Preparing the lethal hit: interplay between exo- and endocytic pathways in cytotoxic T lymphocytes

Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse. We highlight a recently proposed sequential process of fusion events at the IS upon target cell recognition. First, recycling endosomes fuse with the plasma membrane to deliver cargo required for the docking of cytotoxic granules. Second, cytotoxic granules arrive and fuse upon docking in a SNARE-dependent manner. Following fusion, membrane components of the cytotoxic granule are retrieved through endocytosis to ensure the fast, efficient serial killing of target cells that is characteristic of cytotoxic T lymphocytes.     

BAG-6, a jack of all trades in health and disease

BCL2-associated athanogene 6 (BAG-6) (also Bat-3/Scythe) was discovered as a gene product of the major histocompatibility complex class III locus. The Xenopus ortholog Scythe was first identified to act as an anti-apoptotic protein. Subsequent studies unraveled that the large BAG-6 protein contributes to a number of cellular processes, including apoptosis, gene regulation, protein synthesis, protein quality control, and protein degradation. In this context, BAG-6 acts as a multifunctional chaperone, which interacts with its target proteins for shuttling to distinct destinations. Nonetheless, as anticipated from its genomic localization, BAG-6 is involved in a variety of immunological pathways such as macrophage function and T_H1 response. Most recently, BAG-6 was identified on the plasma membrane of dendritic cells and malignantly transformed cells where it serves as cellular ligand for the activating natural killer (NK) cell receptor NKp30 triggering NK cell cytotoxicity. Moreover, target cells were found to secrete soluble variants of BAG-6 and release BAG-6 on the surface of exosomes, which inhibit or activate NK cell cytotoxicity, respectively. These data suggest that the BAG-6 antigen is an important target to shape a directed immune response or to overcome tumor-immune escape strategies established by soluble BAG-6. This review summarizes the currently known functions of BAG-6, a fascinating multicompetent protein, in health and disease.     

Cell-mediated cytotoxicity by natural killer and killer cells, lipid peroxidation and glutathione

In the course of spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral lymphocytes as effector cells, no lipid peroxidation occurred as measured by the production of ethane and thiobarbituric acid-reactive material. Furthermore, impairment of major cellular defense systems of target cells (K562 cells for SCMC, Chang liver cells for ADCC), by decreasing their glutathione content, had no effect on either lipid peroxidation or the cytotoxic response. These findings indicate that peroxidative damage is not a mechanism of NK and K cell-mediated cytotoxicity.     

B7-H6/NKp30 interaction: a mechanism of alerting NK cells against tumors

Natural killer (NK) cells are lymphocytes of the innate immune system that sense target cells through a panel of activating and inhibitory receptors. Together with NKG2D, the natural cytotoxicity receptors (NCRs) are major activating receptors involved in tumor cell detection. Although numerous NKG2D ligands have been identified, characterization of the molecules interacting with the NCRs is still incomplete. The identification of B7-H6 as a counter structure of the NCR NKp30 shed light on the molecular basis of NK cell immunosurveillance. We review here the current knowledge on NKp30 and B7-H6, and we discuss their potential role in anti-tumor immunity.     

Cortisol and immune interferon can interact in the modulation of human natural killer cell activity

This paper reports that cortisol at physiological concentrations minimizes the enhancement of human natural killer (NK) cell activity in vitro by immune interferon (IFN-gamma). This effect may be important for the regulation of NK cytotoxicity in vivo.     

Is the process of localized lysosomal exocytosis responsible for the cytolytic action of killer T-lymphocytes?]

In this paper, we formulate the hypothesis that in the process of target cell lysis a lysosomal enzyme regurgitation, performed by killer cells at the level of the target effector junction, accounts for the target lesion which precedes the lysis (lethal hit). This process of exocytosis, similar to the one described previously in polymorphonuclear neutrophils is supported by cytological studies performed directly on identified killers isolated by micromanipulation. Light and electron microscopy observations confirm a previous report which describes the effector cells rich in lysosomal bodies. In addition, when a killer cell is associated with a target cell to form a conjugate, lysosomes are concentrated near the cell junction and, after incubation at 37 degrees C, acid phosphatases may be detected at the junction. Lysosomal enzyme exocytosis explains why target lysis needs an effector target binding to occur and also the other conditions required for any exocytosis process such as Ca++ in the medium, integrity of the microtubular apparatus, a low level of cyclic AMP and energy dependancy.     

Perforin and its role in T lymphocyte-mediated cytolysis.

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Perforin and its role in T lymphocyte-mediated cytolysis

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack.Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Mechanisms of NK cell activation: CD4+ T cells enter the scene

Natural killer (NK) cells are innate lymphocytes involved in immunosurveillance through their cytotoxic activity and their capacity to secrete inflammatory cytokines. NK cell activation is necessary to initiate effector functions and results from a complex series of molecular and cellular events. We review here the signals that trigger NK cells and discuss recent findings showing that, besides antigen-presenting cells, T cells can play a central role in the initiation of NK cell activation in lymph nodes.     

Insights into NK cell biology from human genetics and disease associations

Rare human primary immunodeficiency disorders with extreme susceptibility to infections in infancy have provided important insights into immune function. Increasingly, however, primary immunodeficiencies are also recognized as a cause of other more common, often discrete, infectious susceptibilities. In a wider context, loss-of-function mutations in immune genes may also cause disorders of immune regulation and predispose to cancer. Here, we review the associations between human diseases and mutations in genetic elements affecting natural killer (NK) cell development and function. Although many such genetic aberrations significantly reduce NK cell numbers or severely impair NK cell responses, inferences regarding the role of NK cells in disease are confounded by the fact that most mutations also affect the development or function of other cell types. Still, data suggest an important role for NK cells in diseases ranging from classical immunodeficiency syndromes with susceptibility to viruses and other intracellular pathogens to cancer, autoimmunity, and hypersensitivity reactions.     

Virus-mediated inhibition of natural cytotoxicity receptor recognition

Natural killer (NK) cells are a part of the innate immune system that functions mainly to kill transformed and infected cells. Their activity is controlled by signals derived from a panel of activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs): NKp30, NKp44, and NKp46 (NCR1 in mice) are prominent among the activating NK cell receptors and they are, notably, the only NK-activating receptors that are able to recognize pathogen-derived ligands. In addition, the NCRs also recognize cellular ligands, the identity of which remains largely unknown. In this review, we summarize the current knowledge regarding viruses that are recognized by the NCRs, focusing on the diverse immune-evasion mechanisms employed by viruses to escape this detection. We also discuss the unique role the NCRs have in regulating NK cell activity with particular emphasis on the in vivo function of NKp46/NCR1.     

Suppression of natural killer cell activity in mouse spleen lymphocytes by several dopamine receptor antagonists

The effects of dopaminergic receptor inhibitors such as thiothixine (D₁/D₂), fluphenazine (D₁/D₂), trifluoperazine (D₁/D₂), pimozide (D₂), flupenthixol (D₁/D₂), (+/–)-SKF 83566 (D₁), and spiperone (D₂) on splenic natural killer (NK) cell cytotoxic activities were assessed in vitro using mouse spleen lymphocytes or enriched NK cells. Both the activities of the splenic NK cell cytotoxicity and the effector-target cell conjugation were suppressed by thiothixine, fluphenazine, and trifluoperazine at concentrations from 2.64 to 14.78 M. In addition, the augmentation of the cytolytic activity of NK cells induced by interferon- or interleukin-2 was antagonized by pretreatment with these neuroleptic compounds. However, neither the splenic NK cell cytotoxicity nor the effector-target cell conjugation were affected by treatment with other neuroleptic compounds such as pimozide, flupenthixol, (+/–)-SKF 83566, and spiperone. Thus, it appears that neuroleptic compounds such as thiothixine, fluphenazine, and trifluoperazine may act through the mechanisms other than a dopaminergic pathway to affect the NK cell-target cell interaction.     

Natural killer cell activation by dendritic cells: balancing inhibitory and activating signals

Natural killer (NK) cells have originally been identified by their spontaneous cytolytic potential against tumor cells, which, however, might result from pre-activation due to prior pathogen exposure. Resting NK cells, on the contrary, require activation by bystander antigen-presenting cells to reach their full functional competence. In this review, we will summarize studies on how dendritic cells (DCs), the most potent type of antigen-presenting cell, communicate with human NK cells to activate them in secondary lymphoid organs and to integrate signals from activated NK cells at sites of inflammation for their own maturation. Furthermore, we will review aspects of the immunological synapse, which mediates this cross-talk. These studies provide the mechanistic understanding of how mature DCs can activate NK cells and survive to go on for the activation of adaptive immunity. This feature of DCs, to activate different waves of immune responses, could be harnessed for immunotherapies, including vaccinations.     

The role of HLA-G in immunity and hematopoiesis

The non-classical HLA class I molecule HLA-G was initially shown to play a major role in feto–maternal tolerance. Since this discovery, it has been established that HLA-G is a tolerogenic molecule which participates to the control of the immune response. In this review, we summarize the recent advances on (1) the multiple structures of HLA-G, which are closely associated with their role in the inhibition of NK cell cytotoxicity, (2) the factors that regulate the expression of HLA-G and its receptors, (3) the mechanism of action of HLA-G at the immunological synapse and through trogocytosis, and (4) the generation of suppressive cells through HLA-G. Moreover, we also review recent findings on the non-immunological functions of HLA-G in erythropoiesis and angiogenesis.     

Interaction of volkensin with HeLa cells: binding,uptake, intracellular localization,degradation and exocytosis

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10^-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.Received 21 April 2004; received after revision 26 May 2004; accepted 9 June 2004     

Cytotoxicity of mouse peritoneal cells after alloimmunization]

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.     

Functional inhibition of isolated pancreatic cells, new technic for the detection of macrophage cytotoxicity]

It is usually accepted that macrophages "activated" by lymphokines may be found cytotoxic against tumoral target cells but show no detectable cytotoxicity in in vitro tests using normal non tumoral cells as target cells. These data have been obtained mainly with the chromium-release test. The present paper describes a new test using normal isolated pancreatic cells as target cells and evaluating the effect of activated or non-activated macrophages on the insulin secretion response to glucose stimulation. The results show a striking decrease in this response following an 18-hr incubation of pancreatic islet cells with activated macrophages, as compared to that of the same cells incubated with control macrophages. This is clear evidence that activated macrophages may alter normal cells and suggests that their cytotoxic properties are not restricted to tumoral target cells.     

Putting a brake on synaptic vesicle endocytosis

In chemical synapses, action potentials evoke synaptic vesicle fusion with the presynaptic membrane at the active zone to release neurotransmitter. Synaptic vesicle endocytosis (SVE) then follows exocytosis to recapture vesicle proteins and lipid components for recycling and the maintenance of membrane homeostasis. Therefore, SVE plays an essential role during neurotransmission and is one of the most precisely regulated biological processes. Four modes of SVE have been characterized and both positive and negative regulators have been identified. However, our understanding of SVE regulation remains unclear, especially the identity of negative regulators and their mechanisms of action. Here, we review the current knowledge of proteins that function as inhibitors of SVE and their modes of action in different forms of endocytosis. We also propose possible physiological roles of such negative regulation. We believe that a better understanding of SVE regulation, especially the inhibitory mechanisms, will shed light on neurotransmission in health and disease.