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

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 (>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 (>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.

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NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Blood ◽

10.1182/blood.v114.22.277.277 ◽

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

Journal of Experimental Medicine ◽

10.1084/jem.185.10.1735 ◽

1997 ◽

Vol 185 (10) ◽

pp. 1735-1742 ◽

Cited By ~ 10

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.156.2.492 ◽

1982 ◽

Vol 156 (2) ◽

pp. 492-505 ◽

Cited By ~ 106

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.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0036 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.3472.3472 ◽

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.

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

Blood ◽

10.1182/blood.v100.9.3304 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3304-3310 ◽

Cited By ~ 15

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.

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Innate immune protein Tag7 stimulates the appearance of cytotoxic NK cells after incubation with lymphocytes

Доклады Академии наук ◽

10.31857/s0869-56524846777-780 ◽

2019 ◽

Vol 484 (6) ◽

pp. 777-780

Author(s):

T. N. Sharapova ◽

E. A. Romanova ◽

L. P. Sashchenko ◽

N. V. Gnuchev ◽

D. V. Yashin

Keyword(s):

Tumor Cell ◽

Nk Cells ◽

Cell Lines ◽

Lymphocyte Subpopulations ◽

Innate Immune ◽

Tumor Cell Lines ◽

Cytotoxic Lymphocyte

Tag7 (PGRP-S) is an innate immune protein that is involved in the antibacterial and antitumor defense and stimulates the maturation of cytotoxic lymphocyte subpopulations. It was found that the incubation of lymphocytes with Tag7 for 3 days promotes the appearance of cytotoxic NK cells that are active against a number of tumor cell lines.

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Correlation Between Defective NK Activity and Low Expression of CD16, NKp44 and NKG2D in Beta Thalassemia Patients

Blood ◽

10.1182/blood.v112.11.5425.5425 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5425-5425

Author(s):

Belkis Atasever ◽

Serap Erdem Kuruca ◽

Zeynep Karakas ◽

Batu Erman ◽

Arzu Ergen ◽

...

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

K562 Cells ◽

Tnf Alpha ◽

Beta Thalassemia ◽

Nk Activity ◽

Ifn Gamma ◽

Cytolytic Function

Abstract Beta thalassemia patients have a major global impact on health and mortality and are characterized by absence of beta globin chain production. In most patients, multiple blood transfusions can induce differences of immune response Therefore, they are often associated with bone marrow expansion and immunodeficiency in terms of lymphocyte subsets and cytokine levels in the peripheral blood and presence of alloantibodies. We have previously shown that children with beta thalassemia major have had decreased NK cells. Natural killer (NK) cells are lymphocyte subpopulations that are important effectors of innate immune responses against infectious pathogens and tumor cells. The cytotoxic activity of NK cells is regulated by the equilibrium between positive and negative signals from multiple receptors expressed on their cell surface; signals that can trigger the cytolytic machinery as well as cytokine or chemokine secretion. The activator receptors of NK cells are natural cytotoxicity receptors (NCR) and NKG2D. NCR are represented by NKp46, NKp44, and NKp30. These receptors, upon engagement by their specific ligands, induce a strong activation of NK-mediated cytotoxic activity. NKp44, a triggering receptor selectively expressed by activated NK cells. NK cells can make cytolytic function by regulating pro-inflammatory cytokines as IFN-gamma, TNF-alpha and IL10. This study was carried out to investigate details NK cell function of 27 transfusion-dependent children with beta thalassemia. Data from 18 age- and sex-balanced children served as controls. For this purpose, we analyzed their cytolytic function against K562 cells in both pure NK cells (CD56+CD16+CD3−) and PBMC. Before and after the assessment of NK activity, we have examined the levels of NK activating receptors expressed on NK cells. The expression levels of the activation receptors (NKp30, NKp44, NKG2D) on CD56+CD16+CD3− NK cells was quantified by multicolour immunofluorescent analysis using flow cytometry. In addition, supernatant IL2, IL12, IFN-gamma, TNF-alpha, TFG-beta, IL10 levels after induced K562 cells were measured by ELISA. We observed that beta thalassemia patients had lower NK activity than controls. Before the assessment of NK activity, we found that NKG2D (2064.03+/−638.64/molecule, p<0.04) and NKp44 (1057.03+/−211.21/molecule, p<0.01) surface density was reduced in a statistically significant manner in beta thalassemia patients. This phenotype correlated with low cytolytic activity. No statistically significant differences were found in the expression of NKp30. In our experimental setting where NK cells encountered K562 targets, samples from patients had significantly increased TGF-beta (544.25+/−521.5 pg/ml, p<0.03), IL10 (16.14+/−11.1 pg/ml p<0.04) when compared with controls. In addition, expression of CD16 of NK cells that induced against K562 only (12924.47+/−6913.37/molecule, p<0.006) significantly increased in controls. As a result, our findings demonstrate that environmental factors such as ineffective cytokine production and functionally defective monocytes, may cause low NK activity in beta thalassemia patients.

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Decreased tumor surveillance in perforin-deficient mice.

Journal of Experimental Medicine ◽

10.1084/jem.184.5.1781 ◽

1996 ◽

Vol 184 (5) ◽

pp. 1781-1790 ◽

Cited By ~ 336

Author(s):

M E van den Broek ◽

D Kägi ◽

F Ossendorp ◽

R Toes ◽

S Vamvakas ◽

...

Keyword(s):

Tumor Cell ◽

Nk Cells ◽

Mhc Class I ◽

Cell Lines ◽

Tumor Cell Lines ◽

Tumor Surveillance ◽

Induced Tumors ◽

Deficient Mice

Immune surveillance against tumors usually depends on T cell recognition of tumor antigens presented by major histocompatibility complex (MHC) molecules, whereas MHC class I- tumors may be controlled by natural killer (NK) cells. Perforin-dependent cytotoxicity is a major effector function of CD8+ MHC class I-restricted T cells and of NK cells. Here, we used perforin-deficient C57BL/6 (PKO) mice to study involvement of perforin and Fas ligand in tumor surveillance in vivo. We induced tumors in PKO and normal C57BL/6 mice by (a) injection of different syngeneic tumor cell lines of different tissue origin in naive and primed mice; (b) administration of the chemical carcinogens methylcholanthrene (MCA) or 12-O-tetradecanoylphorbol-13-acetate (TPA) plus 7,12-dimethylbenzanthracene (DMBA), or (c) by injection of acutely oncogenic Moloney sarcoma virus. The first set of models analyzes the defense against a tumor load given at once, whereas the last two sets give information on immune defense against tumors at the very moment of their generation. Most of the tumor cell lines tested were eliminated 10-100-fold better by C57BL/6 mice in an unprimed situation; after priming, the differences were more pronounced. Lymphoma cells transfected with Fas were controlled 10-fold better by PKO and C57BL/6 mice when compared to untransfected control cells, indicating some role for FasL in tumor control. MCA-induced tumors arose more rapidly and with a higher incidence in PKO mice compared to C57BL/6 or CD8-deficient mice. DMBA+TPA-induced skin papillomas arose with similar high incidence and comparable kinetics in both mouse strains. C57BL/6 and PKO mice have a similar incidence of Moloney murine sarcoma and leukemia virus-induced sarcomas, but tumors are larger and regression is retarded in PKO mice. Thus, perforin-dependent cytotoxicity is not only a crucial mechanism of both cytotoxic T lymphocyte- and NK-dependent resistance to injected tumor cell lines, but also operates during viral and chemical carcinogenesis in vivo. Experiments addressing the role of Fas-dependent cytotoxicity by studying resistance to tumor cell lines that were stably transfected with Fas neither provided evidence for a major role of Fas nor excluded a minor contribution of Fas in tumor surveillance.

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Development of a Novel MICA/B-Specific CAR As a Pan-Tumor Targeting Strategy for Off-the-Shelf, Cell-Based Cancer Immunotherapy

Blood ◽

10.1182/blood-2020-141095 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 5-6

Author(s):

Ryan Bjordahl ◽

John Goulding ◽

Mochtar Pribadi ◽

Robert Blum ◽

Chiawei Chang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

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Cell Immunotherapy

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p<0.0001) and 93% in the liver (p<0.006) for CAR-MICA/B iNK cells vs non-CAR controls (Figure 1F). Additionally, CAR-MICA/B iNK cells were effective at controlling Nalm6-MICA progression in a disseminated leukemia model, suggesting potential application against both hematopoietic and solid tumors. Ongoing work is focused on extending these studies into disease-specific models of endogenous MICA/B expression to further advance CAR-MICA/B iNK cells in both solid and hematologic cancers. In summary, these preclinical data support the development and translation of an off-the-shelf NK cell immunotherapy targeting the conserved α3 domain of MICA/B with potential therapeutic application to multiple hematopoietic and solid tumor types. Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

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