Antibodies to mouse lung capillary endothelium

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM⁺ spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG⁺ spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.     

Monoclonal antibodies against rat saliva and salivary gland antigens

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.     

Comparison of the T cell receptors on insulin-specific hybridomas from insulin transgenic and nontransgenic mice. Loss of a subpopulation of self-reactive clones.

Transgenic mice expressing the human insulin gene do not produce insulin-specific antibody after injection of human insulin. Nevertheless, they have some peripheral T cells that proliferate to human insulin in vitro. To investigate the nature of these T cells, human insulin-specific T cell hybridomas were produced from transgenic and nontransgenic mice. Transgenic hybridomas required more insulin to achieve maximum responses and they produced lower levels of lymphokines than nontransgenic hybridomas. The majority of nontransgenic hybridomas recognized only human and pork insulin whereas transgenic hybridomas recognized beef, sheep, and/or horse insulin in addition to human and pork insulin. The TCR expressed by transgenic and nontransgenic hybridomas were determined by Northern analysis. Both types of hybridomas used several different V alpha and V beta gene families and no favored association between V alpha and V beta gene usage was detected in either type. V beta 1 was used by 7 of 16 nontransgenic hybridomas but only by 1 of 16 transgenic hybridomas. V beta 6 receptors were predominantly expressed by the transgenic hybridomas and all V beta 6-bearing hybridomas recognized beef as well as human insulin. The differences in Ag reactivity and TCR gene usage suggest that V beta 1-bearing human insulin-reactive T cells were clonally deleted or inactivated in the transgenic animal. Other clones, representing a minor subpopulation in nontransgenic mice, were recovered from transgenic mice.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Generation and characterization of novel stromal specific antibodies

Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic. From five fusions clones were identified with predominant reactivity with： 1） fibroblasts and endothelial cells; or 2） broad stromal elements （fibroblast, endothelium, epithelium, follicular dendritic cells）. A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.     

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.     

Comparison of a quadroma and its parent hybridomas in fed batch culture

A quadroma (#22 × 63), formed by the fusion of two hybridomas, and its parent hybridomas (#22 and FMC 63) were each grown in fed batch cultures in order to examine the change in antibody productivity over time of the quadroma compared to its parent hybridomas. The growth rate, glucose uptake rate and lactate production rate of the quadroma were found to be intermediate between those of its parent cells of origin. The specific antibody productivity and internal antibody content of the quadroma followed the same decreasing trends over time as those seen in both parent hybridomas. Losses in specific antibody production rate and antibody content, however, occurred at a faster rate for the quadroma than for either of its parent hybridomas. Although the growth of a non-producing subpopulation is presumed to account for the drop in antibody production, there was no direct correlation between the percentage of high antibody containing cells, as determined by flow cytometry, and the specific antibody production rate.     

Presence of an antigen-specific T cell subset that forms IgE-suppressive factor and IgG-suppressive factor on antigenic stimulation

B6D2F1 mice were given three i.v. injections of ovalbumin (OA), and antigen-specific T cell clones were established from their spleen cells. One of the FcR+ T cell clones formed IgE-binding factors on incubation with OA-pulsed syngeneic macrophages. Neither soluble antigen nor macrophages alone induced factor formation. T cell hybridomas were constructed by fusion of the antigen-specific T cell clone with BW 5147 cells. Among 11 T cell hybridomas established, six clones produced IgE-binding factors on incubation with OA-pulsed BDF1 macrophages. Mouse IgE also induced the same hybridoma to form IgE-binding factors. The majority of IgE-binding factors formed by two T hybridomas and by those produced by the parent T cell clone had affinity for peanut agglutinin but for neither lentil lectin nor Con A. These hybridomas and the original T cell clone spontaneously released glycosylation-inhibiting factor, which inhibits the assembly of N-linked oligosaccharide(s) on IgE-binding factors. On antigenic stimulation, the T cell hybridomas produced both IgE-binding factors and IgG-binding factors. The IgE-binding factors consisted of three species with m.w. of 60,000, 30,000, and 15,000. Both the 60K and 15K IgE-binding factors selectively suppressed the IgE response of DNP-OA-primed rat mesenteric lymph node cells, whereas IgG-binding factors selectively suppressed the IgG response. The results indicate that antigen-primed FcR+ T cells produced IgE-suppressive factors and IgG-suppressive factors on antigenic stimulation. However, the T cell hybridomas were not committed to suppressive activity. When the hybridomas were stimulated by antigen in the presence of glycosylation-enhancing factor (GEF), the 60K, 30K, and 15K IgE-binding factors formed by the cells selectively potentiated the IgE response. IgG-binding factors formed by the cells in the presence of GEF failed to suppress the IgG response. It appears that antigen-specific FcR+ T cells regulate the antibody response through the formation of Ig-binding factors, but that the function of the cells could be switched from suppression to enhancement, depending on the environment of the cells.     

A strategy for the production of human monoclonal antibodies

The EBV-hybridoma system will be, at present, the best method for rescuing low-frequency tumor-reactive B-cell clones in cancer patients to produce human monoclonal antibodies reactive with tumor-related antigens. To improve this system, we established a nobel fusion partner, 3HL3-6J(C5), which produced hybridomas efficiently (greater than 2 x 10(-5)) after fusion with EBV-transformed B cell lines. TAPC-301 and C5TK1, anti-SRBC IgM and anti-HBs IgG producer, respectively, which provided by Prof. Y. Ono, were used as standard EBV-transformed B cell lines. Their hybridomas were good antibody producers (greater than 10 micrograms/10(6) cells/24 hrs) and their cloning was relatively easy. The fusion rate was improved further when electrofusion was carried out instead of the conventional PEG fusion. Using PBL from a hepatoma patient, human monoclonal autoantibodies reactive with some cytoskeleton structure were easily produced by means of this system.     

Studies on the mechanism of long term survival of Taenia taeniaeformis in rats.

An attempt was made to determine if blocking antibody is involved in protecting cysticerci of Taenia taeniaeformis against a host immune response. Immunoflourescence microscopy confirmed that host antibody is presnet on the parasite surface within the capsule. To test if the larvae can still survive after such a coat of blocking antibody is removed, the larvae were trysinised and then implanted into recipients. The results indicate that blocking antibody could be involved in the survival of 1 year old established larvae. Untrypsinised larvae were normal 14 days after implantation into control or immunised rats. Trypsinised larvae implanted in control rats were alive but showed on intense cell adherence on their surface. On the other hand, trypsinised larvae implanted into immunised rats were dead and completely encapsulated. However, experiments with 1 month old larvae were inconclusive.     

Immunological studies of aging. Normal B-cell repertoire in aged mice: studies at a clonal level

As previously reported, old mice produce lower avidity plaque-forming cells (PFC) after immunization with 2,4,6-trinitrophenyl-Ficoll (TNP-F) than do young mice. However, if spleen cells from TNP-F-immunized old mice are incubated with hapten to elute auto-anti-idiotype antibody then high avidity PFC, comparable to those in young mice, are detected. To further evaluate the effect of age on the B-cell repertoire anti-2,4,6-trinitrophenyl-bovine gamma globulin (TNP-BGG) hybridomas were prepared from young (6 to 8 weeks old) and old (18 to 24 months old) mice which had been primed and boosted with TNP-BGG. The monoclonal antibodies (MoAb's) were TNP-specific. Spleens from old and young mice were comparable with respect to the incidence of immunoglobulin-secreting hybridomas obtained, the incidence of TNP-BGG-specific hybridomas obtained, and the isotype distribution of the anti-TNP-BGG hybridomas. The avidities for TNP-BGG of the IgG1 anti-TNP-BGG MoAb's obtained from old and young donors were also comparable. The overall results thus suggest that old and young mice have similar B-cell repertoires and that differences in the antibodies produced are due to regulatory influences.     

Demonstration of a novel tumor-killing factor secreted from human macrophage-monocyte hybridomas

We constructed human macrophage-monocyte hybridomas between a thymidine kinase-deficient human macrophage-like cell line, designated as TAM-2, and human peripheral blood monocytes in order for the study of cytokines from human monocytes. The hybrid and macrophage-monocyte nature of the growing cells was confirmed by the following facts: 1) All of the hybridomas established possess TK activity, whereas the TAM-2 cells are TK negative. 2) Most but not all of the hybridomas express the MaG-1 Ag which was shown to be a human macrophage-granulocyte specific Ag, but not T- and B-specific Ag. In the assay for cytokine, a few of the hybridomas produced a novel tumor-killing factor (TKF) after stimulation with PMA, polypeptone, and retinoic acid. Chemical nature of the TKF from the 3-63 hybrid clone was characterized and compared to those of well-known TNF and lymphotoxin. The TKF from a hybridoma was basic protein and had binding capacity to Con A-Sepharose, whereas TNF had an opposite nature. Moreover, TKF activity was not neutralized by both a murine monoclonal antibody against human TNF and rabbit antisera against human lymphotoxin. Thus, these results strongly indicate that the TKF is a novel TKF produced by human monocytic cells.     

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

Inhibition of IgE production in B hybridomas by IgE class-specific suppressor factor from T hybridomas

The function of IgE class-specific suppressor factor (IgE-TsF) from T hybridomas was studied by employing IgE-producing B hybridomas. IgE-TsF was obtained from IgE class-specific T hybridomas, which had been established by the fusion of a phosphorylcholine-conjugated Mycobacterium-primed T cell population with the T lymphoma cell line BW5147. The absorption experiments showed that IgE-TsF from T hybridomas was composed of the binding site(s) for IgE and I region gene products as observed in conventional IgE-TsF. Incubation of IgE-producing B hybridomas with IgE-TsF for 1 hr at 37 degrees C resulted in the reduction of the number of IgE-secreting cells when assessed by a reverse plaque assay. The proportions of surface IgE-positive cells were concomitantly reduced. After 24 hr incubation with IgE-TsF, the number of cytoplasmic IgE-positive cells was reduced, showing that IgE synthesis was inhibited by IgE-TsF. Antigen-specific TsF from phosphorylcholine-specific T hybridomas did not show any inhibitory effect, and IgE-TsF did not block the antibody production of IgM-producing B hybridomas. Precapping of IgE receptors by anti-epsilon antibody or the simultaneous addition of soluble IgE with IgE-TsF abrogated the suppressive function, suggesting that IgE-TsF acted directly on B epsilon cells through binding with IgE receptors.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.