Natural Killer Cell Cytotoxic Function Research Articles

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

Abstract B032: Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers

Abstract We investigated the interplay between immunosuppressive TGFβ signaling and immunogenic DNA repair in response to immune checkpoint inhibition (ICI) in a new model of breast cancer. Transforming growth factor beta (TGFβ) is a canonical suppressor. Cancer cells evade TGFβ either by selectively overcoming TGFβ proliferative barrier or completely shutting down signaling. Loss of TGFβ signaling is accompanied by high immunosuppressive TGFβ activity. We have shown that loss of TGFβ is critical to genomic integrity. It endorses classical DNA repair and inhibits error-prone repair. We described a score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers, in which the TGFβ target signature is low and DNA repair signature is high, use error-prone DNA repair, are sensitive to chemoradiation and have a greater fraction of the genome altered compared to low βAlt cancers, which maintain TGFβ signaling. We established a novel murine tumor derived transplant model (mTDT) of triple-negative breast cancer, similar to a human PDX, in immunocompetent BALB/cJ mice. Transcriptomics analysis showed that mTDT range from high to low βAlt and from immune poor to immune rich, which was confirmed by multiplex immunstaining. Unsupervised clustering of mTDT based on βAlt score demonstrated that low βAlt was associated with immune rich tumors while high βAlt was associated with immune poor tumors. A similar relationship is evident in the TCGA breast cancers. We then asked how mTDT respond to ICI. Mice bearing mTDT were randomized to monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Neither anti-PD-L1 or TGFβ inhibition affected survival, alone or in combination, whereas radiation significantly increased survival. Unexpectedly immune poor, high βAlt mTDT were the most responsive. To evaluate biological correlates, mice were stratified as a function of treatment as responders, in which tumor growth was controlled for 7 days post-treatment, or non-responders, in which tumor growth was uncontrolled. Tumor infiltrating CD45+ cells increased (P=0.02) as did circulating CD8 T cells (P<0.001) in responders. Natural killer (NK) cells were also significantly expanded in responders (P<0.001). TGFβ broadly downregulates chemokine receptor expression necessary to recruit NK cells and impairs NK cell cytotoxic effector function. We depleted NK cells to test whether they were necessary for the pronounced response to triple treatment. NK depletion was completely abrogated response (P<0.00001) indicating NK cells can provoke an ICI response in high βAlt mammary tumors devoid of lymphocytes. This preclinical model and human breast cancer shows that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors, and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICI and anti-tumor immunity. Citation Format: Jade Moore, Jim Gkantalis, Ines Guix, Colin Foster, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B032.

Ginsenoside Rh2 enhances immune surveillance of natural killer (NK) cells via inhibition of ERp5 in breast cancer

BackgroundOne critical component of the immune system that prevents breast cancer cells from forming distant metastasis is natural killer (NK) cells participating in immune responses to tumors. Ginsenoside Rh2 (GRh2) as one of the major active ingredients of ginseng has been employed in treatment of cancers, but the function of GRh2 in modulating the development of breast cancer remains elusive. PurposeThis study was to dissect the effect of GRh2 against breast cancer and its potential mechanisms associated with NK cells, both in vitro and in vivo. MethodsMDA-MB-231 and 4T1 cells were used to establish in situ and hematogenous mouse models. MDA-MB-231 and MCF-7 were respectively co-cultured with NK92MI cells or primary NK cells in vitro. Anti-tumor efficacy of GRh2 was verified by immunohistochemistry (IHC), Cell Counting Kit-8 (CCK8), high resolution micro-computed tomography (micro-CT) scanning of lungs and hematoxylin and eosin (H&E) staining. Lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry, in vivo depletion of NK cells, enzyme-linked immunosorbent assay (ELISA), western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence and cell transfection were performed for investigating the anti-tumor mechanisms of GRh2. Molecular docking, microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were employed to determine the binding between endoplasmic reticulum protein 5 (ERp5) and GRh2. ResultsWe demonstrated that GRh2 exerted prominent impacts on retarding the growth and metastasis of breast cancer through boosting the cytotoxic function of NK cells, as validated by the elevated release of perforin, granzyme B and interferon-γ (IFN-γ). Mechanistical studies revealed that GRh2 was capable of diminishing the expression of ERp5 and GRh2 directly bound to ERp5 in MDA-MB-231 cells as well as on a recombinant protein level. GRh2 prevented the formation of soluble MICA (sMICA) and upregulated the expression level of MICA in vivo and in vitro. Importantly, the reduced lung metastasis of breast cancer by GRh2 was almost abolished upon the depletion of NK cells. Moreover, GRh2 was able to insert into the binding pocket of ERp5 directly. ConclusionWe firstly demonstrated that GRh2 played a pivotal role in augmenting NK cell activity by virtue of modulating the NKG2D-MICA signaling axis via directly binding to ERp5, and may be further optimized to a therapeutic agent for the treatment of breast cancer.

Effector and cytolytic function of natural killer cells in anticancer immunity.

Adaptive immune cells play an important role in mounting antigen-specific antitumor immunity. The contribution of innate immune cells such as monocytes, macrophages, natural killer (NK) cells, dendritic cells, and gamma-delta T cells is well studied in cancer immunology. NK cells are innate lymphoid cells that show effector and regulatory function in a contact-dependent and contact-independent manner. The cytotoxic function of NK cells plays an important role in killing the infected and transformed host cells and controlling infection and tumor growth. However, several studies have also ascribed the role of NK cells in inducing pathophysiology in autoimmune diseases, promoting immune tolerance in the uterus, and antitumor function in the tumor microenvironment. We discuss the fundamentals of NK cell biology, its distribution in different organs, cellular and molecular interactions, and its cytotoxic and noncytotoxic functions in cancer biology. We also highlight the use of NK cell-based adoptive cellular therapy in cancer.

Cyp4a12-mediated retinol metabolism in stellate cells is the antihepatic fibrosis mechanism of the Chinese medicine Fuzheng Huayu recipe

BackgroundHepatic stellate cells (HSCs), which contain multiple retinol-containing lipid droplets, are important profibrotic cells in liver fibrosis. Under Cyp4a12a/b oxidation, HSC activation was accompanied by the downregulation of genes involved in retinol metabolism, inducing RAE-1 production. By eliminating activated HSCs, NK cells expressing the activating receptor NKG2D are recruited to alleviate fibrosis. FZHY was found to significantly reduce the severity of liver fibrosis by inhibiting the activation and proliferation of HSCs. The molecular processes that govern retinol metabolism, on the other hand, are largely unexplored. This study focused on the regulation of Cyp4a12a/b by FZHY to elucidate the antifibrotic molecular mechanisms underlying the effect of FZHY on retinol metabolism.MethodsTo investigate mechanisms and altered pathways of FZHY against carbon tetrachloride (CCl4)-induced liver fibrosis based on transcriptomics data. Bioinformatics analysis was used to screen its pharmacological targets. The predicted targets were confirmed by a series of in vitro and in vivo experiments, including mass spectrometry, in situ hybridization, immunofluorescence assays and real-time PCR. Then, the results were further characterized by recombinant adenovirus vectors that were constructed and transfected into the cultured primary HSCs.ResultsTranscriptomics revealed that Cyp4a12a/b is nearly completely lost in liver fibrosis, and these effects might be partially reversed by FZHY therapy recovery. In vitro and in vivo studies indicated that Cyp4a12a/b deletion disrupted retinol metabolism and lowered Rae-1 expression. Activated HSCs successfully escape recognition and elimination by natural killer (NK) cells as a result of reduced Rae-1. Notablely, we discovered that FZHY may restore the Cyp4a12a/b capability, allowing the recovery of the cytotoxic function of NK cells against HSCs, and thereby reducing hepatic fibrosis by suppressing HSC activation.ConclusionOur findings revealed a new role for Cyp4a in retinol metabolism in the development of hepatic fibrosis, and they highlight Cyp4a12/Rae-1 signals as possible therapeutic targets for antifibrotic medicines.

The Important Roles of Natural Killer Cells in Liver Fibrosis.

Liver fibrosis accompanies the development of various chronic liver diseases and promotes their progression. It is characterized by the abnormal accumulation of extracellular matrix proteins (ECM) and impaired ECM degradation. Activated hepatic stellate cells (HSCs) are the major cellular source of ECM-producing myofibroblasts. If liver fibrosis is uncontrolled, it may lead to cirrhosis and even liver cancer, primarily hepatocellular carcinoma (HCC). Natural killer (NK) cells are a key component of innate immunity and have miscellaneous roles in liver health and disease. Accumulating evidence shows that NK cells play dual roles in the development and progression of liver fibrosis, including profibrotic and anti-fibrotic functions. Regulating NK cells can suppress the activation of HSCs and improve their cytotoxicity against activated HSCs or myofibroblasts to reverse liver fibrosis. Cells such as regulatory T cells (Tregs) and molecules such as prostaglandin E receptor 3 (EP3) can regulate the cytotoxic function of NK cells. In addition, treatments such as alcohol dehydrogenase 3 (ADH3) inhibitors, microRNAs, natural killer group 2, member D (NKG2D) activators, and natural products can enhance NK cell function to inhibit liver fibrosis. In this review, we summarized the cellular and molecular factors that affect the interaction of NK cells with HSCs, as well as the treatments that regulate NK cell function against liver fibrosis. Despite a lot of information about NK cells and their interaction with HSCs, our current knowledge is still insufficient to explain the complex crosstalk between these cells and hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, B cells, and T cells, as well as thrombocytes, regarding the development and progression of liver fibrosis.

IFN-β activates cytotoxic function of human natural killer cells toward IL-27 and poly(I:C) stimulated PC3 and DU145 cells

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-β secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-β produced in response to stimulation with IL-27 and poly(I:C). IFN-β increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

EBV-Upregulated B7-H3 Inhibits NK cell-Mediated Antitumor Function and Contributes to Nasopharyngeal Carcinoma Progression.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy characterized by the presence of prominent infiltration of lymphocytes, including natural killer (NK) cells. Although NK cells can directly target EBV-infected tumor cells without restriction by the MHC, EBV-positive (EBV+) NPC cells often develop resistance mechanisms that allow them to evade immune surveillance by NK cells. Elucidating the mechanisms involved in EBV-induced NK-cell dysfunction will contribute to the design of novel NK cell-based immunotherapies to treat NPC. Herein, we confirmed that the cytotoxic function of NK cells was impaired in EBV+ NPC tissues and found that EBV infection-induced expression of B7-H3 in NPC negatively correlated with NK-cell function. The inhibitory effect of EBV+ tumor expression of B7-H3 on NK-cell function was clarified in vitro and in vivo. Mechanistically, activation of the PI3K/AKT/mTOR signaling pathway via EBV latent membrane protein 1 (LMP1) was responsible for EBV infection-induced upregulation of B7-H3 expression. In an NPC xenograft mouse model with adoptive transfer of primary NK cells, deletion of B7-H3 on tumor cells in combination with anti-PD-L1 treatment restored NK cell-mediated antitumor activity and significantly improved the antitumor efficacy of NK cells. On the basis of our findings, we conclude that EBV infection can inhibit NK cell-mediated antitumor function by inducing upregulation of B7-H3 expression and provide a rationale for NK cell-based immunotherapies in combination of PD-L1 blockade and overcoming the immunosuppression of B7-H3 to treat EBV-associated NPC.

NK cell dysfunction is linked with disease severity in SARS-CoV-2 patients.

SARS‐CoV‐2 first raised from Wuhan City, Hubei Province in November 2019. The respiratory disorder, cough, weakness, fever are the main clinical symptoms of coronavirus disease 2019 (COVID‐19) patients. Natural Killer (NK) cells as a first defense barrier of innate immune system have an essential role in early defense against pulmonary virus. They kill the infected cells by inducing apoptosis or the degranulation of perforin and granzymes. Collectively, NK cells function are coordinated by the transmitted signals from activating and inhibitory receptors. It is clear that the cytotoxic function of NK cells is disrupted in COVID‐19 patients due to the dysregulation of activating and inhibitory receptors. Therefore, better understanding of the activating and inhibitory receptors mechanism could facilitate the treatment strategy in clinic. To improve the efficacy of immunotherapy in COVID‐19 patients, the functional detail of NK cell and manipulation of their key checkpoints are gathered in current review.

Omics approaches to discover pathophysiological pathways contributing to human pain.

Omics approaches to discover pathophysiological pathways contributing to human pain.

A vaccine targeting resistant tumours by dual T cell plus NK cell attack.

Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.

Inhibition of O-GlcNAcylation Decreases the Cytotoxic Function of Natural Killer Cells.

Natural killer (NK) cells mediate killing of malignant and virus-infected cells, a property that is explored as a cell therapy approach in the clinic. Various cell intrinsic and extrinsic factors affect NK cell cytotoxic function, and an improved understanding of the mechanism regulating NK cell function is necessary to accomplish better success with NK cell therapeutics. Here, we explored the role of O-GlcNAcylation, a previously unexplored molecular mechanism regulating NK cell function. O-GlcNAcylation is a post-translational modification mediated by O-GlcNAc transferase (OGT) that adds the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins and O-GlcNAcase (OGA) that removes the sugar. We found that stimulation of NK cells with the cytokines interleukin-2 (IL-2) and IL-15 results in enhanced O-GlcNAcylation of several cellular proteins. Chemical inhibition of O-GlcNAcylation using OSMI-1 was associated with a decreased expression of NK cell receptors (NKG2D, NKG2A, NKp44), cytokines [tumor necrosis factor (TNF)-α, interferon (IFN-γ)], granulysin, soluble Fas ligand, perforin, and granzyme B in NK cells. Importantly, inhibition of O-GlcNAcylation inhibited NK cell cytotoxicity against cancer cells. However, increases in O-GlcNAcylation following OGA inhibition using an OGA inhibitor or shRNA-mediated suppression did not alter NK cell cytotoxicity. Finally, we found that NK cells pretreated with OSMI-1 to inhibit O-GlcNAcylation showed compromised cytotoxic activity against tumor cells in vivo in a lymphoma xenograft mouse model. Overall, this study provides the seminal insight into the role of O-GlcNAcylation in regulating NK cell cytotoxic function.

Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients.

The Effect of Telomerase Inhibition on NK Cell Activity in Acute Myeloid Leukemia.

Purpose: Acute myeloid leukemia (AML) is known to be an invasive and highly lethal hematological malignancy in adults and children. Resistance to the present treatments, including radiotherapy and chemotherapy with their side effects and telomere length shortening are the main cause of the mortality in AML patients. Telomeres sequence which are located at the end of eukaryotic chromosome play pivotal role in genomic stability. Recent studies have shown that apoptosis process is blocked in AML patient by the excessive telomerase activity in cancerous blasts. Therefore, the find of effective ways to prevent disease progression has been considered by the researchers. Natural killer (NK) cells as granular effector cells play a critical role in elimination of abnormal and tumor cells. Given that the cytotoxic function of NK cells is disrupted in the AML patients, we investigated the effect of telomerase inhibitors on NK cell differentiation. Methods: To evaluate telomerase inhibition on NK cell differentiation, the expression of CD105, CD56, CD57, and KIRs was evaluated in CD34+ derived NK cells after incubation of them with BIBR1532. Results: The results showed that the expression of CD105, CD56, CD57, and KIRs receptors reduces after telomerase inhibition. According to these findings, BIBR1532 affected the final differentiation of NK cells. Conclusion: The results revealed that telomerase inhibitor drugs suppress cancer cell progression in a NK cells-independent process.

Radium-223 dichloride causes transient changes in natural killer cell population and cytotoxic function

Rationale Natural killer (NK) cells play an important role in both the innate and adaptive arms of the immune system. While previous studies have demonstrated the effects of ionizing radiation on cytotoxic function of NK cells, little is known about how a chronic exposure to high LET alpha particles emitted by radionuclides will affect both NK population size and function. This study was conducted to determine the effects of 223RaCl2 on splenic NK cell population size and function in Swiss Webster mice. Methods Swiss Webster mice were administered intravenously with 0, 50, or 600 kBq/kg 223RaCl2. Spleens were harvested at 5, 12, and 19 days post-administration. The numbers of splenocytes per spleen were enumerated and flow cytometry was used to assess changes in the distribution of splenocyte subpopulations of B, CD4 and CD8 T lymphocytes, and NK cells. NK functional activity was quantified using YAC-1 target cells and the 51Cr-release assay. Results The total number of splenocytes was unaffected by 223RaCl2. However, significant changes in the distribution of splenocyte subpopulations were observed for NK cells and CD8 T lymphocytes. NK functional activity was enhanced substantially relative to controls at 12 days post-administration, but decreased markedly by day 19. Conclusion NK functional activity is both diminished and enhanced by 223RaCl2 depending on both administered activity and time post-administration. These results suggest that there may be an optimum window of time to combine the 223RaCl2-induced antitumor NK cell response with other cancer therapies that elicit immune activation.

Abstract 1775: Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Even with intensive treatment, up to 50% of patients will relapse. Checkpoint inhibitors, including antibodies targeting programmed death 1 (PD-1), have dramatically improved survival outcomes but only elicit a response in 20% of patients. PD-1 is expressed on both T-cells and Natural Killer (NK) cells, and depletion of NK cells has been linked to poor prognosis in several solid tumors. NK cells are essential to the innate immune response, do not require MHC-1 expression for recognition, and can stimulate an anti-cancer response through release of cytotoxic granules and direct killing of cancer cells. Upregulation of several interleukins including IL-6, which induces the JAK/STAT3 pathway and subsequently inhibits cancer cell apoptosis, is correlated with a poor prognosis in HNSCC patients. Importantly, inhibition of phosphorylated STAT3 (pSTAT3) increases NK-specific cancer cytotoxicity, NK cell cytokine release, and anti-tumor cell chemokines. Therefore, we hypothesize that impaired NK cell cytotoxicity results in a poor response to immunotherapy. We have shown that a common anti-diabetes drug with anti-cancer properties, metformin, downregulates NK pSTAT3 with subsequent increase in cytotoxicity of peripheral NK cells in HNSCC patients. Metformin also decreases pSTAT3 pathway related cytokine release from HNSCC cell lines. However, it remains unclear whether metformin-mediated enhanced NK cytotoxicity was through a direct effect on NK cells or indirect through inhibition of pSTAT3 in tumor cells and subsequent decrease in inhibitory cytokine release. In addition, inhibiting pSTAT3 could lead to further activation of innate immunity through activation of NK cell cytotoxicity complementing current PD-1 inhibitor therapy and improved treatment response. Here we report the contribution of metformin-mediated direct and indirect effects on NK cell cytotoxic functions and the combined effect of metformin and pembrolizumab using patient samples from a clinical trial and ex vivo models. Understanding metformin-mediated regulation of pSTAT3 and downstream pathways will provide necessary insight to potentially overcome resistance to PD-1 inhibitors and identifying novel treatment combinations for this disease. Citation Format: McKenzie Crist, Maria Lehn, Trisha Wise-Draper. Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1775.

Reversing Hypoxia with PLGA-Encapsulated Manganese Dioxide Nanoparticles Improves Natural Killer Cell Response to Tumor Spheroids.

The adoptive transfer of natural killer (NK) cells, which can recognize and obliterate cancer cells, provides a practical alternative to current treatment modalities to improve cancer patients' survival. However, translating NK cell therapies to treat solid tumors has proven challenging due to the tumor microenvironment (TME). Hypoxia in the TME induces immunosuppression that inhibits the cytotoxic function of NK cells. Thus, reversing hypoxia-induced immunosuppression is critical for effective adoptive NK cell immunotherapy. In this study, we use manganese dioxide nanoparticles (MnO2 NPs) to catalyze the degradation of tumor-produced hydrogen peroxide, thereby generating oxygen. For improved biocompatibility and modulation of oxygen production, the MnO2 NPs were encapsulated into poly(lactic-co-glycolic) to produce particles that are 116 nm in size and with a ζ-potential of +17 mV (PLGA-MnO2 NPs). The PLGA-MnO2 NPs showed first-order oxygen production and sustained high oxygen tension compared to equivalent amounts of bare MnO2 NPs in the presence of H2O2. The PLGA-MnO2 NPs were biocompatible, reduced hypoxia after penetration into the core of cancer spheroids, and decreased hypoxia-induced factor 1 α expression. Reducing hypoxia in the spheroid resulted in a decrease in the potent immunosuppressors, adenosine, and lactate, which was confirmed by electrospray ionization mass spectroscopy (ESI-MS). ESI-MS also showed a change in the metabolism of the amino acids aspartate, glutamine, and glutamate after hypoxia reduction in the cancer cells. Notably, the spheroids' microenvironment changes enhanced NK cells' cytotoxicity, which obliterated the spheroids. These results demonstrate that reducing hypoxia-induced immunosuppression in tumors is a potent strategy to increase the potency of cytotoxic immune cells in the TME. The developed NPs are promising new tools to improve adoptive NK cell therapy.

Targeting Siglec-7: A Novel Immunotherapeutic Approach to Potentiate the Cytotoxic Functions of Natural Killer Cells Against Multiple Myeloma

Background: MultipleMyeloma (MM) is a malignant plasma cell disorder, accounting for approximately 10% of all haematological malignancies. The intrinsic ability of the malignant cells to evade the host immune system is one of the key hallmarks of the disease, and is associated with poor overall survival of MM patients. The immune-suppressive functions of sialylated glycans, which are abundantly expressed on the surface of myeloma cell, has not previously been addressed. We hypothesize that hypersialylation of MM cells is a key mechanism enabling evasion of cytotoxic Natural Killer (NK) cells within the MM bone-marrow (BM) niche. Furthermore, we hypothesize that disrupting the immune-suppressive interactions between sialylated glycans on MM cells, and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could pave the way for the development of novel immunotherapeutic strategies for treating MM patients.Methods and Results: Using a recombinant Siglec-7 Fc chimera, we observed that Siglec-7 ligands (Siglec-7L) are highly expressed (>85%) across a panel of MM cell lines (RPMI 8226, H929 and MM1S). Furthermore, Siglec-7L expression was also observed on CD38+ CD138+ primary MM cells isolated from BM aspirate of MM patient. To determine if Siglec-7L expression on MM cells plays a role in immune-evasion, we next analysed the cognate Siglec-7L receptor (Siglec-7R) on primary BM NK cells from MM patients (n=5). Interestingly, immunophenotyping analysis revealed that CD3- NKp46+ NK cells circulating within the BM of MM patients have significant expression of Siglec-7R on their cell surface (78 ±27%).To investigate the immune-suppressive effects of Siglec-7L and Siglec-7R interaction in dampening the cytotoxic functions of NK cells, we treated MM cell lines RPMI-8226, H929 and MM1S with a sialyltransferase inhibitor (SIA) (3Fax-Peracetyl Neu5Ac; dose-200µM) for 5 days. As expected, this treatment resulted in a complete eradication of Siglec-7L from the surface of MM cells. Subsequently, SIA pre-treated cells were co-cultured with the human cytotoxic NK cell line KHYG-1 at E:T ratios of 1:1, 2.5:1 and 5:1 for 12 hours. Flow cytometry based analysis of cell viability using Annexin V and Sytox Green revealed that abolishing the expression of sialylated glycans from the cell surface of MM cells results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI 8226 and H929 (n=3) (Figure 1). However, a significant increase in NK cell mediated killing was not observed for MM1S cells (n=3) pre-treated with SIA, suggesting a role of “classical” NK cell inhibitory signals in NK cell anti-tumor immunity in the case of MM1S.Furthermore to validate our observations we knocked-down (KD) Siglec-7R in KHYG-1 cells using an siRNA approach. KHYG-1 has relatively lower expression of Siglec-7R (20-25%), as compared to peripheral blood NK cells (>90%), and other NK cell lines such as NK-92 (>60%). siRNA targeting Siglec-7R reduced the expression of Siglec-7R from 25% in the scrambled siRNA control to 15% in the Siglec-7R siRNA experimental condition. Upon co-culture of the scrambled siRNA or Siglec-7R siRNA KD KHYG-1 cells with RPMI-8226 at E:T ratio of 0.25:1 and 0.5:1, we observed a 7% increase in NK cell mediated cytotoxicity towards the myeloma cells (n=2) .Conclusion: We observed that sialylated Siglec-7L is abundantly expressed on the surface of MM cells. The cognate receptors of the ligands are significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions may have significant immune-suppressive effects on the cytotoxicity of NK cells. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interaction in MM. This could be achieved by knocking down Siglec-7R in the setting of an autologous or allogeneic blood derived NK cell therapy. A more efficient approach, both therapeutically and logistically, would be the use of Siglec-7R deficient NK cell lines, such as KHYG-1 or NK-92 for the treatment of patients with MM.Conflict Of Interest: Michael O'Dwyer: Founder, Board of Directors, Equity in Onkimmune. Subhashis Sarkar is supported by a fellowship from Onkimmune. [Display omitted] DisclosuresSarkar:Onkimmune: Other: Fellowship Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.