Journal articles on the topic 'Lytic granule'
To see the other types of publications on this topic, follow the link: Lytic granule.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Lytic granule.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
HSU, HSIANG-TING, Dixita Viswanath, Emily Mace, Athanasia Christakou, Martin Wiklund, Björn Önfelt, and Jordan Orange. "Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 131.6. http://dx.doi.org/10.4049/jimmunol.196.supp.131.6.
Abstract:
Abstract NK cell activation triggers serial events leading to lysis of diseased cells: 1) granule convergence, preformed lytic granules rapidly cluster to the microtubule-organizing center (MTOC), 2) granule/MTOC polarization to the immunological synapse, and 3) degranulation, exocytosis of lytic contents onto the target cell. Granule convergence requires LFA-1 signaling and dynein motor function. Yet, it remains elusive how lytic granule convergence contributes to NK cell cytotoxicity. Using Drosophila S2 cells, we regulate NK cell signal inputs to precisely control granule convergence and degranulation. In the absence of LFA-1 signaling, the average distance of lytic granules to the MTOC increased >1.3 fold, indicating diffuse granule localization. Using imaging flow cytometery, we determined that without LFA-1, the synaptic accumulation and degranulation of lytic granules decreased by 3.3 and 2.5 fold, respectively suggesting undirected granule release. Using an ultrasound-guided-acoustic-trap-microscopy system to enforce live single NK cell with multiple target cell contacts, we determined killing efficiency with real-time granule tracking. NK cells activated by CD16 alone without co-engaging LFA-1 showed ~40% lower targeted killing and ~30% higher non-specific killing of neighboring unlabeled “bystander” S2 cells. Finally, we used ciliobrevin D, a dynein inhibitor, to physically block granule convergence in NK cells conjugated with physiologically relevant target cells. Dynein-inhibited NK cells caused ~36% increase in bystander killing. Thus, we demonstrated that NK cells converge lytic granules to improve the efficiency of targeted lytic granule secretion and prevent collateral damage to neighboring tissue.
2
Pattu, Varsha, Mahantappa Halimani, Monika Peuschel, Elmar Krause, and Jens Rettig. "Regulation of lytic granules for normal cytotoxic T lymphocyte function (P1140)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 64.17. http://dx.doi.org/10.4049/jimmunol.190.supp.64.17.
Abstract:
Abstract Cytotoxic T lymphocytes (CTLs) function to kill bacterial and viral infected target cells by releasing cytotoxic components such as perforin and granzymes that are contained within lytic granules, into the infected cell. Release occurs via the fusion of lytic granules at the contact zone between infected cell and CTL, the immunological synapse (IS). Defect in the fusion of lytic granules results in the dysfunction of CTLs, the underlying cause of the fatal disease Familial Hemophagocytic Lymphohistiocytosis (FHL). In order for fusion to occur, lytic granules must first arrive, dock and prime at the IS. Mutations within Munc 13-4, a priming factor, for lytic granules, results in FHL subtype-3. We have identified the presence of additional priming factors in CTLs. Targeted gene knockouts of these priming factors resulted in defects in lytic granule fusion at the immunological synapse. These findings demonstrate the presence of more regulatory mechanisms for the most critical event in CTLs, lytic granule fusion. Using high-resolution microscopy and evanescent wave imaging we aim to dissect the precise regulation of lytic granule fusion, which in turn would give us more insight into the regulation and function of CTLs.
3
Pattu, Varsha, Ulf Matti, Mahantappa Halimani, Lisa Weins, and Jens Rettig. "Identification of the v-SNARE required for lytic granule fusion in cytotoxic T lymphocytes (176.27)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 176.27. http://dx.doi.org/10.4049/jimmunol.188.supp.176.27.
Abstract:
Abstract Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes, which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the SNARE proteins involved in lytic granule fusion in CTLs remain unknown. Using a targeted gene knock in strategy we identified the v-SNARE that is required for lytic granule fusion in CTLs. We first used super resolution microscopy combined with careful quantification to identify the v-SNARE that is localized exclusively to lytic granules in primary mouse CTLs. By specifically cleaving the v-SNARE with a bacterial neurotoxin and by gene knock down we show a complete loss of lytic granule fusion by two independent methods - 1) LAMP1 based degranulation assay and 2) real time imaging of exocytosis using TIRF microscopy. We therefore conclude that the v-SNARE that is localizing to the lytic granules is mediating their final fusion at the IS, thereby delivering the lethal hit needed for target cell killing.
4
Burkhardt, J. K., J. M. McIlvain, M. P. Sheetz, and Y. Argon. "Lytic granules from cytotoxic T cells exhibit kinesin-dependent motility on microtubules in vitro." Journal of Cell Science 104, no. 1 (January 1, 1993): 151–62. http://dx.doi.org/10.1242/jcs.104.1.151.
Abstract:
One major mechanism of cell-mediated cytolysis is the polarized secretion of lytic granules, a process which is highly dependent on microtubules. We isolated lytic granules from murine cytotoxic T cells and tested their ability to bind to and move along microtubules in vitro. In the presence of a motor-containing supernatant, the granules bound to the microtubules and moved along them at an average maximal rate of 1 microns/second. Virtually every granule could bind to microtubules, and about half translocated within a few seconds of binding. Motility required exogenous cytosolic motors, hydrolyzable nucleotides, and an intact granule membrane. Although the motor preparation used to support granule movement contains both plus- and minus-end-directed motor proteins, granule movement was strongly biased toward microtubule plus-ends. Inactivation of cytoplasmic dynein had little effect on granule binding and movement, but immuno-depletion of kinesin from the motor preparation inhibited granule binding by 50%. These results indicate that most granule movement in this assay is mediated by kinesin. The speed and direction of granule movement in vitro are sufficient to account for the release of lytic granules in the intact T cell. This model system should be valuable for studying the interactions of secretory granules with microtubules, and for identifying the regulatory factors involved.
5
Wilton, Katelynn Marie, and Daniel D. Billadeau. "Vasodilator Stimulated Phosphoprotein (VASP)-Mediated Actin Polymerization Drives Natural Killer Cell Granule Convergence." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 170.7. http://dx.doi.org/10.4049/jimmunol.200.supp.170.7.
Abstract:
Abstract Natural killer (NK) cells eliminate transformed and malignant cells through a highly orchestrated series of actin and microtubule cytoskeletal rearrangements, culminating in the secretion of preformed lytic granules. Despite the importance of actin and microtubules, the roles of many molecular cytoskeletal regulators in this process have not yet been ascertained. In this study, we investigated the role of Vasodilator Stimulated Phosphoprotein (VASP), an actin regulatory protein, in NK cell-mediated cytotoxicity. Interestingly, we found that depletion of VASP inhibits NK cell cytotoxicity and that VASP co-localizes with F-actin at the NK cell – target cell cytotoxic synapse. Surprisingly, despite their co-localization, VASP depletion did not affect either F-actin accumulation at the cytotoxic synapse or conjugate formation. Instead, we found that a minority of VASP localized and biochemically co-purified with cytolytic granules. Although VASP knockdown did not affect microtubule organizing center (MTOC) polarization, it did dramatically impact lytic granule convergence to the MTOC. Significantly, VASP depletion decreased F-actin accumulation on cytolytic granules and depolymerization of F-actin with Latrunculin A impaired lytic granule convergence. Taken together, these results demonstrate a novel requirement for VASP-mediated actin polymerization in NK cell granule convergence and cytotoxicity and highlight a role for F-actin in lytic granule convergence.
6
Liu, Dongfang, Tobias Meckel, and Eric Long. "Distinct Roles of Rab27a in Lytic Granule Movement at the Plasma Membrane and in the Cytosol (89.47)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 89.47. http://dx.doi.org/10.4049/jimmunol.184.supp.89.47.
Abstract:
Abstract The small GTPase Rab27a is essential for degranulation by cytotoxic T cells and natural killer (NK) cells. To test its role in granule movement we visualized lytic granules in live, unstimulated NK cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional (2D) total internal reflection fluorescence (TIRF) microscopy. Movement was tracked and quantified by automated image analysis of thousands of granules. Pharmacological inhibitors showed that long-range, directed 3D movement required microtubules (MT) but not actin, whereas 2D movement under the plasma membrane required actin dynamics. The role of Rab27a was examined by shRNA-mediated knockdown in a human NK cell line and by using NK cells from Rab27a-mutant Ashen mice. Fewer lytic granules reached the plasma membrane in the absence of Rab27a. Rab27a promoted actin-dependent, directed movement under the plasma membrane. In contrast, Rab27a reduced tracking along MT and diffusion of lytic granules in the cytosol. Therefore, Rab27a promotes delivery of lytic granules to, and movement at the plasma membrane, and has a distinct role in the cytosol, where it serves as tether to constrain lytic granule movement.
7
Peña, S. V., D. A. Hanson, B. A. Carr, T. J. Goralski, and A. M. Krensky. "Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins." Journal of Immunology 158, no. 6 (March 15, 1997): 2680–88. http://dx.doi.org/10.4049/jimmunol.158.6.2680.
Abstract:
Abstract CTL and NK cells share a common cytolytic mechanism that involves regulated exocytosis of lytic molecules stored within cytoplasmic granules. Here we describe the processing, subcellular localization, and function of a T and NK cell-specific granule protein that shares homology with small, lytic granule-associated molecules. The gene coding for this protein, 519, is expressed late after T cell activation. Antisera raised against a 519/glutathione-S-transferase fusion protein and a series of peptides derived from the 519 protein sequence permitted the identification of two small CTL protein products of 15 and 9 kDa that are exocytosed after stimulation through the TCR. The 9-kDa product is a processed form of 519 and differs from the 15-kDa product in both its amino and carboxyl terminus. While both 519 proteins are found in cytoplasmic granules, the 9-kDa form is also present in dense, highly cytolytic granules. Functional studies indicate that this protein is lytic against tumor cell targets. The cell type- and stage-specific expression pattern of 519 along with its subcellular localization are reminiscent of molecules that play a vital role in granule-mediated cytolysis by CTL and NK cells. Its lytic activity suggests the involvement of 519 in CTL effector function.
8
Sanborn, Keri B., Gregory D. Rak, Saumya Y. Maru, Analisa Difeo, John A. Martignetti, Remi Favier, Pinaki P. Banerjee, and Jordan S. Orange. "Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse (134.13)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 134.13. http://dx.doi.org/10.4049/jimmunol.182.supp.134.13.
Abstract:
Abstract NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for the directed secretion of their contents to occur. We examined the role of myosin IIA in facilitating this pre-final step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a nonsense mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as their final approach to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation. Supported by NIH grants R01 AI067946 (JSO) and T32-GM07229 (KBS).
9
Kurowska, Mathieu, Nicolas Goudin, Nadine T. Nehme, Magali Court, Jérôme Garin, Alain Fischer, Geneviève de Saint Basile, and Gaël Ménasché. "Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex." Blood 119, no. 17 (April 26, 2012): 3879–89. http://dx.doi.org/10.1182/blood-2011-09-382556.
Abstract:
Abstract Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.
10
Halimani, Mahantappa, Varsha Pattu, Christian Junker, Misty Marshall, Eva Schwarz, Elmar Krause, Ulf Matti, Markus Hoth, and Jens Rettig. "The role of syntaxin11 in cytotoxic T lymphocytes (109.26)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 109.26. http://dx.doi.org/10.4049/jimmunol.186.supp.109.26.
Abstract:
Abstract Cytotoxic T Lymphocytes (CTLs) form immunological synapses (IS) with antigen presenting cells in order to kill them. Killing requires release of lytic granules which occurs via exocytosis, and is dependent on SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. Syntaxin11 is a SNARE protein that is predicted to be involved in the fusion of lytic granules in CTLs, since mutations within the coding region of Syntaxin11 lead to Familial Hemophagocytic Lymphohistiocytosis (FHL4), an immune disorder caused by impaired granule exocytosis. We have investigated the contribution of Syntaxin11 to lytic granule release. We used live cell imaging to observe the trafficking of full length Syntaxin11. Using CD3 specific antibodies as a marker for a functional IS, we show that Syntaxin11 accumulates at the IS after CTL polarization and conjugation with the APC has occurred. Syntaxin11 was also associated with Munc18-2, consistent with the role of Munc18-2 as a syntaxin chaperone and as a docking factor. Using a Syntaxin11 antibody we also confirmed the subcellular localization of Syntaxin11 in fixed CTLs with confocal microscopy. Syntaxin11 was associated with Munc18-2 and CD3 at the IS in fixed CTLs, consistent with our observations in live cell imaging of CTLs. These results support the conclusion that Syntaxin11 is involved in lytic granule exocytosis.
11
Berke, G., and D. Rosen. "Highly lytic in vivo primed cytolytic T lymphocytes devoid of lytic granules and BLT-esterase activity acquire these constituents in the presence of T cell growth factors upon blast transformation in vitro." Journal of Immunology 141, no. 5 (September 1, 1988): 1429–36. http://dx.doi.org/10.4049/jimmunol.141.5.1429.
Abstract:
Abstract Demonstration of C-like "rings," lytic granules, and the Ca2+-dependent lytic proteins--perforin/cytolysin--thereof, in certain cytocidal lymphocytes has led to the hypothesis of a mechanism of lytic granule-exocytosis and a common terminal lytic step in lymphocyte and C-induced lysis. However, neither cytolytic granules, nor formation of C-like rings during lysis have been detected in mature, highly potent, peritoneal exudate CTL (PEL) derived directly from the site of allograft rejection or in cytocidal hybridomas derived from them (PEL hybridomas). We now report that when stimulated in vitro in the presence of Con A supernatant, as a source of T cell growth factors (TCGF) or rIL-2, small in vivo primed PEL transform into large, dividing cytolytic T cells (PEL blasts) that express the same lytic specificity of the original PEL in short term lytic assays. The PEL blasts, in contrast to PEL, possess massive quantities of lytic granules, and protease (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) (BLT-esterase) activity as well as non-specific, cell-mediated cytolytic activity in a long term (4-h) assay. These results suggest that the proposed lytic mechanism involving exocytosis of lytic granules, perforin, and BLT-esterases and the formation of 10 to 20-nm lesions may apply to lysis induced by granule-containing effectors such as large granular lymphocytes and TCGF-dependent CTL lines, such as PEL blasts. However, killing by mature, in vivo primed CTL, such as PEL or their hybridomas, appears to be effected through an alternative, contact-induced, self-destruction process(es) of the target not involving secretory lytic granules or the above lesions. Hence, although the expression of lytic granules and BLT-esterase activities in cytolytic lymphocytes devoid of these components is induced by TCGF, these cellular constituents are not necessary for the expression of CTL-mediated target cell lysis by mature effector cells.
12
Hsu, Hsiang-Ting, Emily M. Mace, Alexandre F. Carisey, Dixita I. Viswanath, Athanasia E. Christakou, Martin Wiklund, Björn Önfelt, and Jordan S. Orange. "NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing." Journal of Cell Biology 215, no. 6 (November 30, 2016): 875–89. http://dx.doi.org/10.1083/jcb.201604136.
Abstract:
Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.
13
Krzewski, Konrad, Aleksandra Gil-Krzewska, Victoria Nguyen, Giovanna Peruzzi, and John E. Coligan. "LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity." Blood 121, no. 23 (June 6, 2013): 4672–83. http://dx.doi.org/10.1182/blood-2012-08-453738.
14
Kapnick, Senta, Alex Ritter, Gillian Griffiths, Jennifer Lippincott-Schwartz, and Pamela Schwartzberg. "Dynamic modulation of cortical actin at the immunological synapse controls lytic granule secretion in cytotoxic T lymphocytes." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 151.4. http://dx.doi.org/10.4049/jimmunol.198.supp.151.4.
Abstract:
Abstract CD8+ cytotoxic T lymphocytes (CTLs) are critical for eliminating virally infected cells. Upon target recognition, CTLs reorganize their actin cytoskeleton to facilitate the polarized secretion of specialized granules containing lytic proteins that kill cells. Because a single CTL can kill multiple targets, granule secretion must be tightly regulated, ensuring only appropriate cells are killed, and preserving granules for serial killing. However, how CTLs regulate secretion remains unclear. Here, we used live confocal and TIRF microscopy to evaluate how cortical actin affects granule secretion at the membrane. While previous work has shown that clearance of actin at the synapse precedes granule secretion, we find that after granule fusion, cortical actin recovers and no further secretion is observed. Depolymerization of recovered actin led to resumed granule fusion, suggesting that recovered actin acts as a barrier to prevent further lytic granule secretion. We further show that CTLs from mice unable to secrete granules due to a Rab27a mutation that prevents granule fusion, failed to recover cortical actin at the synapse. This suggests that granule fusion itself triggers actin recovery in CTLs. Thus, cortical actin both regulates, and is regulated by, secretion. Finally, using fluorescent reporters, we correlated the clearance and recovery of actin with PIP2. PIP2 binds numerous actin regulatory proteins, suggesting phosphatidylinositol distribution in the membrane as a potential mechanism through which CTLs regulate the density of cortical actin during killing. Our work provides insight into actin-related mechanisms regulating CTL secretion that may serial killing capacity during immune responses.
15
Andzelm, Milena M., Xi Chen, Konrad Krzewski, Jordan S. Orange, and Jack L. Strominger. "Myosin IIA is required for cytolytic granule exocytosis in human NK cells." Journal of Experimental Medicine 204, no. 10 (September 17, 2007): 2285–91. http://dx.doi.org/10.1084/jem.20071143.
Abstract:
Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector–target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.
16
Giraudo, Claudio, Waldo Spessott, and Margaret McCormick. "SNARE protein requirements for cytotoxic T lymphocyte-mediated cell killing (P1032)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 65.17. http://dx.doi.org/10.4049/jimmunol.190.supp.65.17.
Abstract:
Abstract Cytotoxic T Lymphocytes (CTLs) and Natural Killer (NK) cells selectively kill virally infected or cancer cells by releasing the content of lytic granules at the contact area with target cells called immunological synapse (IS). SNAREs mediate the maturation and exocytosis of “fully-armed” lytic granules at the IS. Deregulation of SNARE-mediated fusion events, such as in Familial Hemophagocytic Lymphohystiocytosis-4 and -5, in which Syntaxin11 and Syntaxin Binding Protein-2 are mutated, respectively; severely impaired lytic granule release and cytotoxicity of CTL and NK-cells. We have identified cognate SNAREs that specifically interact with Syntaxin11 to perform its function during CTL activation and lytic granule release. Biochemical experiments showed that these interacting proteins can form stable SNARE complexes and confirmed the specificity of these interactions. Stimulated Emission Depletion (STED) Superresolution microscopy studies revealed two pools of Syntaxin 11 in CTLs, one localized to a M6PR-containing vesicular compartment, and one localized at the plasma membrane. We have precisely visualized the localization patterns Sytanxin11-interacting partners and other membrane trafficking proteins involved in this process. These results support the conclusion that the identified Syntaxin11-containing SNARE complex plays a crucial role during CTL-mediated cytotoxicity and FHL pathophysiology.
17
Tschopp, J., D. Masson, and S. Schäfer. "Inhibition of the lytic activity of perforin by lipoproteins." Journal of Immunology 137, no. 6 (September 15, 1986): 1950–53. http://dx.doi.org/10.4049/jimmunol.137.6.1950.
Abstract:
Abstract Cytoplasmic granules isolated from cytolytic T lymphocytes (CTL) lyse red blood cells or tumor cell lines in a nonspecific manner. The activity of highly purified granules was inhibited by human or rabbit serum at dilutions as high as 1/10,000. The main inhibitory activity of human serum was isolated by chromatography and was determined to be high density lipoprotein (HDL). HDL not only inhibited at a concentration of 70 ng/ml the lytic activity of isolated granules, but also of the purified, pore-forming protein perforin present in the granules. Purified low density lipoprotein was equally active. Because the CTL granule activity was inhibited by pure egg lecithin vesicles at a concentration equivalent to the phospholipid content of lipoproteins, the lipid portion of lipoproteins is the likely candidate for granule inactivation. Lipoproteins also decreased in a dose-dependent manner the cytotoxic activity of intact cytolytic T cells. However, cytotoxicity was not completely suppressed, and only in the case of CTL exhibiting low efficiency in killing their targets. It is proposed that lipoproteins inactivate perforin and may thereby inhibit a possible lysis of innocent bystander cells.
18
Sanborn, Keri B., Emily M. Mace, Gregory D. Rak, Analisa Difeo, John A. Martignetti, Alessandro Pecci, James B. Bussel, Rémi Favier, and Jordan S. Orange. "Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity." Blood 118, no. 22 (November 24, 2011): 5862–71. http://dx.doi.org/10.1182/blood-2011-03-344846.
Abstract:
Abstract Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.
19
Halimani, Mahantappa, Varsha Pattu, Christian Junker, Misty Marshall, Ulf Matti, Eva Schwarz, Elmar Krause, Markus Hoth, and Jens Rettig. "Function of Syntaxin11 in cytotoxic T lymphocytes (121.9)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 121.9. http://dx.doi.org/10.4049/jimmunol.188.supp.121.9.
Abstract:
Abstract Cytotoxic T Lymphocytes (CTLs) form immunological synapses (IS) with antigen presenting cells to kill them. Killing requires the fusion of lytic granules at the IS to release the containing cytotoxic molecules (perforin and granzymes). The fusion process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. Since mutations within the coding region of syntaxin11 lead to Familial Hemophagocytic Lymphohistiocytosis (FHL4), an immune disorder caused by impaired granule exocytosis. We have investigated the contribution of syntaxin11 to lytic granule release. Using CD3 specific antibodies as a marker for a functional IS, we show that syntaxin11 accumulates at the IS in CTLs. Syntaxin11 was also co-localized with Munc18-2, a docking factor in agreement with the role of Munc18-2 as a syntaxin chaperone in other cell types. Using high resolution nanoscopy (structured illumination microscopy) we determined the subcellular localization of syntaxin11 in CTLs with a syntaxin11 specific antibody. Syntaxin11 was associated with recycling endosomes and mannose 6-phosphate receptor (M6PR) positive compartments. Functional assays using Syntaxin11 specific siRNA (knockdown studies) show a reduction in degranulation and killing efficiency in contrast to control siRNA transfected CTLs. These results support the conclusion that syntaxin11 is required for lytic granule exocytosis.
20
Li, Yu, and Jordan Scott Orange. "Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 74.5. http://dx.doi.org/10.4049/jimmunol.204.supp.74.5.
Abstract:
Abstract NK cells are resistant to autolysis when they kill a target cell using perforin secretion into the lytic immunological synapse. Perforin makes pores in target cell membranes allowing delivery of pro-apoptotic enzymes. Despite the fact that the perforin-containing organelles (lytic granules) are released in close range to both the NK and target cell, the NK cell membrane is protected. How NK cells avoid autolysis during degranulation is perplexing. We demonstrate that NK cells are protected from perforin by their densely packed presynaptic membranes. When treated with 7-ketocholesterol lipid packing is reduced in NK cells, making them susceptible to perforin mediated autolysis after degranulation. Using advanced imaging, we showed that lytic granules themselves have endogenously densely packed membranes. Importantly, during degranulation lytic granule-cell membrane fusion further reinforces local presynaptic membrane packing. This provides enhanced membrane protection at the specific sites of degranulation where NK cells face maximum local concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line utilizes the same strategy to evade NK cellmediated killing. These cells are perforin-resistant owing to a densely packed postsynaptic membrane. By disrupting membrane packing, we effectively switched them to an NK-susceptible state. Overall, we reveal an unexpected role for lipid membranes in NK cell functionality and that degranulation uses lytic granule membrane packing to create local “shields” against autolysis. Furthermore, lipid membrane modulation could alter the susceptibility of perforin-resistant cancer cells and should be considered in cytotoxic cell therapies.
21
Eitler, Jiri, Natalie Wotschel, Nicole Miller, Laurent Boissel, Hans G. Klingemann, Winfried Wels, and Torsten Tonn. "Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting." Journal for ImmunoTherapy of Cancer 9, no. 1 (January 2021): e001334. http://dx.doi.org/10.1136/jitc-2020-001334.
Abstract:
BackgroundOn encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors.MethodsHere, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92.ResultsUnmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals.ConclusionsThese observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarization
22
Darwich, Abbass, Alessandra Silvestri, Mohamed-Reda Benmebarek, Juliette Mouriès, Bruno Cadilha, Alessia Melacarne, Lapo Morelli, et al. "Paralysis of the cytotoxic granule machinery is a new cancer immune evasion mechanism mediated by chitinase 3-like-1." Journal for ImmunoTherapy of Cancer 9, no. 11 (November 2021): e003224. http://dx.doi.org/10.1136/jitc-2021-003224.
Abstract:
BackgroundNatural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step for cancer progression through yet unidentified mechanisms.MethodsNK cell antibody-dependent cellular cytotoxicity (ADCC) is a major determinant of the clinical efficacy of some therapeutic antibodies including the anti-HER2 Trastuzumab. Thus, we screened sera of Trastuzumab-resistant HER2 +patients with breast cancer for molecules that could inhibit NK cell ADCC. We validated our findings in vitro using cytotoxicity assays and confocal imaging of the lytic granule machinery and in vivo using syngeneic and xenograft murine models.ResultsWe found that sera from Trastuzumab-refractory patients could inhibit healthy NK cell ADCC in vitro. These sera contained high levels of the inflammatory protein chitinase 3-like 1 (CHI3L1) compared with sera from responders and healthy controls. We demonstrate that recombinant CHI3L1 inhibits both ADCC and innate NK cell cytotoxicity. Mechanistically, CHI3L1 prevents the correct polarization of the microtubule-organizing center along with the lytic granules to the IS by hindering the receptor of advanced glycation end-products and its downstream JNK signaling. In vivo, CHI3L1 administration drastically impairs the control of NK cell-sensitive tumors, while CHI3L1 blockade synergizes with ADCC to cure mice with HER2 +xenografts.ConclusionOur work highlights a new paradigm of tumor immune escape mediated by CHI3L1 which acts on the cytotoxic machinery and prevents granule polarization. Targeting CHI3L1 could mitigate immune escape and potentiate antibody and cell-based immunotherapies.
23
Li, Yu, and Jordan S. Orange. "Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis." PLOS Biology 19, no. 8 (August 3, 2021): e3001328. http://dx.doi.org/10.1371/journal.pbio.3001328.
Abstract:
Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.
24
Kataoka, T., K. Takaku, J. Magae, N. Shinohara, H. Takayama, S. Kondo, and K. Nagai. "Acidification is essential for maintaining the structure and function of lytic granules of CTL. Effect of concanamycin A, an inhibitor of vacuolar type H(+)-ATPase, on CTL-mediated cytotoxicity." Journal of Immunology 153, no. 9 (November 1, 1994): 3938–47. http://dx.doi.org/10.4049/jimmunol.153.9.3938.
Abstract:
Abstract An inhibitor of vacuolar type H(+)-ATPase, concanamycin A (CMA), inhibited the specific cytolytic activity of a CD8+ CTL clone, OE4. The inhibitory effect was observed when the effector cells, and not the target cells, were pretreated with CMA. CMA did not seem to inhibit early events, inasmuch as effector/target conjugate formation remained unaffected. Although CMA treatment of OE4 resulted in a slight decrease in the efficiency of granule exocytosis in response to anti-CD3 stimulation, the most prominent effect was a marked reduction of perforin activity and DNA degradation activity in lytic granules. Western blotting analysis indicated a drastic decrease in the amount of perforin in CMA-treated cells. Fluorescent microscopic observation of OE4 stained with acridine orange indicated that CMA raised the pH of the lytic granules. Under transmission electron microscopy, striking morphologic changes in cytoplasmic granular structures were observed after CMA treatment of OE4. The lytic granules of OE4 had homogeneously stained large cores and numerous small vesicles that filled peripheral areas. In contrast, the lytic granules of CMA-treated OE4 showed irregular shapes with no small vesicles, but with cores that became rough and loose. Vacuoles with no structure in them were seen occasionally. These results suggest that acidification through vacuolar type H(+)-ATPase is essential to maintain the structure and function of lytic granules.
25
Capuano, Cristina, Rossella Paolini, Rosa Molfetta, Luigi Frati, Angela Santoni, and Ricciarda Galandrini. "PIP2-dependent regulation of Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway." Blood 119, no. 10 (March 8, 2012): 2252–62. http://dx.doi.org/10.1182/blood-2010-12-324160.
Abstract:
Abstract Cytotoxic lymphocytes clear infected and transformed cells by releasing the content of lytic granules at cytolytic synapses, and the ability of cytolytic effectors to kill in an iterative manner has been documented previously. Although bidirectional trafficking of cytolytic machinery components along the endosomal pathway has begun to be elucidated, the molecular mechanisms coordinating granule retrieval remain completely unexplored. In the present study, we focus on the lytic granule priming factor Munc13-4, the mutation of which in familial hemophagocytic lymphohistiocytosis type 3 results in a profound defect of cytotoxic function. We addressed the role of phosphatidylinositol (4,5)-bisphosphate (PIP2) in the regulation of Munc13-4 compartmentalization. We observed that in human natural killer cells, PIP2 is highly enriched in membrane rafts. Granule secretion triggering induces a transient Munc13-4 raft recruitment, followed by AP-2/clathrin–dependent internalization. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ gene silencing leads to the impairment of granule secretion associated with increased levels of raft-associated Munc13-4, which is attributable to a defect in AP-2 membrane recruitment. In such conditions, the ability to subsequently kill multiple targets was significantly impaired. These observations indicate that Munc13-4 reinternalization is required for the maintenance of an intracellular pool that is functional to guarantee the serial killing potential.
26
Ritter, Alex T., Senta M. Kapnick, Sricharan Murugesan, Pamela L. Schwartzberg, Gillian M. Griffiths, and Jennifer Lippincott-Schwartz. "Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes." Proceedings of the National Academy of Sciences 114, no. 32 (July 17, 2017): E6585—E6594. http://dx.doi.org/10.1073/pnas.1710751114.
Abstract:
CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.
27
Wilton, Katelynn M., and Daniel D. Billadeau. "VASP Regulates NK Cell Lytic Granule Convergence." Journal of Immunology 201, no. 10 (October 3, 2018): 2899–909. http://dx.doi.org/10.4049/jimmunol.1800254.
28
Krzewski, Konrad, Aleksandra Gil-Krzewska, James Watts, John Coligan, and Jack Strominger. "Both VAMP4 and VAMP7 are indispensable for NK cell cytotoxicity: the requirement for two R-SNARE proteins in granule exocytosis. (89.24)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 89.24. http://dx.doi.org/10.4049/jimmunol.184.supp.89.24.
Abstract:
Abstract Natural killer (NK) cells kill cancer and virus-infected cells through their cytolytic activity. The last step in NK cell cytotoxicity, fusion of lytic granules with the plasma membrane resulting in exocytosis of granule content, is still poorly understood. Proteins from the SNARE family mediate all membrane fusion events in the cell. Here we show that NK cells express all seven members of the R-SNARE subgroup. Two of these R-SNARE proteins, VAMP4 and VAMP7, are associated with lytic granules during cytotoxic interactions. However, only VAMP7 co-localizes with granules in non-activated cells. The differences in co-localization of these two R-SNARE proteins, with respect to perforin-containing cytotoxic granules, indicate that they have different functions in exocytosis. Using the tumor NK cell line, YTS, we show that RNAi-mediated disruption of expression of either VAMP4 or VAMP7 inhibits release of lytic granules and severely impairs NK cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in cytokine secretion and endocytosis in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK cell activity than VAMP4.
29
Griffiths, G. M., and S. Isaaz. "Granzymes A and B are targeted to the lytic granules of lymphocytes by the mannose-6-phosphate receptor." Journal of Cell Biology 120, no. 4 (February 15, 1993): 885–96. http://dx.doi.org/10.1083/jcb.120.4.885.
Abstract:
To investigate the question of whether lytic granules share a common biogenesis with lysosomes, cloned cytolytic T cell lines were derived from a patient with I-cell disease. The targeting of two soluble lytic granule components, granzymes A and B, was studied in these cells which lack a functional mannose-6-phosphate (Man-6-P) receptor-mediated pathway to lysosomes. Using antibodies and enzymatic substrates to detect the lytic proteins, I-cells were found to constitutively secrete granzymes A and B in contrast to normal cells in which these proteins were stored for regulated secretion. These results suggest that granzymes A and B are normally targeted to the lytic granules of activated lymphocytes by the Man-6-P receptor. In normal cells, the granzymes bear Man-6-P residues, since the oligosaccharide side chains of granzymes A and B, as well as radioactive phosphate on granzyme A from labeled cells, were removed by endoglycosidase H (Endo H). However, in I-cells, granzymes cannot bear Man-6-P and granzyme B acquires complex glycans, becoming Endo H resistant. Although the levels of granzymes A and B in cytolytic I-cell lymphocytes are < 30% of the normal levels, immunolocalization and cell fractionation of granzyme A demonstrated that this reduced amount is correctly localized in the lytic granules. Therefore, a Man-6-P receptor-independent pathway to the lytic granules must also exist. Cathepsin B colocalizes with granzyme A in both normal and I-cells indicating that lysosomal proteins can also use the Man-6-P receptor-independent pathway in these cells. The complete overlap of these lysosomal and lytic markers implies that the lytic granules perform both lysosomal and secretory roles in cytolytic lymphocytes. The secretory role of lytic granules formed by the Man-6-P receptor-independent pathway is intact as assessed by the ability of I-cell lymphocytes to lyse target cells by regulated secretion.
30
Sykulev, Yuri, Allison M. Beal, Nadia Anikeeva, Rajat Varma, Thomas O. Cameron, Philip Norris, and Michael L. Dustin. "Cytolytic synapses control effectiveness of target cell destruction by CTL (35.17)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 35.17. http://dx.doi.org/10.4049/jimmunol.182.supp.35.17.
Abstract:
Abstract We have investigated the stability of cytolytic synapses as well as patterns of cytolytic granule polarization and kinetics of their release by CTL with different potency to kill target cells. Less potent CTL form unstable cytolytic synapses, accounting for a third of the difference in potency providing the best evidence that stable synapse is important for cytotoxicity. To determine what other unknown mechanistic components may account for the distinct potency of target cell killing, we evaluated differences related to various pathways of granule delivery to the secretory domain of these CTL. Visualization of lytic granule delivery shows that the granules can take long or short paths to the secretory domain. The difference in path is dictated by the kinetics of early TCR signaling. Rapid and robust early signaling causes swift granule concentration near the MTOC and subsequent delivery directly to the secretory domain - the shortest and fastest path. Indolent signaling leads to late recruitment of the granules that move first to the periphery of the synapse and then move tangentially to fuse at the outer edge of the secretory domain - a longer path. The short pathway of granule delivery is associated with more efficient killing. Thus, rapid granule delivery to the secretory domain and containment of released granules within a stable synapse determines the potency of CTL to destroy target cells.
31
Tamzalit, Fella, Diana Tran, Weiyang Jin, Vitaly Boyko, Hisham Bazzi, Ariella Kepecs, Lance C. Kam, Kathryn V. Anderson, and Morgan Huse. "Centrioles control the capacity, but not the specificity, of cytotoxic T cell killing." Proceedings of the National Academy of Sciences 117, no. 8 (February 10, 2020): 4310–19. http://dx.doi.org/10.1073/pnas.1913220117.
Abstract:
Immunological synapse formation between cytotoxic T lymphocytes (CTLs) and the target cells they aim to destroy is accompanied by reorientation of the CTL centrosome to a position beneath the synaptic membrane. Centrosome polarization is thought to enhance the potency and specificity of killing by driving lytic granule fusion at the synapse and thereby the release of perforin and granzymes toward the target cell. To test this model, we employed a genetic strategy to delete centrioles, the core structural components of the centrosome. Centriole deletion altered microtubule architecture as expected but surprisingly had no effect on lytic granule polarization and directional secretion. Nevertheless, CTLs lacking centrioles did display substantially reduced killing potential, which was associated with defects in both lytic granule biogenesis and synaptic actin remodeling. These results reveal an unexpected role for the intact centrosome in controlling the capacity but not the specificity of cytotoxic killing.
32
Tuli, Amit, Jerome Thiery, Ashley M. James, Xavier Michelet, Mahak Sharma, Salil Garg, Keri B. Sanborn, Jordan S. Orange, Judy Lieberman, and Michael B. Brenner. "Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity." Molecular Biology of the Cell 24, no. 23 (December 2013): 3721–35. http://dx.doi.org/10.1091/mbc.e13-05-0259.
Abstract:
Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.
33
Garner, R., C. D. Helgason, E. A. Atkinson, M. J. Pinkoski, H. L. Ostergaard, O. Sorensen, A. Fu, P. H. Lapchak, A. Rabinovitch, and J. E. McElhaney. "Characterization of a granule-independent lytic mechanism used by CTL hybridomas." Journal of Immunology 153, no. 12 (December 15, 1994): 5413–21. http://dx.doi.org/10.4049/jimmunol.153.12.5413.
Abstract:
Abstract The mechanism(s) by which CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granzymes) contained within the granules of CTL and NK, have been implicated, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of killing used by two cytolytic hybridomas (PMM-1 and MD90) that express neither perforin nor the granzymes. These characteristics are compared with results obtained by using a representative Ag-dependent, granule-containing T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target cells in the presence of cyclosporin and the absence of calcium. All the effectors could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the target cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent on protein synthesis, and inhibited by the addition of cyclosporin. Although TNF was shown not be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the level of expression of Fas Ag. With the use of L1210 and an L1210 cell line transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effectively inhibited with anti-Fas Ab. Furthermore the lack of sensitivity to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.
34
Radoja, Sasa, and Jennifer Ma. "Phospholipase D1 regulates the extent of TCR-induced lytic granule release by CD8+ CTL (P1390)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 203.12. http://dx.doi.org/10.4049/jimmunol.190.supp.203.12.
Abstract:
Abstract Regulation of lytic activity in CD8+ CTL remains incompletely understood despite the importance of this effector T cell function in immune response to intracellular pathogens and tumors. One important aspect involving the regulation of T cell effector function is the spatio-temporal regulation of DAG metabolism in T cells. Based on the involvement of phospholipase D (PLD) isoforms in DAG metabolism-related regulation of vesicular trafficking in other cells types, we reasoned that these enzymes might regulate lytic function in CD8+ T cells. By combining the use of pharmacological inhibitors and siRNA-mediated knockdown, we showed that PLD1 isoform modulates lytic function in primary mouse CD8+ CTL. Surprisingly, PLD1 knockdown in CTL lead to the enhancement of lytic activity despite its inhibitory effect on conjugate formation. The observed phenotype was not due to increased levels of expression of lytic molecules but was due to an increased release of lytic granule contents in response to TCR engagement. At the same time, overexpression of PLD1 in CTL resulted in the inhibition of lytic activity that coincided with an accumulation at the immunological synapse of both phosphatidic acid (PA), a byproduct of PLD1, and actin. Thus, PLD1 regulates the degree of lytic response in CD8+ CTL, potentially via PA-mediated induction of actin polymerization at the synapse, which could serve as a molecular gate that controls the extent of lytic granule release toward target cells.
35
Greenberg, A. H., N. Khalil, B. Pohajdak, M. Talgoy, P. Henkart, and F. W. Orr. "NK-leukocyte chemotactic factor (NK-LCF): a large granular lymphocyte (LGL) granule-associated chemotactic factor." Journal of Immunology 137, no. 10 (November 15, 1986): 3224–30. http://dx.doi.org/10.4049/jimmunol.137.10.3224.
Abstract:
Abstract A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.
36
Dupuis, M., E. Schaerer, K. H. Krause, and J. Tschopp. "The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes." Journal of Experimental Medicine 177, no. 1 (January 1, 1993): 1–7. http://dx.doi.org/10.1084/jem.177.1.1.
Abstract:
Cytolytic T lymphocytes (CTL), natural killer cells, and lymphokine-activated killer (LAK) cells are cytolytic cells known to release the cytolytic protein perforin and a family of proteases, named granzymes, from cytoplasmic stores upon interaction with target cells. We now report the purification of an additional major 60-kD granule-associated protein (grp 60) from human LAK cells and from mouse cytolytic T cells. The NH2-terminal amino acid sequence of the polypeptide was found to be identical to calreticulin. Calreticulin is a calcium storage protein and carries a COOH-terminal KDEL sequence, known to act as a retention signal for proteins destined to the lumen of the endoplasmic reticulum. In CTLs, however, calreticulin colocalizes with the lytic perforin to the lysosome-like secretory granules, as confirmed by double label immunofluorescence confocal microscopy. Moreover, when the release of granule-associated proteins was triggered by stimulation of the T cell receptor complex, calreticulin was released along with granzymes A and D. Since perforin is activated and becomes lytic in the presence of calcium, we propose that the role of calreticulin is to prevent organelle autolysis due to the protein's calcium chelator capacity.
37
Pattu, Varsha, Bin Qu, Ute Becherer, Ulf Matti, Eva Schwarz, Misty Marshall, Elmar Krause, Markus Hoth, and Jens Rettig. "The SNARE protein syntaxin 7 is required for immunological synapse formation in cytotoxic T lymphocytes (35.24)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 35.24. http://dx.doi.org/10.4049/jimmunol.182.supp.35.24.
Abstract:
Abstract Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse (IS). T cell receptor (TCR) enrichment at the IS is one of the key early events of IS formation. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the individual SNARE proteins that are important for CTL function are not yet known. We identified syntaxin 7 in CTLs by reverse transcriptase PCR and immunocytochemistry. We found that syntaxin 7 is localised to the IS with a similar distribution to the lytic granule marker - perforin. We then blocked the function of syntaxin 7 by deleting its transmembrane domain and observed a complete loss of TCR accumulation at the IS, indicating that no IS is formed. These results imply that syntaxin 7 is required for IS formation in CTLs. Further studies using specific endosome markers will help us determine the mechanism of syntaxin 7 function in CTLs. Source of research support - GRK1326
38
Zurli, Vanessa, Tommaso Montecchi, Raphael Heilig, Isabel Poschke, Michael Volkmar, Giuliana Wimmer, Gioia Boncompagni, et al. "Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells." Science Signaling 13, no. 631 (May 12, 2020): eaaz1965. http://dx.doi.org/10.1126/scisignal.aaz1965.
Abstract:
Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.
39
Amoscato, A. A., A. M. Brumfield, S. B. Sansoni, R. B. Herberman, and W. H. Chambers. "Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity." Journal of Immunology 147, no. 3 (August 1, 1991): 950–58. http://dx.doi.org/10.4049/jimmunol.147.3.950.
Abstract:
Abstract An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.
40
Mentlik, Ashley N., Keri B. Sanborn, Erika L. Holzbaur, and Jordan S. Orange. "Rapid Lytic Granule Convergence to the MTOC in Natural Killer Cells Is Dependent on Dynein But Not Cytolytic Commitment." Molecular Biology of the Cell 21, no. 13 (July 2010): 2241–56. http://dx.doi.org/10.1091/mbc.e09-11-0930.
Abstract:
Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules, we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization, lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization, dependent on dynein motor function, occurred before MTOC polarization, and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion, one that may prepare, but not commit cells for precision secretory function.
41
Inverardi, L., J. C. Witson, S. A. Fuad, R. T. Winkler-Pickett, J. R. Ortaldo, and F. H. Bach. "CD3 negative "small agranular lymphocytes" are natural killer cells." Journal of Immunology 146, no. 11 (June 1, 1991): 4048–52. http://dx.doi.org/10.4049/jimmunol.146.11.4048.
Abstract:
Abstract We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.
42
Haddad, Elias K., Xufeng Wu, John A. Hammer, and Pierre A. Henkart. "Defective Granule Exocytosis in Rab27a-Deficient Lymphocytes from Ashen Mice." Journal of Cell Biology 152, no. 4 (February 19, 2001): 835–42. http://dx.doi.org/10.1083/jcb.152.4.835.
Abstract:
Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL–Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti–CD3-coated beads. However, rapid anti–CD3-induced granule secretion was drastically defective in both CD8+ and CD4+ T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor–stimulated interferon-γ secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.
43
Hudig, D., N. J. Allison, T. M. Pickett, U. Winkler, C. M. Kam, and J. C. Powers. "The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors." Journal of Immunology 147, no. 4 (August 15, 1991): 1360–68. http://dx.doi.org/10.4049/jimmunol.147.4.1360.
Abstract:
Abstract To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,"Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.
44
Neeft, Maaike, Marnix Wieffer, Arjan S. de Jong, Gabriela Negroiu, Corina H. G. Metz, Alexander van Loon, Janice Griffith, et al. "Munc13-4 Is an Effector of Rab27a and Controls Secretion of Lysosomes in Hematopoietic Cells." Molecular Biology of the Cell 16, no. 2 (February 2005): 731–41. http://dx.doi.org/10.1091/mbc.e04-10-0923.
Abstract:
Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Δ608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.
45
Wood, Stephanie M., Marie Meeths, Samuel C. C. Chiang, Anne Grete Bechensteen, Jaap J. Boelens, Carsten Heilmann, Hisanori Horiuchi, et al. "Different NK cell–activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity." Blood 114, no. 19 (November 5, 2009): 4117–27. http://dx.doi.org/10.1182/blood-2009-06-225359.
Abstract:
Abstract The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, individual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that individual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.
46
Baetz, K., S. Isaaz, and G. M. Griffiths. "Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis." Journal of Immunology 154, no. 11 (June 1, 1995): 6122–31. http://dx.doi.org/10.4049/jimmunol.154.11.6122.
Abstract:
Abstract CTLs from patients with Chediak-Higashi syndrome (CHS) are unable to destroy target cells recognized via the TCR. To determine the mechanism responsible for the loss of cytotoxicity, CD8+ CTL clones have been derived from a patient with CHS. Individual CTL clones show poor killing that can be increased in longer assays. However, in the presence of cycloheximide, the small amount of killing observed is abolished, indicating killing arises from newly synthesized proteins, rather than from proteins stored in granules. In this study, we show that the CHS CTL clones express normal levels of the lytic proteins granzyme A, granzyme B, and perforin, which are processed properly during biosynthesis and targeted correctly to giant lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, in that both contain the lysosomal enzyme cathepsin D and the lytic protein granzyme A, and lack the mannose-6-phosphate receptor (MPR). However, unlike normal CTL clones, the CHS CTL clones are unable to secrete their giant granules in which the lytic proteins are stored. After cross-linking the TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release and confocal microscopy. We suggest that the defect in CHS lies in a protein that is involved in membrane fusion and is essential for the secretion of lysosomal compartments in certain hemopoietic cells.
47
Loo, Li Shen, Le-Ann Hwang, Yao Min Ong, Hock Soon Tay, Cheng-Chun Wang, and Wanjin Hong. "A role for endobrevin/VAMP8 in CTL lytic granule exocytosis." European Journal of Immunology 39, no. 12 (October 14, 2009): 3520–28. http://dx.doi.org/10.1002/eji.200939378.
48
Chiang, Samuel C. C., Jakob Theorell, Miriam Entesarian, Marie Meeths, Monika Mastafa, Waleed Al-Herz, Per Frisk, et al. "Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production." Blood 121, no. 8 (February 21, 2013): 1345–56. http://dx.doi.org/10.1182/blood-2012-07-442558.
Abstract:
Key Points Compared with cytotoxic T cells, NK cells share mechanisms for lytic granule release but more stringently control cytokine production. Analysis of CD57bright cytotoxic T-cell function may prove useful in the diagnosis of primary immunodeficiencies.
49
Spessott, Waldo A., Maria L. Sanmillan, Margaret E. McCormick, Vineet V. Kulkarni, and Claudio G. Giraudo. "SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity." Proceedings of the National Academy of Sciences 114, no. 11 (March 6, 2017): E2176—E2185. http://dx.doi.org/10.1073/pnas.1617981114.
Abstract:
The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.
50
Henkart, P. A., G. A. Berrebi, H. Takayama, W. E. Munger, and M. V. Sitkovsky. "Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes." Journal of Immunology 139, no. 7 (October 1, 1987): 2398–405. http://dx.doi.org/10.4049/jimmunol.139.7.2398.
Abstract:
Abstract 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.