 was added as a growth supplement to alpha-estrogen receptor positive murine-murine hybridomas it enhanced MAb secretion by as much as 255%, in a dose-dependant manner. This effect lasted for as long as the alpha-estrogen receptor was detected in the hybridoma cells, was inhibited by tamoxifen and was not observed in alpha-estrogen receptor negative hybridomas. The synthetic estrogen analogue diethylstilbestrol had no effect. Estradiol-17beta should be added to the list of hybridoma-inducing growth factors.     

Inhibition of T cell-dependent antibody production by D-penicillamine

The effects of D-penicillamine (D-Pen) on the production of immunoglobulins (Ig) and anti-microbial antibodies (Ab) by human mononuclear cells (MNC) cultured in vitro were analysed by haemolytic plaque forming cell (PFC) assays. Polyclonal Ig and Ab production, induced by pokeweed mitogen (PWM), was not affected by D-Pen in pharmacologically relevant concentrations, unless Cu2+ was added. Likewise, D-Pen + Cu2+, but not D-Pen alone, affected polyclonal Ig production induced by Epstein-Barr virus. The production of interleukin-2 and B cell growth factor by phytohaemagglutinin-stimulated T cells was not inhibited by D-Pen; again D-Pen + Cu2+ markedly reduced the production of these cytokines. By contrast, D-Pen significantly reduced the antigen-induced antibody production without requirement of additional Cu2+. Addition of cytokine-containing supernatant to MNC treated with D-Pen + Cu2+ tended to increase the PWM-induced Ig responses, but had no effect on the antigen-induced Ab production of MNC cultured with D-Pen. Thus, the mechanisms by which D-Pen suppresses polyclonal and antigen-induced B lymphocyte responses are different.     

Mesenchymal stem cells stimulate antibody secretion in human B cells

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T-cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme-linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co-cultures of MSC and lymphocytes increased IFN-gamma production. MSC produce IL-6, and addition of MSC to spleen cells dramatically increased IL-6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B-cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.     

Hybridoma-derived human suppressor factors: differential effects on mouse lymphocytes

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.     

Delineation of IgM-receptor bearing human T and B lymphocytes using a direct plaque forming cell (PFC) assay.

Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C, added to SRBC monolayers then incubated at 37 degrees C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)5 mu but not by Fab mu fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)mu specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.     

A monoclonal antibody with specificity for murine mu heavy chain which inhibits the formation of antigen-specific direct IgM plaques

A panel of monoclonal rat antibodies binding to mouse mu heavy chain were tested for their ability to inhibit the formation of antigen-specific plaques in the hemolytic plaque assay. Nine antibodies inhibited SRC-specific direct IgM plaques at high concentrations (greater than 20 micrograms/ml). In contrast to all others, however, one antibody inhibited these plaques at much lower concentrations (down to 0.4 microgram/ml) when added to the assay. This antibody also inhibited plaques formed by cells secreting antibodies against trinitrophenyl or phosphorylcholine determinants. IgG plaques with any of the above specificities were not inhibited. IgM secretion was unaffected by the monoclonal anti-mu antibody. Its inhibitory effect on plaque formation rather appears to be a consequence of its ability to inhibit complement dependent, IgM mediated lysis of erythrocytes. This monoclonal anti-IgM antibody therefore provides a convenient reagent to distinguish specific direct IgM plaques from indirect IgG plaques.     

Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons

More than 13,500 initial hybridoma lines derived from fusions of lymphocytes from non-boosted persons were tested for IgG production against Tetanus Toxoid. However, only 2 were found to produce IgG monoclonal antibodies of the desired specificity. Peripheral blood lymphocytes were then taken from actively immunized donors 3, 7, 14 or 60 days after boosting and fused to the HAT-sensitive heteromyeloma cell line CB-F7. A strong enhancement of both IgG-producing hybridomas and specific IgG-producers was detected in fusions of lymphocyte material derived 7 days after booster injection (239 of 731 IgG-producing lines showed anti-TT-specificity). Two months after boosting no more specific IgG-producing hybridomas could be established from the peripheral blood of the donors. Data were discussed regarding an optimized human monoclonal antibody production against bacterial antigens.     

B Cell Hybridoma Presents Both B-Cell and T-Cell Epitopes for Stimulating Antibody Production Via CD23 Pathway

CD23+ B cell hybridoma 17A11, pulsed with IgE:TNP-KLH triggered IgA, IgG, and IgE antibody production via CD23-mediated presentation. Prior anti-CD23 treatment abrogated 95% of the humoral antibody responses. Both B and T cell epitopes were presented by 17A11. B cell epitopes as recognized by IgG but not T cell epitopes were sensitive to treatment with 0.2 M acetic acid. Efficacy of antigen presentation via CD23 on 17A11 was comparable to that mediated via surface immunoglobulins (sIg) on a CD23 negative 4.5 parental fusion partner B cell line. This is the first demonstration that IgE:TNP-KLH pulsed B cell hybridomas present both B-and T-cell epitopes in stimulating IgA, IgG, and IgE antibody production, and raise a pertinent issue whether IgE antibodies produced under pathophysiological conditions may serve as positive feedback signal for sustaining production of different classes of antibodies.     

B Cell Hybridoma Presents Both B-Cell and T-Cell Epitopes for Stimulating Antibody Production Via CD23 Pathway

CD23+ B cell hybridoma 17A11, pulsed with IgE:TNP-KLH triggered IgA, IgG, and IgE antibody production via CD23-mediated presentation. Prior anti-CD23 treatment abrogated 95% of the humoral antibody responses. Both B and T cell epitopes were presented by 17A11. B cell epitopes as recognized by IgG but not T cell epitopes were sensitive to treatment with 0.2 M acetic acid. Efficacy of antigen presentation via CD23 on 17A11 was comparable to that mediated via surface immunoglobulins (sIg) on a CD23 negative 4.5 parental fusion partner B cell line. This is the first demonstration that IgE:TNP-KLH pulsed B cell hybridomas present both B-and T-cell epitopes in stimulating IgA, IgG, and IgE antibody production, and raise a pertinent issue whether IgE antibodies produced under pathophysiological conditions may serve as positive feedback signal for sustaining production of different classes of antibodies.