Loss of Allospecific Activating Receptor Expressing NK Cells Results in Maternal Tolerance of the Aberrant Fetus

Abstract Background The main functions of Natural Killer (NK) cells are early protection against viruses or tumour cells and production of cytokines that regulate immune functions. NK cells are the first lymphoid cells to repopulate the marrow after Stem Cell Transplantation (SCT) and reach normal levels within 1 month after transplant. Acquisition of both, inhibiting and activating receptors on developing NK cells is an important step in their functional maturation. Previous studies showed the beneficial effect of NK alloreactivity in prevention of relapse, especially in the setting of haploidentical SCT. The aim of this study is to compare the reconstitution of the NK cell compartment during the first 3 months after unmanipulated haploidentical peripheral blood SCT (Haplo) and HLA-identical sibling peripheral blood SCT (HLA-id). Patients and Methods 11 adult patients received SCT (7 Haplo and 4 HLA-id) at Gregorio Marañón Hospital (Madrid-Spain) from November 2012 to April 2013. Conditioning regimen comprised fludarabine, cyclophosphamide and busulfan for Haplo SCT and fludarabine and busulfan or fludaribine and melphalan for HLA-id SCT. Prophylaxis for acute graft-versus-host disease consisted of high dose cyclophosphamide on days +3 and +4, cyclosporine A and mycophenolate mofetil for Haplo and Cyclosporine A and methotrexate for HLA-id. Patient´s characteristics and transplant outcomes are shown in table 1. We analysed reconstitution patterns and phenotype of NK at day +15, +30, +60, and +90 after transplantation by multi-color flow cytometry on FC500 Beckman Coulter® cytometer using the following anti-human monoclonal antibodies: CD3 FITC, CD56 ECD, CD45 PC7, NKG2A PC7, NKp30 PC5, NKp44 PE, Nkp46 PC5, and NKG2D PE (Beckman Coulter®). For comparison between the two groups Mann–Whitney U-test was used. Results 2/7 patients who received Haplo SCT died early in the post-transplantation period (day +50 and +66), and were excluded of the analysis because NK cells were not recovered by those days. NK cells reached normal levels by day +30: median 71 cells/µl (21-1089)) after Haplo; median 213.5 cells/µl (113-499) after HLA-id, and remained at high levels through follow up, with no significant differences between the two groups. Similarly to previous studies, a large percentage of NKbright cells was observed at day +30 after Haplo (median 89% of NK cells (55-97%)), a percentage that tended to decrease at day +60 (30% (7-38%)) and +90 (35% (10-45%)). Interestingly the percentage of NKbright cells after HLA-id SCT at day +30 (median 14.5% of NK cells (6-30%)) compared with Haplo, was significantly lower (p=0.016). This was accompanied by a significantly lower expression of inhibitory receptor NKG2A after HLA-id SCT than after Haplo: 59.5% (50-62%) versus 92.5% (50-62%) at day +30; 54% (38-61%) versus 86% (70-88%) versus at day +60 (p=0.016). Activating receptors NKp44 and NKp30 showed a low expression after both types of SCT throughout the first 3 months after transplantation. By contrast, activating receptor NKp46 levels were significantly higher at day +30 after Haplo than after HLA-id SCT (93% (87-98%) versus 50% (37-51%)) (p=0.016). Finally, high and similar proportions of activating receptor NKG2D were observed in both types of SCT. Figure 1 illustrates the recovery of the NK cell receptor phenotype for each type of SCT. Conclusions Our data showed an early and fast recovery of NK cells after Haplo and HLA-id SCT. However, phenotypic maturation of NK cells appears to be different for each type of transplant. NK cells generated after Haplo exhibit a more immature phenotype, characterized by a higher proportion of NKbright cells, and a higher expression of NKG2A at day +30. Interestingly expression of NKp46 was significantly higher after Haplo than after HLA-id SCT. Other authors have reported cytotoxic activity of these NK cells with high expression of NKp46, suggesting that cytotoxicity may be preserved in these immature NK cells. NKp30, NKG2D and NKp44 expression is less affected by the type of SCT. Acknowledgments This work has been partially supported by Project “Evaluación de la reconstitución inmune después del trasplante haploidéntico de progenitores hemopoyéticos sin depleción T” from Fundación Mutua Madrileña. Disclosures: No relevant conflicts of interest to declare.

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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Inflammatory monocytes and NK cells play a crucial role in DNAM-1–dependent control of cytomegalovirus infection

Journal of Experimental Medicine ◽

10.1084/jem.20151899 ◽

2016 ◽

Vol 213 (9) ◽

pp. 1835-1850 ◽

Cited By ~ 23

Author(s):

Tihana Lenac Rovis ◽

Paola Kucan Brlic ◽

Noa Kaynan ◽

Vanda Juranic Lisnic ◽

Ilija Brizic ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cellular Adhesion ◽

Mononuclear Phagocytes ◽

Inhibitory Receptor ◽

Inflammatory Monocytes ◽

Poliovirus Receptor ◽

Accessory Molecule ◽

Activating Receptor

The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIGIT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIGIT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1–PVR pathway.

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Enhanced AIS Based Intrusion Detection System Using Natural Killer Cells

Journal of Cyber Security and Mobility ◽

10.13052/jcsm2245-1439.942 ◽

2021 ◽

Author(s):

B. J. Bejoy ◽

S. Janakiraman

Keyword(s):

Intrusion Detection ◽

Nk Cells ◽

Natural Killer ◽

Intrusion Detection System ◽

Nk Cell ◽

Detection System ◽

Receptor Activation ◽

Cytokine Release ◽

Selection Algorithm ◽

Activating Receptor

Intrusion detection system is used to monitor the system and network activities to identify anomalies and attacks so that integrity, availability, and confidentiality can be preserved. Here an intrusion detection system based on Artificial Immune System is proposed based on Natural Killer (NK) cells with immunological memory. NK cells are created and each NK cells detection radius is determined using the negative selection algorithm and is trained to detect various attacks. Effective cells with high fairness values are proliferated and distributed to the network using clonal selection algorithm. In this paper, two types of NK cell are used-a Heavyweight NK cell (HWNK) and a number of Lightweight NK cells (LWNK). The incoming data is vectorized and Major Histocompatibility Complex Class I (MHC1) is created. Then based on this MHC1, any of the receptors i.e. Activating Receptor or Inhibiting Receptor is activated. If it is the signature of an attack, Activating Receptor is activated. Activating receptor activation results in either cytokine release or apoptosis. Here cytokine release means an alarm is generated informing the administrator and apoptosis stands for dropping of the packet. If Inhibiting Receptor is activated, it’s a normal packet there is no action taken. The technique proposed yields high accuracy, better detection rate and quick response time.

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Abstract 2807: Activated NK cells efficiently kill Glioblastoma through activating receptor-ligand interactions

10.1158/1538-7445.am2014-2807 ◽

2014 ◽

Author(s):

Justyna Kmiecik ◽

Andrea Gras Navarro ◽

Per Øyvind Enger ◽

Lina Leiss ◽

Jacques Zimmer ◽

...

Keyword(s):

Nk Cells ◽

Receptor Ligand ◽

Ligand Interactions ◽

Activating Receptor

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Endothelial cells express NKG2D ligands and desensitize antitumor NK responses

eLife ◽

10.7554/elife.30881 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 28

Author(s):

Thornton W Thompson ◽

Alexander Byungsuk Kim ◽

P Jonathan Li ◽

Jiaxi Wang ◽

Benjamin T Jackson ◽

...

Keyword(s):

Endothelial Cells ◽

Nk Cells ◽

Nk Cell ◽

Intrinsic Signal ◽

Cell Responses ◽

Cellular Environment ◽

Ligand Interactions ◽

Activating Receptor ◽

Pro Inflammatory Cytokines

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.

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A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection

Science ◽

10.1126/science.1258867 ◽

2015 ◽

Vol 348 (6230) ◽

pp. 136-139 ◽

Cited By ~ 148

Author(s):

Weiwen Deng ◽

Benjamin G. Gowen ◽

Li Zhang ◽

Lin Wang ◽

Stephanie Lau ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Killer Cell ◽

Tumor Rejection ◽

Nkg2d Ligand ◽

New Approach ◽

High Affinity ◽

Activating Receptor

Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D-activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, a shed form of MULT1, a high-affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are always inhibitory and suggest a new approach for cancer immunotherapy.

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TGF- downregulates the activating receptor NKG2D on NK cells and CD8+ T cells in glioma patients

Neuro-Oncology ◽

10.1093/neuonc/nop009 ◽

2009 ◽

Vol 12 (1) ◽

pp. 7-13 ◽

Cited By ~ 159

Author(s):

C. A. Crane ◽

S. J. Han ◽

J. J. Barry ◽

B. J. Ahn ◽

L. L. Lanier ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Activating Receptor

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NCR1 is an activating receptor expressed on a subset of canine NK cells

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2016.05.001 ◽

2016 ◽

Vol 177 ◽

pp. 7-15 ◽

Cited By ~ 5

Author(s):

Christine Grøndahl-Rosado ◽

Preben Boysen ◽

Grethe M. Johansen ◽

Hege Brun-Hansen ◽

Anne K. Storset

Keyword(s):

Nk Cells ◽

Activating Receptor

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NK Cells of Acute Myeloid Leukemia Patients Exhibit Exhausted Phenotype with Impaired Functional Activity

Blood ◽

10.1182/blood-2021-151550 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4466-4466

Author(s):

Dmitry Zhigarev ◽

Alexander W. MacFarlane ◽

Christina Diane Drenberg ◽

Reza Nejati ◽

Asya Varshavsky ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Surface Expression ◽

Healthy Controls ◽

Healthy Donors ◽

Activating Receptor ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) accounts for about one third of all leukemias, with half of all leukemia deaths. The low 5-year survival rate and manifestation of this deadly disease in old age reinforce the need for new safe and effective AML therapies. Considering the exceptional role of immune cells in the recognition and elimination of tumor cells, one of these methods is immunotherapy. However, for the development of immunotherapeutic approaches, it is necessary to clearly understand the role of certain effector cells in the pathogenesis of the disease, as well as to have the knowledge about the phenotypic characteristics of these cells. Natural killer (NK) cells play important roles in innate cancer immunosurveillance, and some published data indicate that the antitumor function of NK cells is reduced in AML patients. To expand upon previous work, we performed a comprehensive analysis of the phenotypic and functional characteristics of NK cells in previously untreated AML patients that took into account a wide variety of biomarkers. The goals of this study were to define the phenotypic and functional differences in NK cells from AML patients and healthy donors and determine how these parameters affect outcome of the disease. We used 14-color flow cytometry to assess more than 30 measurable markers on NK cells from the peripheral blood of 33 untreated AML patients and age-matched healthy controls. In addition, NK cells were tested for interferon-γ responses under antibody-dependent cellular cytotoxicity conditions. Results in AML patients were then compared to healthy donors. We found that the NK cells of patients with AML have a significantly lower capacity to secrete IFN-γ and showed numerous signs of an exhausted phenotype, as compared to healthy controls. These included increased surface expression of CD39, PD-1, and LILRB1 on NK cells from AML patients. Interestingly, surface expression of the TIGIT checkpoint receptor did not differ between AML patients and healthy donors, but surface expression of the activating receptor DNAM-1, which shares the same ligands on tumor cells, was decreased. All of these data confirm that the NK cells of AML patients are functionally impaired. We also noted that the frequency of CD57 + NKG2C + KIR + memory-like "adaptive" NK cells was greater in the blood of AML patients. The proportion of adaptive NK cells did not correlate with the age of donors. Phenotypic features of this cell subpopulation from the AML patients included significantly increased expression levels of CD56 and significantly lower expression of the activating receptor DNAM-1. Disclosures Zhigarev: Janssen R&D: Research Funding; Immunitas Therapeutics: Consultancy; Tavotek Biotherapeutics: Consultancy. Drenberg: Janssen R&D: Current Employment. Campbell: Janssen R&D: Research Funding.

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