Current Protocols in Immunology最新文献

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors.

Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies

Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids

Alternate Protocol: Differentiation of human intestinal organoids

Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids

Basic Protocol 4: Immunofluorescence staining of human intestinal organoids

Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures

Support Protocol 1: Production of Wnt3A conditioned medium

Support Protocol 2: Production of Rspo1 conditioned medium

Support Protocol 3: Extraction of RNA from intestinal organoid cultures

Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). Published 2020. U.S. Government.

Basic Protocol 1: SILAC labeling of mouse macrophages

Basic Protocol 2: Stimulation, cell lysis and Western Blotting

Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment

Basic Protocol 4: Quantitative mass spectrometry

Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein–protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors.

Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near-infrared imaging

Alternate Protocol: Visualizing immunoblots using chemiluminescence

Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation

Support Protocol: Covalent conjugation of antibodies to functionalized beads

Basic Protocol 3: Quantifying proteins and post-translational modifications by targeted mass spectrometry

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr¹⁴², which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells

Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells

Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation

Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis

Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation

Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The “Trojan horse” theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells

Basic Protocol 2: Isolation of primary mouse bone marrow–derived dendritic cells

Support Protocol 1: Validation of dendritic cell purity by flow cytometry

Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells

Support Protocol 2: Isolation of primary mouse spleen cells

Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells

Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load

Support Protocol 4: In vivo and in vitro infection with mycobacteria

Basic Protocol 5: Assembly of the BBB co-culture model

Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model

Listeria monocytogenes is a foodborne pathogen that causes serious, often deadly, systemic disease in susceptible individuals such as neonates and the elderly. These facultative intracellular bacteria have been an invaluable tool in immunology research for more than three decades. Intravenous (i.v.) injection is the most commonly used transmission route in mice, but oral models of infection have also been developed in recent years, and these may be more appropriate for many studies. This article includes detailed instructions for use of either foodborne or i.v. inoculation of mice and discusses the rationale for choosing either model. Additionally, a protocol is provided for enrichment of neutrophils and monocytes from the infected liver in a manner that allows for determination of bacterial burden while still providing sufficient cells for use in flow cytometric analysis or in vitro assays. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Foodborne L. monocytogenes infection

Support Protocol 1: Preparing L. monocytogenes for foodborne infection

Basic Protocol 2: Intravenous L. monocytogenes infection

Support Protocol 2: Preparing L. monocytogenes for intravenous infection

Basic Protocol 3: Enrichment of non-parenchymal cells from the infected liver

The anaphylatoxins (AT) C3a and C5a are effector molecules of C3 and C5 exerting multiple biologic functions through binding and activation of their cognate G protein−coupled receptors. C3a interacts with the C3a receptor (C3aR), whereas C5a and its primary degradation product C5a-desArg engage C5aR1 and C5aR2. In the past, analysis of AT expression has been hampered by cross reaction of antibodies designed to recognize the different AT receptors. Furthermore, assessment of effects mediated by cell-specific activation has been difficult. Here, floxed AT receptor reporter mice are described as tools to monitor AT receptor expression in cells and tissues and to study the functions of C3a and C5a by cell-specific deletion of their cognate AT receptors. © 2020 The Authors.

Basic Protocol 1: Genotyping of floxed GFP-C5aR1 knockin mice

Support Protocol 1: Genotyping of LysMcre-C5ar1^-/- mice

Basic Protocol 2: Genotyping of floxed tdTomato-C3aR and -tdTomato-C5aR2 knockin mice

Support Protocol 2: Preparation of genomic DNA

Basic Protocol 3: Determination of C5aR1, C5aR2, and C3aR expression using floxed AT receptor reporter mice

Support Protocol 3: Determination of C3aR expression using a C3aR-specific antibody

Support Protocol 4: Determination of C5aR1, C5aR2, and C3aR mRNA expression in floxed GFP-C5aR1, floxed tdTomato-C5aR2 or -tdTomato C3aR positive cells

Basic Protocol 4: Analysis of C5aR1-driven ERK1/2 phosphorylation in GFP-C5aR1⁺ cells

Basic Protocol 5: Assessment of C3aR functions in cells obtained from floxed tdTomato-C3aR knockin mice- Determination of C3aR internalization

Alternate Protocol: C3a-induced increase in intracellular Ca²⁺

Basic Protocol 6: C5aR2-driven IFN-γ production from NK cells

Support Protocol 5: Isolation of splenic NK cells by FACS

In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: LCMV infection routes in mice

Support Protocol 1: Preparation of LCMV stocks

ASSAYS TO MEASURE LCMV TITERS

Support Protocol 2: Plaque assay

Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer

MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION

Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells

Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells

Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC)

Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells

Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody

Support Protocol 4: Preparation of splenic lymphocytes

Support Protocol 5: Making BHK21-LCMV lysate

Basic Protocol 7: Challenge models

TRANSGENIC MODELS

Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses

Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge

This manuscript describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. A wide variety of animal models have been used to test new vaccines, drugs, and the impact of cigarette exposure. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Aerosol infection of mice with mycobacteria

Basic Protocol 2: Aerosol infection of guinea pig with mycobacteria using a Madison chamber

Alternate Protocol 1: Cigarette exposure prior to infection of mice with mycobacteria

Alternate Protocol 2: Intravenous infection of mice with mycobacteria

Basic Protocol 3: Necropsy methods for animals experimentally infected with mycobacteria

Basic Protocol 4: Following the course of infection

Basic Protocol 5: Measuring the animal immune response to infection

Support Protocol: Cultivation of mycobacteria for use in animal experiments

Extracellular vesicles (EVs) are small, membranous particles that have recently emerged as one the most important mediators of intercellular communication. They can contain a variety of proteins, lipids, and nucleic acids and thus are responsible for modulation of multiple biological processes, including immune response and regulation of immune cells. Immunomodulatory activity of different EVs can be reliably assessed using EVs isolated from cell culture conditioned medium and added to in vitro or ex vivo cultures of immune cells. This article describes protocols for isolation of EVs from cell culture supernatants by differential ultracentrifugation and density gradient centrifugation. It also provides tools and protocols that enable characterization and validation of isolated particles, as well as analysis of interactions between EVs of interest and different subpopulations of human immune cells. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of extracellular vesicles by differential ultracentrifugation

Basic Protocol 2: Isolation of extracellular vesicles by density gradient centrifugation

Support Protocol 1: Imaging of extracellular vesicles using transmission electron microscopy

Support Protocol 2: Detection of extracellular vesicle protein markers by Western blotting

Support Protocol 3: Measurement and counting of extracellular vesicles by nanoparticle tracking analysis

Basic Protocol 3: Analysis of extracellular vesicle uptake or association by different subpopulations of lymphocytes in vitro