Ia- and IgG-Fc receptor-positive accessory cell sustains peripheral T lymphocyte but not thymocyte mitogenesis induced by OKT3 monoclonal antibody

The proliferative responses of human peripheral blood mononuclear cells (PBL) and thymocytes to OKT3 monoclonal antibody have been investigated. The PBL response to OKT3 was maximal after 72 hr while that of thymocytes was inappreciable at all times measured. Unlike phytohemagglutinin, OKT3 was unable to elicit the mitogenesis of adherent cell-depleted T cells in spite of the presence of exogenously added Interleukin 1 and/or Interleukin 2. The addition of autologous or heterologous adherent cells restored the OKT3 mitogenic response of peripheral purified T cells but not of thymocyte cultures. The adherent cell population that was able to sustain the OKT3-elicited T-cell mitogenesis was constituted by Ia-, Fc receptor-positive cells. These data suggest that the adherent cell-T cell interaction is mediated via the Fc portion of the OKT3 molecule. Furthermore, unlike peripheral T cells, T3-positive thymocytes, which represent the more mature. PHA-responsive subset within the thymus, are unable to cooperate with accessory cells when pulsed with OKT3 monoclonal antibody.     

T lymphocyte activation induced in vivo by the first injection of OKT3 monoclonal antibodies

One of the major side effects induced by the in vivo administration of the murine monoclonal antibody OKT3 is a spontaneously reversible clinical syndrome associating in variable proportions depending on the patient: fever, chills, headaches, diarrhea and seldomly meningismus. Sera from 3 renal allograft recipients treated with OKT3 were studied and showed that a massive although transient release of some cytokines namely, Tumor Necrosis Factor alpha, Interleukin 2 and Interferon gamma is observed following the first OKT3 injection.     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

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 T cell activation by monoclonal anti-Thy-1 antibodies

We have analyzed the requirements for T cell activation by monoclonal anti-Thy-1 antibodies (MAb). A large panel of unselected anti-Thy-1 MAb was capable of inducing a strong proliferative response in resting peripheral T cells and a rise in cytoplasmic free calcium ([Ca2+]i) in both peripheral T cells and a T cell hybridoma. Both of these responses required the interaction of a MAb bound to Thy-1 with a second layer of anti-Ig antibody. Induction of T cell proliferation also required an additional signal, which could be provided by PMA. T cell activation in this system was specific for the Thy-1 molecule, independent of the epitope on Thy-1 recognized by a given MAb, with the anti-Ig reagent was also independent of the type of anti-Ig used, as both polyvalent rabbit anti-rat Ig sera and a mouse MAb to rat Ig functioned as effective cross-linkers. All signals provided by the interaction of anti-Thy-1 MAb with anti-Ig preparations could also be reproduced by the simultaneous binding of two MAb recognizing independent epitopes on Thy-1. Although the physiological role of Thy-1 remains unknown, the model system described here should prove to be very useful in further analysis of the steps involved in the polyclonal activation of murine T cells.     

Induction of interleukin 3 but not interleukin 2 or interferon production in the syngeneic mixed lymphocyte reaction

The syngeneic mixed lymphocyte reaction (SMLR) was assayed in the medium containing syngeneic normal mouse serum (NMS), by using nylon-adherent stimulator cells and nonadherent responder T cells, which were prepared from murine spleens in the absence of fetal calf serum (FCS) to avoid any sensitization to xenogeneic protein antigens. The responder cells in this SMLR, without definite background proliferation, generated specific proliferative response to the syngeneic stimulator cells in a dose-related fashion. The SMLR was accompanied by production of interleukin 3 (IL 3) but not interleukin 2 (IL 2) or interferon (IFN). No cytotoxicity against the syngeneic or allogeneic target cells was induced. Correlating with no production of IL 2 or IFN, no natural killer (NK) activity was detected. The proliferation was not inhibited by addition of specific antiserum for IFN-gamma. In contrast, proliferation in the responder cells when incubated with allogeneic stimulator cells was inhibited by anti-IFN-gamma serum and accompanied by production of IL 2 and IFN as well as IL 3, and by augmentation of NK activity and generation of cytotoxic T cells. Cell surface analysis revealed that the cells producing IL 3 in this SMLR system were Thy-1+ Lyt-1+2- helper T cells. Cells responding to the SMLR culture fluids with DNA replication were Thy-1-Lyt-1-2- asialo GM1- no-marker cells, which were the same as a population responsible for partially purified IL 3. On the other hand, when the responder cells were exposed to FCS before culture and assayed for SMLR in the FCS-free NMS medium, variable levels of IL 2 production were induced in response to the stimulator cells. The responder cells generated a high background DNA replication in the absence of syngeneic stimulators, suggesting that this IL 2 production may result from the stimulation of T cells by FCS as a foreign antigen. Overall, these results suggest that the SMLR may be a cellular interaction, in which non-T cells stimulate Lyt-1+2- helper T cells to produce IL 3 but not IL 2 or IFN. This IL 3 can, in turn, induce proliferation of IL 3 responding cells, which appear to be early precursors in lymphocyte differentiation, but no proliferative response or activation of IL 2- and IFN-dependent mature T cells or NK cells.     

Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside

Cell-surface gangliosides are presumed to play a role in cell growth and differentiation. With the use of monoclonal antibodies directed against GD3, a disialoganglioside expressed predominantly by cells of neuroectodermal origin, we have found that GD3 is expressed by a subpopulation of cells of the immune system including: 1) fetal thymocytes in subcortical regions and near vessels, 2) lymph node lymphocytes in interfollicular areas and near vessels, and 3) a small subset of T cells in the peripheral blood. Mouse monoclonal antibodies (two IgGs, one IgM, and F(ab')2 fragments) reacting with GD3 were found to stimulate proliferation of T cells derived from peripheral blood. Proliferation of T cells was observed even in cultures depleted of macrophages, suggesting that activation by anti-GD3 was not dependent on the presence of accessory cells. T cell proliferation was maximum between days 5 and 7 of stimulation and was preceded by expression of interleukin 2 receptors. No stimulation was observed with control antibodies of the identical isotype or with monoclonal antibodies recognizing the gangliosides GD2 or GM2. During stimulation by anti-GD3 monoclonal antibodies, there was an expansion of the GD3+ pool of T cells, but depletion of GD3+ T cells prior to stimulation abrogated the response. Proliferation induced by binding to GD3 could be augmented by exogenous interleukin 2 and phytohemagglutinin. Anti-CD3 (T3) monoclonal antibodies had little or no effect. These results demonstrate that binding to GD3 on the surface of T cells can elicit signals for T cell proliferation.     

Cytochalasin B enhances T cell mitogenesis by promoting expression of an interleukin 2 receptor

Low concentrations of cytochalasin B enhanced the T cell mitogenesis induced by concanavalin A (Con A) and interleukin 2 (IL-2). Mitogenesis was augmented by cytochalasin B given in the Con A-dependent early phase, or through T cell mitogenesis. Cytochalasin B did not enhance T cell mitogenesis when given only in the IL-2-dependent late phase. Use of the monoclonal antibody that directs the IL-2 receptor showed that cytochalasin B increased the expression of the IL-2 receptor induced by Con A. We concluded that cytochalasin b acts on an early phase of T cell mitogenesis and augments the expression of IL-2 receptor which enables certain nonresponsive T cells to respond to IL-2.     

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.     

Ghrelin but not obestatin regulates insulin secretion from INS1 beta cell line via UCP2-dependent mechanism

The mitochondrial UCP2 mediates glucose-stimulated insulin secretion by decreasing intracellular ATP/ADP ratio. Insulin secretion is a tightly regulated process. Ghrelin, as well as obestatin, were intensively studied to determine their ability to modify insulin secretion. Ghrelin is considered to be an inhibitor of insulin release from pancreatic islets, however little is known about the effects of obestatin. In our study we demonstrate the stimulating effects of both peptides on insulin secretion in INS1 cells. Furthermore, we investigate the potential role of UCP2 in mediating the effects of both peptides on insulin secretion. UCP2 mRNA expression was down-regulated by ghrelin in the presence of 26.4 mM glucose, however it was unchanged after obestatin treatment. Our results confirm that UCP2 could be involved in the stimulating effect of ghrelin on insulin release from INS1 cells.     

Inhibition by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell-dependent B cell activation

Anti-CD3 mAb can activate T cells to help in B cell activation as detected by late events, such as maturation of B cells into Ig-secreting cells (IgSC), or by early events, such as B cell surface expression of the activation marker CD23. Two different anti-CD2 mAb each inhibited anti-CD3-induced T cell-dependent B cell activation in a dose-dependent fashion. Neither irradiation of the T cells prior to culture nor depletion of CD8+ cells abrogated the inhibitory effects of anti-CD2 mAb. Despite the ability of these anti-CD2 mAb to inhibit anti-CD3-induced IL2 production, addition of exogenous IL2 to anti-CD2 mAb-containing cultures could not fully reverse the inhibitory effects on IgSC generation. Furthermore, addition of various combinations of IL1, IL2, IL4, and IL6 or crude PBMC or monocyte culture supernatants also could not reverse anti-CD2-driven inhibition. In T cell-depleted cultures, anti-CD2 mAb had no effect on the ability of IL4 to induce B cell CD23 expression, confirming that anti-CD2 mAb had no direct effect on B cells. However, in cultures containing T+ non-T cells, anti-CD2 mAb did partially inhibit IL4-induced B cell CD23 expression. Taken together, these observations demonstrate that certain CD2 ligands can modulate T cell-dependent B cell activation by a mechanism which, at least in part, involves a direct effect by the CD2 ligand on the T cell itself.     

Nonrestricted cytotoxicity mediated by interleukin 2-expanded leukocytes is inhibited by anti-LFA-1 monoclonal antibodies (MoAb) but potentiated by anti-CD3 MoAb

Previous results have shown that in addition to their ability to kill tumor cell lines, peripheral blood leukocytes (PBL) expanded in interleukin 2 (IL-2) can also destroy normal PBL targets. Cold target competition results show that PBL and tumor cells can be destroyed by the same population of IL-2-expanded leukocytes (IEL), with better killing observed for tumor cell targets. Since cytolytic activity of IEL is nonspecific, differential binding of target cells by IEL could determine how well each target cell type can be killed. The binding affinity of IEL, in turn, could be influenced by the accessory molecules expressed on effector and target cells. We tested the effect of MoAb to LFA-1, CD2, CD3, CD4, CD8, and HLA molecules on killing mediated by IEL. Anti-LFA-1 inhibited strongly the killing of normal PBL and to a lesser extent the killing of tumor cells. Anti-CD2, CD3, CD4, CD8, and HLA class I molecules did not inhibit the nonspecific killing; rather, anti-CD3 potentiated the killing of PBL, K562, and Daudi cells. These results support the notion that qualitative and quantitative variations in LFA-1-mediated binding of target cells by IEL could result in differential killing of targets. The possibility of using anti-CD3 to selectively potentiate the killing of tumor cells is discussed.     

T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.     

IL-4 potentiates activated T cell apoptosis via an IL-2-dependent mechanism

Activation-induced cell death (AICD) of T cells is one of the major mechanisms of peripheral tolerance. The regulation of AICD by IL-4 is poorly understood. In this study, we report that AICD in IL-4-deficient T cells is significantly reduced compared with that in wild-type T cells. This impaired AICD correlates with the failure to induce degradation of cellular FLIP. IL-4-mediated enhancement of AICD and cellular FLIP degradation requires a Janus kinase/STAT-6 signaling pathway. Unexpectedly, these effects of IL-4 could be blocked by a neutralizing anti-IL-2 Ab, and addition of rIL-2 could completely restore the defective AICD in IL-4-deficient T cells. Furthermore, IL-4 regulates the T cell thresholds for IL-2 signaling during AICD. These data suggest that IL-4 promotes AICD via an IL-2-dependent mechanism.     

T lymphocyte adhesion to endothelial cells: mechanisms demonstrated by anti-LFA-1 monoclonal antibodies

Adhesion of lymphocytes to vascular endothelium is the first event in the passage of lymphocytes into a chronic inflammatory reaction. To investigate molecular mechanisms of T-EC adhesion, monoclonal antibodies (Mab) against T cell surface antigens have been tested for inhibition of binding. Baseline and phorbol ester-stimulated adhesion were strongly inhibited by either Mab 60.3 (reactive with the beta-chain of the LFA-1, OKM1, and p150,95 molecules) or by Mab TS 1/22 (specific for the alpha-chain of LFA-1). Although the increased binding of phorbol ester-stimulated lymphocytes was inhibited by anti-LFA-1 antibody, there was no increased expression of LFA-1 on phorbol ester-stimulated T cells, as determined by FACS analysis. Maximal inhibition of unstimulated and phorbol ester-stimulated T-EC adhesion was seen at Mab concentrations of 1 microgram/ml. In contrast, LPS- and IL 1-enhanced T-EC adhesion were only weakly inhibited by these antibodies. Mab 60.3 and TS 1/22 did not stain either unstimulated EC or LPS- or IL 1-stimulated EC, as measured by FACS analysis; moreover, preincubation of EC alone with these antibodies did not lead to inhibition of T-EC binding. Adhesion was not affected by Mab against the sheep erythrocyte receptor (LFA-2), a nonpolymorphic HLA class 1 framework antigen, or against LFA-3, the alpha-chain of OKM1, or the alpha-chain of p150,95. These results suggest that the mechanism of binding of lymphocytes to unstimulated endothelium differs from that to stimulated endothelium. LFA-1 appears to be an important adhesion-related molecule for binding to unstimulated endothelium. However, the increased lymphocyte adhesion to IL 1- or LPS-stimulated EC observed in these experiments appears to be relatively independent of LFA-1. The increased adhesion to stimulated EC could be due either to an increase in the avidity or the density of the EC receptor molecules ordinarily involved in unstimulated T-EC binding or to the formation of alternative receptors on the stimulated EC that are not present on unstimulated cells.