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Putting on glutathione destruction inside cancer therapy: Increased ROS-based remedy, ferroptosis, as well as chemo.
The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.Experimental autoimmune encephalomyelitis, originally experimental allergic encephalomyelitis, is the well-known animal model of multiple sclerosis, an immune- mediated, demyelinating, inflammatory chronic disease of the central nervous system. The experimental disease is widely utilized to test new therapies in preclinical studies, to investigate new hypothesis on the possible pathogenic mechanisms of autoimmune reaction directed against the central nervous system or more generally to investigate the interactions between the immune system and the central nervous system that lead to neuroinflammation. The experimental autoimmune encephalomyelitis may be induced following different protocols in mammals, including nonhuman primates, and autoreactive CD4+ T-lymphocytes directed against myelin antigens are the main factors. Here, after introducing the model, we describe the protocol to induce active EAE in inbred mice, we report on a table the different clinical courses of EAE depending on the combination of antigen /mouse strain and we provide indications on how to evaluate the clinics and pathology of this induced disease.Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4+ T helper cells have been shown to strongly contribute to the disease pathogenesis of IBD. Here, we present a detailed protocol of the preclinical model of T-cell transfer colitis, which can easily be utilized in the laboratory to study T helper cell functions in intestinal inflammation.Asthma is a highly prevalent lung disease, characterized by airway dysfunction and chronic inflammation. Asthma occurs in both children and adults, but frequently originates in early life. Heterogeneous asthma phenotypes exist, but Th2 cells are key players in a large proportion of cases, while other CD4+ T cell subsets are also implicated in driving and limiting pathology. In this chapter, we describe methods for establishing allergic airway disease to model asthma in adult and neonatal mice, along with protocols for measuring key disease parameters and quantifying and phenotyping CD4+ T cell subtypes.Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1 mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.During the last decade, the rapid progress in the development of next-generation sequencing (NGS) technologies has provided relevant insights into complex biological systems, ranging from cancer genomics to microbiology. Among NGS technologies, single-cell RNA sequencing is currently used to decipher the complex heterogeneity of several biological samples, including T cells. Even if this technique requires specialized equipment and expertise, nowadays it is broadly applied in research. In this chapter, we will provide an optimized protocol for the isolation of T cells and the preparation of RNA sequencing libraries by using droplet digital technology (ddSEQ, Bio-Rad Laboratories). We will also illustrate a guide to the main steps of data processing and options for data interpretation. This protocol will support users in building a single-cell experimental framework, from sample preparation to data interpretation.The comparison of methylomes from immune cells enables the identification of differentially methylated regions and thereby region-associated gene loci. Those regions can be used to discriminate one immune cell population from the other, as well as help to identify key molecules and major pathways determining the unique phenotypes of immune cell lineages. The combination of bisulfite treatment of genomic DNA and next-generation sequencing provides the basis for studying epigenetic changes in different immune cell populations. Further development of whole-genome bisulfite sequencing resulted in a protocol for sequencing libraries that accept both single- or double-stranded DNA from fixed or nonfixed cells, respectively. Therefore, researchers can include immune cell populations in their methylation studies whose isolation depends on the staining of intracellular molecules.The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.Lentivirus-mediated gene transfer is an efficient method to introduce a variety of transgenes to human T cells. Here we describe a protocol to transduce human CD4+, CD8+, or CD4+ regulatory T cells. To illustrate the method, we use transduction with lentivirus encoding an HLA-A2-specific chimeric antigen receptor (CAR) and a transduction marker as an example. Methods to isolate, transduce, purify, and expand CD4+ and CD8+ T cells as well as regulatory T cells are provided. We also describe how to carry out cytotoxicity or suppression assays to assess the function of the resulting CAR T cell or CAR regulatory T cells, respectively.Electroporation enables the transfection of different cell types including microbial, plant, and animal cells with charged molecules, such as nuclear acids or proteins. During electroporation, an electrical field is applied to the cells leading to a transient permeabilization of the cell membrane allowing exogenous molecules to enter the cells. Here we report the electroporation of human primary CD4+ -T cells with in-vitro transcribed mRNA to facilitate gene editing (knockout) of the CC-chemokine receptor 5 (CCR5), the coreceptor of the human immunodeficiency virus 1 (HIV1) predominantly used during primary infection. Using such strategy of transient expression of a CCR5-specific Transcription-activator-like-effector nuclease (TALEN), we aim to protect helper T cells from de novo HIV infection.Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>107) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 105 cells.Flow cytometric evaluation of phosphorylation status of signal transduction molecules is a useful method to study T-cell signaling pathways. As mutations occurring in TCR complex molecules, common gamma chain family's cytokines, their receptors or molecules involved in these pathways can lead to severe immune system defects, the study of T-cell signal transduction can be applied to both basic and clinical/translational research areas. learn more In the present chapter, we show two different protocols for the study of T- cell response to an antigen-like stimulus and to IL-2.Antibody responses deeply rely on the interaction of antigen-primed B cells and CD4 helper T cells in the context of germinal center reactions, through signals provided by costimulatory molecules and cytokines. B-cell proliferation and differentiation in antibody-secreting plasma cells are processes that critically depend on the helper function of a specific CD4 T-cell subset, known as follicular helper T cells (Tfh). Here, we describe a method that mimics in vitro the cross talk between Tfh and B cells occurring in the germinal center. The procedure is based on setting up a coculture system with B cells and Tfh isolated from blood of healthy donors, or tonsils removed upon surgical intervention, in order to recapitulate in vitro the Tfh-dependent mechanisms leading to B cells' activation, proliferation, and differentiation.
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