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.     

Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin

To characterize the requirements for T cell proliferation, we have studied the response of purified populations of human T cells to varying concentrations of the mitogen phytohemagglutinin (PHA). Concentrations of PHA which induce optimal proliferative responses induce increases in cytosolic free calcium ([Ca2+]i), expression of interleukin 2 (IL 2) receptors, and production of IL 2. As the concentration of PHA is decreased, each of these processes decreases in parallel. At suboptimal concentrations of PHA, the addition of exogenous IL 2 reconstitutes both the proliferative response and the expression of the IL 2 receptor, as measured by immunofluorescence with antibodies directed against the TAC/IL 2 receptor molecule, but without reconstituting the increase in [Ca2+]i. Therefore, the concentration dependence of responses to PHA appears to be secondary to an absence of IL 2 production due to a failure to induce an increase in [Ca2+]i. The addition of the calcium ionophores A23187 and ionomycin or of accessory cells to low concentrations of PHA induces increases in [Ca2+]i and subsequent proliferative responses, suggesting that the two events are linked. The proliferative response can be inhibited by antibodies directed towards IL 2 or the IL 2 receptor, indicating that the proliferative response was at least partially dependent on the production and action of IL 2. This suggests that, although increases in [Ca2+]i are an integral event in the induction of proliferation by PHA, the increase in [Ca2+]i is required for the production but not the action of IL 2. In addition, low concentrations of PHA deliver an additional signal to cells, independent of an increase in [Ca2+]i, which induces IL 2 receptor expression and allows a proliferative response in the presence of exogenous IL 2.     

Platelet-activating factor (PAF) receptor antagonists inhibit mitogen-induced human peripheral blood T-cell proliferation

The addition of L-652,731 and L-653,150, two synthetic PAF-specific receptor antagonists, to 72 hour cultures of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear leukocytes (PBML) caused a dose-dependent inhibition of (3H)-thymidine incorporation into T-cells (IC50: 25 microM and 3.2 microM, respectively). This inhibition was not reversed by exogenous interleukin (IL)-1 and IL-2. PAF receptor antagonists did not affect the expression of IL-2 receptors (TAC-antigen) on T-cells. Exogenous PAF which by itself had no significant effect on PHA-stimulated PBML proliferation, only partially reversed the inhibition of proliferation caused by PAF receptor antagonists. These results may suggest the involvement of endogenously produced PAF in the regulation of immune reactions.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.     

Interrelationship between signals transduced by phytohemagglutinin and interleukin 1.

In the murine cell line LBRM-331A5, phytohemagglutinin (PHA) induces secretion of the T cell growth factor interleukin 2 (IL2). IL1 augments PHA-induced IL2 production. In this cell line, PHA stimulates a number of biochemical changes including phospholipid hydrolysis, increases in cytosolic free calcium [( Ca2+]i), membrane hyperpolarization, cytosolic alkalinization, and tyrosine phosphorylation of specific substrates. Using LBRM cells, we have studied the interrelationship between these events and the secretion of IL2. Increases in [Ca2+]i triggered by PHA or following addition of ionomycin result in membrane hyperpolarization but are not required for PHA-induced cytosolic alkalinization or tyrosine phosphorylation. Addition of IL1 to PHA-stimulated cells did not affect any of the biochemical parameters, although it significantly augmented PHA-induced IL2 secretion. Increasing [Ca2+]i with ionomycin did not trigger IL2 secretion, increases in cytosolic pH, or tyrosine phosphorylation in the presence or absence of IL1. Preventing increases in cytosolic pH did not alter PHA-induced changes in [Ca2+]i or membrane potential. These data are compatible with PHA including activation of phospholipase C and production of inositol phosphates resulting in both release of Ca2+ from internal stores and transmembrane uptake of Ca2+ as well as activation of protein kinase C. However, unlike other growth factor or mitogen-stimulated systems, the changes stimulated by PHA and IL1 in LBRM cells including IL2 secretion are not regulated by a pertussis toxin-sensitive G protein.     

Cell matrix adhesion-related proteins VLA-1 and VLA-2: regulation of expression on T cells

The mitogens phytohemagglutinin (PHA) and concanavalin A inhibited the appearance of the very late activation antigen (VLA)-1, but did not inhibit VLA-2 expression on cultured activated T cells. In contrast to diminished VLA-1 expression, mitogen treatment caused increased cell surface expression of other activation antigens such as T10, HLA-DR, interleukin 2 (IL 2) receptor, and 4F2, and greater cell proliferation. Conversely, when T cells were not repetitively restimulated with mitogen, these less proliferative "postactivated" T cells had elevated VLA-1 expression. The diminished expression of VLA-1 caused by PHA was reversible since subsequent removal of mitogen was associated with increased VLA-1, paralleled by a decrease in interleukin 2 receptor levels. In addition to preventing or delaying the initial appearance of VLA-1, PHA stimulation also was somewhat effective in causing the disappearance of VLA-1 already present, especially on recently established cultures. However, cultures that had either never seen PHA, not seen PHA for several weeks, or been stimulated regularly with PHA, but were several months old, did not lose VLA-1 in response to PHA stimulation, suggesting that a state of insensitivity to PHA effects could be attained. Unlike PHA-stimulated T cells, T cells repetitively restimulated with alloantigen or the monoclonal antibody T3 did not show a marked absence of VLA-1 but rather showed an increased level of VLA-2 relative to VLA-1. Taken together, results of stimulation by either mitogen, alloantigen, or anti-T3 monoclonal antibody support the conclusion that T cell stimulation in general can cause a decreased VLA-1:VLA-2 ratio, whether by decreased VLA-1 or increased VLA-2. These shifts in VLA-1:VLA-2 ratios are probably not simply the result of shifts in the relative proportions of different subpopulations, because similar growth-related changes in this ratio were observed on the T cell line ANITA, which is a homogeneous population of cells. Because both VLA-1 and VLA-2 are differentially regulated on cultured, long term activated T cells depending on stage of activation and growth conditions, and are members of a family of at least five heterodimers that includes cell matrix adhesion molecules, we suggest that these studies will provide clues to novel aspects of T cell growth regulation, perhaps relating to T cell-matrix adhesion.     

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.     

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.     

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.     

Effect of wheat germ agglutinin on T lymphocyte activation

Wheat germ agglutinin (WGA) is low mitogenic or nonmitogenic for human T lymphocytes and inhibits phytohemagglutinin (PHA)-induced mitotic response of the lymphocytes. In this study, the effect of WGA was analyzed in terms of interleukin 2 (IL2) production, expression of IL2 receptor, and IL2 responsiveness of the T lymphocytes. WGA as well as PHA could induce IL2 mRNA and IL2 production and also elevate cytoplasmic free Ca2+ concentration. The IL2 production was reduced by inhibitors of calmodulin and protein kinase C. The IL2 receptor (Tac) expression was induced at about 20% of the lymphocytes by WGA and the expression induced by PHA was not blocked by the addition of WGA. The lymphocytes precultured with WGA for 3 days could proliferate by the addition of IL2 after removal of WGA. The IL2-dependent proliferation of PHA-blasts was blocked by the addition of WGA. These results indicate that WGA inhibits T lymphocyte proliferation by inhibiting the responsiveness of the lymphocytes to IL2 but not by interfering with IL2 production and IL2 receptor expression.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

Transmembrane signaling during interleukin 1-dependent T cell activation. Interactions of signal 1- and signal 2-type mediators with the phosphoinositide-dependent signal transduction mechanism

The murine T lymphoma line, LBRM-33 1A5, requires synergistic signals delivered by phytohemagglutinin (PHA) and interleukin 1 (IL1) for activation to high level interleukin 2 production. The activation signals provided by PHA and IL1 were replaced by the Ca2+ ionophore, ionomycin, and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), respectively. These observations supported a two-signal model for T cell activation involving increases in intracellular Ca2+ concentration ([Ca2+]i) (signal 1) and activation of protein kinase C (signal 2) as necessary and sufficient events. However, biochemical analyses revealed that additional signals were involved in the activation of LBRM-33 cells by both receptor-dependent and -independent mediators. Both signal 1-type mediators, PHA and ionomycin, exerted pleiotropic effects at the concentrations required for synergy with signal 2-type mediators (IL1, TPA). Within 1-2 min of addition, PHA stimulated phospholipid turnover, including hydrolysis of phosphatidylinositol 4,5-bisphosphate, Ca2+ mobilization, and protein kinase C activation. The [Ca2+]i increase induced by PHA was due to influx from both intracellular and extracellular Ca2+ pools. Similarly, ionomycin increased phospholipid turnover, [Ca2+]i, and directly affected protein kinase C activity in LBRM-33 cells. In contrast, the signal 2-type mediators, TPA and IL1, appeared to act by distinct intracellular mechanisms. TPA induced a protracted association of cellular protein kinase C with the plasma membrane, consistent with the two-signal activation model. Furthermore, acute TPA treatment inhibited PHA-stimulated inositol phosphate release and Ca2+ mobilization, suggesting that this mediator partially antagonized signal 1 delivery. IL1, in contrast, neither activated protein kinase C directly nor did it positively modulate the coupling of signal 1-type mediators to [Ca2+]i or protein kinase C via the phosphoinositide pathway. The intracellular signal delivered by IL1 is, therefore, generated through a mechanism distinct from or distal to the activation of protein kinase C. These studies indicate that the two-signal hypothesis, in its simplest form, is inadequate to explain the signals required for the initiation of IL1-dependent T cell activation.     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

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.     

2'Deoxycoformycin and deoxyadenosine affect IL 2 production and IL 2 receptor expression of human T cells

Congenital deficiency of the enzyme adenosine deaminase (ADA) leads to severe combined immunodeficiency. 2'Deoxycoformycin (dCF), a tightly binding inhibitor of ADA, can induce the metabolic state of ADA deficiency. In vivo, the drug causes specific impairment of lymphocyte function and shows strong immunosuppressive properties. However, to decide whether inhibition of the enzyme ADA offers an attractive approach for immunosuppressive therapy, more information is needed about the immunologic mechanisms affected. In human T cells, we investigated the effect of dCF and deoxyadenosine (AdR) on cell activation, interleukin 2 (IL 2) production, and IL 2 receptor induction after allogeneic and lectin-induced stimulation. After allogeneic stimulation, dCF and AdR affected several events in T cellular immune response. Early events in T cell activation showed to be most sensitive to the drugs. Primary MLC was completely inhibited by concentrations as low as 1 microM dCF and 1 microM AdR. The addition of human recombinant IL 2 (rIL 2) could not abrogate the inhibitory effect of the drugs. Apart from activation of T cells, the drugs interfered with proliferation of activated T cells. Two events in activated T cells were affected: IL 2 production and IL 2 receptor expression. In secondary MLC, IL 2 production was markedly reduced in the presence of 9 microM dCF and 60 microM AdR. These concentrations appeared also to affect IL 2 receptor expression in 12-day primary MLC cells stimulated with rIL 2. Lectin stimulation was also affected by the drugs. In phytohemagglutinin (PHA)-stimulated cultures, 9 microM dCF and 60 microM AdR resulted in inhibition of proliferation and IL 2 receptor expression, whereas IL 2 production was normal. It is concluded that dCF and AdR interfere with several events in T cellular immune response such as cell activation, IL 2 production, and IL 2 receptor expression. According to these results, inhibition of the enzyme ADA seems an attractive approach to immunosuppressive therapy.     

Beta-endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes

Lymphocytes stimulated by mitogens or antigens exhibit an enhanced calcium uptake early in the proliferation or activation response. Modulation of this calcium uptake results in alterations of proliferation and immunocompetence. beta-endorphin and other opioids affect several parameters of lymphocyte competence. Limited data are available concerning the mechanism(s) of these effects. This study examines whether a possible opioid mechanism is the modification of the early calcium influx into stimulated lymphocytes. The time course of both concanavalin A (Con A) and phytohemagglutinin (PHA)-stimulated 45Ca2+ uptake into thymocytes was characterized to determine the optimal time for testing the effects of opioids. beta-Endorphin 1-31 significantly enhanced Con A-stimulated 45Ca2+ uptake into rat thymocytes. This peptide had no significant effect on PHA-stimulated 45Ca2+ uptake or on basal thymocyte 45Ca2+ flux. The beta h-endorphin stimulatory effect was titratable in the range of 0.1 nM to 10 microM. Naloxone did not reverse the enhancement. Met-enkephalinamide and other opioid agonists did not duplicate the stimulatory effect. Thus, the beta h-endorphin 1-31 enhancement of Con A-stimulated 45Ca2+ uptake by rat thymocytes does not operate via classical opioid receptor mechanisms. beta h-endorphin 1-31 appears to be acting on a subset of T cells that are responsive to Con A but not to PHA.     

Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms

Human mesenchymal stem cells (MSCs) have immuno-modulatory properties. They inhibit T-cell proliferation to mitogens and alloantigens in vitro and prolong skin graft survival in vivo. We found that MSCs inhibited the proliferation of peripheral blood lymphocytes (PBLs) to phorbol myristate acetate (PMA), suggesting that MSCs exert an inhibitory effect downstream of the receptor level. We analyzed cytokine profiles of PBLs co-cultured with MSCs. MSCs increased interleukin (IL)-2 and soluble IL-2 receptor in mixed lymphocyte cultures (MLCs), while IL-2 and IL-2R decreased in phytohemagglutinin (PHA)-stimulated PBL cultures. MSCs inhibited IL-2 induced proliferation, without absorbing IL-2. IL-10 levels increased in MLCs co-cultured with 10% MSCs, while the levels were not affected in PHA cultures. In MLCs inhibited by MSCs, antibodies against IL-10 further suppressed proliferation but had no effect in PHA cultures. Addition of indomethacin, an inhibitor of prostaglandin-synthesis, restored part of the inhibition by MSCs in PHA cultures. However, indomethacin did not affect MSC-induced inhibition in MLCs. To conclude, our data indicate that MSC-induced suppression is a complex mechanism affecting IL-2 and IL-10 signaling and may function differently, depending on T-cell stimuli. Prostaglandins are important in the inhibition by MSCs when the T cells were activated by PHA, but not alloantigens.     

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.