Current Protocols in Immunology最新文献

The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell–based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector-encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Epitope-specific CD4⁺ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4⁺ T cells under physiological conditions. © 2019 by John Wiley & Sons, Inc.

Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small intestine, bone marrow, mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells, in which ILC2 enrichment is optimized. In addition, we describe in detail the methodologies used to activate ILC2s and the assays necessary for the detection of their effector cytokines. We highlight the differences in ILC2 characterization within distinct tissues that we have recently identified. © 2019 by John Wiley & Sons, Inc.

Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human osteoclasts, allowing study of the effects of specific cells, cytokines, or chemical substances on osteoclast formation and activity without the need for expensive and time-consuming animal experiments. In addition, we provide protocols for specific staining of osteoclasts and for analysis of resorption activity using calcium phosphate–coated surfaces or bone slices. © 2019 by John Wiley & Sons, Inc.

This unit describes methods for quantifying phagocytosis and imaging the immunological synapse between T cells and antigen-presenting cells (APCs), with both techniques delivering valuable information about APC function. These aspects of APC biology have traditionally been challenging to quantify, and imaging flow cytometry, which harnesses the high-throughput nature of flow cytometry combined with the capacity of microscopy to deliver spatial localization, facilitates analysis of these APC functions in a fashion that was previously not possible. Imaging flow cytometry allows large numbers of events to be captured and large amounts of fluorescence data to be quantified at the physical location of markers of interest, both on the cell surface and in intracellular compartments, combining key features of traditional flow cytometry and fluorescence microscopy. © 2019 by John Wiley & Sons, Inc.

Microglia represent 5-10% of cells in the central nervous system and contribute to the development, homeostasis, injury, and repair of neural tissues. As the tissue-resident macrophages of the central nervous system, microglia execute core innate immune functions such as detection of pathogens/damage, cytokine secretion, and phagocytosis. However, additional properties that are specific to microglia and their neural environment are beginning to be appreciated. This article describes approaches for purification of microglia by fluorescence-activated cell sorting using microglia-specific surface markers and for enrichment of microglia by magnetic sorting and immunopanning. Detailed information about culturing primary microglia at various developmental stages is also provided. Throughout, we focus on special considerations for handling microglia and compare the relative strengths or disadvantages of different protocols. © 2018 by John Wiley & Sons, Inc.

LC3-associated phagocytosis (LAP) is an unconventional form of autophagy that relies on parts of the canonical autophagy machinery for its function. LAP is triggered upon receptor-mediated phagocytosis and is characterized by the formation of a single-membrane vesicle decorated with the autophagy protein LC3. In professional phagocytic cells, such as macrophages, the role of LAP in immune processes has been characterized, although how LAP functions at the molecular level remains poorly defined. It is important to point out that as for all autophagic pathways, the study of LAP is still challenging for the scientific community because it is a dynamic and complex process, requiring interactions among several proteins. Here, we describe the most common methods used to monitor and quantify the formation of LC3-coated single-membrane endosomes, or so-called LAPosomes, and to validate the involvement of LAP in immunological processes of human macrophages. © 2018 by John Wiley & Sons, Inc.

Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc.