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1
Vély, F., and E. Vivier. "Mécanismes moléculaires de la cytotoxicité des cellules NK." médecine/sciences 12, no. 4 (1996): 458. http://dx.doi.org/10.4267/10608/764.
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Bogner, Ulrich, Jack R. Wall, and Horst Schleusener. "Cellular and antibody mediated cytotoxicity in autoimmune thyroid disease." Acta Endocrinologica 116, no. 1_Suppl (August 1987): S133—S138. http://dx.doi.org/10.1530/acta.0.114s133.
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Abstract. Antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity was measured in patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD) using a cytotoxicity assay against thyroid target cells. In the ADCC assay, mean ± sd specific lysis produced by sera from patients with HT was 21.7 ± 10% compared t 6.2 ± 3.9% from normal subjects. In the NK assay, cytotoxicity was significantly increased using lymphocytes from HT patients as effector cells. At effector: target (E:T) cell ratios of 50:1 and 25:1, mean specific lysis ± sd was 18.3 ± 14.3% and 14 ± 11.6%, respectively, compared to 3.7 ±2.1 and 3.1 ± 2.1, respectively, for normals. In Graves' disease, 9 of 19 patients had elevated cytotoxicity, whereas no significant changes of ADCC could be found either, as determined in thyrotoxic patients, after 6 months and at the end of a one-year antithyroid drug treatment. Eight of 19 patients showed normal cytotoxicity (mean % specific lysis 2.5 ± 3.1% compared to 2 ± 2.9% in normal controls) and low titres of microsomal antibodies (Mab), 3 patients had significantly increased cytotoxicity (mean specific lysis 27.6 ± 10%) in the presence of high titres of Mab, whereas 8 patients evidenced high values for cytoxicity (mean specific lysis 24.5 ± 14.1%) but low titres of Mab. NK cell activity, determined in euthyroid Graves' disease patients either under antithyroid drug therapy or in remission, was not significantly different than that of normal subjects at all E:T cell ratios. In conclusion, we demonstrated increased ADCC and NK cell activity in Hashimoto's thyroiditis but normal NK cell activity in euthyroid Graves' disease. Like in HT, ADCC is associated with titres of Mab in sera of Graves' disease patients but was also detectable in Mab-negative sera, which led us to suggest that a hitherto unknown cytotoxic antibody exists which is not measurable by passive haemagglutination for Mab.
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Don Yun, Hyun, Martin Felices, Daniel A. Vallera, Peter Hinderlie, Sarah Cooley, Michel Arock, Jason Gotlib, Celalettin Ustun, and Jeffrey S. Miller. "Trispecific killer engager CD16xIL15xCD33 potently induces NK cell activation and cytotoxicity against neoplastic mast cells." Blood Advances 2, no. 13 (July 6, 2018): 1580–84. http://dx.doi.org/10.1182/bloodadvances.2018018176.
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Key Points NK cell natural cytotoxicity and antibody-dependent cellular cytotoxicity of patients with systemic mastocytosis are normal. Trispecific killer engagers (161533 TriKE) target NK cells from normal donors and systemic mastocytosis patients to kill mast cells.
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Fujisaki, Hiroyuki, Harumi Kakuda, Timothy Lockey, Paul W. Eldridge, Wing Leung, and Dario Campana. "Expanded Natural Killer Cells for Cellular Therapy of Acute Myeloid Leukemia." Blood 110, no. 11 (November 16, 2007): 2743. http://dx.doi.org/10.1182/blood.v110.11.2743.2743.
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Abstract Approximately half of the patients with acute myeloid leukemia (AML) harbor occult disease during therapy, leading to overt relapse. Novel treatments are needed to advance cure rates. AML cells are sensitive to natural killer (NK) cell cytotoxicity if they express HLA Class I molecules that do not bind killer-inhibitory receptors (KIR) on NK cells. The demonstration that haploidentical NK cells can expand in vivo and exert anti-AML activity when infused after non-myeloablative conditioning (Miller et al., Blood105: 3051, 2005), provided impetus to further explore their clinical potential and develop ways to increase their efficacy. The success of NK cell therapy depends on: i) mismatch in recipient HLA and donor KIR phenotype, allowing NK cell alloreactivity; ii) infusion of sufficient numbers of NK cells to achieve an effector: target (E:T) ratio that produces a significant leukemia cytoreduction. We found that K562 cells genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) induced expansion of human NK cells (Imai et al., Blood106: 376, 2005). In the present study, we first tested the stimulatory capacity of irradiated K562-mb15-41BBL in 34 additional healthy donors: CD56+ CD3− NK cell expansion after 7–10 days of culture was 5–87 fold (median, 22); after 21 days, NK cells could expand >1000 fold. CD3+ T cells expanded minimally or not at all. NK cells derived from 12 healthy donors were tested against the AML cell lines K562, KG-1, U937 and HL-60. Expanded NK cells were consistently cytotoxic at low E:T ratios. Thus, mean (± SD) cytotoxicity after 4 hrs at 4: 1 was 85.1% ± 8.7% for K562, 83.7% ± 9.4% for KG-1, 78.8% ± 15.2% for U937 and 94.8% ± 5.1% for HL-60. Expanded NK cells were effective even when outnumbered by target cells: at a 0.5: 1 ratio, cytotoxicities were 34.1% ± 14.7% with K562, 51.5% ± 16.5% with KG-1, 24.5% ± 14.8% with U937 and 52.1% ± 9.8% with HL-60. We next tested cytotoxicity of expanded NK cells from 10 donors against primary cells obtained from the bone marrow of 9 newly diagnosed patients with AML. Median cytotoxicity after 4 hrs of culture at a 4: 1 ratio was high, although interdonor variability was observed, with cytoxicities ranging from 22% to 90%. When expanded NK cells were cultured for 7 days with primary AML cells in the presence of bone marrow mesenchymal cells (to prevent spontaneous apoptosis of the AML cells) we could detect cytotoxicity at a 0.01:1 E:T ratio. Expanded NK cells were consistently more cytotoxic than primary NK cells from the same donor. Gene expression studies revealed marked changes in expression of adhesion molecules and cytokine transcripts after expansion. Expanded NK cells exerted considerable antileukemic effect in NOD-SCID-IL2Rgammanull mice engrafted with human AML cells, providing a strong rationale for their clinical testing. To this end, the K562-mb15-41BBL stimulatory cell line is currently being made under cGMP conditions and conditions for large-scale NK cell expansion have been established in support of a pilot protocol in which expanded haploidentical NK cells with be administered to patients with refractory AML.
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Trotta, Rossana, David Ciarlariello, Jessica Dal Col, Hsiaoyin Mao, Li Chen, Edward Briercheck, Jianhua Yu, Jianying Zhang, Danilo Perrotti, and Michael A. Caligiuri. "The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells." Blood 117, no. 8 (February 24, 2011): 2378–84. http://dx.doi.org/10.1182/blood-2010-05-285130.
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Abstract The ability of natural killer (NK) cells to kill malignant or infected cells depends on the integration of signals from different families of cell surface receptors, including cytokine receptors. How such signals then regulate NK-cell cytotoxicity is incompletely understood. Here we analyzed an endogenous inhibitor of protein phosphatase 2A (PP2A) activity called SET, and its role in regulating human NK-cell cytotoxicity and its mechanism of action in human NK cells. RNAi-mediated suppression of SET down-modulates NK-cell cytotoxicity, whereas ectopic overexpression of SET enhances cytotoxicity. SET knockdown inhibits both mRNA and protein granzyme B expression, as well as perforin expression, whereas SET overexpression enhances granzyme B expression. Treatment of NK cells with the PP2A activator 1,9-dideoxy-forskolin also inhibits both granzyme B expression and cytotoxicity. In addition, pretreatment with the PP2A inhibitor okadaic acid rescues declining granzyme B mRNA levels in SET knockdown cells. Down-modulation of SET expression or activation of PP2A also decreases human NK-cell antibody-dependent cellular cytotoxicity. Finally, the induction of granzyme B gene expression by interleukin-2 and interleukin-15 is inhibited by SET knockdown. These data provide evidence that granzyme B gene expression and therefore human NK-cell cytotoxicity can be regulated by the PP2A-SET interplay.
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Maniar, Amudhan, Xiaoyu Zhang, Wei Lin, Brian R. Gastman, C. David Pauza, Scott E. Strome, and Andrei I. Chapoval. "Human γδ T lymphocytes induce robust NK cell–mediated antitumor cytotoxicity through CD137 engagement." Blood 116, no. 10 (September 9, 2010): 1726–33. http://dx.doi.org/10.1182/blood-2009-07-234211.
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AbstractNatural killer (NK) cells are innate effector lymphocytes that control the growth of major histocompatibility complex class I negative tumors. We show here that γδ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell–mediated killing of tumors that are usually resistant to NK cytolysis. The induction of cytotoxicity toward these resistant tumors requires priming of NK cells by immobilized human immunoglobulin G1 and costimulation through CD137L expressed on activated γδ T lymphocytes. This costimulation increases NKG2D expression on the NK-cell surface, which is directly responsible for tumor cell lysis. Moreover, culturing peripheral blood mononuclear cells with zoledronic acid, a γδ T lymphocyte activating agent, enhances NK-cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors. Our data reveal a novel function of human γδ T lymphocytes in the regulation of NK cell–mediated cytotoxicity and provide rationale for the use of strategies to manipulate the CD137 pathway to augment innate antitumor immunity.
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Cao, Xiangshan, and Jianyong Li. "KIR Gene and HLA-Cw Mismatches Affect on NK Cell Killer Activity." Blood 112, no. 11 (November 16, 2008): 4926. http://dx.doi.org/10.1182/blood.v112.11.4926.4926.
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Abstract The KIRs were knew as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. We investigated NK cell activation on the number of matches between cell killer immunoglobulin-like receptor (KIR) gene and HLA-Cw, and the level of inhibitory KIRs expressed on NK cell surface and the cytotoxicity of NK cell against AML leukemic cells in vitro. NK cell were isolated and purified from 27 healthy donors by isolation kit, Target cells were blasts derived from bone marrow of 30 patients with AML.Inhibitory KIRs expression knew as CD158a, CD158b was analyzed by flow cytometry to estimate the percentage of NK cells that could be inhibited by the HLA-Cw ligands..KIR and HLA gene typing were performed by PCR –SSP. from donors and patients respectively. NK cytotoxicity against AML leukemic cells demonstrated by MTT which showed the correlation between NK cytotoxicity and the number of KIR/HLA matches. the NK-susceptible K562 cell line which lacks HLA class I expression, was used as a positive control target in all cytotoxicity assays, autologous non-NK cell was used as negative control target cell. the cytotoxicity assays was performed in E:T 50:1 20:1 10:1 5:1 2.5:1. Results demonstrated the less number of KIR/HLA-Cw matches, the more NK cells are activated..0 match of NK cell/target cell KIR/HLA-Cw, cytotoxicity was (50.66±8.40)%,1 match and 2 matches were (38.28±6.71)%, (19.74±4.15)%, F=20.226, P<0.001. NK cells expressed KIRs also had relationship with cytotoxicity, inhibitory KIRs expressed >50%, the cytotoxicity is 10%, inhibitory KIRs expressed 20%–50%, the cytotoxicity is 20%, inhibitory KIRs <25%, the cytotoxicity is 55%, F=16.276,p<0.001. Therefore these data indicate NK cell kill AML leukemic cells mechanism follow KIR/HLA-Cw mismatch theory, the level of inhibitory KIRs expressed on NK cell surface showed the percentage of NK cells that could be inhibited by the HLA-Cw ligands. Key words: KIR NK cell CD158 HLA-Cw
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Park, Ji-Eun, Seong-Eun Kim, Bhumsuk Keam, Ha-Ram Park, Soyeon Kim, Miso Kim, Tae Min Kim, Junsang Doh, Dong-Wan Kim, and Dae Seog Heo. "Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines." Journal for ImmunoTherapy of Cancer 8, no. 2 (August 2020): e000873. http://dx.doi.org/10.1136/jitc-2020-000873.
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BackgroundAlthough programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines.MethodsVarious cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors.ResultsWe demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype.ConclusionThese results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.
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Billadeau, Daniel D., Kathryn M. Brumbaugh, Christopher J. Dick, Renee A. Schoon, Xose R. Bustelo, and Paul J. Leibson. "The Vav–Rac1 Pathway in Cytotoxic Lymphocytes Regulates the Generation of Cell-mediated Killing." Journal of Experimental Medicine 188, no. 3 (August 3, 1998): 549–59. http://dx.doi.org/10.1084/jem.188.3.549.
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The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell–mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.
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Gismondi, Angela, Loredana Cifaldi, Cinzia Mazza, Silvia Giliani, Silvia Parolini, Stefania Morrone, Jordan Jacobelli, Elisabetta Bandiera, Luigi Notarangelo, and Angela Santoni. "Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect." Blood 104, no. 2 (July 15, 2004): 436–43. http://dx.doi.org/10.1182/blood-2003-07-2621.
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Abstract In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or β2-integrin engagement on NK cells. (Blood. 2004;104:436-443)
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Bjordahl, Ryan, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, et al. "Off-the-Shelf Natural Killer Cell Immunotherapy for Enhanced Antibody Directed Cellular Cytotoxicity." Blood 128, no. 22 (December 2, 2016): 3363. http://dx.doi.org/10.1182/blood.v128.22.3363.3363.
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Abstract Natural Killer (NK) cells play a crucial role in immunosurveillance and form a first line of defense against cancer. In comparison to other lymphocytes, NK cells are unique in their capability to elicit tumoricidal responses without the need for antigen presentation or prior sensitization. Clinical data from bone marrow transplant and allogeneic NK immunotherapy suggest that MHC mismatch is advantageous in promoting graft-versus-leukemia without eliciting graft-versus-host, providing evidence that NK cells hold promisa as an allogeneic, universal immunotherapeutic. Further, the anti-tumor effect of many monoclonal antibodies is mediated through binding of the low-affinity Fc receptor CD16 on NK cells, which induces tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC). Thus, NK cells represent a unique source of effector cells that can be combined with monoclonal antibodies, bispecific engagers or chimeric antigen receptors to direct tumor specificity and enhance cytotoxicity. Despite the significant potential of NK cell therapy, current clinical practices are limited by the need for large numbers of healthy NK cells, lack of in vivo persistence, and a burdensome manufacturing strategy that requires donor cell extraction, modulation, expansion and re-introduction per each patient. The ability to generate universally histocompatible and genetically-enhanced NK cells from continuously renewable human induced pluripotent stem cell (hiPSC) lines offers the potential to develop a true "off-the-shelf" cellular immunotherapy. While NK differentiation from hiPSC has been demonstrated, the clonal derivation of engineered hiPSCs to improve effector function has been challenging and the scalability and robustness of the differentiation method has been limited by skewed development towards primitive hematopoiesis and the cumbersome use of embryoid bodies. Here we highlight our "off-the-shelf" NK cell therapy preclinical program by demonstrating robust and highly scalable generation of functionally mature, genetically targeted and universally histocompatible NK cells. This program utilizes our previously described naïve hiPSC platform where we uniquely create clonal lines of precisely engineered, renewable hiPSCs and drive definitive hematopoiesis in a highly scalable manner. Because hiPSC differentiation is lineage directed, minimal cellular contamination is seen, including the lack of T and B cells, in the final product. Through precise genetic engineering of naïve hiPSC lines, we have engineered HLA-class I deficient NK cells uniformly expressing a high affinity, non-cleavable version of the Fc receptor CD16 (NcHaCD16-NK). The hiPSC-derived NcHaCD16-NKs display markers of maturity, including CD16, KIR, NCRs, and CD94. When compared to conventional cord blood and peripheral blood sourced NK cells, NcHaCD16-NKs exhibit superior cytotoxicity and production of effector cytokines in response to both solid and liquid tumor cell challenge in vitro. NcHaCD16-NKs exhibit augmented cytokine response following Fc-mediated stimulation, demonstrating function competence of the engineered CD16 construct. Because surface expression of CD16 is resistant to activation-induced shedding, NcHaCD16-NKs continuously maintain enhanced ADCC while retaining the capacity for general cytotoxicity. Importantly, the hiPSC-derived hematopoietic cells can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Preliminary data also shows NcHaCD16-NKs elicit preferred specificity for cancer stem cells as defined by expression of ALDH1 and surface markers such as CD24. In conclusion, the outlined preclinical data demonstrate the potential therapeutic utility of NK cells developed via precision genetic engineering of a renewable, scalable hiPSC platform, and highlights the therapeutic value of NcHaCD16-NKs as an ideal ADCC-mediated "off-the-shelf" NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity and preclinical efficacy. Disclosures Bjordahl: Fate Therapeutics, Inc: Employment. Clarke:Fate Therapeutics: Employment. Gaidarova:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics, Inc.: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics, Inc.: Employment. Kim:Fate Therapeutics, Inc.: Employment. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.
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Stebbins, Christopher C., Carsten Watzl, Daniel D. Billadeau, Paul J. Leibson, Deborah N. Burshtyn, and Eric O. Long. "Vav1 Dephosphorylation by the Tyrosine Phosphatase SHP-1 as a Mechanism for Inhibition of Cellular Cytotoxicity." Molecular and Cellular Biology 23, no. 17 (September 1, 2003): 6291–99. http://dx.doi.org/10.1128/mcb.23.17.6291-6299.2003.
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ABSTRACT Here, we present data suggesting a novel mechanism for regulation of natural killer (NK) cell cytotoxicity through inhibitory receptors. Interaction of activation receptors with their ligands on target cells induces cytotoxicity by NK cells. This activation is under negative control by inhibitory receptors that recruit tyrosine phosphatase SHP-1 upon binding major histocompatibility class I on target cells. How SHP-1 blocks the activation pathway is not known. To identify SHP-1 substrates, an HLA-C-specific inhibitory receptor fused to a substrate-trapping mutant of SHP-1 was expressed in NK cells. Phosphorylated Vav1, a regulator of actin cytoskeleton, was the only protein detectably associated with the catalytic site of SHP-1 during NK cell contact with target cells expressing HLA-C. Vav1 trapping was independent of actin polymerization, suggesting that inhibition of cellular cytotoxicity occurs through an early dephosphorylation of Vav1 by SHP-1, which blocks actin-dependent activation signals. Such a mechanism explains how inhibitory receptors can block activating signals induced by different receptors.
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Nagai, Yuya, Meisam Naeimi Kararoudi, Syed Abbas Ali, Philip H. Imus, Marcelo Pereira, Ezgi Elmas, Ivan Borrello, Dean Anthony Lee, and Gabriel Ghiaur. "Impact of CD38 knockout in NK cells on daratumumab-mediated cytotoxicity and cellular metabolism." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15018-e15018. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15018.
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e15018 Background: Multiple myeloma (MM) is a neoplasm of plasma cells. These cells express high levels of CD38 and are sensitive to daratumumab (DARA), a CD38-targeting monoclonal antibody. DARA kills MM cells via several mechanisms including antibody dependent cellular cytotoxicity (ADCC). Although DARA has improved the outcome of patients, CR is not achieved in all the cases. Besides, most patients that achieve CR will experience relapse. A potential explanation for blunted efficacy of DARA is downregulation of CD38 levels on MM cells and impaired ADCC due to depletion of CD38high NK cells during therapy. ATRA upregulates CD38 levels on target cells and was proposed to improve the efficacy of DARA. NK cell supplementation was tested in a preclinical model, but that had only a modest effect on DARA efficacy, likely due to their short life via DARA-mediated elimination. Methods: We generated CD38 knockout (CD38KO) NK cells from healthy donors via Cas9 RNPs and investigate if these cells boost DARA activity against MM. RNA-seq and cellular metabolic analysis were performed to examine the effect of CD38 deletion on NK cell function. Results: Knockout efficiency was 81.9 ± 6.9% (mean ± SD, N = 5). Very low off-target effects were seen by whole genome sequencing. When compared to paired CD38 wild type (CD38WT) NK cells, CD38KO NK cells showed lower conjugation (2.57% vs. 11.85%, N = 3, p = 0.04), less fratricide (97.3% vs. 54.2%, mean viability, N = 3, p = 0.01), and superior persistence in mice (18.16% vs. 0.42%, mean frequency in blood, N = 5, p < 0.01) in the presence of DARA. Additionally, CD38KO NK cells exhibited enhanced ADCC against all tested MM cell lines and primary samples including CD38low cell lines and primary MM cells from a patient with disease relapse on DARA. Transcriptomic and cellular metabolic analysis revealed that CD38KO NK cells have favorable metabolic shift with higher mitochondrial respiratory capacity (N = 3, p < 0.01). Although ATRA upregulates CD38 levels on MM cell lines, we observed that ATRA treatment upregulates CD38 levels on circulating NK cells in patients. ATRA upregulated CD38 levels on CD38WT NK cells, but not on CD38KO NK cells, and increased their fratricide. Thus, CD38WT NK cells showed impaired overall DARA-mediated cytotoxicity against MM cells in the presence of ATRA, even though those MM cells were sensitized to DARA. Conclusions: We present proof of concept that adoptive CD38KO NK cell therapy has the potential to maximize the efficacy of DARA against MM. Further investigation into the role of metabolic reprogramming of CD38KO NK cells is warranted.
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Yamamoto, Kenta, Robert Blum, and Dan S. Kaufman. "ADAM17-Deficient Pluripotent Stem Cell-Derived Natural Killer Cells Possess Improved Antibody-Dependent Cellular Cytotoxicity and Antitumor Activity." Blood 136, Supplement 1 (November 5, 2020): 2. http://dx.doi.org/10.1182/blood-2020-137766.
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Antibody-dependent cellular cytotoxicity (ADCC) is a key pathway that mediates natural killer (NK) cell cytotoxicity against antibody-opsonized target cells. This process helps mediate the therapeutic efficacy of anti-tumor antibodies. On NK cells, ADCC occurs via engagement of antibody-coated target cells with activating receptor FcγRIIIa, or CD16a, leading to proinflammatory cytokine upregulation, degranulation, and target cell death. Upon cellular activation, the CD16a ectodomain is cleaved from the NK cell surface by A Disintegrin and Metalloprotease-17 (ADAM17). Cleavage of the ectodomain prevents further antibody binding and signaling through CD16a, which dampens NK cell activity. Blocking activation-induced ADAM17-mediated CD16a cleavage has been previously demonstrated to augment ADCC activity and provides a novel strategy to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. To further define the ability of ADAM17 to regulate NK cell activity, we have generated and characterized ADAM17-deficient (ADAM17-KO) NK cells derived from CRISPR/Cas9-modified human induced pluripotent stem cells (iPSCs). ADAM17-KO iPSCs successfully differentiate into hematopoietic progenitor cells, then to NK cells that uniformly express typical NK cell surface markers including CD56, CD94, NKG2D, NKp44, and NKp46. ADAM17-KO iPSC-NKs are functional and kill K562 erythroleukemia cells comparable to wildtype iPSC-derived NK cells (WT iPSC-NK cells) and healthy donor-derived peripheral blood NK cells (PB-NK cells) in vitro. Surprisingly, upon differentiation, ADAM17-KO iPSC-NK cells express ~20% lower CD16a surface expression compared to WT iPSC-NK cells, but stably retain CD16a expression after enrichment for CD16a+ cells and over 6 weeks of expansion in culture. WT iPSC-NKs and PB-NKs rapidly lose CD16a surface expression upon stimulation with phorbol esters, while ADAM17 KO iPSC-NK cells maintain over 90% CD16a expression after this stimulation. Additionally, a significantly higher proportion of ADAM17-KO iPSCs express TNF-α (71%) and CD62L (L-Selectin) (36%) - two other known ADAM17 substrates, on the cell surface after stimulation with phorbol esters for 4 hours compared to WT iPSC-NK (7% TNF-α+, 2% L-Selectin+) and PB-NK (2% TNF-α+, 1% L-Selectin+). CD16a+ ADAM17-KO iPSC-NK cells mediate increased CD107a (45%) and IFNγ (39%) expression when co-incubated with RAJI B-lymphoma cells in the presence of the anti-CD20 antibody rituximab, compared to CD16a+ WT iPSC-NK (32% CD107a+, 11% IFNγ) and PB-NK (37% CD107a+, 7% IFNγ) cells. Similarly, CD16a+ ADAM17-KO iPSC-NK cells upregulate increased CD107a (29%) and IFNγ (42%) expression when co-incubated with CAL27 squamous cell carcinoma cells in the presence of the anti-EGFR antibody cetuximab, compared to CD16a+ WT iPSC-NK (12% CD107a+, 8% IFNγ) and PB-NK (14% CD107a+, 6% IFNγ). Long-term (24 hour) cytotoxicity assay against RAJI cells in the presence of rituximab demonstrates higher cytotoxicity in CD16a+ ADAM17-KO iPSC-NK cells compared to CD16a+ WT iPSC-NK and CD16a+ PB-NK cells over time (see associated figure). In vivo studies to determine the therapeutic efficacy of ADAM17-KO iPSC-NK cells compared to WT iPSC-NK and PB-NK cells are ongoing. Together, these studies demonstrate ADAM17-KO iPSC-NK cells derived from a renewable source of gene-edited iPSCs possess enhanced ADCC potential, and provide a promising candidate to be used for standardized, off-the-shelf NK cell-based therapies in conjunction with therapeutic antibodies. Figure Disclosures Blum: Fate Therapeutics: Current Employment. Kaufman:Fate Therapeutics: Consultancy.
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Ehlers, Femke A. I., Nicky A. Beelen, Michel van Gelder, Tom M. J. Evers, Marjolein L. Smidt, Loes F. S. Kooreman, Gerard M. J. Bos, and Lotte Wieten. "ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response." Cancers 13, no. 13 (June 28, 2021): 3232. http://dx.doi.org/10.3390/cancers13133232.
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Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells’ full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.
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Perera Molligoda Arachchige, Arosh Shavinda. "Human NK cells: From development to effector functions." Innate Immunity 27, no. 3 (March 24, 2021): 212–29. http://dx.doi.org/10.1177/17534259211001512.
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NK cells are the major lymphocyte subset of the innate immune system that mediates antiviral and anti-tumor responses. It is well established that they develop mechanisms to distinguish self from non-self during the process of NK cell education. Unlike T and B cells, natural killer cells lack clonotypic receptors and are activated after recognizing their target via germline-encoded receptors through natural cytotoxicity, cytokine stimulation, and Ab-dependent cellular cytotoxicity. Subsequently, they utilize cytotoxic granules, death receptor ligands, and cytokines to perform their effector functions. In this review, we provide a general overview of human NK cells, as opposed to murine NK cells, discussing their ontogeny, maturation, receptor diversity, types of responses, and effector functions. Furthermore, we also describe recent advances in human NK cell biology, including tissue-resident NK cell populations, NK cell memory, and novel approaches used to target NK cells in cancer immunotherapy.
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Caraux, Anouk, Nayoung Kim, Sarah E. Bell, Simona Zompi, Thomas Ranson, Sarah Lesjean-Pottier, Marcos E. Garcia-Ojeda, Martin Turner, and Francesco Colucci. "Phospholipase C-γ2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells." Blood 107, no. 3 (February 1, 2006): 994–1002. http://dx.doi.org/10.1182/blood-2005-06-2428.
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AbstractPhospholipase C-γ2 (PLC-γ2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-γ2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-γ2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-γ2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-γ on stimulation with IL-12 was normal. Plcg2-/- mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection. Our results suggest that exocytosis of cytotoxic granules, but not cellular polarization toward targets, depends on intracellular calcium rise during NK cell cytotoxicity. In vivo, PLC-γ2 regulates selective facets of innate immunity because it is essential for NK cell responses to malignant and virally infected cells but not to bacterial infections.
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Awan, Farrukh T., Rosa Lapalombella, Rossana Trotta, Jonathan P. Butchar, Bo Yu, Don M. Benson, Julie M. Roda, et al. "CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain–engineered monoclonal antibody." Blood 115, no. 6 (February 11, 2010): 1204–13. http://dx.doi.org/10.1182/blood-2009-06-229039.
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Abstract CD19 is a B cell–specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)–domain designed to enhance binding of FcγRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B–dependent mechanism. The NK cell–mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcγ receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell–mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19+ B-cell malignancies.
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Zhao, Xing, Narendiran Rajasekaran, Cariad Chester, Atsushi Yonezawa, Suparna Dutt, Matt Coffey, Zhixu He, and Holbrook E. Kohrt. "Natural Killer Cells Activated By Oncolytic Reovirus Enhance Cetuximab Mediated Antibody Dependent Cellular Cytotoxicity in an in Vitro and In Vivo Model of Colorectal Cancer." Blood 126, no. 23 (December 3, 2015): 3439. http://dx.doi.org/10.1182/blood.v126.23.3439.3439.
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Abstract The naturally occurring oncolytic virus, reovirus, exhibits cytotoxic effects on cancer cells. Reovirus is currently being tested in multiple clinical trials for the treatment of different cancers. In addition, they also activate the innate and adaptive immune responses targeting immune cells like dendritic cells and macrophages. In this study we investigated the direct effect of reovirus on Natural Killer cells (NK cells) and its effect on NK cell mediated antibody dependent cellular cytotoxicity (ADCC) against the EGFR (Epidermal Growth Factor) positive colorectal cancer cell line: DLD-1 (KRAS mutant). NK cells isolated from human PBMCs were cultured with 1pfu of reovirus for 12 hrs and subsequently co-cultured with DLD-1 cells coated with increasing concentrations anti-EGFR antibody cetuximab. ADCC was measured after 4 hrs using a lactate dehydrogenase (LDH) based cytotoxicity assay. We observed that the reovirus treated NK cells (Reo-NK cells) exhibited a ~16-fold increase in cytotoxicity against DLD-1 (16.3% ±1.5, n=3) compared to untreated NK cells (NK), even in the absence of any cetuximab. In the presence of cetuximab, NK cells showed a dose dependent increase in ADCC, with maximum ADCC, observed at 0.1 µg/ml of cetuximab (DLD-1+NK: 33.4%± 7.1, n=3). Interestingly, Reo-NK cells showed maximum ADCC even at 0.01 µg /ml of cetuximab (DLD-1+Reo-NK: 39.1±7.4, DLD-1+NK: 26.7±2.4%, n=3). Reo-NK cells also exhibited an increased expression of activation marker CD69 (Reo-NK: 70.4%, NK: 35.2%) and degranulation maker CD107a (Reo-NK: 14.6%; NK: 4.45%) compared to the untreated NK cells. We further characterized the Reo-NK cells by using the HIMChip microarray platform; a custom Agilent SurePrint HD 8x15k format array containing over 7,000 unique probes for over 4,274 human immune-related genes. In ingenuity pathway analysis, we observed that the Interferon pathway (2.13E-20) and pathway controlling activation of IRF by cytosolic pattern recognition receptors (1.27E-11) were the predominant pathways observed in the Reo-NK cells. These results suggest an interferon-mediated response could be contributing to the increased cytoxicity of the NK cells. In an in vivo study, DLD-1 cells were grown subcutaneously in athymic nude mice and injected intravenously with reovirus (5x 108 pfu), followed by intraperitoneal injection of Cetuximab (200 ug/mice) every week. We observed a significant regression of tumors in the Reovirus+Cetuximab combination group compared to the Reovirus treated (Reovirus+Cet: 349.9 mm3, Reovirus: 623.8 mm3; n=9; P=0.0028) or Cetuximab treated (Reovirus+Cet: 349.9 mm3, Cet: 730.5 mm3;n=9; P= 0.030) groups on day 28 post treatment. Thus, in this study our results demonstrated that human NK cells when treated with reovirus show increases in activation, degranulation and cytotoxicity when compared to untreated NK cells. Further, in the in vivo model we observed increased tumor regression in mice treated with reovirus in combination with cetuximab. We propose that reovirus activated NK cells are a potential candidate for cell based immunotherapy in combination with FDA approved tumor targeting antibodies to treat malignancies, including lymphomas. Further studies are ongoing to investigate the underlying mechanisms that contribute to the increase in cytotoxicity by NK cells treated with reovirus. Disclosures No relevant conflicts of interest to declare.
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Zimmer, Jacques, Lionel Donato, Daniel Hanau, Jean-Pierre Cazenave, Marie-Marthe Tongio, Alessandro Moretta, and Henri de la Salle. "Activity and Phenotype of Natural Killer Cells in Peptide Transporter (TAP)-deficient Patients (Type I Bare Lymphocyte Syndrome)." Journal of Experimental Medicine 187, no. 1 (January 5, 1998): 117–22. http://dx.doi.org/10.1084/jem.187.1.117.
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In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I–deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I− K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)− NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP− NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP− NK lymphocytes showed them to display a normal diverse repertoire of HLA class I–specific NK receptors. These receptors were expressed at normal levels, apart from the CD94–NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP− individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP− patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.
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Hsu, Hsiang-Ting, Emily M. Mace, Alexandre F. Carisey, Dixita I. Viswanath, Athanasia E. Christakou, Martin Wiklund, Björn Önfelt, and Jordan S. Orange. "NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing." Journal of Cell Biology 215, no. 6 (November 30, 2016): 875–89. http://dx.doi.org/10.1083/jcb.201604136.
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Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.
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Lee, Myeong Soo, Hwa Jeong Huh, Hye-Sook Jang, Chang Sub Han, Hoon Ryu, and Hun-Taeg Chung. "Effects of Emitted Qi on In Vitro Natural Killer Cell Cytotoxic Activity." American Journal of Chinese Medicine 29, no. 01 (January 2001): 17–22. http://dx.doi.org/10.1142/s0192415x01000034.
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The present study investigated the effects of Korean Qi-therapy, ChunSoo Energy Healing, on natural killer (NK) cell cytotoxicity in vitro depending on Qi-treatment time and the types of cells treated. NK cell cytotoxicity was assayed by measuring LDH release from tumor target cells (K562 cell lines). NK activity was significantly increased by emitted-Qi treatment of 30 sec duration. Three and 5 minutes of Qi projection created the greatest increase in NK cell activity when mixtures of NK cells and K562 cells were treated (1.81 and 2.12 fold for 4 hr culture; 1.54 and 1.36 for 16 hr culture, respectively). NK cell activity increased significantly in Qi-treated K562 cells alone (1.13 fold, p < 0.05) compared to control. These results are consistent with in vivo Qi-therapy on humans and suggests that emitted-Qi has an acute stimulatory effect on NK cell activity. This study provides direct scientific support that Qi as such may positively affect human cellular immunity.
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Bjordahl, Ryan, Svetlana Gaidarova, Jode P. Goodridge, Sajid Mahmood, Greg Bonello, Megan Robinson, Chelsea Ruller, et al. "FT576: A Novel Multiplexed Engineered Off-the-Shelf Natural Killer Cell Immunotherapy for the Dual-Targeting of CD38 and Bcma for the Treatment of Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 3214. http://dx.doi.org/10.1182/blood-2019-131373.
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Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (<1%). In an in vitro fratricide assay, FT576 NK cells were entirely resistant to daratumumab-induced fratricide, with no detectable specific cytotoxicity when exposed to increasing concentrations of daratumumab. Conversely, peripheral blood NK cells were sensitive to daratumumab-induced fratricide (up to 33% cytotoxicity within 3 hrs of daratumumab exposure). FT576 NK cells demonstrated enhanced cytotoxicity against the MM1.S MM cell line during a long-term cytotoxicity assay compared with control NK cells that lacked CAR expression (62% cytotoxicity for FT576 vs 26% for control). In addition, cellular persistence was greater than NK cells lacking the IL-15RF protein, and FT576 NK cells demonstrated the unique ability to expand in vitro absent of exogenous cytokine support (61-fold expansion vs. 4-fold for IL-15RF negative). Importantly, FT576 NK cells remained ADCC competent, as combination with daratumumab enhanced cytotoxicity against MM cell lines in a 2D cytotoxicity assay. Additionally, FT576 mediated direct cytotoxicity against RPMI-8226 MM spheroids, leading to >99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.
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Grote, Stefan, Frank Traub, Joerg Mittelstaet, Christian Seitz, Andrew Kaiser, Rupert Handgretinger, and Sabine Schleicher. "Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases." Cancers 13, no. 5 (March 5, 2021): 1124. http://dx.doi.org/10.3390/cancers13051124.
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Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. Conclusions: AdCAR NK-92 cells may provide an interesting and promising “off-the-shelf” cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.
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Nguyen, Quoc H., Robert L. Roberts, Bonnie J. Ank, Syh-Jae Lin, Casey K. Lau, and E. Richard Stiehm. "Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12." Clinical Diagnostic Laboratory Immunology 5, no. 1 (January 1, 1998): 98–104. http://dx.doi.org/10.1128/cdli.5.1.98-104.1998.
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ABSTRACT Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.
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Becknell, Brian, Rossana Trotta, Jianhua Yu, Wei Ding, Hsiaoyin C. Mao, Tiffany Hughes, Trent Marburger, and Michael A. Caligiuri. "Efficient and Reproducible Retroviral Infection of Primary Human Natural Killer Cells." Blood 104, no. 11 (November 16, 2004): 1348. http://dx.doi.org/10.1182/blood.v104.11.1348.1348.
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Abstract Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector PINCO, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection - the enhanced green fluorescent protein (EGFP) - along with various genes of interest. This technique results in successful infection of the CD56dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we demonstrate infection of the CD56bright NK subset as well as the NK-92 and NK-L cell lines. Finally, we modify PINCO to express a cytoplasmically truncated murine CD8 molecule in place of GFP. The resulting vector enables us to transduce NK cells with multiple genes of interest simultaneously and provides an alternative purification method to FACS by using magnetic beads. In summary, we have devised an efficient and highly reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology and to genetically manipulate NK cell populations prior to their administration in cancer therapy.
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Alvarez de Mon, M., J. Casas, R. Laguna, ML Toribio, MO de Landazuri, and A. Durantez. "Lymphokine induction of NK-like cytotoxicity in T cells from B-CLL." Blood 67, no. 1 (January 1, 1986): 228–32. http://dx.doi.org/10.1182/blood.v67.1.228.228.
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Abstract T cells from patients with B cell chronic lymphocytic leukemia (B-CLL) exhibit defective natural killer (NK) activity. In this study, we have analyzed the cytotoxic-inducer effects of gamma interferon (gamma-IFN) and supernatants containing interleukin 2 (IL 2 sup). T cells from patients with B-CLL were incubated with gamma-IFN or IL 2 sup. gamma- IFN did not modulate the very low or undetectable levels of NK activity present in the T cell population. However, the IL 2 sup induced a potent cellular cytotoxicity against NK-sensitive and NK-resistant tumoral target cells. This cytotoxic inducer effect (a) was present in lectin-free IL 2 sup and in a 15,000- to 20,000-dalton molecular weight fraction obtained by gel filtration chromatography of this supernatant; (b) was directed against NK-sensitive and NK-resistant target cells; (c) was not correlated with the basal levels of NK activity; and (d) was not associated with a development or augmentation of the proportion of lymphocytes with classic NK cell phenotype. Taken together, these results demonstrate that unstimulated T cells from B-CLL patients, incubated briefly (18 hours) with IL 2 sup but not gamma-IFN, have strong NK-like cytotoxicity, despite the lack of classic NK activity.
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Alvarez de Mon, M., J. Casas, R. Laguna, ML Toribio, MO de Landazuri, and A. Durantez. "Lymphokine induction of NK-like cytotoxicity in T cells from B-CLL." Blood 67, no. 1 (January 1, 1986): 228–32. http://dx.doi.org/10.1182/blood.v67.1.228.bloodjournal671228.
Full textAbstract:
T cells from patients with B cell chronic lymphocytic leukemia (B-CLL) exhibit defective natural killer (NK) activity. In this study, we have analyzed the cytotoxic-inducer effects of gamma interferon (gamma-IFN) and supernatants containing interleukin 2 (IL 2 sup). T cells from patients with B-CLL were incubated with gamma-IFN or IL 2 sup. gamma- IFN did not modulate the very low or undetectable levels of NK activity present in the T cell population. However, the IL 2 sup induced a potent cellular cytotoxicity against NK-sensitive and NK-resistant tumoral target cells. This cytotoxic inducer effect (a) was present in lectin-free IL 2 sup and in a 15,000- to 20,000-dalton molecular weight fraction obtained by gel filtration chromatography of this supernatant; (b) was directed against NK-sensitive and NK-resistant target cells; (c) was not correlated with the basal levels of NK activity; and (d) was not associated with a development or augmentation of the proportion of lymphocytes with classic NK cell phenotype. Taken together, these results demonstrate that unstimulated T cells from B-CLL patients, incubated briefly (18 hours) with IL 2 sup but not gamma-IFN, have strong NK-like cytotoxicity, despite the lack of classic NK activity.
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Brumbaugh, Kathryn M., Bryce A. Binstadt, Daniel D. Billadeau, Renee A. Schoon, Christopher J. Dick, Rosa M. Ten, and Paul J. Leibson. "Functional Role for Syk Tyrosine Kinase in Natural Killer Cell–mediated Natural Cytotoxicity." Journal of Experimental Medicine 186, no. 12 (December 15, 1997): 1965–74. http://dx.doi.org/10.1084/jem.186.12.1965.
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Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.
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Krusch, Matthias, Julia Salih, Ingrid Kumbier, Carolin Fenner, Lothar Kanz, and Helmut R. Salih. "The Role of the PI3K-AKT-mTOR Pathway in NK Cell Anti-Tumor Reactivity." Blood 114, no. 22 (November 20, 2009): 3690. http://dx.doi.org/10.1182/blood.v114.22.3690.3690.
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Abstract Abstract 3690 Poster Board III-626 The phosphatidylinositol 3-kinase – protein kinase B – mammalian target of rapamycin (PI3K – AKT – mTOR) pathway was found to be abnormally activated in many malignancies. Thus, protein kinase (PK) inhibitors (PKI) targeting different signaling molecules of this pathway are presently under clinical evaluation e.g. in sarcoma, multiple myeloma, or renal cell cancer. However, PK are also responsible for most of the signal transduction in immune effector cells and control various effector mechanisms including proliferation, cellular cytotoxicity, and cytokine release. Among those immunoregulatory signaling pathways, the PI3K – AKT – mTOR pathway was found to play a central role in TLR-mediated release of cytokines in macrophages and DC as well as in the regulation of T cell functions. Little is known about the role of this pathway in NK cell-mediated anti-tumor reactivity. Here we analyzed the tumor cell-induced activation of PI3K, AKT, and mTOR in NK cells and the consequences of an inhibition of these molecules by therapeutic PKI for NK cell anti-tumor reactivity. We found that, in response to tumor target cells, PI3K, AKT, and mTOR are consecutively activated in NK cells as revealed by western blot analyses using phospho-specific antibodies. Presence of the specific PI3K-inhbitor BKM-120 concentration-dependently inhibited cytotoxicity and IFN-g production of NK cells, which is in line with available data defining PI3K as a central regulator of NK cell target recognition. The mTOR inhibitors Sirolimus, Temsirolimus, and Everolimus did not alter cytotoxicity but significantly impaired NK cell IFN-γ production. In contrast, Triciribine, a compound which inhibits the phosphorylation and thus activation of AKT, did not influence cytotoxicity and, tantalizingly, even enhanced NK cell IFN-γ production. Thus, after target cell recognition and the activation of proximal PK like PI3K, different and at least partially independent signaling events govern NK cell cytokine production and cellular cytotoxicity. While the activity of PI3K followed by the activation of mitogen-activated PK is known to be crucial for NK cell cytotoxicity, we here identified the AKT – mTOR pathway as a yet unknown central component in the regulation of NK cell IFN-γ production. Moreover, in light of the important role of NK cells in tumor immune surveillance our data indicate that the choise and dosing of the most suitable PKI for a given cancer patient requires careful consideration. In the future it will be critical to define potential differences in immunosuppressive and immunostimulatory side effects of different compounds among the rapidly growing assortment of multi-targeted PKI to enable therapeutic approaches combining targeting of crucial signaling pathways in tumor cells with immunotherapy. Disclosures: No relevant conflicts of interest to declare.
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Wang, Jong-Shyan, and Chia-Kuan Wu. "Systemic hypoxia affects exercise-mediated antitumor cytotoxicity of natural killer cells." Journal of Applied Physiology 107, no. 6 (December 2009): 1817–24. http://dx.doi.org/10.1152/japplphysiol.00687.2009.
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Natural killer cells (NKs) are important to the clearance of transformed cells. This investigation elucidates how systemic hypoxia influences mobilization of the NK subsets and cytotoxicity of NKs to nasopharyngeal carcinoma cells (NPCs) during exercise. Sixteen sedentary men performed six distinct experimental tests in an air-conditioned normobaric hypoxia chamber: high-intensity exercise [HE; up to maximal O2 consumption (V̇o2 max)] under 21% O2; moderate-intensity exercise (ME; 50% V̇o2 max for 30 min) under 12%, 15%, and 21% O2; and breathing 12% and 15% O2 for 30 min at rest. The results demonstrated that 21% O2 HE, but not ME, increased cellular perforin/granzyme B/interferon-γ levels in NKs and interferon-γ concentration in NK-NPC coincubation, and also promoted capacity of NKs to bind to NPCs and NK-induced CD95 expression and phosphatidylserine exposure of NPCs. However, the HE simultaneously increased percentages of the replicative senescent (CD57+ and CD28−) NKs and the NKs with inhibitory receptors (KLRG1+) that entered the bloodstream from peripheral tissues. Breathing 12% and 15% O2 at rest did not influence mobilization of NK subsets and cytotoxicity of NKs to NPCs. Although both 12% and 15% O2 ME increased NK count, perforin/granzyme B/interferon-γ levels, NK-NPC binding, and NK-induced CD95 expression and apoptosis of NPC, only 12% O2 ME increased percentages of the NKs with CD57+/CD28−/KLRG1+ in blood. Therefore, we conclude that systemic hypoxic exposure affects redistribution of NK subsets and anti-NPC cytotoxicity of NKs during exercise in a concentration-dependent manner. Moreover, exposure to 12% O2 promotes the NK cytotoxicity with mobilizing the replicative senescent/inhibitory NKs into the bloodstream during ME.
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Igarashi, Takehito, Jason Wynberg, Ramprasad Srinivasan, Brian Becknell, J. Phillip McCoy, Yoshiyuki Takahashi, Dante A. Suffredini, W. Marston Linehan, Michael A. Caligiuri, and Richard W. Childs. "Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells." Blood 104, no. 1 (July 1, 2004): 170–77. http://dx.doi.org/10.1182/blood-2003-12-4438.
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Abstract Cellular inactivation through killer immunoglobulin-like receptors (KIRs) may allow neoplastic cells to evade host natural killer (NK) cell–mediated immunity. Recently, alloreactive NK cells were shown to mediate antileukemic effects against acute myelogenous leukemia (AML) after mismatched transplantation, when KIR ligand incompatibility existed in the direction of graft-versus-host disease (GVHD). Therefore, we investigated whether solid tumor cells would have similar enhanced susceptibility to allogeneic KIR-incompatible NK cells compared with their KIR-matched autologous or allogeneic counterparts. NK populations enriched and cloned from the blood of cancer patients or healthy donors homozygous for HLA-C alleles in group 1 (C-G1) or group 2 (C-G2) were tested in vitro for cytotoxicity against Epstein-Barr virus–transformed lymphoblastic cell lines (EBV-LCLs), renal cell carcinoma (RCC), and melanoma (MEL) cells with or without a matching KIR-inhibitory HLA-C ligand. Allogeneic NK cells were more cytotoxic to tumor targets mismatched for KIR ligands than their KIR ligand–matched counterparts. Bulk NK populations (CD3–/CD2+/CD56+) expanded 104-fold from patients homozygous for C-G1 or C-G2 had enhanced cytotoxicity against KIR ligand–mismatched tumor cells but only minimal cytotoxicity against KIR ligand–matched targets. Further, NK cell lines from C-G1 or C-G2 homozygous cancer patients or healthy donors expanded but failed to kill autologous or KIR-matched MEL and RCC cells yet had significant cytotoxicity (more than 50% lysis at 20:1 effector-target [E/T] ratio) against allogeneic KIR-mismatched tumor lines. These data suggest immunotherapeutic strategies that use KIR-incompatible allogeneic NK cells might have superior antineoplastic effects against solid tumors compared with approaches using autologous NK cells.
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Krusch, Matthias, Katrin M. Baltz, Tina Baessler, Mercedes Kloss, Ingrid Kumbier, Andrea Peterfi, Lothar Kanz, and Helmut R. Salih. "Expression of Glucocorticoid-Induced TNF Receptor Ligand on Acute Myeloid Leukemia Cells Mediates the Release of Immunosuppressive Cytokines and Impairs NK Cell-Mediated Immune Surveillance." Blood 108, no. 11 (November 16, 2006): 1941. http://dx.doi.org/10.1182/blood.v108.11.1941.1941.
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Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance of hematological malignancies including acute myeloid leukemia (AML). Among the molecules influencing host-tumor interaction are many members of the TNF superfamily, which mediate multiple cellular functions including cellular proliferation, differentiation and cell death. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while solid tumors express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell cytotoxicity and IFN-γ production. Here we analyzed the role of GITR and its ligand in AML. We report for the first time that GITRL is expressed on primary AML cells in 18 of 30 patients as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein induces the release of the immunoregulatory cytokines IL-10 and TNF as determined by ELISA. GITRL-mediated cytokine production of AML cells is abrogated by inhibition of mitogen activated protein kinase (MAPK) pathways as demonstrated by addition of the specific p38 MAPK inhibitor SB202190, the specific JNK inhibitor SP600125 and the specific ERK Inhibitor II. Furthermore, binding of AML-expressed GITRL to GITR on NK cells downregulates cellular cytotoxicity and IFN-γ production in AML-NK cell cocultures, which can be overcome by addition of GITR-blocking antibodies as determined by cytotoxicity assays and ELISA. Thus, our data indicate that GITRL expression in AML substantially influences tumor immunoediting and enables the escape of leukemia cells from NK cell-mediated immunosurveillance.
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Minute, Luna, Alvaro Teijeira, Alfonso R. Sanchez-Paulete, Maria C. Ochoa, Maite Alvarez, Itziar Otano, Iñaki Etxeberrria, et al. "Cellular cytotoxicity is a form of immunogenic cell death." Journal for ImmunoTherapy of Cancer 8, no. 1 (March 2020): e000325. http://dx.doi.org/10.1136/jitc-2019-000325.
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BackgroundThe immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown.MethodsIn this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient inBatf3,Ifnar1andSting1were used to study mechanistic requirements.ResultsWe observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient inBatf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+T lymphocytes.ConclusionThese results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.
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Sconocchia, Giuseppe, Julie A. Titus, Alessandra Mazzoni, Alberto Visintin, Federica Pericle, Stuart W. Hicks, Fabio Malavasi, and David M. Segal. "CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer Cells." Blood 94, no. 11 (December 1, 1999): 3864–71. http://dx.doi.org/10.1182/blood.v94.11.3864.
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Abstract Receptors used by natural killer (NK) cells to mediate natural cytotoxicity are poorly defined, although it is now clear that a number of adhesion molecules can serve this function. CD38 transduces signals on T- and B-cell lines, and we asked whether it could trigger lytic and secretory responses in human NK cells. By using an anti-CD38 monoclonal antibody in reverse antibody-dependent cellular cytotoxicity experiments, it is shown that CD38 engagement triggers cytotoxic responses by activated NK cells, but not by cytotoxic T lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab′)2 caused activated NK cells to release granzymes and cytokines, but did not trigger an increase in intracellular Ca2+. Fresh NK cells acquired CD38-dependent lytic function during activation with interleukin-2 (IL-2), and inhibitor studies suggested that IL-2 stimulated the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells. The induction of proteins that link commonly expressed adhesion molecules to effector mechanisms could provide a paradigm for pathogen recognition by the innate immune system.
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Sconocchia, Giuseppe, Julie A. Titus, Alessandra Mazzoni, Alberto Visintin, Federica Pericle, Stuart W. Hicks, Fabio Malavasi, and David M. Segal. "CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer Cells." Blood 94, no. 11 (December 1, 1999): 3864–71. http://dx.doi.org/10.1182/blood.v94.11.3864.423k14_3864_3871.
Full textAbstract:
Receptors used by natural killer (NK) cells to mediate natural cytotoxicity are poorly defined, although it is now clear that a number of adhesion molecules can serve this function. CD38 transduces signals on T- and B-cell lines, and we asked whether it could trigger lytic and secretory responses in human NK cells. By using an anti-CD38 monoclonal antibody in reverse antibody-dependent cellular cytotoxicity experiments, it is shown that CD38 engagement triggers cytotoxic responses by activated NK cells, but not by cytotoxic T lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab′)2 caused activated NK cells to release granzymes and cytokines, but did not trigger an increase in intracellular Ca2+. Fresh NK cells acquired CD38-dependent lytic function during activation with interleukin-2 (IL-2), and inhibitor studies suggested that IL-2 stimulated the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells. The induction of proteins that link commonly expressed adhesion molecules to effector mechanisms could provide a paradigm for pathogen recognition by the innate immune system.
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Pende, Daniela, Silvia Parolini, Anna Pessino, Simona Sivori, Raffaella Augugliaro, Luigia Morelli, Emanuela Marcenaro, et al. "Identification and Molecular Characterization of Nkp30, a Novel Triggering Receptor Involved in Natural Cytotoxicity Mediated by Human Natural Killer Cells." Journal of Experimental Medicine 190, no. 10 (November 15, 1999): 1505–16. http://dx.doi.org/10.1084/jem.190.10.1505.
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Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3ζ chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.
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Zhang, Meili, Bernard Wen, Olga M. Anton, Zhengsheng Yao, Sigrid Dubois, Wei Ju, Noriko Sato, et al. "IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages." Proceedings of the National Academy of Sciences 115, no. 46 (October 29, 2018): E10915—E10924. http://dx.doi.org/10.1073/pnas.1811615115.
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The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 μg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.
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Lutgendorf, Susan K., Anil K. Sood, Barrie Anderson, Stephanie McGinn, Heena Maiseri, Minh Dao, Joel I. Sorosky, Koen De Geest, Justine Ritchie, and David M. Lubaroff. "Social Support, Psychological Distress, and Natural Killer Cell Activity in Ovarian Cancer." Journal of Clinical Oncology 23, no. 28 (October 1, 2005): 7105–13. http://dx.doi.org/10.1200/jco.2005.10.015.
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Purpose Psychosocial stress has been related to impaired immunity in cancer patients. However, the extent to which these relationships exist in immune cells in the tumor microenvironment in humans has not been explored. We examined relationships among distress, social support, and natural killer (NK) cell activity in ovarian cancer patients in peripheral-blood mononuclear cells (PBMC), ascitic fluid, and tumor-infiltrating lymphocytes (TIL). Patients and Methods Patients awaiting surgery for a pelvic mass suspected of being ovarian cancer completed psychological questionnaires and gave a presurgical sample of peripheral blood. Samples of tumor and ascites were taken during surgery, lymphocytes were then isolated, and NK cytotoxicity and percentage were determined. The final sample, which was confirmed by surgical diagnosis, included 42 patients with epithelial ovarian cancer and 23 patients with benign masses. Results Peripheral NK cell activity was significantly lower among ovarian cancer patients than in patients with benign masses. Among ovarian cancer patients, NK cytotoxicity in TIL was significantly lower than in PBMC or ascitic fluid. Social support was related to higher NK cytotoxicity in PBMC and TIL, adjusting for stage. Distress was related to lower NK cytotoxicity in TIL. A multivariate model indicated independent associations of both distress and social support with NK cell activity in TIL. Conclusion Psychosocial factors, such as social support and distress, are associated with changes in the cellular immune response, not only in peripheral blood, but also at the tumor level. These relationships were more robust in TIL. These findings support the presence of stress influences in the tumor microenvironment.
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Park, Ki Hyun, Ji Hyeong Ryu, Hyunjoo Bae, Sojeong Yun, Joo Hee Jang, Kyungja Han, Byung Sik Cho, Hee-Je Kim, Hyeyoung Lee, and Eun-Jee Oh. "Delayed NK Cell Reconstitution and Reduced NK Activity Increased the Risks of CMV Disease in Allogeneic-Hematopoietic Stem Cell Transplantation." International Journal of Molecular Sciences 21, no. 10 (May 22, 2020): 3663. http://dx.doi.org/10.3390/ijms21103663.
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Cytomegalovirus (CMV) infection has a significant impact in patients after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated natural killer (NK) cell reconstitution and cytotoxic/cytokine production in controlling CMV infection, especially severe CMV disease in HSCT patients. Fifty-eight patients with acute myeloid leukemia (AML) who received allo-HSCT were included. We monitored NK reconstitution and NK function at baseline, 30, 60, 90, 120, 150, and 180 days after HSCT, and compared the results in recipients stratified on post-HSCT CMV reactivation (n = 23), non-reactivation (n = 24) versus CMV disease (n = 11) groups. The CMV disease group had a significantly delayed recovery of CD56dim NK cells and expansion of FcRγ-CD3ζ+NK cells started post-HSCT 150 days. Sequential results of NK cytotoxicity, NK cell-mediated antibody-dependent cellular cytotoxicity (NK-ADCC), and NK-Interferon-gamma (NK-IFNγ) production for 180 days demonstrated delayed recovery and decreased levels in the CMV disease group compared with the other groups. The results within 1 month after CMV viremia also showed a significant decrease in NK function in the CMV disease group compared to the CMV reactivation group. It suggests that NK cells’ maturation and cytotoxic/IFNγ production contributes to CMV protection, thereby revealing the NK phenotype and functional NK monitoring as a biomarker for CMV risk prediction, especially CMV disease.
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Kaur, Kawaljit, Tahmineh Safaie, Meng-Wei Ko, Yuhao Wang, and Anahid Jewett. "ADCC against MICA/B is Mediated against Differentiated Oral and Pancreatic and Not Stem-Like/Poorly Differentiated Tumors by the NK Cells; Loss in Cancer Patients due to Down-Modulation of CD16 Receptor." Cancers 13, no. 2 (January 11, 2021): 239. http://dx.doi.org/10.3390/cancers13020239.
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Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.
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Kaur, Kawaljit, Tahmineh Safaie, Meng-Wei Ko, Yuhao Wang, and Anahid Jewett. "ADCC against MICA/B Is Mediated against Differentiated Oral and Pancreatic and Not Stem-Like/Poorly Differentiated Tumors by the NK Cells; Loss in Cancer Patients due to Down-Modulation of CD16 Receptor." Cancers 13, no. 2 (January 11, 2021): 239. http://dx.doi.org/10.3390/cancers13020239.
Full textAbstract:
Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.
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Münz, Christian. "Natural Killer Cell Responses during Human γ-Herpesvirus Infections." Vaccines 9, no. 6 (June 15, 2021): 655. http://dx.doi.org/10.3390/vaccines9060655.
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Herpesviruses are main sculptors of natural killer (NK) cell repertoires. While the β-herpesvirus human cytomegalovirus (CMV) drives the accumulation of adaptive NKG2C-positive NK cells, the human γ-herpesvirus Epstein–Barr virus (EBV) expands early differentiated NKG2A-positive NK cells. While adaptive NK cells support adaptive immunity by antibody-dependent cellular cytotoxicity, NKG2A-positive NK cells seem to preferentially target lytic EBV replicating B cells. The importance of this restriction of EBV replication during γ-herpesvirus pathogenesis will be discussed. Furthermore, the modification of EBV-driven NK cell expansion by coinfections, including by the other human γ-herpesvirus Kaposi sarcoma-associated herpesvirus (KSHV), will be summarized.
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Levy, Estrella Mariel, María Paula Roberti, and José Mordoh. "Natural Killer Cells in Human Cancer: From Biological Functions to Clinical Applications." Journal of Biomedicine and Biotechnology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/676198.
Full textAbstract:
Natural killer (NK) cells are central components of the innate immunity. In murine models, it has been shown that NK cells can control both local tumor growth and metastasis due to their ability to exert direct cellular cytotoxicity without prior sensitization and to secrete immunostimulatory cytokines like IFN-γ. The latter participates in cancer elimination by inhibiting cellular proliferation and angiogenesis, promoting apoptosis, and stimulating the adaptive immune system, and it is instrumental for enhancing Ag processing and presentation. Nevertheless, NK cells display impaired functionality and capability to infiltrate tumors in cancer patients. Also, NK cells are feasible targets of stimulation to participate in immunotherapeutic approaches like antibody-based strategies and adoptive cell transfer. Thus, multiple attempts currently aim to manipulate NK for utilization in the immunotherapy of cancer.
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Baessler, Tina, Corina Buechele, Matthias Krusch, Benjamin J. Schmiedel, Lothar Kanz, and Helmut R. Salih. "The Role of CD137 and Its Ligand in Chronic Lymphocytic Leukemia (CLL)." Blood 112, no. 11 (November 16, 2008): 3130. http://dx.doi.org/10.1182/blood.v112.11.3130.3130.
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Abstract Tumor immunosurveillance is dependent on the reciprocal interaction between tumor cells and anti-tumor immunity mediated e.g. by NK cells. This has led to the concept of tumor immunoediting, which incorporates the multitude of mechanisms underlying this dual tumor- and immune-sculpting interaction. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells. Very recently the TNFR family member CD137 has been shown to be induced on NK cells by Fc receptor triggering indicating that not only the Fab region but also the Fc part of a given antibody may be responsible for effects attributed to CD137 modulation (Lin et al., Blood2008, 112: 699). Thus we here studied the role of human CD137 and its cognate counterpart, the CD137 ligand (CD137L) in the interaction of CLL with NK cells. High levels of CD137L expression were detected on B-CLL cells in all investigated patients (n=40). Incubation of CLL cells in the presence of an immobilized CD137-Ig fusionprotein significantly induced the release of the immunoregulatory cytokine TNF demonstrating that CLL-expressed CD137L was capable to transduce bidirectional signals. Furthermore, we found that NK cells of CLL patients displayed substantial CD137 expression. While being absent on resting NK cells, CD137 expression was upregulated on the CD56dimCD16+ but not the CD56brightCD16− NK cell subset of healthy donors upon activation e.g. with IL-2 or IL-15. In addition, CD137 was also induced on NK cells after incubation in supernatants of PBMC of CLL patients. Surprisingly, disruption of CD137-CD137L interaction in cocultures of allogenic NK cells with patient CLL cells by blocking CD137 antibody caused a significant increase in NK cell cytotoxicity. The observed inhibitory effect of CD137L on NK cell reactivity was confirmed in cytotoxicity assays using CD137L-transfectants with mock-transfectants as control. Furthermore, blocking CD137-CD137L interaction also substantially enhanced Rituximab-induced antibody dependent cellular cytotoxicity in an allogenic setting. Importantly, CD137 blockade also substantially enhanced CD107a expression as a surrogate marker for granule mobilization on autologous NK cells within PBMC of B-CLL patients, and this effect was observed both in the absence and more pronounced in the presence of Rituximab. Thus, expression of functional CD137L by CLL cells impairs anti-tumor immunity by diminishing both direct and antibody-dependent cellular cytotoxicity of allogenic and autologous NK cells. Modulation of the CD137-CD137L system might therefore be a suitable therapeutic approach in strategies like antibody therapy which rely on a sufficient NK cell anti-tumor response.
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McWilliams, Emily, Jennifer M. Mele, Faraz Fiazuddin, Carolyn Cheney, Natarajan Muthusamy, and Farrukh T. Awan. "Targeting the Tumor Evasion Interaction of NKG2A and Its Ligand HLA-E Increases Natural-Killer Cell Activity in Chronic Lymphocytic Leukemia." Blood 126, no. 23 (December 3, 2015): 1289. http://dx.doi.org/10.1182/blood.v126.23.1289.1289.
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Abstract Chronic Lymphocytic Leukemia (CLL) represents the most frequent adult leukemia, and remains incurable with current standard therapies. Natural Killer (NK) cell count is predictive of CLL disease progression and their dysfunction in mediating cytokine release and direct or antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells is well documented. Detailed mechanistic insight into the etiology of NK-cell dysfunction in CLL patients is currently lacking. CLL B-cells overexpress HLA-E, the natural ligand for heterodimer CD94/NKG2A receptor complex that is expressed on the surface of NK cells, and this interaction suppresses NK cell activation. While NKG2A/CD94/HLA-E interaction is known to assist NK cells in recognizing "self", tumor cells utilize this mechanism to evade effector cell killing. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mab) we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in patients with CLL. We hypothesized that limiting the interaction of HLA-E/NKG2A will reverse NK cell anergy and result in increased direct cytotoxicity of CLL cells. Our results confirm the over expression of HLA-E on CLL B-cells and demonstrate NKG2A expression on CD16+ NK cells from CLL patients. Next, we examined the effect of anti-NKG2A mab on NK cell direct cytotoxicity. Treatment of NK cells, from both healthy donor and CLL patients, with anti-NKG2A mab increased direct cytotoxicity over isotype control on targets at various effector to target ratios of 25:1 (54% vs. 46%, p< 0.05, n= 12), 12:1 (43% vs. 35%, p<0.05, n=14), and 6:1 (31% vs. 23%, p<0.05, n= 12, for anti-NKG2A mediated cytotoxicity vs isotype mediated cytotoxicity respectively). These results were also validated with HLA-E over and underexpressing target cells. Fc-gamma receptor blocking experiments were also performed to confirm the specificity of the interaction. Further studies are being performed to confirm the specific activity of the antibody including its ability to modulate NK cell activation, enhance ADCC, and the impact of anti-NKG2A therapy for reversing ibrutinib mediated NK-cell dysfunction. This work has laid the foundation for the clinical utility of this reagent in patients with relapsed CLL in combination with ibrutinib. Disclosures No relevant conflicts of interest to declare.
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Baychelier, Florence, Alexis Sennepin, Myriam Ermonval, Karim Dorgham, Patrice Debré, and Vincent Vieillard. "Identification of a cellular ligand for the natural cytotoxicity receptor NKp44." Blood 122, no. 17 (October 24, 2013): 2935–42. http://dx.doi.org/10.1182/blood-2013-03-489054.
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Key Points The cellular ligand of the NKp44L is a novel isoform of the mixed-lineage leukemia-5 protein. NKp44L is not expressed on healthy cells, but on tumor and transformed cells, rendering them more sensitive for the NK cytotoxicity.
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Bonnema, J. D., L. M. Karnitz, R. A. Schoon, R. T. Abraham, and P. J. Leibson. "Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity." Journal of Experimental Medicine 180, no. 4 (October 1, 1994): 1427–35. http://dx.doi.org/10.1084/jem.180.4.1427.
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Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.
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Levin, Michele, Janet Ayello, Frances Zhao, Andrew Stier, Lauren Tiffen, William Quish, Carmella vandeVen, Laxmi V. Baxi, Jessica Hochberg, and Mitchell S. Cairo. "Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)." Blood 116, no. 21 (November 19, 2010): 3928. http://dx.doi.org/10.1182/blood.v116.21.3928.3928.
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Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.
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Gantke, Thorsten, Uwe Reusch, Christian Kellner, Kristina Ellwanger, Ivica Fucek, Michael Weichel, Anne Kerber, Matthias Peipp, and Martin Treder. "AFM26 is a novel, highly potent BCMA/CD16A-directed bispecific antibody for high affinity NK-cell engagement in multiple myeloma." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 8045. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.8045.
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8045 Background: Despite recent advances in the treatment of multiple myeloma (MM), novel therapies are needed to achieve long-lasting remissions in a greater number of patients. Natural killer (NK) cells play a key role in the immune response to MM and have been implicated in the clinical efficacy of current standard of care interventions, including IMiDs, proteasome inhibitors, recently approved immunotherapies and autologous stem cell transplantation (ASCT). Numerous strategies are being developed to enhance the natural NK-cell cytotoxicity against myeloma cells, which is frequently dysregulated in MM. Approaches include modulation of activity, through cytokine stimulation or immune checkpoint targeting, and adoptive transfer of culture expanded NK-cells in ASCT-eligible MM. While highly attractive, these approaches are non-targeted, as they rely on the natural cytotoxicity of NK-cells, and may benefit from antigen-specific retargeting and effector activation. AFM26 is a novel tetravalent, bispecific antibody designed to specifically enhance NK-cell anti-MM activity by redirecting NK-cell lysis to BCMA, an antigen expressed on MM cells. Methods: NK-cell engagement and cytotoxicity of AFM26 towards MM cell lines and freshly isolated tumor cells from MM patients was characterized in vitro and compared with classical antibody formats. Results: AFM26 engages NK-cells with superior avidity ( KD: 1-2nM) through bivalent interaction with CD16A (FcγRIIIa) and demonstrates extended cell surface retention that is not affected by high level IgG, as is particularly relevant in MM. Importantly, AFM26 does not induce NK-cell depletion but selectively induces potent and efficacious lysis of MM cells in vitro. Conclusions: In summary, AFM26 is a promising candidate to enhance NK-cell activity and confer tumor-specificity to NK-cells in MM. Differentiation of AFM26 from classical antibody formats and its potential for combination with cellular NK-cell therapies is highlighted.