Immunology

Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.

Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). In this protocol, a robust method to determine low intracellular ROS production using H₂DCFDA was validated by several ROS-specific as well as NOX-2-specific inhibitors. Cells were treated with inhibitors or control substances prior to treatment with the ROS-inducer of interest. H₂DCFDA was added only for the last 30 min of the treatment schedule. To terminate its conversion, we used a ROS-specific inhibitor until analysis by flow cytometry within the FITC-channel (Ex: 488 nm/Em: 519 nm). In summary, this protocol allows the detection of signaling-relevant intracellular ROS production in cell lines and primary immune cells (e.g., Mono Mac 6 cells and Bone marrow-derived dendritic cells, respectively). Using this method in combination with specific inhibitors, we were able to validate even exceptionally low amounts of ROS produced by NOX-2 and relevant for immune-regulatory signaling.

Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S^-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H₂O₂ is detected with antibodies specific for the sulfonic acid (SO₃H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S^-; active) or irreversibly oxidized (SO₃H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.

Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and non-hematopoietic cells and is also present in many biological fluids in a bioactive soluble form. DPP4 expression is modulated by inflammation, and measurements of its activity have been used as biomarker for disease. Here, we describe a method to evaluate the enzymatic activity of DPP4 in vitro and in vivo.

Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR (Stockinger et al., 2014). This protocol is designed for treatment with Kyn or FICZ in mouse embryonic fibroblasts (MEFs) or primary peripheral monocytes.

The production of reactive oxygen species, including H₂O₂, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H₂O₂ release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H₂O₂-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)₂]). The H₂O₂-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H₂O₂ release from cells. This assay can measure H₂O₂ release from whole cells, mitochondria, or the NADPH oxidase.

The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the NLRP3 inflammasome components into HEK293T cells, which enables inflammasome activation and the production of IL-1β upon stimulation with nigericin.

Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.

Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, which can be applied to other organs. In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon microscopy. Analysis of tumor interactions with immune cells using this protocol should aid in a better understanding of the importance of these interactions and their role in developing immunotherapies.

Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and finally, 6) analysis of the proteins by immunoblot and revelation of the glycerophospholipids by iodin fumigation.