Cytotoxic Activity Of Natural Killer Cells Research Articles

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells

BackgroundNeuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal...

Immunomodulatory effects of flavonoids: An experimental study on natural-killer-cell-mediated cytotoxicity against lung cancer and cytotoxic granule secretion profile

Background: A new approach involving immune-cell-mediated cancer therapy has been adopted extensively for the sake of lung cancer treatments by utilizing natural killer (NK) cells. NK cell activity can be enhanced with certain agents, and among them are flavonoids. Thus, this study was conducted to investigate the immunomodulatory roles of apigenin, luteolin and quercetin on NK cell activity against lung cancer cells and on the secretions of perforin and granulysin profile. Methods: The NK-92 cells were grown in complete α-Minimum Essential Medium (MEM). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 media. NK cell activity against lung cancer cells were done using MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The secretions of perforin and granulysin profiles were then analysed using enzyme-linked immunosorbent assay. Results: Apigenin, luteolin and quercetin significantly increased the NK-cell-mediated cytotoxic activity against lung cancer cells at concentrations 12.5 µg/ml and 25µg/ml ( P < 0.001). The secretion levels of perforin and granulysin from NK cells were also significantly enhanced with apigenin and luteolin treatment but not with quercetin. Conclusions: All three flavonoid compounds possessed some significant immunomodulatory actions on NK cell cytotoxic activity and granule secretion profiles towards lung cancer therapy.

Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

The uteroplacental contact zone cytokine influence on NK cell cytotoxicity to trophoblasts

Objective The present study was to estimate the role of cytokines for trophoblast death in NK cells presence. Methods This study involves assessment of NK-92 line NK cell cytotoxic activity against JEG-3 line cells, in presence of cytokines. We also assessed the effect of secretory placenta products on NK cell cytotoxic activity toward JEG-3 line cells. Results Uteroplacental contact zone cytokines are able to enhance trophoblast mortality both by themselves in case of IL-1β, IL-6, IFNγ, IL-4, TGFβ, bFGF, and also through increasing the cytotoxic potential of NK cells in case of IL-1β, IFNγ, IL-8, TGFβ, and GM-CSF. PLGF decreases NK cell cytotoxicity for trophoblasts. Secretory products of first trimester placenta enhance NK cell cytotoxic potential for trophoblasts. Conclusions Cytokines of the uteroplacental contact zone can appear a mechanism ensuring trophoblast mortality dynamics throughout pregnancy.

Enhancing the Immune Surveillance in Multiple Myeloma Via CDK4/6 Inhibition

Deregulation of cyclin D genes is a uniform event in multiple myeloma (MM) and represent a striking addiction as observed in pan-cancer genome-wide CRISPR screening data. However, early stage Cyclin D and other cell cycle kinases inhibitors have shown a lack of single agent activity suggesting that targeting of cell cycle regulation is insufficient to produce a durable response in MM. Recent evidence recognizes the Cyclin D and CDK4 activities within the immune tumor microenvironment, supporting a previously unrecognized immunomodulatory functions of CDK4/6. This is particularly important in MM, a highly heterogeneous disease that resides in a complex ecosystem comprising of immune, endothelial, and stromal cells. We here evaluated the tumor intrinsic and extrinsic effects of CDK4/6 inhibition in MM with the goal to define rationally designed combination strategies to effectively impact MM growth. We have evaluated MM cell sensitivity to CDK4/6 inhibitors (both Palbociclib and Abemaciclib) in a panel of 32 MM cell lines and primary MM patients' samples. As expected, both inhibitors were mostly cytostatic with G0/G1 cell cycle arrest and significant impact on the pRB-E2F axis both in vitro as well as in vivo. In luminescence subcutaneous SCID models engrafted with H929 or MM1S MM cells expressing an E2F-driven luciferase reporter, treatment with low dose Palbociclib or Abemaciclib caused regression of bulky tumors, evidenced by a ~40% reduction in tumor volume at the 14-day end-point and a decreased E2F1 reporter activity. We next studied genome-wide transcriptional response to treatment using RNA-seq analysis in two MM cell lines using multiple doses and duration (24 and 72 hours) to evaluate the effect of short and long exposure to the drug. Gene set enrichment analysis (GSEA) of RNA-seq data confirmed significant downregulation of proliferation and E2F target genes at 24 hours post treatment. Interestingly, after longer exposure to both drugs we observed modulation of signatures for Ag presentation (including upregulation of HLA-A,B, and C; B2MG), innate immune response (ICAM-1 and 2; IL-8 and several CCLs) and interferon inducible genes IFN response (IRF1, IRF9, STAT1, STAT2, STAT4, OSA1, OSA2, MX1). To confirm genomic data and evaluate if CDK4/6i promotes the induction of the senescence-associated secretory phenotype (SASP) program in MM cells, we performed cytokine profile to assess the secretion of 174 soluble factors in the MM cell supernatant upon CDK4/6i. We confirmed a significant increase of chemokines involved in NK cell recruitment (CCL2, CCL4, CCL5), as well as cytokines that promote NK cell proliferation and activation. Moreover, we found that intercellular adhesion molecule-1 (ICAM-1) and the NKG2D ligands ULBP2 and MICA, required for activation of NK cell cytotoxicity and tumor cell targeting, were induced after CDK4/6i in MM cells. Although not secretory per se, these NK cell ligands are part of the transcriptional module linked to the SASP. Overall, these data suggest that, in addition to a more stable cell cycle arrest, CDK4/6i may promote MM cell immune surveillance through induction of the SASP program. To further confirm the immune effects, we tested freshly isolated NK cells from MM patients and healthy donors using in vitro MM-NK cell coculture assay and observed enhanced degranulation and cytokine production (intracellular IFN-γ and TNFα) by NK cells in response to MM cells. We finally investigated the potential of combining CDK4/6i with daratumumab in a standard 4-h ADCC assays using NK cells from MM patients as effector and MM cell lines as target. Daratumumab-mediated ADCC against MM cells was significantly augmented in the combination compared to single agent. In conclusion, we here report a novel anti-MM activity of CDK4/6i which is beyond the previously reported growth arrest. The observed ability to directly modulate the immune system along with decrease in the proliferative potential of MM cells may provide opportunities to develop unique combination approaches in MM. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Fulciniti:NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Legend: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company.

Long noncoding RNA LINC00657 inhibits cervical cancer development by sponging miR-20a-5p and targeting RUNX3.

Long noncoding RNAs act essential regulators in cervical cancer progression. Our study aimed to investigate the underlying function and molecular mechanisms of LINC00657 in cervical cancer. QRT-PCR results indicated that LINC00657 was significantly decreased in cervical cancer. Gain-and loss-of-function experiments were performed in SiHa and HeLa. Functional assays demonstrated that LINC00657 inhibited cervical cancer cell growth, migration and invasion. Moreover, miR-20a-5p was confirmed as a target of LINC00657. Furthermore, miR-20a-5p promoted the development of cervical cancer via targeting RUNX3. DR5 acts as a vital promoter in activating NK cells and is a downstream target of RUNX3. We found that LINC00657 overexpression promoted the cytotoxic activity of NK cells via regulating RUNX3/DR5 axis. Therefore, LINC00657 suppressed cervical cancer progression via inducing miR-20a-5p/RUNX3/DR5 mediated NK cell tolerance. In conclusion, LINC00657 was identified as a novel tumor-suppressor in cervical cancer and could function as a potential therapeutic target for clinical treatment.

The Role of Thyroid Hormone in the Innate and Adaptive Immune Response during Infection.

In the past decades, there has been growing evidence for a functional interaction between the thyroid hormone and the immune system. This article provides an overview of the mechanisms by which thyroid hormones affect the innate and adaptive immune response during infection. The influence of thyroid hormone on the most important players of the innate [neutrophils, macrophages, natural killer (NK) cells, and dendritic cells (DCs)] and adaptive immune system (B- and T-lymphocytes) is reviewed here based on both clinical and preclinical studies. The effects of modulation of the immune system by drugs, such as monoclonal antibodies, tyrosine kinase inhibitors, and interferons on thyroid function, are beyond the scope of this article. Thyroid hormones regulate the activity of neutrophils which is reflected by higher numbers of neutrophils outside the bloodstream and enhanced activity of the respiratory burst following stimulation with thyroid hormone. Hyperthyroidism affects neutrophil function to a larger extent than hypothyroidism. In addition to neutrophil function, macrophage function is strongly affected by thyroid hormones, with triiodothyronine having a pro-inflammatory effect in these cells. NK cell proliferation and cytotoxic activity are also dependent on thyroid hormone levels. Finally, thyroid hormones enhance DC proliferation and maturation. In the adaptive immune system, a hyperthyroid state leads to increased activation of lymphocytes. This effect of thyroid hormone is mediated by various factors including NF-κB and protein kinase C signaling pathways and the β-adrenergic receptor. In general, a hyperthyroid state leads to a more activated immune system whereas hypothyroidism leads to a less activated immune system. © 2020 American Physiological Society. Compr Physiol 10:1277-1287, 2020.

Escape of tumor cells from the NK cell cytotoxic activity

In recent years, NK cells, initially identified as potent cytotoxic effector cells, have revealed an unexpected complexity, both at phenotypic and functional levels. The discovery of different NK cell subsets, characterized by distinct gene expression and phenotypes, was combined with the characterization of the diverse functions NK cells can exert, not only as circulating cells, but also as cells localized or recruited in lymphoid organs and in multiple tissues. Besides the elimination of tumor and virus-infected cells, these functions include the production of cytokines and chemokines, the regulation of innate and adaptive immune cells, the influence on tissue homeostasis. In addition, NK cells display a remarkable functional plasticity, being able to adapt to the environment and to develop a kind of memory. Nevertheless, the powerful cytotoxic activity of NK cells remains one of their most relevant properties, particularly in the antitumor response. In this review, the process of tumor cell recognition and killing mediated by NK cells, starting from the generation of cytolytic granules and recognition of target cell, to the establishment of the NK cell immunological synapse, the release of cytotoxic molecules, and consequent tumor cell death is described. Next, the review focuses on the heterogeneous mechanisms, either intrinsic to tumors or induced by the tumor microenvironment, by which cancer cells can escape the NK cell-mediated attack.

Quantitative and Functional Analysis of PD-1+ NK Cells in Patients With Autoimmune Thyroid Disease.

Natural killer (NK) cells have an important role in innate immunity and in the regulation of immune response. The role of NK cells expressing the programmed cell death protein-1 (PD-1) regulatory receptor has not been explored in patients with autoimmune thyroid disease (AITD). To analyze the levels and function of PD-1+ NK cells in samples from AITD patients. Cases and controls, observational study. Hospital Universitario la Princesa, Spain. Forty patients with AITD, 16 with Hashimoto thyroiditis (HT), 24 with Graves' disease (GD), and 15 healthy controls. Multiparametric flow cytometry analysis of peripheral blood NK cells. In vitro assays of cytotoxic activity of NK cells, and synthesis of cytokines. Levels and function of PD-1+ NK cells in blood samples from AITD patients and controls. Increased levels of NK cells and the CD56dimPD-1+ subset were observed in GD patients. In HT, an enhanced expression of the regulatory receptors NKG2A and NKG2C by CD56brightPD-1+ NK cells was detected. AITD patients showed an increased synthesis of IL-10 by CD56brightPD-1- NK cells, whereas CD56dimPD-1+ cells from GD patients exhibited an enhanced production of interferon-γ. PD-1+ NK cells from patients with GD and HT showed an increased cytotoxic activity. Significant associations were observed in patients with GD or HT between the levels of PD-1+ NK cells and clinical laboratory parameters. The different abnormalities in NK cell subset levels, in the expression of PD-1 and its function in AITD patients' further support the complex role of these cells in this pathogenesis.

Abstract 6686: SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner

Abstract SO-C101 (RLI-15) is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells and reducing the toxicity of IL-15 as such. SO-C101 was previously shown to exhibit a potent anti-metastatic activity in Renca, B16F10 melanoma and delayed tumor growth in T cell-based mouse tumor models (CT26, MC38). Here we investigated the anti-tumor efficacy in predominantly natural killer (NK)-cell based mouse tumor models TC-1 and TRAMP-C2. We showed that SO-C101 monotherapy was effective in the treatment of established TC-1 tumors, which was dependent on the presence of both NK and CD8+ T cells, but not CD4+ T cells. In an early treatment setting SO-C101 significantly decreased the rate of tumor development also in dependence on NK and CD8+ T cells. SO-C101 effectively reduced tumor growth in TRAMP-C2 mice in early and advanced treatment settings. However, only in combination with anti-PD-1 antibody treatment the tumor development was prevented in majority of mice. This effect was durable, and the new tumor development was further significantly delayed after a tumor cell re-challenge, which suggests the involvement of memory T cells despite an important NK cell role in anti-tumor efficacy in these models. The efficacy of SO-C101 and anti-PD-1 treatment was not dependent on CD4+ T cells, but mainly on NK and CD8+ T cells. Interestingly, SO-C101 and anti-PD-1 treatment in double NK/CD8+ T cell-depleted mice decreased tumor growth which suggests an involvement of other immune cell populations in the anti-tumor efficacy. SO-C101 stimulated the proliferation and the cytotoxic activity of NK cells and memory CD8+ T cells without significant expansion of regulatory T cells. These data show the importance of various immune cell populations during SO-C101 monotherapy and the treatment in combination with anti-PD-1 antibodies, and set a base for further complex analysis of SO-C101 behavior. The therapeutic potential of SO-C101 is currently being tested in an ongoing Phase I clinical study in cancer patients. Citation Format: Irena Adkins, Romana Mikyskova, Nada Hradilova, Guy de Martynoff, David Bechard, Ulrich Moebius, Milan Reinis, Radek Spisek. SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6686.

Abstract 933: Pre-clinical characterization of TIM-3 antibody NB002

Abstract Targeting immune checkpoint molecules PD1 and CTLA-4 by activating the immune system have improved the lives of many cancer patients. However, this type of immunotherapy is not effective for all patients, suggesting that there are additional mechanisms of immune evasion. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has been reported to be expressed on exhausted T and NK cells within the tumor microenvironment. The data in many preclinical models suggest that targeting TIM-3 pathway might provide an additional immune modulation of this anti-tumor axis in cancer patients. We have developed a novel monoclonal antibody NB002 against TIM-3,which forms a high-affinity interaction with a unique epitope. NB002 can enhance IFN-gamma secretion and proliferation of these exhausted T cells that has increased TIM-3 expression. Moreover, TIM-3 is constitutively expressed on NK cells. TIM-3 blockade could enhance in vitro cytotoxic activity of NK cells. Altogether, our data shows that NB002 is capable of targeting exhausted T cells and NK cells resulting activation of antitumor immunity. Further preclinical assessment in vivo and in vitro for solid tumors will support clinical development of NB002. Citation Format: Xin Dong, Jannie Dong, Jun Ma, Lianqi Zhao, Haojie Wang, Binbin Wang, Qian Gao, Hui Pan, Dong Wang, Liegang Shao. Pre-clinical characterization of TIM-3 antibody NB002 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 933.

Abstract 3333: EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity

Abstract Purpose: Breg (B regulatory) cells have been implicated in suppression of anti-tumor response in murine and human solid tumors. Prior studies in our laboratory demonstrated that murine EMT-6 breast carcinoma is rejected in B cell deficient mice (BCDM), but not in wild type (WT) mice, due to an enhanced immune response. Tumor infiltrating B cells (TIL-B), and normal splenic B cells co-cultured with EMT-6 (EMT6-B), express LAP/TGF-β, PD-L1, and suppress CD8+ and NK cell proliferation in vitro. Increased CD8+ and NK cell infiltration into the tumor bed is seen in BCDM. BCDM tumor rejection is abrogated by NK depletion. We studied EMT6-B effects on NK cell phenotype, proliferation and cytokine expression in vitro and in vivo. Experimental design: Normal splenic B cells were co-cultured with mitomycin C treated EMT-6, and Breg phenotype, and effects on NK cell phenotype and proliferation and cytokine function were studied in vitro and in vivo. Results: EMT6-B but not WT splenic B cells inhibited CFSE labeled CD49b+ NK cell proliferation in response to IL-15 in dose dependent fashion. EMT-6-B suppressed expression of CD226 and NKG2D activating receptors on NK cells, and increased expression of PD-1 and PD-L1 respectively. EMT6-B suppressed the induction of IFNγ+/CD49b+ NK cells following PMA/ION stimulation as assayed by intracellular flow cytometry (10%±1.2 to 2%±0.5) and also suppressed IL-2 induced induction of IFNγ+/CD49b+ NK cells (70%±5 to 20%±7.8). EMT-6 and CT26 colon carcinoma conditioned B cells, but not C166 endothelial cell conditioned B cells suppressed NK degranulation (IFNγ+/CD107a+ NK cells). EMT6-B inhibited YAC-1 cell induced NK cell degranulation, and killing of YAC-1 targets, more effectively than naïve B cells (40%±6.2 to 18%±5.1) (P=0.05). NK suppression was inhibited by anti-PD-L1, and anti-TGF-β antibody. EMT6-B expressed increased FasL compared to splenic B cells, and induced NK apoptosis as measured by 7AAD/Annexin-V staining. Blocking antibody to FasL decreased NK apoptosis, and restored NK cell proliferation. Infiltrating NK cells in the EMT6 tumor bed demonstrated increased PD-1 compared to splenic and TDLN NK cells, and higher levels of IFN-γ, CD107a, and Granzyme B in BCDM vs. WT mice. EMT6 expressed high levels of CD155 NK inhibitory ligand compared to C166 endothelial cells. The EMT-6 tumor bed contained higher levels of CD19+CD155+ B cells (22% / tumor bed vs 6% / TDLN, vs 9%/ spleen). BCDM expressed higher levels of CD226+/Ki67+ NK cells in spleen compared to WT splenic NK cells. Antibodies to Fas-L, TGF-beta, and PD-L1 partially restored anti-tumor response in WT mice and decreased tumor growth. Conclusions: EMT-6 TIL-B or normal B cells conditioned by EMT-6 coculture acquire an NK suppressive phenotype which inhibits NK cell activation, proliferation, degranulation and cytotoxic function. Targeting of Breg-NK interactions may improve anti-tumor immune response. Citation Format: Yu Zhang, Muhammad Husnain, Zhifen Luo, Mahmood Al Bayati, Joseph D. Rosenblatt. EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3333.

Inhibitory KIR2DL2 Gene: Risk for Deep Endometriosis in Euro-descendants.

Endometriosis (EDT) is an inflammatory disease characterized by implantation/growth of endometrial tissue, glands, and/or stroma, outside the uterus. Reduced NK cell cytotoxic activity has been implicated in its pathogenesis, together with other immunologic alterations. We investigated the influence of KIR gene polymorphisms and their HLA ligand combinations in deep endometriosis (DE) susceptibility. One hundred sixty women with a histological diagnosis of DE and 202 control women without the disease, who underwent laparoscopy, were enrolled. The DE group was subdivided into initial (I/II; n = 60) and advanced stages (III/IV, n = 100). KIR and HLA class I gene polymorphisms were typed by PCR-SSP and sequence-based-typing (SBT), respectively. We observed a significant association of KIR2DL2, an inhibitory gene of B haplotype, conferring risk for DE in Euro-descendants. Positive associations of Bx haplotype and centromeric AB segments were also found. However, no association with KIR-HLA ligand combination was observed. Our data suggest KIR2DL2 gene to be a relevant factor favoring NK inhibition in DE in Euro-descendants, contributing to the defective NK cytotoxic activity and impaired clearance of ectopic endometrial cells in the disease.

Involvement of natural killer cells in the pathogenesis of endometriosis in patients with pelvic pain.

ObjectivesTo detect the involvement of immune cells in the pathogenesis of endometriosis in patients with stable status or pelvic pain.MethodsBlood was collected from patients with endometriosis with and without pelvic pain. Natural killer (NK) and Th17 cells were analyzed by flow cytometry, and secretion of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-7) was verified by enzyme-linked immunosorbent assay. We isolated immune cells from blood by density-gradient centrifugation to investigate the expression of functional molecules including sterile alpha motif domain-containing protein 9 (SAMD9), Ral guanine nucleotide dissociation stimulator-like 2 (RGL2), early growth response protein 1, and Akirin2. We also searched the BIOGPS database for protein expression profiles.ResultsSAMD9 and RGL2 expression levels were significantly upregulated in patients with pelvic pain. Furthermore, lysophosphatidic acid receptor 1 expression was higher in endometrial tissues from patients with pelvic pain, and was mainly localized in stromal and glandular epithelial cells in ectopic lesions.ConclusionNK cells play an important role in the pathogenesis of endometriosis in patients with pelvic pain. Suppressing the cytotoxic activity of NK cells may thus help to reduce the progression of pelvic pain in patients with endometriosis.

Dysfunctional Natural Killer cells expanded from a liver with Hepatocellular Carcinoma show reduced killing against a unique HCC cell line

BackgroundThe liver has one of the largest reserves of NK cells in the body. NK cells in the liver are key defenses against viral hepatitis, liver fibrosis, and tumorigenesis. In the healthy liver, they exhibit higher cytotoxicity and secrete more cytokines compared to those isolated from peripheral blood. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing cancers worldwide. Previously, NK cells have been shown to be dysfunctional in HCC. Additionally, NK cells are reduced in number intratumorally and peripherally and there is a decrease in the cytotoxic CD56dim subpopulation. Many studies have shown a downregulation of key signaling molecules involved in peripheral NK cell cytotoxic activity. We aimed to further investigate the level of dysfunction of NK cells isolated from HCC livers to document the nature of dysfunction of liver‐resident NK cells in eliminating HCC cell lines.MethodsBriefly, samples of tissue were collected from explanted livers and made into single cell suspensions. Next, lymphocytes were isolated using density‐based centrifugation. The resulting fraction was placed into RPMI‐1640 with IL‐2 and IL‐15. After two weeks, samples were taken to determine the number and purity of NK cells by flow cytometry. Target cell lines were labeled with chromium‐51 to test the killing capacities of NK cells isolated from healthy donor PBMC’s or HCC patient livers. NK cells were prepared in serial dilutions and in triplicate to generate a killing curve. After 4 hours of coincubation of effector NK cells with target cells, the released radioactive particles were measured and analyzed.ResultsTwo cell lines, Hep3B (ATCC) and HCO2 (generated in our lab), were used as target cells. Figures 1 and 2 depict the results of separate killing assays using the different target HCC cell lines. The effector cells in each assay consist of NK cells sourced from either healthy donor PBMC’s, cirrhotic patient PBMC’s, or HCC liver. Notably, the results show that even under the constant stimulation of IL‐2 and IL‐15, which are known interleukins that are involved in the proliferation and activation of NK cells and T‐cells, there is a large dysfunction in NK cells sourced from diseased livers. More strikingly, our results show that there is a specific inability of NK cells sourced from HCC livers to eliminate HCO2 cells which have been previously shown in our lab to be sorafenib‐resistant. Our hypothesis is that there is an innate dysfunction that is present in NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines. Additionally, HCO2 cells possess a unique resistance to killing by NK cells isolated and expanded from an HCC liver.ConclusionHere we show that NK cells can be isolated and expanded, ex vivo, from human diseased livers. Additionally, we show that despite being stimulated for over 2 weeks with cytokines, expanded NK cells still show a marked reduction in killing capacity against a variety of HCC cell lines compared to NK cells expanded from PBMC’s from healthy and diseased donors. Our results confirm that there is an innate dysfunction that is present within NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines.HCC cell lines (Hep3B, in circles, HCO2, in triangles) were labeled with Chromium‐51 for 2 hours. Target cells were rinsed with media and cocultured with NK cells at various effector‐to‐target ratios. NK cells were sourced from either PBMC’s from a cirrhotic patient (PBNK, in blue) or a liver with hepatocellular carcinoma (LNK, in green) and expanded ex vivo with IL‐2 and IL‐15.Figure 1

Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro

Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

Tumor-derived soluble CD155 inhibits DNAM-1-mediated antitumor activity of natural killer cells.

CD155 is a ligand for DNAM-1, TIGIT, and CD96 and is involved in tumor immune responses. Unlike mouse cells, human cells express both membranous CD155 and soluble CD155 (sCD155) encoded by splicing isoforms of CD155. However, the role of sCD155 in tumor immunity remains unclear. Here, we show that, after intravenous injection with sCD155-producing B16/BL6 melanoma, the numbers of tumor colonies in wild-type (WT), TIGIT knock-out (KO), or CD96 KO mice, but not DNAM-1 KO mice, were greater than after injection with parental B16/BL6 melanoma. NK cell depletion canceled the difference in the numbers of tumor colonies in WT mice. In vitro assays showed that sCD155 interfered with DNAM-1-mediated NK cell degranulation. In addition, DNAM-1 had greater affinity than TIGIT and CD96 for sCD155, suggesting that sCD155 bound preferentially to DNAM-1. Together, these results demonstrate that sCD155 inhibits DNAM-1-mediated cytotoxic activity of NK cells, thus promoting the lung colonization of B16/BL6 melanoma.

Innate inflammation drives NK cell activation to impair Treg activity

IL-12 and IL-18 synergize to promote TH1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cells increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFNγ, and loss of suppressive function. We generated islet-autoreactive CTL “avatars”, which upon exposure to IL-12 and IL-18, upregulated IFNγ and Granzyme-B leading to increased lymphocytotoxicity of a human β-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.

Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors.

Natural killer (NK) cells are a population of innate lymphoid cells playing a pivotal role in host immune responses against infection and tumor growth. These cells have a powerful cytotoxic activity orchestrated by an intricate network of inhibitory and activating signals. The importance of NK cells in controlling tumor growth and in mediating a robust anti-metastatic effect has been demonstrated in different experimental mouse cancer models. Consistently, high density of tumor-infiltrating NK cells has been linked with a good prognosis in multiple human solid tumors. However, there are also tumors that appear to be refractory to NK cell-mediated killing for the presence of an immunosuppressive microenvironment affecting NK cell function. Immunotherapeutic strategies aimed at restoring and increasing the cytotoxic activity of NK cells in solid tumors, including the adoptive transfer of NK and CAR-NK cells, are currently employed in preclinical and clinical studies. In this review, we outline recent advances supporting the direct role of NK cells in controlling expansion of solid tumors and their prognostic value in human cancers. We summarize the mechanisms adopted by cancer cells and the tumor microenvironment to affect NK cell function, and finally we evaluate current strategies to augment the antitumor function of NK cells for the treatment of solid tumors.

Natural killer cell function changes by the Japanese Kampo Medicine Juzentaihoto in General fatigue patients

BackgroundPhysical/mental fatigue can alter behavior and reduce cognitive abilities. Even in healthy people, general fatigue correlates with NK cell function. Juzentaihoto (JTT) is a popular Kampo medicine used to treat general fatigue. In many animal models, JTT enhances immune functions, such as cytotoxic T-lymphocyte and NK cell activities. This is the first clinical study has examined the effect of JTT in patients with general fatigue. PurposeWe investigated whether peripheral NK cell populations and functions are altered by JTT treatment in patients with general fatigue. Patients and MethodsThe open-label unblinded study included ten patients (two males, eight females; median age 36.5 years, range 29–46 years) with general fatigue (Performance Status, PS 0–2) and. As controls, six healthy volunteers (two males, four females; median age [range]: 39 [36–45] years) were also enrolled in this study and provided informed consent before sampling. All patients were administered JTT three times per day before each meal for 28 consecutive days and were evaluated twice weekly for 42 days to assess compliance and adverse events. The primary endpoint was fatigue score obtained by a subjective numerical rating scale and PS. The secondary endpoint was the effects of JTT on circulating lymphocytes based on the expression of surface receptors in specific lymphocytic subsets. ResultsExpression of activation markers significantly increased from day 0–28 and decreased after 42 days. Inhibitory markers were upregulated in the cluster of high cytokine-producing ability but were downregulated in the fraction with high cytotoxicity. Increased expression of functional markers was observed following interleukin-2 stimulation in vitro. ConclusionsJTT induces cytotoxic activity of NK cells in patients with general fatigue. NK cells may maintain a balanced state by increasing inhibitory functions. Oral administration of JTT may improve immune function by activating NK cells.