Regulatory T-Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Immunological Techniques for Analysing Treg
Chapter 1: Analysis of the In Vivo Function of Follicular Regulatory T (TFR) Cells in the Regulation of Antibody Response
1 Introduction
2 Materials
2.1 Mice
2.2 Immunization
2.3 Cell Purification and Enrichment
2.4 Cell Sorting
2.5 Flow Cytometry
2.6 ELISA
3 Methods
3.1 Immunization of Mice
3.2 Cell Purification and Enrichment
3.3 Sorting TFH and TFR Cells
3.4 Adoptive Transfer
3.5 Flow Cytometry
3.6 ELISA of Serum Antibody Titers
4 Notes
References
Chapter 2: Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation
1 Introduction
2 Materials
2.1 Common Reagents, Materials, and Solutions
2.2 Mice
2.3 Material for Adoptive Transfer of Treg and Tconv Cells and Bone Marrow Transfer
3 Methods
3.1 Adoptive Transfer
3.1.1 Harvesting Lymphoid Organs and Preparing Single-Cell Suspensions
3.1.2 Fluorescent Labelling and Cell Sorting
3.1.3 Preparation of Sorted Cells for Injection into Recipient Mice
3.1.4 Analysis of CD4+ T Cell Populations in Recipient Mice
3.2 BM Chimeras
3.2.1 Recipient Mice Irradiation Protocol (Day -1)
3.2.2 BM Collection (Day 0)
3.2.3 BM Cells Preparation (Day 0)
Complement Pretreatment
T-Ccell-Mediated Complement Depletion
3.2.4 BM Transfer into Recipient Mice (Day 0)
3.2.5 Analysis of BM Reconstitution (5-6 Weeks After Transfer)
3.2.6 Analysis of Tconv and Treg in BM Chimera
4 Notes
References
Chapter 3: Depletion of Treg by the Diphtheria Toxin System
1 Introduction
2 Materials
2.1 DT-Mediated Treg Depletion
2.2 Tissue Dissociation and Extraction of T Cells
2.3 Flow Cytometry
3 Methods
3.1 Systemic Depletion of Treg
3.2 Selective Depletion of Skin-Resident Treg
3.3 Preparing Single-Cell Suspensions from Mouse Ear Skin
3.4 Preparing Single-Cell Suspensions from Skin-dLN
3.5 Flow Cytometry
4 Notes
References
Chapter 4: Analysis of Peripherally Derived Treg in the Intestine
1 Introduction
2 Materials
2.1 Mice and Antibiotics
2.2 Isolation of Lamina Propria Cells
2.3 Cell Staining and Flow Cytometry
3 Methods
3.1 Isolation of Lamina Propria Cells from the Colon
3.2 Cell Staining and Flow Cytometry
4 Notes
References
Chapter 5: Isolation and Analysis of Tumor-Infiltrating Treg
1 Introduction
2 Materials
2.1 Buffers
2.2 Digestion Enzyme Solution (Option 1)
2.3 Digestion Enzyme Solution (Option 2)
2.4 Antibody Staining
2.4.1 FACS Blocking Solution
2.4.2 FACS Permeabilization Reagents
2.4.3 Antibody Cocktail Example
3 Methods
3.1 Tumor Dissociation
3.2 Density Gradient Centrifugation
3.3 Analysis of Isolated Regulatory T Cells via Flow Cytometry
3.4 Quantification of Tumor-Infiltrating Lymphocytes
4 Notes
5 Suggested Reagents (Table 2)
References
Part II: Molecular Techniques for Analysing Treg
Chapter 6: Differentiation of Peripheral Treg
1 Introduction
2 Materials
2.1 Adoptive T Cell Transfer into DEREG Mice
2.2 Oral Ovalbumin Administration to DO11.10 Mice
2.3 CD4+ Cell Enrichment Through MACS Purification
3 Methods
3.1 Differentiation of pTreg from Adoptively Transferred Polyclonal Naïve CD4+ T Cells
3.2 Differentiation of PTreg from Monoclonal CD4+ T Cells Through Oral Ovalbumin Administration
3.3 Sample Enrichment in CD4+ T Cells Through MACS Purification (Optional)
4 Notes
References
Chapter 7: Functional Analysis of Foxp3 and Its Mutants by Retroviral Transduction of Murine Primary CD4+ T Cells
1 Introduction
2 Materials
2.1 Construction of Retroviral Vectors
2.2 Preparation of Retroviral Supernatant
2.3 Retroviral Transduction of Murine Primary T Cells
2.4 Flow Cytometry
3 Methods
3.1 Construction of Retroviral Vectors Expressing Mutant Foxp3
3.2 Preparation of Retroviral Supernatant
3.3 Retroviral Transduction of Murine CD4+ T Cells with Foxp3 and Its Mutants
3.3.1 Isolation of Naïve CD4+ T Cells
3.3.2 Preparation of Splenic Antigen-Presenting Cells (APCs)
3.3.3 In Vitro Stimulation of Naïve CD4+ T Cells and Retroviral Transduction
3.4 Flow Cytometric Analysis of CD4+ Tconv Cells Transduced with WT or Mutant Foxp3
3.4.1 Cell Surface Staining
3.4.2 Intracellular Staining of Foxp3 and CTLA-4
3.4.3 Data Analysis and Interpretation
4 Notes
References
Chapter 8: Genetic Tools for Analyzing Foxp3+ Treg Cells: Fluorochrome-Based Transcriptional Reporters and Genetic Fate-Mapping
1 Introduction
2 Materials
2.1 Mouse Lines
2.2 Flow Cytometry: Device Specifications
2.3 Magnetic Bead Separation Before Flow Cytometric Analysis (Optional)
2.4 Genotyping
2.5 Flow Cytometric Staining
3 Methods
3.1 Breeding of Foxp3RFP/GFP Mice
3.1.1 Zygosity of Experimental Foxp3RFP/GFP Mice
3.1.2 Foxp3RFP/GFP Mice and Cre/loxP
3.2 Genotyping of Foxp3RFP/GFP x R26-YFP Mice
3.2.1 Genotyping by Flow Cytometry
3.2.2 Genotyping by PCR
3.3 Flow Cytometric Analysis
3.3.1 Sample Preparation
3.3.2 Multicolor Flow Cytometry
3.3.3 Magnetic Bead Separation
Enrichment for CD25+ Cells
3.3.4 Analysis of transcription factors
3.3.5 Fluorescence Compensation of Fluorochrome Reporter
3.4 Application Examples
3.4.1 tTreg Cell Lineage Tracing in Thymus and Peripheral Tissues
3.4.2 tTreg Cell Ablation In Vivo
4 Notes
References
Chapter 9: Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations
1 Introduction
1.1 Gene Expression and DNA Methylation
1.2 Detection of DNA Methylation
1.3 Control of Gene Expression in Treg Cells
2 Materials
3 Methods
3.1 Isolation of gDNA
3.2 Bisulfite Conversion of gDNA
3.3 PCR Amplification and Cloning
3.3.1 Design of Bisulfite Primers
3.3.2 PCR Amplification
3.3.3 Isolation of DNA from Agarose Gel
3.4 Cloning
3.4.1 Restriction Digestion of the Vector and BS PCR Product for Cloning
3.4.2 Ligation
3.4.3 Transformation of Competent Bacteria
3.5 Isolation of Plasmid DNA
3.5.1 Miniprep Plasmid DNA Isolation
3.5.2 Restriction Digestion
3.6 Sequence Analysis
3.7 Analyze Data
4 Notes
References
Chapter 10: Protein Ubiquitination Assay
1 Introduction
2 Materials
2.1 His Pull-Down
2.2 Immunoprecipitation
2.3 Expression of MBP and His Proteins
2.4 MBP Pull-Down
2.5 Western Blotting
3 Methods
3.1 His Pull-Down
3.2 Immunoprecipitation
3.3 MBP Pull-Down Assay
References
Part III: Human Treg Analysis and Clinical Application
Chapter 11: Phenotypic and Functional Studies of Human Treg Cell Subpopulations
1 Introduction
2 Defining the Phenotype of Treg Cell Subpopulations
3 Materials
3.1 Common Reagents
3.2 Isolating Treg Cells
3.2.1 Flow Cytometric Sorting (Fig. 1)
3.2.2 Magnet-Based Isolation
3.3 Expanding Treg Cells
3.4 Suppression Assay
3.5 Cytokine Secretion Assay
4 Methods
4.1 Isolating Treg Cells
4.1.1 Flow Cytometric Sorting
4.1.2 (Alternative Protocol) Hybrid or Magnetic Beads Only
Hybrid: CD4+/CD25+ T Cell Enrichment
Treg Cell Isolation
4.2 Expanding Treg Cells
4.3 Suppression Assay
4.4 Cytokine Secretion Assay
References
Chapter 12: In Vitro Analysis of CTLA-4-Mediated Transendocytosis by Regulatory T Cells
1 Introduction
2 Materials
2.1 Purification of Regulatory T Cells (Tregs)
2.2 Expansion of Regulatory T Cells (Tregs)
2.3 Transendocytosis Assay
3 Methods
3.1 Purification of CD4+ T Cells
3.2 Purification of CD25+ Tregs from CD4+ T Cells
3.3 In Vitro Expansion of CD4+ CD25 Bright CD127- Tregs
3.4 CTLA-4 Transendocytosis Assays
3.4.1 Labeling of DG-75-Ligand-Expressing Lines with Cell-Trace Violet
3.4.2 Coculture of DG-75 with Tregs to Observe Transendocytosis
3.5 Effect of Time on CTLA-4-Mediated Transendocytosis
3.6 Effect of Treg:APC Ratio on CTLA-4-Mediated Transendocytosis
4 Notes
References
Chapter 13: Isolation and Functional Characterization of Regulatory CD4+ T Cells from the Inflamed Joints of Patients with Rhe...
1 Introduction
2 Materials
2.1 Isolation of Synovial Fluid Mononuclear Cells (SFMCs)
2.2 Tregs Enrichment Pre-sorting
2.3 FACS Sorting of Tregs
2.4 T Cell Suppression Assay
2.5 Monocyte Modulation Assay
2.6 Teff, Tregs, and Monocytes ``Three-Way´´ Suppression Assay
3 Methods
3.1 Synovial Fluid Mononuclear Cell (SFMC) Isolation
3.2 Treg Enrichment Pre-sorting
3.3 FACS Sorting of Synovial Tregs and Teffs
3.4 Teff Labelling for Suppression Assay
3.5 Teff:Treg Suppression Assay (No APC Present)
3.6 Teff, Tregs, and Monocytes ``Three-Way´´ Suppression Assay
3.7 Treg Modulation of CD14+ Monocytes
4 Notes
References
Chapter 14: A GMP Protocol for the Manufacture of Tregs for Clinical Application
Abbreviations
1 Introduction
2 Materials
2.1 Common Reagents/Equipment
3 Methods
3.1 Blood Procurement
3.2 Volume Reduction
3.3 CD8 Depletion
3.4 CD25 Enrichment
3.5 Expansion of Tregs by Polyclonal Stimulation
3.6 Harvest and Bead Removal
3.7 Preparation of Bulk Drug Product (DP)
3.8 Cryopreservation
4 Quality Control (QC) Measures
4.1 Sterility
4.2 Endotoxin
4.3 Mycoplasma
4.4 Suppression Assay
4.5 Cell Count
4.6 Phenotypic Markers
4.7 ExpAct Treg Bead Detection Test
5 Notes
References
Part IV: Single Cell and High-Dimensional Techniques for Analysing Treg
Chapter 15: Regulatory T-Cell Phenotyping Using CyTOF
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Viability and Surface Staining
2.3 Fixation and Intracellular Staining
2.4 DNA Intercalation and Data Acquisition
3 Methods
3.1 Cell Preparation
3.2 Viability Staining
3.3 Surface Staining
3.4 Fixation and Intracellular Staining
3.5 DNA Intercalation and Data Acquisition
3.6 Data Processing
4 Notes
References
Chapter 16: High-Dimensional Single-Cell Profiling of Tumor-Infiltrating CD4+ Regulatory T Cells
1 Introduction
2 Materials
2.1 Cells
2.2 Reagents
2.3 Plastic Components
2.4 Instruments
2.5 Software
3 Methods
3.1 Multicolor Flow Cytometric Panel Design
3.2 Staining Protocol
3.3 High-Dimensional Single-Cell Data Preprocessing
3.4 Computational Analysis: Phenograph Clustering and UMAP Analysis
4 Notes
References
Chapter 17: Single-Cell Transcriptome Analysis of Treg
1 Introduction
2 Materials
2.1 Single-Cell Preparation of PBMCs and Dead Cell Removal
2.2 (Alternative) Flow Cytometric Sorting for Purifying Alive T Cells from Mice (see Note 1)
2.3 scRNA-seq Library Preparation with Chromium
2.4 Bioinformatic Analysis
3 Methods
3.1 Single-Cell Preparation of PBMCs and Dead Cell Removal
3.2 Dead Cell Removal
3.3 (Alternative) Flow Cytometric Sorting for Purifying Alive T Cells from Mice
3.4 scRNA-seq Library Preparation with Chromium Next GEM Single Cell 3′ Reagent Kits v3.1
3.4.1 Single-Cell Encapsulation and Droplet Generation
3.4.2 Transfer Droplets and RT Reaction
3.4.3 Post-RT Cleanup
3.4.4 cDNA Amplification, Cleanup, and Quality Control
3.4.5 Library Construction: Fragmentation, End Repair, and A-Tailing
3.4.6 Library Construction: Adaptor Ligation
3.4.7 Sample Index PCR
3.4.8 Post-library Construction QC, Quantification, and Sequencing
3.4.9 Sequencing
3.5 Bioinformatic Analysis
3.5.1 Generation of Feature-Barcode Matrices Using the 10x Genomics CellRanger Pipeline
3.5.2 Statistical Analysis and Visualization Using R
4 Notes
References
Index