Current Protocols in Immunology最新文献

Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice cultures allow growth of three-dimensional brain tissue that conserves the cellular architecture. This unit describes the preparation and culturing of organotypic brain slices from mice. In three basic protocols, the general procedure, the specific preparation of chopper slices, and slicing of whole-brain vibratome sections are described. Support protocols explain the use of postnatal or adult mice, the preparation of coronal or sagittal slices, the preparation of co-cultures, post-processing of slices, the use of extra filter membranes, and the re-use of membrane inserts. This unit provides an easy-to-understand methodological introduction to brain slice cultures. © 2018 by John Wiley & Sons, Inc.

Bone marrow transplantation is the standard of care for a host of diseases such as leukemia and multiple myeloma, as well as genetically inherited metabolic diseases affecting the central nervous system. In mouse models, bone marrow transplantation has proven a valuable tool for understanding the hematopoietic system and the homing of hematopoietic cells to their target organs. Many techniques have been developed to create chimeric mice, animals with a hematopoietic system derived from a genetic background that differs from the rest of the body. Current genetic tools allow for virtually limitless possibilities in the choice of donor mice. This protocol describes methods of bone marrow transplantation in mouse models for studies of the brain under basal and pathological conditions. Specific points to be addressed include the preparation of recipient mice by irradiation or chemotherapy; the choice, isolation, and injection of donor cells; and analytical methods such as fluorescence-activated cell sorting and immunostaining. © 2018 by John Wiley & Sons, Inc.

Spinal cord injury (SCI) leads to irreversible devastating neurological disabilities. Accumulated evidence in the literature indicates that the inflammatory response that occurs in the spinal cord following injury contributes importantly to spread tissue damage to healthy regions adjacent to the lesion site, and consequently, to increase neurological deficits. Therefore, targeting inflammation could lead to the development of new therapies to prevent tissue damage and neurological impairments after SCI. Inflammation is regulated, in part, by the expression of pro-inflammatory and anti-inflammatory cytokines synthesized, mainly, by glial cells. Hence, methodologies that could ease the quantification of multiple cytokines and immune cells from spinal cord tissue samples are needed to assess the potential of new anti-inflammatory therapies. In the present unit, we describe how to induce contusion injuries in the mouse spinal cord, as well as, two useful methodologies to assess neuroinflammation in lesioned spinal cord tissue samples. © 2018 by John Wiley & Sons, Inc.

CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level. This approach enables rapid target discovery and validation in both mouse and human primary T cells. © 2018 by John Wiley & Sons, Inc.

The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease. This article outlines a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley & Sons, Inc.

This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathways © 2018 by John Wiley & Sons, Inc.

Vitiligo is an autoimmune skin disease in which the pigment-producing melanocytes are destroyed by autoreactive CD8⁺ T cells. As a result, patients develop disfiguring white spots on the skin. This article discusses the first mouse model of vitiligo that develops epidermal depigmentation, similar to disease in human patients. To achieve epidermal depigmentation, mice are genetically engineered to retain melanocytes in the skin epidermis. Induction of disease occurs by adoptive transfer of melanocyte-specific CD8⁺ T cells into recipient mice and the subsequent activation of these T cells using a viral vector. Depigmentation of the epidermis occurs within 5 to 7 weeks in a patchy pattern similar to patients with vitiligo. This article describes the methods of vitiligo induction, quantification of lesion progression and regression, processing of the skin for detailed analysis, and how to use this model to inform clinical studies. © 2018 by John Wiley & Sons, Inc.

Statistical tests and graphs are an important part of any immunological research publication or presentation, but not all immunologists have the statistical expertise to choose the best methods to evaluate and represent their experiments. These protocols provide a brief overview of the statistical methods most relevant to immunology researchers, so they can either analyze their own data or better understand their statistician collaborators. Protocols cover the basics of t‐tests, ANOVA, nonparametric tests, linear and nonlinear regression models, categorical response models, statistical design of experiments, and advanced methods like re‐sampling tests, Bayesian statistics, and methods for high‐throughput ‐omics data. Each topic receives a brief overview of the theory and usage, followed by brief instructions and code examples for R software. These protocols should be useful to biologists who are learning statistics for the first time and biologists who would like a refresher on basic statistical methods.

Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8⁺ T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc.