Natural Killer Cell Surveillance Research Articles

The immunoglobulin superfamily ligand B7H6 subjects T cell responses to NK cell surveillance.

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

ELFN1-AS1 promotes GDF15-mediated immune escape of colorectal cancer from NK cells by facilitating GCN5 and SND1 association

The ability of colorectal cancer (CRC) cells to escape from natural killer (NK) cell immune surveillance leads to anti-tumor treatment failure. The long non-coding RNA (lncRNA) ELFN1-AS1 is aberrantly expressed in multiple tumors suggesting a role as an oncogene in cancer development. However, whether ELFN1-AS1 regulates immune surveillance in CRC is unclear. Here, we determined that ELFN1-AS1 enhanced the ability of CRC cells to escape from NK cell surveillance in vitro and in vivo. In addition, we confirmed that ELFN1-AS1 in CRC cells attenuated the activity of NK cell by down-regulating NKG2D and GZMB via the GDF15/JNK pathway. Furthermore, mechanistic investigations demonstrated that ELFN1-AS1 enhanced the interaction between the GCN5 and SND1 protein and this influenced H3k9ac enrichment at the GDF15 promotor to stimulate GDF15 production in CRC cells. Taken together, our findings indicate that ELFN1-AS1 in CRC cells suppresses NK cell cytotoxicity and ELFN1-AS1 is a potential therapeutic target for CRC.

Abstract 4077: Dual-targeted CAR-NK cell therapy: optimized CAR design to prevent antigen escape and elicit a deep and durable response in multiple myeloma

Abstract Introduction: Multiple myeloma (MM) is an incurable hematologic malignancy and new strategies that offer a chance of obtaining long-term progression free survival are urgently needed. MM is associated with profound immune alterations and dysfunction of natural killer (NK) cells have been demonstrated to be crucial factors in MM progression. Myeloma cells are susceptible to killing by natural killer (NK) cells but acquire the ability to elude NK cell surveillance by avoiding recognition and suppressing NK cell function. Given the role of NK cells in the pathogenesis of MM, interest in harnessing NK cells to treat the cancer has been energized by the remarkable success of other adoptive cell therapies such as chimeric antigen receptor (CAR)-T cells. However, relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating the identification of additional targets. GPRC5D, expressed on MM cells from primary marrow samples with a distribution independent of BCMA. Methods: To prevent BCMA-antigen escape and elicit a deeper and more durable response in MM, we developed a new multiplexed edited NK cell configuration to restore anti-myeloma NK cell immunity, consisting of anti-BCMA VHH and anti-GPRC5D VHH antibodies, NKG2D and 2B4 co-stimulation signaling domains, and IL-15. Results: Dual targeting BCMA/GPRC5D CAR-NK showed potent in vitro killing of both BCMA+ and GPRC5D+ myeloma cells. Utilizing a repeated rounds of cancer cell clearance assay, BCMA/GPRC5D CAR NK cells showed remarkable persistence and antigen-mediated expansion of CAR-NK cells after more than 4 rounds of tumor cell re-challenges. Moreover, in comparison with single targeted BCMA CAR-NK cells, Dual targeting BCMA/GPRC5D CAR-NK cells effectively lysed BCMA negative target cells. In addition, in BCMA-antigenic escape model, it achieved more sustained tumor control than single targeting BCMA CAR-NK cells. PD-1/PD-L1 axis inhibition has been reported to enhance NK cell activity against MM cells by augmenting NK cell trafficking, immune complex formation, and cytotoxicity against PD-L1-expressing MM cells. We also demonstrated that combination of BCMA/GPRC5D CAR-NK with anti-PDL1-IL15 also showed more persistent tumor control. Conclusions: Our studies demonstrate the development of dual targeting BCMA/GPRC5D CAR NK cells may represent a highly effective off-the-shelf therapeutic product as a monotherapy or in combinations with other immune-regulating agents. Citation Format: Cuiqing Yang, Yifang Wang, Tingting Liu, Chao Wang, Huanyu Wang, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhuoxiao Cao. Dual-targeted CAR-NK cell therapy: optimized CAR design to prevent antigen escape and elicit a deep and durable response in multiple myeloma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4077.

Immune checkpoint HLA-E:CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance.

Circulating tumor cells (CTCs), shed by primary malignancies, function as "seeds" for distant metastasis. However, it is still largely unknown how CTCs escape immune surveillance. Here, we characterize the transcriptomes of human pancreatic ductal adenocarcinoma CTCs, primary, and metastatic lesions at single-cell scale. Cell-interaction analysis and functional studies invitro and invivo reveal that CTCs and natural killer (NK) cells interact via the immune checkpoint molecule pair HLA-E:CD94-NKG2A. Disruption of this interaction by blockade of NKG2A or knockdown of HLA-E expression enhances NK-mediated tumor cell killing invitro and prevents tumor metastasis invivo. Mechanistic studies indicate that platelet-derived RGS18 promotes the expression of HLA-E through AKT-GSK3β-CREB signaling, and overexpression of RGS18 facilitates pancreatic tumor hepatic metastasis. In conclusion, platelet-derived RGS18 protects CTCs from NK-mediated immune surveillance by engaging the immune checkpoint HLA-E:CD94-NKG2A. Interruption of the suppressive signaling prevents tumor metastasis invivo by immune elimination of CTCs.

Abstract C015: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC

Abstract T-cell based immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be a powerful way to enhance T cell infiltration into tumors and reactivate a different type of innate Natural Killer (NK) cell anti-tumor immunity that can mediate tumor control. Here, we found that the pancreas tumor microenvironment (TME) suppresses NK cell surveillance following therapy-induced senescence (TIS) with MEK and CDK4/6 inhibitor treatment through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity enhanced the production of pro-inflammatory SASP factors and led to NK and T cell infiltration and immune-mediated tumor control in transplanted and genetically engineered PDAC mouse models. Mechanistically, EZH2 suppression induced expression of SASP factors CCL2 and CXCL9/10 necessary for lymophocyte chemotaxis into the pancreas TME. EZH2 levels were also associated with reduced NK cell numbers, SASP expression, and overall survival in a PDAC patient cohort. Taken together these results demonstrate that EZH2 mediates repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK and T cell surveillance in PDAC to achieve immune-mediated tumor responses. Citation Format: Loretah Chibaya, Katherine C. Murphy, Yvette Lopez Diaz, Kelly D. DeMarco, Haibo Liu, Sneha Gopalan, Melissa Faulkner, Junhui Li, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Elisa de Stanchina, Karl Simin, Lihua J. Zhu, Thomas G. Fazzio, Scott W. Lowe, Marcus Ruscetti. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C015.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Increased Expression of Mitochondrial UQCRC1 in Pancreatic Cancer Impairs Antitumor Immunity of Natural Killer Cells via Elevating Extracellular ATP.

Pancreatic cancer (PC) is one of the most lethal malignancies characterized by a highly immunosuppressive tumor microenvironment (TME). Previously, we have reported that ubiquinol-cytochrome c reductase core protein I (UQCRC1), a key component of mitochondrial complex III, is generally upregulated in PC and produces extracellular ATP (eATP) to promote PC progression. Here, we sought to investigate whether the oncogenic property of UQCRC1 is generated through its effects on natural killer (NK) cells in the TME. We found that UQCRC1 overexpression in PC cells inhibited cytotoxicity of NK cells, as well as the infiltration of NK cells toward PC, whereas knockdown of UQCRC1 enhanced the cytotoxicity and chemotaxis of NK cells. Adoptive NK cell therapy in the subcutaneous mouse model and CIBERSORTx analysis with human PC specimens confirmed UQCRC1 elicited immunosuppressive effects on NK cells. Such UQCRC1-induced impairment of NK cells was mediated by eATP and its metabolite adenosine via P2Y11R and A2AR, respectively. Mechanistically, we found the UQCRC1/eATP axis reduced the expression of chemokine CCL5 in cancer cells and altered the balance of activating receptor DNAM-1 and inhibitory receptor CD96 on NK-92MI cells, resulting in decreased chemotaxis and exhausted phenotype of NK-92MI cells. Taken together, our study provides the evidence to support a novel mechanism by which energy metabolism change in cancer cells remodels the TME and impedes NK cell surveillance. It also suggests that targeting UQCRC1 may be a potential combined strategy for PC immunotherapy.

Harnessing natural killer cells for the treatment of multiple myeloma

Multiple myeloma (MM) is hematologic malignancy that is associated with profound immune alterations. Myeloma cells are susceptible to killing by natural killer (NK) cells but acquire the ability to elude NK cell surveillance by avoiding recognition and suppressing NK cell function. Major advances in the treatment of multiple myeloma have been achieved by effective new drugs that redirect NK cells and enhance their function. Despite significant progress, myeloma remains incurable and novel treatment approaches are required. Strategies to take advantage of the intrinsic antitumor properties of NK cells to treat MM represent a novel immunotherapeutic approach. One such strategy is adoptive NK cell therapy that consist of infusions of NK cells that have been propagated ex vivo. Adoptive NK cell therapy encompasses contemporary genetic engineering strategies such as chimeric antigen receptor (CAR)-engineered NK cells. An alternative approach involves the use of pharmacologic agents to enhance NK cell activity against myeloma. NK cell-modulating therapies can be used to bolster the function of endogenous NK cells or to enhance the efficacy of adoptively infused NK cells. Here, we review the mechanistic complexities influencing NK cell activity in MM and highlight a variety of innovative NK cell-based strategies being developed for the treatment of MM.

TM4SF5-mediated liver malignancy involves NK cell exhaustion-like phenotypes

Aberrant extracellular matrix and immune cell alterations within the tumor microenvironment promote the pathological progression of liver carcinogenesis. Although transmembrane 4 L six family member 5 (TM4SF5) is involved in liver fibrosis and cancer, its mechanism avoiding immune surveillance during carcinogenesis remains unknown. We investigated how TM4SF5-mediated signaling caused immune evasion using in vitro primary cells and in vivo liver tissues from genetic or chemically induced mouse models. TM4SF5-transgenic and diethylnitrosamine (DEN)-induced liver cancer mouse models exhibited fibrotic and cancerous livers, respectively, with enhanced TM4SF5, pY705STAT3, collagen I, and laminin γ2 levels. These TM4SF5-mediated effects were abolished by TM4SF5 inhibitor, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC). TM4SF5-dependent tumorigenesis involved natural killer (NK) cell exhaustion-like phenotypes including the reduction of NK cell number or function, which were blocked with TSAHC treatment. TM4SF5 expression in cancer cells downregulated stimulatory ligands and receptors for NK cell cytotoxicity, including SLAMF6, SLAMF7, MICA/B, and others. TM4SF5 suppression or inhibition reduced STAT3 signaling activity and recovered the receptor levels and NK cell surveillance, leading to reduced fibrotic and cancerous phenotypes, and longer survival. Altogether, these findings suggest that TM4SF5-mediated STAT3 activity for extracellular matrix modulation is involved in the progression of liver disease to HCC and that TM4SF5 appears to suppress NK cells during liver carcinogenesis.

Abstract PO-124: EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses

Abstract T cell-targeting immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that cellular senescence and its associated secretory phenotype (SASP), which leads to production of proinflammatory cytokines, chemokines, and growth factors, can be a powerful way to reactivate a different type of Natural Killer (NK) cell immunity. Recently, we found that RAS pathway targeting MEK and CDK4/6 inhibitors can induce senescence in KRAS mutant lung tumor and PDAC models; however, therapy-induced senescence (TIS) led to NK cell-mediated tumor regressions only in the lungs. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for PDAC immunotherapy. Syngeneic murine KRAS-driven lung tumor and PDAC cells were transplanted into lungs, pancreas, or liver of C57BL/6 mice. Following 2-week treatment with the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P) to induce senescence, immune responses were assessed by flow cytometry, and the SASP profile assessed by RNA- and ATAC-seq analysis. An NK1.1-targeted antibody was also used to deplete NK cells and evaluate treatment efficacy. shRNA-mediated EZH2 knockdown and treatment with EZH2 methyltransferase inhibitors in murine and human PDAC cells and mouse models was used to determine the role of EZH2 in SASP regulation by qPCR and cytokine array. The effects of EZH2 blockade on NK cell immunity were determined in co-culture migration and cytotoxicity assays in vitro and in PDAC transplant models in vivo by flow cytometry. The efficacy of T/P treatment in combination with EZH2-targeting shRNAs or inhibitors was measured by ultrasound tumor measurements and survival in PDAC-bearing animals. Tumors propagated in the pancreas TME display transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15,-18) and chemotaxis (CCL2,-7,-8; CXCL9,-10) following TIS, and do not undergo anti-tumor NK immune surveillance as compared to tumors grown in the lungs and liver. We identified induction of EZH2 and repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell infiltration and anti-tumor cytotoxicity in PDAC models. Remarkably, EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in PDAC-bearing mice that were reversed following NK cell depletion. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with TIS could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor control. Citation Format: Loretah Chibaya, Yvette Lopez-Diaz, Haibo Liu, Katherine C. Murphy, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Lakhena Leang, Elisa de Stanchina, Lihua J. Zhu, Scott W. Lowe, Marcus Ruscetti. EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-124.

Regulation of MHC I Molecules in Glioblastoma Cells and the Sensitizing of NK Cells.

Immunotherapy has been established as an important area in the therapy of malignant diseases. Immunogenicity sufficient for immune recognition and subsequent elimination can be bypassed by tumors through altered and/or reduced expression levels of major histocompatibility complex class I (MHC I) molecules. Natural killer (NK) cells can eliminate tumor cells in a MHC I antigen presentation-independent manner by an array of activating and inhibitory receptors, which are promising candidates for immunotherapy. Here we summarize the latest findings in recognizing and regulating MHC I molecules that affect NK cell surveillance of glioblastoma cells.

Role of Natural Killer Cells in Uveal Melanoma.

Simple SummaryMetastatic Uveal Melanoma (MUM) is a lethal malignancy with no durable treatment available to date. A vast majority of patients with MUM present with liver metastasis. The liver harbors metastatic disease with an apparent lack of a cytotoxic T cell response. It is becoming evident that MUM is not an immunologically silent malignancy and the investigation of non-T cell anti-tumor immunity is warranted. In this review, we highlight the relevance of Natural Killer (NK) cells in the biology and treatment of MUM. Potent anti-NK cell immunosuppression employed by uveal melanoma alludes to its vulnerability to NK cell cytotoxicity. On the contrary, micro-metastasis in the liver survive for several years within close vicinity of a plethora of circulating and liver-resident NK cells. This review provides unique perspectives into the potential role of NK cells in control or progression of uveal melanoma.Uveal melanoma has a high mortality rate following metastasis to the liver. Despite advances in systemic immune therapy, treatment of metastatic uveal melanoma (MUM) has failed to achieve long term durable responses. Barriers to success with immune therapy include the immune regulatory nature of uveal melanoma as well as the immune tolerant environment of the liver. To adequately harness the anti-tumor potential of the immune system, non-T cell-based approaches need to be explored. Natural Killer (NK) cells possess potent ability to target tumor cells via innate and adaptive responses. In this review, we discuss evidence that highlights the role of NK cell surveillance and targeting of uveal melanoma. We also discuss the repertoire of intra-hepatic NK cells. The human liver has a vast and diverse lymphoid population and NK cells comprise 50% of the hepatic lymphocytes. Hepatic NK cells share a common niche with uveal melanoma micro-metastasis within the liver sinusoids. It is, therefore, crucial to understand and investigate the role of intra-hepatic NK cells in the control or progression of MUM.

Neuroblast senescence in the aged brain augments natural killer cell cytotoxicity leading to impaired neurogenesis and cognition.

Normal aging is accompanied by escalating systemic inflammation. Yet the potential impact of immune homeostasis on neurogenesis and cognitive decline during brain aging have not been previously addressed. Here we report that natural killer (NK) cells of the innate immune system reside in the dentate gyrus neurogenic niche of aged brains in humans and mice. In situ expansion of these cells contributes to their abundance, which dramatically exceeds that of other immune subsets. Neuroblasts within the aged dentate gyrus display a senescence-associated secretory phenotype and reinforce NK cell activities and surveillance functions, which result in NK cell elimination of aged neuroblasts. Genetic or antibody-mediated depletion of NK cells leads to sustained improvements in neurogenesis and cognitive function during normal aging. These results demonstrate that NK cell accumulation in the aging brain impairs neurogenesis, which may serve as a therapeutic target to improve cognition in the aged population.

Cancer cells educate natural killer cells to a metastasis-promoting cell state.

Natural killer (NK) cells have potent antitumor and antimetastatic activity. It is incompletely understood how cancer cells escape NK cell surveillance. Using ex vivo and in vivo models of metastasis, we establish that keratin-14+ breast cancer cells are vulnerable to NK cells. We then discovered that exposure to cancer cells causes NK cells to lose their cytotoxic ability and promote metastatic outgrowth. Gene expression comparisons revealed that healthy NK cells have an active NK cell molecular phenotype, whereas tumor-exposed (teNK) cells resemble resting NK cells. Receptor-ligand analysis between teNK cells and tumor cells revealed multiple potential targets. We next showed that treatment with antibodies targeting TIGIT, antibodies targeting KLRG1, or small-molecule inhibitors of DNA methyltransferases (DMNT) each reduced colony formation. Combinations of DNMT inhibitors with anti-TIGIT or anti-KLRG1 antibodies further reduced metastatic potential. We propose that NK-directed therapies targeting these pathways would be effective in the adjuvant setting to prevent metastatic recurrence.

Post-translational Mechanisms Regulating NK Cell Activating Receptors and Their Ligands in Cancer: Potential Targets for Therapeutic Intervention

Efficient clearance of transformed cells by Natural Killer (NK) cells is regulated by several activating receptors, including NKG2D, NCRs, and DNAM-1. Expression of these receptors as well as their specific “induced self” ligands is finely regulated during malignant transformation through the integration of different mechanisms acting on transcriptional, post-transcriptional, and post-translational levels. Among post-translational mechanisms, the release of activating ligands in the extracellular milieu through protease-mediated cleavage or by extracellular vesicle secretion represents some relevant cancer immune escape processes. Moreover, covalent modifications including ubiquitination and SUMOylation also contribute to negative regulation of NKG2D and DNAM-1 ligand surface expression resulting either in ligand intracellular retention and/or ligand degradation. All these mechanisms greatly impact on NK cell mediated recognition and killing of cancer cells and may be targeted to potentiate NK cell surveillance against tumors. Our mini review summarizes the main post-translational mechanisms regulating the expression of activating receptors and their ligands with particular emphasis on the contribution of ligand shedding and of ubiquitin and ubiquitin-like modifications in reducing target cell susceptibility to NK cell-mediated killing. Strategies aimed at inhibiting shedding of activating ligands and their modifications in order to preserve ligand expression on cancer cells will be also discussed.

"Acquired" NKG2D Ligand Stimulates NK Cell-mediated Tumor Immunosurveillance.

Engagement of activating receptor NKG2D to its ligand mediates natural killer (NK) cell activation and enhances cytotoxicity. NKG2D ligands (NKG2DLs) are frequently expressed on the tumor cell surface. However, the expression patterns of different NKG2DLs vary between tumor cells. Downregulation of certain ligand enables the tumor cells to escape NK cell-mediated immunosurveillance. By generating tumor cell lines with high expression of NKG2D ligand MULT1, we aimed to explore the function of NKG2DLs diversity on the activation and regulation of NKG2D signaling pathway. NK cells were potently activated by the "acquired" MULT1 expression on MOVCAR 5009 cells. Further, the progression of the tumor was significantly inhibited in mice inoculated with MULT1-expressing MOVCAR 5009 cells. Also, the pulmonary metastasis of MULT1-expressing B16-F0 cells was also significantly reduced in vivo. Our results implied that "acquired" NKG2D ligands enhance antitumor responses of NK cells, providing insights for designing novel therapeutic strategies and drugs to enhance NK cell surveillance over malignances.

MYC Functions As a Master Switch for Natural Killer Cell-Mediated Immune Surveillance of Lymphoid Malignancies

Background: The MYC oncogene drives T and B cell lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Such lymphomas are said to be “oncogene-addicted” to MYC. In order to develop targeted therapies for MYC-driven cancers, it is vital to understand how MYC regulates both cell autonomous and non-cell-autonomous processes, including host immunity.Approach: We have used a particularly tractable approach for studying the role of oncogenic MYC on the host immune system during lymphomagenesis through a tetracycline (tet)-system regulated transgenic mouse model of MYC-driven T cell lymphoma (SRα-tTA/Tet-O-MYC mice; Felsher and Bishop, Molecular Cell, 1999). By delineating the global immunological changes during primary MYC-driven T cell lymphomagenesis in SRα-tTA/Tet-O-MYC mice using mass cytometry (CyTOF) and CIBERSORT, we identified anti-tumor immune subsets that can be developed as therapies to treat MYC-driven lymphomas.Results: Amongst the immune subsets evaluated, our results demonstrated a specific systemic suppression of natural killer (NK) cell-mediated surveillance in SRα-tTA/Tet-O-MYC mice bearing overt MYC-driven T cell lymphomas. Inactivation of lymphoma-intrinsic MYC restores NK cell-mediated immune surveillance, suggesting that the regulation of NK cell-mediated surveillance may be integral to MYC-induced lymphomagenesis. We observed that lymphoma-specific MYC transcriptionally represses STAT1/2 and secretion of Type I Interferons (IFNs) required for normal NK cell homeostasis and NK cell-mediated immune surveillance in the tumor microenvironment. Concordantly, treating T cell lymphoma-bearing SRα-tTA/Tet-O-MYC mice with Type I IFN improves survival by rescuing NK cell production, and in part overriding MYC-mediated suppression of NK surveillance. We showed that human lymphomas with both high levels of MYC and low levels of STAT1/2 have lower NK surveillance, and are associated with poor prognosis. Finally, we established the therapeutic implications of our findings by showing that adoptive transfer of NK cells significantly delays lymphoma initiation, and recurrence after MYC inactivation; suggesting that NK cell-based therapy may be effective against MYC-driven lymphomas.Conclusion: Subversion of NK cell surveillance is integral to MYC-induced lymphomagenesis. Our studies provide a rationale for further developing the NK subset as a cell-based immunotherapy to effectively treat MYC-driven lymphomas in the future. DisclosuresNo relevant conflicts of interest to declare.

Brief Report: Association of Natural Killer Cell Ligand Polymorphism HLA–C Asn80Lys With the Development of Anti‐SSA/Ro–Associated Congenital Heart Block

Fetal exposure to maternal anti-SSA/Ro antibodies is necessary but not sufficient for the development of autoimmune congenital heart block (CHB), suggesting that other factors, such as fetal genetic predisposition, are important. Given the previously described association between major histocompatibility complex alleles and CHB risk, we undertook the present study to test the hypothesis that a variant form of HLA-C Asn80Lys, which binds with high affinity to an inhibitory killer cell immunoglobulin-like receptor (KIR) and thus renders natural killer (NK) cells incapable of restricting inflammation, contributes to the development of CHB. Members of 192 pedigrees in the US and Europe (194 cases of CHB, 91 unaffected siblings, 152 fathers, 167 mothers) and 1,073 out-of-study controls were genotyped on the Immunochip single-nucleotide polymorphism microarray. Imputation was used to identify associations at HLA-C Asn80Lys (Asn, C1; Lys, C2) and KIR. Tests for association were performed using logistic regression. McNemar's test and the pedigree disequilibrium test (PDT) were used for matched analyses between affected and unaffected children. Compared with out-of-study controls of the same sex, the C2 allele was less frequent in the mothers (odds ratio [OR] 0.63, P = 0.0014) and more frequent in the fathers (OR 1.40, P = 0.0123), yielding a significant sex-by-C2 interaction (P = 0.0002). The C2 allele was more frequent in affected siblings than in unaffected siblings (OR 3.67, P = 0.0025), which was consistent with the PDT results (P = 0.016); these results were observed in both sexes and across the US and European cohorts. There was no difference in the frequency of the inhibitory KIR genotype (KIR AA) between affected and unaffected children (P = 0.55). These data establish C2 as a novel genetic risk factor associated with CHB. This observation supports a model in which fetuses with C2 ligand expression and maternal anti-SSA/Ro positivity may have impaired NK cell surveillance, resulting in unchecked cardiac inflammation and scarring.

Dysfunctional Natural Killer Cells in the Aftermath of Cancer Surgery.

The physiological changes that occur immediately following cancer surgeries initiate a chain of events that ultimately result in a short pro-, followed by a prolonged anti-, inflammatory period. Natural Killer (NK) cells are severely affected during this period in the recovering cancer patient. NK cells play a crucial role in anti-tumour immunity because of their innate ability to differentiate between malignant versus normal cells. Therefore, an opportunity arises in the aftermath of cancer surgery for residual cancer cells, including distant metastases, to gain a foothold in the absence of NK cell surveillance. Here, we describe the post-operative environment and how the release of sympathetic stress-related factors (e.g., cortisol, prostaglandins, catecholamines), anti-inflammatory cytokines (e.g., IL-6, TGF-β), and myeloid derived suppressor cells, mediate NK cell dysfunction. A snapshot of current and recently completed clinical trials specifically addressing NK cell dysfunction post-surgery is also discussed. In collecting and summarizing results from these different aspects of the surgical stress response, a comprehensive view of the NK cell suppressive effects of surgery is presented. Peri-operative therapies to mitigate NK cell suppression in the post-operative period could improve curative outcomes following cancer surgery.

MicroRNA-29b mediates altered innate immune development in acute leukemia.

Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell-depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer.