Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

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.

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Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1373 ◽

2015 ◽

Vol 26 (7) ◽

pp. 1273-1285 ◽

Cited By ~ 23

Author(s):

Anne Reversat ◽

Maria-Isabel Yuseff ◽

Danielle Lankar ◽

Odile Malbec ◽

Dorian Obino ◽

...

Keyword(s):

T Lymphocytes ◽

B Cell ◽

Mhc Class Ii ◽

Cell Receptor ◽

B Cell Receptor ◽

Microtubule Organizing Center ◽

Antigen Uptake ◽

Immune Synapse ◽

Organizing Center ◽

Dependent Transport

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.

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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 ◽

10.1073/pnas.1710751114 ◽

2017 ◽

Vol 114 (32) ◽

pp. E6585-E6594 ◽

Cited By ~ 35

Author(s):

Alex T. Ritter ◽

Senta M. Kapnick ◽

Sricharan Murugesan ◽

Pamela L. Schwartzberg ◽

Gillian M. Griffiths ◽

...

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Immunological Synapse ◽

Cortical Actin ◽

Immune Synapse ◽

Superresolution Microscopy ◽

Lytic Granules ◽

Infected Cells ◽

Granule Secretion ◽

Lytic Granule

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.

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Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells.

The Journal of Cell Biology ◽

10.1083/jcb.95.1.137 ◽

1982 ◽

Vol 95 (1) ◽

pp. 137-143 ◽

Cited By ~ 201

Author(s):

B Geiger ◽

D Rosen ◽

G Berke

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Specific Binding ◽

Target Cells ◽

Microtubule Organizing Center ◽

Organizing Center ◽

Tubulin Antibodies ◽

Membrane Antigens ◽

Immunofluorescence Labeling ◽

Microtubular System

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL-TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.

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Rab27a Is Required for Regulated Secretion in Cytotoxic T Lymphocytes

The Journal of Cell Biology ◽

10.1083/jcb.152.4.825 ◽

2001 ◽

Vol 152 (4) ◽

pp. 825-834 ◽

Cited By ~ 290

Author(s):

Jane C. Stinchcombe ◽

Duarte C. Barral ◽

Emilie H. Mules ◽

Sarah Booth ◽

Alistair N. Hume ◽

...

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Immunological Synapse ◽

Target Cells ◽

Rab Proteins ◽

Loss Of Function ◽

Α Subunit ◽

Lytic Granules ◽

Granzyme A ◽

Geranylgeranyl Transferase

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

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Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

Science Signaling ◽

10.1126/scisignal.aaz1965 ◽

2020 ◽

Vol 13 (631) ◽

pp. eaaz1965 ◽

Cited By ~ 4

Author(s):

Vanessa Zurli ◽

Tommaso Montecchi ◽

Raphael Heilig ◽

Isabel Poschke ◽

Michael Volkmar ◽

...

Keyword(s):

T Cells ◽

Cytotoxic T Cells ◽

Critical Role ◽

Underlying Mechanism ◽

Cell Receptor ◽

Microtubule Organizing Center ◽

Lytic Granules ◽

Organizing Center ◽

Lytic Granule ◽

Insight Into

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.

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Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

Molecular Biology of the Cell ◽

10.1091/mbc.e13-05-0259 ◽

2013 ◽

Vol 24 (23) ◽

pp. 3721-3735 ◽

Cited By ~ 33

Author(s):

Amit Tuli ◽

Jerome Thiery ◽

Ashley M. James ◽

Xavier Michelet ◽

Mahak Sharma ◽

...

Keyword(s):

Natural Killer ◽

Target Cell ◽

Nk Cell ◽

Killer Cell ◽

Critical Factor ◽

Target Cells ◽

Interacting Protein ◽

Immune Synapse ◽

Lytic Granules ◽

Lytic Granule

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.

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Protease-Dependent Uncoating of a Complex Retrovirus

Journal of Virology ◽

10.1128/jvi.79.14.9244-9253.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 9244-9253 ◽

Cited By ~ 26

Author(s):

Jacqueline Lehmann-Che ◽

Marie-Lou Giron ◽

Olivier Delelis ◽

Martin Löchelt ◽

Patricia Bittoun ◽

...

Keyword(s):

Nuclear Import ◽

Viral Genome ◽

Productive Infection ◽

Wild Type Virus ◽

Target Cells ◽

Wild Type ◽

Microtubule Organizing Center ◽

Viral Protease ◽

Virus Life Cycle ◽

Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

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Cdc42-interacting protein–4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse

Journal of Experimental Medicine ◽

10.1084/jem.20061893 ◽

2007 ◽

Vol 204 (10) ◽

pp. 2305-2320 ◽

Cited By ~ 73

Author(s):

Pinaki P. Banerjee ◽

Rahul Pandey ◽

Rena Zheng ◽

Megan M. Suhoski ◽

Linda Monaco-Shawver ◽

...

Keyword(s):

Actin Filament ◽

Cell Activation ◽

Immunological Synapse ◽

Nk Cell ◽

Filamentous Actin ◽

Microtubule Organizing Center ◽

Interacting Protein ◽

Lytic Granules ◽

Organizing Center ◽

Microtubule Networks

An essential function of the immunological synapse (IS) is directed secretion. NK cells are especially adept at this activity, as they direct lytic granules to the synapse for secretion, which enables cytotoxicity and facilitates host defense. This initially requires rearrangement of the actin cytoskeleton and, subsequently, microtubule-dependent trafficking of the lytic granules. As these two steps are sequential, specific linkages between them are likely to serve as critical regulators of cytotoxicity. We studied Cdc42-interacting protein–4 (CIP4), which constitutively interacts with tubulin and microtubules but focuses to the microtubule organizing center (MTOC) after NK cell activation, when it is able to associate with Wiskott-Aldrich syndrome protein (WASp) and the actin filament–rich IS. WASp deficiency, overexpression of CIP4, or parts of CIP4 interfere with this union and block normal CIP4 localization, MTOC polarization to the IS, and cytotoxicity. Reduction of endogenous CIP4 expression using small interfering RNA similarly inhibits MTOC polarization and cytotoxic activity but does not impair actin filament accumulation at the IS, or Cdc42 activation. Thus, CIP4 is an important cytoskeletal adaptor that functions after filamentous actin accumulation and Cdc42 activation to enable MTOC polarization and NK cell cytotoxicity.

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HLA restriction of human cytotoxic T lymphocytes specific for influenza virus. Poor recognition of virus associated with HLA A2.

Journal of Experimental Medicine ◽

10.1084/jem.148.6.1458 ◽

1978 ◽

Vol 148 (6) ◽

pp. 1458-1467 ◽

Cited By ~ 57

Author(s):

A McMichael

Keyword(s):

Influenza Virus ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Influenza A ◽

Human Peripheral Blood ◽

Peripheral Blood Lymphocytes ◽

The Other ◽

Target Cells ◽

Surface Molecules ◽

Human Peripheral Blood Lymphocytes

Cytotoxic T lymphocytes (CTL), specific for influenza A/X31 virus, were generated from human peripheral blood lymphocytes. These CTL lysed target cells that were infected with the same virus and that shared HLA A or B locus antigens. Minimal lysis was observed when HLA-D antigens were shared. Not all HLA A and B antigens were equally effective. Efficient lysis of target cells was seen when HLA A1, A3, B7, B8, B27 and BW21 were shared with the CTL, but when HLA A2 was the only shared antigen lysis was usually minimal. This deficiency in CTL function associated with HLA A2 was not absolute. It is suggested that the function of this antigen might be influenced by other surface molecules on the cell and in particular the other HLA products.

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