Cytotoxic T-cells : methods and protocols

Preface
Contents
Contributors
Chapter 1: Flow Cytometry Instrument Setting as a Crucial Checkpoint for Optimal T-Cell Analysis and Sorting
	1 Introduction
		1.1 Polychromatic Flow Cytometry
		1.2 Lymphocytes
	2 Materials
		2.1 PMT Setting and Quality Check
		2.2 Antibody Titration
		2.3 LIVE/DEAD Staining
		2.4 Compensation Control Preparation
		2.5 Cell Surface Immunophenotyping
		2.6 Cell Proliferation Assay
		2.7 Setting and Optimization of Sorting Conditions
	3 Methods
		3.1 Doublet Discrimination
		3.2 Dead Cell Exclusion
		3.3 Definition of the Background/Unspecific Signal
		3.4 Choice of the Best Fluorophore
		3.5 PMT Setting and Daily Quality Check
		3.6 Antibody Titration for Surface Markers
		3.7 LIVE/DEAD Staining
		3.8 Compensation Control Preparation
			3.8.1 CompBeads Preparation
			3.8.2 ArC Beads Preparation
		3.9 Cell Surface Immunophenotyping from hPBMCs
		3.10 T-Cell Proliferation Assay by CFSE Labeling
		3.11 Setting and Optimization of T Cell Sorting
	4 Notes
	References
Chapter 2: Phenotypical and Functional Characterization of Cytotoxic Unconventional T-Cells
	1 Introduction
	2 Materials
		2.1 Phenotypical Characterization of Unconventional T-Cells from Peripheral Whole Blood or PBMCs
		2.2 DNTs Purification from PBMCs
		2.3 Intracellular Cytokine Staining on  DNTs
		2.4 In Vitro Expansion of Double-Negative T-Cells
	3 Methods
		3.1 Phenotypical Characterization of Unconventional T-cells from Peripheral Blood or PBMCs
			3.1.1 Gating Strategy
		3.2 DNTs Purification from PBMCs
		3.3 Intracellular Cytokine Staining on  DNTs
			3.3.1 Gating Strategy
		3.4 In Vitro Expansion of Double-Negative T-Cells
	4 Notes
	References
Chapter 3: Tumor Infiltrating T Cell Cytotoxicity Assay
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Isolation of Total Lymphocytes from Peripheral Blood
		2.3 Tumor Tissue Disruption and Isolation of Total Tumor-Infiltrating Lymphocytes (TILs)
		2.4 Magnetic Labeling of CD8+ T-Cells
		2.5 Manual Cell Separation of CD8+ T-Cells
		2.6 Checking the Purity of the Isolated CD8+ T-Cells
		2.7 Preparation of Target Cells
		2.8 Coculture
		2.9 FACS Acquisition After Coculture
		2.10 Equipment
	3 Methods
		3.1 Isolation of Total Peripheral Blood Mononuclear Cells (PBMCs)
		3.2 Manual Disruption of Tumors
		3.3 Isolation of the Effector CD8+ T-Cells from PBMCs and Tumor Cell Suspensions
			3.3.1 Magnetic Staining of PBMCs and Tumor Cell Suspensions
			3.3.2 Manual Cell Separation
			3.3.3 Checking the Purity of the Enriched Effector CD8+ T-Cells from Blood and Tumor
		3.4 Isolation of the Target Cells from the Tumor (CD45- Cells)
			3.4.1 Magnetic Staining of Tumor Cell Suspension
			3.4.2 Manual Separation of Tumor Cells
			3.4.3 Checking the Purity of the CD45- Flow-through Cells
		3.5 Preparing the Target Cells for Coculture with Effector Cells
		3.6 Cytotoxicity Assay: Coculture of Target and Effector Cells
		3.7 FACS Acquisition and Gating Strategy
		3.8 Data Analysis
	4 Notes
	References
Chapter 4: Antigen-Specific In Vivo Killing Assay
	1 Introduction
	2 Materials
		2.1 Mice and Reagents
		2.2 Immunization Solution
		2.3 Splenocyte Isolation and Peptide pulsing
		2.4 Splenocyte Isolation and FACS Acquisition
		2.5 Equipment
	3 Methods
		3.1 Day 1: Mouse Immunization
		3.2 Day 8: Injection of Splenocytes Pulsed and Non-pulsed Splenocytes
			3.2.1 Isolation of Target Splenocytes for Pulsing with Peptide and Labeling with  CFSE
			3.2.2 Peptide Pulsing of Isolated Target Cells
			3.2.3 Labeling Target Cells with  CFSE
			3.2.4 Injecting Labeled Target Cells
		3.3 Day 9: Re-isolation of Target Spleen Cells from Recipient Mice and FACS Acquisition
		3.4 Data Analysis
	4 Notes
	References
Chapter 5: CTL ELISPOT Assay and T Cell Detection
	1 Introduction
		1.1 Principle
		1.2 ELISPOT  Pros
		1.3 ELISPOT  Cons
		1.4 Positive Controls and Antigen Formats
	2 Materials
	3 Methods
		3.1 Interferon γ Elispot Assay
		3.2 Perforin Elispot Assay
	4 Notes
	References
Chapter 6: Methods for Characterization of Senescent Circulating and Tumor-Infiltrating T-Cells: An Overview from Multicolor F...
	1 The Role of T-Cell in Immunosenescence
	2 Senescent T-Cell Characterization by Classical Markers
	3 Emerging New Markers for Senescent T-Cell Characterization by Multicolor Flow Cytometry
	4 Single-Cell RNA Seq Strategies to Dissect T-Cell Heterogeneity in Immunosenescence
	5 Conclusions and Future Perspectives
	References
Chapter 7: In Vivo and In Vitro Models for the Study of Bone Remodeling and the Role of Immune Cells
	1 Introduction
	2 In Vivo Studies
		2.1 Methods for the Study of Bone Remodeling in Animal Models
			2.1.1 Micro-computed Tomographic Imaging
			2.1.2 Histomorphometric Analysis
				Dynamic Histomorphometry
				Static Histomorphometry
			2.1.3 Mechanical Tests
				Sample Preparation
				Bending Tests
			2.1.4 Bone Turnover Markers
				C- and N-Terminal Telopeptides of Type I Collagen
				N- and C-Terminal Pro-peptides of Type 1 Collagen
				Tartrate-Resistant Acid Phosphatase (TRAP)
				Bone-Specific Alkaline Phosphatase (bone ALP)
				Osteocalcin
	3 In Vivo Studies Models
		3.1 B cell-Deficient Mice Are Osteopenic
		3.2 T-cell-Deficient Nude Mice Display Decreased Basal  Mass
		3.3 In Vivo T-Cell Depletion
		3.4 Germ-Free  Mice
		3.5 Fracture Healing and Immune Cells
	4 In Vitro Models
		4.1 Osteoclastogenesis Assays
			4.1.1 Murine Samples
			4.1.2 Human Samples
		4.2 Osteoblastogenesis from Bone Marrow Samples
			4.2.1 Murine Samples
			4.2.2 Human Samples
	5 Notes
	References
Chapter 8: Characterization of Cytotoxic T Lymphocytes (CTL) by Tissue Microarray
	1 Introduction
	2 Materials
		2.1 TMA Construction
		2.2 Immunohistochemistry (Paraffin)
		2.3 Immunohistochemistry (Frozen)
		2.4 Immunofluorescence
	3 Methods
		3.1 Fixation and Paraffin Embedding of Tissue
			3.1.1 Fixation Protocol
			3.1.2 Alternative Fixation of Tissue
			3.1.3 Paraffin Embedding Protocol
		3.2 Tissue Microarray Construction
			3.2.1 Protocol Construction with Manual Method
		3.3 Tissue Microarray Sectioning
		3.4 Preparation of Paraffin Sections for Immunohistochemistry
			3.4.1 Sectioning Protocol
			3.4.2 Deparaffinization and Re-hydration of Tissue Slide
		3.5 Antigen Retrieval
		3.6 Immunohistochemical Staining of Paraffin-Embedded Tissues
		3.7 Preparation of Frozen Tissues
		3.8 Sectioning of Frozen Tissues
		3.9 Fixation for IHC and ICC
			3.9.1 4% Paraformaldehyde (See Note 13)
			3.9.2 Ethanol
			3.9.3 Methanol (See Note 14)
			3.9.4 Acetone (See Note 15)
		3.10 Permeabilization for IHC and ICC
			3.10.1 Solvents
			3.10.2 Detergents
		3.11 Standard Immunohistochemical Staining Procedure for Frozen Sections
		3.12 Evaluation of IHC
		3.13 Staining for Immunofluorescence
		3.14 Double Immunofluorescence (Sequential Protocol)
		3.15 Evaluation of Immunofluorescence
	4 Notes
	References
Chapter 9: A Review of Proteomics Strategies to Study T-Cell Activation and Function in Cancer Disease
	1 Role of T-Cell in Cancer
	2 Proteomics Strategies to Study Cytotoxic T-Cells in Cancer
	3 New Frontiers in Tumors Treatment
	4 Proteomics Strategies to Study Cancer Immunotherapy
	5 Conclusions and Perspectives
	References
Chapter 10: Methods to Study Posttranslational Modification Patterns in Cytotoxic T-Cells and Cancer
	1 Role of Protein Posttranslational Modifications (PTMs) in Cancer
	2 PTMs Regulate T-Cell Activation and Suppression in Many Cancer Types
	3 Different Strategies to Study PTMs
		3.1 Immunoblotting
		3.2 Mass Spectrometry
	4 Immuno-enzymatic ASSAYS
	5 Conclusions and Perspectives
	References
Chapter 11: Whole Exome Sequencing for the Identification of Mutations in CD8+ T-Cells
	1 Introduction
		1.1 Library Preparation, Cluster Generation, and Sequencing
		1.2 Data Pre-processing
		1.3 Handling of Aligned Exome Sequencing Data and Variant Detection
		1.4 Post VCF analysis
			1.4.1 Variant Annotation
			1.4.2 Variant Filtration and Prioritization
	2 Materials
		2.1 Library Preparation, Cluster Generation, and Sequencing
		2.2 Files and Software Needed for Data Pre-processing
		2.3 Handling of Aligned Exome Sequencing Data and Variant Detection
		2.4 Post VCF Analysis
	3 Methods
		3.1 Library Preparation, Cluster Generation, and Sequencing
			3.1.1 DNA Fragmentation
			3.1.2 End Repair of Fragmented  DNA
			3.1.3 Adenylate 3′  Ends
			3.1.4 Ligate Adapters
			3.1.5 Enrichment of Adapter-Modified DNA Fragments by PCR
			3.1.6 First Hybridization
			3.1.7 First  Wash
			3.1.8 Second Hybridization
			3.1.9 Second  Wash
			3.1.10 PCR Amplification
			3.1.11 Enriched Library Validation and qPCR Method for Library Quantification
		3.2 Data Pre-processing
		3.3 Handling of Aligned Exome Sequencing Data and Variant Detection
		3.4 Post VCF Analysis
	4 Notes
	References
Chapter 12: Handling High-Throughput Omics Data for Systems Genetics Analysis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Handling Genomics and Transcriptomics Data
		3.2 Building Gene Co-expression Networks
		3.3 Network Mapping
	References
Chapter 13: Analysis of miRNA Expression Using Digital PCR and the QuantStudio 3D Digital PCR System
	1 Introduction
		1.1 Preliminary Considerations
			1.1.1 Sample Handling, Processing, and Storage
			1.1.2 RNA Extraction
			1.1.3 Retro-Transcription (miRNA Specific vs. Universal)
			1.1.4 miRNA Normalization in Serum
	2 Materials
		2.1 Materials
		2.2 Instruments and Software
	3 Methods
		3.1 dPCR Analysis
		3.2 The MIQE Guidelines for dPCR
		3.3 Further Applications of dPCR
	4 Notes
	References
Chapter 14: Cytotoxic T Lymphocytes (CTLs) and Kidney Transplantation: An Overview
	1 Introduction
	2 The Phenotypic Identity and Properties of Human CTLs CD8+ T Cells
	3 The Metabolic Regulation of CTLs
	4 CTLs and T cell-Mediated Rejection (TCMR)
	5 The Human Transcriptome of CTLs in Renal TCMR versus CD4+T Cells and NK Cells
	6 Immunological Profile of CTLs in Acute Rejection and Borderline Changes
	7 CTLs and Transplantation Tolerance
	8 CTLs and Immunosuppressive Therapies
	9 Concluding Remarks
	References
Chapter 15: Protein-Bound Uremic Toxins and Immunity
	1 Introduction
		1.1 Microbiota Dysbiosis in Uremia
		1.2 Immune Dysfunction in Uremia
	2 Microbial Derived Uremic Toxins: Indoxyl Sulfate and p-Cresyl Sulfate
		2.1 Effects of p-Cresyl Sulfate on Immune Response
		2.2 Effects of Indoxyl Sulfate on Immune Response
	3 Conclusions
	References
Chapter 16: Gut Microbiota, the Immune System, and Cytotoxic T Lymphocytes
	1 Overview On Gut Microbiota and Galt
	2 Immune System in Health: How the Gut Microbiota Promotes the Immune Tolerance
		2.1 Microbial Commensals and Metabolites
		2.2 Treg Cells
		2.3 Intestinal DCs
		2.4 Immunoglobulin A (IgA)
	3 Immune System and Gut Microbiota in Disease: Chronic Intestinal Inflammatory Disorders, Celiac Disease, and Tumor Immunogeni...
		3.1 Break of Tolerance: Inflammatory Bowel Disease, Celiac Disease
		3.2 Excess of Tolerance: Modulating the Cytotoxic Response Toward Tumors and Vaccines
	4 Conclusions
	References
Index