Immunology Today, voL 7, No. 9, 1986

reviewsSignal transduction and intracellular events in T-lymphocyte activation

The important role of a novel signal transduction pathway has the cellular activation and profiferation of many cell types. The key event in this pathway is the hydrolysis of a distinct membrane-associated inositol phospholipid and the resulting generation of two defined products that act as second messengers in cell activation. Sufficient evidence has accumulated to indicate that this signal transduction pathway plays an important role in T-cell activation. In this article, Noah Isakov and his colleagues review briefly the general characteristics of this pathway and describe in more detail experimental evidence that establishes its critical role in T-cell activation.

recently been established in

T-cell activation and proliferation are initiated, under physiological conditions, by the interaction between an antigen associated with a membrane-bound major histocompatibility complex (MHC) molecule, and a corresponding cell surface receptor. The receptor consists of an antigen-specific disulfide-linked heterodimer covalently linked to nonpolymorphic proteins of the T3 complex. Experimentally, this physiological interaction can be mimicked by mitogenic plant lectins, antibodies to defined cell surface proteins such as the T-cell receptor, T3, sheep erythrocyte receptor (T1 1) or Thy-1 and, in the case of activated T cells, by a well-defined growth factor, interleukin 2 (IL-2). The consequences of these cell surface interactions can be measured by convenient end-point assays that detect: (1) such T-cell functions as proliferation, cytolysis of target cells and production of IL-2 or other lymphokines; and (2) T-cell activation markers such as la antigens and cell surface receptors for IL-2 and transferrin. These functions and markers acquired de novo activation of genes, mRNA transcription and synthesis of new proteins. Hence, mechanisms must exist to transduce signals originating at the cell's surface into its nucleus. Although the interactions of ligands with T-cell surface receptors as well as the endpoints of the activation events have been widely studied, very little is known about the biochemical events that occur between these two extreme points. For example, we do not know whether the same signal transduction pathways are set in motion by multiple activating signals (antigens, antibodies, lectins or growth factors) acting via distinct cell surface receptors, or whether T-cell subsets, each with individual, genetically programmed functions have differing signal requirements to activate such functions. Beyond the basic interest in understanding these signal transduction mechanisms, clarifying their nature on the biochemical level may serve a practical purpose, namely, the development of pharmacological agents to downregulate (e.g. in autoimmune reactions) or stimulate (e.g. in immunodeficiencies) T-cell activation and proliferation. Department of Immunology, ScrippsClinic and ResearchFoundation, La Jolla, California 92037, USA ~) 1986, Elsevier Science Publishers B.V., Amsterdam 0167-4919/86/$02.00

NoahIsakov,WolfgangScholzand AmnonAltman Inositol phospholipidsand signaltransduction Turnover of membrane phospholipids is a characteristic event in many cell types undergoing stimulation by external ligands. The critical component in this signal transduction system is phosphatidylinositol 4,5bisphosphate (PtdlnsP2), normally found in the cell membrane in minute quantities (reviewed in Ref. 1 and 2). Interaction of a ligand with its cell surface receptor stimulates a phosphodiesterase (phospholipase C) to hydrolyse PtdlnsP2 (Fig. 1). GTP-binding proteins, which are known to play an important regulatory role in another signal transduction pathway, i.e., the adenylate cyclase system which uses cyclic AMP as a second messenger, may constitute the coupling mechanism between receptor occupancy and phospholipase C stimulation 3. The products of hydrolysed PtdlnsP2 are diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). These two second messengers trigger two parallel pathways that act in concert to elicit a physiological response (Fig. 1). DG activates the enzyme protein kinase C (PKC) by increasing its affinity for Ca2+. This Ca2+- and phospholipid-dependent ubiquitous enzyme is translocated upon activation from the cytosol to the membrane and catalyses the phosphorylation of numerous protein substrates at seryl and threonyl residues by using ATP as a phosphate donor. One of the substrates for PKC may be a Na+/H+ exchanger whose action results in an increase of intracellular pH, an event that accompanies, and seems to be causally related to, cellular activation and growth in many systems (see below). The second product of PtdlnsP2 hydrolysis, IP3, releases Ca2+ from intracellular stores, probably the endoplasmic reticulum, thereby increasing the concentration of free intracellular Ca2+ ([Ca2+]i). This event may activate Ca2+-dependent protein kinases, resulting in protein phosphorylation. When cells are stimulated by external ligands, both pathways, i.e., the activation of PKC and the increase in [Ca2+]i, are set in motion as a result of PtdlnsP2 hydrolysis and generation of the two relevant second messengers. However, experimentally, these two synergistically acting pathways can be dissociated and studied separately by using chemicals that activate only one or the other. Thus, several tumor promoters (TP) mimic the effects of DG and activate PKC without inducing an increase in [Ca2+]i. On the other hand, calcium ionophores (CI) can mimic the physiological signal of IP3 and increase [Ca2+]i. However, since PKC is a Ca2+-dependent enzyme that can be activated irreversibly by a Ca2+-dependent protease, a sufficient increase in [Ca2+]i may directly activate PKC in the absence of PtdlnsP2 hydrolysis and DG generation. This may be the reason why CI can cause an increase in intracellular pH and cellular proliferation,

271

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although these events do not require increased [Ca2+]~ per se.

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Fig.2. Partialpurificationof PKCfrom humanleukemiaT-ceilcytosolon a DE52 column. UnstimulatedJurkatcellswerewashedin serum-fleeroediumand resuspended in an EGTA-containingbuffer, disrupted by sonication and centrifuged at 100 000g for I h. Thesupematant(cytosolfraction)wasco//ectedandloadedon a DE52 column. Elution was performed with a linear gradient of NaG and enzymaticactivity was determinedin the presence(e) or absence(o) of Ca2+, diolein and phosphatidylserine.

enzyme consists of two functional domains; a regulatory hydrophobic domain which binds to the cell membrane upon cell activation, and a hydrophilic domain which possessesthe catalytically active center. PKC activation is usually linked to the agonist-receptor induced PtdlnsP2 hydrolysis, although it can be activated irreversibly in an alternative manner by a Ca2+-dependent protease, calpain 6~. Upon proteolysis by calpain, the two domains are separated to form a Ca2+ and phospholipid independent catalytic fragment 6-8. Activation of PKC seems to consist of a DG-induced increase in the affinity of the enzyme for Ca2+ and formation of a quaternary complex consisting of PKC, DG, phospholipid and Ca2 + . Among the membrane phospholipids, phosphatidylserine is the most efficient in PKC activation. Since the cell membrane consists of several phospholipids possessing various degrees of stimulatory or inhibitory effects on PKC, their asymmetric distribution in the membrane lipid bilayer may determine the activation step. Although PKC has been isolated from a heterogeneous population of human peripheral blood lymphocytes9'1°, it has not been characterized extensively in human T cells. Recently, we partly characterized PKC prepared from cytosols of the human leukemicT-cell line Jurkat, or peripheral blood-derived resting T cells purified by sheep en/throcyte rosetting (Isakov and Altman, unpublished). After fractionation by DE-52 ion exchange chromatography, the enzyme eluted at 0.02-0.05 M NaCI (Fig. 2). As expected, optimal activity of the enzyme

Immunology Today, vol. 7, No. 9, 1986

in a cell-free system, with 32p-ATP used as a phosphate donor and calf thymus histone H1 as a substrate, depended on the presence of three cofactors; Ca 2+, a synthetic DG, and a phospholipid (phosphatidylserine). Omission of any of these cofactors substantially decreased or totally eliminated PKC activity. Stimulation of resting T cells with TP (see Fig. 3) induced rapid translocation of PKC from the cytosol to the particulate fraction. This translocation did not occur upon stimulation with CI, which induces T-cell proliferation. Similar properties have been reported for PKC isolated from the murine thymoma line EL4 (Ref. 11). In recent experiments, stimulation of an IL-2dependent T-cell line, CTLL, with the respective growth factor also induced a rapid cytosol to membrane PKC translocation 12. It remains to be seen, first, whether a physiological stimulus, i.e., antigen, induces such translocation and, second, whether PKC activation and translocation occur similarly in resting and activated T cells. As noted earlier, PKC phosphorylates a wide range of substrates in vivo or in cell-free systems. Several of these {arget proteins have already been identified, and additional information about these and others is an important step in elucidating PKC's exact physiologic role and sites of action. In T cells, the relevant proteins are thelL-2 receptor (IL-2-R), which is phosphorylated at Ser 247 (Ref. 13 and B. Gallis, unpublished), and the T3 complex in which the gamma chain and, to a lesser extent the delta chain, are being phosphorylated 14. However, phosphorylation of these T-cell surface proteins by PKC remains to be shown more directly, since phorbol esters (which activate PKC, see below) were used in these studies. If drugs that selectively inhibit PKC activity were available, the role of this enzyme in T- (and other) cell activation events might become clearer; however, no known drugs are really adequate at present. Phospholipid interacting drugs such as phenothiazines and local anesthetics are primarily inhibitors of calmodulin, but also block PKC activation in a cell-free system 9.1s. Such drugs were found to block lymphocyte mitogenesis in vitro ~6. For more direct investigation of the drug's effects on PKC as a possible inhibitor of T-cell function, we added increasing concentrations of the phenothiazine chlorpromazine to cultured T cells and analysed activation of the cells and of PKC. Chlorpromazine clearly inhibited phytohemagglutinin (PHA)-induced or 12-0tetradecanoylphorbol-13-acetate (TPA)-induced T-cell proliferation and IL-2-R expression. In parallel (albeit at higher concentrations), the drug also inhibited cytosol to membrane PKC translocation in vivo in phorbol esterstimulated human T cells, or phorbol ester-induced enzyme activation in a cell-free system. Although the contribution of PKC inhibition to the overall inhibition of T-cell activation by phenothiazines (or other drugs) is difficult to assess, this effect of the drugs may be important in understanding their immunosuppressive effects.

Effects of tumor promoters on T cells TP, in particular the phorbol esters, have been widely studied in recent years and their pleiotropic effects in several cellular systems have been described. The multiple effects of TP, or at least of the phorbol esters, are thought to rest on their structural similarity to DG, the physiological activator of PKC. TP can replace DG and directly activate PKC in vitro. Moreover, recent studies

re ,iew$established that PKC is, in fact, the cellular receptor for most of the known TP17-19. Thus, both TP phorbol ester binding sites and active PKC are similarly distributed in different tissues and in subcellular fractions within the cells; both co-purify following chromatography, and an excellent correlation exists between the relative abilities of phorbol esters to stimulate tumor formation, activate

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I m m u n o l o g y Today, vol. 7, No. 9, 1986

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Red Fluorescence Intensity (Phycoerythrin Conjugated Anti-Leu 4) Fig. 4. T-cell activation and cell surface regulation. Human PBLwere stimulated for 3 days with various mitogens and separated by sheep erythrocyte rosetting. T cell-enriched populations were stained for cell surface markers by reaction with anti-lL2 receptor antibody (anti-Tac), followed by fluoresceinated rabbit anti-mouse Ig, or phycoerythrin-conjugated anti-Leu-4 which reacts with the 7-3antigen. Cells were analysed on a FACS,and the relative intensity of green (vertical axis) versusred (horizontal axis) fluorescence was tested on a log scale and plotted as a two-parameter display on three levels of intensity.

274

PKC and bind to cellular receptors. Various effects of TP on T lymphocytes, mainly those of TPA, have been described. As mentioned earlier, TPA induced a cytosol to membrane translocation of PKC in the murine EL4 thymoma 11. The ability of TPA to synergize with plant lectins, e.g., Con A and PHA, in inducing the production and secretion of IL-2 and other lymphokines by T cells is well known2°-22; this effect is caused by an increase in the transcription of the specific genes 23-2s. Additionally, TPA alone induces a high level of IL-2 production by a subline of EL4 cells26. It appears that the EL4 cells are at a particular level of activation that requires only the signal provided by TPA to produce IL-2, whereas most other T cells require an additional signal that plant lectins or CI can provide. TP also supply a sufficient signal for inducing transcription of the IL-2-R gene 25'27'28 and its subsequent expression on the cell surfacez9,3° (Fig. 4). The ability of TP-stimulated T cells to respond to picomolar concentrations of IL-2 indicates that at least some of the TP-induced IL-2-R are of the high affinity class31. Teleocidin, an indole alkaloid TP that is structurally unrelated to the phorbol esters (or DG), has similar effects on T cells, indicating that structural similarity of TP to DG (the physiological activator of PKC) is not a prerequisite for their biological activity32. TP can stimulate proliferation of resting human T cells32-39. These proliferating T cells display transferrin receptors and IL-2-R32. For more direct study of the relationship between this mitogenic effect and the activation of PKC, we analysed TPA-induced PKC activation in human T cells. Fig. 3 displays the mitogenic effect

on resting T cells and the translocation of PKC from their cytosols to their membranes in vivo. Also shown is activation of T cell-derived PKC in vitro as a function of TPA concentration. Similar concentrations of TPA, 1-2 ng/ml (approximately 1.6-3.0 nM), induced half-maximal effects in all three assay systems, suggesting that T-cell proliferation is related to PKC activation. Interestingly, when human T cells are induced to proliferate by TP, no transcription of the IL-2 gene or secretion of IL-2 is detectable32; moreover, this proliferation cannot be blocked by a neutralizing anti-lL-2 antibody 2s. These findings indicate that, in addition to the conventional, IL-2-dependent pathway of T-cell proliferation, another IL-2-independent pathway can operate, probably via the activation of PKC. The activation of PKC by TP leads to phosphorylation of the IL-2-R~3 and the T3 complex ~4 in human T cells. However, in addition to these regulatory activities at the post-transcriptional level, PKC activation also exerts transcriptional control as shown by the ability of TP to induce IL-2-R specific mRNA 2s'z7-z8. We (Fig. 4) 32 and others ~4'35'36 found that TP can downregulate T3 expression on human T cells, and it remains to be determined whether this event reflects only internalization of the T3 molecules or also modulation of T3-related gene(s) transcription. Thus, analysis of the regulatory effects of PKC on T-cell activation at several levels should be an active area of research in the near future. Increase in the intracellular pH Growth factor-induced activation of various cell types

Immunology Today, vol. 7, No. 9, 1986

reviewsWith regard to T cells, stimulation of quiescent rat or murine thymocytes with lectins, TPA or C! results in a common and sustained increase in pHi by 0.1-0.15 pH units 39~1. The effects of Con A and TPA, or CI and TPA are additive 39. This alkalinization is sensitive to amiloride 4°,43,44, a known inhibitor of the Na+/H ÷ exchanger. The addition of IL-2 to IL-2-dependent T-cell lines can increase the pHi ~. However, T-cell proliferation occurs in the presence of amiloride concentrations that prevent this increase, and even in HCO3--free medium (thus eliminating a role for an alternative HC03- transport system in cell alkalinization; Ref. 44), suggesting that neither cellular alkalinization nor activation of the Na+/ H ÷ exchanger is essential for IL-2-driven T-cell proliferation. Although this may be true for activated T cells

is followed by increase in the intracellular pH (pHi), suggesting that cytoplasmic alkalinization is important to cell activation 37. This alkalinization is achieved through activation of a membrane Na÷/H + exchanger. Normally activated by physiological ligands, this increase in pHi can be induced by plant lectins (PHA, Con A), TP and C138~°. Since TP elicits this increase in pHi without a c o n c o m i t a n t increase in [Ca2+]i 38'4°'41, current thinking is that: (1) PKC activation is responsible for cellular alkalinization; and (2) increased [Ca2+]i is not essential for this event. However, since increased [Ca2+]i could activate PKC by itself, or at least synergize with other cofactors in inducing such activation, the observed cellular alkalinization induced by CI may result from PKC activation. It has been suggested that activated PKC may directly phosphorylate the Na+/H + exchanger 42.

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275

-reviews already displaying IL-2 receptors, resting T cells might differ. In the latter, cellular alkalinization may be a prerequisite for subsequent activation, a possibility that has yet to be examined.

276

Heterogeneity of signal requirements in T-cell activation Increased [Ca~+]i is a critical component of signal transduction by various cell-surface receptors. Studies by several groups demonstrated that the increase in [Ca2+] i is also an important signal in T-cell activation. This area has been reviewed recently 4s. Antigens, antibodies to the clonotypic T-cell receptor or the T3 complex, as well as anti-T1 1 antibodies and Cl can all, under appropriate conditions, stimulate increased [Ca2+]i and T-lymphocyte proliferation. The conclusion that the increase in [CaL+]i plays an important role in T-cell triggering via the antigen receptor is supported by the finding that stimulation of Jurkat cells by an anti-T-cell receptor antibody or Cl results in phosphorylation of the same cytoplasmic proteins 46. As discussed above, it is quite likely that T-cell activation by Cl is, in fact, linked to PKC activation. Physiological [Ca2+] i in resting cells presumably is not sufficient for PKC activation. However, activation of PKC by DG or TP increases the affinity of PKC for Ca 2÷, thus allowing it to be partially or fully activated by basal [Ca2+]i levels of resting cells. Alternatively, in the absence of DG or TP stimulation, PKC could be activated by increasing [Ca2+]i , e.g., with Cl. Increase in the [Ca2+] i was indeed found to synergize with TP in PKC activation 47'48. At optimal concentrations, Cl alone can trigger IL-2-R expression (Fig. 4) and human T-cell proliferation 49's° in the apparent absence of IL-2 production s°. This is similar to the previously mentioned effect of TP on resting human T cells, and may involve an IL-2-independent, PKC-related proliferative pathway. Although TP or CI can activate T cells independently, at concentrations low enough that neither is mitogenic by itself, their combination results in a strong proliferative response comparable to that induced by PHA or ConA, and high level IL-2 production sl-s4. T cells in any of several subsets can be triggered, although each possesses a unique functional genetic program. That is, cytotoxic T cells are programmed to lyse appropriate target cells, and helper or suppressor T lymphocytes exert positive or negative regulatory influences, respectively, via the production of lymphokines or direct cell contact. Some recent studies addressed the question of heterogeneity among T-cell subsets with respect to activation signal requirements. In human T cells, either TP or Cl provides a sufficient stimulus to trigger IL-2-R expression and proliferation 2s'33's° (Fig. 4). However, as noted above, this proliferative response is probably IL-2-independent. Therefore, addition of exogenous IL-2 to TP- or Cl-stimulated T cells results in a synergistic response reflecting the concerted action of the IL-2-dependent and -independent pathways. The situation is, however, different in murine T cells, whose stimulation with appropriate TP or CI concentrations induces IL-2-R and enables the cells to proliferate in response to exogenous IL-2, but does not stimulate IL-2-independent proliferation. The basis for this difference is not known, but may reflect quantitatively or qualitatively different requirements for activation signals by human and murine T cells. For example, the activation threshold may be higher in murine T cells than in their human counterparts. In recent experiments, the L3T4 +

Immunology Today, voL 7, No. 9, 1986

subset of murine T cells became responsive to exogenous IL-2 upon stimulation with TP or CI. In contrast, induction of IL-2 responsiveness in Lyt 2 + cells required a combined stimulus by TP plus CI ss. The signal provided by TPA or CI seems sufficient for the induction of IL-2-R in mouse or human T cells, yet neither signal alone can induce IL-2 production (or IL-2 mRNA). Only a combination of the two signals triggers IL-2 production 52-54. Thus, induction of the IL-2-R and IL-2 genes seem to have different signal requirements. We have recently examined the role of PKC activation and the Ca 2+ signal in the reactivation of resting, alloantigen-activated mouse T cells s3. Such cells, after 14 days of stimulation in a primary mixed lymphocyte culture, manifested low levels of alloantigen-specific reactivity. Using specific alloantigen or IL-2 these bulk cultures were reactivated to proliferate, display IL-2-R, secrete IL-2 and lyse allogeneic target cells (Fig. 5). We found that a combination of TP plus Cl, but neither alone, mimicked alloantigen (or IL-2) in this respect s3. This led us to examine whether activation of cytolytic cells can be triggered directly by TP plus Cl. To address this question, we used limiting dilution techniques to clone alloreactive helper or cytotoxic T cells and analyse their activation signal requirements separately. As expected, TP plus Cl induced proliferation and IL-2 secretion by a helper T-cell clone. However, the same combination did not activate the cytotoxic T-cell clone. When both clones were mixed, cytolytic activity was induced by TP + CI, an effect that was abolished by adding a monoclonal antibody to the murine IL-2-R s3 (Fig. 5). The conclusion from these, and similar s6, studies is that TP + Cl can mimic specific antigen in that they are capable of directly triggering helper T cells but not cytotoxic T cells. Activation of the latter is secondary to the release of IL-2 by helper T cells, and its subsequent binding to IL-2-R on the cytotoxic cells. Thus, these studies demonstrate different signal requirements for activation of T-cell help or cytotoxicity.

Conclusions and future directions The studies reviewed here and elsewhere 4s clearly indicate that the signal transduction system, which uses the two hydrolysis products (DG and IP3) of the membrane phospholipid PtdlnsP2 as second messengers, plays an important role in the activation of T lymphocytes. This concept is similar to those reported for B lymphocytes s7 and many other cellular systems (reviewed in Ref. 1 and 2). The way is now open for a detailed analysis of the intracellular steps involved in T-cell activation on the biochemical level. The following questions should be addressed. Is the same signal transduction pathway triggered after stimulation by different ligands (antigens, lectins or antibodies to T-cell surface determinants)? Are different signals required for activation of distinct genetic programs in various T-cell subsets? What, on the biochemical level, is the difference between tolerogenic and immunogenic signals for T cet!~? At what level (i.e., transcriptional or post-transcriptivna'., .~ T-cell activation regulated by PKC? What are the immunologicallyrelevant target proteins phosphorylated by PKC upon T-cell activation? Do putative biochemical defects in this signal transduction system correlate with phenotypic abnormalities of T-cell responses? And, finally, is this particular pathway essential for T cells to perform their various tasks, or could alternative signal transduction system(s) be important in T-cell activation? In this re-

Immunology Today, vot 7, No. 9, 1986

T#VI#WS-spect, identifying drugs that selectively block PtdlnsP2generated activation signals at defined points, and obtaining T-cells clones with mutations at such points, would be extremely helpful. The studies performed to date have addressed some of these questions. However, many more answers are needed to complete the puzzle of T-cell activation to the point where we can fully understand and control these events experimentally or therapeutically. This is Publication No. 43101MM from the Department of Immunology, Scripps Clinic and Research Foundation. The work reported herein was supported, in part, by NIH grants CA-35299, AM-35411, Leukemia Society of America Scholarship (A.A.) and Special Fellowship (N.I.), and the Deutsche Forschungsgemeinschaft (W.S.). The expert technical help of J. Cardenas and P. Marshall, the editorial assistance of K. Occhipinti and P. Minnick, and the continuous support of Dr F. J. Dixon are greatly appreciated. References

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