Optimizing Lentiviral Transduction of Human Natural Killer Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4714-4714 ◽  
Author(s):  
Su Su ◽  
Dawn M Betters ◽  
Muthalagu Ramanathan ◽  
Keyvan Keyvanfar ◽  
Aleah Smith ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Phenotype ◽  
Human Renal Cell Carcinoma ◽  
Hiv 1

Abstract Abstract 4714 The development of an efficient method to genetically modify natural killer (NK) cells could be used to characterize NK cell differentiation, acquisition of self-tolerance, tumor trafficking in vivo, as well as to manipulate NK cells to enhance their activity against infectious diseases and tumors. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on LV transduction of either fresh or in vitro expanded human NK cells or its effects on NK cell phenotype and cytolytic function. In this study, we used an HIV-based LV expressing enhanced green fluorescence protein (EGFP) driven by a murine stem cell virus long terminal repeat (MSCV-LTR) promoter to transduce CD3− and CD56+ and/or CD16+ human NK cells that were either resting, IL-2 activated, or expanded in vitro using an irradiated EBV-LCL feeder cell line. We observed that resting NK cells were difficult to transduce with LVs, even at high multiplicities of infection (MOI), with transduction efficiencies (TE) in the range of only 3–14%. The efficiency of LV transduction improved when the NK cells were pre-stimulated in vitro with IL-2: TE improved to 21±0.2% in NK cells cultured for 24 hours in media containing IL-2 (200 U/mL) and 28.7±12.9% in NK cells that underwent in vitro expansion over 9 days prior to transduction using irradiated EBV-LCL feeder cells and media containing IL-2 (200U/mL). Subsequently, we evaluated incremental MOIs (3-200) to optimize LV transduction of expanded NK cells; optimal transduction was achieved using a spinoculation protocol at a MOI of 25 which resulted in the highest transduction efficiencies with the least amount of cell death. Increasing the MOI above this level resulted in a small increase in transduction, but was offset by an increase in NK cell apoptosis/death. Using a one-round, non-spinoculation protocol and an MOI of 30, we obtained a median transduction efficiency of 29% (range 16–41) with excellent retention of NK cell viability. This optimized protocol was used to transduce expanded NK cells with a LV vector encoding an shRNA targeting a region of the NK cell inhibitory receptor transcript NKG2A. Following transduction, surface expression of NKG2A decreased significantly on expanded NK cells compared to non-transduced expanded NK cells and “scramble transduced” LV controls; at a MOI of 10, the MFI of NKG2A on expanded human NK cells decreased 35% compared to non-transduced and LV transduced scramble controls (median MFI 428, 673, 659 in shRNA, non-transduced and scramble LV control transduced NK cells respectively). A comparison of transduction efficiencies using LVs expressing EGFP driven by MSCV-LTR, EF1a, and Ubi promoters showed MSCV-LTR mediated the highest level of gene expression in expanded NK cells. Transduced NK cells maintained stable EGFP transgene expression in vitro, which peaked 5 days following LV transduction and remained stable for an additional 9 days. The phenotype of lentiviral transduced NK cells was similar to non-transduced NK cells. Specifically, expression of CD56, CD16, granzyme A and B, perforin, the inhibitory receptors NKG2A, KIR3DL1, KIR3DL2, and KIR2DL1/DL2, and the activating receptors NKG2D, NCRs NKp46, and NKp30 were not altered in either fresh or expanded NK cells following LV transduction, although we did observe a significant reduction in NKp44 expression in LV transduced cells (22% compared to 50% on untransduced NK cells; 0.02). Furthermore, NK cell function, as assessed by cytokine production and cytotoxicity vs tumor targets was not altered in LV transduced NK cells. A 51Cr release cytotoxicity assay showed GFP+ NK cells, flow sorted following LV transduction of expanded NK cells, had similar cytotoxicity against K562 cells and human renal cell carcinoma cells (RCC) compared to non-transduced expanded NK cell controls (figures). In conclusion, we show that an HIV-1 based lentiviral vector driven by a MSCV-LTR, mediated efficient and stable gene transfer in IL-2 activated and in vitro expanded human NK cells. This study provides valuable insights for methods to optimize the long-term expression of LV transduced genes in human NK cells which could be used to improve their anti-tumor function in vivo. Target: K562 cells Target: RCC cell line Disclosures: No relevant conflicts of interest to declare.

scholarly journals In VivoActivation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits AcuteIn VivoHIV-1 Infection in Humanized Mice

2015 ◽  
Vol 89 (12) ◽  
pp. 6264-6274 ◽  
Author(s):  
Kieran Seay ◽  
Candice Church ◽  
Jian Hua Zheng ◽  
Kathryn Deneroff ◽  
Christina Ochsenbauer ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Humanized Mice ◽  
Nsg Mice ◽  
Acute Hiv ◽  
Interleukin 15 ◽  
Hiv 1

ABSTRACTNatural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acutein vivoHIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells.In vitrostimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acutein vivoHIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ−/−(NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productivein vivoinfection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression ofin vivoHIV-1 infection. Thus, thein vivoactivation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acutein vivoHIV-1 infection. These results indicate thatin vivoactivation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection.IMPORTANCEEpidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, andin vitrostudies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated thatin vivoactivation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression ofin vivoHIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus,in vivoimmunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.

scholarly journals A cloned cell line mediating natural killer cell function inhibits immunoglobulin secretion.

1982 ◽  
Vol 156 (2) ◽  
pp. 658-663 ◽  
Author(s):  
G Nabel ◽  
W J Allard ◽  
H Cantor
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
B Lymphocytes ◽  
Cell Function ◽  
Killer Cell ◽  
Major Histocompatibility Complex Gene ◽  
Cell Surface Antigens

We previously described a cloned cell line that combines information for a unique display of cell surface antigens and specialized function similar to activated natural killer (NK) cells. In addition to conventional cellular targets such as the YAC-1 and MBL-2 lymphomas, this cloned line also lysed lipopolysaccharide-activated B lymphocytes. To determine whether some NK cells can inhibit B cell function, we tested the ability of NK-like clones to suppress Ig secretion in vitro and in vivo. These cloned cells suppressed Ig secretion when they constituted as few as 0.2% of the total cell population and inhibition did not require identity at the H-2 locus. We suggest that some NK cells might recognize non-major histocompatibility complex gene products on activated B lymphocytes and lyse these cells, and this might represent a fundamental cell-cell interaction that regulates antibody secretion by activated B cells.

scholarly journals Mechanosensation and Mechanotransduction in Natural Killer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Federica Maggi ◽  
Maria Beatrice Morelli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Tyrosine Phosphatase ◽  
Cell Interaction ◽  
Structural Basis ◽  
Adhesive Interactions

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

scholarly journals RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii194-ii195
Author(s):  
Nazanin Majd ◽  
Maha Rizk ◽  
Solveig Ericson ◽  
Kris Grzegorzewski ◽  
Sharmila Koppisetti ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
In Vitro Cytotoxicity ◽  
Orthotopic Mouse Model ◽  
Hematopoietic Stem ◽  
Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 1184-1191 ◽  
Author(s):  
Nobuyoshi Hanaoka ◽  
Tatsuya Kawaguchi ◽  
Kentaro Horikawa ◽  
Shoichi Nagakura ◽  
Hiroaki Mitsuya ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nk Cells ◽  
Natural Killer ◽  
Blood Cells ◽  
Cell Injury ◽  
Nk Cell ◽  
K562 Cells ◽  
Cytotoxic Cells ◽  
Immune Mediated

AbstractThe mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.

Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1612-1621 ◽  
Author(s):  
Lei Yao ◽  
Cecilia Sgadari ◽  
Keizo Furuke ◽  
Eda T. Bloom ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Primary Cultures ◽  
Interleukin 12 ◽  
Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

Inflammation restraining effects of prostaglandin E2 on natural killer–dendritic cell (NK-DC) interaction are imprinted during DC maturation

Blood ◽  
2011 ◽  
Vol 118 (9) ◽  
pp. 2473-2482 ◽  
Author(s):  
Catharina H. M. J. Van Elssen ◽  
Joris Vanderlocht ◽  
Tammy Oth ◽  
Birgit L. M. G. Senden-Gijsbers ◽  
Wilfred T. V. Germeraad ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
T Helper 1 ◽  
Ifn Γ ◽  
Dc Maturation

Abstract Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer–dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood–derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44+ NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

Natural-killer cells and dendritic cells: “l'union fait la force”

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2252-2258 ◽  
Author(s):  
Thierry Walzer ◽  
Marc Dalod ◽  
Scott H. Robbins ◽  
Laurence Zitvogel ◽  
Eric Vivier
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Activation ◽  
Nk Cell ◽  
Interleukin 12 ◽  
Cell Contact ◽  
Ifn Γ

AbstractSeveral recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-α/β production by activated DCs enhance, in turn, NK-cell IFN-γ production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.

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 ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3928-3928
Author(s):  
Michele Levin ◽  
Janet Ayello ◽  
Frances Zhao ◽  
Andrew Stier ◽  
Lauren Tiffen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Activation Marker

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.

scholarly journals Functional Dichotomy in Natural Killer Cell Signaling

2001 ◽  
Vol 193 (12) ◽  
pp. 1413-1424 ◽  
Author(s):  
Francesco Colucci ◽  
Eleftheria Rosmaraki ◽  
Søren Bregenholt ◽  
Sandrine I. Samson ◽  
Vincenzo Di Bartolo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Surface Receptors ◽  
Nk T Cells ◽  
Nk Cell Differentiation ◽  
Ifn Γ

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen–receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1−/− mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1−/− mice produced normal amounts of interferon (IFN)-γ in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1−/− NK cells resulted in normal IFN-γ production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1−/− NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-γ production.

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