JAK1 and JAK2 Modulate Tumor Cell Susceptibility To Natural Killer (NK) Cells Through Regulation Of PDL1 Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3472-3472
Author(s):  
Roberto Bellucci ◽  
Allison Martin ◽  
Davide Bommarito ◽  
Kathy S. Wang ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Nk Cell ◽  
Cell Lysis ◽  
Cell Killing ◽  
Tumor Cell Lines ◽  
Ifn Γ

Abstract NK cells are the primary effectors of the innate immune response against infections pathogens and malignant transformation through their efficient cytolytic activity and cytokine secretion. Nevertheless, tumor cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not yet completely understood. To identify novel pathways that modulate tumor cell resistance to NK cells, we previously developed a cell-cell interaction based screening approach using a large sub-set of a lentiviral shRNA library containing multiple independent shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 significantly increased secretion of INF-γ from NK cells and increased tumor cell susceptibility to NK cell lysis. To examine the role of the JAK signaling pathway in the modulation of tumor cell susceptibility to NK lysis, we analyzed down-stream signaling pathways in several cell lines (IM9, U937, K562, RPMI, MM1S KM12BM) and primary tumor cells (AML, MM, ALL). In the absence of NK cells, silencing JAK1 or JAK2 did not affect the basal activation of STAT proteins (STAT1(pY701), STAT1(pS727), STAT3(pY705), STAT3(pS727), STAT4(pY693), STAT5(pY694), STAT6(pY641)) or AKT(pS473) and ERK1/2(pT202/pY204) or expression of activating or inhibitory ligands on tumor cells. Because JAK1 and JAK2 transduce signals downstream of the IFN-γ receptor, we hypothesized that JAKs may play a role in tumor cell evasion of NK cell activities such as cytolysis and IFN-γ secretion. To test this hypothesis we pre-incubated various tumor cell lines or primary tumor cells with activated NK supernatant or recombinant human IFN-γ. Tumor cell activation in this fashion resulted in activation of STAT1 (pSTAT1(pY701)) but none of the other STATs, ERK or AKT. As expected, STAT1 activation was blocked when JAK1 or JAK2 were silenced or inhibited by a JAK inhibitor. Silencing of STAT1 with 2 independent shRNAs also resulted in increased tumor susceptibility to NK cell cytolysis in 3 different tumor cell lines tested. To confirm that IFN-γ secreted by activated NK cells induced resistance in tumor cell targets we used a blocking IFN-γ antibody (D9D10). 10μg/ml D9D10 completely blocked STAT1 phosphorylation and in different experiments using U937, IM-9, KM12BM, MM1S and RPMI we found that D9D10 significantly increased specific NK target cell lysis by 51.8%, 78.5%, 25.1%, 20.6% and 28.5% compared to IgG1 isotype controls. Similar results were obtained whit different primary tumor cells. To determine whether IFN-γ stimulation affected expression of ligands involved in NK cell recognition of tumor cells, we analyzed the effect of activated NK supernatant or IFN-γ on the expression of MHC Class I, β2M, HLA-C, HLA-A2, NKG2D, NKP44, NKP46, NKP30 ligands using chimeric FC proteins, MICA/B, DNAM-1 ligands (CD112, CD155), 2B4 ligand (CD48), TRAIL ligands (TRAIL-R1, TRAIL-R2), Fas ligand (CD95) and PD1 ligands (PDL1, PDL2, B7H3, B7H4). The basal expression of these ligands varied among the various tumor cell lines or primary tumors tested but the only ligand that was significantly up-regulated in every tumor sample tested was PDL1. PDL1 expression by tumor cells is known to inhibit T cell immunity. To test whether increased levels of PDL1 could also inhibit NK cell killing, we co-cultured primary NK cells with U937, IM9, KM12BM, RPMI, K562, MM1S, primary MM, AML and ALL cells with or without 10μg/ml anti-PDL1 antibody (recombinant mab with Fc mutated to eliminate FcR-mediated effects). Blocking PDL1 significantly increased NK cell killing of U937, IM9, KM12BM, RPMI, MM, AML and ALL (p=0.03, p=0.02, p=0.03, p=0.005, p=0.009, p=0.03 and p=0.02 respectively). NK cell killing activity did not further increase when a JAK inhibitor was added to the co-culture. These results show that NK cell secretion of IFN-γ results in IFN receptor signaling and activation of JAK1, JAK2 and STAT1 in the tumor cell targets, followed by rapid up-regulation of PDL1 expression and increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents subsequent PDL1 up-regulation resulting in increased susceptibility of tumor cells to NK cell activity suggesting that JAK pathway inhibitors may work synergistically with other immunotherapy regimens by eliminating IFN-induced PDL1 mediated immunoinhibition. Disclosures: Freeman: Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

1982 ◽  
Vol 156 (2) ◽  
pp. 492-505 ◽  
Author(s):  
S L Helfand ◽  
J Werkmeister ◽  
J C Roder
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Target Cells ◽  
Tumor Cell Lines

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

36 Molecular events regulating solid tumor cell responses to natural killer cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A37-A37
Author(s):  
Michal Sheffer ◽  
Constantine Mitsiades
Keyword(s):  
Tumor Cell ◽  
Antigen Presentation ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Nk Cell ◽  
Tumor Cell Lines ◽  
Cell Responses ◽  
Genome Scale

BackgroundNatural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies.MethodsTo systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach),1 correlating cytotoxicity scores for each cell line with the CCLE transcriptional data2 (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells.3 4Figure 1 schematically depicts the two screens.ResultsBased on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Widespread analysis of CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells, compared with epithelial-like cell lines. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA.Abstract 36 Figure 1Overview of PRISM and CRISPR studies a, Schematic depiction of PRISM study. b, Schematic depiction of CRISPR screens. c, Histogram of gene fold changes (z-scores). Listed are selected genes with most prominent p-values across more than one screen.ConclusionsThis study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The integration of PRISM and CRISPR identified potential regulators of tumor cell response to NK cell, which upon further validation, may serve as biomarkers in future NK cell-based studies. Moreover, NK cells may complement T-cells, killing tumor cells that do not respond to immune checkpoint inhibitors.AcknowledgementsThis work was supported by Stand Up To Cancer (SU2C) Convergence 2.0 Grant; SU2C Phillip A. Sharp Award for Innovation in Collaboration; Claudia Adams Barr Program in Innovative Basic Cancer Research; Human Frontier Science Program Fellowship; and Leukemia and Lymphoma Society Scholar Award.ReferencesYu C, et al., High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat Biotechnol 2016. 34(4): p. 419–23.Ghandi M, et al. Next-generation characterization of the cancer cell line encyclopedia. Nature 2019. 569(7757): p. 503–508.Doench JG, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016. 34(2): p. 184–191.Shalem O, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014;343(6166): p. 84–87.

scholarly journals NK cells promote cancer immunoediting through tumor-intrinsic loss of interferon-stimulated gene expression

2020 ◽  
Author(s):  
Yung Yu Wong ◽  
Luke Riggan ◽  
Edgar Perez-Reyes ◽  
Christopher Huerta ◽  
Matt Moldenhauer ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Metastatic Cancer ◽  
Cancer Immunoediting ◽  
Ifn Γ

AbstractNatural killer (NK) cells are innate lymphocytes that constantly patrol host tissues against transformed cells in a process known as cancer immunosurveillance. Previous evidence in mice has demonstrated that NK cell-derived IFN-γ can promote immunoevasion by sculpting the immunogenicity of developing tumors in a process known as cancer immunoediting. This process involves the elimination of highly immunogenic “unedited” tumor cells followed by the eventual escape of less immunogenic “edited” tumor cell variants that are able to escape recognition or elimination by the immune system. Here, we show that NK cell-edited fibrosarcomas decrease the expression of 17 conserved IFN-γ-inducible genes compared to unedited tumor cells. High expression of 3 of these identified genes (Psmb8, Trim21, Parp12) in human tumor samples correlates with enhanced survival in breast cancer, melanoma, and sarcoma patients. While NK cell-edited fibrosarcomas displayed resistance to IFN-γ growth suppression in vitro, functional knockouts of individual interferon stimulated genes (ISGs) were not required for outgrowth of unedited tumor cell lines in vitro and in vivo compared to complete loss of IFN signaling. Furthermore, knockout of IFN-γ-intrinsic signaling via deletion of Ifngr in edited B16 F10 and E0771 LMB metastatic cancer cell lines did not impact host survival following lung metastasis. Together, these results suggest that unedited tumors can be selected for decreased IFN-γ signaling to evade immune responses in vivo, and as a consequence, tumor-extrinsic IFN signaling may be more important for potentiating durable anti-tumor responses to advanced solid tumors.

Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell–mediated cytotoxicity

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3304-3310 ◽  
Author(s):  
Theresa A. Caragine ◽  
Masaki Imai ◽  
Alan B. Frey ◽  
Stephen Tomlinson
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Human Tumor ◽  
Mammary Adenocarcinoma ◽  
Tumor Cell Lines ◽  
Human Tumor Cell

Abstract Crry is a rodent membrane–bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell–mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell–mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell–mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell–mediated ADCC could be reversed by treatment with anti–Crry F(ab)2. In addition, anti–Crry F(ab)2 enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition.

Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3714-3714 ◽  
Author(s):  
Lei Wu ◽  
Peter Schafer ◽  
George Muller ◽  
David Stirling ◽  
J. Blake Bartlett
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Dependent Manner ◽  
Activation Markers ◽  
Early Results ◽  
Ifn Γ ◽  
Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

Expression and secretion of VEGF in solid tumor cells is mediated by the mammalian target of rapamycin (mTOR)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14123-14123
Author(s):  
E. M. Lackner ◽  
M. T. Krauth ◽  
R. Kondo ◽  
L. Rebuzzi ◽  
K. Eigenberger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Tumor Cell Lines ◽  
Neoplastic Cells ◽  
Malignant Effusions ◽  
Vegf Protein

14123 Background: Tumor progression and metastasis formation are often associated with enhanced angiogenesis and with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and a mediator of vascular permeability. We here describe that VEGF is produced and secreted by neoplastic cells in various solid tumors and its production mediated through mTOR. Methods and Results: As assessed by ELISA, the VEGF protein was detected in supernatants of cell lines derived from breast cancer (MDA-MB231), pancreatic carcinoma (BxPC-3), lung cancer (A-427), colon carcinoma (HCT8), and cholangiocellular carcinoma (EGI-1). In addition, VEGF was detected in supernatants of primary tumor cells obtained from malignant effusions in various malignancies (breast cancer, n=4; pancreatic cancer, n=1; ovarial cancer, n=1; parotic carcinoma, n=1; oesophageal carcinoma, n=1). In each case, VEGF protein was detectable in neoplastic cells by immunocytochemistry, and was found to accumulate in supernatants of cultured tumor cells over time, suggesting constant production and secretion. Correspondingly, as assessed by RT-PCR, primary tumor cells as well as the cell lines tested were found to express VEGF mRNA in a constitutive manner. Since mTOR is a well known regulator of VEGF synthesis, we applied rapamycin on primary neoplastic cells and on tumor cell lines. Rapamycin (20–200 nM) was found to counteract the production and secretion of VEGF in all tumor cells tested (VEGF in supernatants in cultures supplemented with rapamycin at 100 nM compared to control=100% on day 6: MDA-MB231: 11.8±0.2%; BxPC-3: 23.6±18.8%; A-427: 30.1±3.4%; HCT8 17.2±0.5%; EGI-1 28.4±1.1%; p<0.05). By contrast, neither rapamycin nor VEGF were found to modulate growth of primary tumor cells or the growth of the tumor cell lines tested. Conclusions: Various human tumor cells express and secrete VEGF. VEGF production is mediated through mTOR. These observations may have implications for the design of new treatment approaches attempting to counteract VEGF production/secretion and thus VEGF-dependent angiogenesis and effusion- formation in solid tumors. No significant financial relationships to disclose.

Patulin Acts Synergistically with Doxorubicin and Bortezomib in Cytotoxicity against Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2664-2664
Author(s):  
Alex Hessel ◽  
Malefa Tselanyane ◽  
Fengrong Wang ◽  
Ebenezer David ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Primary Tumor ◽  
Dose Effect ◽  
Tumor Cell Lines ◽  
Synergism And Antagonism

Abstract Patulin (4-Hydroxy-4H-furo 3,2-C-pyran-2(6H)-one) is a first-in-class mycotoxin under development as a novel chemotherapeutic agent. The mechanism of action of Patulin has been reported to include activation of mitogen activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). We have previously shown Patulin to have activity against myeloma, leukemia, and lymphoma cell lines, as well as primary tumor cells in clinical samples from patients with these diagnoses (Wang BLOOD 2007). Moreover, we have shown that Patulin specifically and potently targets tumor cells over normal cells and effectively killed primary tumor cells of patients with refractory illness. The aim of this study was to determine whether Patulin acts synergistically with the topoisomerase inhibitor doxorubicin or the proteasome inhibitor bortezomib. We hypothesize that drugs with non-cross-reactive modes of action could be complementary. Human tumor cell lines from B-cell lymphoma (DAUDI), T-Cell leukemia and lymphoma (Jurkat and H9, respectively), and myeloid leukemia (HL60) malignancies were tested for their sensitivity to single agents Patulin, bortezomib, and doxorubicin as well as Patulin in combination with the latter two agents (Patulin and bortezomib, P + B; Patulin and doxorubicin, P + D). Cells were treated with a range of concentrations of each single agent and the drugs in combination over 24 hours. Following treatment, cell metabolic activity was assessed using a microculture tetrazolium (MTT) assay and cell viability was assessed by flow cytometry using Annexin V and propidium iodide (PI) staining. Dose-effect curves, median effect plots, and combination index (CI) values were generated in the Compusyn software program for each target cell population. Median-effect doses (IC50s) of individual drugs were interpolated using the y-intercept of median-effect plots. Three dose-effect data points were used to create a range of CI values at different fractions of affect (fa). The lower and upper values of the CI range were used to characterize drug combinations as synergistic, antagonistic, or additive based on Chou’s Symbols for Synergism and Antagonism using CI analysis (Table 1). The role for ROS in the mechanism of action of Patulin was confirmed by flow cytometry showing increased levels of ROS in cell lines following Patulin exposure. Preincubation of cell lines with N-acetyl cysteine (NAC) or concurrent exposure to Patulin and NAC abrogated the cytotoxic activity of the mycotoxin. H9 cells were most sensitive to the effects of Patulin, with an IC50 of 1.2 μM. Combinations of P + B acted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cells; however, P + B also demonstrated moderate antagonism against the Jurkat and H9 cell lines (Table 2). Likewise, combinations of P + D interacted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cell lines while simultaneously demonstrating strong antagonism against the H9 cell line. Patulin kills leukemia and lymphoma cells via generation of intracellular ROS. Synergy of Patulin with either bortezomib or doxorubicin in leukemia and lymphoma cell lines indicates a distinct mechanism of action for the mycotoxin and compared to other chemotherapeutics and supports the rationale for continued development of Patulin as a novel chemotherapeutic mycotoxin. TABLE 1. Chou’s Symbols for Synergism and Antagonism using CI Analysis CI Description &lt; 0.1 Very Strong Synergism 0.1–0.3 Strong Synergism 0.3–0.7 Synergism 0.7–0.85 Moderate Synergism 0.85–0.90 Slight Synergism 0.90–1.10 Nearly Additive 1.10–1.20 Slight Antagonism 1.20–1.45 Moderate Antagonism 1.45–3.3 Antagonism 3.3–10 Strong Antagonism &gt; 10 Very Strong Antagonism TABLE 2. IC50 of single agents patulin, bortezomib, and doxorubicin and CI in hematological cancer cell lines Tumor cell line IC50 of patulin (μM) IC50 of bortezomib (nM) IC50 of doxorubicin (μM) CI: P + B CI: P + D T-Cell Jurkat 1.16 5600 5.2 0.6 -- 1.2 0.1 -- 1.2 H9 1.2 14 0.33 0.4 -- &gt; 1 0.3 -- &gt; 1 B-Cell DAUDI 0.98 1.1 0.19 &lt; 0.1 -- 0.1 &lt; 0.1 -- 0.3 Myeloid HL60 1.7 0.42 0.07 &lt; 0.1 -- 0.1 &lt; 0.1

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 449-449
Author(s):  
Marc Schmitz ◽  
Senming Zhao ◽  
Yvonne Deuse ◽  
Knut Schaekel ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Human Blood ◽  
Nk Cell ◽  
Tnf Alpha ◽  
Tumor Cell Lines ◽  
Specific Lysis ◽  
Tumoricidal Activity ◽  
Ifn Gamma

Abstract Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity2002;17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood;2002;100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (&gt;93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (&gt;90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 277-277
Author(s):  
Roberto Bellucci ◽  
Hong-Nam Nguyen ◽  
Allison Martin ◽  
Anna C. Schinzel ◽  
Stefan Heinrichs ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cell Activity ◽  
Tumor Cell Lines ◽  
Nk Cell Activity ◽  
Myeloma Cells ◽  
Increased Sensitivity

Abstract Abstract 277 NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures: No relevant conflicts of interest to declare.

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