scholarly journals CD38 contributes to human natural killer cell responses through a role in immune synapse formation

2018 ◽  
Author(s):  
Mathieu Le Gars ◽  
Christof Seiler ◽  
Alexander W. Kay ◽  
Nicholas L. Bayless ◽  
Elsa Sola ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Synapse Formation ◽  
Cell Function ◽  
Nk Cell ◽  
Critical Role ◽  
Target Cells ◽  
Immune Synapse ◽  
Ifn Γ

AbstractNatural killer (NK) cells use a diverse array of activating and inhibitory surface receptors to detect threats and provide an early line of defense against viral infections and cancer. Here, we demonstrate that the cell surface protein CD38 is a key human NK cell functional receptor through a role in immune synapse formation. CD38 expression marks a mature subset of human NK cells with a high functional capacity. NK cells expressing high levels of CD38 display enhanced killing and IFN-γ secretion in response to influenza virus-infected and tumor cells. Inhibition of CD38 enzymatic activity does not influence NK cell function, but blockade of CD38 and its ligand CD31 abrogates killing and IFN-γ expression in response to influenza-infected cells. Blockade of CD38 on NK cells similarly inhibits killing of tumor cells. CD38 localizes and accumulates at the immune synapse between NK cells and their targets, and blocking CD38 severely abrogates the ability of NK cells to form conjugates and immune synapses with target cells. Thus, CD38 plays a critical role in NK cell immune synapse formation. These findings open new avenues in immunotherapeutic development for cancer and infection by revealing a critical role for CD38 in NK cell function.

Ex Vivo Expansion of Cord Blood Natural Killer Cells Overcomes Impaired Immune Synapse Formation and Effector Function in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2905-2905
Author(s):  
Dongxia Xing ◽  
Alan G Ramsay ◽  
John G. Gribben ◽  
William K Decker ◽  
Jared K Burt ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Synapse Formation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Immune Synapse ◽  
Low Cytotoxicity

Abstract Natural killer (NK) cells play an important role in host immunity by eradicating pathogen-infected and tumor cells. Peripheral blood-derived NK cell therapy has shown promise in clinical trials for acute myeloid leukemia. Cord blood (CB) is another potentially rich source of NK cells. However NK cells isolated directly from CB have poor cytolytic activity. We investigated the mechanism for the low cytolytic activity of CB NK cells and whether the defect could be overcome by ex vivo expansion. NK cell killing of the target cells is achieved by formation of a mature immune synapse, followed by secretion of lytic granules containing perforin and granzymes. F-actin polymerization at the NK-tumor cell conjugates is a hallmark of immune synapse formation which can be detected and quantitated by confocal microscopy. We hypothesized that CB NK cells exhibit low cytotoxicity against leukemia target cells due to a defect in the formation of the immune synapse. We confirmed that unmanipulated CB NK cells exhibit low cytotoxicity against AML blasts (5% at an E:T ratio 20:1) in comparison to peripheral blood (PB) NK cells (35% at an E:T ratio 20:1). Evaluation of the natural cytotoxicity receptors (NCRs) showed normal expression of the NKp30 and the NK46 receptors. We then investigated whether this poor cytolytic capability is accompanied by poor immune synapse formation with tumor cells. Surprisingly, both CB and PB NK cells expressed comparable levels of perforin and demonstrated similar perforin polarization to the immune synapse. F-actin polarization was observed in only 12% (range 9– 21%) of the CB NK cell/K562 conjugates versus 85% (range 68–87%) of PB NK cell/K562 conjugates (P<0.001). Remarkably, this impairment could be reversed by ex vivo expansion of CB NK cells with rIL-2. Expanded CB NK cells formed an increased percentage of immune synapses with K562 tumor cells 65% (range 60–71%) and primary human AML blasts 48% (range 39 –55%) which was comparable to the levels generated with PB NK cells. The present data reinforce the conclusion that formation of the activating NK cell immune synapse is required for cytotoxic activity. Furthermore, natural cytotoxicity receptors (NCR) and Kir receptors including KIR2DL1/S1 and KIR2DL2 were preserved on the expanded CB NK population. Moreover, the expanded CB NK cells were able to lyse AML targets in vitro (29% at 20:1 E:T ratio). Finally, we demonstrated that ex vivo expanded CB NK cells efficiently kill human AML in an NOD/SCID null mouse model. A 50 % reduction in AML tumor burden was documented in comparison to control groups by 6 weeks post NK infusion (P<0.05). Our results suggest that ex vivo expansion of CB NK cells is a feasible and potentially effective strategy for the treatment of AML.

scholarly journals The Ly-49D Receptor Activates Murine Natural Killer Cells

1996 ◽  
Vol 184 (6) ◽  
pp. 2119-2128 ◽  
Author(s):  
L.H. Mason ◽  
S.K. Anderson ◽  
W.M. Yokoyama ◽  
H.R.C. Smith ◽  
R. Winkler-Pickett ◽  
...  
Keyword(s):  
Gene Family ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Subset ◽  
Class I ◽  
Target Cells ◽  
Color Flow ◽  
Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1612-1621 ◽  
Author(s):  
Lei Yao ◽  
Cecilia Sgadari ◽  
Keizo Furuke ◽  
Eda T. Bloom ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Primary Cultures ◽  
Interleukin 12 ◽  
Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3183-3190 ◽  
Author(s):  
Kathy S. Wang ◽  
David A. Frank ◽  
Jerome Ritz
Keyword(s):  
T Lymphocytes ◽  
Nk Cells ◽  
Natural Killer ◽  
Critical Role ◽  
Interleukin 2 ◽  
Interleukin 12 ◽  
Target Cells ◽  
Functional Responses ◽  
Antitumor Effects ◽  
Ifn Γ

Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-γ by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rβ1 and IL-12Rβ2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2–primed NK cells show enhanced functional responses to IL-12 as measured by IFN-γ production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer.

scholarly journals The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody

Blood ◽  
2010 ◽  
Vol 116 (13) ◽  
pp. 2286-2294 ◽  
Author(s):  
Don M. Benson ◽  
Courtney E. Bakan ◽  
Anjali Mishra ◽  
Craig C. Hofmeister ◽  
Yvonne Efebera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Killer Cell ◽  
Cell Immune Response ◽  
Cell Versus

Abstract T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti–PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1+ MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

scholarly journals Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells

2017 ◽  
Vol 9 (5) ◽  
pp. 511-525 ◽  
Author(s):  
Sophie M. Poznanski ◽  
Amanda J. Lee ◽  
Tina Nham ◽  
Evan Lusty ◽  
Margaret J. Larché ◽  
...  
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Critical Role ◽  
Interleukin 12 ◽  
Tnf Α ◽  
Ifn Γ

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation.

scholarly journals Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15

2021 ◽  
Author(s):  
Melanie A MacMullan ◽  
Pin Wang ◽  
Nicholas Alexander Graham
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Critical Role ◽  
Cytokine Signaling ◽  
Cytotoxic Lymphocytes ◽  
Signaling Events ◽  
Bioinformatic Approaches ◽  
Ribosomal S6 Kinase

Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 minutes, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-sites were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 Ser5, PAK2 Ser141, and STK17B Ser12. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

scholarly journals TXNIP Regulates Natural Killer Cell-Mediated Innate Immunity by Inhibiting IFN-γ Production during Bacterial Infection

2020 ◽  
Vol 21 (24) ◽  
pp. 9499
Author(s):  
Dong Oh Kim ◽  
Jae-Eun Byun ◽  
Won Sam Kim ◽  
Mi Jeong Kim ◽  
Jung Ha Choi ◽  
...  
Keyword(s):  
Bacterial Infection ◽  
Nk Cells ◽  
Natural Killer ◽  
Transforming Growth Factor ◽  
Nk Cell ◽  
Critical Role ◽  
Killer Cell ◽  
Transforming Growth Factor Β ◽  
Primary Mouse ◽  
Ifn Γ

The function of natural killer (NK) cell-derived interferon-γ (IFN-γ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-γ in NK cells during bacterial infection. TXNIP inhibited the production of IFN-γ and the activation of transforming growth factor β-activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip−/− (KO) NK cells enhanced the activation of macrophages by inducing IFN-γ production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-γ is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-γ during bacterial infection.

Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2663-2663
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Suppression ◽  
Direct Contact ◽  
Myeloid Leukemia ◽  
Synapse Formation ◽  
Nk Cell ◽  
Immune Synapse ◽  
Acute Myeloid

Abstract Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glycosylation Affects Ligand Binding and Function of the Activating Natural Killer Cell Receptor 2B4 (CD244) Protein

2011 ◽  
Vol 286 (27) ◽  
pp. 24142-24149 ◽  
Author(s):  
Stefanie Margraf-Schönfeld ◽  
Carolin Böhm ◽  
Carsten Watzl
Keyword(s):  
Ligand Binding ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Negative Impact ◽  
Killer Cell ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Responses

2B4 (CD244) is an important activating receptor for the regulation of natural killer (NK) cell responses. Here we show that 2B4 is heavily and differentially glycosylated in primary human NK cells and NK cell lines. The differential glycosylation could be attributed to sialic acid residues on N- and O-linked carbohydrates. Using a recombinant fusion protein of the extracellular domain of 2B4, we demonstrate that N-linked glycosylation of 2B4 is essential for the binding to its ligand CD48. In contrast, sialylation of 2B4 has a negative impact on ligand binding, as the interaction between 2B4 and CD48 is increased after the removal of sialic acids. This was confirmed in a functional assay system, where the desialylation of NK cells or the inhibition of O-linked glycosylation resulted in increased 2B4-mediated lysis of CD48-expressing tumor target cells. These data demonstrate that glycosylation has an important impact on 2B4-mediated NK cell function and suggest that regulated changes in glycosylation during NK cell development and activation might be involved in the regulation of NK cell responses.

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