T-Cell Receptor Signaling: Methods and Protocols (Methods in Molecular Biology, 2111)

Preface
Contents
Contributors
Chapter 1: Exploration of T-Cell Diversity Using Mass Cytometry
	1 Introduction
	2 Materials
		2.1 Antibody Conjugation
		2.2 Staining
		2.3 Acquisition and Normalization
		2.4 Analysis
	3 Methods
		3.1 Panel Design
		3.2 Antibody Conjugation
		3.3 Antibody Titration
		3.4 Staining
		3.5 Acquisition and Normalization
		3.6 Analysis
			3.6.1 Predefined Analysis
			3.6.2 Semi-Biased Analysis
	4 Notes
	References
Chapter 2: A Carrier Strategy for Mass Cytometry Analysis of Small Numbers of Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Carrier Cells
		2.2 Preparation of PBMCs
		2.3 Mass Cytometry Cell Staining
	3 Methods
		3.1 Carrier Cell Labeling
		3.2 Thawing PBMCs
		3.3 Mix Carrier Cells and PBMCs
		3.4 Cisplatin Labeling
		3.5 Surface Staining of PBMCs
		3.6 Intercalator Staining
		3.7 Loading Sample on CyTOF
		3.8 High-Dimensional Data Analysis
	4 Notes
	References
Chapter 3: Simultaneous Measurement of Surface Proteins and Gene Expression from Single Cells
	1 Introduction
	2 Materials
		2.1 Single-Cell RNA-seq Material
			2.1.1 Reagent and Supply
			2.1.2 Equipment
		2.2 Other Materials for CITE-seq
	3 Methods
		3.1 Single-Cell RNA-seq Prep
		3.2 ADT Library Construction
			3.2.1 Live Cell Staining
			3.2.2 Run 10x Genomics (Single Cell 3′ Reagent Kits v2) as Described in the Link at Subheading 3.1 Single-Cell RNA-seq Prep Un...
			3.2.3 ADT-Derived cDNA and mRNA-Derived cDNA Separation (See Note 9)
			3.2.4 Final ADT Library Construction
				Purify ADT-Derived cDNAs
				Amplify ADT-Derived cDNAs
				Final ADT Library Construction
				QC for ADT Library
		3.3 Sequencing
		3.4 Analysis
	4 Notes
	References
Chapter 4: Analysis of Transcriptional Profiling of Immune Cells at the Single-Cell Level
	1 Introduction
		1.1 T-Cell Receptors and V(D)J Recombination
		1.2 Transcriptional Profiling at the Single-Cell Level
		1.3 Immune Cell Profiling with 10xGenomics 5 Prime V(D)J Preparation
		1.4 Tools for Visualization Analysis of Single-Cell TCR Sequencing Data
	2 Materials
		2.1 Computer
		2.2 Data Matrix Files
	3 Methods
		3.1 Formatting V(D)J Data Matrices to Use for Annotating the Expression Data
		3.2 Loading the 5′ Expression Dataset and the V(D)J Matrix to R as a Seurat Object
		3.3 Combining the Gene Expression and V(D)J Seurat Objects
		3.4 Annotating Each VDJ Cell According to Which TRAV Gene ItExpresses
	4 Notes
	References
Chapter 5: CRISPR/Cas9-Based Genetic Screening to Study T-Cell Function
	1 Introduction
	2 Materials
		2.1 Generating a Jurkat T-Cell Line Expressing Functional Cas9
		2.2 Lentiviral Packaging of sgRNA Library
		2.3 T-Cell Activation-Based Screening
		2.4 Genomic DNA Extraction
	3 Methods
		3.1 Generating a Jurkat T-Cell Line Expressing Functional Cas9
			3.1.1 Packaging of the Cas9 Lentivirus
			3.1.2 Transduction of Jurkat T Cells with Cas9 Lentivirus
			3.1.3 Testing the Function Activity of Transduced Cas9 in Jurkat Cells
		3.2 Packaging and Transducing Lentiviruses Encoding the sgRNA Library
			3.2.1 Amplification of sgRNA Library
			3.2.2 Production and Titering of Lentiviruses Encoding the sgRNA Library
			3.2.3 Infection of Cas9+ Jurkat Cells with Lentiviruses Encoding the sgRNA Library
		3.3 T-Cell Activation-Based Screening
			3.3.1 T-Cell Activation
			3.3.2 CD69 Staining and FACS Sample Preparation
		3.4 Genomic DNA Extraction for High-Throughput Sequencing
	4 Notes
	References
Chapter 6: Preferential Expansion of CD4+Foxp3+ Regulatory T Cells (Tregs) In Vitro by Tumor Necrosis Factor
	1 Introduction
	2 Materials
		2.1 Single Cell Suspension Preparation and CD4+ T-Cell Enrichment
		2.2 Culture of Unfractionated CD4+ T Cells with TNF
	3 Methods
		3.1 Collection of Spleen and Lymph Nodes and Single-Cell Suspension Preparation
		3.2 The Enrichment of CD4+ T Cells
		3.3 CFSE Division Assay on CD4+ T Cells Following Stimulation with TNF
		3.4 Immunostaining and Flow Cytometry Analysis of TNF-Expanded Tregs
	4 Notes
	References
Chapter 7: In Vitro Differentiation of CD4+ T Cell Effector and Regulatory Subsets
	1 Introduction
	2 Materials
		2.1 Coating Plates for T Cell Activation
		2.2 Lymphocyte Harvest
		2.3 Magnetic Selection
		2.4 Naïve T Cell FACS Sort
		2.5 T Cell Plating and Activation/Polarization
	3 Methods
		3.1 Coating Plates for T Cell Activation
		3.2 Lymphocyte Harvest
		3.3 Magnetic Selection (See Note 11)
		3.4 Naïve T Cell FACS Sort
		3.5 T Cell Plating for Activation and Polarization
	4 Notes
	References
Chapter 8: CD4+ T-Cell Differentiation In Vitro
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Materials for Naive CD4+ T-Cell Sorting
		2.3 Materials for Plate Coating and Cell Culture
		2.4 Materials for T-Cell Differentiation and Examination
	3 Methods
		3.1 Isolation of Naïve CD4+ T Cells
		3.2 Differentiation of Th and Treg Cells
		3.3 Analysis of Differentiation of Th Cells In Vitro
			3.3.1 Intracellular Staining and Flow Cytometry Analysis
			3.3.2 mRNA Extraction and Real-Time Quantitative PCR Analysis
	4 Note
	References
Chapter 9: Characterization of Immune Cell Subset Expansion in Response to Therapeutic Treatment in Mice
	1 Introduction
	2 Materials
		2.1 Plastics
		2.2 Antibodies
		2.3 Blocking Agents
		2.4 Viability Dye
		2.5 Buffers
		2.6 Instruments
	3 Methods
		3.1 Animal Treatments
		3.2 Preparation of Single-Cell Suspensions
		3.3 Staining of Surface Antigens with Fluorescent Antibodies
		3.4 Fixation, Permeabilization, and Staining of Intracellular Markers with Fluorescent Antibodies
		3.5 Process Immune Cells via Flow Cytometry
		3.6 Analyze Flow Cytometry Data
	4 Notes
	References
Chapter 10: Primary T-Cell Transduction to Study Follicular Helper T-Cell Differentiation
	1 Introduction
	2 Materials
		2.1 Construction of Retroviral Vector
		2.2 Mice
		2.3 Recombinant Retrovirus Preparation and Cell Culture
		2.4 Adoptive Transfer, Immunization, and Flow Cytometry Analysis
	3 Methods
		3.1 Construction of Retroviral Expression Vector
		3.2 Preparation of Recombinant Retrovirus, in Vitro Culture of Primary CD4+ T Cells, and Spinfection of the Cultured Primary C...
			3.2.1 Preparation of Recombinant Retrovirus
			3.2.2 Purification of Mouse CD4+ T Cells (See Note 4)
			3.2.3 Stimulation of Primary CD4+ T Cells (See Note 6)
			3.2.4 Spinfection and Rest of CD4+ T Cells
		3.3 Adoptive Transfer and OVA-Alum Immunization
			3.3.1 Adoptive Transfer of CD4+ T Cells
			3.3.2 OVA-Alum Immunization of Recipient Mice
		3.4 Analysis of Transduced Cells
			3.4.1 Cell Preparation from Spleen
			3.4.2 Surface Staining
			3.4.3 Flow Cytometer Analysis
	4 Notes
	References
Chapter 11: In Vitro Generation of Stem Cell Memory-Like T Cells from Activated T Cells
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Cell Culture
		2.3 Antibodies (Ab) and Fluorophores
		2.4 Equipment
		2.5 Mice
	3 Methods
		3.1 Human CD8+ T-Cell Isolation
		3.2 Co-culturing of Human CD8+ T Cells with Autologous LCL
		3.3 Isolation of EBV-Specific T Cells with Central Memory Phenotypes
		3.4 Co-culturing of EBV-Specific T Cells with OP9-hDLL1 Cells
		3.5 Isolation and Analysis of iTSCM Cells
		3.6 Adoptive iTSCM Cell Transfer to Tumor-Bearing NSG Mice
	4 Notes
	References
Chapter 12: Artificial Antigen Presentosomes for T Cell Activation
	1 Introduction
	2 Materials
		2.1 Plasmids and E. coli Strains
		2.2 Antibodies
		2.3 Protein Purification
		2.4 Flow Cytometry
		2.5 ELISA
	3 Methods
		3.1 Recombinant Protein Production
		3.2 Refolding and FPLC Purification
		3.3 Direct and Competitive ELISA
		3.4 Immunoprecipitation
		3.5 AAP Construction
		3.6 Antigen-Specific T Cell Activation and Proliferation
	4 Notes
	References
Chapter 13: Imaging Chimeric Antigen Receptor (CAR) Activation
	1 Introduction
	2 Materials
	3 Methods
		3.1 Construction of Jurkat CAR T Cells
		3.2 Preparation of Small Unilamellar Vesicles (SUV) for Making Membranes (We follow our previous protocol for making SUV and  ...
		3.3 Preparation of Antigen-Functionalized Supported Lipid Bilayers (See Note 7)
		3.4 Imaging CAR T Cell Activation
	4 Notes
	References
Chapter 14: Assessing the Impact of Phytochemicals on Immune Checkpoints: Implications for Cancer Immunotherapy
	1 Introduction
		1.1 Phytochemicals
		1.2 Cancer Treatment Strategies
		1.3 Immune Checkpoints
		1.4 Impact of Natural Products on T Cell Checkpoints
	2 Materials
		2.1 Cell Culture
		2.2 Determination of PD-L1 and/or PD-L2 Expression
		2.3 T Cell Isolation from Mice
		2.4 T Cell Proliferation: Tritiated Thymidine Incorporation
		2.5 T Cell Proliferation: Oregon Green 488 Staining
		2.6 Interleukin-2 (IL-2) Production
		2.7 T Cell-Cancer Cell Co-cultures
	3 Methods
		3.1 Cell Culture: Adherent Cancer Cells
		3.2 Cell Culture: Non-adherent Jurkat T Cells
		3.3 Cell Counting
		3.4 Determination of PD-L1 Expression
		3.5 T Cell Isolation from Mice
		3.6 T Cell Proliferation: Tritiated Thymidine Incorporation
		3.7 T Cell Proliferation: Oregon Green 488 Staining
		3.8 IL-2 Production
		3.9 T Cell-Cancer Cell Co-cultures
	4 Notes
	References
Chapter 15: Assessment of Immune Protective T Cell Repertoire in Humans Immunized with Novel Tuberculosis Vaccines
	1 Introduction
	2 Basic Methods
		2.1 Media, Buffers, and Other Solutions
		2.2 Peptide Pool Preparation
	3 Methods
		3.1 Human IFN-γ ELISpot Assay by Using Fresh PBMC to Determine T Cell Reactivity to Peptide Pools Spanning the Protein Express...
		3.2 Three-Dimensional Matrix Design for the Preparation of New Peptide Pools to Identify T Cell-Reactive Single Peptide
		3.3 Human IFN-g ELISpot Assay by Using Frozen PBMC to Determine Maximum T Cell-Reactive Single Peptide
		3.4 Ag-Specific T Cell Expansion in PBMC to Identify Single Peptide Reactive CD4 and CD8 T Cells
		3.5 Intracellular Cytokine Staining and Flow Cytometry to Identify Single Peptide Reactive CD4 and CD8 T Cells in Expanded Ag-...
		3.6 Mycobacterial Growth Inhibition Assay (MGIA) Using BCG to Evaluate Mycobacterial Growth Inhibition by Vaccine-Induced T Ce...
		3.7 HLA-Associated Recognition of Identified Single Peptides by Using Immune Epitope Database (IEDB) and Clinical Relevance of...
	4 Notes
	References
Chapter 16: Retroviral Gene Transduction into T Cell Progenitors for Analysis of T Cell Development in the Thymus
	1 Introduction
	2 Materials
		2.1 Preparation of Retroviral Supernatant
		2.2 Gene Transduction into Fetal T Cell Progenitors
		2.3 Gene Transduction into Adult T Cell Progenitors
	3 Methods
		3.1 Preparation of Retroviral Supernatant
		3.2 Gene Transduction into Fetal T Cell Progenitors
		3.3 Gene Transduction into Adult T Cell Progenitors
	4 Notes
	References
Chapter 17: Testing the Efficiency and Kinetics of Negative Selection Using Thymic Slices
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Low-Melting Point (LMP) Agarose
		3.2 Dissection of Thymus for Slices Preparation
		3.3 Embedding the Thymus in LMP Agarose
		3.4 Cutting Slices with Vibratome
		3.5 Harvesting and Labeling of Thymocytes
		3.6 Overlaying Thymocytes on Slices
		3.7 Slice Dissociation and Flow Cytometry Analysis
		3.8 Analyzing the Flow Cytometry Data and Calculating the Cell Loss
	4 Notes
	References
Chapter 18: Investigating T Cell Receptor Signals In Situ by Two-Photon Microscopy of Thymocytes Expressing Genetic Reporters ...
	1 Introduction
	2 Materials
		2.1 5-Fluorouracil (5-FU) Injection
		2.2 BM Harvest
		2.3 Retroviral Transduction of BM Cells
		2.4 Neonatal Injection
		2.5 Thymic Lobe Harvesting, Slicing, and Preparation for Imaging
		2.6 Two-Photon Imaging of Thymic Tissue and Analysis
	3 Methods
		3.1 5-FU Injection
		3.2 BM Harvest
		3.3 Retroviral Transduction of BM Cells
		3.4 Neonatal Injection
		3.5 Thymic Lobe Harvesting, Slicing, and Preparation for Imaging
		3.6 Two-Photon Imaging of Thymic Tissue and Analysis
	4 Notes
	References
Chapter 19: An Integrated Strategy for Identifying Targets of Ubiquitin-Mediated Degradation in CD4+ T Cells
	1 Introduction
	2 Materials
		2.1 T Cell Preparation
		2.2 K-ε-GG Pulldown
		2.3 Whole-Cell Proteome Preparation
		2.4 RNA-Seq Preparation
		2.5 Data Analysis
		2.6 Validation
	3 Methods
		3.1 Preparing Samples for Mass Spectrometry and RNA Sequencing
		3.2 K-ε-GG Pulldown
		3.3 Whole-Cell Proteome Preparation
		3.4 RNA-Seq Preparation
		3.5 RNA-Seq Data Processing
		3.6 Whole-Cell Proteome Analysis
		3.7 K-ε-GG Enrichment Analysis
		3.8 Validating Substrate Ubiquitination
		3.9 Validating Substrate Degradation
	4 Notes
	References
Chapter 20: Radioisotope-Based Protocol for Determination of Central Carbon Metabolism in T Cells
	1 Introduction
	2 Materials
		2.1 Radiolabeled Tracers (Store at -20 C or 4 C for Short-Term Storage)
		2.2 Other Reagents and Equipment
	3 Methods
		3.1 Pre-coat 48-Well Plates at Day -1
		3.2 T Cell Isolation at Day 0
		3.3 Incubate T Cells in Flux Reactions at Day 1 or Other Selected Time Points
		3.4 3H-Based Glycolysis and FAO (in 48 Well Plate) (See Note 2)
		3.5 14C-Based Glutamine, Pyruvate, and Glucose Oxidation (in Septum Glass Vials)
		3.6 Prepare Scintillation Solution of 3H-Based Samples at Day 2
		3.7 Prepare Scintillation Solution of 14C-Based Samples at Day 2
		3.8 Read the Results
	4 Notes
	References
Chapter 21: Studying Peripheral T Cell Homeostasis in Mice: A Concise Technical Review
	1 Introduction
		1.1 Homeostasis of T Cells in Nonlymphopenic Hosts
		1.2 Homeostasis of T Lymphocytes in Lymphopenic Hosts
		1.3 Studying T Cell Homeostasis in Mice
		1.4 Source of T Cells to Study T Cell Homeostasis
		1.5 Mouse Models to Study T Cell Homeostasis
	2 Materials
		2.1 Buffers and Equipment
	3 Methods
		3.1 Tissue Collection and Preparation of Cell Suspension
		3.2 Cell Counting and Viability Analysis
		3.3 Enumeration of LN Cells
		3.4 T Cell Enrichment with the Stem Cell Kit
			3.4.1 Kit Reagents Stored at 4 C
			3.4.2 Procedure
		3.5 Evaluation of T Cell Purity
		3.6 Evaluation of T Cell Recovery
			3.6.1 Labelling of Lymphocytes with CFSE or CTV
		3.7 Adoptive Transfer in Lymphopenic and Genetically Modified Recipients
		3.8 Evaluation of Transferred T Cells by Flow Cytometry
			3.8.1 Sacrifice of Mice
			3.8.2 Antibody Staining
			3.8.3 Flow Cytometry
	4 Results and Discussion
	References
Chapter 22: Detection, Expansion, and Isolation of Human MAIT Cells
	1 Introduction
	2 Material
		2.1 Detection of MAIT Cells
		2.2 Expansion of MAIT Cells
		2.3 Isolation of MAIT Cells
	3 Method
		3.1 Detection of MAIT Cells
			3.1.1 Preparation of Human Peripheral Blood Mononuclear Cells (PBMCs)
			3.1.2 Detection of Human MAIT Cells by Flow Cytometry
		3.2 Expansion of Human MAIT Cells
			3.2.1 Preparation of Artificial Antigen-Presenting Cells
			3.2.2 Expansion of Human MAIT Cells
		3.3 Magnetic Bead Sorting of MAIT Cells
	4 Notes
	References
Index