Notes

Advancements within SUMO-based unsafe effects of homologous recombination.
While the zebrafish has for some time been regarded as a powerful model organism with which to study early events in hematopoiesis, recent evidence suggests that it also ideal for unraveling the molecular requirements for T cell development in the thymus. Like mammals, zebrafish possess an adaptive immune system, comprising B lymphocytes as well as both the γδ and αβ lineages of T cells, which develop in the thymus. Moreover, the molecular processes underlying T cell development in zebrafish appear to be remarkably conserved. Thus, findings in the zebrafish model will be of high relevance to the equivalent processes in mammals. Finally, molecular processes can be interrogated in zebrafish far more rapidly than is possible in mammals because the zebrafish possesses many unique advantages. Here, we describe these unique attributes and the methods by which they can be exploited to investigate the role of novel genes in T cell development.Not only is human T cell development characterized by unique changes in surface marker expression, but it also requires specific growth factors and conditions to mimic and study T cell development in vitro. In this chapter, we provide an overview of the specific aspects that need attention when performing T cell differentiation cultures with human hematopoietic and T cell progenitors.During their development, human T cells undergo similar genomic changes and pass through the same developmental checkpoints as developing thymocytes in the mouse. The difference between both species, however, is that some of these developmental stages are characterized by different phenotypic markers, and as a result, evidence emerges that the molecular regulation of human T cell development subtly differs from the mouse (Taghon et al., Curr Top Microbiol Immunol 36075-97, 2021; Haddad et al., Immunity 24217-230, 2006; Hao et al., Blood 1111318-1326, 2008; Taghon and Rothenberg, Semin Immunopathol 30383-398, 2008). In this chapter, we describe in detail how the different stages of human T cell development can be characterized and isolated using specific surface markers.When a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. Identifying signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and molecular associations between numerous proteins within the developing T cell. Given the unique, temporal, and developmental changes that occur during development, isolating and analyzing small populations of thymocytes are necessary to get a complete picture of the development process. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.Negative selection removes potentially harmful T cell precursors from the conventional T cell pool. This process can involve the induction of apoptosis, anergy, receptor editing, or deviation into a regulatory T cell lineage. As such, this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate these various forms of negative selection are not clear. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes fetal thymic organ culture methods designed to analyze the signals that determine these unique cell fates.Intra-thymic injection is a powerful tool for adoptive transfer of cells, cellular tag reagents for tracking recent thymic emigrants (RTEs), or other substances directly into the thymus. The traditional approach developed decades ago requires an invasive surgery to open the thoracic cavity and visualize the thymus. Subsequently, a technique was developed requiring only a small skin incision needed to identify the precise injection site. Nevertheless, both techniques require surgical intervention, and this can lead to elevated animal stress levels and pain which necessitates analgesic medication administration. Here we describe a less invasive technique allowing in situ visualization and transfer of cell suspensions or substances into the thymus via an ultrasound-guided intra-thymic injection approach.Quantitative real-time PCR and next-generation sequencing (NGS) are invaluable techniques to analyze T cell receptor (Tcr) gene rearrangements in mouse lymphocyte populations. Although these approaches are powerful, they also have limitations that must be accounted for in experimental design and data interpretation. Here, we provide relevant background required for understanding these limitations and then outline established quantitative real-time PCR and NGS methods that can be used for analysis of mouse Tcra and Tcrb gene rearrangements in mice.For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.T cell development occurs in the thymus and is coordinated temporally and spatially within the highly complex thymic microenvironment. Therefore, T cell selection and maturation events cannot be fully recapitulated using traditional two-dimensional tissue culture in vitro. The thymic slice system provides a highly versatile system for studying T cell development ex vivo while preserving three-dimensional thymic architecture. Using the thymic slice system, T cell selection and maturation events can be visualized by live imaging and quantified by flow cytometry. Here we describe the process for generating slices from neonatal and adult mice.T cells and innate lymphoid cells (ILCs) share expression of many key transcription factors during development and at mature stage, resulting in striking functional similarities between these lineages. Taking into account ILC contribution is thus necessary to appreciate T cell functions during immune responses. Furthermore, understanding ILC development and functions helps to understand T cells. Here we provide methods and protocols to isolate pure populations of multipotent precursors to T cells and innate lymphoid cells (ILCs) from adult mouse bone marrow, using flow cytometric sorting. These include precursors to all lymphocytes (viz., LMPPs and ALPs) and multipotent precursors to ILCs that have been recently refined (viz., specified EILPs, committed EILPs, and ILCPs).T cells develop in the thymus from bone marrow precursors, and genetic manipulation is an indispensable tool to explore their development in vivo. Retroviral transduction of T cell precursors in the bone marrow can be used to specifically eliminate or enforce gene expression. Here, we describe a fast and efficient method to ectopically express a gene in T cell precursors through retroviral transduction and transplant into recipient mice, which will enable laboratories to evaluate gene function in T cell development in vivo.The thymus is compartmentalized into the cortex and the medulla. Cortical and medullary thymic epithelial cells (TECs) characterize T cell-producing and T cell-selecting functions of cortical and medullary microenvironments in the thymus. Enzymatic digestion of the thymus and flow cytometric isolation of TECs and their subpopulations are useful for molecular and cellular characterization of TECs. However, the cellularity of cTECs and mTECs isolated from mouse thymus is limited. In this chapter, we describe the method for isolation of a large number of TECs using enlarged mouse thymus, which enables biochemical and proteomic analysis of TEC subpopulations.Expansion of T cell subsets in vitro is a valuable tool for exploration of effector function and differentiation. Bisindolylmaleimide I nmr Here we provide protocols for in vitro differentiation of CD4 and CD8 T cell subsets from naïve T cells for functional studies.Bone marrow chimeras are widely used in immunological studies, to dissect the contributions of hematopoietic and non-hematopoietic cells in immune cell development or functions, to quantify the impact of a given mutation, or in preclinical studies for hematopoietic stem cell transplantation. Here we describe a set of procedures for the generation of bone marrow chimeras.Many analytical or cell culture procedures require homogeneous starting cell populations that cannot be obtained directly from organ dissection. Here, we describe two enrichment procedures to achieve this goal and discuss their respective advantages in specific experimental contexts.T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multiparameter flow cytometry-based cell sorting are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with relatively simple flow cytometry screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a multi-laser flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.Genetics approaches have been instrumental to deciphering T cell development in the thymus, including gene disruption by homologous recombination and more recently Crispr-based gene editing and transgenic gene expression, especially of specific T cell antigen receptors (TCR). This brief chapter describes commonly used tools and strategies to modify the genome of thymocytes, including mouse strains with lineage- and stage-specific expression of the Cre recombinase used for conditional allele inactivation or expressing unique antigen receptor specificities.
Here's my website: https://www.selleckchem.com/products/gf109203x.html