Janine Moncuit

Immunoglobulin G-binding factors (IgG-BF) inhibit IgG secretion by, as well as proliferation of, hybridoma B cells

Mouse immunoglobulin G-binding factors (IgG-BF) produced either by activated T cells (ATC) or by hybridoma T cells (T2D4) directly inhibit the in vitro IgG secretion by hybridoma B cells. This inhibition affects IgG1, IgG2a and IgG2b and can be detected as early as after 2 h incubation of the cells with IgG-BF eluted from non-equilibrium pH gradient electrophoresis gels. Moreover, IgG-BF also exert a strong growth-inhibitory effect on hybridoma B cells without any detectable immediate cytotoxicity. These results provide an experimental basis to analyze the molecular and biological effects induced by IgG-BF on B cells.

Detection and quantification of secreted soluble FcγRIIA in human sera by an enzyme-linked immunosorbent assay

Abstract FcγRIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon. We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble FcγRIIA. It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of FcγRIIA as detector antibodies. The enzymatic reaction was amplified using an NADH/NAD + amplification system. As little as 0.8–1.5 ng/ml (20–38 pM) of purified recombinant secreted FcγRIIA could be detected. The serum levels of secreted sFcγRIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors. The mean value was 11.9 ng/ml ± 6.55 (297 pM ± 163) and the median value was 10.6 ng/ml (265 pM) (range: 0–764 pM).

Software for the quantitative evaluation of in vitro monoclonal antibody production from ELISA data

Software has been developed which permits the quantitation of monoclonal antibodies secreted by B cell hybridomas. This program does not require the user to enter a large number of complex parameters and can be easily used without any previous computer experience. It fits all the experimental and standard curves by determining overlapping linear domains using the linear least-squares method. The program is based on logarithmic interpolations for determining Ig concentrations comparing experimental samples to Ig concentrations in standards. It provides a complete print-out of the data with editing options and is written in BASIC EDEX 4.0 Commodore computer language. It permits the accurate quantification of minute amounts of monoclonal antibodies and can be used to detect the inhibitory or enhancing effects of lymphokines or cytokines on Ig secretion by hybridoma B cells.

A sensitive method for testing the effect of immunoglobulin binding factor on Ig secretion by hybridoma B cells

A microtiter plate assay to detect the effect of immunoglobulin binding factor (IBF) on Ig secretion by hybridoma B cells is described. This technique permits the analysis of Ig secretion by only 200-400 hybridoma B cells using a PFC assay and an ELISA, and therefore increases the IBF/cell number ratio. This increase allows the detection of a strong IBF-mediated inhibitory effect on Ig secretion by hybridoma B cells, which is otherwise difficult to obtain reproducibly. The technique is simple, does not require any transferring step and is convenient, since it permits large numbers of samples to be tested. It can be extended to test IBF for all isotypes or other lymphokines that affect Ig secretion.

Molecular mass analysis of murine immunosuppressive immunoglobulin G-binding factors (IgG-BFs) produced by T-cell hybrids

Induced and constitutive murine IgG‐binding factors (IgG‐BFs) have been purified by affinity chromatography from supernatants of T‐cells preincubated with or without murine monoclonal IgG1 and IgG2b, respectively. IgG‐BF M r values have been studied by SDS‐polyacrylamide gel electrophoresis (PAGE) after treatment with SDS under conditions which do not noticeably alter their immunosuppressive activities on the secondary in vitro IgG antibody response. Suppression was recovered at M r values of 80000, 40000 and 20000. When induced IgG‐BF was tested, the isotype‐specific suppressive activity was found only at 40 kDa. The 20‐kDa moiety appeared to derive from the 40‐kDa component and the material found at 80 kDa exerted non‐specific immunosuppressive effects. We conclude therefore that isotype‐specific IgG‐BF has an apparent M r of 40000.

Soluble Fcgamma receptor, Fc gammaRIIa2, is present in two forms in human serum and is increased in patients: with stage C chronic lymphocytic leukemia

Human Fcgamma receptor type II (FcgammaRII/CD32) can be produced in a soluble form, termed FcgammaRIIa2, which contains the extra- and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-encoding exon. We show that human sera contain two forms of FcgammaRIIa2. A full-length secreted protein, which has a 32 kDa backbone, can be detected only in some sera while all sera contain a C-terminal truncated form, lacking part of the intracytoplasmic tail, and exhibiting a 24 kDa protein backbone. The 24 kDa form is significantly increased in sera from stage C patients with B chronic lymphocytic leukemia, compared to healthy donors, stage A and B CLL patients, or CLL patients in complete remission.

In vitro inhibition of tumor B cell growth by IgG-BF-producing FcγRII+T cell hybridoma and by immunoglobulin G-binding factors

The growth-modulating effect on mouse hybridoma B cells of IgG-BF-producing FcγRII+mouse T cell hybridomas and of the IgG-BF isolated from the culture supernatants of these cells has been examined. Cocultures of IgG-secreting hybridoma B cells with IgG-BF-producing T hybridomas or with partially purified IgG-BF demonstrated a reproducible inhibition of the tumor B cell growth. The inhibition was due to a cytostatic and not to a cytotoxic effect. Hybridoma B cells cultured in liquid medium in the presence of soluble IgG-BF, or cocultured in semisolid agarose assays with IgG-BF-producing hybridoma T cells did not undergo immediate cytolysis but were prevented from proliferating. Thus, our data indicate that IgG-BF-producing FcγRII+T cells interfere with the proliferation of transformed B cells, possibly through soluble IgG-BF.

Structurally different anthracyclines provoke different effects on cell cycle and tumor B cell differentiation

Previously we have detected a stimulatory effect on immunoglobulin (IgG) synthesis when hybridoma cells were treated with doxorubicin. In order to determine whether this is a general property of anthracycline, we have selected three analogs--doxorubicin (DOX), pirarubicin (THP-DOX) and aclarubicin (ACR)--which differ mainly in the methylation state of their amino sugars. Cell cycle analysis by flow cytometry and drug localization by scanning confocal microscopy were also performed. The results show that when cells (UN2 hybridoma B cells), were exposed to subtoxic doses of DOX or THP (with unmethylated amino sugars), a strong increases in IgG secretion, heavy (H) and light (L) chain synthesis and the corresponding mRNA levels were induced. Furthermore these two drugs arrested the cells in the G2/M phase of the cell cycle. In contrast, exposure to ACR (with its methylated amino sugar) at similar subtoxic doses induced a blockade of cells in the G1 phase with no increase of IgG synthesis, at the subtoxic doses used, all three drugs could still be detected in the nucleus as well as in the cytoplasm, as determined by confocal laser microscopy. Thus, the relationship between cell cycle blockade, IgG stimulation and anthracycline structure is suggested by these results.

T CELL HYBRIDOMAS SECRETING SUPPRESSOR FACTOR

Publisher Summary This chapter discusses the T cell hybridomas secreting suppressor factor. Fusion of myeloma cells with spleen cells from immunized mice gives rise to B cell hybridomas producing antibodies against the sensitizing antigen. The fusion of T lymphoma cells with spleen cells can yield to hybrid cell lines with T cell surface markers. Such hybridomas may constitute a source of continus cells lines exerting certain T cell functions such as cytotoxic, helper, or suppressor activities. In an experiment described in the chapter, it was found that crude supernatants of T 1 inhibited from 21% to 27 % of the plaque forming cells response while supernatants of BW 5147 exerted no inhibition. It was also observed that when supernatants of T 1 were passed on immunoadsorbents of Sepharose beads coated with rabbit IgG, no suppressive activity was found in the effluent, while the acid eluted material inhibited from 49 % to 60 % of the PFC. When the supernatants of T 1 were passed on Sepharose beads coated with rabbit IgM, the suppressive activity was found in the effluent but not in the acid eluate. The same results were obtained with supernatants of T 2 hybrids and of T 1 clones.