Human T lymphocyte activation by monoclonal antibodies; OKT3, but not UCHT1, triggers mitogenesis via an interleukin 2-dependent mechanism

OKT3 and UCHT1 monoclonal antibodies, which recognize the same human T cell surface antigen, induce proliferation in T lymphocytes. In this report, we compared the mechanism by which these antibodies trigger DNA synthesis in human peripheral blood mononuclear cell (PBMC) cultures. Whereas PBMC from all donors tested were mitogenically inducible by OKT3, cells from only 25 of 40 donors were responsive to UCHT1 . UCHT1 treatment of PBMC from responders, but not from nonresponders, resulted in the expression by T cells of membrane binding sites reactive with anti-Tac monoclonal antibody, which specifies the human interleukin 2 (IL 2) receptor. UCHT1 -induced PBMC supernatants from nonresponders, but unexpectedly, also from responders, contained no measurable IL 2 activity. In keeping with this finding, anti-Tac monoclonal antibody failed to suppress UCHT1 -triggered [3H]thymidine incorporation into PBMC from responsive donors. By contrast, OKT3 treatment of PBMC from all donors led to the emergence of IL 2 receptors, and substantial IL 2 production, and the resultant DNA synthesis was inhibitable by anti-Tac antibody. These data indicate that the interaction of OKT3 and UCHT1 monoclonal antibodies with the same T cell structure leads to the induction of proliferation via two different mechanisms: one dependent on the availability of IL 2 (OKT3) and one independent on the production and processing of this lymphokine ( UCHT1 ). PBMC unresponsiveness to UCHT1 could therefore not be related to a dysfunction in IL 2 synthesis or IL 2 receptor display.     

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.     

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.     

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.     

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.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

Early events in lymphocyte activation as defined by three new monoclonal antibodies

Three new lymphocyte activation antigens are described whose kinetics of appearance place them very early in the activation pathway. The 78,000 dalton early antigen (Ea) 1 is present at low levels on resting lymphocytes, and its expression is enhanced twofold to threefold within 3 hr of stimulation. Ea2, a nondisulfide-bonded 86,000 and 73,000 dalton heterodimer, is first detectable 3 hr after activation and peaks by 9 hr. Its presence on all but a few cell lines, plus the variable association with a lower m.w. (28,000) structure, suggest that it may serve as a receptor for a growth factor. Neither Ea1 nor Ea2 are restricted to lymphocytes. The 31,000 dalton Ea3 antigen is induced only by PHA but not by other means of activation, and may pre-exist within the cell. The Ea3 antibody blocks PHA-induced but not OKT3-induced mitogenesis, suggesting differences in the pathways of activation by these two stimuli. These reagents, and OKT3, were used to define the cyclosporine A (CSA)-sensitive stage of lymphocyte activation. CSA blocks at a point before the biosynthesis of Ea1 and after that of T3/T cell receptor loss from the cell surface, at a point close to Ea2 biosynthesis.     

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.     

Early events during the activation of human lymphocytes by the mitogenic monoclonal antibody OKT3

The effects of the mitogenic monoclonal antibody OKT3 on the metabolic changes preceding DNA synthesis during the activation of human peripheral blood mononuclear cells were compared with those induced by PHA. The aspects studied included uridine transport, the incorporation of inositol into phospholipids, Na+-dependent amino acid uptake, and protein synthesis. All four parameters were increased in response to the ligation of the T lymphocyte receptor recognized by OKT3. These changes were apparent as early as the corresponding changes induced by PHA. However, the increases in uridine uptake and inositol incorporation were disproportionately reduced when compared to those caused by PHA, and no evidence of high-dose inhibition was seen in cells activated by OKT3. This suggests that at least some lectin-induced changes in metabolism are mediated through additional mechanisms, probably involving distinct receptors.     

Regulation of in vivo immunological reactions by monoclonal antibody against lymphocyte activation antigen

In vivo effects of a monoclonal antibody that recognizes rat lymphocyte activation antigen were studied. Spleen cells obtained from sheep red blood cell (SRBC)-immunized rats developed strong PFC response against SRBC. However, the 5C6-F4 treatment resulted in the inhibition of subsequent development of PFC response. The suppression of PFC response was due to the inhibition of generation of helper T cells, but not due to the preferential induction of suppressor cells. In addition, 5C6-F4 antibody was also found to inhibit the clinical expression of collagen-induced rat arthritis and the synovial inflammation in collagen-induced arthritis rats. Furthermore, the in vivo generation of cytotoxic cells against syngeneic tumor cells was also inhibited by 5C6-F4 antibody. The in vivo administration of 5C6-F4 antibody did not cause any pathological changes in brain, lung, liver, kidney, spleen, thymus, and lymph nodes.     

Enumeration of T lymphocyte subsets by monoclonal antibodies in young and aged humans

The percentage and absolute number of peripheral blood mononuclear cells reactive with monoclonal antibodies identifying mature thymocytes and T cells (T3+), helper/inducer cells (T4+), and suppressor/cytotoxic cells (T8+) was determined in 19 young (mean age 35 yr) and 31 elderly (mean age 72 yr) individuals. The percent representation but not the absolute number of T cells (T3+) declined significantly (p less than 0.001) in the elderly, and the decline was attributable to both an absolute and relative decrease in the representation of the subpopulation of cytotoxic/suppressor (T8+) cells. The percentage and number of helper/inducer (T4+) T cells was comparable in both age groups.     

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.     

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.     

Regulation by anti-CD2 monoclonal antibody of the activation of a human T cell clone induced by anti-CD3 or anti-T cell receptor antibodies

In this study the effect of anti-cluster designation (CD) 2 monoclonal antibodies (mAb) on the activation of a cloned human T cell line, HY837, after triggering the CD3/T cell receptor (TcR) complex by anti-CD3 or anti-TcR mAb is described. HY837, which reacts with a series of mAb directed at different epitopes on the TcR, could be induced to proliferation and interleukin 2 (IL-2) production by soluble mAb directed at the CD3/TcR complex in the absence of accessory cells. mAb directed at the CD2 epitope T11-1 were shown to block the IL-2 production by HY837, as well as the expression of the IL-2 receptor, induced by anti-CD3 mAb, resulting in the inhibition of the proliferative response. The effect of anti-CD2 mAb on the proliferative response of HY837, induced by anti-CD3 mAb, was not due to a competition for Fc binding sites. In contrast, the proliferative responses and IL-2 production of HY837, induced by mAb directed at the TcR, were shown to be enhanced by the action of the anti-CD2 mAb. These results indicate that effects mediated by anti-CD3/TcR mAb cannot always be extrapolated to antigen-mediated effects and show that anti-CD2 mAb may regulate the T cell response, induced by mAb directed at the CD3/TcR complex, depending on which part of this complex is triggered during activation.     

Effects of anti-Lyt-2 and anti-L3T4 monoclonal antibodies on the function of cytotoxic T lymphocyte/helper T lymphocyte hybrid T cell clones

CTL/HTL hybrid clones provide a unique system that allows detailed analysis of the role of Lyt-2, L3T4, and other structures involved in T cell functions. We have demonstrated previously that the fusion of cloned murine CTL and helper T lymphocytes with defined specificity generated hybrid cells that expressed both Lyt-2 and L3T4 as well as two TCR. Data obtained with these hybrid clones demonstrated that cytolysis is closely linked to the CTL TCR. We have analyzed the effects of anti-Lyt-2 and anti-L3T4 as well as anti-TCR mAb on cytolysis, proliferation, and lymphokine release by a number of hybrid clones. We found that anti-Lyt-2 and anti-L3T4 mAb were able to inhibit both proliferation and lymphokine release by the hybrid clones in response to stimulation of either the CTL or helper T lymphocyte parent TCR. In contrast, only anti-Lyt-2 and anti-CTL TCR mAb were able to block cytolysis of target cells bearing the Ag recognized by the CTL TCR. These results provide further evidence that cytolysis is closely linked to the CTL TCR and that Lyt-2 and L3T4 have more than a passive role as accessory molecules on the surface of T lymphocytes.     

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.     

Restriction of the human in vivo immune response against the mouse monoclonal antibody OKT3

The murine monoclonal antibody OKT3 (IgG2a) was administered prophylactically to 17 renal allograft recipients (5 mg/day, i.v.), either alone or in association with corticosteroids (0.25 mg/kg/day) and azathioprine (3 mg/kg/day). In all patients the kinetics of the IgM and IgG anti-OKT3 response was monitored by means of immunofluorescence and ELISA. All patients treated with OKT3 alone showed a rapid and strong sensitization that completely neutralized the therapeutic effectiveness of the monoclonal antibody. The anti-OKT3 sensitization was manifested by accelerated OKT3 clearance and abrupt reappearance of circulating OKT3+ cells before the end of treatment. This immune response was significantly delayed and reduced in its incidence and intensity in patients receiving low dose corticosteroids and azathioprine in association to OKT3; mainly IgM anti-OKT3 antibodies that did not accelerate OKT3 clearance were then observed. The fine specificity of the antibodies produced was studied, using patients whole sera and various mouse IgG2a-affinity chromatography-purified serum fractions. The results obtained showed that the anti-OKT3 response was remarkably restricted to two main categories of antibodies: a) anti-idiotypic antibodies that inhibited OKT3 binding to T cells and abrogated its therapeutic activity and b) anti-mouse IgG2a (anti-isotypic) antibodies that did not neutralize OKT3 immunosuppressive activity. These results suggest that OKT3-immunized patients might still be sensitive to the immunosuppressive effect of other anti-T cell monoclonals that do not share the OKT3 idiotype and possibly isotype.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.     

Neutrophil activation detected by monoclonal antibodies

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.