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.

Highly resolved chemical imaging of living cells by using synchrotron infrared microspectrometry.

Using synchrotron radiation as an ultra-bright infrared source, we have been able to map the distributions of functional groups such as proteins, lipids, and nucleic acids inside a single living cell with a spatial resolution of a few microns. In particular, we have mapped the changes in the lipid and protein distributions in both the final stages of cell division and also during necrosis.

Chemical imaging of nucleic acids, proteins and lipids of a single living cell. Application of synchrotron infrared microspectrometry in cell biology.

Hybridoma B-cells have been used as models to evaluate the performance of synchrotron infrared microscopy to obtain chemical images of a single living cell. Chemical mapping of nucleic acids, proteins and lipids at a resolution of a few microns, close to the diffraction limit in the mid-infrared region are shown.

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.

Fc receptors as targets for immunotherapy.

Human membrane and soluble Fc epsilon receptors (Fc epsilon RI, Fc epsilon RII/CD23) and Fc gamma receptors (Fc gamma RI/CD64, Fc gamma RII/CD32, Fc gamma RIII/CD16) have been implicated in a number of diseases. Their functional roles such as capture and clearance of immune complexes, antibody-dependent cell cytotoxicity, or cytokine or inflammatory mediator release, make them potential targets for immuno-intervention. In the present review, we will describe how membrane and soluble human Fc epsilon R and Fc gamma R have been already used as targets/tools for immuno-interventions by using monoclonal and bispecific engineered antibodies. Some therapeutic uses of these molecules both in cancer, infectious, and auto-immune diseases are presented.

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

Natural and recombinant soluble low-affinity Fc gamma R: detection, purification, and functional activities.

Studies on the identification, cloning, and biochemical characterization of natural and recombinant human and mouse low-affinity soluble Fc gamma R (sFc gamma R) have been developed using various methods. RT-PCR and/or biochemical analyses have demonstrated that low-affinity sFc gamma R (i) are generated by enzymatic cleavage of membrane-associated receptors or by an alternative splicing of the transmembrane region encoding exon and (ii) comprise only the extracellular domains or these domains plus the intracellular region of the membrane-associated molecules, respectively. Functional studies indicated that recombinant sFc gamma R bind mouse and human IgG subclasses with a binding profile identical to that of their membrane counterparts and inhibit Fc gamma R-mediated functions such as immune complex binding or ADCC. In addition, it has been demonstrated that a mouse recombinant truncated sFc gamma RII inhibits antibody responses to T-dependent antigens as well as B-cell proliferation and that a human recombinant truncated sFc gamma RIIIB blocks the Ig production by activated human peripheral blood mononuclear cells. Finally, different immunoassays devised to detect and quantitate circulating sFc gamma R showed that sFc gamma R serum levels vary in circumstances such as injections of protein antigens, in parasitic infections, in tumor-bearing mice, in patients with multiple myeloma (MM), or upon infusions of IgG or Fc gamma fragments in MM or immune thrombocytopenic purpura patients. The use of recombinant sFc gamma R, as well as the availability of monoclonal and polyclonal antibodies directed against different regions of these molecules, makes it possible to characterize further the biological effects of sFc gamma R and their biochemical and immunochemical characteristics, as well as to define their putative ligands on cell membranes.

Detection and quantification of secreted soluble Fc gamma RIIA in human sera by an enzyme-linked immunosorbent assay.

Fc gamma RIIA 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 Fc gamma RIIA. It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of Fc gamma RIIA 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 Fc gamma RIIA could be detected. The serum levels of secreted sFc gamma RIIA 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).

Release of soluble Fc gamma RII/CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion?

Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.

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

The growth-modulating effect on mouse hybridoma B cells of IgG-BF-producing Fc gamma RII+ 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 Fc gamma RII+ T cells interfere with the proliferation of transformed B cells, possibly through soluble IgG-BF.

Regulation of IgG production by suppressor Fc gamma RII+ T hybridomas.

In this work, we analyzed the immunoglobulin heavy (H) and light (L) chain production by two variant B hybridomas, UN2.C3 and UN2.C17.K1 co-cultured with cells from a Fc gamma RII+, IgG-binding factor (IgG-BF)-producer T hybridoma (T2D4.C1) or with cells of a Fc gamma RII-, IgG-BF-nonproducer variant (D10C5). We showed that only the Fc gamma RII+ hybridoma directly inhibits the IgG secretion by UN2.C3 through a soluble mediator. This inhibition affects the H and L chain synthesis as well as the H and L chain-encoding mRNA steady state. No apparent cytotoxic effect could be detected. In contrast, the production of kappa chain by an H chain-negative variant (UN2.C17.K1) was unaffected. This indicates that a complete IgG molecule is required to observe the inhibitory effect induced by T2D4.C1. The pattern of effector/target cell interactions observed in our work suggests that the soluble factor involved in the suppression of IgG production is IgG-BF, able to transiently modify the IgG gene expression in target cells.

Regulatory effects of IgG-BF on hybridoma B cells. molecular characterization of variant cell lines.

Immunoglobulin G-binding factors (IgG-BF) produced by mouse T cells or hybridoma T cells (T2D4) were used to manipulate in vitro mouse hybridoma B cells. Both IgG production by, and proliferation of, these cells was inhibited by IgG-BF, or during co-cultures with IgG-BF-producing T2D4 cells. Thus, treatment of tumor B cells, besides its potential therapeutic use, represents an invaluable model for studying the regulation of Ig production by IgG-BF at a molecular level. To further analyze the molecular events induced by IgG-BF in B cell hybridomas, a set of variant clones of a hybridoma cell line (UN2) was isolated and variants were characterized for their Ig production and their Fc gamma R expression.

Regulation of hybridoma B-cell proliferation by immunoglobulin-binding factor.

Immunoglobulin-binding factors (IBF), which are cytokines able to suppress the immunoglobulin production by normal and transformed B cells, have been tested for their ability to interfere with hybridoma B-cell growth. Coculture experiments performed in soft agar between hybridoma B cells and hybridoma T cells secreting IgG-BF indicated that IgG-BF act as a growth regulatory molecule able to inhibit strongly the development of hybridoma B-cell colonies; this cytostatic effect, which appeared not to be a direct cytotoxic effect, was confirmed when using semi-purified IgG-BF in both soft agar and liquid medium cultures of hybridoma B cells. Thus, IBF appear to act as both immunoregulatory and growth regulatory factors. Some of the molecular consequences of this dual effect and its possible role in certain B-cell lymphoproliferative diseases such as multiple myeloma are discussed.

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.

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.

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.

Size and charge heterogeneity of murine IgG-binding factors (IgG-BF).

Size and charge of murine IgG-binding factors (IgG-BF) were determined. Four different sources were used to produce the factors: a) cells of a T cell hybrid (T2D4) constitutively secreting IgG-BF upon incubation in serum-free medium, b) T2D4 cells incubated with mouse monoclonal IgG1 antibody in order to induce in vitro the production of isotype-specific IgG1-BF, c) T2D4 cells induced in vivo by passage as ascites in nude mice and incubated in serum-free medium, and d) in vivo alloantigen-activated T cells (ATC) incubated in serum-free medium. IgG-BF were affinity purified on Sepharose beads coated with rabbit or mouse IgG and identified by their biologic activities, i.e., inhibition of in vitro secondary IgG antibody production to SRBC and inhibition of rosette formation between Fc gamma receptor-positive spleen cells and rabbit IgG-sensitized erythrocytes. IgG-BF produced by either of these cell sources was found to be heterogeneous in both size and charge. In each case, IgG-BF activities were recovered in three fractions of apparent Mr-74,000 to 78,000, 35,000 to 40,000, and 19,000 to 23,000-and in four fractions of pI-4.7 (or 5.3, depending on experimental conditions), 6.5, 7.7, and 8.4. Moreover, IgG-BF translated in vitro from T2D4 poly A RNA by using rabbit reticulocyte lysate exhibited the same heterogeneity. Thus, IgG-BF contain different proteins exerting similar biologic activities.

Receptors for immunoglobulin isotypes (FcR) on murine T cells. II. Multiple FcR induction on hybridoma T cell clones.

In the preceding report (Eur. J. Immunol. 1985. 15: 662), we described a variety of receptors for the Fc portion of the different isotypes of mouse immunoglobulins (FcR), that were found to be expressed on hybridoma T cell clones. In the present work, we wondered whether the expression of these T cell FcR would be regulated by environmental influences such as the presence of corresponding ligands. We found that exposing the cells to the bulk of serum immunoglobulins in vivo, or to purified monoclonal immunoglobulins in vitro both resulted in FcR induction. The expression of all constitutive receptors, i.e. Fcgamma 1/2bR, Fcgamma3R, FcalphaR and FcepsilonR, could be increased upon incubation with IgG1, IgG2b, IgG3, IgA and IgE, respectively. After induction, the specificity of FcR was not modified. Two FcR were detectable only upon induction. These were Fcgamma2a/2b/1R, induced by IgG2a and FcmuR, induced by IgM. Interestingly, FcR detectable after induction only were short-lived at the membrane. Ten to 15 h after induction they were not detected any more, whereas the expression of constitutive FcR remained elevated for at least 24 h following induction. Therefore, depending on the concentration of immunoglobulins in the environment, and depending on whether they are short lived or long lived, FcR can modulate their expression on the membrane of T cells. Such a versatility might be an efficient means to contribute to isotypic regulation through the release of regulatory immunoglobulin-binding factors. factors.