Suppressor Activity of T Cells Bearing Fc Receptors for IgG on Lymphoid Colony Formation

Human T lymphocytes, upon phytohaemagglutinin stimulation, are able to form colonies in semisolid media. Peripheral T cells bearing Fc receptors (T_G) were studied for their possible regulatory activity on lymphoid colony development. Although no substantial differences were observed between the cloning efficiency of unfractionated T cells (thus including T_G cells) and T cells depleted of T_G, a sharp suppression of colony formation occurred when positively selected T_G cells were readded to T_G-depleted suspensions. Therefore, T_G suppressor activity seems to be strictly dependent upon cell interaction with ox IgG immune complexes used for T_G cell isolation. Different experimental approaches failed to demonstrate, although did not exclude, that suppression in this system is mediated by soluble factors, γ-irradiation of T_G cells abrogated their suppressor capacity.     

Modulation of Pokeweed Mitogen-Induced Human B-Cell Differentiation by Aggregated IgG

The modulatory effect of human heat-aggregated IgG on human B-cell differentiation induced by pokeweed mitogen was investigated with three experimental protocols. Pulse exposure to aggregated IgG, followed by extensive washings before culture, of peripheral blood mononuclear cell suspensions rigorously depleted of platelets and containing less than 4% monocytes resulted in a selective decrease of the numbers of IgG-containing cells and IgG-secreting cells, whereas a simultaneous decrease of the numbers of cells producing IgG and, to a lesser extent, of those producing IgM or IgA was observed when the pulsed suspensions contained platelets and more than 4% monocytes. This non-isotype-specific suppression was shown to be more pronounced when aggregated IgG and platelets were present in the cell suspensions throughout the cultures. The results suggest that two distinct suppressor pathways can be triggered by aggregated IgG. The first one is restricted to cells producing the matching isotype, in the absence of platelets, with few monocytes in the cell suspensions. The second one leads to a nonspecific suppression of the three major Ig classes. It requires the presence of platelets and/or a high percentage of monocytes and, although it remains to be demonstrated, is probably mediated by prostaglandin E2.     

Effects of Ethanol on Human Monocyte IgG Fc Receptors

The IgG Fc receptor function of human monocytes (Mo) exposed to ethanol in vitro was assessed by a rosette assay in which IgG-coated sheep red blood cells were used as test particles. Pre-incubation of Mo in autologous serum with ethanol (55 mM and above) for 15 min at 37 degrees C, caused a significantly lower percentage of Mo-forming rosettes. This reduction by ethanol was not observed when Mo were preincubated with ethanol in fetal calf serum or in serum-free conditions, but was present when Mo were pre-incubated with ethanol in serum-free media to which IgG (10-150 micrograms/ml) had been added. Donor-dependent differences were observed in the reduction of Mo-forming rosettes in the presence of autologous serum and ethanol. Mo allowed to form rosettes without ethanol were exposed to ethanol during a subsequent phagocytosis process. A dose-dependent inhibition of the internalization of test particles was found in Mo from all donors.     

Concanavalin A Induction of Suppressor Activity in the T-Helper Subset Defined by Monoclonal Antibodies

Human monocyte-depleted peripheral blood mononuclear cells were separated in T4+ and T8+ populations by a panning technique. Petri dishes coated with goat anti-mouse antibodies were plated by peripheral blood mononuclear cells coated by monoclonal antibodies, either T4 or T8. The cell populations were separated into adherent and non-adherent populations based on binding to the goat anti-mouse-coated plastic dishes. The purity of the adherent populations was 96%. T4+ and T8+ populations were used as effector cells in the concanavalin A-induced suppressor test. The T4+ population revealed a pronounced suppressor activity similar to that exhibited by the T8+ population. This finding was independent of two different sources of monoclonal antibodies, T4/T8 and OKT4/OKT8. The registered suppressor activity in the monoclonal antibody-defined helper population could not be explained either by a switch of the membrane phenotype from T4+ to T8+ cells or by an increased interleukin 2 consumption of the concanavalin A-treated cells.     

Induction of Suppressor Cell Activity by Human Pregnancy Serum

ABSTRACT: Third trimester pregnancy serum caused a dose-dependent inhibition of the one-way allogeneic mixed leukocyte reaction (MLR) through an effect that occurred during the first 48 hours of culture. Pregnancy serum also inhibited the mitogenic responsiveness of normal mononuclear leukocytes to concanavalin A (Con A) while the responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were less affected. Preincubation of normal peripheral blood mononuclear cells (PBMC) for 48 hours with 10% pregnancy serum enhanced a suppressor activity transferable with cells. These pregnancy serum-induced effector cells suppressed the MLR only when they were autologous to the responder population (p < 0.05). The same suppressor cell preparation inhibited the proliferative responses of autologous PBMC to Con A (p < 0.001) and PWM (p < 0.05). These data suggest that one or more factors in pregnancy serum can induce or enhance suppressor cell activity in vitro. A comparable increase in suppressor cell activity in vivo may be responsible for blocking maternal rejection of the fetus and for the observed improvement in clinical activity of rheumatoid arthritis during pregnancy.     

Binding of Aggregated IgG by Human B Lymphocytes Independent of Fc Receptors

We present data concerning the mechanism of the binding of aggregated IgG and of IgG-sensitized erythrocytes by different human lymphocyte-like cells. The results showed that the two phenomena are related to different subpopulations of lymphocytes. By fractionation and depletion studies of B lymphocytes, T lymphocytes, and lymphocyte-like cells binding IgG-sensitized erythrocytes (EA-RFC), it was demonstrated that binding of aggregated IgG is related to B lymphocytes, whereas binding of IgG-sensitized erythrocytes depends on another subpopulation of lymphocyte-like cells. Whereas binding of IgG-sensitized erythrocytes by EA-RFCs depends entirely on the Fc fragment, binding studies with IgG fragments suggested that the interaction of aggregated IgG with B lymphocytes is different.     

Receptors for IgG (Fc receptors) on human lymphocytes: re-evaluation by multiple techniques

This study examines whether receptors for IgG (Fc receptors), as identified by different methods, are found on identical human lymphocyte subpopulations, and the relationship of Fc receptors to surface immunoglobulin (SIg) and receptor for complement (C'). Fc receptors were identified by two rosetting techniques (antibody-sensitized human erythrocytes, HuEA, or sheep erythrocytes, ShEA) and two immunofluorescent techniques (heat-aggregated IgG of human origin, HuAgg, or of guinea-pig origin, GPAgg).

When lymphocytes depleted of cells rosetting with ShEA were compared to HuEA-depleted lymphocytes, the two subpopulations appeared to be significantly different. However, when lymphocytes were depleted of cells rosetting with ShEA which had been sensitized with lower concentrations of antibody, the subpopulation so depleted appeared to be virtually identical to HuEA-depleted lymphocytes. These studies suggest that more than one lymphocyte subpopulation has Fc receptors and that each subpopulation can, in part, be identified and distinguished from the other by the capacity to bind IgG at differing concentrations.

In particular, these experiments may serve to resolve the controversy concerning the presence of Fc receptors on lymphocytes bearing surface immunoglobulin (SIg). Depletion of lymphocytes rosetting with ShEA removed most of the SIg-bearing lymphocytes; depletion of cells rosetting with ShEA which had been sensitized with lower concentrations of IgG antibody, however, failed to deplete SIg-bearing lymphocytes even though other Fc-bearing populations were completely depleted. These results suggest that SIg-bearing lymphocytes (B lymphocytes) do have Fc receptors but that high concentrations of IgG are needed to demonstrate them.

     

Suppression of Mitogen-Induced Lymphoproliferation by Soluble IgG Fc Receptors in Retroplacental Serum in Normal Human Pregnancy

A competitive ELISA was used to quantify soluble IgG Fc receptors (FcR) in retroplacental serum (RPS) and peripheral serum (PS) from 10 women after uncomplicated full-term deliveries. The RPS contained significantly higher amounts of soluble FcR than did PS from the same individuals. RPS suppressed phytohaemagglutinin-stimulated lymphoproliferation as compared with PS, and a positive correlation ( r =0.66) was found between the degree of suppression and the difference in soluble FcR level between RPS and PS. Absorption of sera with Sepharose 4B coupled with heat-aggregated IgG strongly reduced the immunosuppressive activity, whereas absorption with Sepharose coupled with IgG F(ab')₂ fragments did not. When IgG-binding material was eluted from Sepharose beads and added to cell cultures, the immunosuppressive activity was restored. The data indicate that soluble FcR at physiological levels have immunosuppressive properties. FcR-mediated immunosuppression may be of importance for maintenance of local immunosuppression during pregnancy.     

Purification of A Suppressor Lymphokine (SL) from a Human T-Cell Line

Human T leukemia cell line 81-66-45 spontaneously releases into the medium a suppressor lymphokine (SL), able to inhibit PHA-stimulated normal peripheral blood T cell proliferation. Ion exchange and gel filtration chromatography were used successfully to isolate and purify this immunosuppressive lymphokine from culture supernatants. When the purified suppressor lymphokine was characterized with SDS-polyacrylamide gel electrophoresis under reducing conditions, it was found to be a single protein chain of 66,000 daltons. Titration curves of the purified suppressor lymphokine indicated that the inhibitory activity is dose dependent. The suppressor lymphokine is cytostatic and its addition to the peripheral blood lymphocytes (PBL) did not change the cell number or cell viability. This factor was stable at pH 2.0-8.5 and at 56° C for 30 minutes. The structural relationship of this lymphokine with other T cell factors is discussed.     

Purification of A Suppressor Lymphokine (SL) from a Human T-Cell Line

Human T leukemia cell line 81-66-45 spontaneously releases into the medium a suppressor lymphokine (SL), able to inhibit PHA-stimulated normal peripheral blood T cell proliferation. Ion exchange and gel filtration chromatography were used successfully to isolate and purify this immunosuppressive lymphokine from culture supernatants. When the purified suppressor lymphokine was characterized with SDS-polyacrylamide gel electrophoresis under reducing conditions, it was found to be a single protein chain of 66,000 daltons. Titration curves of the purified suppressor lymphokine indicated that the inhibitory activity is dose dependent. The suppressor lymphokine is cytostatic and its addition to the peripheral blood lymphocytes (PBL) did not change the cell number or cell viability. This factor was stable at pH 2.0–8.5 and at 56° C for 30 minutes. The structural relationship of this lymphokine with other T cell factors is discussed.     

Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI)

Earlier studies from this and other laboratories have provided indirect evidence for the involvement of the C gamma 2 domain of human IgG in the binding of IgG to the high affinity monocyte Fc receptor (FcRI). Two approaches have been used to extend these studies and to further localize the site of interaction on human IgG. Firstly, monoclonal antibodies (MAbs) directed against different epitopes on IgG were assayed for their capacity to inhibit the binding of radiolabelled IgG to human monocytes or U937 cells. The capacity of the MAbs to interact with their respective epitopes on FcR-bound IgG was also studied using indirect radiobinding and immunofluorescence assays. Secondly, a number of IgGs from several different species and fragments of human IgGs were assayed for their ability to inhibit the binding of radiolabelled IgG to human monocytes. The amino acid sequences of those IgGs exhibiting relatively tight, intermediate or weak binding to monocyte FcRs were compared. On the basis of these studies a possible monocyte FcR-binding site on human IgG is postulated, involving the lower hinge region of IgG (residues Leu 234-Ser 239) with possible involvement of the nearby N-proximal bend and two beta-strands (Gly 316-Lys 338).     

Phorbol Ester Induction of Ia and a Shared B-Cell/T-Cell Phenotype in Normal but Not Autoimmune (lpr) Mice

Tumour-promoting phorbol diesters are mitogenic for lymphocytes and induce differentiation of B and T cell lines as well as promyelocytic leukaemia cells. This paper demonstrates that 12-O-tetradecanoyl-13-acetate (PMA), when cocultured with normal murine bone marrow cells (BMC), significantly augments an antigenic cell-surface determinant called 14D10. This antigen is present constitutively in the majority of bone-marrow lymphocytes of autoimmune lpr mice. PMA has little enhancing effect when cocultured with lpr BMC. In addition, Ia antigenic determinants are increased by PMA in normal but not lpr BMC. Retinoic acid (RA) and PMA act synergistically both to increase 14D10 and to enhance the stimulatory ability of target lymphocytes as measured by proliferation in an autologous mixed lymphocyte reaction (AMLR). We suggest that lpr mice have persistent expression of gene products like 14D10 that are usually repressed in normal adult mice. These gene products can be activated in normal mouse bone marrow by PMA which acts through a Ca2+-dependent phospholipid-dependent C protein kinase. The in vivo enhanced expression of 14D10 in lpr mice suggests activation by some mechanism or factors yet to be described.     

Studies on the Role of CD43 in Human B-Cell Activation and Differentiation

The monoclonal antibody (MoAb) B1B6 to human leucocyte sialoglycoprotein, CD43, induces aggregation of T cells and delivers progression signals early during activation of both T and B cells in the presence of primary activators of protein kinase C. In this report we further studied the role of CD43 in human B-cell activation and differentiation. About 5-10% of resting tonsillar B cells are CD43+. In the presence of TPA or antibodies to CDw40, the proportions of CD43+ cells drastically increased. The expression was optimal on day 3 of culture, when up to 80% and 50%, respectively, were CD43+. Whereas MoAb B1B6 together with TPA induced a three- to fivefold higher proliferative response as compared to TPA alone, antibodies to CDw40 did not synergize with MoAb B1B6 in B-cell proliferation. Tonsillar populations depleted of CD43+ B cells responded with lower proliferation to TPA alone or to TPA and B1B6 or anti-CDw40 antibodies. MoAb B1B6 did not affect the production of IgM or IgG as induced by pokeweed mitogen in the presence of autologous T cells, from either peripheral blood or tonsillar B cells. Neither did it affect the IgG production from the CD43+ BSF-2 sensitive Epstein-Barr virus-transformed lymphoblastoid cell line CESS. The results show that CD43 is upregulated on B cells during activation. Furthermore, CD43+ B cells are included in the population which responds to signals delivered by TPA, anti-CD43 or anti-CDw40 antibodies, and the proliferation of this population is not merely due to an expansion of the small population of CD43+ cells present among these cells. Moreover, the epitopes recognized by MoAb B1B6 are not involved in the differentiation of and ultimate Ig-secretion from activated B cells.     

Correlation of IgG Fc Receptors on Granulocytes with Serum Immune Complex Level in Systemic Lupus Erythematosus

The expression of FcγRI, FcγRII and FcγRIII on granulocytes in the blood of patients with systemic lupus erythematosus was investigated. The relationship between the receptor expression and serum immune complex (IC) concentration was analysed. The decrease in mean fluorescence intensity of both FCγRII and FcγRIII of patients' granulocytes stained by specific monoclonal antibodies (using MoAb IV. 3 and 3G8) was significant. The detected decrease of FcγRII was inversely correlated with the high circulating IC level in patients' sera.     

Effect of rIFN-γ on Antibody-Mediated Cytotoxicity via Human Monocyte IgG Fc Receptor II (CD32)

Human monocytes and macrophages express an isoform of IgG Fe receptor II (FcγRII), FcγRII. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR). respectively. We investigated the effect of recombinant (r)IFN-γ on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), Using human erythrocytes (E) sensitized with mlgGl as target cells, FcγRII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased FcγRII expression, and FcγRll-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rlFN-γ (40 h) reversed this decrease. Short-term rlFN-γ-cultured cells, and fresh cells express similar numbers of FcγRII, and exhibit comparable FcγRII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rlFN-γ either from day 0 or from day 7, did not affect expression or functional activity of FcγRII. Furthermore, the effects were observed in both HR and LR individuals.
Our results show that rlFN-γ has strong effects on FcγRII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.     

Down-Regulation of CD59 (Protectin) Expression on Human Colorectal Adenocarcinoma Cell Lines by Levamisole

The vulnerability of tumour cells to complement-mediated immune attack is regulated by membrane associated molecules. Recently, we have shown that the expression of the membrane attack complex inhibitor CD59 is enhanced on colonic adenocarcinoma cells compared to normal colonic epithelial cells. CD59 was shown, in the same study, to protect the tumour cells from complement-mediated lysis. Levamisole (LMS), used in conjunction with 5-fluorouracil as adjuvant therapy, reduces the incidence of colon cancer relapse following surgical resection. This led to our investigation of the effect of LMS on CD59 expression and function on the human colorectal cell lines HT29 and Caco-2. When cultured in the presence of 10 μM LMS, the cells reduced their expression of CD59 in a time-dependent manner. LMS treated HT29 ceils were more sensitive to lysis by complement than control cells, and the reduction in CD59 expression was shown to be partly responsible for this. A reduction in CD59 expression will augment complement-mediated immune surveillance and may contribute to LMSs anti-tumour activity in vivo .     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Independent Segregation of Two Functional Markers Expressed on the Same B-Cell Subset in the Mouse: The Mls Determinants and LPS Receptors

Mice of the C3H/Tif strain display a mixed leukocyte reaction (MLR) with all H-2k strains carrying any of the known alleles of the Mls locus. In particular, C3H/Tif is incompatible with the related substrain C3H/HeJ, from which it also differs at the locus responsible for the recognition of lipopolysaccharides (LPS) as B-cell mitogens, and at the Mod-1 locus. Our genetic analysis indicates that the MLR incompatibility between these strains is not H-2-linked and segregates as controlled by a single locus, most probably identical to Mls, for which the C3H/Tif strain expresses a previously unidentified allele, Mlse. Moreover, segregation data show that this locus assorts independently of LPS responsiveness and that neither marker is closely linked to the Mod-1 locus in linkage group II.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b.