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.

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.

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.

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.

Public H-2 specificities are target determinants for alloreactive cytotoxic T lymphocytes.

The specificity of the cross-killing exerted by cytotoxic T lymphocytes (CTL's) generated against H-2 region products was investigated in a 51Cr release assay on a panel of target cells from a number of different H-2 haplotypes. Their pattern of reaction shows that: (1) The target cells, expressing public specificities (H-2.28, H-2.1, H-2.3 or H-2.8) which should, theoretically, be recognized by the CTL's were killed, while those expressing no public specificities, recognized according to the H-2 chart by the CTL's were not killed. (2) The CTL's generated against the H-2.28 specificity expressed on the D region products cross react with target cells expressing this specificity in their K region products and vice versa. The same phenomenon was observed with the H-2.1 specificity. These results provide evidence that public specificities are targets for CTL's. Antiserum reacting against the public specificity recognized by the CTL's was found to block the cross-killing, however, to the same extent as antisera directed against any specificity (private or public) expressed on the same molecule as the target determinant. Finally both the inhibition studies using anti-H-2 antisera and the direct cytotoxic assays showed that the public specificities of the H-2.28 family carried by the H-2.D and H-2.L molecules were recognized by different subpopulations of CTL's.

Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells.

The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.

Characterization of suppressive immunoglobulin-binding factor (IBF). III. Biochemical and immunochemical characteristics of IBF produced by activated T cells.

The biochemical characteristics of immunoglobulin binding factor produced by activated cells (ATC) were investigated. For this purpose, supernatants of ATC were purified by affinity chromatography on insolubilized IgG and the eluted material was iodinated (125I), treated with mercaptoethanol; and run on SDS polyacrylamide gels. The radioactivity was found in two peaks corresponding to m.w. of 38,000 d and 18,000 d. This result extends and confirms our previous findings that IBF produced by ATC is identical to IBF produced by L-5178-Y internally labeled thymoma cells. The effect of various pH, temperatures, and proteolytic and glycolytic enzymes on the binding properties of 3H-leucine-or H-fucose-labeled IBF to IgG and on the polyacrylamide gel profiles was also studied. By all these criteria, IBF appeared to be a glycoprotein in which the presence of the 38,000 to 40,000 d chain is necessary for the binding to IgG. In the attempt to study the relationships between IBF and I-region products, purified IBF produced by ATC was incubated with anti-Ia immunoadsorbent, and the eluted material was iodinated and run on gels. The 38,000 d and 18,000 d chains characteristic of IBF were found to be specifically retained on the relevant immunoadsorbent. These data favor the hypothesis that IBF bears or is associated with Ia determinants.

Characterization of suppressive immunoglobulin-binding factor (IBF). II. Purification and molecular weight determination of IBF produced by L-5178-Y theta-positive lymphoma.

L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes. IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d. These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,00 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.

The private specificity H-2.4 and the public specificity H-2.28 of the D region are expressed on two independent polypeptide chains.

The antigenic specificities H-2.4 (private) and H-2.28 (public) in the H-2a haplotype are controlled by the D region of H-2 as defined by the available recombinants. In previous studies we have demonstrated by the antibody-induced redistribution method that the antisera against these specificities contain antibodies against at least two different polypeptide chains. We here report the results of the indirect immunoprecipitation of radiolabeled antigens after solubilization with Nonidet-P40. The antisera against the two specificities precipitated from such extracts two different and independent polypeptide chains, indicating that the products of the D region, as presently defined, comprise at least two different molecules. The molecular weight of both chains is approximately 45 000, which is similar to other molecules bearing private H-2 antigenic specificities. Consequently, the chromosomal segment presently defined by recombination studies as the D region, must contain another locus, controlling the second polypeptide chain which is detectable by anti-H-2.28 antisera, besides the H-2D locus which controls the polypeptide chain bearing the private specificity H-2.4 as well as most of the public specificities.

Modifications of the thymocyte membrane during redistribution of concanavalin A receptors.

The modifications of the thymocyte membrane which are induced by concanavalin A (Con A) were studied by means of the variation of electrophoretic mobility which the lectin induces on the cell. The electrophoretic mobility of thymocytes is increased when Con A is used under conditions expected to induce a cap formation and the subsequent endocytosis. This increase persists as long as the lectin is present in the medium and disappears as soon as the lectin is eliminated. The redistribution of the Con A receptors into spots and caps may partially explain this electrical modification. The ionized groups of the thymocyte membrane are drastically modified during these phenomena with: 1) a diminution by 61% in the density of the sialic carboxyl groups, 2) a decrease of 40% in the density of phosphate groups and of 60% in the density of amino groups, 3) a 20 times higher density of unidentified negatively charged groups. The electrophoretic mobility of normal human blood lymphocytes is similarly increased by Con A. A marginal difference exists in the shape of the dose response curves obtained when normal and cancerous thymocytes react with increasing doses of Con A. No measurable electrical modification was observed during redistribution of H-2 and Thy-1.2 antigens on EL4 cells. Some experiments suggest a possible correlation between the mitogenicity of the lectin and a measurable modification of the cell electrophoretic mobility.