Natural Killer Cell-mediated Cytolysis Research Articles

Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

Ciclopirox olamine sensitizes leukemia cells to natural killer cell-mediated cytolysis by upregulating NKG2DLs via the Akt signaling pathway

The activating receptor natural killer group 2D (NKG2D) expressed by Natural killer (NK) cells functions as a “master-switch” in governing the awakening status of NK cells. The NKG2D-mediated cytotoxicity has been declared to be related with the expression levels of NKG2D ligands (NKG2DLs) expressed on tumor cells. Therefore, selective induction of NKG2DLs could be a reliable approach to enhance the efficacy of NK cell-mediated immunotherapy. Our existing study demonstrated that Ciclopirox Olamine (CPX), an off-patent antifungal agent, effectively elevated the expression of NKG2DLs on leukemia cells and sensitized leukemia cells to NK-cell mediated cytolysis. Induction of ROS production and AKT phosphorylation by CPX is essential for the up-regulation of NKG2DLs expressions. Inhibition of AKT by using AKT inhibitor MK2206 decreased both NKG2DLs expressions and NK cell cytotoxicity. These data indicated that increased sensitivity of CPX-treated leukemia cells to NK cell cytolysis was attributed to higher NKG2DLs expressions, resulting from activated AKT signaling pathway. Our findings support the ongoing development of CPX as an anti-tumor agent and suggest its promising immunotherapeutic value in the medication of leukemia.

Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

BackgroundThe rich biodiversity of medicinal plants and their importance as sources of novel therapeutics and lead compounds warrant further research. Despite advances in debulking surgery and chemotherapy, the risks of recurrence of ovarian cancer and resistance to therapy are significant and the clinical outcomes of ovarian cancer remain poor or even incurable. ObjectiveThis study aims to investigate the effects of leaf extracts from a medicinal plant Leea indica and its selected phytoconstituents on human ovarian cancer cells and in combination with oxaliplatin and natural killer (NK) cells.MethodsFresh, healthy leaves of L. indica were harvested and extracted in 70% methanol by maceration. The crude extract was partitioned with n-hexane, dichloromethane and ethyl acetate. Selected extracts and compounds were analyzed for their effects on cell viability of human ovarian cancer cells, NK cell cytotoxicity, and stress ligands expression for NK cell receptors. They were also evaluated for their effects on TNF-α and IL-1β production by enzyme-linked immunosorbent assay in lipopolysaccharide-stimulated human U937 macrophages.ResultsLeaf extracts of L. indica increased the susceptibility of human ovarian tumor cells to NK cell-mediated cytotoxicity. Treatment of cancer cells with methyl gallate but not gallic acid upregulated the expression of stress ligands. Tumor cells pretreated with combination of methyl gallate and low concentration of oxaliplatin displayed increased levels of stress ligands expression and concomitantly enhanced susceptibility to NK cell-mediated cytolysis. Further, NK cells completely abrogated the growth of methyl gallate-pretreated ovarian cancer cells. The leaf extracts suppressed TNF-α and IL-1β production in human U937 macrophages. Methyl gallate was more potent than gallic acid in down-regulating these cytokine levels.ConclusionsWe demonstrated for the first time that leaf extracts of L. indica and its phytoconstituent methyl gallate enhanced the susceptibility of ovarian tumor cells to NK cell cytolysis. These results suggest that the combined effect of methyl gallate, oxaliplatin and NK cells in ovarian cancer cells warrants further investigation, for example for refractory ovarian cancer. Our work is a step towards better scientific understanding of the traditional anticancer use of L. indica.

Directing CAR NK Cells via the Metabolic Incorporation of CAR Ligands into Malignant Cell Glycans.

The abundance of sialic acid-containing glycans in the glycocalyx of malignant cells enables immune evasion. Here, we leverage the biosynthetic pathways that permit pervasive sialylation to incorporate a chimeric antigen receptor (CAR) ligand into malignant cell glycans, and demonstrate that this increases the susceptibility of malignant cells to the cytolytic activity of CAR-expressing natural killer (NK) cells. Specifically, we applied a C-9-functionalized nonnatural sialic acid [i.e., fluorescein sialic acid (FL-SA)] to modify malignant cell glycans. We confirm the metabolic incorporation of FL-SA into plasma membrane-associated glycans. The preparation of anti-fluorescein CAR NK cells permitted studies demonstrating that treating malignant cells with FL-SA increased susceptibility to CAR NK cell-mediated cytolysis. Furthermore, we observed that the specificity of the anti-fluorescein CAR NK cells is enhanced for fluorescein-labeled cells, and an increased release of cytokines from the CAR NK cells upon incubation with FL-SA-treated cells. The results arising from this study demonstrate that CAR ligands can be metabolically incorporated into malignant cells, and we reason that such strategies could be leveraged to tackle the issue of antigen heterogeneity that limits the clinical efficacy of CAR T/NK cell therapies.

Metastatic colorectal cancer cells harness nonsense-mediated mRNA decay for immune evasion

Abstract Immunosuppression microenvironment allows primary tumor growth, while tumor-inherent factors that bring about immune evasion during metastasis remain elusive. Here, we observed a stronger nonsense-mediated mRNA decay (NMD) activity with a higher expression of up-frameshift protein 1 (UPF1) in colorectal cancer (CRC) metastasis than in matched primary cancer cells. In metastatic CRC SW620 cells, the higher UPF1 expression was found to arise from the increased stability of UPF1 by ubiquitin specific peptidase 10 (USP10)-mediated deubiquitination. In contrast, in primary CRC SW480 cells, autophagy promotes proteolysis of ubiquitinated UPF1 and inhibits NMD activity. Interestingly, in SW620 cells NMD selectively controlled a cluster of immune-related genes including ICAM1. Furthermore, NMD inhibition exposed SW620 cells but not SW480 cells to natural killer (NK) cell-mediated cytolysis that depended on ICAM1. Conclusion NMD activity can be recommissioned in CRC metastasis cells to escape from NK-dependent immune surveillance, and inhibiting NMD to boost anti-tumor immunity could be a novel strategy to treat CRC metastasis.

Artemisinin sensitizes tumor cells to NK cell-mediated cytolysis

The antimalarial drug Artemisinin has been reported to possess direct anti-tumor effects on various types of tumor cells. However, its anti-tumor potential has not been fully revealed, and its effects on tumor susceptibility to immune surveillance by the host are still unknown. Natural killer (NK) cells are the first line in tumor surveillance by the host, and have been recognized as a promising target for tumor immunotherapy. Here, we reported that Artemisinin sensitized tumor cells to NK cell cytolysis. Both human K562 and Raji tumor cells, and mouse YAC-1 tumor cells were more susceptible to human or mouse NK cell cytolysis in vitro after Artemisinin pretreatment. Conjugation formation between tumor cells and NK cells was increased after pretreatment with Artemisinin. Such effects on tumor cells by Artemisinin might not be the results of tumor recognition by NK cells, since major ligands of NK cell surface receptors were not affected. Mechanistically, although Artemisinin didn’t induce tumor cell apoptosis, Artemisinin enriched apoptosis-related gene sets in these tumor cells, which might predispose tumor cells to apoptosis upon NK cell cytolysis. Moreover, NK cell numbers, percentages, maturation and functions were preserved in the presence of Artemisinin in vitro, suggesting that Artemisinin displays detrimental effects only on tumor cells but not on immune cells. These data reveal a novel anti-tumor mechanism of Artemisinin and demonstrate that Artemisinin could be a promising drug candidate for cancer treatment.

Parvifoline AA Promotes Susceptibility of Hepatocarcinoma to Natural Killer Cell-Mediated Cytolysis by Targeting Peroxiredoxin

Natural killer (NK) cells play a crucial role in the surveillance of malignant cells. The engagement of NK group 2 member D (NKG2D) receptor with its ligands on target cells represents a promising therapeutic strategy against cancers. Here, we report that parvifolineAA (PAA), a natural ent-kaurane diterpenoid, markedly stimulates the expression of NKG2D ligands on hepatocellular carcinoma (HCC) cells, considerably enhancing their recognition and lysis by NK cells. We determined that PAA covalently binds to the conserved cysteine site of peroxiredoxins I/II (Prxs-I/II) and inhibits their catalytic activity, subsequently activating the ROS/ERK axis and the immunogenicity of HCC toward NK cells. Robust tumor growth inhibition by PAA dependent on NK cell activation was detected invivo. Our data suggest Prxs-I/II as a promising cancer immune therapeutic target and provide a compelling rationale for further development of the inhibitor PAA as a sensitizer agent for NK cell-mediated HCC immunotherapy.

A Preliminary Study on the Expression of CD160 on NK Cells and Its Mechanism of Mediating NK Killing Effect

To investigate the expression of CD160 on the surface of human natural killer (NK) cells and its possible relationship with hematological malignancies. CD160 expression on human leukemia cell line NK92 cells was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The proliferation characteristics and cell surface markers of this cell line were determined. Cytotoxicity of NK92 against 2 human myeloid leukemia cell lines, K562 and THP-1 was analyzed ex vivo. CD160 blocking antibody CL1-R2 was employed to clarify its role in NK cell mediated cytolysis. Then, the expression of CD160 on NK cells in peripheral blood from various patients with hematological malignancies were measured by flow cytometry. The mRNA and protein levels of CD160 expressions on NK92 cells were confirmed by RT-PCR and Western blot, respectively. The flow cytometry results demonstrated that the strong positive expression of CD160 could be detected on the NK92 cell surface. NK92 could effectively kill K562 and THP-1 cells, while the cytolysis effect was abrogated in the presence of CD160 blocking antibody CL1-R2. The high levels of HVEM were expressed on both target cells, but the HLA class I molecules were absent on K562. The expression of CD160 on CD3-CD56+ NK cells in peripheral blood from patients with acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients was significant lower than that in the normal controls (P<0.05). The cytolysis function of human NK cells is mediated partially by CD160 molecule. The decrease of CD160 expression on NK cells from patients with various hematological malignancies implies that down-regulation of CD160 expression may be a novel mechanism of tumor immune escape.

HIL-15-gene modified human natural killer cells (NKL-IL15) exhibit anti-human leukemia functions.

Natural killer (NK) cells can kill transformed cells and represent anti-tumor activities for improving the immunotherapy of cancer. In previous works, we established human interleukin-15 (hIL-15) gene-modified NKL cells (NKL-IL15) and demonstrated their efficiency against human hepatocarcinoma cells (HCCs) in vitro and in vivo. To further assess the applicability of NKL-IL15 cells in adoptive cellular immunotherapy for human leukemia, here we report their natural cytotoxicity against leukemia in vitro and in vivo. Flow cytometry, ELISA and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays. Leukemia xenograft NOD/SCID mice were established by subcutaneous injection with K562 cells, and then treated with irradiated NKL cells. We found NKL-IL15 cells displayed a significant high cytolysis activity against both human leukemia cell lines and primary leukemia cells from patients, accompanied with up-regulated expression of molecules related to NK cell cytotoxicity such as perforin, granzyme B and NKp80. Moreover, cytokines secreted by NKL-IL15 cells, including TNF-α and IFN-γ, could induce the expression of NKG2D ligands on target cells, which increased the susceptibility of leukemia cells to NK cell-mediated cytolysis. Encouragingly, NKL-IL15 cells significantly inhibited the growth of leukemia cells in xenografted NOD/SCID mice and prolonged the survival of tumor-bearing mice dramatically. Furthermore, NKL-IL15 cells displayed stimulatory effects on hPBMCs, indicating the immunesuppressive status of leukemia patients could be improved by NKL-IL15 cell treatment. These results provided evidence that IL-15 gene-modification could augment NK cell-mediated anti-human leukemia function, which would improve primary NK cell-based immunotherapy for leukemia in future.

Modulation of innate and adaptive cellular immunity relevant to HIV-1 vaccine design by seminal plasma.

Mucosal exposure to HIV-1 infection generally occurs in the presence of semen. Immunomodulation by seminal plasma is well described in the reproductive biology literature. Little is known, however, about the impact of seminal plasma on innate and adaptive anti-HIV-1 cellular immunity. The study investigated the effects of seminal plasma on immune responses considered important for prophylactic HIV-1 vaccine development, namely innate and adaptive cellular immunity mediated by natural killer (NK) cells and T cells, respectively. The ability of seminal plasma to modulate direct, antibody-dependent and cytokine-stimulated NK cell activation was assessed utilizing intracellular cytokine staining. Direct and antibody-dependent cellular cytotoxicity was assessed using lactate dehydrogenase release assays. The effects of seminal plasma on T-cell activation upon stimulation with staphylococcus enterotoxin B or HIV-1 Gag peptides were assessed by intracellular cytokine staining. The impact of seminal plasma on redirected cytolysis mediated by T cells was measured using lactate dehydrogenase release assays. Both direct and antibody-dependent NK cell activation were dramatically impaired by the presence of either HIV-1-uninfected or HIV-1-infected seminal plasma in a dose-dependent manner. Additionally, seminal plasma suppressed both direct and antibody-dependent NK cell-mediated cytolysis, including anti-HIV-1 antibody-dependent cytolysis of gp120-pulsed CEM.NKr-CCR5 cells. Finally, seminal plasma attenuated both HIV-1 Gag-specific and staphylococcus enterotoxin B-induced CTL activation. Semen contains potent immunosuppressors of both NK cell and CD8 T-cell-mediated anti-HIV-1 immune responses. This could impede attempts to provide vaccine-induced immunity to HIV-1.

Mesenchymal Stem Cells Derived from Human Bone Marrow and Adipose Tissue Maintain Their Immunosuppressive Properties After Chondrogenic Differentiation: Role of HLA-G.

Mesenchymal stem cells (MSC) have emerged as alternative sources of stem cells for regenerative medicine because of their multipotency and strong immune-regulatory properties. Also, human leukocyte antigen G (HLA-G) is an important mediator of MSC-mediated immunomodulation. However, it is unclear whether MSC retain their immune-privileged potential after differentiation. As promising candidates for cartilage tissue engineering, the immunogenic and immunomodulatory properties of chondro-differentiated MSC (chondro-MSC) require in-depth exploration. In the present study, we used the alginate/hyaluronic acid (Alg/HA) hydrogel scaffold and induced both bone marrow- and adipose tissue-derived MSC into chondrocytes in three-dimensional condition. Then, MSC before and after chondrocyte differentiation were treated or not with interferon γ and tumor necrosis factor α mimicking inflammatory conditions and were compared side by side using flow cytometry, mixed lymphocyte reaction, and immunostaining assays. Results showed that chondro-MSC were hypoimmunogenic and could exert immunosuppression on HLA-mismatched peripheral blood mononuclear cells as well as undifferentiated MSC did. This alloproliferation inhibition mediated by MSC or chondro-MSC was dose dependent. Meanwhile, chondro-MSC exerted inhibition on natural killer cell-mediated cytolysis. Also, we showed that HLA-G expression was upregulated in chondro-MSC under hypoxia context and could be boosted in allogenic settings. Besides, the Alg/HA hydrogel scaffold was hypoimmunogenic and its addition for supporting MSC chondrocyte differentiation did not modify the immune properties of MSC. Finally, considering their chondro-regenerative potential and their retained immunosuppressive capacity, MSC constitute promising allogenic sources of stem cells for cartilage repair.

A bispecific protein rG7S-MICA recruits natural killer cells and enhances NKG2D-mediated immunosurveillance against hepatocellular carcinoma

MHC class I-related chain A (MICA) is a principal immunoligand of the natural killer (NK) cell receptor NK group 2, member D (NKG2D) and plays a key role in NK cell-mediated immune recognition. Shedding of MICA from tumor cells leads to immunosuppression. To reconstitute the immunosurveilance function of NK cells, we constructed a fusion protein rG7S-MICA and explored its potential anti-tumor activity against hepatocellular carcinoma (HCC). rG7S-MICA consists of human MICA and a single-chain antibody fragment (scFv) targeting the tumor-associated antigen cluster of differentiation 24 (CD24). In vitro, rG7S-MICA engaged both NK cells and CD24+ human HCC cells, and triggered NK cell-mediated cytolysis. Furthermore, in CD24+ HCC-bearing nude mice, rG7S-MICA specifically targeted to the tumor tissue, where it effectively recruited NK cells and induced the release of cytokines, and showed superior anti-tumor activity. In conclusion, rG7S-MICA provides a new approach for HCC-targeting immunotherapy and has attracting potentials for clinical applications.

Platelets in cancer metastasis: To help the "villain" to do evil.

Cancer progress is accompanied by platelet activation and thrombotic complications. Platelets are a dangerous alliance of cancer cells, and are a close engager in multiple processes of cancer metastasis. Platelet adhesion to cancer cells forms a protective cloak that helps cancer cells to escape immune surveillance and natural killer cell-mediated cytolysis. Platelets facilitate tethering and arrest of disseminated cancer cells in the vasculature, enhance invasive potentials and thus extravasation of cancer cells. Moreover, platelets recruit monocytes and granulocytes to the sites of cancer cell arrest, and collaborate with them to establish a pro-metastatic microenvironment and metastatic niches. Platelets also secret a number of growth factors to stimulate cancer cell proliferation, release various angiogenic regulators to regulate tumor angiogenesis and subsequently promote cancer growth and progress. Albeit platelets are helping the "villain" cancer to do evil, the close engagements of platelets in cancer metastasis and progress can be used as the intervention targets for new anti-cancer therapeutic developments. Platelet-targeted anti-cancer strategy may bring in novel anti-cancer treatments that can synergize the therapeutic effects of chemotherapies and surgical treatments of cancer.

Podocalyxin-like protein 1 functions as an immunomodulatory molecule in breast cancer cells

Podocalyxin-like protein 1 (PCLP1), a CD34-related sialomucin involved in the regulation of cellular morphology and adhesion, is expressed by a number of normal cells and various tumor cells. In breast malignancies PCLP1 overexpression has been associated with the most aggressive, metastatic cancers and poor prognosis. These observations suggest that PCLP1 expression could provide a mechanism to evade the immune response, thereby promoting metastatic progression of cancer. In the present work, we aimed to determine the effect of PCLP1 overexpressed in MCF7 breast cancer cells on natural killer (NK) cell cytotoxicity, dendritic cell maturation, and agonist-induced T cell proliferation. The results showed that PCLP1 expressed in MCF7 breast cancer cells confers resistance to NK cell-mediated cytolysis and impairs T cell proliferation. Furthermore, PCLP1 decreased the levels of NK cell activating receptors NKG2D, NKp30, NKp44, NKp46, DNAM-1, and CD16 on cell surface in a contact-dependent manner. Moreover, NK cells acquired PCLP1 from MCF7 cells by a process known as trogocytosis. These data reveal a new function of PCLP1 expressed on tumor cells as an immunomodulatory molecule, which may represent a mechanism to evade the immune response.

Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

IB-03 * IDH MUTANT GLIOMAS ARE RESISTANT TO NATURAL KILLER CELL-MEDIATED CYTOLYSIS

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are common in gliomas. 50-80% of WHO II/III gliomas possess IDH1/IDH2 mutation. Methylation-related transcriptional repression is a feature of IDH mutant (IDHmut) tumors. Epigenetic repression of natural killer (NK) cell ligands is a common occurrence in cancer, but it is unknown if this occurs in primary brain tumors. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut gliomas, and correlate this with decreased susceptibility of IDHmut cells to natural killer (NK) cell-mediated cytolysis in vitro. Our data suggest that NK cell-astrocyte contact is required for production of Th1 cytokines IFN-γ and TNF-α, and that production of these cytokines is limited in IDHmut astrocytes. We also show that NKG2DL-specific antibody blockade can result in significant reduction in NK-cell mediated cytolysis in vitro. Furthermore, we infect IDHmut cells with JDNI7 virus containing ULBP3 plasmid DNA to induce overexpression of this ligand. Upon ULBP3 overexpression, these cells are significantly more sensitive to NK-cell mediated lysis. Taken together, our data suggest a potential immune surveillance role for the NKG2DL, ULBP3, during IDHmut gliomagenesis.

Heterologous expression of mutated HLA-G1 reduces alloreactivity of human dermal fibroblasts.

To engineer a stable HLA-G molecule and evaluate its immunomodulatory properties in transgenic human dermal fibroblasts (HDFs). A mutated HLA-G1 (mHLA-G1) molecule was generated by modifying the endoplasmic reticulum retrieval motif and 3'-untranslated region miRNA-binding sites of HLA-G1. Immunomodulatory properties of transgenic HDF-mHLA-G1 were evaluated in vitro. Stable mHLA-G1 expressing HDF cells were successfully generated and flow cytometry analysis revealed that mHLA-G1 efficiently localized to the cell surface. Natural killer cell-mediated cytolysis of HDF-mHLA-G1/green fluorescent protein (GFP) was reduced by 73% compared with HDF-GDP. HDF-mHLA-G1/GFP decreased phytohemagglutinin-activated peripheral blood mononuclear cell proliferation by 30% versus HDF-GFP. We are the first to successfully create a human fibroblast source with reduced alloreactivity using a novel mHLA-G1 construct. This approach may be extended to other cell types including human embryonic stem cells for use in allogeneic transplantation for cell-based regenerative medicine applications.

Abstract 3669: IDH mutant glial cell resistance to natural killer cell cytotoxicity

Abstract Mutations in IDH 1 and 2 are common in gliomas. Methylation-related epigenetic repression of gene expression is a feature of IDH mutant (IDHmut) tumors. Epigenetic repression of NK cell ligands is a common occurrence in cancer, but it is unknown if this occurs in gliomas. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut tumors, and correlate this with decreased susceptibility to natural killer (NK) cell-mediated cytolysis in vitro We compared IDHmut and IDHwt gene expression values for annotated immune response genes and examined NKG2DL HM450 mean methylation values in glioma patients using TCGA data. cDNA was synthesized from cultured IDHwt or IDHmut astrocytes. NKG2DL expression was assayed using primers for NKG2D ligands ULBP1, ULBP2, ULBP3 and RAET1G. IDHwt astrocytes, U87 cells and IDHmut astrocytes were cultured with activated NK cells. Cytotoxicity was measured by flow cytometry. mRNA expression values for NKG2DLs ULBP1 and ULBP3 were significantly reduced in IDHmut gliomas. Conversely, methylation values were significantly increased. NKG2DL hierarchical clustering of gene expression of shows genotype-based expression clusters. IDHmt astrocytes expressed a two to eight-fold reduction in the relative gene expression of NKG2DLs compared with IDHwt astrocyte. Cytotoxicity is significantly lower in IDHmut astrocytes/NK cell co-cultures compared with IDHmut/NK or U87/NK co-cultures. Our data demonstrates evidence for the transcriptional repression of NKG2DLs in IDHmut gliomas in silico and in vitro. Reduced NKG2D levels in IDHmut cells correlates with impaired NK-mediated cytotoxicity and suggests a role for NK cell resistance in IDH mutant tumors. Further studies are in progress to utilize HSV vectors that express ULBP3 to determine whether restoration of ULBP3 expression in cancer cells will enhance susceptibility to NK activation and tumor cell killing. Results of these experiments will be reported. Citation Format: Xiaoran Zhang, Yigang Chang, Aofei Li, Joseph C. Glorioso, Paola Grandi, Mike Lotze, Nduka Amankulor. IDH mutant glial cell resistance to natural killer cell cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2014-3669

Isocitrate dehydrogenase mutant glial cell resist natural killer cell cytotoxicity (TUM2P.906)

Abstract Introduction: Mutations in IDH 1 and 2 are common in gliomas. Methylation-related epigenetic repression of gene expression is a feature of IDH mutant (IDHmut) tumors. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut tumors, and correlate this with decreased susceptibility to natural killer (NK) cell-mediated cytolysis in vitro Methods: Microarray: We compared IDHmut and IDHwt gene expression and NKG2DL mean methylation values in glioma patients using TCGA data. RT-PCR: NKG2DL expression was assayed using primers for NKG2D ligands. Cytotoxicity: Astrocytes were cultured with activated NK cells. Cytotoxicity was measured by flow cytometry. Results: mRNA expression values for NKG2DLs ULBP1 and ULBP3 were significantly reduced in IDHmut gliomas. Conversely, methylation values were significantly increased. NKG2DL hierarchical clustering of gene expression of shows genotype-based expression clusters. IDHmt astrocytes expressed a two to eight-fold reduction in the relative gene expression of NKG2DLs compared with IDHwt astrocytes. Cytotoxicity is significantly lower in IDHmut astrocytes/NK cell co-cultures compared with IDHmut/NK or U87/NK co-cultures. Conclusions: Our findings demonstrate evidence for the transcriptional repression of NKG2DLs in IDHmut gliomas in silico and in vitro. Reduced NKG2D levels in IDHmut cells correlates with impaired NK-mediated cytotoxicity and suggests a role for NK cell resistance in IDH mutant tumors.

Properties of virus-induced NK cells correlating with their ability to kill activated CD4 T cells (IRM7P.489)

Abstract Activated natural killer (NK) cells induced during viral infections of mice can regulate adaptive immunity by lysing activated CD4 T cells. This seems to be a general property of interferon-inducing viruses, but the efficiency of this process is in part dependent on the nature of the instigating virus. Here we examined the differential expression of activating and inhibitory receptors on NK cells from lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VACV)-infected mice, because NK cells from VACV-infected mice were far less efficient at killing activated CD4 T cells than those from LCMV-infected mice. Relative to expression levels on LCMV-activated NK cells, NKG2A expression was higher in VACV-induced NK cells while NKG2D was lower. NK cells activated by infections with LCMV, Pichinde virus (PV), mouse hepatitis virus (MHV), and murine cytomegalovirus (MCMV) displayed similar expression patterns for all of the examined receptors, whereas VACV-induced cells had a distinct expression pattern. Another major difference between VACV-induced NK cells and NK cells activated by the other viruses was in the expression of granzyme B, an essential component of NK cell-mediated cytolysis. VACV-induced NK cells expressed granzyme B at lower levels than NK cells from mice infected with LCMV, PV, MHV A59, or MCMV. These results indicate that the NK cell receptor expression and the cytolytic activity of NK cells can vary with the infecting virus.