Protocols in Current Issue
0 Q&A 206 Views May 20, 2023

P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.

0 Q&A 177 Views May 20, 2023

T cells localized to the kidneys and vasculature/perivascular adipose tissue (PVAT) play an important role in hypertension and vascular injury. CD4+, CD8+, and γδ T-cell subtypes are programmed to produce interleukin (IL)-17 or interferon-γ (IFNγ), and naïve T cells can be induced to produce IL-17 via the IL-23 receptor. Importantly, both IL-17 and IFNγ have been demonstrated to contribute to hypertension. Therefore, profiling cytokine-producing T-cell subtypes in tissues relevant to hypertension provides useful information regarding immune activation. Here, we describe a protocol to obtain single-cell suspensions from the spleen, mesenteric lymph nodes, mesenteric vessels and PVAT, lungs, and kidneys, and profile IL-17A- and IFNγ-producing T cells using flow cytometry. This protocol is different from cytokine assays such as ELISA or ELISpot in that no prior cell sorting is required, and various T-cell subsets can be identified and individually assessed for cytokine production simultaneously within an individual sample. This is advantageous as sample processing is kept to a minimum, yet many tissues and T-cell subsets can be screened for cytokine production in a single experiment. In brief, single-cell suspensions are activated in vitro with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Golgi cytokine export is inhibited with monensin. Cells are then stained for viability and extracellular marker expression. They are then fixed and permeabilized with paraformaldehyde and saponin. Finally, antibodies against IL-17 and IFNγ are incubated with the cell suspensions to report cytokine production. T-cell cytokine production and marker expression is then determined by running samples on a flow cytometer. While other groups have published methods to perform T-cell intracellular cytokine staining for flow cytometry, this protocol is the first to describe a highly reproducible method to activate, phenotype, and determine cytokine production by CD4, CD8, and γδ T cells isolated from PVAT. Additionally, this protocol can be easily modified to investigate other intracellular and extracellular markers of interest, allowing for efficient T-cell phenotyping.

Protocols in Past Issues
0 Q&A 199 Views May 5, 2023

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such as Staphylococcus aureus to overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such as S. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and both Lactococcus lactis and S. aureus.

0 Q&A 531 Views Apr 20, 2023

Chimeric antigen receptor (CAR)-T therapy launched a new era for cancer treatments, displaying outstanding effectiveness in relapsed or refractory B-cell malignancies. Demonstrating the tumor-killing ability of CAR-Ts in mouse xenograft models serves as a golden criterium in preclinical research. Here, we describe a detailed method for evaluating CAR-T’s function in immune-deficient mice bearing Raji B cell–induced tumors. It includes generating CD19 CAR-T cells from healthy donors, injecting tumor cells and CAR-T cells into mice, and monitoring tumor growth and CAR-T state. This protocol provides a practical guide to evaluate CAR-T’s function in vivo within eight weeks.

Graphical overview

0 Q&A 491 Views Apr 5, 2023

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

0 Q&A 745 Views Mar 20, 2023

E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs, including the brain, lung, heart, and colon. The effect of flavored fourth-generation pod-based E-cigs (JUUL) on murine gut inflammation is modulated by both flavor and exposure period. Exposure of mice to JUUL mango and JUUL mint for one month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after one month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after three months of exposure. In this protocol, we detail the process of RNA isolation from the mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon.

0 Q&A 390 Views Mar 5, 2023

A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.

0 Q&A 370 Views Feb 20, 2023

Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow. We set out to remove this hurdle by genetically engineering antibodies directly in the immunoglobulin (Ig) locus of hybridoma cells. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and homology-directed repair (HDR) to modify antibody’s format [mAb or antigen-binding fragment (Fab’)] and isotype. This protocol describes a straightforward approach, with little hands-on time, leading to stable cell lines secreting high levels of engineered antibodies. Parental hybridoma cells are maintained in culture, transfected with a guide RNA (gRNA) targeting the site of interest in the Ig locus and an HDR template to knock in the desired insert and an antibiotic resistance gene. By applying antibiotic pressure, resistant clones are expanded and characterized at the genetic and protein level for their ability to produce modified mAbs instead of the parental protein. Finally, the modified antibody is characterized in functional assays. To demonstrate the versatility of our strategy, we illustrate this protocol with examples where we have (i) exchanged the constant heavy region of the antibody, creating chimeric mAb of a novel isotype, (ii) truncated the antibody to create an antigenic peptide-fused Fab’ fragment to produce a dendritic cell–targeted vaccine, and (iii) modified both the constant heavy (CH)1 domain of the heavy chain (HC) and the constant kappa (Cκ) light chain (LC) to introduce site-selective modification tags for further derivatization of the purified protein. Only standard laboratory equipment is required, which facilitates its application across various labs. We hope that this protocol will further disseminate our technology and help other researchers.

Graphical abstract

0 Q&A 362 Views Feb 5, 2023

Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host’s early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The pro-inflammatory cytokine interferon gamma (IFNγ), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity.

To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium ( This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi.

We generated bone marrow–derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM with for one hour. Shortly before infection, cells were stained with CellROXTM Deep Red reagent. In its reduced form, CellROXTM is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time.

We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures.

Graphical abstract

0 Q&A 306 Views Jan 5, 2023

Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.

0 Q&A 667 Views Jan 5, 2023

Traditional drug safety assessments often fail to predict complications in humans, especially when the drug targets the immune system. Rodent-based preclinical animal models are often ill-suited for predicting immunotherapy-mediated adverse events in humans, in part because of the fundamental differences in immunological responses between species and the human relevant expression profile of the target antigen, if it is expected to be present in normal, healthy tissue. While human-relevant cell-based models of tissues and organs promise to bridge this gap, conventional in vitro two-dimensional models fail to provide the complexity required to model the biological mechanisms of immunotherapeutic effects. Also, like animal models, they fail to recapitulate physiologically relevant levels and patterns of organ-specific proteins, crucial for capturing pharmacology and safety liabilities. Organ-on-Chip models aim to overcome these limitations by combining micro-engineering with cultured primary human cells to recreate the complex multifactorial microenvironment and functions of native tissues and organs. In this protocol, we show the unprecedented capability of two human Organs-on-Chip models to evaluate the safety profile of T cell–bispecific antibodies (TCBs) targeting tumor antigens. These novel tools broaden the research options available for a mechanistic understanding of engineered therapeutic antibodies and for assessing safety in tissues susceptible to adverse events.

Graphical abstract

Figure 1. Graphical representation of the major steps in target-dependent T cell–bispecific antibodies engagement and immunomodulation, as performed in the Colon Intestine-Chip

0 Q&A 482 Views Dec 5, 2022

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies.

Graphical abstract

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