Stimulation Of Natural Killer Cells Research Articles

NK cell cytotoxicity towards pluripotent stem cells and their neural progeny: impacts of activating and inhibitory receptors and KIR/HLA mismatch.

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2 and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2 or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2 stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Combinatorial immunotherapy of anti-MCAM chimeric antigen receptor modified expanded natural killer cells and NKTR-255 against neuroblastoma

Pediatric patients with recurrent metastatic neuroblastoma (NB) have a dismal 5-year survival. Novel therapeutic approaches are urgently needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is expressed in a variety of pediatric solid tumors, including NB, and constitutes anovel target for immunotherapy. Here, we developed a chimeric antigen receptor (CAR) expressing natural killer (NK) cell-targeting MCAM by non-viral electroporation of CAR mRNA into exvivo expanded NK cells. Expression of anti-MCAM CAR significantly enhanced NK cell cytotoxic activity compared to mock NK cells against MCAMhigh but not MCAMlow/knockout NB cells invitro. Anti-MCAM-CAR-NK cell treatment significantly decreased tumor growth and prolonged animal survival in an NB xenograft mouse model. NKTR-255, a polymer-conjugated recombinant human interleukin-15 agonist, significantly stimulated NK cell proliferation and expansion and further enhanced the in vitro cytotoxic activity and invivo anti-tumor efficacyofanti-MCAM-CAR-NK cells against NB. Our preclinical studies demonstrate that exvivo expanded andmodified anti-MCAM-CAR-NK cells alone and/or incombination with NKTR-255 are promising novel alternative therapeutic approaches to targeting MCAMhigh malignant NB.

Chronic stimulation desensitizes β2-adrenergic receptor responses in natural killer cells.

Adrenergic receptors (ARs) are preferentially expressed by innate lymphocytes such as natural killer (NK) cells. Here, we study the effect of epinephrine-mediated stimulation of the β2-adrenergic receptor (β2AR) on the function of human NK cells. Epinephrine stimulation inhibited early NK cell signaling events and blocked the function of the integrin LFA-1. This reduced the adhesion of NK cells to ICAM-1, explaining how NK cells are mobilized into the peripheral blood upon epinephrine release during acute stress or exercise. Additionally, epinephrine stimulation transiently reduced NK cell degranulation, serial killing, and cytokine production and affected metabolic changes upon NK cell activation via the cAMP-protein kinase A (PKA) pathway. Repeated exposure to β2AR agonists resulted in the desensitization of the β2AR via a PKA feedback loop-initiated G-protein switch. Therefore, acute epinephrine stimulation of chronically β2AR stimulated NK cells no longer resulted in inhibited signaling and reduced LFA-1 activity. Sustained stimulation by long-acting β2-agonists (LABA) not only inhibited NK cell functions but also resulted in desensitization of the β2AR. However, peripheral NK cells from LABA-treated asthma patients still reacted unchanged to epinephrine stimulation, demonstrating that local LABA administration does not result in detectable systemic effects on NK cells.

TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant.

High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. Ex-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-κb pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. Allo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.

TLR Agonists Modify NK Cell Activation and Increase Its Cytotoxicity in Acute Lymphoblastic Leukemia.

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.

Immunostimulatory Effects of Korean Mineral-Rich Seawaters on Cyclophosphamide-Induced Immunosuppression in Mice.

Deep seawater (DS), obtained from a depth over 200 m, has health benefits due to its rich nutrients and minerals, and intake of DS has shown diverse immunomodulatory effects in allergies and cancer. Therefore, the immunostimulatory effects of Korean mineral-rich seawaters were examined in a cyclophosphamide (CPA)-induced immunosuppression model. Three samples of Korean seawater, namely DS from the East Sea off the coasts of Pohang (PDS) and Uljin (UDS), and seawater from the West Sea off the coast of Boryeong (BS), were collected. The seawaters were abundant in several minerals (calcium, iron, zinc, selenium, etc.). Mice were orally administered the seawaters for 42 days, followed by CPA-induced immunosuppression. The CPA induction reduced the weight of the spleen and lymph nodes; however, the administration of seawaters increased the weight of the lymphoid organs, accompanied by stimulation of natural killer cells' activity and NF-kB-mediated cytokine production (IFNγ, TNFα, IL1β, IL6, and IL12). The mouse-derived splenocytes showed lymphoproliferation without cytotoxicity in the seawater groups. Histopathological analysis revealed that the seawaters improved the CPA-induced atrophic changes by promoting lymphoproliferation in the spleen and lymph nodes. These results provide useful information for the use of Korean mineral-rich seawaters, particularly PDS and UDS, as alternative immunostimulants under immunosuppressive conditions.

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation.

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.

Regulation of NK cell effector function by Acly

Abstract Natural Killer (NK) cells, crucial for viral immunity and tumor clearance, are influenced by metabolism. A prior study revealed that cytokine stimulation boosts the citrate-malate shuttle and cytosolic acetyl-CoA through ATP citrate lyase (ACLY) in NK cells. Acetyl-CoA is vital for fatty acid synthesis and protein acetylation, including histones. To explore the role of ACLY in NK cell function, we generated an inducible NK-specific Acly knockout mouse model. ACLY loss in NKp46+ NK cells did not alter maturation or IFN-γ production in naïve NK cells. However, ACLY-deficient NK cells exhibited notable proliferation defects in homeostatic and IL-15-stimulated conditions, associated with impaired glycolysis. The stimulation and priming of NK cells through IL-15 is an important mechanism for enhancing effector and anti-tumor functions. In addition to proliferation defect, IL-15-primed ACLY-deficient NK cells showed reduced IFN-γ responses to DAP12-associated activating receptors, specifically NKG2D, Ly49H, and Ly49D. RNA-seq revealed ACLY-deficient NK cells failed to upregulate DAP12 expression with IL-15 stimulation, confirmed at the protein level. These findings suggest the importance of ACLY in NK cell proliferation and potentially DAP12-driven effector functions. Ongoing experiments are focused on investigating how ACLY and cytosolic acetyl-CoA are linked to DAP12 expression, and the effects of this during in vivo effector functions.

Therapeutic Inducers of Natural Killer cell Killing (ThINKK): preclinical assessment of safety and efficacy in allogeneic hematopoietic stem cell transplant settings

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients, but cure rates are still dismal. To prevent leukemia relapse following HSCT, we aim to...

Epigenetic drug screen of natural killer cells identified compounds controlling immune training and tolerance

Abstract Natural killer (NK) cells are cytotoxic innate lymphocytes that provide defense against pathogens and malignancy. New evidence identified that NK cells are capable of memory-like immune responses in certain settings. This innate immune memory is thought to be imprinted through epigenetic modifications. However, precise epigenic pathways or strategies to modulate long-term NK cell activity are not well defined. In this study, we performed a screen of an epigenetic library containing 160 drug compounds with well-characterized epigenetic regulatory mechanisms, to identify drugs and pathways that could train or tolerize NK cell activation. One compound that we identified was the H3K27 methyltransferase Ezh2, which regulated NK cell lineage commitment from bone marrow hematopoietic stem cells and altered the phenotype of differentiated NK cells to increased cytotoxicity. This outcome provides an option to train or induce stronger cytotoxic effects on NK cells. In contrast, another compound we identified was the histone deacetylase inhibitor, Givinostat, which did not alter NK cell commitment from bone marrow stem cells, but significantly decreased NK cell function in the periphery resulting in a more tolerant innate immune state to secondary NK cell stimulation. Identification and exploration of these epigenome altering drugs may offer further insight into downstream pathways of NK cell function or provide novel therapies for tolerizing or priming innate immune responses.

Membrane Fluctuation Model for Understanding the Effect of Receptor Nanoclustering on the Activation of Natural Killer Cells through Biomechanical Feedback.

We investigated the role of ligand clustering and density in the activation of natural killer (NK) cells. To that end, we designed reductionist arrays of nanopatterned ligands arranged with different cluster geometries and densities and probed their effects on NK cell activation. We used these arrays as an artificial microenvironment for the stimulation of NK cells and studied the effect of the array geometry on the NK cell immune response. We found that ligand density significantly regulated NK cell activation while ligand clustering had an impact only at a specific density threshold. We also rationalized these findings by introducing a theoretical membrane fluctuation model that considers biomechanical feedback between ligand-receptor bonds and the cell membrane. These findings provide important insight into NK cell mechanobiology, which is fundamentally important and essential for designing immunotherapeutic strategies targeting cancer.

Dynamic Hybrid Module-Driven NK Cell Stimulation and Release for Tumor Immunotherapy.

Natural killer (NK) cells have become a powerful candidate for adoptive tumor immunotherapy, while their therapeutic efficacy in solid tumors remains unsatisfactory. Here, we developed a hybrid module with an injectable hydrogel and hydroxyapatite (HAp) nanobelts for the controlled delivery of NK cells to enhance the therapy of solid tumors. Surface-functionalized HAp nanobelts modified with agonistic antibodies against NKG2D and 4-1BB and cytokines IL-2 and IL-21 support survival and dynamic activation. Thus, the HAp-modified chitosan (CS) thermos-sensitive hydrogel not only improved the retention of NK cells for more than 20 days in vivo but also increased NK cell function by more than one-fold. The unique architecture of this biomaterial complex protects NK cells from the hostile tumor environment and improves antitumor efficacy. The generation of a transient inflammatory niche for NK cells through a biocompatible hydrogel reservoir may be a conversion pathway to prevent cancer recurrence of resectable tumors.

MADD regulates natural killer cell degranulation through Rab27a activation.

Natural killer (NK) cells have the ability to lyse other cells through the release of lytic granules (LGs). This is in part mediated by the small GTPase Rab27a, which was first identified to play a crucial role in degranulation through the study of individuals harboring mutations in the gene encoding Rab27a. However, the guanine nucleotide exchange factor (GEF) regulating the activation of Rab27a in cytotoxic lymphocytes was unknown. Here, we show that knockout of MADD significantly decreased the levels of GTP-bound Rab27a in both resting and stimulated NK cells, and MADD-deficient NK cells and CD8+ T cells displayed severely reduced degranulation and cytolytic ability, similar to that seen with Rab27a deficiency. Although MADD colocalized with Rab27a on LGs and was enriched at the cytolytic synapse, the loss of MADD did not impact Rab27a association with LGs nor their recruitment to the cytolytic synapse. Together, our results demonstrate an important role for MADD in cytotoxic lymphocyte killing.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Anti-Inflammatory Effects and Immunomodulatory Efficacy of Unitein (Fermented Glycine max, Panax ginseng, and Chenpi Mixture) in RAW 264.7 Macrophages and Mice Splenocytes along with Component Analysis

Inflammation and the immune system are intricately linked, with the immune system serving as a vital defense mechanism in the human body. Consequently, there is great emphasis placed on the regulation of both the body’s inflammatory response and the immune system. This study investigated the anti-inflammatory properties and immunomodulatory effects of Unitein (fermented Glycine max, Panax ginseng, and chenpi mixture) in both RAW 264.7 macrophages and mice splenocytes while also comprehensively analyzing its components. To this end, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was employed to evaluate the cytotoxicity of the samples to RAW 264.7 cells. The Griess assay was utilized to determine nitrite concentrations, while the enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of inflammation-related cytokines in RAW 264.7 cells and isolated mouse splenocytes. Additionally, quantitative polymerase chain reaction was employed to quantify the mRNA expression of inflammation-related genes in the cells, while the lactate dehydrogenase assay was used to analyze natural killer (NK) cell activity. Our results revealed that Unitein exhibited no toxicity to RAW 264.7 cells at concentrations below 1.5 mg/mL. However, Unitein effectively suppressed the release of proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ, as well as enzymes COX-2 and iNOS from RAW 264.7 cells, while concurrently promoting the release of anti-inflammatory cytokines IκB-α, IL-10, and IL-4. Furthermore, Unitein inhibited the increase in TNF-α, IL-1β, IFN-γ, IL-12, nuclear factor (NF)-κB p50, NF-κB p65, and iNOS induced by lipopolysaccharides (LPSs) in isolated mouse splenocytes. Notably, Unitein also exhibited the potential to stimulate NK cell activity. Metabolite analysis indicated that Unitein contained more active compounds with anti-inflammatory and immunity-enhancing properties than did unfermented Unitein. This study highlights the potential therapeutic role of Unitein in mitigating inflammation and enhancing immune responses, thereby deepening our understanding of its biological activities and underlying mechanisms. These findings underscore the significance of Unitein as a potential candidate for the development of novel anti-inflammatory and immunomodulatory interventions.

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Degranulation assay to evaluate NK cell natural and antibody-dependent cell-mediated cytotoxicity against A549 tumor spheroids.

Adoptive natural killer (NK) cell-based immunotherapy is a promising treatment approach in cancer that is showing notable efficacy against hematological malignancies. However, the success of NK cell immunotherapy in patients with solid tumors is limited due to several barriers, which include the immunosuppressive tumor microenvironment (TME), heterogeneity of tumor cells and poor NK cell infiltration into the tumor. Advances in 3D in vitro culture technologies have opened new avenues for the development of more physiological human cancer models that mimic important tumor features which are absent in traditional 2D studies and may be essential for the improvement of immunotherapies against solid tumors. Here, we describe a comprehensive protocol to generate tumor spheroids from the A549 lung carcinoma cell line, then establish co-cultures with NK cells to, ultimately, determine NK cell functional response with a degranulation assay, a surrogate of NK cell cytotoxicity against tumor spheroids. Additionally, we studied degranulation by stimulating NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) with cetuximab, an IgG1 monoclonal antibody used in cancer therapy. Likewise, other monoclonal antibodies or combination treatments could also be studied in this 3D co-culture system, providing very valuable information to define effective combinations of therapeutic agents able to generate NK cells with high cytotoxic potential that could lead to more successful adoptive NK cell-based therapies for the treatment of solid tumors.

Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 μM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

Suppression of NK Cell Activation by JAK3 Inhibition: Implication in the Treatment of Autoimmune Diseases.

Natural killer (NK) cell is an essential cytotoxic lymphocyte in our innate immunity. Activation of NK cells is of paramount importance in defending against pathogens, suppressing autoantibody production and regulating other immune cells. Common gamma chain (γc) cytokines, including IL-2, IL-15, and IL-21, are defined as essential regulators for NK cell homeostasis and development. However, it is inconclusive whether γc cytokine-driven NK cell activation plays a protective or pathogenic role in the development of autoimmunity. In this study, we investigate and correlate the differential effects of γc cytokines in NK cell expansion and activation. IL-2 and IL-15 are mainly responsible for NK cell activation, while IL-21 preferentially stimulates NK cell proliferation. Blockade of Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway by either JAK inhibitors or antibodies targeting γc receptor subunits reverses the γc cytokine-induced NK cell activation, leading to suppression of its autoimmunity-like phenotype in vitro. These results underline the mechanisms of how γc cytokines trigger autoimmune phenotype in NK cells as a potential target to autoimmune diseases.

Abstract C055: Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma

Abstract Background: Tumor-induced immunosuppression and cell proliferation are the hallmarks of progression of malignant tumors including high-grade serous ovarian carcinoma (HGSOC), a fatal malignancy of women. Natural killer (NK) cell plays critical roles in killing tumor cells in the tumor vicinity as it does not require antigen presentation. Our earlier studies showed the frequency of tumor infiltrating NK cells is significantly low as compared with that of tumor stroma suggesting that tumor-induced factors may be involved in inhibition of NK cell infiltration, and the tumor continue to metastasize. Cytokines including IL-10/IL-15 are used to stimulate NK cells and tumor continuously secretes IL-10 or IL-15 and it is possible that excessive exposure to these cytokines induces cytokine inducible SH2-containing protein (CISH), a marker of NK cell exhaustion. We have shown earlier that root powder of Ashwagandha (ASH), an anti-stress natural herb improved NK cell population and reduced tumor progression. The goal of this study was to determine whether Withaferin A (WFA), an active ingredient of ASH, targets the induction of CISH or cyclin D1 (CCND1), a stimulator cell growth and prevented tumor progression. Materials & methods: Study-1: An exploratory study was conducted with clinical specimens which included normal ovaries (n=5) and ovarian high grade serous carcinoma (HGSC) at both early and late stages (n=5 each) and expression levels of CISH and CCND1 were examined. Study-2: A prospective study using the laying hen model of OVCA supplemented with or without 2% dietary ASH root powder for 90 days (n=7 each;normal, early and late-stage). Normal and tumor ovarian tissues were collected at the end of the study and used for analysis. Both the clinical and hen specimens were used for immunohistochemistry, western blotting and gene expression assays to determine expression levels of CISH and CCND1. In vitro studies were also conducted with WFA on the OVCAR3, an HGSC cell line, where multiple doses were tested, and expression of markers previously mentioned were examined. Data were collected and analyzed by ANOVA and unpaired t-test and considered significant when P<0.05. Results: Frequency of CISH-expressing cells in both clinical and hen specimens in normal, early and late-stages were localized. Compared to normal, increased expression of both CISH and CCND1 was seen in early-stages and increased further through late stages. Similarly, strong expression of both CISH and CCND1 was seen in proteomic and genomic studies. ASH supplementation is hens compared to untreated showed a significant decrease (P<0.001) in CISH expression as well as in CCND1 gene expression studies. In vitro studies WFA treatment significantly decreased expression of both markers (P<0.05). Conclusion: The results of this study suggest that both CISH and CCND1 are involved in progression of OVCA and dietary supplementation with ASH or treatment with WFA may be an effective bi-targeted immunotherapy against HGSOC. Support: Ovarian Cancer Spore, Roswell Park Citation Format: Jasmin C. Acosta, Animesh Barua. Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C055.