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.

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.

scholarly journals Unraveling the functional relevance of the Ubiquitin-specific peptidase 22 for necroptotic cell death

2021 ◽  
Author(s):  
◽  
Jens Rödig
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Cell Death Signaling

Ubiquitination is regarded as one of the key post-translational modifications in nearly all biological processes, endowed with numerous layers of complexity. Deubiquitinating enzymes (DUBs) dynamically counterbalance ubiquitination events by deconjugating ubiquitin signals from substrates. Dysregulation of the ubiquitin code and its negative regulators drive various pathologies, such as neurological disorders and cancer. The DUB ubiquitin-specific peptidase 22 (USP22) is well-known for its essential role in the human Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, mediating the removal of monoubiquitination events from Histone 2A and 2B (H2A and -B), thereby regulating gene transcription. In cancer, USP22 was initially described as a part of an 11-gene expression signature profile, predicting tumor metastasis, reoccurrence and death after therapy in a wide range of tumor cells. However, novel roles for USP22 have emerged recently, accrediting USP22 essential roles in regulating tumor development as well as apoptotic cell death signaling. One of the hallmarks of cancer is the evasion of cell death, especially apoptosis, a form of programmed cell death (PCD). Necroptosis, a regulated form of necrosis, is regarded as an attractive therapeutic strategy to overcome apoptosis-resistance in tumor cells, although a profound understanding of the exact signaling cascade still remains elusive. Nevertheless, several ubiquitination and deubiquitination events are described in fine-tuning necroptotic signaling. In this study, we describe a novel role for USP22 in regulating necroptotic cell death signaling in human tumor cell lines. USP22 depletion significantly delayed TNFa/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFa-induced nuclear factor-kappa B (NF-KB) signaling or TNFa-mediated extrinsic apoptosis. Intriguingly, re-expression of USP22 wildtype in the USP22 knockout background could re-sensitize HT-29 cells to TBZ-induced necroptosis, whereas re-constitution with the catalytic inactive mutant USP22 Cys185Ser did not rescue susceptibility to TBZ-induced necroptosis, confirming the USP22 DUB-function a pivotal role in regulating necroptotic cell death. USP22 depletion facilitated ubiquitination and unexpectedly also phosphorylation of Receptor-interacting protein kinase 3 (RIPK3) during necroptosis induction, as shown by Tandem Ubiquitin Binding Entities (TUBE) pulldowns and in vivo (de)ubiquitination immunoprecipitations. To substantiate our findings, we performed mass-spectrometric ubiquitin remnant profiling and identified the three novel USP22-regulated RIPK3 ubiquitination sites Lysine (K) 42, K351 and K518 upon TBZ-induced necroptosis. Further assessment of these ubiquitination sites unraveled, that mutation of K518 in RIPK3 reduced necroptosis-associated RIPK3 ubiquitination and additionally affected RIPK3 phosphorylation upon necroptosis induction. At the same time, genetic knock-in of RIPK3 K518R sensitizes tumor cells to TNFa-induced necroptotic cell death and amplified necrosome formation. In summary we identified USP22 as a new regulator of TBZ-induced necroptosis in various human tumor cell lines and further unraveled the distinctive role of DUBs and (de)ubiquitination events in controlling programmed cell death signaling.

scholarly journals Preferential Interaction of a Novel Tumor Surface Protein (p38.5) with Naive Natural Killer Cells

1997 ◽  
Vol 185 (10) ◽  
pp. 1735-1742 ◽  
Author(s):  
Ballabh Das ◽  
Mary O. Mondragon ◽  
Shi-Zhen Tao ◽  
Allen J. Norin
Keyword(s):  
T Lymphocytes ◽  
Membrane Protein ◽  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Surface Protein ◽  
Dependent Manner ◽  
Tumor Cell Lines

A receptor–ligand interaction exclusive to natural killer (NK) cell–mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand–cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3−CD5−CD16+) relative to T lymphocytes (CD3+CD5+ CD16−). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell–mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.

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 The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans

2009 ◽  
Vol 206 (7) ◽  
pp. 1495-1503 ◽  
Author(s):  
Cameron S. Brandt ◽  
Myriam Baratin ◽  
Eugene C. Yi ◽  
Jacob Kennedy ◽  
Zeren Gao ◽  
...  
Keyword(s):  
Immune System ◽  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Human Tumor ◽  
Cell Surface Molecule ◽  
B7 Family

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.

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.

Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients

2007 ◽  
Vol 73 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Malin Wickström ◽  
Katarina Danielsson ◽  
Linda Rickardson ◽  
Joachim Gullbo ◽  
Peter Nygren ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
Human Tumor Cells ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines

Interaction of Human Tumor Cells with Human Platelets and the Coagulation System

1983 ◽  
Vol 50 (03) ◽  
pp. 726-730 ◽  
Author(s):  
Hamid Al-Mondhiry ◽  
Virginia McGarvey ◽  
Kim Leitzel
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Human Platelets ◽  
Factor Vii ◽  
Coagulation System ◽  
Breast Adenocarcinoma ◽  
Activate Factor ◽  
Tumor Cell Lines

SummaryThis paper reports studies on the interaction between human platelets, the plasma coagulation system, and two human tumor cell lines grown in tissue culture: Melanoma and breast adenocarcinoma. The interaction was monitored through the use of 125I- labelled fibrinogen, which measures both thrombin activity generated by cell-plasma interaction and fibrin/fibrinogen binding to platelets and tumor cells. Each tumor cell line activates both the platelets and the coagulation system simultaneously resulting in the generation of thrombin or thrombin-like activity. The melanoma cells activate the coagulation system through “the extrinsic pathway” with a tissue factor-like effect on factor VII, but the breast tumor seems to activate factor X directly. Both tumor cell lines activate platelets to “make available” a platelet- derived procoagulant material necessary for the conversion of prothrombin to thrombin. The tumor-derived procoagulant activity and the platelet aggregating potential of cells do not seem to be inter-related, and they are not specific to malignant cells.

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