Natural Killer Cells Display an Activated Phenotype but Reduced Effector Functions in Obese Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3430-3430
Author(s):  
Sebastien Viel ◽  
Laurie Besson ◽  
Emily Charrier ◽  
Jacques Bienvenu ◽  
Emmanuel Disse ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Granzyme B ◽  
K562 Cells ◽  
Obese Patients ◽  
Activation Markers ◽  
Significant Difference ◽  
The Impact

Abstract The impact of adiposity on the immune system remains largely unexplored. While obesity has been suggested to be a predisposing or adverse prognostic factor in certain neoplastic diseases it is not yet clear to what extent this may involve the innate or adaptative immune systems. Adipose tissue produces a large number of secreted molecules, or adipocytokines, which may have immunomodulatory functions. This project aimed to determine whether phenotypical and/or functional properties of circulating natural killer (NK) cells were influenced by body mass index (BMI). In a preliminary study, 47 patients with no history of hematological malignancy were included, including 14 healthy volunteers with a normal BMI (18.5-25), 10 patients considered to be overweight (25 < BMI < 30), 11 patients considered as obese (BMI > 30) and 12 patients who were previously obese and had lost weight. Peripheral blood was analyzed by flow cytometry for the following markers: activating receptors (CD16, C161, DNAM-1, 2B4, NKG2C, NKG2D, NKp46, NKp30), inhibitor receptors (NKG2A, KIR2DL1, KIR2DL2, KIR3DL1), activation markers (CD69, granzyme B, NKG2C), maturation markers (CD56, CD57, CD94, CX3CR1) and cytotoxicity markers (perforin, NKG7). Moreover the capacity of NK cells to degranulate and to produce several cytokines (TNF, IFN-g) or chemokines (MIP1-b) in response to stimulation by K562 cells or Rituximab coated -tumor B cells was evaluated. Results showed a positive correlation between BMI and total number of circulating NK cells, with a significant difference between lean patients and obese patients. Immunophenotypic analyses showed that NKp46 and CD94 expression (measured by Mean Fluorescence Intensity) were both significantly reduced with increased BMI. NK cells from obese patients also show signs of activation, characterized by an elevation of the expression of CD69 and granzyme B and a reduction of the expression of CD16. The ability of NK cells to be activated in the presence of cell lines was also reduced in obese patients: NK cell secretion of IFN-g and MIP-1b in the presence of Granta cells or MIP-1b in the presence of K562 decreased linearly with increasing BMI. NK cell degranulation upon co-culture with K562 cells was also negatively correlated with BMI. In these different assays pre-obese and ex-obese patients scored intermediate between lean and obese patients. Overall these results suggest in vivo activation and exhaustion of NK cells in obese patients. These cells are thus potentially less likely to participate as effector cells in immunotherapeutic regimens. Disclosures No relevant conflicts of interest to declare.

T/NK Lymphocytosis in CML Ph+ Patients During Dasatinib Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3279-3279
Author(s):  
Antonella Russo Rossi ◽  
Alessandra Ricco ◽  
Paola Carluccio ◽  
Mario Delia ◽  
Manuela Leo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Cytotoxic T Cell ◽  
Molecular Response ◽  
Imatinib Treatment ◽  
Long Term Outcome ◽  
Significant Difference ◽  
The Impact

Abstract Abstract 3279 Poster Board III-1 Background. Some authors reported that Natural Killer (NK) cells from CML patients are defective in NK cell activity and NK cell number decrease as the disease progresses to the advanced phase and probably the abnormal BCR/ABL gene causes abnormal NK cell differentiation.TK inhibitors reduce BCR/ABL transcription and could restore NK cell numbers and/or function.Moreover Dasatinib,by the blockade of SRC Kinases,could affect the development of NK cells as well as T-lymphocytes.Our aim was to verify the impact of Dasatinib treatment on T CD8+ and NK cells modulation. Methods. We evaluated 24 patients with CML resistant/intollerant to Imatinib and treated with Dasatinib at a starting dose of 70 mg/BID or 100 mg/QD.Blood count were monitored; lymphocytosis has been definited by an increased number of peripheral blood lymphocyte counts ≥ 3.0×10(e)9/L and by the predominance of LGLs in peripheral blood smear. Immunophenotyping was done with flow-cytometry using antibodies against the following antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD16, and CD56. Results. With a median of 19 mo. of Dasatinib therapy (range 3–43), 15/24 cases (62.5%) developed peripheral blood lymphocytosis. Median onset of lymphocytosis was 3 months after the initiation of Dasatinib therapy (range 1–12) and duration was 14 months (range 6–40).Lymphocytosis was CD3+/CD8+/Cytotoxic T Cell in 9 patients (60%) and CD3-/CD16+/CD56+/NK Cell in 6 patients (40%).In all 15 patients no symptoms or signs suggestive of LGL leukemia or viral infections were documented.There was no significant difference in terms of the frequency of severe adverse events, including pleural effusion between patients with and without lymphocytosis. 11(73%) of the 15 patients who developed lymphocytosis achieved MMolR and 4/11 presented Bcr/Abl mutation at the time of imatinib treatment (F317L, E255K, F359V, E255K),whereas only 3(33%) of the 9 patients without lymphocytosis achieved MMolR and 1/3 presented Bcr/Abl mutation (F359V). Moreover molecular response was earlier in the group of patients with lymphocytosis (8vs12 mo.). Conclusions. The development of lymphocytosis in our patients seems to be associated to an improved response to dasatinib in terms of molecular response and time to response. The assessment of higher frequency lymphocytosis requires further analysis; a larger patients'cohort should be needed to explore the biological role of lymphocytosis and the impact on the long term outcome in patients treated with dasatinib. Disclosures: No relevant conflicts of interest to declare.

scholarly journals α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

2021 ◽  
Vol 22 (2) ◽  
pp. 656
Author(s):  
Hantae Jo ◽  
Byungsun Cha ◽  
Haneul Kim ◽  
Sofia Brito ◽  
Byeong Mun Kwak ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Granzyme B ◽  
Akt Pathway ◽  
Cell Cytotoxicity ◽  
Anticancer Effects ◽  
Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

scholarly journals Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 156-164
Author(s):  
V Pistoia ◽  
S Zupo ◽  
A Corcione ◽  
S Roncella ◽  
L Matera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Suspensions ◽  
Normal Peripheral Blood ◽  
Colony Stimulating Activity ◽  
Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

scholarly journals A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 659-659 ◽  
Author(s):  
Jan Davidson-Moncada ◽  
Noriko Sato ◽  
Robert F Hoyt ◽  
Robert N Reger ◽  
Marvin Thomas ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Raji Cell ◽  
K562 Cells ◽  
Ct Imaging ◽  
Pet Ct ◽  
Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals Dysregulation of Natural Killer Cells in Obesity

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 573 ◽  
Author(s):  
Donal O’Shea ◽  
Andrew E. Hogan
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Biology ◽  
Viral Infections ◽  
Nk Cell ◽  
Viral Immunity ◽  
Host Protection ◽  
Cell Functions ◽  
Innate Lymphocytes ◽  
The Impact

Natural killer (NK) cells are a population of lymphocytes which classically form part of the innate immune system. They are defined as innate lymphocytes, due to their ability to kill infected or transformed cells without prior activation. In addition to their cytotoxic abilities, NK cells are also rapid producers of inflammatory cytokines such as interferon gamma (IFN-γ) and are therefore a critical component of early immune responses. Due to these unique abilities, NK cells are a very important component of host protection, especially anti-tumour and anti-viral immunity. Obesity is a worldwide epidemic, with over 600 million adults and 124 million children now classified as obese. It is well established that individuals who are obese are at a higher risk of many acute and chronic conditions, including cancer and viral infections. Over the past 10 years, many studies have investigated the impact of obesity on NK cell biology, detailing systemic dysregulation of NK cell functions. More recently, several studies have investigated the role of NK cells in the homeostasis of adipose tissue and the pathophysiology of obesity. In this review, we will discuss in detail these studies and focus on emerging data detailing the metabolic mechanisms altering NK cells in obesity.

Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3244-3244
Author(s):  
Gabriele Multhoff ◽  
Catharina Gross ◽  
Anne Dickinson ◽  
Ernst Holler
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Hla Class I ◽  
K562 Cells ◽  
Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

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.

Enhancement of Natural Killer Cell Interferon-Gamma Production By the Derivatives of the Natural Product Phyllanthusmins Via TLR-Mediated NF-Kb and STAT3 Signaling Pathways

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1031-1031
Author(s):  
Luxi Chen ◽  
Long Yi ◽  
Yuning Ren ◽  
Jianying Zhang ◽  
A. Douglas Kinghorn ◽  
...  
Keyword(s):  
Nk Cells ◽  
Signaling Pathways ◽  
Natural Product ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Killer Cell ◽  
Immune Defense ◽  
Ifn Gamma ◽  
Stat3 Signaling

Abstract Natural killer (NK) cells are known to play a pivotal role in regulating immune defense against tumors and viruses. However, very few studies describe the effects of natural products or their derivatives on NK cell function. We previously showed that the natural product lignan phyllanthusmin C enhances human NK cell IFN-gamma production. In this study, we successfully synthesized dozens of phyllanthusmin derivatives and screened their activities to enhance IFN-gamma production by NK cells. We found that three of these derivatives possessed capacities to induce IFN-gamma production by NK cells. Among them, derivative No. 17 had the highest efficacy and was significantly superior (P < 0.05) to that of the original phyllanthusmin C. These three phyllanthusmin derivatives had no adverse effect on human NK cell survival or proliferation. All derivatives alone or in combination with IL-12 or IL-15 induced human NK cell interferon (IFN)-gamma in comparison with the corresponding vehicle control or cytokine alone (P < 0.05). These derivatives stimulated IFN-gamma production in both CD56bright and CD56dim human NK cell subsets (P < 0.01). Mechanistically, immunoblotting assays and chromatin immunoprecipitation (ChIP) analyses combined with promoter-reporter luciferase assays revealed that these phyllanthusmin derivatives induced the phosphorylation of NF-κB and STAT3, resulting in their increased binding on theIFN G promoter, which was dependent on the Toll-like receptor (TLR) 1 and the TLR3 signaling pathways, respectively. STAT3 knockdown with lentivirus shRNA and inhibition of NF-kB signaling with a specific inhibitor (TPCK) significantly attenuated IFN-gamma production induced by these phyllanthusmin derivatives in human NK cells (P < 0.05). Blockade of TLR1 or TLR3 with their corresponding neutralizing antibodies nearly abolished activation of NF-κB or STAT3 as well as IFN-γ induction by the phyllanthusmin derivatives (P < 0.05). In conclusion, we have successfully synthesized and screened phyllanthusmin derivatives and identified three compounds that induce IFN-gamma production in human NK cells, one of which had optimal potential. The induction of human NK cell IFN-gamma production by phyllanthusmin derivatives is mediated by TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering their potential for use as agents for cancer prevention or treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for the involvement of CD56 molecules in alloantigen-specific recognition by human natural killer cells.

1991 ◽  
Vol 173 (6) ◽  
pp. 1451-1461 ◽  
Author(s):  
N Suzuki ◽  
T Suzuki ◽  
E G Engleman
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Target Molecules

In recent reports we have described the generation of natural killer (NK) lines devoid of CD3/TCR structures but with apparent specificity for allogeneic target cells. Using one such NK line as an immunogen, we now report the generation of two monoclonal antibodies (mAbs), designated 2-13 and 5-38, which bind selectively to the majority of CD3-, CD16+, CD56+ lymphocytes and inhibit the lysis of specific allogeneic target cells by a panel of alloreactive NK lines. By contrast, these mAbs had no effect on classical NK cell mediated lysis of K562 cells or major histocompatibility-restricted T cell-mediated cytolysis. Immunoprecipitation of radiolabeled NK lines followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the target molecules of both mAbs have a molecular mass of approximately 180 kD. Leu 19, a well-described anti-CD56 mAb, precipitated a 180 kD protein from NK cells, and the binding of Leu 19 to NK cells was blocked by pretreatment with both 2-13 and 5-38. However, in contrast to these mAbs, Leu 19 had no effect on the cytolytic activity of allospecific NK cells. Sequential immunoprecipitation analysis revealed that all three mAbs recognized distinct molecular species of CD56. We interpret these findings as indicating that multiple isoforms of CD56 are differentially expressed on NK lines and play critical roles in the recognition/interaction of these cells with their specific allogeneic targets.

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