Invariant Natural Killer T-Cells: Methods and Protocols (Methods in Molecular Biology, 2388)

✍ Scribed by Chaohong Liu (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 199
• Category: Library

✦ Synopsis

This detailed book focuses on various aspects of Invariant Natural Killer T-Cells (iNKT), which are known to contribute to homeostasis and autoimmunity and can also cause various pathological responses such as allergy, infection, excessive autoimmune response, and cancer. The volume explores techniques for identification and isolation of iNKT cells, iNKT cell activation and transformation, as well as proliferation and differentiation and much more. Written for the highly successful Methods in Molecular Biology series, 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.
Authoritative and practical, Invariant Natural Killer T-Cells: Methods and Protocols is an ideal guide for researchers working with and studying iNKT cells, perhaps to provide a deeper knowledge of the human immune system.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Genetic Analysis of iNKT Cell Development and Function
1 Introduction
1.1 General Development of NKT Cells
1.2 Type I NKT Cells or iNKT Cells
1.3 Type II NKT Cells
1.4 Detection of NKT Cells
2 Materials
3 Methods
3.1 Prepare Solutions
3.2 Cell Collection
4 Notes
References
Chapter 2: Genetic Studies of Natural Glycosphingolipid Ligands for NKT Cells
Abbreviations
1 Introduction
2 Materials
2.1 Mice and Embryonic Stem (ES) Cells
2.2 GSLs (Chemically Synthesized, Note 1)
2.3 NKT Cell Hybridoma Cells (Note 2)
2.4 Antibodies and Glycolipid Tetramers
3 Methods
3.1 Generation of Mice Deficient of FUT1, FUT2, and Sec1 (Note 3)
3.2 Generation of Mice Deficient of iGb3 Synthase (A3galt2)
3.3 Generation of Mice Deficient of FUT1/FUT2/Sec1/A3galt2
4 Notes
References
Chapter 3: Retroviral Transduction of NKT Hybridoma Cells
1 Introduction
2 Materials
3 Methods
3.1 Retroviral Package in HEK 293T
3.2 Concentration of Retrovirus by Centrifugation (See Note 5)
3.3 Transduction of DN32.D3 by Retrovirus (See Note 9)
3.4 Determination of Transduction Efficiency by Flow Cytometry (See Note 13)
4 Notes
References
Chapter 4: Methods for Studying Mouse and Human Invariant Natural Killer T Cells
1 Introduction
2 Materials
2.1 Generation of Mouse iNKT Cells
2.1.1 Isolate and Expand mMNC-iNKT Cells
2.1.2 Generate mHSC-iNKT Cells
2.2 Generation of Human iNKT Cells
2.2.1 Isolate and Expand hPBMC-iNKT Cells
2.2.2 Generate hHSC-iNKT Cells
2.3 Characterization of Mouse or Human iNKT Cells
2.3.1 Phenotype Analysis of Mouse or Human iNKT Cells
2.3.2 Function Analysis of Mouse or Human iNKT Cells
3 Methods
3.1 Generation of mMNC-iNKT Cells
3.1.1 Prepare MNCs from Mouse Spleen and Liver (Fig. 1a)
3.1.2 Magnetic Separation of mMNC-iNKT Cells
3.1.3 In Vitro Expansion of mMNC-iNKT Cells
3.2 Generation of Mouse HSC-iNKT Cells (mHSC-iNKTs)
3.2.1 Generate HSC-iNKT Mouse (See Note 12) (Fig. 1c)
3.2.2 Isolate and Expand mHSC-iNKT Cells (Refer Subheading 3.1)
3.3 Generation of hPBMC-iNKT Cells
3.3.1 Isolate PBMCs from Human Peripheral Blood (Fig. 2a)
3.3.2 Magnetic Separation of hPBMC-iNKT Cells
3.3.3 In Vitro Expansion of hPBMC-iNKT Cells
3.4 Generation of Human HSC-iNKT Cells
3.4.1 Generate HSC-iNKT Humanized Mouse (Fig. 2c) (See Note 21)
3.4.2 hHSC-iNKT Cells from Humanized Mouse (Fig. 2d)
3.4.3 In Vitro Expansion of hHSC-iNKT Cells
3.5 Characterization of Mouse or Human iNKT Cells
3.5.1 iNKT Cell Phenotype Analysis
3.5.2 iNKT Cell Function Analysis
4 Notes
References
Chapter 5: Isolation and Detection of Murine iNKT Cells in Different Organs
1 Introduction
1.1 The Characteristics of iNKT Cells
1.2 The Function of iNKT
1.3 iNKT Cells Development
1.4 The Subtypes of iNKT Cells
2 Materials
2.1 Mice
2.2 Materials and Solutions
2.3 Equipment
2.4 Software
3 Methods
3.1 Preparation of Tissue Samples from C57BL/6, CD1d-/-, and Jα18-/- Mice
3.2 Isolation of Splenocytes
3.3 Isolation of Thymocytes
3.4 Harvest Lymphocytes from the Liver
3.5 Detection iNKT Cells by Flow Cytometry
3.6 Representative Results
3.7 Discussion
4 Notes
References
Chapter 6: Identifying, Isolation, and Functional Use of Human Liver iNKT Cells
1 Introduction
2 Materials
3 Methods
3.1 Sample Collection and Preparation
3.2 Liver Cells Preparation
3.3 Isolation of Mononuclear Cells from Liver Sample
3.4 One Tube Innate T Cells for Identifying iNKT Cells and MAIT Cells
3.5 Gating Strategy and Sample Analysis
3.6 Cytokine Production by Innate T Cells
3.7 Functional Assay of iNKT Cells
4 Notes
References
Chapter 7: Isolation and Characterization Methods of Human Invariant NKT Cells
1 Introduction
2 Materials
2.1 Isolation of Invariant NKT Cells by Immunomagnetic Beads
2.2 Isolation of Invariant NKT Cells by Flow Cytometry
2.3 Recognition and Quantitation of Invariant NKT Cells
3 Methods
3.1 Isolation of Invariant NKT Cells by Immunomagnetic Beads
3.2 Isolation of Invariant NKT Cells by Flow Cytometry
3.3 Recognition and Quantitation of Invariant NKT Cells
4 Notes
References
Chapter 8: Detection of Mouse Type I NKT (iNKT) Cells by Flow Cytometry
1 Introduction
2 Materials
2.1 Mouse Tissue Experiment Materials
2.1.1 Mice
2.1.2 Media and Buffers
2.1.3 Instruments and Plasticwares
2.1.4 Antibodies, Staining Kit, and Other Fluorochrome-Labeled Reagents
3 Methods
3.1 Detection of Type I NKT Cells from Mouse Tissue by Flow Cytometry
3.1.1 Preparation of Single-Cell Suspension from a Mouse Spleen
3.1.2 Preparation of Single-Cell Suspension from the Thymus and Lymph Nodes
3.1.3 Staining Dead Cells
3.1.4 Cell Surface Protein Staining
3.1.5 Intranuclear Transcription Factor Staining
3.1.6 Data Acquisition
3.1.7 Data Analysis
Type I NKT Cell Identification Gating Strategy
Identification of Type I NKT Cell Subsets
4 Notes
References
Chapter 9: Identification of Rare Thymic NKT Cell Precursors by Multiparameter Flow Cytometry
1 Introduction
2 Materials
2.1 Equipment
3 Methods
3.1 Staining Design
3.2 Isolation of Thymic NKT Cells
3.2.1 Preparing the Thymocyte Suspension and Enriching for NKT Cells by Depleting CD8-Positive Cells
3.2.2 Enriching NKT Cells from CD8-Depleted Thymocytes by Positive Selection
3.2.3 Surface Staining
4 Notes
References
Chapter 10: iNKT Cel Transfer: The Use of Cell Sorting Combined with Flow Cytometry Validation Approach
1 Introduction
2 Materials
2.1 Mouse Strains
2.2 General Reagents
2.3 Equipment
3 Methods
3.1 Isolation of Splenocytes from WT Mice (Donor) and iNKT Cells Staining
3.2 WT iNKT Cell Sorting and Cell Transfer into Jα18-/- Mice (Recipient)
3.3 iNKTs Detection by Flow Cytometry in Liver of Jα18-/- Mice After WT Cells Transference
4 Notes
References
Chapter 11: Expansion of Human iNKT Cells Ex Vivo
1 Introduction
2 Materials
2.1 Preparation of Human PBMCs from Human Peripheral Blood
2.2 Expansion of iNKT Cells
2.3 Expansion of DCs
2.4 Assessment of the Purity of iNKT Cells in the Cell Products
2.5 Measurement of Cytokine Secreted by iNKT Cells
3 Methods
3.1 Preparation of Human PBMCs from Human Peripheral Blood
3.2 Culture of Human iNKT Cells
3.3 Assessment of iNKT Cell Population by FACS
3.4 Assessment of the Antitumor Functionality of iNKT Cells
4 Notes
References
Chapter 12: The Expansion and Cytotoxicity Detection of Human iNKT Cells
1 Introduction
2 Materials
2.1 Expansion of Human iNKT Cells In Vitro
2.2 Isolation of Human iNKT Cells
2.3 Cytotoxicity Assay of Human iNKT Cells
3 Methods
3.1 Human iNKT Cells Expansion from Human Peripheral Blood Mononuclear Cells (PBMCs)
3.1.1 Preparation of Human Peripheral Blood Mononuclear Cells (PBMCs)
3.1.2 Human iNKT Cells Expansion in Response to α-GalCer/IL-2 In Vitro
3.2 Cytotoxicity Assay of Human iNKT Cells
3.2.1 Enrichment of Human iNKT Cells
3.2.2 Prepare of Targets
3.2.3 Cytotoxicity Assay
4 Notes
References
Chapter 13: Ex Vivo Expansion of Th2-Polarizing Immunotherapeutic iNKT Cells from Human Peripheral Blood
1 Introduction
2 Materials
2.1 iNKT Cell Expansion and Culture
2.2 Flow Cytometry and Cell Sorting
2.3 Functional Assay: Antitumor Cytotoxicity
3 Methods
3.1 iNKT Expansion
3.2 iNKT Yield and Phenotype Assessment by Flow Cytometry
3.3 iNKT Functional Assay: 51Cr Release Antitumor Cytotoxicity Assay
4 Notes
References
Chapter 14: Intravital Microscopy Imaging of Invariant Natural Killer T-Cell Dynamics in the Liver Using CXCR6-eGFP Transgenic...
1 Introduction
2 Materials
2.1 Mice
2.2 Reagents
2.3 Equipment
2.4 Reagent and Equipment Setup
3 Methods
3.1 Preoperative Preparation of Mouse
3.2 Surgery for Liver IVM
3.3 IVM Observation
4 Notes
References
Chapter 15: In Vivo Cytotoxicity by α-GalCer-transactivated NK Cells
1 Introduction
2 Materials
2.1 General Instruments, Plasticware, and Buffers
2.2 Mice
2.3 Materials and Reagents for Treatment Procedures
2.4 Instruments and Materials for Tissue Processing
2.5 Labeling and Injection of Target Cells
2.6 Immunophenotyping (If Applicable)
3 Methods
3.1 General Considerations
3.2 Treatment of Recipient Mice
3.3 Isolation of Splenocytes from Donor Mice
3.4 Labeling and Injection of Target Cells
3.5 Processing of Recipient Mouse Tissues
3.5.1 Liver MNC Isolation
3.5.2 Lung MNC Isolation
3.6 Data Acquisition and Analysis
3.7 Verifying the Contribution of NK Cells to Enhanced β2M-/- Target Cell Clearance in α-GalCer-Treated Recipients
4 Notes
References
Chapter 16: Redirecting iNKT Cell Antitumor Immunity with α-GalCer/CD1d-scFv Fusion Proteins
1 Introduction
2 Materials
2.1 Recombinant CD1d Fusion Proteins
2.2 CD1d Tetramer
2.3 Antibodies Used for Flow Cytometry Analysis
2.4 Buffers
3 Methods
3.1 Administration of α-GalCer/CD1d-scFv Fusion Proteins
3.2 Preparation of Single-Cell Suspension
3.3 Surface Staining
3.4 Fixation and Permeabilization
3.5 Intracellular Staining
4 Notes
References
Chapter 17: Investigating the Dynamic Changes in iNKT Cell Metabolic Profiles During Development
1 Introduction
2 Materials
2.1 Preparation of an Enriched Single-Cell Suspension from the Mouse Thymus
2.2 Flow Cytometric Assessment of iNKT Cell Metabolism
2.3 Agilent SeahorseSeahorse Metabolic Flux Analysis of iNKT Cells
2.4 Transcriptome-Based Bioinformatics to Assess iNKT Cell Metabolism
3 Methods
3.1 Preparation of iNKT Cells from the Thymus for Flow Cytometry and Cell Sorting
3.2 Flow Cytometric Assessment of iNKT Cell Metabolism
3.3 Agilent SeahorseSeahorse Analysis of iNKT Cell Glycolysis and OXPHOSOxidative phosphorylation (OXPHOS)
3.4 Transcriptome-Based Bioinformatic Assessment of iNKT Cell Metabolism
4 Notes
References
Index