T cell antigen receptor triggered exocytosis in cytotoxic T lymphocytes is inhibited by soluble, but not immobilized monoclonal antibodies to Lyt-2 antigen

The effect of monoclonal antibodies (mAb) to surface antigens on the T cell antigen receptor (TcR)-triggered exocytosis of intracellular granules in cytotoxic T lymphocytes (CTL) was studied. Soluble anti-LFA-1, anti-TcR, and anti-Lyt-2 mAb inhibited both CTL-inflicted 51Cr-release from the target cell (TC) and TC-stimulated exocytosis of granules from cloned CTL. Soluble anti-TcR and anti-Lyt-2 mAb but not soluble anti-LFA-1 mAb inhibited exocytosis, which was triggered by solid-phase anti-TcR mAb. Immobilized anti-Lyt-2 did not inhibit secretion triggered by immobilized anti-TcR mAb; immobilized anti-LFA-1 mAb had an modest inhibiting effect. Inhibition of exocytosis by soluble anti-Lyt-2 mAb was greater when stimulating anti-TcR mAb were immobilized at a lower density on a plastic surface. When the requirement for TcR cross-linking was bypassed by synergistic action of phorbol ester and ionophore A23187, no inhibition of exocytosis by soluble anti-Lyt-2 mAb was detected. The obtained data point to steric hindrance as the most likely explanation of the inhibition of TcR-triggered CTL activation by anti-Lyt-2 mAb.     

Antigen receptor-triggered secretion of a trypsin-type esterase from cytotoxic T lymphocytes

Exocytosis of cytolysin-containing granules from cytotoxic T lymphocytes (CTL) was studied with the use of granule enzyme (BLT esterase) as a convenient biochemical marker. Using cloned CTL, we demonstrate here that BLT esterase secretion into the supernatant is specifically triggered by antigen-bearing target cells and that this secretion is inhibited by soluble monoclonal antibodies against the antigen-specific T cell receptor (TcR). Immobilized anti-receptor antibodies induced efficient enzyme secretion in the absence of target cells, thus implying a direct involvement of TcR complex in triggering exocytosis of granules. These results support the role of the granule exocytosis in CTL functions and provide a quantitative and direct assay of a rapid CTL functional response to antigenic stimulation.     

Modulation of cytolytic T lymphocyte functions by an inhibitor of serine/threonine phosphatase, okadaic acid. Enhancement of cytolytic T lymphocyte-mediated cytotoxicity.

A specific and potent inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), and its inactive analog, tetramethyl ether (OA-TME), were tested in the cytotoxicity and granule exocytosis assays of CTL activation. At low concentrations OA enhanced, whereas at higher concentrations OA inhibited, CTL responses. The Ag-specific and retargeted cytotoxicity, granule exocytosis induced by target cell (TC), anti-TCR mAb, or PMA and A23187, and conjugate formation with TC were inhibited by pretreatment of CTL with OA as expected if protein phosphatases and protein dephosphorylation were indeed involved in the TCR-mediated signal transduction and effector responses of CTL. Cytotoxicity and granule exocytosis were unaffected by pretreatment of CTL with OA-TME. The inhibitory effect of OA on the exocytic response of CTL induced by TC and anti-TCR mAb can be dissociated from the inhibition of the response to PMA and A23187, suggesting the involvement of a serine and/or threonine protein phosphatase in the early events of transmembrane signaling. At lower concentrations, OA, but not OA-TME, was able to enhance the Ag-specific cytotoxicity and TC-induced exocytosis from CTL clones. The enhancement of these TCR-mediated responses of CTL was observed only if the activation was induced by the Ag on the TC surface, because OA did not enhance either the anti-TCR mAb-induced exocytosis of granules from the CTL clone or lysis of the Ag-nonbearing TC by CTL in a retargeting assay. The biphasic character of the effects of OA on CTL-TC interactions suggests the existence of at least two functionally distinct phosphatases in CTL. The ability of OA to enhance the Ag-specific response is unique and indicates the presence of an inhibitory phosphoprotein phosphatase that should be considered as a participant in the down-regulation of the cell-cell interactions between CTL and TC. The inhibitory effects of OA on both TC-induced and anti-TCR mAb-triggered CTL responses at higher concentrations point to the importance of yet another phosphatase in the CTL-TC interactions and in the TCR-mediated transmembrane signaling. The use of OA may help to decipher the details of biochemical changes involved in T lymphocyte effector functions.     

Locus of inhibitory action of cAMP-dependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway

The mechanism of the cAMP involvement in regulation of cellular functions was studied here using a novel functional assay (antigen receptor-triggered exocytosis of granules) of cloned cytotoxic T lymphocytes (CTL). We suggest that cAMP-dependent protein kinase, protein kinase A, counteracts the protein kinase C and Ca2+-mediated stimulatory T-cell antigen receptor (TcR)-triggered biochemical pathway. This suggestion is supported by experimental results which satisfy criteria for protein kinase A involvement in cellular functions. Pretreatment of CTL with cholera toxin induces cAMP accumulation in CTL, partially inhibits TcR-triggered "lethal hit" delivery to the target cell, and almost completely blocks TcR-triggered exocytosis of granules from CTL. Other agents that raise the intracellular level of cAMP, including forskolin and isobutylmethylxanthine (IBMX) also inhibit TcR-triggered CTL activation. Involvement of cAMP-dependent protein kinase in an inhibitory pathway is suggested by the synergistic effects of cyclic nucleotide analogs 8-bromo-cAMP and N6-benzoyl-cAMP in inhibition of TcR-triggered exocytosis. Forskolin and IBMX inhibited TcR-triggered phosphoinositide turnover in CTL, suggesting that cAMP affected very early events in signal transduction that follow TcR cross-linking by a ligand. The ability of IBMX to inhibit CTL activation when the TcR cross-linking step was by-passed by the combination of phorbol myristate acetate and ionophore A23187 suggests that the locus of inhibitory effect of cAMP is at both the early and late stages of the TcR-triggered transmembrane signaling pathway.     

Stepwise maturation of lytic granules during differentiation and activation of human CD8+ T lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1(+) lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.     

The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Triggering of CD8+ cytotoxic T lymphocytes via CD3-epsilon differs from triggering via alpha/beta T cell receptor. CD3-epsilon-induced cytotoxicity occurs in the absence of protein kinase C and does not result in exocytosis of serine esterases.

The requirement for protein kinase C (PKC) during triggering of murine CD8+ CTL was investigated. To this, CTL were depleted for PKC by pretreatment with PMA. This procedure neither influenced alpha/beta-TCR, CD3-epsilon, CD8, CD2, and lymphocyte function-associated Ag-1 expression, nor CTL-target cell conjugate formation. Although cytolytic effector function of PKC-depleted CTL triggered via alpha/beta-TCR structures was completely inhibited, target cell lysis induced via CD3-epsilon remained unaffected. Furthermore this PKC-independent cytolysis pathway was not associated with the release of serine esterases. Analyses at the clonal level revealed that PKC depletion blocked the cytolytic response of up to 95% of alpha/beta-TCR triggered CTL clones. The data suggest the existence of a distinct signaling pathway triggered via CD3-epsilon that is not associated with exocytosis of serine esterases and probably independent of PKC.     

The dual role of the cAMP-dependent protein kinase C alpha subunit in T-cell receptor-triggered T-lymphocytes effector functions.

In order to directly evaluate the role of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit in T-cell receptor- (TCR) triggered cytotoxic T-lymphocytes (CTL) effector functions, cells were studied after pretreatment with antisense oligomers complementary to mRNA for the C alpha or C beta subunits. C alpha subunit is shown to be predominantly expressed in CTL. In some experiments the pretreatment of the CTL with the C alpha antisense, but not with the control or C beta antisense oligomers, resulted in the inhibition of cAMP-independent PKA activity without significantly affecting the level of total cAMP-inducible PKA activity. In parallel assays, CTL which were pretreated with the C alpha antisense oligomer had enhanced antigen-bearing target cell-triggered-, anti-TCR monoclonal antibody-triggered-, and phorbol 12-myristate 13-acetate/A23187-triggered exocytosis of granules, as well as enhanced antigen-specific cytotoxicity. In contrast, the TCR-triggered gamma-interferon mRNA expression and gamma-interferon secretion were inhibited in C alpha antisense-pretreated CTL. These results suggest that the C alpha subunit of PKA may have a dual role in regulation of T-lymphocytes effector functions: (i) it may down-regulate TCR-triggered protein-synthesis independent responses such as cytotoxicity and exocytosis, thereby counteracting TCR-triggered activation even in the absence of the second messenger, cAMP, and (ii) the C alpha subunit activity is likely to be required for the nuclear and/or cytoplasmic events in CTL's activation involved in lymphokine synthesis and secretion.     

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

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

Functional and biochemical characterization of a calcium-ionophore-induced state of unresponsiveness in a cytolytic T cell clone.

To characterize the requirements for the induction of an anergic state in immunocompetent cells we examined the effect of an increase in intracellular calcium concentration on the subsequent responsiveness of cytolytic T cells to antigenic stimulation in vitro. Pretreatment of a murine cytolytic T cell clone with the calcium-ionophore A23187 resulted in the induction of an anergic state characterized by a decrease in cytolytic activity and granule exocytosis upon Ag-specific stimulation. Furthermore, IFN-gamma synthesis declined whereas de novo synthesis of a yet unidentified protein with a molecular mass of 33 kDa as well as proliferative response of cells in response to exogenous IL-2 were unaffected. This state of partial unresponsiveness 1) could be prevented by concomitant pretreatment of cells with cyclosporin A or protein synthesis inhibitors and 2) was reversible within 48 h. Biochemical analysis of TCR-induced intracellular activation revealed a block in signal transduction before the activation of protein kinase C because cellular unresponsiveness could be bypassed by the phorbol ester PMA plus the calcium-ionophore A23187. However, phosphatidylinositol turnover was markedly inhibited in unresponsive cells that also did not show a calcium influx on stimulation with concanavalin A. We conclude that a rise in intracellular calcium in cytolytic T cells might not only be necessary for cellular activation but may also trigger the induction of a partial unresponsiveness to antigenic stimulation due to an inhibition in the early phase of signal transduction.     

Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.     

Physical and functional association of the T cell receptor and the T3 molecular complex on cytotoxic T cell clones that are differentially inhibitable by anti-T3 antibodies

To examine the hypothesis that the antigen-specific T cell receptor (TcR) can function independently from the T3 complex on cytolytic T lymphocyte (CTL) clones, the physical and functional association of the T3 molecular complex and the T cell receptor has been examined on CTL clones that are differentially susceptible to inhibition by anti-T3 antibodies. From a panel of nine DPw2-specific CTL clones derived from the same donor, two clones (8.4 and 8.8) that were the most disparate in their susceptibility to inhibition by anti-T3 antibody were chosen for study. No significant differences were found between 8.4 and 8.8 for: (1) the levels of cell surface expression of the T3 complex and the TcR; (2) the ability to modulate T3 cell surface molecules; and (3) the capacity of the TcR to comodulate with the T3 complex. Modulation of the T3 complex from clone 8.4 did not significantly affect cytolytic activity, and incubation of modulated 8.4 with additional anti-T3 antibody did not inhibit cytolytic activity. Although no T3 function for clone 8.4 could be demonstrated by simply blocking cytolytic activity with anti-T3 antibody, addition of limiting quantities of anti-T11 (but not anti-T4, anti-Tac, or anti-LFA-1) antibodies plus anti-T3 produced a marked synergistic inhibition of cytolysis. These results suggest that: (1) CTL clones that are resistant to inhibition by anti-T3 antibodies actually have a physical and functional association between the T3 complex and the TcR; and (2) the ability to demonstrate a functional role for T3 by antibody blocking may, in some cases, require limiting the involvement of the T11 molecule in CTL-target interactions. The most likely explanation for the observed heterogeneity in susceptibility to blocking by anti-T3 antibodies is, therefore, thought to be that individual CTL clones possess TcR with differential avidity for specific targets.     

Protein kinase C delta localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity

Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.     

CTLs contain and use intracellular stores of FasL distinct from cytolytic granules

CTL lyse target cells through the release of cytolytic granule contents and cell surface expression of Fas ligand (FasL). Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. We demonstrate that murine CTLs undergo two waves of FasL cell surface expression after stimulation. The first wave is from a pre-existing pool of FasL, and the second wave requires new protein synthesis. Signaling for FasL expression appears to be finely tuned as a weak signal preferentially induced surface translocation of the stored FasL, whereas a strong signal preferentially triggered the expression of de novo synthesized FasL. The early FasL is differentially regulated from degranulation, as there were multiple circumstances whereby rapid FasL cell surface expression and FasL-dependent killing occurred in the absence of detectable degranulation. Furthermore, we found through confocal microscopy that stored FasL resides in vesicles distinct from cytolytic granules. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation.     

Regulation by glutathione of the effect of lymphokines on differentiation of primary activated lymphocytes. Influence of glutathione on cytotoxic activity of CD3-AK-.

By activating murine lymphocytes with anti-CD3 antibodies for 1 to 2 days, we generated a subset of activated killer cells, namely CD3-AK-. CD3-AK- mediated the slow lysis (20-h 125I-UdR release assay) of allogeneic P815 but had little effect on syngeneic HFL/b cells. Addition of IL-2 (murine or human) or an IL-2 inducer such as PMA in the assay medium induced the cytolytic activity of CD3-AK- on HFL/b. The activating effect of murine IL-2 and PMA on CD3-AK- was decreased by anti-murine IL-2 mAb. Although anti-murine IL-4 mAb alone did not show any effect, it enhanced the inhibitory effect of anti-IL-2 mAb, suggesting that IL-2 and IL-4 may have a synergistic effect on the cytolytic activity of CD3-AK-. Incubation of CD3-AK- with L-buthionine-(SR)-sulfoximine (BSO), an inhibitor of de novo glutathione (GSH) synthesis, decreased cellular GSH levels and inhibited the cytolytic activity of CD3-AK-, in a concentration-dependent manner. This inhibitory effect of BSO was not primarily due to a general cytotoxic effect and was positively correlated with the requirement for IL-2 for the CD3-AK(-)-mediated killing of the target cells. Incubation of CD3-AK- with GSH or 2-ME, which increased the level of cellular GSH, reversed the inhibitory effect of BSO. These results suggest that cellular GSH may regulate the effect of lymphokine(s) such as IL-2 and thus affect the differentiation of activated primary cytotoxic lymphocytes.     

The use of monoclonal antibodies in the study of the interaction between adrenal medullary cell membranes and chromaffin granules

Simultaneous incubation of bovine adrenal medullary plasma membranes (PM) with chromaffin granules (CG) resulted in the release of the soluble granular content. The molecular mechanism of this process was studied with several monoclonal antibodies (mAb) raised against different plasma membrane components. Specific inhibition of the catecholamine secretion was obtained upon incubation with the monoclonal antibody UIA/NEU/VI B17. The corresponding antigen had an apparent molecular weight of 54000 Dalton. These results suggest a specific recognition between proteins located on the plasma membrane and chromaffin granule membrane, the interaction of which mediates exocytosis.     

Immunomodulating properties of a novel series of protein kinase C activators. The bryostatins

Low concentrations of the protein kinase C activators, bryostatins 1 and 2 synergized with recombinant B cell stimulatory factor-1 in triggering differentiation (granule enzyme expression) and cytotoxic T lymphocyte (CTL) development in naive, resting lymph node T cells. Bryostatin greatly enhances efficiency of recombinant interleukin-2 in triggering development of in vivo primed CTL during in vitro incubation, thereby providing experimental evidence for the efficacious use of lower concentrations of recombinant interleukin-2 for in vivo tumor rejection studies. Both bryostatins 1 and 2 were able to trigger cytotoxicity of CTL clones against antigen-nonbearing target cells and inhibited CTL cytotoxicity against Ag-specific target cells. Bryostatin 1 and 2 synergize with Ca2+ ionophores in triggering the exocytosis of cytolytic granules from CTL at very low concentrations. In view of the lack of tumor promoting activity of the bryostatins, the possible use of these agents in vivo is discussed.     

Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis.

The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.     

Cytotoxic T lymphocyte unresponsiveness induced by prolonged treatment with immobilized anti-CD3 antibody. Association of impairment of cytolytic activity with temporary depletion of intracellular protein kinase C

In our study we investigated the effect of pretreatment of bulk CTL and CTL clones with immobilized anti-CD3 antibody (Ab) or PMA. Primary CTL and CTL clones were cultured in dishes coated with anti-CD3 Ab or in medium containing PMA (5 nM) and assayed for Ag-specific or Ag-nonspecific "redirected" cytolysis using FcR+ P815 cells as targets. Cytotoxic activity of bulk CTL and five of six CTL clones tested in this study were inhibited by prolonged (longer than 6 h) pretreatment with immobilized anti-CD3 Ab or PMA, whereas proliferation of CTL clones or expression of surface CD3 molecules were not. The intracellular granule enzyme (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) activity of CTL clones was not reduced under these suppressive conditions, indicating that the incompetence of CTL is not merely due to depletion of cytolytic granules by chronic stimulation. The suppressed cytotoxicity could be recovered by culturing CTL without perturbation of CD3 molecules for 24 h. In one exceptional clone, BM10-37, pretreatment with immobilized anti-CD3 Ab or PMA did not suppress the cytotoxic activity. Immunostaining of intracellular protein kinase C (PKC) revealed that PKC was depleted after prolonged treatment with immobilized anti-CD3 Ab or PMA in those susceptible CTL clones but not in the resistant BM10-37. These findings lead us to conclude that prolonged stimulation of CD3 of CTL results in depletion of PKC and that PKC may be essential for signal transduction to deliver a lethal hit to the target cells.