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丁香实验推荐阅读
Studying MHC Class II Peptide Loading and Editing In Vitro

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of major histocompatibility complex (MHC) class II molecu ...

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Assembly of Matched Alpha/Beta Subunits to HLA Class II Peptide Receptors

Human antigen presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are composed of polymorphic α and β subunits. The combination of polymorphic α- and β-chains results in cis (encoded on the same chromosome) or trans (encoded on different chromos ...

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PulseChase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading

Pulse–chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval (“pulse”), during which all newly synthesi ...

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Gene Transduction in Human Monocyte-Derived Dendritic Cells Using Lentiviral Vectors

Monocyte-derived dendritic cells (MDDCs) are widely used in the field of human immunology. Although a variety of gene delivery procedures have been used in MDDC, it has remained difficult to achieve robust gene transductions. In this chapter, we describe a procedure for high efficiency gene t ...

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Analysis of Proteasome Generated Antigenic Peptides by Mass Spectrometry

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle ionization method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for de ...

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Preparing Antigens Suitable for Cross-presentation Assays In Vitro and In Vivo

Cross-presentation is defined as the ability of certain professional antigen-presenting cells to take up, process and present extracellular antigens on major histocompatibility class I (MHC-I) molecules to CD8+ T cells. The stimulation of naive cytotoxic CD8+ T cells by this process, t ...

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Isolation of a Specialized, Antigen-Loaded Early Endosomal Subpopulation by Flow Cytometry

Isolation and characterization of antigen-containing endosomes remains difficult utilizing standard purification techniques. Here, we describe a method, which allows isolation of antigen-loaded endosomes, that is based on flow cytometrical analysis and sorting. We spec ...

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Monitoring the Intracellular Routing of Internalized Antigens by Immunofluorescence Microscopy

Professional antigen-presenting cells such as dendritic cells (DCs) and macrophages internalize extracellular antigens, process them intracellularly, and present the resulting antigen-derived peptides in the context of MHC I or MHC II molecules. Since the intracellular rou ...

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Monitoring Dendritic Cell Activation and Maturation

Since the 1997 discovery that the first identified human homologue of Drosophila Toll could activate the innate immune system, the innate arm of immunity has rapidly taken on a new light as an important player in the recognition of pathogens and damaged self. The recognition of danger by dendritic ...

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Preparation of Dendritic Cells by In Vitro Cultures

In vitro cultures of bone marrow-derived precursors are a convenient method for generating dendritic cells (DC). This method additionally overcomes the problem of low availability of certain DC types, DC heterogeneity, and laborious procedures encountered using ex vivo isolation ...

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The Purification of Large Numbers of Antigen Presenting Dendritic Cells from Mouse Spleen

Dendritic cells (DC) are found at low frequency in lymphoid and non-lymphoid tissues. Different DC subsets are adept at different roles in immunity in diverse scenarios of attack by infectious agents, as well as in the maintenance of self-tolerance. A key element in the ability of DC to initiate adap ...

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Tracking Antigen-Specific CD8+ T Cells Using MHC Class I Multimers

The tracking of epitope-specific T cells is a useful approach for the study of adaptive immune responses. This protocol describes how Major Histocompatibility Complex Class I (MHC-I) multimers can be used to stain, enrich, and enumerate (rare) populations of CD8+ T cells specific for a given ant ...

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Production of CD4+ and CD8+ T Cell Hybridomas

T cell hybridomas are very useful tools to investigate antigen presenting cell (APC) function. They were developed based on the fusion technology that led to monoclonal antibody section. Antigen-specific primary T cells are generated and fused to an immortal thymoma line. Unfused thymo ...

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Cloning CD8+ Cytolytic T Lymphocytes

CD8+ T lymphocyte cloning has resulted in many fundamental advances: structural elucidation of peptide-MHC recognition, spatiotemporal dissection of the thymic positive and negative selection processes and is further expected, TCRs being the key molecules controlling T cell a ...

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Evaluating CD8+ T Cell Responses In Vitro

The 51Cr-release assay described in the 1960s has been for decades the gold standard cytolytic assay and remains in use in many laboratories. Whereas other radioactive tests were later on described, they never fully replaced the 51Cr-release assay. More thorough understanding of CTL biol ...

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Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and effici ...

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Bioinformatics Identification of Antigenic Peptide: Predicting the Specificity of Major MHC Class I and II Pathway Players

Bioinformatics methods for immunology have become increasingly used over the last decade and now form an integrated part of most epitope discovery projects. This wide usage has led to the confusion of defining which of the many methods to use for what problems. In this chapter, an overview is given f ...

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Recombinant Poxviruses: Versatile Tools for Immunological Assays

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, ...

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Purification, Preparation, and Use of ChaperonePeptide Complexes for Tumor Immunotherapy

The molecular chaperone heat-shock protein 70 (Hsp70) possesses immune stimulatory properties that have been employed in the preparation of anticancer vaccines. Hsp70 binds antigenic peptides in the cytoplasm of cancer cells. Hsp70 thus serves as a convenient, non-discriminati ...

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Identifying Source Proteins for MHC Class I-Presented Peptides

Identification of antigenic peptides recognized by cytolytic T lymphocytes (CTL) is a prerequisite for the development of targeted cancer immunotherapy approaches. This chapter provides a global approach for the identification of peptides recognized by CTL. It implies the iden ...

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