T-Follicular Helper Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Identification of Tfh and Tfr Cells in Humans and Mice
	Chapter 1: Complete Visualization of T Follicular Helper Cells in Germinal Centers by Light Sheet Fluorescence Microscopy
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Preparation for T and B Cell Adoptive Transfer
			2.3 Immunization
			2.4 Lymph Node Preparation for Imaging
			2.5 Lymph Node Imaging
			2.6 Image Analysis
		3 Methods
			3.1 Preparation of Cells for Adoptive Transfer
			3.2 Adoptive Cell Transfer to Recipient Mice and Immunization
			3.3 Preparation for Popliteal Lymph Node Imaging
			3.4 Light Sheet Fluorescent Microscopy Image Acquisition
			3.5 Image Analysis
		4 Notes
		References
	Chapter 2: In Vivo Imaging of Tfh Cells
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Cell Transfer
			2.3 Immunization
			2.4 Surgery
			2.5 Image Acquisition
			2.6 Image Analysis
		3 Methods
			3.1 Cell Transfer and Immunization
			3.2 Preparation of Popliteal Lymph Node for Imaging
			3.3 Imaging
			3.4 Image Analysis
		4 Notes
		References
	Chapter 3: Circulating T Follicular Helper Subsets in Human Blood
		1 Introduction
		2 Materials
			2.1 Analysis of cTfh Cell Subsets in Human Blood Samples
			2.2 Analysis of B Helper Functions
		3 Methods
			3.1 Analysis of cTfh Cell Subsets in Human Blood Samples
				3.1.1 Staining of Blood Cells
				3.1.2 Acquisition and Analysis of Flow Data
			3.2 Assessment of B Helper Capacity
				3.2.1 Obtaining PBMCs from Fresh Blood Samples
				3.2.2 Sorting Blood B Cell Subsets
				3.2.3 Sorting cTfh Cells
				3.2.4 T and B Cell Co-Culture and Readouts
		4 Notes
		References
	Chapter 4: Identification of Human Blood and Tissue T Follicular Regulatory (Tfr) Cells by Flow Cytometry
		1 Introduction
		2 Materials
			2.1 Cell Preparation
				2.1.1 Peripheral Blood
				2.1.2 Tissue
			2.2 Surface Staining
			2.3 Intracellular Staining
		3 Methods
			3.1 Cell Suspension Preparation
				3.1.1 Isolation of Mononuclear Cells from Peripheral Blood
				3.1.2 Isolation of Mononuclear Cells from Human Lymph Nodes
				3.1.3 Isolation of Mononuclear Cells from Human Tonsillar Tissue
			3.2 Surface Staining
			3.3 Intracellular Staining
			3.4 Analysis
		4 Notes
		References
	Chapter 5: Using Mass Cytometry to Address Tfh and Tfr Heterogeneity
		1 Introduction
		2 Materials
		3 Methods
			3.1 Day 1: Staining
			3.2 Day 2: Sample Collection
			3.3 Post-processing and Analysis
		4 Notes
		References
	Chapter 6: Identification of T Peripheral Helper (Tph) Cells
		1 Introduction
		2 Materials
			2.1 Cells
			2.2 Cell Isolation or Culture
			2.3 Staining for Flow Cytometry
			2.4 Staining for Mass Cytometry
			2.5 Equipment
		3 Methods
			3.1 Thawing Cells
			3.2 Identifying Tph Cells Using Flow Cytometry
				3.2.1 Staining Protocol
				3.2.2 Gating Strategy
			3.3 Identifying Tph Cells Using Mass Cytometry
				3.3.1 Staining Protocol (Note 6)
				3.3.2 Manual Gating Strategy
				3.3.3 Identifying Tph Cells in Dimensional Reduction Analyses
			3.4 Assessment of Tph Cell Function
				3.4.1 Isolation of CD4+ T Cells
				3.4.2 Flow Cytometry Staining for Cell Sorting
				3.4.3 Gating Strategy to Sort Tph Cells
				3.4.4 Intracellular Staining on Sorted Cells and Gating Strategy
				3.4.5 T Cell Coculture with Memory B Cells
		4 Notes
		References
	Chapter 7: Identification of Non-classical Follicular T Cells
		1 Introduction
		2 Materials
			2.1 Flow Cytometry
				2.1.1 Reagents
				2.1.2 Tetramers and Antibodies
				2.1.3 Instruments
			2.2 Confocal Microscopy
				2.2.1 Reagents
				2.2.2 Tetramer and Antibodies
				2.2.3 Instruments
		3 Methods
			3.1 Flow Cytometry (All Steps Except for Subheading 3.1.2 Must Be Carried Out at 4 C)
				3.1.1 Cell Suspension Preparation
				3.1.2 Cell Stimulation
				3.1.3 Blocking, Live/Dead and Surface Staining
				3.1.4 Fixation and Permeabilization
				3.1.5 Cytokine Staining
				3.1.6 Flow Cytometry Analysis
			3.2 Confocal Microscopy
				3.2.1 CD1d-Tetramer Staining
				3.2.2 Fixation and Cryoprotection
				3.2.3 Preparation of Tissue Sections
				3.2.4 Antibody Staining
				3.2.5 Mounting and Imaging
		4 Notes
		References
	Chapter 8: Identification of Follicular T Cells in the Gut
		1 Introduction
		2 Materials
			2.1 Mouse Strains
			2.2 General Laboratory Equipment
			2.3 Reagents, Solvents and Solutions
		3 Methods
			3.1 Cell Isolation
				3.1.1 Cell Isolation by Tissue Meshing through 40 μm Cell-Strainer
				3.1.2 Cell Isolation by Microdissection of PP
				3.1.3 Cell Isolation by Microdissection of PP in Presence of P2RX7 Inhibitor KN-62
			3.2 Flow Cytometry Based Annexin V Apoptosis Assay for Determining Apoptotic and Necrotic Cells
		4 Notes
		References
Part II: Development and Function of Tfh and Tfr Cells
	Chapter 9: In Vivo Migration and Tfh Cell Interactions
		1 Introduction
		2 Materials
			2.1 Cell Isolation
			2.2 Cell Transfer and Immunization
			2.3 Surgery
			2.4 Image Acquisition
			2.5 Data Analysis
		3 Methods
			3.1 Cell Isolation
			3.2 Adoptive Transfer and Immunization
			3.3 Surgery
			3.4 Intravital Imaging
			3.5 Data Analysis
				3.5.1 T Cells Migration
				3.5.2 T-B Cell Interactions
		4 Notes
		References
	Chapter 10: A Method to Analyze Models of the Dynamics of Germinal Centers
		1 Introduction
		2 Model Analysis
			2.1 Finite Differences Method for Local Sensitivity Analysis
			2.2 Analytic Method for Sensitivity Analysis
			2.3 Global Sensitivity Analysis: Latin Hypercube Sampling (LHS)
			2.4 Global System Sensitivity
			2.5 Software and Calculations
		3 Example Model
		Appendix
		References
	Chapter 11: Identification of Antigen Presenting Cell Subsets Supporting Human Tfh Differentiation
		1 Introduction
		2 Materials
			2.1 Tfh Polarization of Blood Naive CD4+ T Cells
				2.1.1 Blood Naive CD4+ T Cell Isolation
				2.1.2 Staining Naive CD4+ T Cells with a Proliferation Dye
				2.1.3 CD4+ T Cells and APCs Polarization by Tonsil APC Co-culture
				2.1.4 Detection of Effector Molecules in the Supernatant
				2.1.5 Detection of Effector Molecules by Intracellular Flow Cytometry
			2.2 Stimulation of Tfh Effector Functions
				2.2.1 Tonsil Cells Extraction by Mechanical Tissue Dissociation
				2.2.2 Tonsil Tfh Cell Sorting
				2.2.3 Tfh and APCs Co-culture
				2.2.4 Assessing Tfh Effector Functions after Co-culture
		3 Methods
			3.1 Tfh Polarization of Blood Naive CD4+ T Cells
				3.1.1 Blood Naive CD4+ T Cell Isolation
				3.1.2 Staining of Naive CD4+ T Cells with a Proliferation Dye
				3.1.3 Allogeneic Co-culture with APCs
				3.1.4 Detection of Effector Molecules in the Supernatant
				3.1.5 Detection of Effector Molecules by Intracellular Flow Cytometry
			3.2 Stimulation of Tfh Effector Functions
				3.2.1 Tonsil Cells Extraction by Mechanical Tissue Dissociation
				3.2.2 Tonsil Tfh Cell Sorting
				3.2.3 Tfh and APCs Co-culture
				3.2.4 Assessing Tfh Effector Functions After Co-culture
		4 Notes
		References
	Chapter 12: The Use of Peptide-MHCII Tetramers to Identify Antigen-Specific T Follicular Helper and T Follicular Regulatory Ce...
		1 Introduction
		2 Materials
			2.1 Immunization
			2.2 Cell Preparation
			2.3 Cell Staining Components
			2.4 Flow Cytometer and Analysis
		3 Methods
			3.1 Mouse Immunization
			3.2 Collection of Draining Lymph Nodes and Cell Preparation
			3.3 Cell Staining
			3.4 Flow Cytometry Gating Strategies
		4 Notes
		References
	Chapter 13: Assessment of the TCR Repertoire of Human Circulating T Follicular Helper Cells
		1 Introduction
		2 Materials
			2.1 Reagents and Equipment
			2.2 Buffers
		3 Methods
			3.1 PBMC Isolation
			3.2 CD14+ (Monocytes) and CD4+ Cells Enrichment
			3.3 Staining and Sorting for cTFH Cells or cTFH Subsets
			3.4 Antigen Pulsing of Antigen Presenting Cells
			3.5 CFSE Labeling and Stimulation of cTFH Cells or cTFH Subsets
			3.6 Sorting for Antigen-Specific cTFH and Dry Cell Pellet Preparation
			3.7 Internal Control
			3.8 Genomic DNA Extraction and Sample Preparation for ImmunoSEQ Assay
			3.9 Analysis of Sequencing  Data
		4 Notes
		References
	Chapter 14: Assessing In Vivo T Cell-Dependent Antigen-Specific Antibody Responses
		1 Introduction
		2 Materials
		3 Methods
			3.1 Food Allergy or Gut (Intra-gastric or i.g.) Immunization Protocol
			3.2 Immunization with Sheep Red Blood Cells (SRBC)
			3.3 Airway Challenge (Allergic Airway Disease) Model
			3.4 Mouse Bleeding and Serum Preparation
			3.5 ELISA for Ag-Specific Antibodies (Total IgG, IgG Subtypes or IgA), Using Ovalbumin as an Example
			3.6 Antigen Challenge for Antibody Affinity Tests
			3.7 ELISA Protocol for Measuring Ab Affinity with Hapten-Specific Antibodies (Total IgG, IgG subtypes, or IgA), Using NP as an...
			3.8 Antigen-Specific Mouse IgE ELISA Protocol
			3.9 Preparation of SRBC Membrane Antigens for ELISA
		4 Notes
		References
	Chapter 15: Tfh-Mediated and Tfr-Suppressed Antigen-Driven IgG and IgE Assays
		1 Introduction
		2 Materials
			2.1 HDM Solution
			2.2 NP-OVA Emulsion
			2.3 Mice
			2.4 Cell Purification
			2.5 Assay Plating
			2.6 Antibody ELISA
			2.7 Flow Cytometry
		3 Methods
			3.1 Preparation of  HDM
			3.2 Preparation of NP-OVA/CFA Vaccine
			3.3 Immunization of Mice with  HDM
			3.4 Tfh, Tfr, and B Cell Sorting
			3.5 Cell Plating
			3.6 Flow Cytometry to Assess B Cell Function
			3.7 IgE Elisa to Assess B Cell Function
		4 Notes
		References
Part III: Tfh and Tfr Cells in Infectious and Inflammatory Diseases
	Chapter 16: Assessment of the Impact of Cytokines on TFH, TREG, and TFR Cell Populations After Influenza Infection
		1 Introduction
		2 Materials
			2.1 Generation of Bone Marrow Chimeras
			2.2 Infection
			2.3 Tissue Processing and Flow Cytometry
		3 Methods
			3.1 Generation of Bone Marrow Chimeras
				3.1.1 Irradiation
				3.1.2 BM Harvesting from Donor Mice
			3.2 Infections
			3.3 Tissue Processing and Flow Cytometry
			3.4 Gating Strategy
		4 Notes
		References
	Chapter 17: Evaluation of Human Circulating T Follicular Helper Cells in Influenza- and SARS-CoV-2-Specific B Cell Immunity
		1 Introduction
		2 Materials
			2.1 Whole Blood Cell Surface Staining
			2.2 Data Acquisition and Analysis
		3 Methods
			3.1 Staining of Whole Human Blood for cTFH Cells and B Cells
			3.2 Gating and Analysis of CD4+ T Cell and B Cell Subsets
		4 Notes
		References
	Chapter 18: Detection of T Follicular Helper Cells and T Follicular Regulatory Cells in Experimental Sjögren´s Syndrome
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Media and Buffers
			2.3 Equipment
			2.4 ELISA and ELISpot
			2.5 Flow Cytometry
			2.6 Confocal Microscopy
		3 Methods
			3.1 ESS Model Establishment
			3.2 ESS Pathology Assessment
				3.2.1 Saliva Flow Rate Measurement
				3.2.2 Serum Autoantibodies Detection
				3.2.3 Histopathological Analysis
			3.3 Phenotypic Analyses of Tfh and Tfr Cells by Flow Cytometry in Naïve Controls and ESS Mice
			3.4 Detection of Cytokine Production by ELISpot Assay
			3.5 Confocal Microscopic Analysis of Tfh and Tfr Cells in the Lymphoid Organs in Naïve Controls and ESS Mice
		4 Notes
		References
	Chapter 19: Identification of Human T Follicular Cells in Ectopic Lymphoid Structures
		1 Introduction
		2 Materials
			2.1 Cell Collection and Preparation
			2.2 Staining for Flow Cytometry
				2.2.1 Surface Staining
			2.3 Immunohistochemistry Staining
		3 Methods
			3.1 Isolation of Mononuclear Cells from Minor Salivary Glands
			3.2 Sample Staining for Flow Cytometry
				3.2.1 Surface Staining
				3.2.2 Flow Cytometry Analysis
			3.3 Sample Staining for Immunohistochemistry
				3.3.1 Immunohistochemistry Analysis
		4 Notes
		References
	Chapter 20: T Follicular Helper Cells in Asthma Through Murine Models of Allergic Airway Disease
		1 Introduction
		2 Materials
			2.1 Mouse Model of Allergic Airway Disease
				2.1.1 Chronic Allergen Exposure Model of Allergic Airway Disease
				2.1.2 Sensitization and Challenge Model of Allergic Airway Disease
			2.2 Processing Tissue Samples
			2.3 Flow Cytometry Analysis of  TFH
			2.4 Identifying Tissue Resident  TFH
			2.5 Assessment of TFH-Induced Antibody Response by HDM-Specific ELISA
		3 Methods
			3.1 Generating Allergic Airway Disease
				3.1.1 Chronic Allergen Exposure Model of Allergic Airway Disease
				3.1.2 Sensitization and Challenge Model of Allergic Airway Disease
			3.2 Tissue Processing
				3.2.1 Lung Tissue
				3.2.2 Lymph  Node
			3.3 Flow Cytometry Staining of  TFH
				3.3.1 Basic Identification of TFH During Allergic Disease
				3.3.2 Cytokine Assessment of  TFH
			3.4 Identification of Tissue-Located  TFH
				3.4.1 Staining Intravascular Cells
			3.5 Assessment of TFH-Induced Antibody Responses
				3.5.1 Preparation of Lung Homogenate
				3.5.2 Preparation of Serum Samples
				3.5.3 Allergen-Specific Antibody Enzyme-Linked Immunosorbent Assay (ELISA)
		4 Notes
		References
	Chapter 21: Therapeutic Modulation of T Follicular Helper Cells by Low-Dose IL-2 Treatment
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Immunization
			2.3 IL-2 Treatment
			2.4 Cell Preparation
			2.5 FACS Analysis
		3 Methods
			3.1 Immunization and IL-2 Treatment
			3.2 Cell Preparation
			3.3 Cell Staining
				3.3.1 Tfh Cell Frequency Analysis (See Note 5)
				3.3.2 GC Reaction Analysis
				3.3.3 Tfh Cell Cytokine Production Analysis
		4 Notes
		References
	Chapter 22: 3D Tissue Explant and Single-Cell Suspension Organoid Culture Systems for Ex Vivo Drug Testing on Human Tonsil-Der...
		1 Introduction
		2 Materials
			2.1 Consumables and Equipment
			2.2 Cell Preparation and Culture
			2.3 Flow Cytometry
		3 Methods
			3.1 Setup of Cell and Tissue Cultures
				3.1.1 Preparation of Collagen Sponges and Plates
				3.1.2 Dissection of Adenoid Tissue for HLH and HLAC Cultures
				3.1.3 Culture and Drug Treatment of HLH and  HLAC
			3.2 Tfh Cell Subset Analysis by Flow Cytometry
				3.2.1 Preparations (See Note 24)
				3.2.2 Prepare Cells for Flow Cytometry Staining
				3.2.3 Surface Staining
				3.2.4 Viability Staining
				3.2.5 Fixation and Intracellular Staining
				3.2.6 Acquisition and Data Analysis
		4 Notes
		References
Index