CAR-NK Cells: From Natural Basis to Design for Kill

Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an ‘off-the-shelf product’ because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.

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NK Cell Regulation in Cervical Cancer and Strategies for Immunotherapy

Cells ◽

10.3390/cells10113104 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3104

Author(s):

Adriana Gutiérrez-Hoya ◽

Isabel Soto-Cruz

Keyword(s):

Cervical Cancer ◽

Nk Cells ◽

Nk Cell ◽

Hpv Infection ◽

Cytolytic Activity ◽

Cellular Immunotherapy ◽

Antigen Receptors ◽

Cytokines And Chemokines ◽

Hpv Status ◽

Infected Cells

Cervical cancer is one of the most prevalent gynaecological malignancies worldwide and is related to human papillomavirus (HPV) infection, viral persistence, progression, and invasion. Therefore, the immune response is linked to HPV status. Natural killer (NK) cells play a central role against virus-infected cells and tumours through a delicate balance between activating and inhibitory receptors and secretion of cytokines and chemokines. These cells also play a crucial role in tumour immunosurveillance. For these reasons, there is growing interest in harnessing NK cells as an immunotherapy for cervical cancer. These studies are diverse and include many strategies such as transferring activated autologous or allogeneic NK cells, improving the activation and cytolytic activity of NK cells using cytokines or analogues and modifying chimeric antigen receptors to increase specificity and targeting NK cells. However, research regarding the application of NK cells in immunotherapy is limited. This article focuses on recent discoveries about using NK cells to prevent and treat cervical cancer and the possibility of cellular immunotherapy becoming one of the best strategies to exploit the immune system to fight tumours.

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Enhancing network activation in natural killer cells: predictions from in silico modeling

Integrative Biology ◽

10.1093/intbio/zyaa008 ◽

2020 ◽

Vol 12 (5) ◽

pp. 109-121

Author(s):

Sahak Z Makaryan ◽

Stacey D Finley

Keyword(s):

Nk Cells ◽

Natural Killer ◽

In Silico ◽

Intracellular Signaling ◽

Cell Activation ◽

Nk Cell ◽

Mechanistic Model ◽

Cell Killing ◽

Published Data ◽

Network Activation

Abstract Natural killer (NK) cells are part of the innate immune system and are capable of killing diseased cells. As a result, NK cells are being used for adoptive cell therapies for cancer patients. The activation of NK cell stimulatory receptors leads to a cascade of intracellular phosphorylation reactions, which activates key signaling species that facilitate the secretion of cytolytic molecules required for cell killing. Strategies that maximize the activation of such intracellular species can increase the likelihood of NK cell killing upon contact with a cancer cell and thereby improve efficacy of NK cell-based therapies. However, due to the complexity of intracellular signaling, it is difficult to deduce a priori which strategies can enhance species activation. Therefore, we constructed a mechanistic model of the CD16, 2B4 and NKG2D signaling pathways in NK cells to simulate strategies that enhance signaling. The model predictions were fit to published data and validated with a separate dataset. Model simulations demonstrate strong network activation when the CD16 pathway is stimulated. The magnitude of species activation is most sensitive to the receptor’s initial concentration and the rate at which the receptor is activated. Co-stimulation of CD16 and NKG2D in silico required fewer ligands to achieve half-maximal activation than other combinations, suggesting co-stimulating these pathways is most effective in activating the species. We applied the model to predict the effects of perturbing the signaling network and found two strategies that can potently enhance network activation. When the availability of ligands is low, it is more influential to engineer NK cell receptors that are resistant to proteolytic cleavage. In contrast, for high ligand concentrations, inhibiting phosphatase activity leads to sustained species activation. The work presented here establishes a framework for understanding the complex, nonlinear aspects of NK cell signaling and provides detailed strategies for enhancing NK cell activation.

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Nonstochastic Coexpression of Activation Receptors on Murine Natural Killer Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.8.1341 ◽

2000 ◽

Vol 191 (8) ◽

pp. 1341-1354 ◽

Cited By ~ 97

Author(s):

Hamish R.C. Smith ◽

Hubert H. Chuang ◽

Lawrence L. Wang ◽

Margarita Salcedo ◽

Jonathan W. Heusel ◽

...

Keyword(s):

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Inhibitory Receptors ◽

Antigen Receptors ◽

Killer Cells ◽

Histocompatibility Complex ◽

Nk Cell Cytotoxicity

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti–Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3+ T cells, indicating that Ly-49H may be an NK cell–specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1+ cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

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CAR-NK Cell: A New Paradigm in Tumor Immunotherapy

Frontiers in Oncology ◽

10.3389/fonc.2021.673276 ◽

2021 ◽

Vol 11 ◽

Author(s):

Faroogh Marofi ◽

Alaa S. Al-Awad ◽

Heshu Sulaiman Rahman ◽

Alexander Markov ◽

Walid Kamal Abdelbasset ◽

...

Keyword(s):

Nk Cells ◽

Immune Escape ◽

Nk Cell ◽

Tumor Immunotherapy ◽

Antigen Receptors ◽

New Paradigm ◽

Immune Effector ◽

Effector Cells ◽

Therapeutic Competence

The tumor microenvironment (TME) is greatly multifaceted and immune escape is an imperative attribute of tumors fostering tumor progression and metastasis. Based on reports, the restricted achievement attained by T cell immunotherapy reflects the prominence of emerging other innovative immunotherapeutics, in particular, natural killer (NK) cells-based treatments. Human NK cells act as the foremost innate immune effector cells against tumors and are vastly heterogeneous in the TME. Currently, there exists a rapidly evolving interest in the progress of chimeric antigen receptor (CAR)-engineered NK cells for tumor immunotherapy. CAR-NK cells superiorities over CAR-T cells in terms of better safety (e.g., absence or minimal cytokine release syndrome (CRS) and graft-versus-host disease (GVHD), engaging various mechanisms for stimulating cytotoxic function, and high feasibility for ‘off-the-shelf’ manufacturing. These effector cells could be modified to target various antigens, improve proliferation and persistence in vivo, upturn infiltration into tumors, and defeat resistant TME, which in turn, result in a desired anti-tumor response. More importantly, CAR-NK cells represent antigen receptors against tumor-associated antigens (TAAs), thereby redirecting the effector NK cells and supporting tumor-related immunosurveillance. In the current review, we focus on recent progress in the therapeutic competence of CAR-NK cells in solid tumors and offer a concise summary of the present hurdles affecting therapeutic outcomes of CAR-NK cell-based tumor immunotherapies.

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CD137 Expression Is Enhanced in EBV-Infected T or NK Cells by Viral Protein LMP1 and Mediates Anti-Apoptotic Intracellular Signaling through NF-ĸB Activation.

Blood ◽

10.1182/blood.v114.22.1929.1929 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1929-1929

Author(s):

Ayako Arai ◽

Ken-ichi Imadome ◽

Mayumi Takahashi ◽

Koichi Naka ◽

Tetsuya Fukuda ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Intracellular Signaling ◽

Mononuclear Cells ◽

Nk Cell ◽

Jurkat Cells ◽

Rt Pcr ◽

Infected Cells ◽

Ebv Dna

Abstract Abstract 1929 Poster Board I-952 Epstein-Barr virus (EBV) can infect not only B cells but also T or NK cells uncommonly and causes lymphoid malignancies, such as extranodal NK/T-cell lymphoma nasal type (ENKL), aggressive NK-cell leukemia, and EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), which is also known as chronic active EBV infection. However, why and how EBV infects T or NK cells and the mechanism of action responsible for these EBV-induced malignancies have not been elucidated to date. To clarify the molecular mechanism underlying development of EBV-T/NK-LPD, we focused on costimulatory receptor CD137, which is expressed on the surface of activated T cells and plays a pivotal role in their proliferation, survival, and differentiation. We investigated CD137 expression on the surface of EBV-infected T/NK cells (EB-T/NK cells) by flow cytometry. First, three EBV-positive T and NK cell lines, SNT8, SNK6, and SNT16, were obtained for examination. These cell lines had been established from primary lesions of ENKL patients (SNT8 and SNK6) and peripheral blood of an EBV-T/NK-LPD patient (SNT16). CD137 expression was confirmed on the cell surface of these cells, whereas the EBV-negative T and NK cell line, Jurkat and KHYG1 cells, respectively, were negative for CD137. Next, we investigated expression on the surface of EB-T/NK cells derived from EBV-T/NK-LPD patients. EBV-T/NK-LPD was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in the peripheral blood (PB), and detection of EBV-infected T or NK cells. To detect the infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. Next, we measured the EBV-DNA titer of each fraction by quantitative RT-PCR. Nine patients (aged 8–41 years; 4 male, 5 female; 4 T and 5 NK cell types) were diagnosed with EBV-T/NK-LPD. Then, we examined surface CD137 expression of the infected cells of each patient. Expression was detected in 7 of 9 patients. Control cells (PB mononuclear cells of a healthy donor, who was negative for EBV-DNA titer in the PB) did not express the molecule. We also examined transcription of CD137 mRNA by RT-PCR assay and detected it in all the 12 EB-T/NK-cell samples described above. From these results we concluded that CD137 expression was induced at the level of both mRNA and protein in EB-T/NK cells. To investigate the molecular mechanism of CD137 overexpression in EBV-T/NK cells, we examined the influence of viral proteins on CD137 expression. EB-T/NK cells express EBV-encoded proteins, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We cotransfected expression plasmids for these proteins with a luciferase reporter plasmid containing the CD137 gene promoter in Jurkat cells and performed a luciferase assay. LMP1 significantly upregulated the CD137 promoter activity, although the other molecules did not. Furthermore, in a transient expression assay of these viral proteins using Jurkat cells, transcription of endogenous mRNA of CD137 was upregulated only in the LMP1 transfectant. These results indicate that LMP1 may transactivate CD137 transcription and expression in EBV-T/NK cells. Next, we investigated the role of CD137 in developing EBV-T/NK-LPD. We cultured the above-mentioned CD137-expressing EBV-T/NK cells on CHO cells that stably express human CD137L on the cell surface. NF-ĸB activation was detected in CD137-positive EBV-T/NK cells that were cocultured with CD137L-expressing CHO cells. We confirmed that both p50 and p52 translocated to the nucleus, indicating that both canonical and non-canonical pathways for NF-ĸB activation were activated downstream of CD137. Finally, we investigated the role of CD137-mediated NF-ĸB activation in the development of EBV-T/NK-LPD. We cocultured EB-T/NK cells on CHO-wt or CHO-CD137L with VP-16 for 48 h and determined apoptosis by measuring DiCO6 uptake. We noted that stimulation of CD137 significantly suppressed VP-16-induced apoptosis of these cells. Together, these results indicate that EBV-infected T/NK-cells express CD137 on the cell surface, which may be induced by LMP1 and activate the anti-apoptotic intracellular signaling pathway through NF-ĸB activation. This pathway may contribute to immortalization of the infected cells and development of EBV-T/NK-LPD. Disclosures: No relevant conflicts of interest to declare.

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CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas

Blood ◽

10.1182/blood.2020006619 ◽

2020 ◽

Vol 136 (20) ◽

pp. 2308-2318 ◽

Cited By ~ 12

Author(s):

Margery Gang ◽

Nancy D. Marin ◽

Pamela Wong ◽

Carly C. Neal ◽

Lynne Marsala ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Interferon Γ ◽

Interleukin 12 ◽

Cellular Immunotherapy ◽

Antigen Receptors ◽

Cell Responses ◽

Xenograft Models ◽

Activation Motif

Abstract Natural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell–resistant tumors.

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Maternal natural killer cells at the intersection between reproduction and mucosal immunity

Mucosal Immunology ◽

10.1038/s41385-020-00374-3 ◽

2021 ◽

Author(s):

Evgeniya V. Shmeleva ◽

Francesco Colucci

Keyword(s):

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Fetal Growth ◽

Immune Cells ◽

Mucosal Immunity ◽

Nk Cell ◽

Cell Responses ◽

Recent Developments ◽

Mucosal Lining

AbstractMany maternal immune cells populate the decidua, which is the mucosal lining of the uterus transformed during pregnancy. Here, abundant natural killer (NK) cells and macrophages help the uterine vasculature adapt to fetal demands for gas and nutrients, thereby supporting fetal growth. Fetal trophoblast cells budding off the forming placenta and invading deep into maternal tissues come into contact with these and other immune cells. Besides their homeostatic functions, decidual NK cells can respond to pathogens during infection, but in doing so, they may become conflicted between destroying the invader and sustaining fetoplacental growth. We review how maternal NK cells balance their double duty both in the local microenvironment of the uterus and systemically, during toxoplasmosis, influenza, cytomegalovirus, malaria and other infections that threat pregnancy. We also discuss recent developments in the understanding of NK-cell responses to SARS-Cov-2 infection and the possible dangers of COVID-19 during pregnancy.

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Enhancing network activation in Natural Killer cells: Predictions from in silico modeling

10.1101/395756 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sahak Z. Makaryan ◽

Stacey D. Finley

Keyword(s):

Nk Cells ◽

Natural Killer ◽

In Silico ◽

Intracellular Signaling ◽

Cell Activation ◽

Nk Cell ◽

Mechanistic Model ◽

Published Data ◽

Cell Therapies ◽

Network Activation

ABSTRACTNatural killer (NK) cells are part of the innate immune system and are capable of killing diseased cells. As a result, NK cells are being used for adoptive cell therapies for cancer patients. The activation of NK cell stimulatory receptors leads to a cascade of intracellular phosphorylation reactions, which activates key signaling species that facilitate the secretion of cytolytic molecules required for cell killing. Strategies that maximize the activation of such intracellular species can increase the likelihood of NK cell activation upon contact with a cancer cell, and thereby improve efficacy of NK cell-based therapies. However, due to the complexity of intracellular signaling, it is difficult to deduce a priori which strategies can enhance species activation. Therefore, we constructed a mechanistic model of the CD16, 2B4 and NKG2D signaling pathways in NK cells to simulate strategies that enhance signaling. The model predictions were fit to published data and validated with a separate dataset. Model simulations demonstrate strong network activation when the CD16 pathway is stimulated. The magnitude of species activation is most sensitive to the receptor concentration and the rate at which the receptor is deactivated. Co-stimulation of CD16 and NKG2D in silico required fewer ligands to achieve half-maximal activation than other combinations, suggesting co-stimulating these pathways is most effective in activating the species. We applied the model to predict the effects of perturbing the signaling network and found two strategies that can potently enhance network activation. When the availability of ligands is low, it is more influential to engineer NK cell receptors that are resistant to proteolytic cleavage. In contrast, for high ligand concentrations, inhibiting phosphatase activity leads to more activation. The work presented here establishes a framework for understanding the complex, nonlinear aspects of NK cell signaling and provides detailed strategies for enhancing NK cell activation.

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NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation

Science Signaling ◽

10.1126/scisignal.abe2740 ◽

2021 ◽

Vol 14 (684) ◽

pp. eabe2740

Author(s):

Quentin Verron ◽

Elin Forslund ◽

Ludwig Brandt ◽

Mattias Leino ◽

Thomas W. Frisk ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Intracellular Signaling ◽

Large Scale ◽

Spatial Organization ◽

Nk Cell ◽

Microcontact Printing ◽

Cytotoxic T Cells ◽

Target Cells ◽

Immune Synapses

Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell–activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures. Live-cell imaging and analysis of fixed human NK cells in this reductionist system showed that the spatial distribution of activating ligands influenced the formation, stability, and outcome of NK cell synapses. Whereas engagement of LFA-1 alone promoted synapse initiation, combined engagement of LFA-1 and CD16 was required for the formation of mature synapses and degranulation. Organizing LFA-1 and CD16 ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS shape exhibited similar arrangements of the lytic machinery. However, degranulation only occurred in regions containing ligands that therefore induced local signaling, suggesting the existence of a late checkpoint for degranulation. Our results demonstrate that the spatial organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.

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CD58 Immunobiology at a Glance

Frontiers in Immunology ◽

10.3389/fimmu.2021.705260 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yalu Zhang ◽

Qiaofei Liu ◽

Sen Yang ◽

Quan Liao

Keyword(s):

Nk Cells ◽

Intracellular Signaling ◽

Inflammatory Responses ◽

Nk Cell ◽

Costimulatory Molecule ◽

Soluble Form ◽

Target Cells ◽

Lymphocyte Function

The glycoprotein CD58, also known as lymphocyte-function antigen 3 (LFA-3), is a costimulatory receptor distributed on a broad range of human tissue cells. Its natural ligand CD2 is primarily expressed on the surface of T/NK cells. The CD2-CD58 interaction is an important component of the immunological synapse (IS) that induces activation and proliferation of T/NK cells and triggers a series of intracellular signaling in T/NK cells and target cells, respectively, in addition to promoting cell adhesion and recognition. Furthermore, a soluble form of CD58 (sCD58) is also present in cellular supernatant in vitro and in local tissues in vivo. The sCD58 is involved in T/NK cell-mediated immune responses as an immunosuppressive factor by affecting CD2-CD58 interaction. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment, allowing sCD58 as a novel immunotherapeutic target. Recently, the crucial roles of costimulatory molecule CD58 in immunomodulation seem to be reattracting the interests of investigators. In particular, the CD2-CD58 interaction is involved in the regulation of antiviral responses, inflammatory responses in autoimmune diseases, immune rejection of transplantation, and immune evasion of tumor cells. In this review, we provide a comprehensive summary of CD58 immunobiology.

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