T-Cell Development: Methods and Protocols

Preface to the Second Edition
Contents
Contributors
Part I: Background Information
	Chapter 1: A Beginner´s Guide to T Cell Development
		1 T Cells and T Cell Antigen Recognition
		2 An Overview of T Cell Development
			2.1 A Brief Thymic Tour
			2.2 Early T Cell Development: The Common Core
			2.3 Emergence of the αβ Lineage
			2.4 Selection of αβ T Cells: Basic Principles
			2.5 Signaling Positive Selection
			2.6 Central Tolerance and Negative Selection
			2.7 Positive Versus Negative Selection Signals and Mechanisms
			2.8 Differentiation of αβ Lineage Cells and Thymic Egress
			2.9 High-Avidity Cells and Acquisition of Effector Properties
		3 Thymic Architecture
		4 Tracking Developing Thymocytes
			4.1 Early T Cell Development
			4.2 αβ T Cell Development
		5 Concluding Remarks
		References
	Chapter 2: Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing...
		1 Introduction
		2 cTEC Characteristics and Functional Roles
		3 mTEC Subset Characteristics and Functional Roles
		4 Single-Cell Assessment of TEC Heterogeneity and Diversity
			4.1 Reclassification of the mTEC Compartment into Four Major Groups
			4.2 TRA Signature, Another Trait of TEC Heterogeneity
			4.3 TEC Transcriptional Changes During Development and Aging
		5 The Use of Lineage Tracing to Identify TEC Progenitor Populations and Parent-Daughter Relationships
		6 Conclusions
		References
	Chapter 3: Early Development of Innate Lymphoid Cells
		1 Introduction
		2 Progenitors in Early ILC Development
			2.1 Lymphoid Progenitors: LMPP, CLP, and ALP
			2.2 EILP
			2.3 ILCP
			2.4 Other Described Early ILC Progenitors
				2.4.1 CHILP
				2.4.2 αLP
				2.4.3 Committed and/or Lineage-Biased ILC Precursors
			2.5 Central Versus Peripheral ILC Progenitors
		3 Possible Mechanisms That Separate Innate Lymphoid Cell Lineage from T Cell Lineage
		4 Lineage Specification in Early ILC Development
			4.1 Killer Versus Helper ILC
			4.2 LTi Cell Versus Helper ILC
		5 Differences Between Fetal and Adult Early ILC Development
		6 Critical Transcription Factors in Early ILC Development
			6.1 ID2
			6.2 TCF-1
			6.3 GATA-3
			6.4 TOX
			6.5 NFIL3
		7 Concluding Remarks
		References
	Chapter 4: Development of γδ T Cells: Soldiers on the Front Lines of Immune Battles
		1 Introduction
		2 Antigen Recognition by γδ T Cells
		3 Links Between Vγ Usage and Behavior
		4 Development
			4.1 Phenotypic Characterization of γδ Cells During Fetal Ontogeny
			4.2 Cross Talk Between αβ and γδ Progenitors
			4.3 Control of αβ/γδ Lineage Commitment
			4.4 Acquisition of Effector Fate During Development in the Thymus
		5 Methodologies to Assess γδ T Cell Functions
		6 Conclusion
		References
	Chapter 5: Principles of Advanced Flow Cytometry: A Practical Guide
		1 Principles of Fluorescent Flow Cytometry
		2 Basic Aspects of Technology
			2.1 Conventional Flow Cytometers
			2.2 Spectral Flow Cytometers
		3 Common Misunderstandings of Flow Cytometry Concepts
			3.1 Autofluorescence Needs to Be Minimized
			3.2 Isotype Control Staining Is the Best Negative Control
			3.3 The More Fluorescent Reagent (Antibody) Is Used, the Better
			3.4 Fluorescent Spillover Compensation Should Be Avoided
			3.5 Use of Stained Cells Versus Stained Compensation Beads
			3.6 Use of Similar, but Not Identical Fluorescent Reagents for Spillover Compensation
			3.7 Understanding the Difference Between Fluorescent Spillover and Spectral Spreading Error
			3.8 Optimized Multicolor Immunofluorescent Panels
		4 Step-by-Step Guide of Creating a High-Dimensional Flow Cytometry Experiment on a Conventional Flow Cytometer
			4.1 Hypothesis
			4.2 Panel Design
			4.3 PMT Voltage Optimization
			4.4 Antibody Titration
			4.5 Compensation Optimization
			4.6 Data Acquisition and Analysis
		5 General Principles of Creating a High-Dimensional Flow Cytometry Experiment on a Spectral Flow Cytometer
			5.1 Hypothesis
			5.2 Panel Design
			5.3 PMT Voltage Optimization
			5.4 Antibody Titration
			5.5 Compensation with Spectral Unmixing
			5.6 Data Acquisition and Analysis
		References
Part II: Core Approaches and Strategies
	Chapter 6: Genetic Strategies to Study T Cell Development
		1 Introduction
		2 Lineage- and Stage-Specific Gene Disruption
		3 Fixing TCR Specificity to Analyze Selection Events
		4 Perspectives
		References
	Chapter 7: A Simplified Approach to Evaluating T Cell Development by Flow Cytometry
		1 Introduction
		2 Materials
			2.1 Removal and Preparation of Lymphoid Organs
			2.2 Cell Surface or Intracellular Staining with Fluorochrome-Conjugated Antibodies
		3 Methods
			3.1 Removal of Lymphoid Organs and Preparation of Cells
			3.2 Cell Surface Staining with Fluorochrome-Conjugated Antibodies
			3.3 Intracellular Staining for the FoxP3 (or Other Transcriptional Factors)
		4 Notes
		References
	Chapter 8: Purification of Thymocyte and T Cell Subsets
		1 Introduction
		2 Materials
		3 Methods
			3.1 Obtaining Single-Cell Suspensions
			3.2 Magnetic Bead Purification of T Cell Subsets by Depletion
			3.3 Magnetic Bead Enhancement Pre-flow Cytometric Sorting
			3.4 Isolation of Double Negative Thymocytes with Bead Purification
			3.5 Flow Cytometric Sorting
				3.5.1 Flow Cytometric Sorting: General Procedure
				3.5.2 Secondary Protocol for Sorting Populations 0.01%
		4 Notes
	Chapter 9: Generation of Bone Marrow Chimeras
		1 Introduction
		2 Materials
			2.1 Preparation of Recipient Mice (See Note 1)
			2.2 Donor Bone Marrow Preparation
			2.3 T Cell Depletion (Optional; See Note 3)
			2.4 Bone Marrow Injection
		3 Method
			3.1 Prepare Recipient Mice
			3.2 Prepare Bone Marrow Cell Suspension from Donor Mice
			3.3 T Cell Depletion (Optional)
			3.4 Bone Marrow Injection
		4 Notes
		References
	Chapter 10: Generation of Murine T Cell Effector Populations In Vitro
		1 Introduction
		2 Materials
			2.1 Blocking and Activating Antibodies
			2.2 Cytokines
			2.3 Magnetic Cell Subset Purification
			2.4 Medium Components
			2.5 Media
			2.6 Instruments and Accessories
			2.7 Antibodies for Flow Cytometry (Sorting or Purity)
			2.8 Cells
			2.9 Reagents for Cytokine Analysis
		3 Methods
			3.1 Preparation of Naïve T Cells
			3.2 Preparation of T Cell Depleted Spleen as a Source of APCs
			3.3 Alternative Protocol: Cultures Without APCs
			3.4 Preparation of Condition Mixes
			3.5 Culture Setup with APCs
			3.6 Culture Setup Without APCs
			3.7 Media Refresh and Expansion (See Note 13)
			3.8 Analysis at 96 h (d4 of Culture)
			3.9 Notes
		References
Part III: Specific Techniques
	Chapter 11: Large-Scale Isolation of Mouse Thymic Epithelial Cells
		1 Introduction
		2 Materials
			2.1 Dissociation of Stromal Cells from Thymus
			2.2 Enrichment of Thymic Stroma Cells
			2.3 Cell Surface Staining for Flow Cytometric Analysis and Cell Sorting
		3 Methods
			3.1 Dissociation of Stromal Cells from Thymus
			3.2 Enrichment of Thymic Stroma Cells
			3.3 Cell Surface Staining for Flow Cytometric Analysis
		4 Notes
		References
	Chapter 12: Generation of Retrogenic Mice to Investigate T Cell Development
		1 Introduction
		2 Materials
			2.1 Preparation of Retroviral Supernatant
			2.2 Preparation of Recipient Mice (See Note 4 and Chapter 9)
			2.3 Preparation of Donor Mice
			2.4 Isolation of Bone Marrow Cells from Donor Mice
			2.5 Bone Marrow Cells Activation and Transduction
			2.6 Bone Marrow Injection
		3 Methods (See Note 5)
			3.1 Preparation of Retroviral Supernatants
			3.2 Donor Mice Preparation and 5-FU Injection (Day -4) (See Note 13)
			3.3 Bone Marrow Cell Harvesting and Transduction
			3.4 Preparation of Recipient Mice and Bone Marrow Transplantation
		4 Notes
		References
	Chapter 13: Purification of Bone Marrow Precursors to T Cells and ILCs
		1 Introduction
			1.1 Multipotent Lymphoid Precursors
			1.2 Multipotent ILC Precursors
			1.3 Additional ILC Precursor Populations
			1.4 Strategies of Isolation of Bone Marrow Precursors to ILCs and T Cells by Flow Cytometry
			1.5 Description of Procedure
		2 Materials
			2.1 Bone Marrow Isolation and Single-Cell Suspension
			2.2 Pre-sort Depletion
			2.3 Antibody Staining
			2.4 Fluorescence-Activated Cell Sorting (FACS) or Flow Cytometric Cell Sorting
		3 Methods
			3.1 Bone Marrow Isolation and Single-Cell Suspension
			3.2 Pre-sort Depletion
			3.3 Antibody Staining
			3.4 Fluorescence-Activated Cell Sorting (FACS)
		4 Notes
		References
	Chapter 14: Studying T Cell Development in Neonatal and Adult Thymic Slices
		1 Introduction
		2 Materials
			2.1 Dissecting a Mouse Thymus
			2.2 Preparation of a Mouse Thymus for Slicing
			2.3 Embedding and Vibratome Slicing a Thymic Lobe
				2.3.1 Embedding a Thymic Lobe in Low-Melt Agarose
				2.3.2 Slicing a Thymic Lobe Using a Vibratome
			2.4 Plating Thymic Slices for Organotypic Culture
			2.5 Thymocyte and Peptide Overlay onto Adult Thymic Slices
				2.5.1 Peptide Overlay onto Adult Thymic Slices (for Negative Selection)
				2.5.2 Thymocyte Overlay onto Adult Thymic Slices
			2.6 Preparation of Thymic Slices for Flow Cytometry Analysis
			2.7 Visualizing Thymic Slices Using Two-Photon Imaging
				2.7.1 Preparation of Thymic Slices for Two-Photon Imaging
				2.7.2 Two-Photon Microscope Setup
		3 Methods
			3.1 Dissecting a Mouse Thymus
			3.2 Preparation of a Mouse Thymus for Slicing
			3.3 Embedding and Vibratome Slicing a Thymic Lobe
				3.3.1 Embedding a Thymic Lobe in Low-Melt Agarose
				3.3.2 Slicing a Thymic Lobe Using a Vibratome
			3.4 Plating Thymic Slices for Organotypic Culture
			3.5 Thymocyte and Peptide Overlay onto Adult Thymic Slices
				3.5.1 Peptide Overlay onto Thymic Slices (for Negative Selection)
				3.5.2 Thymocyte Overlay onto Adult Thymic Slices
			3.6 Preparation of Thymic Slices for Flow Cytometry Analysis
			3.7 Visualizing Thymic Slices Using Two-Photon Imaging
				3.7.1 Preparation of Thymic Slices for Two-Photon Imaging
				3.7.2 Two-Photon Microscope Setup
		4 Notes
		References
	Chapter 15: Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines
		1 Introduction
			1.1 Design and Construction of MICe-IL-7/FLT3L/SCF Plasmid
			1.2 OP9-DL4 Cells Transduced to Express Human IL-7, FLT3L, and SCF
		2 Materials
		3 Methods
			3.1 Isolation of CD34+ Mononuclear Cells from Umbilical Cord Blood (CB)
			3.2 Initiation and Maintenance of Coculture
		4 Notes
		References
	Chapter 16: Methods for Study of Mouse T Cell Receptor α and β Gene Rearrangements
		1 Introduction
			1.1 Biochemistry of Tcra and Tcrb Gene Rearrangements
			1.2 Structure and Recombination of Tcrb Loci
			1.3 Structure and Recombination of Tcra Loci
			1.4 Developmental Control of Tcra and Tcrb Recombination
			1.5 Molecular Analysis of Tcra and Tcrb Gene Rearrangements
			1.6 Quantification of Tcrb Gene Rearrangements
			1.7 Next-Generation Sequencing Analysis of Tcrb Gene Rearrangements
			1.8 Next-Generation Sequencing Analysis of Tcra Gene Rearrangements
		2 Materials
			2.1 TaqMan Quantitative Real-Time qPCR for Tcrb Rearrangements
			2.2 5′-RACE-Based NGS of Tcra Rearrangements
		3 Methods
			3.1 TaqMan Quantitative Real-Time qPCR for Tcrb Rearrangements
			3.2 5′-RACE-Based NGS of Tcra Gene Rearrangements
		4 Notes
		References
	Chapter 17: Ultrasound-Guided Intra-thymic Cell Injection
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Single-Cell Suspension of Thymocytes
			3.2 Magnetic Bead Enrichment of DN Thymocytes
			3.3 Ultrasound-Guided Intra-thymic Injection
		4 Notes
		References
	Chapter 18: Fetal Thymic Organ Culture and Negative Selection
		1 Introduction
		2 Materials
		3 Methods
			3.1 Timed Matings
			3.2 Preparing the Culture Dishes
			3.3 Removing Fetal Thymic Lobes
			3.4 Feeding
			3.5 Analysis
		4 Notes
		References
	Chapter 19: In Vitro Analysis of Thymocyte Signaling
		1 Introduction
		2 Materials
			2.1 Tissue Preparation
			2.2 Stimulation with Peptide-Pulsed APC (See Note 3)
			2.3 Stimulation with Peptide/MHC I Tetramers
			2.4 Confocal Microscopy
			2.5 IP-FCM
		3 Methods
			3.1 Harvesting Thymi (See Note 5)
			3.2 Stimulation with APC
				3.2.1 Peptide-Loaded APC
				3.2.2 Stimulation with Peptide-Loaded APC
				3.2.3 Stimulation with Peptide-MHCI or Peptide-MHCII Tetramers
			3.3 Confocal Microscopy
				3.3.1 Mounting Cells
				3.3.2 Permeabilization/Blocking
				3.3.3 Staining
				3.3.4 Mounting the Coverslip
			3.4 IP-FCM
				3.4.1 Generation of Ab-Coupled CML Beads
				3.4.2 Sample Lysis
				3.4.3 For FCM Staining of Beads
				3.4.4 By FCM Acquisition of Data
		4 Notes
		References
	Chapter 20: Identification and Purification of Human T Cell Precursors
		1 Introduction
		2 Materials
			2.1 Extraction of Human Thymocytes
			2.2 Lymphoprep Density Gradient for Isolation of Viable Mononuclear Cells
			2.3 MACS Direct CD34 Progenitor Cell Isolation Kit
			2.4 Flow Cytometric Cell Sorting
			2.5 CD3+ and CD8+ Depletion with Dynabeads
			2.6 Anti TCR-γδ Microbead Kit
		3 Methods
			3.1 Human Thymocyte Cell Suspension
			3.2 Purification of Viable Cells by Lymphoprep Density Gradient
			3.3 Isolation of Extrathymic CD34-Positive Cells Using Magnetic-Activated Cell Sorting (MACS)
			3.4 Purification of CD34+ Precursors by Fluorescence-Activated Cell Sorting (FACS)
			3.5 Isolation of Intrathymic CD34+ Cells
			3.6 Isolation of Immature Single Positive Cells (CD4+CD8-CD3-)
			3.7 Isolation of Double Positive and Single Positive (SP) Thymocytes
			3.8 Isolation of TCR γδ Cells
		4 Notes
		References
	Chapter 21: In Vitro Model Systems to Study Human T Cell Development
		1 Introduction
		2 Materials
			2.1 Freezing and Thawing of Human T Cell Progenitors
			2.2 Viral Transduction of Human T Cell Progenitors
			2.3 Preparation of OP9 Feeder Layers
			2.4 Initiation and Maintenance of OP9 Co-culture Experiments
			2.5 Culturing of MS5-hDLL4 Stromal Cells
			2.6 Preparation of ATO Complete Medium (R-B27 with Cytokines)
			2.7 Assembling ATOs
			2.8 Maintenance of ATO Cultures
			2.9 Collecting Cells from ATOs for Analysis
			2.10 Analysis of OP9 Co-culture and ATO Experiments
		3 Methods
			3.1 Freezing and Thawing of Human T Cell Progenitors
			3.2 Culture Progenitor Cells and Transduction
			3.3 Preparation of OP9 Feeder Layers
			3.4 Initiation and Maintenance of OP9 Co-culture Experiments
			3.5 Culturing of MS5-hDLL4 Stromal Cells
			3.6 Preparation of ATO Complete Medium (R-B27 Complete with Cytokines)
			3.7 Assembling ATOs
			3.8 Maintenance of ATO Cultures
			3.9 Collecting Cells from ATOs for Analysis
			3.10 Analysis of OP9 Co-culture and ATO Experiments
		4 Notes
		References
	Chapter 22: Zebrafish: A Tractable Model for Analysis of T Cell Development
		1 Introduction
		2 Materials
			2.1 Zebrafish Facility, Husbandry, and Culture of Embryos
			2.2 Zebrafish Lines
			2.3 Morpholino Design and Preparation
			2.4 CRISPR Design and Preparation
			2.5 Microinjection
			2.6 Transient Rescue Experiments
			2.7 Western Blot [25, 26]
			2.8 Whole-Mount TUNEL Assays
			2.9 Whole-Mount In Situ Hybridization
			2.10 Mounting Embryos for Observation
			2.11 Fluorescence-Activated Cell Sorting (FACS)
		3 Methods
			3.1 Identifying Zebrafish Ortholog
			3.2 Preparation of Microinjection Plates/Needles
			3.3 Preparation of Zebrafish Embryos
			3.4 Dechorionation
			3.5 FACS Sorting of Lineage-Marked T Cell Progenitors
			3.6 Morpholino Microinjection
			3.7 Western Blot Analysis
			3.8 Fluorescent Microscopy
			3.9 Whole-Mount TUNEL Assays
			3.10 Whole-Mount In Situ Hybridization
			3.11 Using Zebrafish to Screen for Human Disease-Causing Alleles in SCID
			3.12 CRISPR
			3.13 Insights into Knockdown Versus Knockout
		4 Notes
		References
Index