Immunoregulation through CD8 (Ly-2): state of aggregation with the alpha/beta/CD3 T cell receptor controls interleukin 2-dependent T cell growth

The activation of T lymphocytes by an antigenic challenge requires that the CD3 T cell receptor alpha/beta (TcR) is bound to an appropriate ligand, i.e. major histocompatibility complex antigen, on an antigen-presenting cell. In addition, numerous studies suggest that the accessory molecules CD4 and CD8 participate in the recognition process, and may have inhibitory as well as augmenting effects depending on the ways in which they participate. In the present study we attempt to define the conditions by which CD8 exerts enhancing and inhibitory effects on resting CD8+ T cell activation, and which parameters of activation are regulated through participation of CD8/CD4. We find that experimental procedures leading to TcR-CD8 aggregation induce T cell activation whereas experimental procedures preventing TcR-CD8 aggregation inhibit T cell activation. CD8/CD4-induced variations in the extent of T cell activation are apparent as variations in interleukin 2 (IL 2)-dependent growth and in the number of blastoid cells bearing IL 2 receptors. Inhibition of CD8+ T cell activation is successful only if the majority, if not all CD8 molecules are occupied by soluble antibody. This latter finding argues against the suggestion of other groups that CD8 may be a receptor for negative signaling. Rather, the results support the alternative notion that a basal level of TcR-CD8 aggregation, existing in the resting state or induced by TcR-ligand interaction, is an essential prerequisite for CD8+ T cell activation. Enhancement or depression of this basal level of aggregation causes facilitation or inhibition, respectively, of activation. This may be a mechanism for the regulation of IL 2-dependent clonal expansion of T cells in immune responses.     

Impaired T cell signal transduction through CD28 in a patient with idiopathic thrombocytopenia.

We describe an infant whose peripheral blood mononuclear cells were unable to proliferate or synthesize IL-2 in response to a mitogenic combination of antibodies directed against CD2 and CD28. This peculiar defect, which has been stable to date, was attributed to an impairment in CD28-mediated T cell activation, because further comitogenic combinations containing anti-CD28 monoclonals also failed to induce normal proliferation of the patient's T cells. In contrast, proliferation after membrane stimulation (with anti-CD2, recombinant IL-2, or certain lectins) or transmembrane activation (with phorbol ester and calcium ionophore) was normal, suggesting that his lymphocytes did not have a general membrane or intracellular signalling impairment. A T cell line derived from the patient confirmed the existence of a severe defect in CD28-mediated T cell proliferation, but also showed a profound impairment in CD3-induced T cell proliferation. Other cell surface molecules like CD2 and CD25 were, in contrast, capable of transducing normal proliferation signals. As all relevant molecules were detectable by cytofluorography and immunoprecipitation, we conclude that the patient's lymphocytes had an intrinsic defect in the delivery of CD28-mediated signals which, in the absence of monocytes, also affected CD3-mediated proliferation. The study of this novel kind of immunodeficiency may help to unravel the complex interactions that take place among CD2, CD3 and CD28 during T cell activation. The presence of an idiopathic thrombocytopenia in the patient suggests the intriguing possibility of a role for CD28 in the maintenance of peripheral blood platelets levels, although alternative interpretations are not ruled out.     

CD45 phosphotyrosine phosphatase and p56lck protein tyrosine kinase: a functional complex crucial in T cell signal transduction.

Tyrosine phosphorylation of several intracellular proteins is observed very early during T cell activation. p56lck, a non-receptor src-like protein tyrosine kinase (PTK) which is associated with the intracellular domains of CD4 and CD8 co-receptors, has been implicated in these early signal transduction events. Furthermore, recent experiments indicate that the receptor phosphotyrosine phosphatase, CD45, might be important in the regulation of p56lck PTK activity and that its expression is required for the generation of second messenger molecules following TCR triggering. Here, using co-capping experiments and double indirect immunofluorescence microscopy in functional human T lymphocytes, a specific co-distribution of a significant fraction of p56lck with CD45, but not with several other cell surface proteins, has been revealed. This is the first demonstration of a physical interaction between a receptor phosphotyrosine phosphatase and a PTK under physiologically relevant conditions. In addition, after antibody-induced capping of CD4, both a co-localization of p56lck and CD4, and concomitantly a significant increase in intracellular phosphotyrosine at the sites of CD4 caps were observed. In strong contrast to these results, co-clustering of CD4 with CD45 did not result in any detectable intracellular phosphotyrosine at the cap sites. These data indicate that CD45 can act on CD4-associated phosphoproteins in viable human T lymphocytes. Further, this provides evidence that p56lck PTK is a substrate of CD45 phosphotyrosine phosphatase in vivo and thereby supports the idea that CD45 is an early regulator of T cell activation involved in the modulation of the coupling of receptor-triggered events to intracellular signalling pathways.     

T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C-independent staurosporine-sensitive step.

The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of gamma and epsilon subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 gamma and epsilon and also of zeta, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.     

Signals involved in T cell activation. T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies

Monoclonal antibodies (mAb) directed against the T cell differentiation antigen CD28 (Tp44) induce proliferation of resting T lymphocytes in the presence of phorbol esters. Moreover, it has been reported that such antibodies augment and sustain T cell proliferation induced by soluble antigens, phytohemagglutinin and anti-CD3 mAb. Recently, we have shown that in monocyte-depleted T cell suspensions, anti-CD28 mAb 9.3 and Kolt-2 were able to circumvent the requirement for interleukin 2(IL2) in T cell proliferation induced by soluble anti-CD3 antibodies. Apart from the synergy of anti-CD28 antibodies with phorbol myristate acetate and anti-CD3 antibodies, we found that anti-CD28 mAb were able to induce T cell mitogenesis in combination with an E rosette-blocking anti-CD2 antibody. In this report, we show that antibodies directed against different epitopes on the CD2 antigen can synergize with anti-CD28 mAb. Furthermore, we demonstrate that proliferation induced through the synergistic action of anti-CD28 mAb with anti-CD2 antibodies can be induced in the absence of accessory cells and is accompanied by the production of IL2 and the expression of IL2 receptors. We were unable to induce detectable Ca2+ mobilization through the simultaneous binding of anti-CD28 and anti-CD2 mAb. Taken together, these data show that IL2-dependent proliferation can be induced through the simultaneous binding of anti-CD28 and anti-CD2 antibodies, possibly through phosphatidyl inositol-independent pathways. The observations may provide further insight into the activation mechanisms of human T cells.     

Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli.

To investigate the transforming potential of hepatitis C virus (HCV), HCV core protein was produced in BALB/3T3 A31-I-1 cells. The cells expressing HCV core gene cooperatively with the v-H-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent and serum-independent growth. The cells producing HCV core protein showed enhanced growth against stimulus of growth factor. In addition, antisense oligodeoxynucleotides against mRNA encoding HCV core protein suppressed the growth of HCV core-producing cells. Furthermore, HCV core protein activated mitogen-activated protein kinase and serum response element, which respond to growth stimuli. From these results, we concluded that HCV core protein is involved in the acquisition of cell growth advantage.     

Suppression of T cell responses through competition for T cell growth factor (interleukin 2)

Addition of activated T cells or of cells from a cytotoxic T cell clone to mixed leukocyte cultures abolished the generation of cytolytic activity in these cultures. The cytolytic T cell response is completely restored if T cell growth factor (interleukin 2) is added, indicating that the suppression is due to absorption or consumption of IL2 by the added cells.     

Identification of the intracytoplasmic region essential for signal transduction through a B cell activation molecule, CD40.

CD40 is a 45-kDa glycoprotein expressed on human B lineage cells. Anti-CD40 induces the proliferation of B cells and the extracellular region of CD40 is related to those of a certain kind of growth factor receptors. Therefore, it has been proposed that CD40 might be a receptor for a molecule involved in the growth regulation of B cells. The cDNA coding for CD40 was transfected into the murine B lymphoma cell line M12 and the murine thymoma cell line EL4. The growth of both M12 and EL4 transfectants expressing human CD40 was inhibited by anti-CD40. Phorbol 12-myristate 13-acetate (PMA) augmented the growth inhibitory effects of anti-CD40 on transfectants. The CD40 molecule was constitutively phosphorylated not only in human tonsil B cells but also in transfectants expressing CD40. PMA augmented the phosphorylation of CD40 in these cells. These results indicate that in spite of the growth inhibitory effect of anti-CD40, the augmentative effect of PMA is conserved in CD40+ transfectants and suggest that the transfectant might be useful for the study of signal transduction mechanism through CD40. To investigate which part of the CD40 molecule is important for signal transduction, transfectants expressing mutant CD40 cDNA were established and their growth response to anti-CD40 was evaluated. The mutant molecule, which had an Ala for Thr substitution at position 234, and the deletion mutants lacking Thr234 were inactive in growth signal transduction, indicating that Thr234 itself or the region around Thr234 is essential for signal transduction through CD40.     

Multiple signal transduction pathways activated through the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.     

T cell activation via the CD2 molecule is associated with protein kinase C translocation from the cytosol to the plasma membrane

T cell activation via the CD2 molecule involves phospholipase C and phosphoinositide hydrolysis. Here we demonstrate that the triggering of subclones of the human T leukemia Jurkat cell line by anti-CD2 as well as anti-CD3 monoclonal antibodies is able to induce activation (i.e. translocation from cytosol to cell membrane) of protein kinase C (PKC), which is dependent on the formation of 1,2-diacylglycerol from inositol 4-5-bisphosphate. The kinetics of PKC translocation parallels the rise in intracellular calcium following both CD2 and CD3 stimulations. These results further demonstrate that CD2 and CD3 activation pathways use similar signal transduction mechanisms.     

Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme,protein kinase C-β

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC-α was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC-α proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC-β was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC-β. Neither the phorbol ester-induced direct activation of PKC nor the specific activity of the plasma membrane-bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC-β was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration-dependent manner, the activation of lysophosphatid acyltransferase-catalyzed elevated incorporation of cis-polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin-2 (IL-2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030- or BMA 031-stimulated cells, expression of high-affinity IL-2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high-affinity IL-2 receptors might be regulated by a signal-transducing pathway involving activation and translocation of PKC-α. Lysophosphatid acyltransferase-catalyzed incorporation of cis-polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC-β, which is specifically inhibited by CsA. Neutralization of PKC-β by introducing anti-PKC-β antibodies prevented IL-2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC-β and regulation of IL-2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC-β by CsA may result in inhibition of IL-2 gene expression in human lymphocytes.     

The T cell receptor associated CD3-ϵ protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif

Signal transduction through the Tcell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-? chain. Since both CD3-?and the ζ, chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-? in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-? in vivo. Induction of CD3-? phosphorylation followed similar kinetics to that of the ζ, chain phosphorylation. In contrast to ζ, CD3-? phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-? phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-? by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.     

Synergy of tumor necrosis factor with protein kinase C activators on T cell activation

The ability of tumor necrosis factor (TNF)-alpha to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co-mitogenic with low doses of anti-CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187. TNF was co-mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl-phorbol 12-myristate 13-acetate. Furthermore, H-7 a known inhibitor of PKC, inhibited the proliferative response of T cells induced by esters plus TNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells. This proliferative effect of TNF was inhibited by an anti-interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL2 binding to its receptor. Although PKC activation induced CD25 (IL2R) expression but very little IL2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high-affinity IL2R on purified T cells in the presence of phorbol 12,13-dibutyrate. Our results indicate that TNF is co-mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role on T cell activation increasing the number of effective IL2/IL2R interactions when these are limiting.     

The role of protein kinase C in early activation vs. growth of T lymphocytes

Protein kinase C (PKC) has been implicated in the signaling of a number of cellular responses including activation of T cells. In an approach to study the role of PKC in both early activation and interleukin 2 (IL2)-dependent growth, we have used in vivo phosphorylation of endogenous substrates as a marker for PKC activation. In the present report the requirements for CD3-specific activation of PKC are defined, and it is shown that the conditions required for optimal activation coincide with the induction of a proliferative response. Moreover, it is also shown that IL2 receptor interactions induce only low levels of PKC substrate phosphorylation and that IL2 does not cooperate with CD3-specific signals on the level of PKC activation.     

Comparison of interleukin 2 and 12-O-tetradecanoyl-phorbol 13-acetate as signals for protein kinase C activation in purified human T lymphocytes

Interleukin 2 (IL2) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti-CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes. TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti-CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti-CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti-CD3 mAb-treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation. In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high-affinity IL2 receptors.     

Functional analysis of CD82 in the early phase of T cell activation: roles in cell adhesion and signal transduction

To define T cell co-stimulatory molecules that work in the early phase of T cell activation, we established monoclonal antibodies (mAb) that inhibit or enhance T cell activation by the histiocytic leukemia cell line U937. One of the mAb, 53H5, which recognized both T cells and U937, was identified to bind to CD82 by expression cloning. Functional analyses of CD82 revealed that 1) CD82 needs to exist on both T cells and U937 for the full activation of T cells; 2) CD82 expression is up-regulated on both T cells and U937 by stimulation such as CD3 ligation or treatment with phorbol 12-myristate 13-acetate; 3) overexpression of CD82 enhances both homotypic and heterotypic cell adhesion betweenT cells and U937; 4) CD82 signal co-stimulates T cells and the signal works synergistically with the CD28-mediated T cell co-stimulation signal; 5) in mixed leukocyte reactions using U937 as stimulator cells, CD82 overexpression on U937 correlates with the higher allogeneicity of U937 cells. These results indicate that CD82 co-stimulates T cells not only by sending intra-T cell signals that work synergistically with CD28 signals but also by inducing enhanced T cell-antigen-presenting cell interaction.     

The regulation of T cell growth: requirements for the activation and replication of antigen-specific interleukin 2 producing T cells

The antigenic requirements for stimulating the production of interleukin 2 (IL2) and the growth of IL2 producing T cells was evaluated using antigen-(Keyhole limpet hemocyanin) specific F1 T helper cell lines. The results demonstrate that both the induction and growth of these cells requires specific antigen and antigen-presenting cells of the correct I-A type. Evidence is presented that argues that soluble macrophage derived factors released as a result of T cell-macrophage interaction are insufficient, even in the presence of antigen, to promote growth of IL2 producing cells. We thus conclude that the direct interaction of IL2 producing cells with an antigen presenting cell is obligatory for activation and growth. These results suggest that T cell proliferation, and as a consequence the magnitude of T cell mediated immune responses, is limited by the availability of "approximately" presented antigen to IL2 producing T cells. The requirement for antigen presenting cells in antigen-driven responses is in contrast with mitogen-induced T cell activation, in which soluble factors can substitute for the accessory cells.     

Does co-aggregation of the CD45 and CD3 antigens inhibit T cell antigen receptor complex-mediated activation of phospholipase C and protein kinase C?

The binding of agonistic monoclonal antibodies (mAb) to the CD3 antigen in T cells induces a rapid increase in tyrosine phosphorylation, inositive phosphate (IP) production, a rise in intracellular calcium and protein kinase C (PKC) activation. These intracellular signals have been implicated in the control of interleukin-2 and interleukin-2R receptor gene expression, thereby regulating T cell proliferation. Previous studies have shown that co-ligation of the CD45 and CD3 antigens inhibits CD3-induced tyrosine phosphorylation, IP production, calcium signals and T cell proliferation. It has therefore been suggested that the CD45 antigen uncouples the T cell receptor (TcR) from mitogenic signal pathways. In this study co-ligation of the CD3 and CD45 antigens with precisely constructed bispecific mAb did not inhibit CD3-induced T cell proliferation, IP production, calcium signals, diacylglycerol production or PKC activation. Furthermore, co-ligation of CD3 and CD45 antigens already cross-linked with IgM mAb did not lead to inhibition of CD3-induced calcium signals. Inhibitions of CD3-induced intracellular signals were observed following co-ligation of IgG CD45 and CD3 mAb with anti-IgG (F(ab')2 fragments. However, comparable inhibitions were also noted following co-ligation of CD3 with other abundant cell-surface antigens such as CD5 and LFA-1, and inhibitions were only observed when the CD3 mAb used required cross-linking to induce signals. These results suggested that the inhibitory effects of CD45 IgG mAb were not specific and were caused by the prevention of CD3-CD3 cross-linking following CD3 antigen co-ligation with other cell surface molecules. These findings are inconsistent with a specific inhibitory role for the CD45 phosphotyrosine phosphatase in uncoupling the TcR from mitogenic signal pathways.     

Tuning of T cell clone size and activation threshold by control of CD25 expression through mitogen-activated protein kinase pathways

BACKGROUND: Following T cell receptor (TcR) engagement, CD25 expression is upregulated by T cells and controls their proliferation. It is not known how CD25 expression levels differentially influence T helper (Th) 1 and Th2 cell physiology. METHOD: CD25 upregulation, and other T cell functions, were studied in human Th1 and Th2 clones following stimulation with various stimuli. The effects of pharmacological substances were then evaluated to identify the signaling pathways controlling CD25 upregulation. RESULTS: Upon TcR engagement, one Th2 clone was induced to express substantially more CD25 than a Th1 clone, although both clones downregulated CD3 with similar dose responses. It was also found that the amount of antigen needed to elicit proliferation and cytokine production was considerably lowered in the presence of interleukin-2 (IL-2) for the Th2 cells, while for the Th1 cells the threshold of activation was not modified by the presence of IL-2. It was then shown that PP2 and cyclosporin A strongly inhibited CD25 expression in both clones, while wortmannin and Ro-31-8220 had more limited effects. In contrast, mitogen-activated protein kinase (MAPK) inhibitors had strikingly different effects on CD25, blocking its expression in the Th2 cells, while augmenting it, or leaving it unaffected, in the Th1 cells. CONCLUSION: These unexpected observations suggested that in some T cells TcR-mediated activation of the MAPK pathways may inhibit CD25 expression rather than promoting it. Absence of this negative control mechanism may endow Th2 cells with a growth advantage over Th1 cells and their effector functions may be elicited at lower antigen doses.     

Phosphatases in concert with kinases set the gain for signal transduction through the T cell receptor

The 'tunable activation thresholds' model for signal transduction through the T cell receptor (TCR)/CD3 signaling complex proposes that rapid cycles of phosphorylation and dephosphorylation are integral to regulating the frequency of protein-protein interaction, thus having considerable influence over the activation of downstream signaling pathways. Co-temporal activation of kinases and phosphatases could serve to modulate the ongoing signaling response, depending on the relative balance of their opposing enzymatic activities. Although recent reports have addressed the mechanisms by which specific kinase/phosphatase pairs contribute to the initiation and termination of signaling, we sought a more global understanding of the ability of the kinase/phosphatase balance to regulate, or "tune", the very proximal steps of TCR signaling in primary human T cells. Herein, we provide biochemical evidence that phosphotyrosine induction via the TCR is subject to fine-tuning based on the overall activity of kinases and phosphatases relative to one another, leading to cycles of phosphorylation and dephosphorylation, with implications for developing the next generation of immunotherapeutic agents.