Fc receptor heterogeneity: immunofluorescent studies of B, T, and "third population" lymphocytes in human blood with rabbit IgG b4/anti-b4 complexes.

IgG Fc receptors on human peripheral blood lymphocytes (PBL) were characterized by immunofluorescence studies with defined rabbit IgG b4 allotype/anti-allotype complexes. Three discrete types of Fc receptor-bearing cells, totaling approximately 33% of PBL, were identified. Fc receptors of the three types differed in their sensitivity to trypsin and in either absolute or localized density (topography) as determined by variable requirements for anti-IgC cross-linking in order to visualize bound complexes microscopically. The question of additional heterogeneity related to differences in individual Fc receptor affinity for complexed IgG was not approached in this study. Ten to 15% of PBL had pronase-sensitive, trypsin-resistant Fc receptors readily detected by direct immunofluorescence by using large fluorescein-conjugated complexes prepared near equivalence. Double label and lymphocyte fractionation experiments established this population to be largely distinct from suface IgM+ B cells and T cells, and identical to EA Ripley rosette-forming cells. Approximately 50% of surface IgM+ B cells and approximately 10% of T cells had lower density Fc receptors identified by indirect immunofluorescence with small complexes prepared in antigen excess or by cross-linking fluorescein-conjugated complexes with anti-rabbit IgG anti-serum. An additional approximately 15% peripheral T and B cells had very low density Fc receptors detectable by complexing the IgG on the cell surface by sequential incubations of cells with b4 IgG and anti-b4. Fc receptors on B and T cells were sensitive to both pronase and trypsin digestion. The heterogeneity of IgG Fc receptors on different lymphocyte subpopulations as defined by these these experiments may be of relevance for further analysis of normal and abnormal immune function.     

Antigen-antibody complexes bound to B-lymphocyte Fc gamma receptors regulate B-lymphocyte differentiation

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.     

Suppression of adult B cell differentiation in pokeweed mitogen-stimulated cultures by Fc(IgG) receptor-negative T cells from cord blood.

Unfractionated T lymphocytes from cord blood suppressed adult B cell differentiation into immunoglobulin-producing cells in pokeweed mitogen-stimulated co-culture system. Cord blood T cells were fractionated into T cells bearing Fc receptors for IgG (Tgamma cells) and T cells lacking Fc receptors for IgG(Tnon-gamma cells) by rosette formation with ox erythrocytes coated by the IgG fraction of rabbit antisera followed by Ficoll-Hypaque gradient sedimentation. T gamma cells from cord blood, even though isolated after the interaction with immune complexes, showed no suppressor activity on adult B cell differentiation, whereas Tnon-gamma cells exerted strong suppression to a similar extent to that by unfractionated cord T cells. The suppressor activity on B cell differentiation by Tnon-gamma cell as well as by unfractioned T cells from cord blood was completely abrogated by irradiation with 2000 rads. These results indicated that, contrary to suppressor function found in adult T cells, the suppressor activity in cord T cells might be exerted by a T cell subset lacking Fc receptors for IgG(Tnon-gamma cells).     

Influence of immunoglobulin isotypes and lymphoid cell phenotype on the transfer of immune complexes to follicular dendritic cells

Follicular dendritic cells (FDC) are located only inside lymph follicles and are characterized mainly by their capacity to retain high amounts of immune complexes by their Fc or C3b receptors. In this work, we examine the influence of immunoglobulin isotypes and the subset of lymphoid cells (B or T) upon the transfer of immune complexes from lymphocytes to FDC. FDC isolated from mice lymph nodes by enzymatic digestion are able to fix, through Fc receptors, gold-labeled immune complexes presented by lymphoid cells. As demonstrated by electron microscopy, this transfer requires the establishment of close contacts between both cell types. Using different cell selection techniques we show that B lymphoid cells take up immune complexes more efficiently than do T lymphoid cells and transfer a larger number of them to FDC. This transfer mechanism is dependent on the immunoglobulin isotype: immune complexes constituted of IgG2a, IgG2b, and IgG1 isotypes are better transferred to FDC than those constituted of IgG3 and IgM.     

Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin.

Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.     

IL-4 (B cell stimulatory factor 1) overcomes Fc gamma receptor-mediated inhibition of mouse B lymphocyte proliferation without affecting inhibition of c-myc mRNA induction

Mouse B cells are stimulated to proliferate by Fab'2 fragments of rabbit anti-mouse Ig antibodies. Proliferation is inhibited, however, in the presence of IgG anti-mouse Ig. We have previously shown that this inhibition is mediated by binding of the IgG anti-Ig to receptors for Fc gamma R on B cells. This report describes conditions under which IgG anti-mu or anti-delta will induce proliferation despite Fc gamma R engagement. Culture supernatants of Con A-stimulated, Il-4-secreting Th cell lines, but not of Il-2-secreting Th cell lines, will co-stimulate with IgG anti-Ig to induce small B cells to incorporate [3H]TdR. This co-mitogenic activity is inhibitable by anti-IL-4 antibodies and can also be induced by Il-4 affinity purified from the T cell supernatants or by supernatants containing rIl-4. B cells precultured with Il-4 for 18 h, while still expressing normal levels of Fc gamma R, also proliferate to IgG anti-Ig. We have previously shown that Fc gamma R-mIg cross-linking will inhibit mIg-dependent increases in c-myc mRNA levels. We investigated whether Il-4 allows B cells to respond to IgG anti-Ig by elevating c-myc. The data show that Il-4 has little effect on c-myc mRNA levels in either IgG or Fab'2 anti-Ig-containing cultures.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Fc receptors on monocytes cause OKT3-treated lymphocytes to internalize T3 and to secrete IL-2

Monocytes cause OKT3-treated T cells to secrete IL-2 and to lose cell surface T3. We have studied the fate of the "lost" T3. Immunofluorescence microscopy on permeabilized cells showed that monocytes induce T cells to internalize T3. Furthermore, experiments with radioiodinated T cells showed that the internalized T3 was not degraded and exhibited an unaltered polypeptide composition for at least 16 hr. The role of Fc receptors in inducing internalization was also investigated. Fc receptors were depleted from monocytes by allowing the phagocytes to spread on immune complexes. Such depleted monocytes exhibit a fourfold reduction in their ability to promote both internalization of T3 and production of IL-2. A comparable reduction is seen if F(ab')2 fragments of OKT3 were employed in place of intact IgG. Furthermore, monocyte Fc receptors that have been blocked by heat-aggregated human IgG also show much reduced capability for induction of OKT3-mediated T-cell proliferation. We therefore conclude that Fc receptors bind to the Fc domain of OKT3 and thereby cause OKT3-treated T cells to internalize T3 and become activated.     

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.     

Fc receptors on cultured myeloma and hybridoma cells

The specificity of the Fc gamma receptors on the X63.Ag8.653 nonproducing myeloma cell line has been examined for binding to IgG1-, IgG2a-, and IgG2b-containing antigen-antibody complexes. Complexes containing each of these subclasses bind, and the binding of each is inhibited by the others. Trypsin treatment did not inhibit the binding of any of these subclasses. Furthermore, the monoclonal anti-Fc receptor antibody 2.4G2 inhibits the binding of all three subclasses. These results, together with those of other investigators, suggest that there is a single FcR for IgG1, IgG2a, and IgG2b on mouse B cells which differs in its specificity from the macrophage Fc gamma R. This is confirmed by the fact that a mutant IgG2b myeloma protein which binds to the macrophage Fc gamma 1/gamma 2b receptor does not bind to the Fc gamma R on X63.Ag8.653.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

Studies were carried out to determine whether the mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity (ADCC) to herpes simplex virus (HSV)-infected target cells has surface Fc receptors which participate in the reaction. The F (ab')2 fragment of human IgG antibody was inactive both in ADCC and in complement-mediated cytolysis, but retained the capacity to neutralize infectious virus, to agglutinate erythrocytes coated with viral antigens, and to bind to the surface of virus-infected cells. Treatment of sensitized virus-infected target cells with staphylococcus protein A, which has affinity for the Fc epitope of IgG, strongly reduced their susceptibility to lysis by ADCC in a dose-dependent relationship. These findings indicate that the Fc portion of IgG antibody to the virus is necessary for cytotoxicity. Treatment of blood mononuclear cells with either heat-aggregated gamma-globulin or HSV immune complexes inhibited effector cell activity. The presence of "third party" cellular immune complexes also strongly inhibited ADCC. Adsorption of mononuclear cells to plastic surfaces coated with soluble third party immune complexes resulted in a significant reduction in effector cell activity. These findings demonstrate that the ADCC effector cell possesses surface Fc receptors which are utilized in the ADCC reaction. The presence of Fc receptors on the surface of the effector cell indicates that it is a K cell rather than a null cell.     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.     

Receptor-mediated binding of C1q on pulmonary endothelial cells

Normal undamaged pulmonary endothelial cells appear to be immunologically privileged in that they do not express receptors for the Fc portion of IgG nor for C3b. However, these receptors become unmasked on endothelial cells injured by viral infection or exposure to white cell lysates. We now present evidence to indicate that C1q binds to specific receptors on the surface of normal healthy endothelial cells. The binding is dose-dependent, reversible and saturable. Furthermore our data show that binding of C1q to endothelial cells is via the collagenous portion of the molecule not via the globular head regions. Thus binding of C1q to endothelium would have the effect of exposing Fc receptors that could then bind to IgG of circulating immune complexes. That Fc receptors are in fact exposed is shown by rosette formation with antibody sensitized erythrocytes. With 2C1r-2C1s-associated C1q, no binding occurred using C1 fixation and transfer assays. Our results indicate that C1q binding to endothelium provides a means for localizing immune complexes on pulmonary vessels and may be important in the initiation and progression of the inflammatory response.     

Characterization of murine lymphocyte IgE receptors by flow microfluorometry

A flow microfluorometric technique has been developed to analyze IgE receptors on splenic and mesenteric lymph node mononuclear cells from BALB/c mice. Our data show that 1) the binding of DIBADL cross-linked IgE dimers to IgE receptors is specific in that it is inhibited by monomeric rat and mouse IgE but not by mouse or rabbit IgG or by the monoclonal anti-Fc gamma R antibody 2.4G2, and conversely, the binding of DIBADL cross-linked IgG dimers is inhibited by monomeric IgG or 2.4G2 but not by rat or mouse IgE; 2) the binding of IgE dimers is saturable on cells from uninfected and Nippostrongylus brasiliensis (Nb)-infected mice; 3) IgE dimer binding is detectable on most splenic B lymphocytes from uninfected and Nb-infected mice, but not on T lymphocytes from uninfected mice, and on few, if any, T lymphocytes from Nb-infected mice; 4) Nb infection causes a parallel increase in the percentages of B lymphocytes and cells expressing IgE receptors and Fc gamma R; 5) Nb infection leads to a marked increase in B lymphocyte IgE receptor expression, has little if any effect on IgE receptor affinity, and causes only minor changes in Fc gamma R expression; and 6) in vivo activation of B lymphocytes by a goat antibody to mouse IgD decreases IgE receptor expression considerably, but has a minimal effect on Fc gamma R expression. Thus, there are separate receptors for IgE and IgG on murine B lymphocytes, and the effect of Nb infection or anti-IgD treatment on their expression is different.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.     

Isotype Specificity and Heterogeneity of Fcγ-Receptors on Guinea Pig Splenic B and T Lymphocytes

The isotype specificity of Fc-receptors for IgG (Fcγ-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fcγ-R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette-forming T cells bear essentially the Fcγ-R monspecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.     

T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.