Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

Abstract Abstract 246 Natural killer (NK) cells are an innate component of immune system that can produce a graft vs. leukemia (GVL) effect after stem cell transplantation. NK cells derived from acute myeloid leukemia (AML) patients are defective in their cytolytic function against leukemic cells. In order to better understand the mechanism of this defect, we performed functional assays examining immunological synapse formation of AML patient NK cells with autologous and allogeneic primary AML cells acting as antigen-presenting cells (APCs). Confocal microscopy was used to image and score F-actin polymerization at the immunological synapse between patient NK cells and leukemic cells. Accumulation of F-actin beneath the area of the NK: APC contact site is a hallmark of NK lytic synapses and allows signaling molecules to regulate appropriate activation and effector function. AML patient derived NK cells (AML-NK cells) formed significantly fewer synapses with autologous leukemia cells than healthy donor NK cells (12% versus 30%, n = 16. p > 0.001). Moreover, AML-NK cells were defective in their ability to recruit the key receptor NKG2D and the signaling molecule phosphotyrosine to immunological synapse contact sites. Signaling through the costimulatory ligand4-1BB-L (CD137L) has been shown to activate T cells, enhance antitumor responses and has multiple immunomodulatory effects on dendritic cells and NK cells. We postulated that AML-NK cells could be activated for enhanced cytolytic activity using artificial APCs generated to express CD137L. To test this, we setup co-culture assays using AML-NK cells and artificial CD137L-APCs before subsequent examination of immunological synapse function with AML blasts. Stimulated AML-NK cells that formed cell conjugate interactions with AML blasts, showed a significant increase in formation of immunological synapses compared to unstimulated AML-NK cells. The number of AML-NK/AML blast immunological synapses increased 16 hours after stimulation and peaked at approximately 72 hours. CD137L stimulation of AML-NK cells was also associated with increased cytotoxic function against primary AML cells (n = 6, p <0.01). Furthermore, CD137L stimulation increased recruitment of tyrosine-phosphorylated proteins at AML-NK immunological synapses compared with unstimulated control experiments (RRI 4.1 versus 2.3, n = 3, p < 0.01). Taken together, our data suggests that immune functional suppression of AML-NK cells in leukemia patients can be reversed by CD137L activation signaling, resulting in enhanced F-actin synapse formation, phosphotyrosine signaling, and cytolytic function. Thus, enhanced recruitment of signaling molecules to the NKIS may represent a novel immunomodulatory function of CD137L in the NK cell–mediated killing of AML cells. These findings should aid development of new immune based therapies for leukemia. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Defective cytotoxic lymphocyte degranulation in syntaxin-11–deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Blood ◽

10.1182/blood-2007-02-074468 ◽

2007 ◽

Vol 110 (6) ◽

pp. 1906-1915 ◽

Cited By ~ 205

Author(s):

Yenan T. Bryceson ◽

Eva Rudd ◽

Chengyun Zheng ◽

Josefine Edner ◽

Daoxin Ma ◽

...

Keyword(s):

Nk Cells ◽

Hemophagocytic Lymphohistiocytosis ◽

Nk Cell ◽

Severe Disease ◽

Target Cells ◽

Loss Of Function ◽

Cytotoxic Lymphocyte ◽

Familial Hemophagocytic Lymphohistiocytosis ◽

Genes Encoding ◽

Syntaxin 11

Abstract Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8+ T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.

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The Role of the Cytoskeleton in Regulating the Natural Killer Cell Immune Response in Health and Disease: From Signaling Dynamics to Function

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.609532 ◽

2021 ◽

Vol 9 ◽

Author(s):

Aviad Ben-Shmuel ◽

Batel Sabag ◽

Guy Biber ◽

Mira Barda-Saad

Keyword(s):

Immune Response ◽

Nk Cells ◽

Signaling Pathways ◽

Natural Killer ◽

Immunological Synapse ◽

Nk Cell ◽

Cell Immune Response ◽

Effector Functions ◽

Health And Disease

Natural killer (NK) cells are innate lymphoid cells, which play key roles in elimination of virally infected and malignant cells. The balance between activating and inhibitory signals derived from NK surface receptors govern the NK cell immune response. The cytoskeleton facilitates most NK cell effector functions, such as motility, infiltration, conjugation with target cells, immunological synapse assembly, and cytotoxicity. Though many studies have characterized signaling pathways that promote actin reorganization in immune cells, it is not completely clear how particular cytoskeletal architectures at the immunological synapse promote effector functions, and how cytoskeletal dynamics impact downstream signaling pathways and activation. Moreover, pioneering studies employing advanced imaging techniques have only begun to uncover the architectural complexity dictating the NK cell activation threshold; it is becoming clear that a distinct organization of the cytoskeleton and signaling receptors at the NK immunological synapse plays a decisive role in activation and tolerance. Here, we review the roles of the actin cytoskeleton in NK cells. We focus on how actin dynamics impact cytolytic granule secretion, NK cell motility, and NK cell infiltration through tissues into inflammatory sites. We will also describe the additional cytoskeletal components, non-muscle Myosin II and microtubules that play pivotal roles in NK cell activity. Furthermore, special emphasis will be placed on the role of the cytoskeleton in assembly of immunological synapses, and how mutations or downregulation of cytoskeletal accessory proteins impact NK cell function in health and disease.

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Natural killer cells: origin, phenotype, function

Medical Immunology (Russia) ◽

10.15789/1563-0625-nkc-2330 ◽

2021 ◽

Vol 23 (6) ◽

pp. 1207-1228

Author(s):

E. V. Tyshchuk ◽

V. A. Mikhailova ◽

S. A. Selkov ◽

D. I. Sokolov

Keyword(s):

Bone Marrow ◽

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Immunological Synapse ◽

Nk Cell ◽

Cell Receptor ◽

Killer Cells ◽

Activated State

Natural killer cells (NK) are innate immune lymphocytes produced in the bone marrow. Isolation of NK cells as a separate population of lymphocytes is related to discovery of their ability to induce the death of tumor cells without prior sensitization. In this review, an attempt was made to systematize the numerous data on the biology of NK cells presented in the literature. The authors consider the stages of NK cells` differentiation from a common lymphoid progenitor (CLP) in the bone marrow, describe two functionally different populations of mature NK cells – CD56brightCDl6- and CD56dimCD16+. In addition, the role of cytokines and chemokines in the development of NK cells is discussed. The review includes data on the spectrum of molecules expressed by NK cells: adhesion molecules (LFA-1, LFA-2, LFA-3; αMβ2, αXβ2, L-selectin, VLA-4, VLA-5; PECAM-1; CEACAM-1), cytokine receptors (IL-1R, IL-2ra, IL-2Rb/IL-2Rc, IL-6Rα, IL-7Ra, IL-8R, IL-10R, IL-12Rβ1, IL-15ra, IL-18R, IL-21ra, IFNGR2, TGFBR, c-Kit, CXCR1, CXCR3, CXCR4, CCR4, CCR5, CCR6, CCR7, IChemR23, CX3CR1), as well as receptors that regulate the activity of NK cells (LILRB1, LILRB2, LILRB4; KIR2DL1-5; KIR2DS1-5; KIR3DL1-3; KIR3DS1; NKG2A, NKG2C, NKG2D; Siglec7, Siglec9; CD16; NKRP-1; TIGIT; TACTILE; NKp30, NKp44, NKp46, NKp80; LAIR-1; PD-1; TIM-3; 2B4; TLR1-9). The authors also examine the mechanisms of implementing cytotoxic activity by NK cells, including cytotoxicity, via expression of MHC-I-specific receptors, CD16 Fc receptors, receptors and ligands of apoptosis (Fas-FasL and TRAIL-TRAILR) as well as other receptors. The review describes in detail the structure of immunological synapse between the NK cell and target cell, receptor interactions, and the role of the cytoskeleton in its formation. The data are summarized on the variants of exocytosis of lytic granules by NK cells, including complete or partial fusion of vesicles with the plasma membrane, exocytosis of vesicles containing perforin and FasL, and the formation of microvesicles containing granzyme B. The review also describes data on ability of NK cells to maintain activated state for a long time, as well as to maintain contact with several targets at the same time. In addition to the functions inherent in natural killers as cells of innate immunity, the authors point out their ability to exhibit the features of cells of adaptive immunity. In general, a variety of mechanisms that regulate the activity of NK cells may complement the specific functions of lymphocytes, thus making the immune system more efficient.

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Hemophagocytic lymphohistiocytosis in syntaxin-11–deficient mice: T-cell exhaustion limits fatal disease

Blood ◽

10.1182/blood-2012-07-441139 ◽

2013 ◽

Vol 121 (4) ◽

pp. 604-613 ◽

Cited By ~ 50

Author(s):

Tamara Kögl ◽

Jürgen Müller ◽

Birthe Jessen ◽

Annette Schmitt-Graeff ◽

Gritta Janka ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Hemophagocytic Lymphohistiocytosis ◽

Cytolytic Activity ◽

T Cell Exhaustion ◽

Inhibitory Receptors ◽

Cell Exhaustion ◽

Syntaxin 11 ◽

Deficient Mice

Abstract Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

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Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.3029.3029 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3029-3029

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

William Decker ◽

Sufang Li ◽

Simon Robinson ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Ex Vivo ◽

Immune Therapy ◽

Cytolytic Activity ◽

Lymphocyte Function ◽

Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

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Inhibitory Receptor Signaling Destabilizes Immunological Synapse Formation in Primary NK Cells

Frontiers in Immunology ◽

10.3389/fimmu.2013.00410 ◽

2013 ◽

Vol 4 ◽

Cited By ~ 8

Author(s):

Thushara P. Abeyweera ◽

Molly Kaissar ◽

Morgan Huse

Keyword(s):

Nk Cells ◽

Synapse Formation ◽

Immunological Synapse ◽

Receptor Signaling ◽

Inhibitory Receptor

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Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy

Communications Biology ◽

10.1038/s42003-021-02669-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Nora Trinks ◽

Sebastian Reinhard ◽

Matthias Drobny ◽

Linda Heilig ◽

Jürgen Löffler ◽

...

Keyword(s):

Nk Cells ◽

Fluorescence Imaging ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Super Resolution ◽

Cell Types ◽

Lytic Granules ◽

Isotropic Expansion ◽

Confocal Fluorescence Imaging

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

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Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1794.1794 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1794-1794 ◽

Cited By ~ 1

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

Simon Robinson ◽

Catherine M. Bollard ◽

Nina Shah ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Actin Polymerization ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Immune Dysfunction ◽

Lymphocytic Leukemia ◽

Immune Synapse

Abstract Abstract 1794 Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6). Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide's anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Modulation of Siglec-7 Signaling via in situ Created High-affinity cis-Ligands

10.1101/2020.07.15.203125 ◽

2020 ◽

Author(s):

Senlian Hong ◽

Chenhua Yu ◽

Shi Yujie ◽

Peng Wang ◽

Digantkumar G. Chapla ◽

...

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Immunological Synapse ◽

De Novo ◽

Immune Checkpoints ◽

Target Cells ◽

High Affinity ◽

Affinity Ligand ◽

Adaptive Immune Cells ◽

Nk Cytotoxicity

AbstractSialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

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