A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p<0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

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Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽

10.1182/blood.v84.3.841.841 ◽

1994 ◽

Vol 84 (3) ◽

pp. 841-846 ◽

Cited By ~ 37

Author(s):

MR Silva ◽

R Hoffman ◽

EF Srour ◽

JL Ascensao

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Mononuclear Cells ◽

Viral Infections ◽

Nk Cell ◽

Interleukin 2 ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Human Natural Killer Cells

Abstract Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

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Rituximab-Mediated ADCC Is Augmented by Concomitant Interference with Inhibitory Self-Recognition by Human NK Cells.

Blood ◽

10.1182/blood.v106.11.2456.2456 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2456-2456

Author(s):

Liat Binyamin ◽

R. Katherine Alpaugh ◽

Kerry S. Campbell ◽

Hossein Borghaei ◽

Louis M. Weiner

Keyword(s):

B Cells ◽

Nk Cells ◽

Mononuclear Cells ◽

Cell Function ◽

Nk Cell ◽

Tumor Cell Death ◽

Peripheral Blood Mononuclear ◽

Self Recognition ◽

Missing Self ◽

Anti Cd20

Abstract The anti-CD20 monoclonal antibody, rituximab is widely used in the treatment of non-Hodgkin lymphomas. However, clinical responses to rituximab are variable. It has been demonstrated that rituximab can lead to tumor cell death by engaging the cellular immune system through antibody dependent cellular cytotoxicity (ADCC). NK cells have been shown to play a critical rule in eliminating rituximab coated B-cells, and the efficiency of killing depends on the interaction between the Fc portion of rituximab and the FcγRIII (CD16) activating receptor on NK cells. NK cell function is regulated by a complex balance of inhibitory and activating signals that enable the cells to survey their surrounding and selectively target and kill targets that do not display a “self” ligand (the “missing self hypothesis”). We hypothesized that interference with inhibitory self-recognition would augment rituximab-induced NK cell-mediated ADCC. Initial studies with the 721.221 B51 (HLA Bw4+) CD20+ cell line and NK92.26.5 cells transduced with human CD16 suggested that interference with KIR3DL1 recognition of Bw4 augmented tumor lysis in the presence of rituximab. To further test this hypothesis we employed human NK cells and autologous EBV transformed B cells from normal volunteers, and blocked the KIR3DL1 inhibitory receptor on NK cells using (Fab′)2 fragments of the DX9 antibody, in conjunction with rituximab exposure. Inhibitory blockade promoted rituximab-mediated cytotoxicity by peripheral blood mononuclear cells in three separate HLABw4+, KIR3DL1+ volunteers. These results suggest that manipulating the balance between inhibitory and activating receptors on NK cells might be applied to improve ADCC and ultimately lead to an improvement in response rates to rituximab and related lymphoma-directed antibodies that mediate ADCC. Supported by R01CA50633.

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Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽

10.1182/blood.v84.3.841.bloodjournal843841 ◽

1994 ◽

Vol 84 (3) ◽

pp. 841-846 ◽

Cited By ~ 1

Author(s):

MR Silva ◽

R Hoffman ◽

EF Srour ◽

JL Ascensao

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Mononuclear Cells ◽

Viral Infections ◽

Nk Cell ◽

Interleukin 2 ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Human Natural Killer Cells

Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

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Evaluation of NK Cell Function by Flowcytometric Measurement and Impedance Based Assay Using Real-Time Cell Electronic Sensing System

BioMed Research International ◽

10.1155/2013/210726 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Ki-Hyun Park ◽

Hyesun Park ◽

Myungshin Kim ◽

Yonggoo Kim ◽

Kyungja Han ◽

...

Keyword(s):

Nk Cells ◽

Real Time ◽

Cell Activation ◽

Mononuclear Cells ◽

Cell Function ◽

Nk Cell ◽

Target Cells ◽

Peripheral Blood Mononuclear ◽

Electronic Sensing ◽

Nk Cytotoxicity

Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1αβ, IFN-γ, and TNF-α, and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions.

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Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

Cancers ◽

10.3390/cancers13030577 ◽

2021 ◽

Vol 13 (3) ◽

pp. 577

Author(s):

Adrián Fernández ◽

Alfonso Navarro-Zapata ◽

Adela Escudero ◽

Nerea Matamala ◽

Beatriz Ruz-Caracuel ◽

...

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Fold Increase ◽

Clinical Grade ◽

Transcriptome Profile ◽

Expansion Process ◽

Peripheral Blood Mononuclear ◽

Activation Status

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

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“Adherent” versus Other Isolation Strategies for Expanding Purified, Potent, and Activated Human NK Cells for Cancer Immunotherapy

BioMed Research International ◽

10.1155/2015/869547 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Senthamil R. Selvan ◽

John P. Dowling

Keyword(s):

Nk Cells ◽

Large Scale ◽

Mononuclear Cells ◽

Adoptive Cell Therapy ◽

Cost Effective ◽

Future Research ◽

Peripheral Blood Mononuclear ◽

Simple Method ◽

Antitumor Potential ◽

Healthy Donors

Natural killer (NK) cells have long been hypothesized to play a central role in the development of new immunotherapies to combat a variety of cancers due to their intrinsic ability to lyse tumor cells. For the past several decades, various isolation and expansion methods have been developed to harness the full antitumor potential of NK cells. These protocols have varied greatly between laboratories and several have been optimized for large-scale clinical use despite associated complexity and high cost. Here, we present a simple method of “adherent” enrichment and expansion of NK cells, developed using both healthy donors’ and cancer patients’ peripheral blood mononuclear cells (PBMCs), and compare its effectiveness with various published protocols to highlight the pros and cons of their use in adoptive cell therapy. By building upon the concepts and data presented, future research can be adapted to provide simple, cost-effective, reproducible, and translatable procedures for personalized treatment with NK cells.

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Aberrant anti-viral response of natural killer cells in severe asthma

European Respiratory Journal ◽

10.1183/13993003.02422-2018 ◽

2020 ◽

Vol 55 (5) ◽

pp. 1802422

Author(s):

Justine Devulder ◽

Cécile Chenivesse ◽

Valérie Ledroit ◽

Stéphanie Fry ◽

Pierre-Emmanuel Lobert ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Severe Asthma ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Asthma Patients ◽

Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

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NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽

10.1182/blood.v110.11.4879.4879 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4879-4879

Author(s):

Juan Tong ◽

Huilan Liu ◽

Liangquan Geng ◽

Zimin Sun ◽

Baolin Tang ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Nk Cell ◽

Linear Regression Analysis ◽

Cell Activity ◽

Target Cells ◽

Peripheral Blood Stem Cells ◽

Peripheral Blood Mononuclear ◽

Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

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

Blood ◽

10.1182/blood.v112.11.5425.5425 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5425-5425

Author(s):

Belkis Atasever ◽

Serap Erdem Kuruca ◽

Zeynep Karakas ◽

Batu Erman ◽

Arzu Ergen ◽

...

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

K562 Cells ◽

Tnf Alpha ◽

Beta Thalassemia ◽

Nk Activity ◽

Ifn Gamma ◽

Cytolytic Function

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

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Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides

Human & Experimental Toxicology ◽

10.1191/0960327104ht477oa ◽

2004 ◽

Vol 23 (10) ◽

pp. 463-471 ◽

Cited By ~ 34

Author(s):

Adrian Reed ◽

Leticia Dzon ◽

Bommanna G Loganathan ◽

Margaret M Whalen

Keyword(s):

T Cells ◽

Nk Cells ◽

Tumor Cells ◽

Organochlorine Pesticides ◽

Mononuclear Cells ◽

Viral Infections ◽

Nk Cell ◽

Cell Preparation ◽

Lytic Function ◽

Cytotoxic Function

Organochlorine pesticides are used worldwide. To our knowledge there have been no studies dealing with the effects of these agents under in vitro conditions on human natural killer (NK) cell cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of organochlorine pesticides and some of their breakdown products on the ability of human NK cells to lyse tumor cells. A total of 11 compounds were tested. The compounds were tested in both purified NK cells as well as a cell preparation that contained other mononuclear cells (predominantly T cells) and NK lymphocytes (referred to as T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 hour to 6 days. Exposure of highly purified NK cells to 5 μ M α-chlordane, γ-chlordane, 4,4'-DDT, heptachlor, oxychlordane, or pentachlorophenol (PCP) inhibited their ability to destroy K562 tumor-cells by 88±5, 92±8, 61±13%, 64±10%, 69±11%, 76±12%, respectively, after a 24h exposure. The loss of cytotoxic function seen with α-and γ-chlordane remained essentially constant out to 6 days, while that seen with 4,4'-DDT, oxychordane and PCP increased with longer exposures (6 d). PCP was the most effective of the compounds tested at decreasing NK function. Of the compounds that caused decreased lytic function when tested in purified NK cells, only PCP and oxychordane decreased the lytic function of the T/NK cell preparation after any exposure. The results provide evidence of relative toxic potential for the 11 compounds and their immunomodulatory effects on other mononuclear cells (such as T-cells, B-cells, and monocytes) as well as NK lymphocyte function.

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