Current Protocols in Immunology最新文献

Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8⁺ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone-marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona-fide cross-presenting DCs. We also describe methods to assess cross-presentation by DCs using the activation of primary CD8⁺ T cells as a readout. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of bone marrow progenitor cells

Basic Protocol 2: In vitro differentiation of dendritic cells with GM-CSF

Support Protocol 1: Preparation of conditioned medium from GM-CSF producing J558L cells

Basic Protocol 3: In vitro differentiation of dendritic cells with Flt3L

Support Protocol 2: Preparation of Flt3L containing medium from B16-Flt3L cells

Basic Protocol 4: Expansion of cDC1s in vivo for use in ex vivo experiments

Basic Protocol 5: Characterizing resting and activated dendritic cells

Basic Protocol 6: Dendritic cell stimulation, antigenic cargo, and fixation

Support Protocol 3: Preparation of model antigen coated microbeads

Support Protocol 4: Preparation of apoptotic cells

Support Protocol 5: Preparation of recombinant bacteria

Basic Protocol 7: Immunocytochemistry immunofluorescence (ICC/IF)

Support Protocol 6: Preparation of Alcian blue-coated coverslips

Basic Protocol 8: CD8⁺ T cell activation to assess cross-presentation

Support Protocol 7: Isolation and labeling of CD8⁺ T cells with CFSE

Sjögren's syndrome (SS) is a systemic autoimmune disease affecting multiple organ systems. Salivary and lacrimal gland involvement cause dry mouth and dry eye and are the most common clinical presentations of the disease. Patients with SS also have autoantibodies targeting multiple nuclear and cytoplasmic antigens. Innate immune activation plays a critical role in SS pathogenesis. This article describes the activation of specific innate immune pathways in mice to study SS salivary gland manifestations. Methodologies for evaluating salivary gland inflammation and salivary function are described. This article also describes protocols for in-house assays to measure autoantibody titers in serum. © 2020 Wiley Periodicals LLC

Basic Protocol 1: Acceleration of Sjögren's syndrome by activating the toll-like receptor 3 pathway

Basic Protocol 2: Induction of Sjögren's syndrome by activating the stimulator of interferon genes pathway

Alternate Protocol: Acceleration of Sjögren's syndrome by the administration of Freund's incomplete adjuvant

Support Protocol 1: Evaluating salivary gland function

Support Protocol 2: Evaluating salivary gland inflammation

Support Protocol 3: Measuring autoantibody titers by indirect immunofluorescence

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction–independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Generation and culture of cell monolayers in Transwells

Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function

Support Protocol: Immunofluorescent staining of monolayers

Basic Protocol 3: Multiplex flux assay

Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long-term cultures as three-dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co-culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/colonoid-immune cell co-cultures with separate apical and basolateral compartments. Co-cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Seeding enteroid fragments onto Transwells for monolayer formation

Alternate Protocol: Seeding enteroid fragments for monolayer formation using trituration

Basic Protocol 2: Isolation of monocytes and derivation of immune cells from human peripheral blood

Basic Protocol 3: Isolation of neutrophils from human peripheral blood

Basic Protocol 4: Assembly of enteroid/macrophage or enteroid/neutrophil co-culture

Cellular interactions are often essential to regulate immune cell activities during an immune response. To understand the details of this process, it is necessary to study individual receptor/ligand interactions in a quantitative fashion. However, this is often very difficult or even impossible when using real cells for stimulation. Here, we present a method to use cell-sized latex beads for such studies. These beads can be coated with agonistic antibodies or specific ligands in a defined and quantifiable fashion. This creates the possibility of titrating the strength of the stimulation for a specific receptor in a three-dimensional system. Using natural killer (NK) cells as an example, we demonstrate how these beads can be used to stimulate NK cell responses. © 2020 The Authors.

Basic Protocol 1: Covalent coating of latex beads with antibodies

Basic Protocol 2: Quantification of the amount of antibodies on the beads with the QIFIKIT^®

Alternate Protocol 1: Covalent coating of latex beads with streptavidin to bind biotinylated proteins

Alternate Protocol 2: Quantification of the amount of protein on the beads with the QIFIKIT^®

Support Protocol: Functional testing of the beads in a natural killer cell degranulation assay

Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC.

Basic Protocol: Cecal ligation and puncture in the mouse

This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti-ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL-1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenge in a sepsis model. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Priming and activation of inflammasomes in mouse macrophages

Basic Protocol 2: Activation of human NLRP1 inflammasome by DPP8/9 inhibitor talabostat

Basic Protocol 3: Purification and detection of ASC pyroptosome

Alternate Protocol 1: Detection of ASC speck by immunofluorescence staining

Basic Protocol 4: Activation of canonical NLRP3 inflammasome in mice by intraperitoneal delivery of MSU crystals

Alternate Protocol 2: Activation of noncanonical NLRP3 inflammasome in mice by intraperitoneal delivery of LPS

Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC

Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs

Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells

Basic Protocol 2: ELISpot assay for enumeration of memory B cell−derived antibody-secreting cells

Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role that NK cells play is analogous to that of cytotoxic T cells in that they provide rapid responses to virus-infected cells and responses to tumor formation. Unmodified NK cells have long been used in various immunotherapies to treat different tumors, with only marginal success. However, in the last few years, NK cells modified to express chimeric antigen receptors (CAR-NK cells) have emerged as particularly ideal cellular platforms for antigen-specific antitumor agents. Unlike CAR-T cells, they do not elicit allogeneic responses or graft-versus-host disease and therefore can be administered to recipients with differing MHC expression. This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC

Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells

Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells

Support Protocol: Production of lentiviral supernatant