Activation requirements for normal T cells: accessory cell-dependent and -independent stimulation by anti-receptor antibodies

We have examined the requirements for the activation of normal T cells by two anti-T cell receptor antibody preparations, including a rabbit antiserum, R3497, which binds to all normal T cells, and a rat monoclonal antibody, KJ16-133, which binds to about 20% of T cells. The requirements for stimulation of T cells by both antibodies were similar. Soluble antibodies in the absence of accessory cells (AC) failed to induce either proliferation or the expression of IL 2 receptors, and the addition of either IL 2 or PMA failed to synergize with these soluble antibodies for an AC-independent proliferative response. Activation could only be achieved in the presence of Fc receptor-positive AC, although Fc receptor expression alone appeared not to be sufficient for AC activity because some Fc receptor-positive cells did not function in this capacity. Activation with anti-receptor antibody conjugated to Sepharose 4B beads could be demonstrated in the presence of some exogenous cofactors, such as IL 2 and PMA, but not in the presence of recombinant IL 1. When activation by soluble antibody plus AC was compared to activation by bead-conjugated antibody + recombinant IL 2, it was found that the former favored the stimulation of Lyt-2+ cells. The effects of the addition of anti-L3T4 monoclonal antibody was also examined in this system. Anti-L3T4 inhibited the response of L3T4+ cells when used in the presence of Ia+ as well as Ia- AC, and it also inhibited activation in a system in which KJ16-133 conjugated to Sepharose was used in the absence of AC. Because anti-L3T4 had an inhibitory effect in the presence of Ia- AC as well as in the absence of any AC, it is concluded that L3T4 does not necessarily function by interacting with Ia on the surface of AC, and may directly transmit down-regulatory signals when bound by anti-L3T4.     

Accessory cell function in the Con A response: role of Ia-positive and Ia-negative accessory cells

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing interleukin 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to 27%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed-type hypersensitivity

The in vivo effects of monoclonal GK1.5 antibody, directed against the L3T4a determinant expressed on Class II-restricted T cells, on the induction and expression of murine delayed-type hypersensitivity (DTH) responses were examined. Development and expression of both hapten (2,4-dinitrofluorobenzene and 2,4,6-trinitrochlorobenzene)- and protein antigen poly(Glu60Ala30Tyr10)-specific DTH are significantly inhibited by injection of monoclonal anti-L3T4a antibody. The inhibitory effects of anti-L3T4a were most pronounced when administered during the afferent (induction) phase of the DTH response, leading to the functional inhibition of the generation of both polyclonal lymph node T-proliferative cells (Tprlf) and DTH effector cells (TDH). The in vivo inhibitory effect is apparently unrelated to preferential induction of suppressor T cells as GK1.5 inhibited DTH induction in cyclophosphamide-treated as well as normal recipients. L3T4a expression on the various T-cell subsets involved in DTH induction and elicitation was also examined. The data show that three functionally distinct, antigen-specific T-cell subsets, Tprlf, TDH, and Th cells involved in DTH induction, bear the Lyt 1+2-, L3T4+ phenotype. Possible mechanisms where in vivo injection of anti-L3T4a inhibits Class II-restricted T-cell subsets involved in DTH induction and expression, including immune depletion and inhibition of T-cell-receptor/ligand interactions, are discussed.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

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.     

L3T4+ T cells regulate Abelson virus-induced lymphomagenesis

To evaluate the role of T cells in regulation of lymphomagenesis, experiments were performed using Abelson murine leukemia virus (AMuLV). In vitro transformation of bone marrow target cells by this B lymphotropic retrovirus was inhibited by peripheral lymph node cells from naive mice. The inhibitory activity depended on Thy-1+ L3T4+ cells but did not require Lyt-2+ cells. In vivo depletion of L3T4+ T cells with a mAb (GK1.5) altered the course of AMuLV-induced lymphoma. L3T4 depletion of naturally resistant C57BL/6 mice resulted in dramatic susceptibility to lymphoma induction. Lymphoma cells from anti-L3T4-treated C57BL/6 mice infected with AMuLV displayed the B lineage transformation marker P1606C3. These studies reveal an important immunologic component of Abelson disease resistance involving L3T4+ T cells.     

Selective activation of helper and cytolytic T-cell functions of L3T4+ clones with either antireceptor antibody or phorbol ester and ionophore

After activation with specific antigen and antigen presenting cells (APC) L3T4+ inducer T-cell clones can lyse Ia+ APC. The present study characterizes the mechanism of activation and specificity of L3T4+ inducer cell-mediated cytolytic function. Two methods that bypass the physiological stimulus of antigen presented on Ia+ APC were used to activate L3T4+ clones. The first method utilized an antireceptor monoclonal antibody (MAb), KJ16.133, to activate KJ16.133+ clones. The activated clones expressed nonspecific cytolytic activity, killing target cells irrespective of their H-2 haplotype or their ability to express cell surface Ia molecules. The crosslinking of bound KJ16.133 antibody greatly enhanced cytolytic activity. This activation is receptor specific because KJ16.133- clones were not activated under identical conditions. The second method of activation was provided by a synergistic action of phorbol-12-myristate-13-acetate (PMA) and ionophore A23187. These agents nonspecifically activated all L3T4+ clones tested. The simultaneous presence of the two agents is required for maximal activation. Again, the activated clones expressed potent nonspecific cytolytic activity. These observations demonstrated that L3T4+ inducer T-cell-mediated killing can be separated into two stages: an activation step, which can be specifically and nonspecifically triggered and an effector phase which causes nonspecific lysis of bystander targets. The induction of nonspecific cytolytic activity by antireceptor MAb was inhibited by anti-L3T4 MAb (GK1.5). In contrast, activation of nonspecific cytolytic activity by treatment with PMA plus A23187 was not inhibited by anti-L3T4 MAb. Under the above activation conditions, antireceptor MAb selectively induced the secretion of IL-3 and expression of nonspecific cytolytic activity. However, there was little or no concomitant proliferation and production of IL-2. In contrast, activation by PMA plus A23187 coordinately induces expression of nonspecific cytolytic activity, secretion of lymphokines (IL-3 and IL-2), and cell proliferation. Thus, the anticlonotypic activation preferentially induces certain functions whereas activation with PMA plus A23187 is not selective.     

Sodium periodate-induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1

T lymphocytes oxidized with the mitogen sodium periodate undergo a proliferative response when cultured in the presence of Ia+ accessory cells. However, the exact role(s) the accessory cells play in such a response has not been clearly defined. We have evaluated the role of Ia and the requirement for interleukin 1 (IL 1) in periodate mitogenicity by using the Ia+ cloned tumor cell lines P388AD (Ia+, IL 1 inducible) and P388NA (Ia+, IL 1 noninducible) as accessory cells. P388AD but not P388NA was able to supply accessory cell function to periodate-treated T cells, suggesting that Ia expression alone was not sufficient to reconstitute a response. Monoclonal anti-I-Ad and anti-I-Ed antibody blocked the accessory cell function of P388AD. In addition, monoclonal antibody GK 1.5, directed against the T cell determinant L3T4a, blocked the P388AD/periodate-treated T cell interaction, confirming that this interaction was restricted by class II molecules. Although Ia expression was required, the response was not major histocompatibility complex (MHC) restricted, because allogeneic as well as syngeneic macrophages were capable of supplying accessory cell function to periodate-treated T cells. Exogenous IL 1 alone was able to trigger periodate-treated T cells, suggesting that Ia was required for the induction of IL 1 synthesis by the accessory cells. Furthermore, purified IL 2, devoid of IL 1 activity, was able to fully reconstitute the proliferative response of accessory cell-depleted oxidized T cells to a level equal to that of whole spleen accessory cells or P388AD. These data suggest that periodate-treated T cells can proliferate with IL 1 alone and that Ia+ accessory cells in periodate-mediated T cell mitogenicity may function in the release of IL 1 and the induction of IL 2 synthesis by the T cells.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

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.     

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.     

IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function.

IL-10, a newly designated cytokine primarily produced by the Th2 subset of CD4+ T lymphocytes and Ly-1+ B lymphocytes, has recently been hypothesized to inhibit cytokine production by Th1 T cell clones by blocking accessory cell- (AC) dependent costimulatory function. To evaluate the effect of IL-10 on Con A-induced proliferative responses of resting murine T cells, purified T cells were cultured with different types of AC. The addition of IL-10 produced a 70 to 90% inhibition of resting T lymphocyte proliferation when purified populations of macrophages were used as AC, but had no effect on the AC function of T-depleted spleen cells, activated B cells, dendritic cells, or L cells. The inhibitory effects of IL-10 were inversely related to the concentration of mitogen and could be reversed by the addition of the neutralizing anti-IL-10 mAb, SXC1. The inhibition of macrophage AC function was not related to the induction of a suppressor cytokine as stimulation by mixtures of macrophages and limiting numbers of dendritic cells was not inhibited. The decrease in proliferative responses was primarily secondary to inhibition of IL-2 production although the failure of exogenous IL-2 to completely reconstitute the response suggested that IL-10 may also exert inhibitory effects on the induction of expression of a functional IL-2R. These results are most consistent with a model in which IL-10 inhibits the induction of expression on macrophages of a critical costimulatory molecule that may be constitutively expressed on other types of AC.     

Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4) was hapten-specific (anti-ABA + Iak), the other (4.35F2) was alloreactive (anti-Iak). Activation of these clones by antigen, concanavalin A (Con A) or by the F23.1 antibody was studied by assaying the production of interleukin 3 (IL 3). Both soluble and solid phase-coupled F23.1 induced T cell activation in the complete absence of class II MHC, immobilized antibody (either Sepharose-coupled or plastic-adsorbed) being more effective. The induction of IL 3 production by suboptimal doses of either Con A or plastic-adsorbed F23.1 was inhibited by the anti-L3T4 antibody GK1.5, as was the response to F23.1 coupled to Sepharose-4B beads. However, the responses to optimal or superoptimal doses of these stimuli were not inhibited. In contrast, weak responses to non-TCR cross-linking stimuli such as phorbol myristate acetate (PMA) or low concentrations of soluble F23.1 were not inhibited by GK1.5 (the latter response was usually slightly enhanced). These results show that anti-L3T4 antibodies are not inherently inhibitory, but require both ligation and cross-linking of the TCR for their effect. We propose a model whereby L3T4 interacts with the TCR during T cell activation. Anti-L3T4 antibodies sterically hinder the formation of TCR complexes and so prevent activation. However, by increasing the epitope density of the activating ligand, the avidity of the T cell/ligand interaction can be increased sufficiently to prevent this disruption.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of Lyt-2+ (CD8+) and L3T4+ (CD4+) T cell subsets by anti-receptor antibody

The mAb F23.1, specific for V beta 8-related determinants on the TCR, was used to study the requirements for TCR cross-linking and for accessory cells (AC) in the induction of proliferation or IL-2 responsiveness in L3T4+ (CD4+) and Lyt-2+ (CD8+) T cells. T cells were exposed in vitro to soluble native F23.1 antibody, to heteroconjugates composed of the Fab fragments of F23.1 linked to Fab fragments of antibodies specific for Ia determinants on AC, or to F23.1 immobilized on an insoluble matrix. Soluble F23.1 antibody-induced proliferation in naive T cells only in the presence of both AC and exogenous IL-2, and these responses were confined to Lyt-2+ T cells. In contrast, heteroconjugates capable of crosslinking F23.1+ TCR to AC surface Ia determinants were capable of inducing proliferation in both L3T4+ and Lyt-2+ T cells in the absence of added lymphokine. Moreover, binding to and presumably multi-valent crosslinking of the TCR by immobilized F23.1 was sufficient to induce proliferation in both Lyt-2+ and L3T4+ T cells in the absence of AC or exogenous IL-2. Further, it was found that the conditions necessary for T cell growth factor secretion paralleled closely those required for induction of T cell proliferation in the absence of added lymphokine, suggesting that production of endogenous lymphokine might be the limiting process for triggering of T cell proliferation. Taken together, these findings suggest that under optimal conditions of TCR cross-linking, TCR occupancy and cross-linking is sufficient to deliver all of the signals necessary to initiate proliferation in naive populations of both L3T4+ and Lyt-2+ T cells. However, when conditions for TCR signaling are suboptimal, as may be the case for normal Ag-mediated stimulation, a role for second signals delivered by AC or exogenous lymphokines can become critical for T cell activation.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

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.     

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.