Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors

Previous studies indicated that Ca++ ionophores and phorbol esters in synergy could substitute for the initial activation step of normal T lymphocytes or T cell clones leading to increased expression of receptors for the growth factor interleukin 2 (IL 2) and secretion of interleukins, with the mitogenic signal for T cell proliferation being dependent on the presence of IL 2. In this study, the question was addressed as to whether T lymphocytes activated through the Ca++ ionophore ionomycin and the phorbol ester 12-o-tetradecanoyl phorbol 3-acetate (TPA) also acquired the competence to kill relevant target cells. The results indicate that T lymphocytes from primed mice proliferate and lyse the relevant allogeneic target cells after in vitro stimulation with ionomycin plus TPA, and that T lymphocyte preparations enriched for a subpopulation bearing the Lyt-2 marker are dependent on exogeneous sources of IL 2 to proliferate and become competent killer cells, whereas preparations enriched for subpopulations bearing the L3T4 marker grow independently of exogenous IL 2.     

Distinction between antigen receptor and IL 2 receptor triggering events in the activation of alloreactive T cell clones with calcium ionophore and phorbol ester

A previous study indicated that Ca++ ionophores in conjunction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) could induce normal T lymphocytes to express receptors for the T cell growth factor, interleukin 2 (IL 2), to secrete IL 2, and to proliferate (1). Here we used long-term alloreactive Lyt-2+ cytotoxic or T4+ "helper" T cell clones. In response to their specific alloantigen, all of the clones secreted IFN-gamma but only the T4+ clone secreted IL 2 and proliferated in response to the appropriate alloantigen in the absence of exogenous IL 2. The Ca++ ionophore ionomycin and TPA, used in conjunction, mimicked the effect of specific alloantigen on these T cell clones, i.e., they induced the secretion of IFN-gamma in all clones and the secretion of IL 2 in the T4+ clone. In the absence of exogenous IL 2, a proliferative response was induced only for the IL 2 secreting clone. Increased sensitivity to exogenous IL 2 for some T cell clones was also observed after either alloantigen or ionomycin and TPA treatment; this could be correlated with an increase in the expression of IL 2 receptors 6 hr after a pulse with ionomycin and TPA. These results suggest that, for a given T cell clone, activation of the Ca++ -dependent protein kinase c can replace the antigen-receptor triggering events leading to interleukin secretion and increased expression of IL 2 receptors but cannot substitute for the IL 2 dependent triggering of the IL 2 receptor.     

Activation signals in human lymphocytes: interleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C

The mitogenic activation of resting T lymphocytes involves two distinct cellular events, the synthesis of the ultimate mitogen interleukin 2 and the synthesis and expression of receptors for it. In order to get more detailed information on the mechanisms associated with these activating steps (the effects of different stimuli, leading to activation of protein kinase C were investigated in human lymphocytes). The anti-T-cell receptor (TCR) and anti-CD3 monoclonal antibodies (BMA 031 and BMA 030, respectively), as well as the combination of the phorbol ester, TPA, with a calcium ionophore-induced interleukin 2 synthesis and subsequent proliferation in human peripheral blood lymphocytes. Incubation of cells with synthetic diacylglycerols and calcium ionophores proved to be effective in expression of high affinity interleukin receptors, no detectable amounts of interleukin 2 were, however, synthetized. When diacylglycerols were, however, added repetitively, interleukin 2 was also produced. Both anti-TCR/CD3 antibodies and TPA or DiC8 caused activation and translocation of protein kinase C from the cytosol to the plasma membrane. Significant differences, however, were observed between the time kinetics of the translocation of the enzyme. In plasma membranes of TPA-stimulated cells activation of protein kinase C was detectable up to 4 hr. In contrast, the highest specific activity of protein kinase C was measured in the plasma membranes after 15 min of DiC8 addition to cells. Anti-CD3 monoclonal antibodies activated protein kinase C in a biphasic manner. Shortly after binding of BMA 030 to the T cell antigen receptor/CD3 complex the activity of protein kinase C was increased in the plasma membrane, then it declined to control levels followed by a second long-lasting activation of the enzyme up to 4 hr. These results suggest different signal requirements for different activation steps. While for synthesis and expression of interleukin 2 receptors a short term activation of protein kinase C is sufficient, long-term activation of the enzyme is necessary for interleukin 2 synthesis in human lymphocytes.     

Interleukin 2 responses of lpr and normal L3T4-/Lyt-2- T cells induced by TPA plus A23187

The major population of cells that accumulate abnormally in MRL/Mp-lpr/lpr lymphoid tissue is Thy-1+, L3T4-, and Lyt-2-. To clarify the functional potential of these cells, we examined their proliferation, interleukin 2 (IL 2) receptor expression, and IL 2 secretion by using as stimulants the combination of 12-O-tetradecanoylphorbol-2-acetate and A23187 (a calcium ionophore). Although the lpr T cells were capable of responding to these stimulants, the nature of the response and of the concentrations of ligand required differed sharply from the responses of normal adult T cells, and of adult L3T4-Lyt-2- thymocytes. There was a strong similarity but not identity when responses of 16 day fetal thymocytes were compared with those of lpr L3T4-Lyt-2- cells. The unusual functional properties of the lpr cells, such as high A23187 dose requirement for maximal proliferation, low percentage of IL 2 receptor-expressing cells, and low levels of IL 2 secretion, suggested that these cells are arrested at a stage of development similar to that of 16-day fetal thymocytes and before adult L3T4-/Lyt-2- thymocytes.     

Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells

The macrophage-derived product, interleukin 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of interleukin 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of 31 pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.     

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.     

Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes

Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells.     

Study of the biological activities of toxic shock syndrome toxin-1. I. Proliferative response and interleukin 2 production by T cells stimulated with the toxin

The mitogenic and interleukin 2 (IL 2) production-inducing effects of toxic shock syndrome toxin-1 (TSST-1) on murine lymphocytes were investigated. TSST-1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome. TSST-1 was mitogenic for splenic T cells and peanut agglutinin (PNA)-negative thymocytes, but not for T cell-depleted spleen cells, PNA-positive thymocytes or IL 2-dependent CTLL 2-cells. A factor mitogenic for CTCC-2 cells with a molecular weight of 30-35 kdaltons was obtained by stimulating spleen cells with TSST-1 and it was absorbed by CTLL-2 cells, indicating that the factor is IL 2. For substantial amounts of IL 2 to be produced, 10 ng or more of TSST-1 per ml and 48 hr or more of incubation were required. Removal of T cells abrogated the IL 2 production by spleen cells. T cells obtained by the nylon wool column method alone produced IL 2 on TSST-1 stimulation in the presence of either macrophages or a macrophage lysate containing interleukin 1. However, T cells obtained by a combination of the nylon wool column method and anti-Ia antibody treatment produced IL 2 in the presence of macrophages but not of the macrophage lysate, indicating that IL 2 production by TSST-1-stimulated T cells is absolutely dependent on the presence of accessory cells.     

Interleukin 4 drives phytohemagglutinin-activated T cells through several cell cycles: No synergism between interleukin 2 and interleukin 4

Cell kinetic studies of T cells stimulated with the interleukin 2 (Il-2), Il-4, or both lymphokines were performed with conventional [3H]thymidine incorporation and with the bivariate BrdU/Hoechst technique. Il-2 and Il-4 are able to drive phytohemagglutinin-activated T cells through more than one cell cycle. Neither synergistic nor inhibitory effect on T-cell proliferation was seen for the stimulation with both Il-2 and Il-4 as compared with the effect of Il-2 alone. The quantitative data of the cell cycle distribution of phytohemagglutinin-activated T cells suggest that the population of Il-4-responsive cells is at least an overlapping population, if not a real subset of the population of the Il-2-responsive cells.     

Comparison of phorbol ester/calcium ionophore and phytohemagglutinin-induced signaling in human T lymphocytes. Demonstration of interleukin 2-independent transferrin receptor gene expression

The proliferation of human T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T cell growth factor (interleukin 2 (IL-2), IL-2 receptors, and transferrin receptors (TFR). We examined the time course of accumulation of mRNA for these genes in T cells stimulated with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore, ionomycin, and compared their expression to T cells stimulated with phytohemagglutinin. In cells treated with PDB/ionomycin, maximum expression was observed at 3 hr for IL-2 mRNA and at 6 hr for TFR mRNA, whereas the level of IL-2 receptor mRNA reached a peak 24 to 48 hr after stimulation. In phytohemagglutinin-stimulated T cells IL-2 mRNA was detectable within 3 hr but peaked later at 12 hr; the level of IL-2 receptor mRNA similarly peaked 24 to 48 hr later. Accumulation of TFR mRNA in phytohemagglutinin-stimulated T cells, however, was not detectable at 6 hr and reached a peak only between 12 to 24 hr. The early accumulation of TFR mRNA in PDB/ionomycin-stimulated T cells seemed, in part, independent of the interaction of IL-2 with its own receptor, because TFR mRNA was detectable as early as 1 hr after stimulation and addition of cycloheximide before addition of PDB/ionomycin did not abolish the PDB/ionomycin-induced accumulation of TFR mRNA. In addition, either PDB or ionomycin used alone induced the expression of TFR mRNA but not IL-2 mRNA. These results indicated that the combination of PDB/ionomycin accelerated the expression of IL-2 and TFR genes in T cells compared to phytohemagglutinin and triggered an IL-2-independent pathway for the induction of TFR mRNA.     

Teleocidin and phorbol ester tumor promoters exert similar mitogenic effects on human lymphocytes

The tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) affects a wide variety of cellular functions via its binding to protein kinase C (PKC). The TPA molecule contains a diacylglycerol (DAG)-like structure, which may explain its ability to mimic DAG in PKC activation. Teleocidin (TCD) is a different tumor promoter which can compete with TPA in binding to its cell surface receptors even though structurally unrelated to TPA or DAG. Since TCD may use an additional receptor system and/or be distinguished from TPA in its effect on cells, we compared the effects of TPA and TCD on human peripheral blood lymphocytes (PBL). Both tumor promoters preferentially enhanced cell proliferation of sheep erythrocyte-rosetted lymphocytes, which were enriched for T cells. Additionally, TPA and TCD both induced a high density of cell surface receptors for interleukin 2 (IL2) and transferrin, but not synthesis or production of IL2. However, either of the tumor promoters synergized with T cell mitogens to induce high level IL2 production by PBL. In dose response and kinetic studies, matching concentrations of TPA and TCD induced similar effects in PBL. The results thus demonstrate that TPA and TCD are alike in mitogenic capacity, and suggest that structural similarity between the tumor promoter and DAG, the physiological activator of PKC, is not an essential property for promoting tumors or affecting a wide variety of cellular functions.     

Phorbol myristate acetate and in vitro T lymphocyte function. II. Influence of PMA and supernatants from PMA-treated P388D1 cells on the proliferation of cloned T cells

Neither the culture supernatants from P388D1 cells pulsed with phorbol myristate acetate (PMA), nor PMA itself in concentrations ranging from 10(-6) M to 10(-9) M, are directly mitogenic for murine T lymphocyte clones, yet both markedly augment the antigen-driven proliferation of many, but not all, cloned T lymphocytes. The component of PMA-induced P388D1 supernatant responsible for its co-mitogenic activity is probably PMA, rather than interleukin 1. For responsive clones, the co-mitogenic effect of PMA requires stimulator cells that display the specific allogeneic determinants recognized by the clones. This response persists after T cells are removed from the stimulating population, ruling out induction of mitogenic lymphokines from stimulator T cells by PMA as a primary mechanism for augmentation of clonal proliferation. Both splenocytes and thymocytes cooperate with PMA for enhanced clonal expansion, but heat treatment (45 degrees C, 45 min) of thymocytes destroys their cooperative capacity. PMA can also potentiate the lymphokine-driven proliferation of cloned T cells, indicating that PMA can, under certain conditions, influence T cell clonal expansion by a direct action on T lymphocytes.     

Changes in gene expression induced by a phorbol diester: expression of IL 2 receptor, T3, and T cell antigen receptor

Phorbol esters cause an apparent differentiation of human T leukemic cell lines. It was shown previously that TPA induces the expression of the interleukin 2 (IL 2) receptor and the T3 complex on some T cell lines, including CCRF-CEM. We demonstrate that expression of the IL 2 receptor correlated with an induction of the 3.5 and 1.5 kb IL 2 receptor mRNA. In addition, the TPA-induced expression of the T3 polypeptides was found to be accompanied by induction of a putative T cell antigen receptor heterodimer on CEM cells. This was demonstrated by the co-precipitation of the T cell receptor with T3 from digitonin-solubilized cells. The cells expressed high levels of T3 delta- and T cell receptor beta-chain mRNA in the absence of TPA. The effect of TPA was to cause a rapid accumulation of T cell receptor alpha-chain mRNA. This suggested that the alpha-chain gene was rearranged before TPA induction and that expression of the T cell receptor/T3 complex on the cell surface was regulated by the level of alpha-chain expression. It was also shown that cloned sublines of CEM cells which expressed different T cell antigen phenotypes differed in their response to TPA.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.     

Role of membrane potential in the response of human T lymphocytes to phytohemagglutinin

We have analyzed the role of membrane potential on T cell activation and cell proliferation. Depolarization of T lymphocytes, by increasing the extracellular concentration of K+ during a 1-hr exposure to PHA, results in a marked inhibition of cell proliferation. In parallel, depolarization of T cells prevented the normal increase in [Ca2+]i seen after PHA binding. In depolarized cells, PHA failed to induce IL 2 secretion, but, in contrast, IL 2 receptor expression was triggered normally and the cells were subsequently responsive to exogenous IL 2. Increasing [Ca2+]i in depolarized cells with the ionophore ionomycin, or bypassing the requirement for an increase in [Ca2+]i with TPA, restored the PHA-induced proliferative response in depolarized cells. These data confirm that a membrane potential-sensitive step, namely, Ca2+ influx and the resulting change in [Ca2+]i, is triggered by PHA. The inhibitory effects of depolarization are mediated through the impairment of IL 2 secretion, but not IL 2 receptor expression. T cell proliferation can therefore be regulated by altering membrane potential, which in turn modulates the extent of the change in [Ca2+]i. This study suggests a role for transmembrane potential in the regulation of the T cell proliferative response.     

5-Azacytidine induced inhibition of mitogenic response by agents that activate or augment activation of human peripheral blood T lymphocytes

Previous work from our laboratory demonstrated the mitogenic response of human peripheral blood T lymphocytes by phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, or interleukin 2 (IL-2). Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA could be responsible for the inhibiting effect of 5-azacytidine (5-aza-CR) on DNA methylation. In this study, we first demonstrated the inhibition of mitogenic response by agents, such as PHA, PMA and ionomycin, or IL-2, that activate or augment activation of human peripheral blood T cells by treatment of the analog 5-azacytidine. Over 1 microM of 5-azacytidine, we detected significant inhibition of proliferative response and over 5 microM of 5-azacytidine toxic effect of cell viability. We found no significant change of T cell subsets after treatment of 5-azacytidine.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. I. Functional characterization by monoclonal antibodies

Three rat monoclonal antibodies (MAb) capable of stimulating interleukin 2 (IL 2) production by a variant subline of EL4 thymoma cells (EL4-6.1) have been produced. The stimulatory capacity of these MAb (designated RL73, RL119, and RL388) was originally found to be dependent on the presence of irradiated peritoneal exudate cells; however, this requirement could be replaced by the cellfree supernatant of the "macrophage-like" cell line P388D1 or by biochemically purified human interleukin 1 (IL 1). A number of other rat MAb directed against cell surface structures did not stimulate IL 1-dependent IL 2 production by EL4-6.1 cells; however, certain MAb directed against Thy-1 as well as the lectin phytohemagglutin did have this capacity. Furthermore, the stimulatory activity of MAb RL73, RL119, and RL388 appeared to be restricted to the EL4-6.1 variant line, because neither the parental EL4 line from which it was derived nor a series of ovalbumin-specific T-T hybrids responded to these MAb. The cell surface antigens recognized by MAb RL73, RL119, and RL388 were present on a wide variety of T cell lines and T-T hybrids, as well as on lines of B cell, macrophage, and fibroblast origin. Interestingly, the MAb reacted with the majority (approximately 85%) of thymocytes but not (or only to a very small extent) with resting T lymphocytes. After stimulation by concanavalin A, however, the three MAb reacted strongly with activated T lymphoblasts. The latter data suggest that MAb RL73, RL119, and RL388 may react with cell surface structures that are normally expressed as a consequence of lymphocyte activation.     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.