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.     

Similar molecular requirements for antigen receptor-triggered secretion of interferon and granule enzymes by cytolytic T lymphocytes

At least two biologically significant responses are triggered by the crosslinking of the T-cell receptor (TcR) on the surface of cloned cytotoxic T lymphocytes (CTL): synthesis and secretion of macrophage-activating factor(s) (MAF) that can be attributed to interferon-gamma (IFN) and release of preformed cytolytic granules. We directly compared the molecular requirements for synthesis and secretion of IFN and secretion of granule enzymes triggered in the same cell by the same activating ligand (antigen or monoclonal antibody (mAb) to TcR). An increase in the surface density of activating ligand (immobilized anti-TcR mAb) enhanced both secretion of IFN and secretion of granules. Secretion of IFN occurred immediately after synthesis: only low (but detectable) levels of IFN were detected in cell cytosolic or particulate fractions isolated from Percoll gradients of lysed CTL, while very high levels of IFN were found in the stimulated CTL culture fluids. Inhibitors of RNA synthesis and protein synthesis blocked secretion of IFN, but did not inhibit release of preformed cytolytic granules. The requirement for TcR crosslinking in triggering both secretion of granules and secretion of IFN from CTL was pharmacologically reproduced by the synergistic action of PMA, a protein kinase C activator, and the Ca2+ ionophore A23187. Both secretion of IFN and secretion of granules were absolutely dependent upon extracellular Ca2+: EGTA completely blocked both TcR- and PMA/A23187-induced secretion of IFN and exocytosis of granules. These studies suggest that similar molecular mechanisms are involved in secretion of newly synthesized IFN and secretion of preformed cytolytic granules. One notable difference between the molecular requirements for the two secretory events was a much lower concentration requirement for PMA for IFN synthesis and secretion than for granule secretion in the synergistic interactions with A23187. Implications of these studies for the exocytosis model of cell-mediated cytotoxicity are discussed.     

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.     

The role of Ca2+ and Mg2+ in the cytotoxic T lymphocyte reaction and in the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-serine esterase by human T cell clones

Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon Ag-specific activation. BLT-serine esterase secretion could also be induced by the Fc gamma+ target cell Daudi in the presence of mAb specific for the TCR/CD3 complex, CD2, or the T cell activation Ag Tp 103. In addition, anti-CD3 and a mitogenic combination of anti-CD2 mAb, induced secretion of BLT-serine esterase in the absence of target cells, whereas anti-Tp 103 failed to do so. The secreted BLT-serine esterase activity induced by the various ligands was inhibited by the serine esterase inhibitors PMSF and m-ABA, but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone. Significant BLT-serine esterase activity was induced by target cells or soluble anti-CD3 in the absence of extracellular Ca2+ ions, provided that extracellular Mg2+ ions were present. The cytotoxic activities by the human CTL clones were completely blocked under these conditions. All ligands that induced BLT-serine esterase secretion in the absence of extracellular Ca2+, induced a transient rise of intracellular Ca2+. Soluble anti-CD3 mAb did not induce a transient rise in intracellular Ca2+ or secretion of BLT serine esterase in CTL preincubated for 2 h with 5 mM EGTA. These findings indicate that mobilization of intracellular Ca2+ in human CTL clones is required for induction of secretion of BLT-serine esterase.     

Mechanism of killing by virus-induced cytotoxic T lymphocytes elicited in vivo.

The mechanism of lysis by in vivo-induced cytotoxic T lymphocytes (CTL) was examined with virus-specific CTL from mice infected with lymphocytic choriomeningitis virus (LCMV). LCMV-induced T cells were shown to have greater than 10 times the serine esterase activity of T cells from normal mice, and high levels of serine esterase were located in the LCMV-induced CD8+ cell population. Serine esterase was also induced in purified T-cell preparations isolated from mice infected with other viruses (mouse hepatitis, Pichinde, and vaccinia). In contrast, the interferon inducer poly(I.C) only marginally enhanced serine esterase in T cells. Serine esterase activity was released from the LCMV-induced T cells upon incubation with syngeneic but not allogeneic LCMV-infected target cells. Both cytotoxicity and the release of serine esterase were calcium dependent. Serine esterase released from disrupted LCMV-induced T cells was in the form of the fast-sedimenting particles, suggesting its inclusion in granules. Competitive substrates for serine esterase blocked killing by LCMV-specific CTL, but serine esterase-containing granules isolated from LCMV-induced CTL, in contrast to granules isolated from a rat natural killer cell tumor line, did not display detectable hemolytic activity. Fragmentation of target cell DNA was observed during the lytic process mediated by LCMV-specific CTL, and the release of the DNA label [125I]iododeoxyuridine from target cells and the accompanying fragmentation of DNA also were calcium dependent. These data support the hypothesis that the mechanism of killing by in vivo-induced T cells involves a calcium-dependent secretion of serine esterase-containing granules and a target cell death by a process involving nuclear degradation and DNA fragmentation.     

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.     

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.     

Cross-talk with Ca(2+) influx does not underlie the role of extracellular signal-regulated kinases in cytotoxic T lymphocyte lytic granule exocytosis

One important mechanism cytotoxic T lymphocytes use to kill target cells is exocytosis of lytic granules that contain cytotoxic agents such as perforin and granzyme. Ca(2+) influx and activation of protein kinase C have been known for many years to be key signals for granule exocytosis. Recent work has suggested that activation of extracellular signal-regulated kinases (ERK), members of the mitogen-activated protein kinase (MAP kinase) family, may be a third required signal. We surmised that the involvement of ERK in lytic granule exocytosis could be mediated through cross-talk with Ca(2+) influx, rather than constituting an independent signal. We tested this idea using TALL-104 human leukemic CTLs as a model system and discovered the following. 1) ERK inhibition caused a modest decrease in the amplitude of increases in intracellular Ca(2+) concentration, but this effect cannot account for the profound inhibition of granule exocytosis. 2) Ca(2+) influx can activate ERK in TALL-104 cells, but this effect does not contribute to ERK activation stimulated by solid phase anti-CD3 monoclonal antibodies. We conclude that cross-talk between ERK signaling and Ca(2+) does not mediate the role of ERK in CTL lytic granule exocytosis.     

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.     

Role of accessory molecules in signal transduction of cytolytic T lymphocyte by anti-T cell receptor and anti-Ly-6.2C monoclonal antibodies

Accessory molecules present on the cell surface of cytolytic T lymphocytes (CTL) play an important role in their activation. Antigen-specific recognition by CTL is inhibited by antibodies against Lyt-2, L3T4, or LFA-1 molecules. Presently it is not known whether these molecules function by binding a ligand such as class I or class II on the target cell or by delivering a signal that down-regulates T cell activation. In the present study we utilized anti-T cell antibodies including anti-T3 and anti-T cell receptor (alpha/beta) as well as an anti-Ly-6.2C monoclonal antibody to activate CTL clones to kill irrelevant targets or secrete BLT esterase. The redirected lysis assay system utilizes the fact that heteroconjugates between anti-T3, and anti-T cell receptor, or anti-Ly-6.2C and anti-trinitrophenyl can trigger CTL lysis of trinitrophenyl-coupled targets that did not express antigen. In this system anti-Lyt-2 antibodies as well as anti-LFA-1 antibodies inhibited triggering via T cell receptor-related molecules but not via the anti-Ly-6.2C heteroconjugate. In addition, the anti-Lyt-2 was shown to inhibit conjugate formation in the heteroaggregate assay system suggesting that the anti-Lyt-2 antibodies acted early in inhibiting CTL activity. Similar results were observed in a system in which the CTL clones were triggered to secrete a BLT-esterase-like activity in the absence of target cells. Anti-T3 coated on plastic was shown to activate BLT-esterase secretion. This secretion was inhibited by anti-Lyt-2 and anti-LFA-1. Thus, it would appear that both the Lyt-2 molecule and the LFA-1 molecule act as signal-transducing elements involved in CTL activation. In particular, the Lyt-2 molecule appears to preferentially function in receptor-mediated T cell activation.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

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.     

Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.     

Nicotinamide and 3-aminobenzamide interfere with receptor-mediated transmembrane signaling in murine cytotoxic T cells: independence of Golgi reorientation from calcium mobilization and inositol phosphate generation.

The two competitive inhibitors of ADP-ribosylation, nicotinamide and 3-aminobenzamide, have been reported to interfere with TNF-induced cell apoptosis, and there is evidence that they inhibit killer-induced target cell lysis as well. There are very few drugs known to specifically interfere with target apoptosis induced by killer cells. We therefore sought to explore the effects these inhibitors have on CTL-mediated cell lysis. Here we show that TcR-mediated transmembrane signaling in CTL, measured by Ca2+ mobilization and generation of inositol phosphates, is inhibited by nicotinamide. The possibility that all cell functions are suppressed by the drug is excluded by the finding that constitutive secretion of BLT serine esterase is not inhibited, whereas stimulated secretion of this enzyme is suppressed. We also show that nicotinamide does not interfere with CTL target cell binding or reorientation of the Golgi apparatus toward the target binding site. It is concluded that nicotinamide inhibits transmembrane signaling in CTL and thereby interferes with delivery of the lethal hit to targets.     

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)     

Docking of lytic granules at the immunological synapse in human CTL requires Vti1b-dependent pairing with CD3 endosomes

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8(+) T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

Reorientation and fusion of cytotoxic T lymphocyte granules after interaction with target cells as determined by high resolution cinemicrography

The interaction of murine cytotoxic T lymphocyte (CTL) clones with human lymphoblastoid target cells was studied in thin preparations by using high resolution cinemicrography. CTL not bound to target cells were morphologically polar, possessing a broad leading edge containing the nucleus, and a tapered tail containing a large number of granules. The CTL were observed to move by the extension of pseudopods from the leading edge. Initial contact with a target cell was made via the leading edge of the CTL. If the human target cell expressed the appropriate HLA antigen, distinct morphologic changes occurred in the CTL as early as 2 min after initial contact. The CTL rounded up, and the nucleus moved from a position adjacent to the zone of contact to be replaced by the cytoplasmic granules. Redistribution of the granules was completed as early as 10 min after initial contact was made. These morphologic changes did not occur when the CTL made contact with other CTL, or with target cells that did not express the appropriate HLA antigens. In studies that make use of Nomarski optics, an apparent fusion of CTL cytoplasmic granules with the membrane in the vicinity of the target cell contact area was observed 4 min after binding, and before granule reorientation was complete. These data provide direct evidence for the occurrence of both reorientation of the cytoplasmic contents and granule fusion in CTL with a time course similar to that of administration of the lethal hit.     

Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

A unique SNARE machinery for exocytosis of cytotoxic granules and platelets granules

Abstract

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells target infected or transformed cells with perforin-containing cytotoxic granules through immune synapses, while platelets secrete several types of granules which contents are essential for thrombosis and hemostasis. Recent work has culminated in the notion that an exocytic SNARE complex, based on a very similar set of components, is primarily responsible for exocytosis of the diverse granules in these different cell types. Granule exocytosis is, in particular, uniquely dependent on the atypical Q-SNARE syntaxin 11, its interacting partners of the Sec/Munc (SM) family, and is regulated by Rab27a. Mutations in these exocytic components underlie disease manifestations of familial hemophagocytic lymphohistiocytosis (FHL) subtypes, characterized by hyperactivation of the immune system, as well as platelet granule secretion defects. Here we discuss the key discoveries that led to the converging notion of the syntaxin 11-based exocytosis machinery for cytotoxic granules and platelet-derived granules.