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.     

Modulation induction of the T3 antigen by OKT3 antibody is monocyte dependent

We investigated the influence of monocytes on the susceptibility of the T3 antigen on human T cells to modulation induction by OKT3 antibody. In the absence of monocytes, the T3 antigen was only minimally susceptible to modulation. After the addition of 20% monocytes to the culture, however, complete modulation was readily observed. Furthermore, we found that even in the absence of OKT3 antibody, monocytes were able to down-regulate the expression of the T3 antigen, although to a lesser extent. The ability of monocytes to enhance antigenic modulation proved to be a more general phenomenon. Each individual T cell antigen, however, differed in its susceptibility to modulation by antibody, monocytes, or both, thereby establishing its own characteristic pattern. In addition, after complete modulation of the T3 antigen, the addition of monocytes to the culture thereafter had a distinct inhibitory effect on the reexpression of the T3 antigen. Monocyte enhancement of T3 modulation is significantly reduced when using the OKT3 F(ab')2 fragment, as is OKT3 mitogenesis. After pulsing the monocytes with OKT3 antibody before adding them to the culture, T3 modulation became nearly complete even in the absence of added OKT3 antibody. Monocyte-induced modulation proved not to be MHC restricted, thus allowing for comparative analysis of this effect between monocytes and other cell types. A moderate, however, incomplete modulation enhancement was observed with the human monocyte cell line U937 and with Daudi cells. This finding proved to coincide with the distinct ability of these cell lines to bind OKT3 antibody by their Fc receptors, as was the case with monocytes. In contrast, neither Fc receptor binding nor T3 modulation enhancement was observed with the cell lines Cess and G7. In addition, no effective T3 modulation was observed with glutaraldehyde-fixed monocytes. The overall results seem to indicate that effective modulation of the T3 antigen by OKT3 antibody requires the active participation of Fc receptors on monocytes.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG

Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand

Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.     

The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3

The means by which normal human serum inhibits the activation of PBMC by OKT3 has been investigated. The Fc portion of intact IgG is shown to be the major inhibitor in human serum of this OKT3-induced stimulation. Furthermore, inhibition by IgG subclasses correlated with their ability to bind to the monocyte Fc receptor, i.e., IgG1 and IgG3 produced greater inhibition than IgG2 and IgG4, and this inhibition was competitive. In contrast, hypogammaglobulinemic serum, IgA, IgM, and F(ab')2 of IgG were not inhibitory relative to intact IgG or Fc of IgG. Because of the similarities between T3 and the idiotype-defined T cell receptor for antigen, these investigations suggest that IgG might modulate the interactions between the T cell recognition complex and anti-idiotype antibody, thus regulating the idiotype network.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.     

Human T cell activation induced by a monoclonal mouse IgG3 anti-CD3 antibody (RIV9) requires binding of the Fc part of the antibody to the monocytic 72-kDa high-affinity Fc receptor (FcRI)

The mitogenic activity of anti-CD3 mouse monoclonal antibodies (mAb) in cultures of human peripheral blood mononuclear cells (PBMC) depends on the ability of the mAb to interact with CD3 molecules on the T cells, and with Fc receptors (FcR) on monocytes. Two types of FcR with distinct specificity for murine (m) IgG subclasses are involved: a 72-kDa receptor (FcRI) binds mIgG2a and a 40-kDa receptor (FcRII) binds mIgG1. In this study we examined the mitogenic activity of mIgG3 anti-CD3 mAb RIV9. In cultures of human PBMC, the mAb induced T cell proliferation and interleukin 2 production. We found that subjects, unresponsive to mIgG2a anti-CD3 (e.g., OKT3), were also RIV9 nonresponders. In contrast, nonresponders to mIgG1 anti-CD3 (e.g., anti-Leu4) had a normal response to RIV9. Our results therefore suggested that anti-CD3 mAb of the mIgG2a and mIgG3 subclass bind to the same monocytic FcR. Human monomeric IgG, which has been shown to bind to FcRI only, blocked T cell proliferation induced by mIgG2a and mIgG3 anti-CD3, but had no effect on T cell proliferation induced by mIgG1 anti-CD3. In contrast, a mAb (IV.3) to FcRII, which blocks ligand binding of the receptor, blocked the mitogenic activity of mIgG1 anti-CD3 antibodies, but had no effect on T cell proliferation induced by mIgG3 anti-CD3 or by mIgG2a anti-CD3. Binding of RIV9 to FcR of responder monocytes could be demonstrated in immunofluorescence. Monocytes from the RIV9 nonresponder subjects however were unable to bind the Fc portion of this antibody. The binding of fluorescein (FITC)-conjugated mIgG3 or FITC-conjugated mIgG2a to responder monocytes could be inhibited by human monomeric IgG and by mIgG2a and mIgG3, but not by the mAb to FcRII. The results demonstrate that mIgG3 binds to FcRI on human monocytes and that this binding is needed for the mitogenic activity of mIgG3 anti-CD3.     

Phagocytosis of concanavalin A-treated erythrocytes is mediated by the Fc gamma receptor

Impaired Fc gamma receptor-mediated phagocytosis has been reported in monocytes from HLA-DR2- and -DR3-positive disease-free individuals compared to normals without these B cell alloantigens. We have noted, however, a decrease in the ingestion of concanavalin A (Con A)-treated rabbit erythrocytes (E-Con A) in the same immunogenetically defined groups (DR2 vs Other: 2.94 +/- 0.84 erythrocytes/monocyte vs 4.16 +/- 1.37, p less than 0.003; DR3 vs Other: 3.35 +/- 1.51 vs 4.16 +/- 1.37, p less than 0.04). These data raised the possibility that carbohydrate-lectin interactions might trigger ingestion mediated by the Fc gamma receptor. To test this hypothesis, we performed receptor modulation and monosaccharide blocking experiments. Modulation of the Fc gamma receptor off the apical cell surface of monocytes by adherence to solid-phase IgG aggregates specifically reduced internalization of E-Con A and IgG-sensitized erythrocytes (EA) to 9.1% and 10.6% of control, respectively (p less than 0.001). Internalization of wheat germ agglutinin-treated erythrocytes, tannic acid-treated erythrocytes, and zymosan was not inhibited. In reciprocal modulation experiments using solid-phase Con A, no effects on phagocytosis of any particle was observed. alpha-Methyl mannoside, 0.1 M in PBS, did not inhibit the internalization of EA but blocked ingestion of E-Con A by 97% (p less than 0.001). Other monosaccharides had little or no effect on the ingestion of any of the phagocytic probes. These data demonstrate that a mechanism integrally involving the Fc gamma receptor mediates the ingestion of E-Con A by human monocytes. This Fc receptor has an oligosaccharide(s) with an exposed mannose which may be functionally significant. Whereas the mannose moiety does not play a crucial role in the interaction of the Fc gamma receptor with the Fc portion of IgG, engagement of the receptor via mannose can initiate internalization. Our findings raise the possibility that nonimmune functions may utilize classical immune system receptors through carbohydrate interactions. Furthermore, the ability of the Fc gamma receptor to trigger internalization is defective in HLA-DR2 and -DR3 normals, whether the receptor is ligated at its classical ligand-binding site or by way of its carbohydrate moieties.     

Cross-linking Fc receptors on monocytes triggers IL-6 production. Role in anti-CD3-induced T cell activation

IL-6 is a multifunctional cytokine which is produced by a variety of cells. Therefore it was examined whether anti-CD3-induced T cell activation was associated with the induction of functionally relevant IL-6 in human monocyte accessory cells. Significantly increased amounts of IL-6 were detected in supernatants of anti-CD3-treated PBMC. Stimulation of FACS-sorted greater than 98% pure monocyte accessory cells, but not of highly purified T cells with anti-CD3, resulted in an increased IL-6 production. Furthermore, anti-CD3 significantly enhanced IL-6 mRNA expression in monocyte accessory cells. IL-6 production was not limited to anti-CD3, inasmuch as equivalent IL-6 stimulation could be achieved with a mouse IgG2a isotype control antibody. In contrast to solid phase-bound mouse IgG2a, the soluble form of this antibody failed to induce IL-6 secretion indicating a requirement for Fc gamma RI receptor cross-linking. Moreover, this property may be specific for the Fc gamma RI receptor inasmuch as mouse IgG1 antibodies binding to the Fc gamma RII receptor did not significantly enhance IL-6 production. The role of IL-6 being an additional signal in T cell activation was confirmed by the finding that an anti-IL-6 antiserum was able to suppress anti-CD3-induced T cell activation. These data indicate that binding of anti-CD3 to Fc gamma RI may generate an activation signal towards the monocyte accessory cell leading to the production and secretion of monocyte IL-6, which in turn augments T cell activation, and also may be relevant to a variety of antibody-mediated immune responses against viral and bacterial infections.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.