KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2449-2449
Author(s):  
Frits van Rhee ◽  
Susann M. Szmania ◽  
JuMei Shi ◽  
Michele Cottler-Fox ◽  
Justin W. Dyniewski ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Nk Cell ◽  
Allogeneic Transplantation ◽  
Myeloma Cell ◽  
Myeloma Cells

Abstract Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma. Figure Figure

scholarly journals TIM-3 Expression Is Downregulated on Human NK Cells in Response to Cancer Targets in Synergy with Activation

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2417 ◽  
Author(s):  
Tram N. Dao ◽  
Sagar Utturkar ◽  
Nadia Atallah Lanman ◽  
Sandro Matosevic
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Interferon Gamma ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Dysfunction ◽  
Nk Cell Receptors ◽  
Ifn Γ

Among natural killer (NK) cell receptors, the T-cell immunoglobulin and mucin-containing domain (TIM-3) has been associated with both inhibitory and activating functions, depending on context and activation pathway. Ex vivo and in vitro, expression of TIM-3 is inducible and depends on activation stimulus. Here, we report that TIM-3 expression can be downregulated on NK cells under specific conditions. When NK cells are exposed to cancer targets, they synergize with stimulation conditions to induce a substantial decrease in TIM-3 expression on their surface. We found that such downregulation occurs following prior NK activation. Downregulated TIM-3 expression correlated to lower cytotoxicity and lower interferon gamma (IFN-γ) expression, fueling the notion that TIM-3 might function as a benchmark for human NK cell dysfunction.

Adoptive Infusion of Allogeneic KIR Ligand Mismatched NK Cells Reduce GVHD in an MHC Matched Allogeneic Stem Cell Transplantation Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1310-1310
Author(s):  
Andreas Lundqvist ◽  
Leigh Samsel ◽  
Michael Eckhaus ◽  
Ramaprasad Srinivasan ◽  
Yoshiyuki Takahashi ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Murine Model ◽  
Nk Cell ◽  
Allogeneic Transplantation ◽  
Allogeneic Bone ◽  
Median Score ◽  
The Impact

Abstract Retrospective data suggest NK cells play a role in protecting recipients from graft versus host disease (GVHD) in the setting of killer IgG-like receptor (KIR) ligand incompatibility. In humans, this protective effect is most evident with MHC mismatched transplantation, usually following in vivo or in vitro T-cell depletion. In MHC mismatched murine transplant models, lethal GVHD is reduced following the adoptive infusion of KIR ligand mismatched NK cells; it is unknown whether NK cells can mediate similar protective effects following MHC matched transplantation. Therefore, we investigated the impact of adoptively infusing KIR ligand mismatched NK cells on GVHD in an MHC matched T-cell replete murine model of allogeneic transplantation. Balb/C recipient mice underwent allogeneic bone marrow (8 x 106 cells) and splenocyte (15 x 106 cells) transplantation from B10.d2 donors following 950cGy of irradiation. Allogeneic B10.d2 donor NK cells were first isolated by negative depletion using magnetic beads selecting for CD4, CD5, CD8a, CD19, Gr-1 and Ter-119, and then expanded over 4-6 days in vitro in DMEM media containing 10% FCS and 500U/ml of IL-2. NK cell subsets (KIR ligand matched vs. KIR ligand mismatched) were then isolated by flow cytometry into Ly49I/C+ NK cells (KIR ligand mismatched in the GVHD direction for Balb/C recipients) and Ly49A/G+ NK cells (KIR ligand matched for Balb/C recipients). On day +4, recipient mice received a single tail vein injection with either KIR ligand matched, KIR ligand mismatched or unsorted “bulk” NK cells (0.5–1.0 x 106 NK cells). All (9/9) control transplant recipients (no adoptive NK cell infusion) as well as recipients of Ly49A/G (KIR ligand matched) NK cells (13/13) developed skin GVHD, in contrast to 4/7 (57%, p=0.03) recipients of bulk NK cells and only a minority (13% [1/8], p < 0.01) of animals receiving KIR ligand mismatched NK cells. Using a cumulative clinical GVHD scoring system (total score = 9), overall GVHD was decreased in recipients of KIR ligand mismatched NK cells (median score = 0 at day +45) compared to mice that received KIR ligand matched NK cells (median score = 3; p = 0.15) or no NK cells (median score = 3; p= 0.12); no significant difference in survival was observed between cohorts. This murine model provides the first in vivo evidence that adoptively infused KIR ligand mismatched allogeneic NK cells reduce GVHD following T-cell replete MHC matched allogeneic transplantation. The impact of infusing multiple doses of KIR ligand mismatched NK cells on GVHD and their ability to induce a graft-vs-tumor effect in tumor bearing Balb/c mice is currently being evaluated.

scholarly journals Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation

2021 ◽  
Vol 12 ◽  
Author(s):  
Jessica M. Sierra ◽  
Florencia Secchiari ◽  
Sol Y. Nuñez ◽  
Ximena L. Raffo Iraolagoitia ◽  
Andrea Ziblat ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Nk Cells ◽  
Cancer Patients ◽  
Nk Cell ◽  
The Cancer Genome Atlas ◽  
T Cell Proliferation ◽  
Dependent Manner ◽  
Effector Functions

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Adoptively-Infused NK Cells Maintain Their Antitumor Effects in Vivo in the Presence of CyclosporineA (CSA)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2563-2563
Author(s):  
Hisayuki Yokoyama ◽  
Andreas Lundqvist ◽  
Maria Berg ◽  
Muthalagu Ramanathan ◽  
Rebecca Lopez ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Median Survival ◽  
Nk Cell ◽  
Stimulation Index ◽  
Surface Expression ◽  
Inhibition Of Proliferation

Abstract Animal models show infusions of donor NK cells given after allogeneic HCT can prevent GVHD while simultaneously mediating a graft-vs-tumor effect. However, it is unclear whether adoptively infused NK cells can mediate these beneficial effects in the presence of CSA, which is commonly given after HCT to prevent GVHD. In this study, we analyzed the in vitro effects of pharmacological concentrations of CSA on NK cell phenotype, cell proliferation, and tumor cytotoxicity. We also evaluated in vivo whether CSA administration would reduce the anti-tumor effects of adoptively infused NK cells in tumor bearing mice. PBMCs collected from healthy donors were labeled with CFSE then were stimulated in vitro with IL-2 for 7 days in the presence or absence of CSA (1000ng/ml). CFSE proliferation assays on fresh PBMC showed CSA inhibited IL-2 stimulated CD3+ T-cell proliferation more than CD3−/CD56+ NK cell proliferation (mean percentage inhibition of proliferation 49.4% vs. 22.2% for T cells and NK cells respectively; p<0.05). CD3−/CD56+ NK cells were then isolated from PBMCs of healthy donors and expanded in vitro with irradiated EBV-LCL and IL-2 for 10 days. In contrast to T-cells, CSA only minimally inhibited IL-2 induced proliferation of expanded NK cells (mean 9.5% inhibition of proliferation by CFSE staining). T cells and NK cells were next isolated from PBMCs and stimulated with either OKT3, PMA-Ionomycin (PI), or IL-2. A [3H] TdR uptake assay showed T cell proliferation was inhibited at a substantially higher level by CSA (mean stimulation index for OKT3: 0.03, for PI: 0.35, for IL-2: 0.55) compared to that of expanded NK cells (mean stimulation index for IL-2: 0.82, p<0.05). Furthermore, an ELISA assay showed CSA treatment reduced IL-2 induced secretion of INF-g by T cells more than expanded NK cells (mean reduction in INF-g secretion in T cells of 94.7 % vs. 36.5 % in NK cells, p<0.05). Compared to controls, culturing in vitro expanded NK cells in CSA did not alter surface expression of the activating receptors NKp30, NKp42, and NKG2D but did reduce surface expression of NKp44 and TRAIL (mean reduction in surface expression 36% and 36.3% respectively). Cytotoxic granule release assessed by CD107a staining was inhibited by CSA in CD8+ melanoma specific T cells co-cultured with melanoma cells (mean 12.2 % inhibition) in contrast to NK cells co-cultured with K562 cells where CSA increased CD107a expression a mean 29.7% (p<0.05). Furthermore, at a 20:1 E:T ratio, 51Cr cytotoxity assays showed CSA did not reduce the cytotoxicity of in vitro expanded NK cells against renal cell carcinoma (RCC) cells (58% mean lysis) compared to NK cells cultured in control media (55% mean; p=n.s.). In contrast, melanoma specific T-cell killing of tumor targets was significantly lower in CTL cultures containing CSA compared to control media (38.0% vs. 50.2% respectively P<0.05). Next, we assessed the impact of CSA administration on the anti-tumor effects of adoptive NK cell infusions in tumor bearing animals where syngeneic NK cell infusions following bortezomib treatment have been shown to delay tumor progression and prolong survival. BALB/c mice injected with 100,000 luciferase transfected RENCA tumor cells i.v. received 3 weekly treatments with the combination of bortezomib (5ug/mouse i.v.) and 2×106 syngeneic NK cells i.v. with or without daily administration of CSA (15mg/kg sc). Bioluminescence imaging in controls that did not receive CSA showed tumor growth was slower and survival was prolonged in mice receiving adoptive NK cell infusions (median survival 53 days) compared to mice that did not receive NK cells (median survival 30.2 days; p<0.05). CSA administration did not impair the anti-tumor effects of adoptive NK cell infusions; mice receiving CSA and adoptive NK cell infusions had similar tumor growth and survival as recipients of NK cells without CSA (median survival 47 vs. 53 days; p=n.s.). These results show that CSA is significantly more immunosuppressive to T cells compared to NK cells and provide evidence that the anti-tumor effects of adoptively infused in vitro expanded NK cells are maintained even in the presence CSA.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Natural killer cell behavior in lymph nodes revealed by static and real-time imaging

2006 ◽  
Vol 203 (3) ◽  
pp. 619-631 ◽  
Author(s):  
Marc Bajénoff ◽  
Béatrice Breart ◽  
Alex Y.C. Huang ◽  
Hai Qi ◽  
Julie Cazareth ◽  
...  
Keyword(s):  
T Cell ◽  
Lymph Nodes ◽  
Nk Cells ◽  
Natural Killer ◽  
T Cell Activation ◽  
Cell Activation ◽  
Leishmania Major ◽  
Nk Cell ◽  
Killer Cell

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-γ and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.

Valproic Acid Upregulates NKG2D Ligand Expression and Potentially Enhances Natural Killer Cell–mediated Cytolysis of Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5061-5061
Author(s):  
Jumei Shi ◽  
Yi Tao ◽  
Xiaosong Wu ◽  
Xiaojing Hu ◽  
Yang Shao ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Nkg2d Ligand ◽  
Myeloma Cells ◽  
Nkg2d Ligands ◽  
Ligand Expression

Abstract Abstract 5061 Objective: Multiple myeloma (MM) is a plasma cell malignancy. Although high-dose chemotherapy supported by autologous hematopoietic stem-cell transplantation can produce higher response rates and longer survival than standard chemotherapy, MM remains largely incurable by current therapeutic strategies. Clinical and preclinical data have been demonstrated that natural killer (NK) cells have an anti-myeloma effect. However, low NK cell activity affected the successful utilization of NK cell adoptive immunotherapy for myeloma. In this study, we investigated the effect of valproic acid (VPA), as a histone deacetylase inhibitor, on the expression of NKG2D ligands on human myeloma cell lines and primary myeloma cells derived from patients. We also evaluated the sensitivity of myeloma cells to NK cell-mediated lysis before and after treatment with VPA, and the possible underlying mechanisms. Method: ARK, OPM2 myeloma cell lines and primary myeloma cells (n = 4) from patients with myeloma were cultured with 0.5–3mM VPA for 24 to 72 hours. Cells cultured without VPA were as control groups. To evaluate the effect of VPA on NKG2D ligand expression of myeloma cells, mRNA and protein expression of NKG2D ligands (including MICA/B, ULBP1, ULBP2 and ULBP3) of myeloma cells were tested by Real time-PCR and flow cytometry. 51Cr release assay and NKG2D antibody blocking experiments were combined to examine the susceptibility of myeloma cells to NK cell-mediated lysis in response to VPA and the possible killing mechanisms. Results: ARK and OPM-2 myeloma cell lines were treated with various concentrations of VPA. The most prominent upregulations of mRNA expressions of MICA/B, ULBP2 and ULBP 3 were observed in the two tested myeloma cell lines. The enhancement of cell surface expression of MICA/B, ULBP2 and ULBP 3 was detected by flow cytometry after cells treatment with VPA. We also found that VPA upregulated MICA/B expression on OPM2 MM cell line in a time- and dose-dependent manner. These data suggest that VPA could upregulate expression of NKG2D ligands in myeloma cell line at both the mRNA and protein levels. A similar increase of surface expression of NKG2D ligands was obtained in 2–3 mM VPA treated primary MM cells (n=4). NK cell function was studied by 51Cr release assay. VPA treatment of OPM2 showed higher sensitivity to NK cell lysis than untreated cells (specific killing: 72.8±6.2% vs 32.1±3.7% at E/T 10:1, p <0.01). The killing of VPA treatment of patient myeloma cells by NK cells also significantly increased compared with control cells (specific killing: 46.7±5.6% vs 21.6±4.5% at E/T 10:1, n=4, p <0.01). The enhancing effect of VPA was blocked by pretreatment of OPM2 cells with a combination of anti-MICA/B, anti-ULBP1,2,3 mAbs or pretreatment of NK cells with anti-NKG2D mAbs, indicating the contribution of NKG2D-NKG2D ligands interations to VPA-modulated lysis of myeloma. We also found that constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not AKT signaling pathway may be involved in VPA-induced higher NKG2D ligand expression. Conclusion: Histone deacetylase inhibitor valproic acid could upregulate cell surface expression of NKG2D ligands, rendering myeloma cells more sensitive to NK cells, and thereby enhance NK cell–mediated lysis of myeloma. Our findings imply that regulation of the expression of NKG2D ligands by treatment with histone deacetylase inhibitors may be an attractive strategy for immunotherapy of myeloma. Acknowledgments: This work was supported by grants from National Natural Science Foundation of China (81071856 and 30973450 to JS) and funds from Shanghai Tenth People's Hospital (10RD103 and 11SC103 to JS). Disclosures: No relevant conflicts of interest to declare.

Role of NOTCH1 in Aggressive NK-Cell Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4231-4231
Author(s):  
Hideki Makishima ◽  
Masao Ota ◽  
Takefumi Suzuki ◽  
Kozo Nakayama ◽  
Fumihiro Ishida
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Western Blotting ◽  
Nk Cell ◽  
Cell Leukemia ◽  
Expression Levels ◽  
Healthy Donors ◽  
Enhanced Expression

Abstract Back ground: Aggressive NK-cell leukemia (ANKL) is a malignant NK-cell proliferative disorder resistant to most chemotherapies and frequently shows rapidly progressive clinical course. ANKL is characterized with hepatosplenomegaly, hemophagocytic syndrome, and strong association with EBV, and is mostly observed among Asian populations. Oncogenic mechanism of ANKL is not clear. NOTCH1 is a gene encoding a transmembrane receptor that regulates T-cell development and is a major oncogene in T-cell lymphoblastic leukemia (T-ALL). Recently, the potential involvement of NOTCH signaling in the development of NK-cells has been reported. We discuss here that the NOTCH1 protein might play a significant role in the pathogenesis of ANKL. Patients, materials, and methods: 6 ANKL cases, 7 Chronic NK-cell lymphocytosis (CNKL) cases, and 3 NK tumor cell lines (NKL, NK-92, and KHYG) derived from ANKL patients were enrolled. Chromosomal translocation (7;9) was not found in any subjects. The expression levels of NOTCH1 intracellular subunit (NOTCH-IC) were examined with flowcytometry and western blotting. NK-cell lines were cultured with g-secretase inhibitor (GSI) (L-685,458) for evaluation of the effect. Cell proliferation and apoptosis were measured with XTT assay and annexinV positivity, respectively. The mutation analysis of NOTCH1 genome was performed with nested PCR and direct sequencing. mRNA expression of NOTCH1 gene was measured with real time PCR using the corresponding TaqMan probes. Results: NOTCH-IC was detected in CD2+CD3− fractions from healthy donors, CNKL patients, ANKL patients, NK-cell lines, and Cos7 cells transfected with NOTCH1 gene coding NOTCH-IC, but not in CD2-CD3− fractions. The expression levels were significantly high in ANKL patients, compared to healthy donors (P < 0.05). With western blotting, cleaved form (110kDa) of NOTCH1 protein was recognized in all NK-cell lines. NOTCH-IC expression was reduced with the treatment of GSI in NKL and NK-92, but not in KHYG. Cell proliferation was inhibited with GSI in NKL and NK-92, whereas the inhibitory effect was not seen in KHYG. There was no significant difference in the increase ratio of apoptotic cells after the treatment of GSI among 3 NK-cell lines. Mutations in HD, TAD, or PEST domain, where frequent mutations are detected in T-ALL, were not found in NK-cell lines, and no enhanced expression of NOTCH1 mRNA in ANKL cells, compared with healthy donor NK-cells. Conclusion: These results indicate that NOTCH-IC was increased in ANKL cells and might be an important role in the proliferation. Genomic or transcriptional dysregulation would not be the cause for enhanced expression of NOTCH1 in ANKL contrast with T-ALL. Further investigation is required for the relationship with NOTCH1 ligands. In the future, NOTCH1 might be a novel therapeutic target in ANKL.

scholarly journals Indications of the Protective Role of Natural Killer Cells in Human Cutaneous Leishmaniasis in an Area of Endemicity

1998 ◽  
Vol 66 (6) ◽  
pp. 2698-2704 ◽  
Author(s):  
Kerima Maasho ◽  
Fabio Sanchez ◽  
Erwin Schurr ◽  
Asrat Hailu ◽  
Hannah Akuffo
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cutaneous Leishmaniasis ◽  
Natural Killer ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Proliferating Cells ◽  
Peripheral Blood Mononuclear

ABSTRACT The role of natural versus acquired immunity to Leishmania aethiopica infection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded to Leishmania antigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3− CD16/56+ natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia whereLeishmania is endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro with L. aethiopica antigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association ofNRAMP1 alleles with the studied phenotype and susceptibility to L. aethiopica-induced leishmaniasis was also evaluated. The results show that Leishmania antigens can induce NK cell and CD8+-T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4+-T-cell proliferation. We conclude from these observations that NK cells and CD8+ cells proliferating in response toLeishmania stimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to the NRAMP1 host resistance gene.

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