A transforming growth factor beta-like immunosuppressive factor in immunoglobulin G-binding factor.

Immunoglobulin G-binding factors (IgG-BF), which are produced by cells of the immune system, inhibit antibody production. In this paper, we show that transforming growth factor-beta (TGF-beta) suppresses secondary in vitro anti-sheep red blood cell responses of mouse splenocytes and lipopolysaccharide- or anti-IgM-stimulated mouse B cell responses in a way similar to, and with the same kinetics as, rodent IgG-BF. Moreover, the immunosuppressive activity of IgG-BF was totally neutralized by polyclonal and monoclonal anti-TGF-beta antibodies and it eluted with TGF-beta by gel exclusion chromatography, suggesting that a TGF-beta-like immunosuppressive factor is present in IgG-BF. We also show that TGF-beta behaves as an IgG-BF since it binds to insolubilized IgG, but not to insolubilized F(ab')2 or bovine serum albumin. Altogether, the data support the concept of a biological role for TGF-beta in the IgG-mediated negative feedback of antibody responses.

Separate cytotoxic T lymphocyte subsets recognize the different H-2 specificities.

Using a monolayer adsorption technique, the fine specificity of cytotoxic effector T lymphocytes (CTL) generated against allogeneic or semi-allogeneic H-2 haplotypes was investigated. The results show that: (a) CTL reacting with the private specificity expressed on an H-2.K molecule can be separated from those reacting with the public specificities expressed on the same molecule and (b) the CTL that recognize cross-reacting H-2 determinants (public specificities) can also be separated into several subpopulations. These data support the hypothesis that an allogeneic stimulation induces a large number of independent T cell clones that react with H-2 determinants.

Relationship between HL-A antigens and beta-2-microglobulin as studied by immunofluorescence on the lymphocyte membrane.

The antibody-induced redistribution of beta2-microglobulin (beta2-micro) and HL-A antigens on the surface of living lymphocytes was studied by immunofluorescence. When all beta2-micro was redistributed on the lymphocyte membrane by specific rabbit antibodies and goat antirabbit Ig conjugates, the HL-A antigens were no more detectable with anti-HL-A conjugates outside the beta2-micro caps already formed. However, the redistribution of HL-A antigens fails to provoke the redistribution of all detectable beta2-micro molecules. These results suggest that HL-A antigens may be associated with beta2-micro at the cell surface, but that all beta2-micro molecules are not bound to HL-A antigens.

Independence of H-2K and H-2D antigenic determinants on the surface of mouse lymphocytes.

At 37 degrees C, fluorescein-conjugated anti-H-2 alloantibodies specifically induce, at the surface of living mouse lymphocytes, the redistribution of the corresponding H-2 antigens, which cluster as patches and sometimes single caps at one pole of the cell. This aggregation is inhibited at 0 degrees C and the H-2 antigens, stained by fluorescent antibodies in the cold, appear evenly spread over the cell surface. This phenomenon was used to define the relationships between the membrane structures bearing the antigens coded by the H-2K and the H-2D genes of the H-2 region. Monospecific anti-H-2 antibodies coupled to either tetramethyl rhodamine isothiocyanate or fluorescein isothiocyanate were used to induce the redistribution of H-2D and H-2K antigens of the H-2(b) and H-2(k) haplotype at the surface of lymph node cells from homozygous and F(1) hybrid mice. It was observed that the diffuse distribution of H-2K antigens labeled at 0 degrees C was not affected by the prior antibody-induced aggregation of H-2D antigens and vice versa. The results were the same for H-2 antigens governed by genes located either in cis or in trans position. These data indicate that the H-2K and H-2D antigens migrate independently at the cell surface, and suggest that the gene products from the D and the K end of the H-2 region are expressed on independent molecules or structures at the cell membrane.

Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.

B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.

Characterization of soluble gp130 released by melanoma cell lines: A polyvalent antagonist of cytokines from the interleukin 6 family.

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.

Soluble Fc gamma receptors.

Soluble Fc gamma receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (Fc gamma RII) or by proteolytic cleavage at the cell membrane (Fc gamma RII and Fc gamma RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations of soluble Fc gamma RIII are correlated with the stage of the disease.

The carbohydrate moieties of suppressor IgG-binding factor released by murine T cells.

The carbohydrate moieties of murine IgG-binding factor (IgG-BF) were studied using lectins binding N-glycosylated sequences such as Concanavalin A (Con A), Lens culinaris agglutinin (LcA), and wheat germ agglutinin (WGA), and lectins binding O-glycosylated sequences such as peanut agglutinin (PNA) and Helix pomatia Agglutinin (HpA). Sources of IgG-BF were: (1) supernatants from T2D4, a T cell hybridoma constitutively producing IgG-BF, and (2) factor purified by affinity chromatography on rabbit IgG-Sepharose, using T2D4 supernatants or supernatants of alloantigen-activated T cells (ATC) as starting material. The presence of IgG-BF was assessed by its ability to inhibit secondary anti-sheep red blood cell (SRBC) IgG antibody responses in vitro and to inhibit rosette formation between Fc gamma receptor (Fc gamma R)-positive spleen cells and erythrocytes sensitized with rabbit anti-Forssman IgG antibodies. Fractionation on immobilized lectins showed that IgG-BF: (1) is completely adsorbed by WGA and PNA and partially by Con A, LcA and HpA, and (2) can be eluted from the five different lectins using the competitor sugars. When produced in the presence of tunicamycin, an inhibitor of N-glycosylation, IgG-BF still binds to HpA which has affinity for O-glycosylated carbohydrate chains. These results indicate that IgG-BF is a glycoprotein with N- and O-glycosylated carbohydrate moieties.

Identification of the Fc gamma RII-related component of murine IgG-BF.

Suppressor murine IgG-BF produced by the T cell hybrid (T2D4) expressing low affinity Fc gamma R (Fc gamma RII) contain four biologically active polypeptides of pI 5.2, 6.3, 7.7 and 8.3, respectively. They were fractionated by affinity chromatography on immunoadsorbents coupled with F(ab')2 fragments of the monoclonal anti-Fc gamma RII antibody 2.4G2 and by hydrophobic interaction chromatography. Both methods led to the identification of biologically active IgG-BF which react with 2.4G2 and of IgG-BF which do not react with 2.4G2. Molecules bearing the epitope recognized by 2.4G2 had an apparent pI of 5.3 while the pI of those which did not express this epitope were 6.3, 7.8 and 8.5, respectively. Therefore, one IgG-BF polypeptide of pI 5.3 is probably related to Fc gamma RII.

21.1.1, a novel activation marker of T and B cells.

A new rat mAb designated mAb 21.1.1 was raised against a T cell hybridoma of mouse origin, T2D4. This antibody, an IgG2b, immunoprecipitates from the membrane extracts of iodinated T2D4 cells a 56-kDa glycoprotein of apparent pI 4.6 which gives a 34-kDa polypeptide after treatment with endoglycosidase F. MAb 21.1.1 reacts with an antigen expressed on murine mitogen-activated thymocytes and T cells, and on B cells stimulated by anti-IgM antibodies. Cells isolated from the spleen, lymph nodes and bone marrow are negative, as are purified resting B cells or T cells. This antigen is strongly expressed on most day-16 fetal thymocytes whereas adult thymocytes are almost negative. mAb 21.1.1 may be useful for the study of activation and differentiation of T and B cells.

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 Mr 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 Mr 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 Mr of 40000.

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.

Murine soluble Fc gamma receptors/IgG-binding factors (IgG-BF): analysis of the relation to Fc gamma RII and production of milligram quantities of biologically active recombinant IgG-BF.

The production of soluble forms of low-affinity Fc gamma R by cells expressing recombinant or natural membrane Fc gamma RII, and the structural relationships between these soluble receptors and membrane Fc gamma RII are described. We show that 37-40 kD soluble Fc gamma RII, corresponding to the two N-terminal domains of Fc gamma RII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane Fc gamma RII. Moreover, we describe methods to produce and purify to homogeneity large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated Fc gamma RII cDNA. These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography. By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts. Like natural IgG-BF, the 95-99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells.

Mechanism of inhibition of lipopolysaccharide-stimulated mouse B-cell responses by transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF beta 1) is a pleiotropic cytokine which inhibits growth of many cell types and positively or negatively regulates the production of Ig isotypes. By using mouse resting B cells stimulated by lipopolysaccharide (LPS), we investigated whether the effect of TGF beta 1 on Ig production is related to its effect on cell growth. We show that low doses of TGF beta 1 stimulate IgG3 and IgG2b production whereas higher doses inhibit IgM, IgG3, IgG1 and IgG2b secretion and cell proliferation. TGF beta 1 titration curves and kinetics experiments suggested that the inhibitory effect on Ig secretion and B-cell growth are closely related. We defined the phase at which TGF beta 1 exerts its anti-proliferative effect on mouse B cells. TGF beta 1 does not modify the increase in expression of class II antigens which occurs before transition from G0 to G1. However, it partially inhibits the induction of expression of low-affinity Fc gamma RII and cell enlargement which both begin during the early G1 phase, and it totally blocks induction of the expression of transferrin receptors, a marker of the late G1 phase. Thus, TGF beta 1 blocks LPS-stimulated mouse B cells in the early G1 phase, and this results in inhibition of Ig production.

Ligands and biological activities of soluble Fc gamma receptors.

Soluble forms of low-affinity Fc gamma receptors (sFc gamma R) circulate in biologic fluids. Their plasmatic levels vary in immunological disorders or related diseases. They are produced by enzymatic cleavage of the membrane receptors or by alternative splicing. They bind IgG with the same isotype specificity as their cell surface counterparts and thus modulate Fc-dependent immune functions. Recent data suggest that they also bind non-Ig ligands present on leukocytes. Functional implications of these findings are discussed.

Prognostic value of cytokine and soluble Fc gamma receptor assays in oncology.

The clinical course of a cancer is influenced by the interaction of tumour cells with the patient's immune system. It is thus conceivable that immunological parameters may be used as markers of prognostic or predictive value. We report here that increased serum levels of IL-6 is a signal of poor prognosis and predicts unresponsiveness to immunotherapy in patients with metastatic melanoma. In cervical cancer, IL-6 produced by infiltrating macrophages is a marker of invasive cancer. In patients with multiple myeloma, the plasmatic levels of soluble Fc gamma receptors are markers of the disease, sCD16 being drastically decreased and sCD32 being slightly increased.

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.

Soluble Fc gamma receptors: interaction with ligands and biological consequences.

Soluble Fc gamma receptors are produced by cleavage of the membrane receptors or by alternative splicing. They are found in biologic fluids. After a brief description of the structure and mode of production of soluble Fc gamma R, we address the question of ligands and function of the soluble Fc gamma R by using recombinant molecules and transgenic animals. We show that soluble Fc gamma R are not only IgG-binding factors which interfere with, and block, Fc-dependent immune reactions but also molecules that interact, in vitro, with non-Ig-ligands such as CR3 and CR4 and are trigger or regulate immune functions via these receptors.

Functions of Fc receptors on human dendritic Langerhans cells.

Immature dendritic cells are antigen presenting cells highly specialized for capturing and processing foreign protein antigens. These cells express Fc gamma RII and Fc epsilon RI which, by their ability to internalize and use the endocytic pathway, increase their capacity to process antigens. Immature dendritic cells, such as epidermal Langerhans cells, also release soluble forms of Fc gamma RII. These latter molecules are likely to compete with the membrane-associated Fc gamma R to diminish or abrogate the capacity of dendritic cells to present immune complexes, as suggested by our in vitro experiments using both human and mouse epidermal Langerhans cells. However, when dendritic cells mature in vitro and become efficient stimulators of resting T cells, they rapidly down-regulate and sometimes completely abolish the expression of their membrane-associated Fc gamma R and Fc epsilon RI. Consequently, they lose or at least strongly diminish their capacity to capture immune complexes. At this stage, the release of soluble Fc gamma R by dendritic cells is also markedly diminished. One can hypothesize that the membrane-associated Fc gamma RII and the soluble Fc gamma RII are molecules expressed when dendritic cells are potent capturing and processing cells, the soluble Fc gamma RII molecule acting by competition as a negative regulatory element on the Fc gamma RII-mediated internalization of IgG-containing immune complexes. Thus, the expression of membrane-associated Fc gamma R and Fc epsilon RI, as well as the release of soluble Fc gamma R, would seem to characterize the immature stage of dendritic cells.

Fc gamma-receptor III (CD 16) is involved in NK-B cell interaction.

CD16, the low affinity receptor for monomeric IgG (Fc gamma RIIIA), is a well characterized activation molecule on NK cells. In this study we investigated the role of CD16 in NK cell-mediated regulation of immunoglobulin production. Cocultures of the CD16+ human NK clone CNK6 and highly purified SAC/IL-2-activated B lymphocytes with various CD16 antibodies showed significantly diminished NK-enhanced immunoglobulin production in a dose-dependent manner, indicating that CD16 is relevant in NK-B cell interaction. Similarly, recombinant soluble CD16 incubated with B cells before cultures, suppressed the NK cell-stimulated B cell antibody response. Enhanced immunoglobulin production was also inhibited by Fc-specific F(ab')2 anti-body fragments. Coculture of NK cells with B lymphocytes resulted in induction of mRNA for IFN-gamma and TNF-alpha. The accumulation of mRNA for these cytokines was prevented by addition of CD16 and Fc-specific antibodies. It is proposed that interaction of CD16 on NK cells with B cell bound immunoglobulin leads to induction of cytokines in NK cells which stimulate immunoglobulin production by B cells.