This volume provides protocols to successfully apply cutting-edge technologies to characterize the biology of T cells at an unprecedented level of complexity. Chapters guide readers through flow cytometry and fluorescence-activated cell sorting, the behaviour of single T cells after adoptive cell transfer (ACT), single cell gene expression by multiplex PCR, lentiviral transduction approaches, protocols to derive large numbers of early-differentiated memory T cells by using dedicated cytokines cocktails, approaches to measure telomerase activity in terminally differentiated T cells, and approaches to define Treg cells at the phenotypic and functional level. The final part of the book is dedicated to the analysis of the differentiation and effector functions of innate T cells, namely the well-known γ/δ T cells, and the recently identified CD8+ mucosal associated invariant T (MAIT) cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, T-Cell Differentiation: Methods and Protocols aims to provide protocols that are fundamental to monitor the T cell compartment at the level of single cells in pathological and immunotherapy conditions.
Table of Contents
The Vast Universe of T Cell Diversity: Subsets of Memory Cells and their Differentiation.- Basic Aspects of T Helper Cell Differentiation.- FACS Analysis of Memory T lymphocytes.- Intravital Microscopy Analysis of Hepatic T cell dynamics.- Analysis of T Cell Activation by Confocal Microscopy.- Phenotypic and Functional Analysis of Antigen-specific T Cell Exhaustion.- pMHC Multiplexing Strategy to Detect High Numbers of T Cell Responses in Parallel.- Differentiation of Diverse Progenies of Memory T cells from Naïve CD8+ T Cell Precursors.- Gammaretroviral Production and T Cell Transduction To Genetically Retarget Primary T Cells Against Cancer.- Measuring Telomerase Activity in Senescent Human T Cells upon Genetic Modification.- Strategies for T Helper Cell Subset Differentiation from Naïve Precursors.- Phenotypic and Functional Analysis of the Suppressive Function of Human Regulatory T Cells.- Approaches to Detect microRNA Expression in T Cell Subsets and T Cell Differentiation.- Next-generation Sequencing Analysis of Long Non-Coding RNAs in CD4+ T Cell Differentiation.- Highly Multiplexed, Single Cell Transcriptomic Analysis of T-cells by Microfluidic PCR.- Single-cell RNA Sequencing of Human T Cells.- Extensive Phenotypic Analysis, Transcription Factor Profiling, and Effector Cytokine Production of Human MAIT Cells by Flow Cytometry.- Developmental and Functional Assays to Study Murine and Human gd T Cells.