Natural Killer Cells: Methods and Protocols (Methods in Molecular Biology, 1441)

✍ Scribed by Srinivas S. Somanchi (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 372
• Category: Library

✦ Synopsis

This volume contains collection of Natural Killer Cell methodologies relevant for both basic and translational research. These methodologies present new developments in the natural killer (NK) cell field, such as understanding the influence of NK cells metabolism on its function, identifying complexity of NK cell subsets through mass cytometry, and determining the emergence of memory NK cells in murine model of MCMV infection. Methods that study NK cell migration and cytotoxicity through endpoint analysis or live single cell imaging are also discussed. Chapters also describe methods pertaining to translational application of NK cells, such as ex vivo expansion of NK cells on K562 cell lines genetically modified to express either membrane bound IL-15 or membrane bound IL-21, large scale NK cell culture, current techniques for engineering NK cells to express chimeric antigen receptors or chemokine receptors using retroviral vectors,electroporation of mRNA, and the natural phenomenon of trogocytosis. Written in the highly successful Methods in Molecular Biology series format, these chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting edge and thorough, Natural Killer Cells: Methods and Protocols is a valuable resource for researchers who not only want to understand mechanisms that govern NK cell behavior and diversity, but also for those who want to understand how to systematically evaluate NK cells for adoptive immunotherapy applications.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Tracking Effector and Memory NK Cells During MCMV Infection
1 Introduction
2 Materials
2.1 Reagents
2.2 Mice
2.3 Preparation of MCMV Stocks
2.4 MCMV Plaque Assay
2.5 Adoptive Transfer
3 Methods
3.1 Preparation of Viral Stocks from Balb/c Salivary Glands
3.2 Generating MEFs for Plaque Assay
3.3 Plaque Assay to Quantify MCMV Titers
3.4 Adoptive Transfer of CD45.1+ Ly49H+ NK Cells and MCMV Infection
3.5 Tracking the Expansion, Contraction, and Memory Phases of Transferred Ly49H+ NK Cells
4 Notes
References
Chapter 2: Application of Mass Cytometry (CyTOF) for Functional and Phenotypic Analysis of Natural Killer Cells
1 Introduction
2 Materials
2.1 Antibody Conjugation
2.2 Mass Cytometry Labeling
2.3 Cell Stimulation
2.4 Running CyTOF Mass Cytometry
3 Methods
3.1 Antibody Conjugation Using MaxPar Metal Labeling Kit
3.2 Surface and Intracellular Labeling of Cells for Mass Cytometry
3.3 Running Samples on a CyTOF Mass Cytometer
3.4 Data Analysis
4 Notes
References
Chapter 3: Assessment of NK Cell Metabolism
1 Introduction
2 Materials
2.1 Magnetic Bead Isolation of NK Cells
2.2 Culture of NK Cells with Metabolic Inhibitors (Table 1)
2.3 Assays to Detect Intracellular IFN-γ
2.4 Detection of Intracellular ATP Levels
2.5 Extracellular Flux Assays
3 Methods
3.1 NK Cell Enrichment
3.2 Stimulation of NK Cells
3.2.1 Antibody Stimulation of NK Cells
3.2.2 Cytokine Stimulation of NK Cells
3.2.3 Staining Cells for Intracellular IFN-γ
3.3 Measurement of Intracellular ATP in NK Cells
3.4 Extracellular Flux Assay
3.4.1 Preparation of Instrument and Plate
3.4.2 Plating NK Cells and Preparing Sensor Cartridge
3.5 Data Acquisition and Analysis
4 Notes
References
Chapter 4: Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells
1 Introduction
2 Materials
2.1 Premade Reagents
2.2 Instrument Requirements and Software
2.3 Primers and Probes
3 Methods
3.1 SNP Genotyping Methodology: FCGR3A-V158F and FCGR2C- C/T
3.2 CNV Genotyping Methodology: FCGR3A and FCGR2C
4 Notes
References
Chapter 5: Measurement of Average Telomere Length in Ex Vivo Expanded Natural Killer Cells by Fluorescence In Situ Hybridization (FISH) and Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Telomere Length Assay
3.1.1 Day 1
3.1.2 Day 2
3.2 Analysis and Interpretation of Results
3.2.1 Flow Cytometry
3.2.2 Data Analysis and Calculation of Relative Telomere Length
4 Notes
References
Chapter 6: In Vitro Assessment of Human Natural Killer Cell Migration and Invasion
1 Introduction
2 Materials
2.1 Cells
2.2 Assay Plates
2.3 Reagents
2.4 Instruments
3 Methods
3.1 Matrigel Invasion Assay
3.2 Detection of Transmigrated Cells
4 Notes
References
Chapter 7: Microfluidic-Based Live-Cell Analysis of NK Cell Migration In Vitro
1 Introduction
2 Materials and Facilities
2.1 Microfluidic Device Design and Mask Fabrication by Photo-­Lithography
2.2 Poly-dimethylsiloxane (PDMS) Device Fabrication by Soft Lithography
2.3 Fibronectin Coating of the Microfluidic Device
2.4 Microscopy and Image Acquisitions
2.5 Data Analyses
2.6 Natural Killer Cells Purification, Activation, and Expansion In Vitro
2.7 Culture of Bone Marrow-Derived DC In Vitro
3 Methods
3.1 Microfluidic Device Design
3.2 Fabrication: Photolithography and Soft Lithography ( See Note 3)
3.3 Fibronectin Coating and Blocking of Devices
3.4 Culture of Activated NK Cells
3.5 Culture of Bone Marrow-Derived DC In Vitro
3.6 Migration Studies Using the Microfluidic-­Based Setup
3.7 Images Acquisitions and Data Analyses
4 Notes
References
Chapter 8: Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response
1 Introduction
2 Materials
2.1 Fabrication of Microwell Chips
2.2 Fabrication of Microwell Chip Holder
2.3 Setup for Live-­Cell Time-Lapse Imaging
2.4 Preparing the Microwell Chip for Imaging
2.5 Cleaning the Microchip for Reuse
3 Methods
3.1 Fabrication of Silicon-Glass Microwell Array Chips
3.2 Fabrication of Microwell Chip Holder and Gasket
3.3 Preparing the Microwell Chip for Imaging
3.4 Fluorescence Labeling of NK and Target Cells
3.5 Loading of NK and Target Cells on the Microchip
3.6 Setup for Time-­Lapse Imaging
3.7 Cleaning the Microchip for Reuse
3.8 Analysis and Quantification of NK Cell Data
3.8.1 Cell Tracking and Quantification
3.8.2 Analysis of Target Cell Death by Calcein Fluorescence Decay
4 Notes
References
Chapter 9: Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method
1 Introduction
2 Materials
3 Methods
3.1 Cytotoxicity Assay Setup
3.1.1 Preparing NK Cells
3.1.2 Calcein AM Staining of Target Cells and Assay Setup
3.2 Acquisition and Analysis of Data by Image Cytometer
3.2.1 Imaging/Acquiring Data
3.2.2 Data Analysis and Percent Specific Lysis
4 Notes
References
Chapter 10: Analysis of Intracellular Ca2+ Mobilization in Human NK Cell Subsets by Flow Cytometry
1 Introduction
2 Materials
2.1 Cells, Media, and Solutions
2.2 Antibodies and Fluorescent Reagents for Ca2+ Flux Detection
2.2.1 Stimulating Mouse IgG1 Isotype mAbs (See Note 4)
2.2.2 Fluorochrome-
2.2.3 Fluorescent Reagents
2.3 Flow Cytometry Hardware and Software
2.4 Other Material
3 Methods
3.1 Preparation of Peripheral Blood Mononuclear Cells
3.2 Staining, Stimulation, and Acquisition
3.2.1 Ca2+ Flux Detection
3.2.2 Control Samples for Compensation of Spectral Overlap
3.3 Flow Cytometry Compensation and Analysis
4 Notes
References
Chapter 11: Using NK Cell Lipid Raft Fractionation to Understand the Role of Lipid Rafts in NK Cell Receptor Signaling
1 Introduction
2 Materials
2.1 Density Gradient Ultracentrifugation
2.2 Raft Fractionation by β-Octylglucoside Selective Solubility
2.3 Protein Purification from  DRM Fractions
3 Methods
3.1 Density Gradient Ultracentrifugation
3.2 Raft Fractionation by β-Octylglucoside Selective Solubility
3.3 Protein Purification from DRM/Rafts
4 Notes
References
Chapter 12: High- and Super-Resolution Microscopy Imaging of the NK Cell Immunological Synapse
1 Introduction
2 Materials
3 Methods
3.1 NK Cell Culture
3.2 Primary NK Cells Isolation and Purification
3.3 Coating Slides with Activating Antibody
3.4 Preparing NK Cells and Activation on Coverslips
3.5 Antibody and Phalloidin Staining
3.6 Mounting Coverslips on Slides
3.7 Image Acquisition
3.8 Processing and Analyzing Images (See Note 9)
4 Notes
References
Chapter 13: The Planar Lipid Bilayer System Serves as a Reductionist Approach for Studying NK Cell Immunological Synapses and Their Functions
1 Introduction
2 Materials
2.1 Lipid Preparation
2.2 Determination of Protein Seeding Density on the Lipid Bilayer
2.3 Assembling the Supported Lipid Bilayer
3 Methods
3.1 Lipid Preparation
3.2 Determination of Protein Seeding Density on the Lipid Bilayer
3.3 Assembling the Supported Lipid Bilayer
4 Notes
References
Chapter 14: Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Genetically Engineered K562 Feeder Cells
3.2 Isolation of Peripheral Blood Mononuclear Cells
3.3 Culture of NK Cells from Isolated PBMCs
3.4 Receptor Expression on Cultured NK Cells
4 Notes
References
Chapter 15: Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21
1 Introduction
2 Materials
2.1 Isolation of PBMCs
2.2 RosetteSep Purification of NK Cells
2.3 CD3 Depletion of PBMCs
2.4 NK Cell Expansion and Phenotype
2.5 Assessment of NK Cell Cytotoxicity
3 Methods
3.1 Isolation and Purification of NK Cells
3.1.1 Isolation of PBMCs
3.1.2 RosetteSep™ Purification of NK Cells
3.1.3 CD3 Depletion of PBMCs
3.2 Culture and Irradiation of K562 mbIL21
3.3 NK Cell Expansion
3.3.1 Stimulation 1
3.3.2 Stimulation 2
3.4 Phenotype Analysis and Calculating NK Cell Fold Expansion
3.4.1 Phenotype by Flow Cytometry
3.4.2 Calculating NK Cell Fold Expansion
3.5 Assessment of NK Cell Cytotoxicity
4 Notes
References
Chapter 16: Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy
1 Introduction
2 Materials
3 Methods
3.1 Expansion of K562mb15-­41BBL Cells in G-Rex Flasks
3.2 Ficoll Gradient Separation of Cryopreserved Peripheral Blood or Apheresis Cells
3.3 Harvest and Irradiate of K562mbIL15-­41BBL Feeder Cells
3.4 Expansion of NK Cells in G-Rex 100s
3.5 Transduction of Human NK Cells with Retroviral Vectors
4 Notes
References
Chapter 17: Gene Modification of Human Natural Killer Cells Using a Retroviral Vector
1 Introduction
2 Materials
2.1 Separation of Mononuclear Cells and NK Cells Selection
2.2 NK Cells Activation and Expansion
2.3 NK-Cell Transduction
3 Methods
3.1 Separation of Mononuclear Cells
3.2 NK-Cell Selection (See Note 4)
3.3 NK-Cell Activation
3.3.1 Day −1
3.3.2 Day 0
3.3.3 Day 3
3.4 NK-Cell Transduction
3.4.1 Day 4
3.5 NK-Cell Expansion
3.5.1 Day 6
3.5.2 Day 7
3.5.3 Day 10
3.5.4 Day 12
4 Notes
References
Chapter 18: Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy
1 Introduction
2 Materials
2.1 Expansion of NK Cells
2.1.1 Expansion of NK Cells by Co-culture with the Irradiated Feeder Cells
2.1.2 NK Isolation
2.2 Nucleofection of CAR mRNA
2.2.1 Generation of CAR mRNA
2.2.2 Nucleofection
2.2.3 Flow Cytometric Analysis of CAR Expressing in NK Cells
2.3 Functional Analysis of CAR-Modified Expanded NK Cells In Vitro
2.3.1 In Vitro Cytotoxicity
2.3.2 Tumor Cell Recovery
2.3.3 Intracellular CD107a and IFN-γ Assays
3 Methods
3.1 Expansion of NK Cells
3.1.1 Expansion of NK Cells by Co-culture with the Irradiated Feeder Cells
3.1.2 NK Isolation
3.2 Nucleofection of CAR mRNA
3.2.1 Production of CAR mRNA
3.2.2 Nucleofection
3.2.3 Flow Cytometric Analysis of CAR Expressing in NK Cells
3.3 Functional Analysis of CAR-Modified Expanded NK Cells In Vitro
3.3.1 In Vitro Cytotoxicity
3.3.2 Tumor Cell Recovery
3.3.3 Intracellular CD107a and IFN-γ Assays
4 Notes
References
Chapter 19: mRNA Transfection to Improve NK Cell Homing to Tumors
1 Introduction
2 Materials
2.1 Electroporation Components
2.2 Phenotype of Chemokine Receptor Expression Components
2.3 In Vitro Migration Assay Components
3 Methods
3.1 Electroporation of NK Cells
3.2 mRNA Titration and Phenotype of Chemokine Receptor Expression
3.3 In Vitro Migration Assay
4 Notes
References
Chapter 20: In Vitro Generation of Human NK Cells Expressing Chimeric Antigen Receptor Through Differentiation of Gene-­Modified Hematopoietic Stem Cells
1 Introduction
2 Materials
2.1 Isolation and Cryopreservation of Umbilical Cord Blood HSC
2.2 Lentiviral Transduction of HSC
2.3 Differentiation of HSC to NK Cell Lineage
2.3.1 Co-culture with OP9-DL1 Stromal Cells
2.3.2 Feeder-Free Differentiation Protocol
3 Methods
3.1 Isolation and Cryopreservation of Umbilical Cord Blood HSC (See Note 1)
3.2 Lentiviral Transduction of HSC
3.2.1 High-Titer Lentivirus for Modification of HSC
3.2.2 Lentiviral Transduction of HSC
3.3 Culture and Passaging of OP9-DL1 Stromal Cells
3.4 Differentiation of HSC to NK Cell Lineage
3.4.1 Differentiation by Co-culture with OP9-DL1 Stromal Cells
3.4.2 Feeder-Free Differentiation Culture (Alternate Protocol)
4 Notes
References
Chapter 21: Engineering Receptor Expression on Natural Killer Cells Through Trogocytosis
1 Introduction
2 Materials
3 Methods
3.1 Trogocytosis Co-culture
3.1.1 Preparing ‘Acceptor’ NK Cells
3.1.2 Preparing ‘Donor’ Cells
3.1.3 Trogocytosis Co-culture Setup
3.1.4 Acid Wash
3.2 Flow Cytometry
3.2.1 Data Acquisition
3.2.2 Gating Scheme for Analysis
3.3 Assessing Function and Persistence of Acquired Receptors
3.3.1 In Vitro Migration Assay
3.3.2 Persistence of Acquired Receptors
4 Notes
References
Chapter 22: Electroporation of siRNA to Silence Gene Expression in Primary NK Cells
1 Introduction
2 Materials
2.1 Isolation of NK Cells
2.2 siRNA Electroporation
2.3 RT-PCR
3 Methods
3.1 Isolation of Primary NK Cells
3.2 Electroporation of NK Cells with siRNA
3.3 Assessment of Gene Silencing
4 Notes
References
Chapter 23: Mouse Xenograft Model for Intraperitoneal Administration of NK Cell Immunotherapy for Ovarian Cancer
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Mice
2.3 Injection of Tumor into Mice
2.4 Animal Imaging
2.5 Cytokines
2.6 Analysis of NK Cell Survival
3 Methods
3.1 Establishment of IP Tumor and Imaging
3.2 Delivery of NK Cells and Cytokine Injections
3.3 Monitoring NK Cell Engraftment and Survival in Blood
3.4 Intraperitoneal Washes to Measure NK Cell Persistence
4 Notes
References
Chapter 24: Aerosol Delivery of Interleukin-2 in Combination with Adoptive Transfer of Natural Killer Cells for the Treatment of Lung Metastasis: Methodology and Effect
1 Introduction
2 Materials
3 Methods
3.1 NK Cells Activation and Expansion
3.2 Development of Osteosarcoma Tumor Model
3.3 NK Cell and Aerosolized IL2 Treatment
3.4 Detection of NK Cells in the Lung Upon Aerosol IL-2 Treatment
3.4.1 Detection of NK Cells in the Lungs of Animals Without Lung Tumors Upon Aerosol IL-2 Treatment
3.4.2 Detection of NK Cells in the Lungs of Animals with Lung Tumors Upon Aerosol IL-2 Treatment
3.5 Methodology to Evaluate the Systemic Effects of Aerosol IL-2
3.6 Detection of Serum IL-2 Levels After Aerosol and Systemic IL-2 Delivery
4 Notes
References
Chapter 25: Noninvasive Imaging of Natural Killer Cell-Mediated Apoptosis in a Mouse Tumor Model
1 Introduction
2 Materials
2.1 Cell Lines and Culture Medium
2.2 Establishment of Caspase-3 Biosensor and Rluc Cell Line
2.3 In Vivo Experiments: Mice, Imaging Reagents, and Instruments
3 Methods
3.1 NK92 Cell Culture
3.2 Expression of Caspase-3 Biosensor, Rluc, mCherry in Tumor Cells
3.3 In Vitro Bioluminescence Imaging
3.4 Noninvasive In Vivo Imaging of Caspase-3 Activation
4 Notes
References
Chapter 26: Noninvasive In Vivo Fluorescence Imaging of NK Cells in Preclinical Models of Adoptive Immunotherapy
1 Introduction
2 Materials
3 Methods
3.1 Staining NK Cells with DiR
3.2 Infusion of DiR Stained NK Cells
3.3 Fluorescence In Vivo Imaging
4 Notes
References
Chapter 27: In Vivo 19F-Magnetic Resonance Imaging of Adoptively Transferred NK Cells
1 Introduction
2 Materials
3 Methods
3.1 Establishing Murine Intracranial Tumor Model
3.1.1 Placing Guide Screws in Cranium
3.1.2 Intracranial Injection of Tumor Cells
3.2 In Vivo Infusion of 19F Labeled NK Cells
3.2.1 Labeling NK Cells with 19F
3.2.2 Intracranial NK Cell Infusion
3.3 In Vivo MRI Imaging of 19F Labeled NK Cells
3.4 Determining 19F Labeling of NK Cells
3.5 Effect of 19F Labeling on NK Cell Function
4 Notes
References
Chapter 28: Generation of BiKEs and TriKEs to Improve NK Cell-­Mediated Targeting of Tumor Cells
1 Overview
1.1 Mediation of NK Cell Function Through CD16 and ADCC
1.2 Potential Advantages of BiKEs and TriKEs Over Conventional Antibodies
1.3 Bi- and Tri-specific Reagents Targeting NK Cells and Tumors
2 Methodology
2.1 Selection of Variable Fragment Source and Linkers
2.2 Selection of Vector and Expression System
2.3 Cloning the BiKE/TriKE Components into Expression Vector
2.4 Expression and Isolation of the BiKEs/ TriKEs
2.5 Testing the BiKEs and TriKEs
3 Future Directions
References
Chapter 29: Regulatory Considerations for NK Cells Used in Human Immunotherapy Applications
1 Introduction
2 Chemistry, Manufacturing, and Control (CMC)
3 Standard Operating Procedures (SOPs)
4 Specific Issues in Manufacturing NK Cells that Affect Product Safety
4.1 The Use of Products Not Approved for Use in Humans
4.1.1 Qualification of Source Leukocytes
4.1.2 Qualification of Feeder Cell Lines
4.2 Cell Culture
4.2.1 Media
4.2.2 Serum vs. Serum-Free
4.2.3 Antibiotics
4.2.4 Closed Systems
4.2.5 Final Formulation
4.2.6 Cryopreservation
4.3 Release Criteria
4.3.1 Identity/Purity: Inclusion of Desired Cells
4.3.2 Identity/Purity: Exclusion of Undesirable Cells
4.3.3 Sterility Testing
4.3.4 Potency
5 Notes
References
Index