2.4G2, a monoclonal antibody to macrophage Fc gamma receptors, reacts with murine T cell Fc gamma receptors and IgG-binding factors.

In order to determine whether T cell Fc gamma receptors (Fc gamma R) and IgG-binding factors (IgG-BF) are structurally related, we searched for common antigens on these molecules. We found that the anti-macrophage Fc gamma 1/gamma 2bR monoclonal antibody 2.4G2 binds to similar determinant(s) on the macrophage-like J774 cells and on the Fc gamma R+ hybridoma T cells T2D4. On the T cell membrane, these determinants are associated with Fc gamma 1/gamma 2bR. They are absent on Fc gamma R- hybridoma T cells or the FcR- BW5147 thymoma cells. 2.4G2-reactive molecules were also detected in soluble material either extracted or released in the supernatant of Fc gamma R+ hybridoma T cells. 2.4G2-reactive molecules released in the supernatant of T2D4 cells could be absorbed on Sepharose beads coupled to rabbit IgG and they were recovered by acid elution. Conversely, Sepharose beads coupled to 2.4G2 retained molecules which had affinity for rabbit IgG, which suppressed an in vitro antibody response and which had the same molecular weight as IgG-BF. These results indicate that T cell Fc gamma R and IgG-BF share common epitopes and that 2.4G2 can serve as an anti-IgG-BF antibody.

The role of carbohydrates in the IgG binding and suppressive activities of shed human Fc receptors.

IgG Fc receptor (Fc gamma R) shed from human peripheral mononuclear cells and purified by IgG affinity chromatography bound to ConA, Pisum sativum, Ulex europeus and peanut lectins. The soluble Fc gamma R was not absorbed by wheat germ, soyabean, lentil, Sophora japonica or Helix pomotia lectins. Out of 14 different sugars, none interfered with the interaction between IgG Fc and membrane Fc gamma R. These results show that shed Fc gamma R had glucosyl, mannosyl, fucosyl and galactosyl groups, but that these components were not part of the IgG-binding site(s). The shed FcR had IgG-binding factor (BF) activity; it suppressed secondary in vitro antibody production. In accordance with the binding studies, IgG-BF activity could be adsorbed by ConA and PNA, but not by lentil lectins. The presence of 10 mM galactose or alpha-methylmannoside had no influence on biological activity, suggesting that, although present on human shed Fc gamma R, these sugars do not play a major role in its suppressive activity.

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.

Receptors for immunoglobulin isotypes (FcR) on murine T cells. I. Multiple FcR expression on T lymphocytes and hybridoma T cell clones.

T cell receptors for the Fc portion of the various isotypes of mouse immunoglobulins (FcR) were examined by rosette formation, using as indicator cells erythrocytes coated with monoclonal antibodies of all known isotypes of serum immunoglobulins. Three populations of mouse T cells were studied: normal thymocytes, activated T cells (ATC), generated by educating thymocytes in lethally irradiated allogeneic hosts, and hybridoma T cells, derived from somatic hybridization of ATC with the FcR-negative thymoma BW.5147. We found that many different FcR could be distinguished by their specificity for a single isotype or for a combination of several isotypes; ATC and hybridoma T cells expressed several such receptors that, at least in cloned cells, could be demonstrated to be borne by individual cells; hybridoma T cells of independent origin bore indistinguishable receptors whereas ATC expressed markedly different FcR and upon overnight incubation at 37 degrees C, immunoglobulins were found to bind onto the cell surface, even though no corresponding constitutive FcR was detected. The same was observed with hybridoma T cells and with thymocytes. It follows that a single T cell can express several FcR. Altogether, these FcR are capable of binding all known isotypes of serum immunoglobulins. They differ from one T cell to another.

Isolation and partial characterization of messenger RNA, from murine T cell hybrids, coding for suppressive immunoglobulin G-binding factor.

Poly A RNA has been isolated from a murine T cell hybridoma ( T2D4 ) that spontaneously secretes suppressive immunoglobulin G-binding factor ( IgGBF ). Translation products, obtained from a rabbit reticulocyte lysate translation system and after injection into Xenopus laevis oocytes, contain material with the biologic activity, the affinity, and the m.w. of murine IgGBF ; it suppresses secondary in vitro IgG antibody production in a dose-dependent fashion. The suppressive factor binds to IgG but not to IgM immunoadsorbents and, after mild NaDodSO4 treatment, dissociates in NaDodSO4 polyacrylamide gels into two peaks at 78 and 40 kD. Translation products from two non- IgGBF -secreting cell lines (BW-5147, a T lymphoma line, and A9, a fibroblast cell line) fail to exert any suppressive activity. On sucrose gradients, the RNA responsible for the biologic activity was found in one major peak located at 11S. IgGBF synthesized in a cellfree translation system by using poly A RNA and sucrose gradient fractions was also characterized by immunoprecipitation with Fc fragments of [35S]methionine-labeled proteins. On NaDodSO4 polyacrylamide gels, it migrates in one peak located at 37 kD. We conclude that IgGBF is coded for by 11S poly A RNA and that no post-translational modifications (other than proteolytic cleavage) are necessary to obtain a biologically active factor with Ig-binding properties.

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.

Control of isotype expression by helper T-cell clones and suppressor cells.

We report here experiments demonstrating the profound influence of T lymphocytes on isotype expression by B lymphocytes. It was shown that in a secondary in vitro response, T helper cells from primed spleen predominantly induced an IgG1 plaque-forming cell (PFC) response, while T helper cells from primed lymph node induced IgG1, IgG2a and IgG2b PFC responses. Under the same experimental conditions, T helper cell clones were able to induce IgG1, IgG2a and IgG2b responses; therefore, T helper cells are not involved in controlling preferential isotype expression. Stimulated spleen cells were shown to contain T suppressor cells which were able to limit the expression of IgG2a and IgG2b responses. Under certain circumstances, IgG1-specific suppressor cells were also demonstrated. A T-cell hybridoma, T2D4, spontaneously produced the IgG-binding factor, thereby suppressing the expression of the three subclasses studied. Exposure of these cells to IgG1 myeloma protein led to selective enhancement for the production of molecules binding to IgG1, and suppression of the IgG1 PFC response. Similar observations were made with IgG2a and IgG2b myeloma proteins, and the specificity of these isotype suppressive factors was demonstrated. The general significance of these observations is discussed.

Isotype regulation of antibody production: T-cell hybrids can be selectively induced to produce IgG1 and IgG2 subclass-specific suppressive immunoglobulin-binding factors.

T2D4, a T-cell hybrid, spontaneously secretes suppressive immunoglobulin factor(s); when incubated with purified monoclonal mouse immunoglobulins, this hybrid produces high levels of immunoglobulin-binding factors specific for the subclass of the inducing immunoglobulin. Thus, we were able to induce the production of IgG1- or IgG2-specific inhibitory factors by the same T2D4 T-cell hybrid. These subclass-specific suppressive factors bind selectively to the IgG1 or IgG2 subclasses and inhibit specifically the secretion of antibodies of the corresponding subclass. Our results favor a model of negative regulation of isotype expression in which a given isotype triggers suppressor mechanism(s) specifically inhibiting its production.

Multiple CTL subsets generated by H-2.L locus products stimulation: evidence for an antigenic determinant private to H-2.Ld.

Analysis of the fine specificity of CTL subpopulations raised by an H-2.L locus products stimulation (H-2dm2 anti-H-2d) was performed by absorption experiments by using monolayers of macrophages of H-2m, H-2q, H-2b, and H-2k haplotypes. The results show the existence of four CTL subsets. The pattern of reactivity of three of them could be correlated with that of antibodies present in H-2dm2 anti-H-2d antisera (anti-H-2.64, anti-H-2.65, and anti-H-2.Kk). The fourth CTL subset reacted with a specificity unique to H-2.Ld molecules (a private specificity?), absent on cells from H-2m, H-2q, H-2b, and H-2k haplotypes, and undescribed as yet by serologic methods. These data support the hypothesis that the H-2.L locus products are comparable in their antigenic properties to those of the H-2.K and H-2.D loci.

Interferon enhances the expression of Fc gamma receptors.

Murine T2D4 cells derived from a T cell hybrid line were incubated with partially purified or electrophoretically pure mouse interferon and tested for the expression of Fc gamma R as assessed by a) counting the number of cells forming rosettes with IgG-sensitized sheep erythrocytes, and b) incubating the cells with heat-aggregated rabbit IgG and then determining either the number of cells stained with fluorescein conjugated goat anti-rabbit IgG or the extent of labeling by using radioactive iodinated staphylococcus protein A. Although interferon induced a rapid increase in Fc gamma R expression on the Fc gamma R-positive T2D4 cells, it did not induce either Fc gamma R on the Fc gamma R negative BW5147 cells or Fc gamma R on either cell line. Human leukocyte interferon enhanced the expression of Fc gamma R on human Burkitt cells (Daudi) but did not affect the expression of Fc gamma R on mouse cells. We suggest that interferon may influence several effector functions of the immune system by modulating Fc receptor expression.