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
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.
- NSF-PAR ID:
- 10168877
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Immunology
- Volume:
- 130
- Issue:
- 1
- ISSN:
- 1934-3671
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Polyclonal stimulation of memory B cells using unfractionated PBMCsAlternate Protocol : Stimulation of small PBMC numbers using 96‐well plates with U‐bottom wellsBasic Protocol 2 : ELISpot assay for enumeration of memory B cell−derived antibody‐secreting cells -
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Abstract Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed in
E. coli , most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.This article was corrected on 19 July 2022. See the end of the full text for details.
Basic Protocol 1 : Lentivirus production and expression line creationSupport Protocol 1 : Six‐well assay for estimation of production cell line yieldSupport Protocol 2 : Universal ELISA for quantifying proteins with fused leucine zippers and His‐tagsBasic Protocol 2 : Cultures for production of Class II MHC proteinsBasic Protocol 3 : Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatographyAlternate Protocol 1 : IMAC purification of His‐tagged Class II MHCSupport Protocol 3 : Protein concentration measurements and adjustmentsSupport Protocol 4 : Polishing purification by anion‐exchange chromatographySupport Protocol 5 : Estimating biotinylation percentage by streptavidin precipitationBasic Protocol 4 : Peptide exchangeBasic Protocol 5 : Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometryAlternate Protocol 2 : Native isoelectric focusing to validate MHC‐II peptide loadingBasic Protocol 6 : MultimerizationBasic Protocol 7 : Staining cells with Class II MHC tetramers -
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Abstract Staphylococcus aureus is a Gram‐positive bacterium that colonizes almost every organ in humans and mice and is a leading cause of diseases worldwide.S. aureus infections can be challenging to treat due to widespread antibiotic resistance and their ability to cause tissue damage. The primary modes of transmission ofS. aureus are via direct contact with a colonized or infected individual or invasive spread from a colonization niche in the same individual.S. aureus can cause a myriad of diseases, including skin and soft tissue infections (SSTIs), osteomyelitis, pneumonia, endocarditis, and sepsis.S. aureus infection is characterized by the formation of purulent lesions known as abscesses, which are rich in live and dead neutrophils, macrophages, and surrounded by a capsule containing fibrin and collagen. Different strains ofS. aureus produce varying amounts of toxins that evade and/or elicit immune responses. Therefore, animal models ofS. aureus infection provide a unique opportunity to understand the dynamics of organ‐specific immune responses and modifications in the pathogen that could favor the establishment of the pathogen. With advances in in vivo imaging of fluorescent transgenic mice, combined with fluorescent/bioluminescent bacteria, we can use mouse models to better understand the immune response to these types of infections. By understanding the host and bacterial dynamics within various organ systems, we can develop therapeutics to eliminate these pathogens. This module describes in vivo mouse models of both local and systemicS. aureus infection. © 2021 Wiley Periodicals LLC.This article was corrected on 20 July 2022. See the end of the full text for details.
Basic Protocol 1 : Murine model ofStaphylococcus aureus subcutaneous infectionAlternate Protocol : Murine tape stripping skin infection modelBasic Protocol 2 : Sample collection to determine skin structure, production of inflammatory mediators, and bacterial loadBasic Protocol 3 : Murine model of post‐traumaticStaphylococcus aureus osteomyelitisBasic Protocol 4 : Intravenous infection of the retro‐orbital sinusSupport Protocol : Preparation of the bacterial inoculum -
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Abstract New genetic engineering techniques have advanced the field of plant molecular biology, and
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Abstract 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 cellsBasic Protocol 2 : In vitro differentiation of dendritic cells with GM‐CSFSupport Protocol 1 : Preparation of conditioned medium from GM‐CSF producing J558L cellsBasic Protocol 3 : In vitro differentiation of dendritic cells with Flt3LSupport Protocol 2 : Preparation of Flt3L containing medium from B16‐Flt3L cellsBasic Protocol 4 : Expansion of cDC1s in vivo for use in ex vivo experimentsBasic Protocol 5 : Characterizing resting and activated dendritic cellsBasic Protocol 6 : Dendritic cell stimulation, antigenic cargo, and fixationSupport Protocol 3 : Preparation of model antigen coated microbeadsSupport Protocol 4 : Preparation of apoptotic cellsSupport Protocol 5 : Preparation of recombinant bacteriaBasic Protocol 7 : Immunocytochemistry immunofluorescence (ICC/IF)Support Protocol 6 : Preparation of Alcian blue‐coated coverslipsBasic Protocol 8 : CD8+T cell activation to assess cross‐presentationSupport Protocol 7 : Isolation and labeling of CD8+T cells with CFSE
