免疫学

Elevations in cytosolic calcium (Ca²⁺) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca²⁺ indicators remains the gold standard for visualization and quantification of intracellular Ca²⁺ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca²⁺ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca²⁺ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability – an essential requirement for recording dynamic changes in cytosolic Ca²⁺ levels – and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca²⁺ signals over time using Fiji (ImageJ) software.

Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (e.g., DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues.

Graphic abstract:

Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4⁺ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4⁺ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

B lymphocyte activation is regulated by its membrane-bound B cell receptors (BCRs) upon recognizing diverse antigens. It is hypothesized that antigen binding would trigger conformational changes within BCRs, followed by a series of downstream signaling activation. To measure the BCR conformational changes in live cells, a fluorescent site-specific labeling technique is preferred. Genetically encoded fluorescent tags visualize the location of the target proteins. However, these fluorescent proteins are large (~30 kDa) and would potentially perturb the conformation of BCRs. Here, we describe the general procedures of utilizing short tag-based site-specific labeling methodologies combining with fluorescence resonance energy transfer (FRET) assay to monitor the conformational changes within BCR extracellular domains upon antigen engagement.

Testicular macrophages (tMΦ) are the most abundant immune cells residing in the testis, an immune-privileged organ. TMΦ are known to exhibit different functions, such as protecting spermatozoa from auto-immune attack by producing immunosuppressive cytokines and trophic roles in supporting spermatogenesis and male sex hormone production. They also contribute to fetal testicular development. Recently, we characterized two distinct tMΦ populations based on their morphology, localization, cell surface markers, and gene expression profiling. Here, we focus and describe in detail the phenotypical distinction of these two tMΦ populations by fluorescence-activated cell sorting (FACS) using multicolor panel antibodies combining with high-resolution immunofluorescence (IF) imaging. These two techniques enable to classify two tMΦ populations: interstitial tMΦ and peritubular tMΦ.

Growing evidence suggests the involvement of TLR4, a receptor in the innate immune system, in muscle loss in uremia. Recently, we have evaluated TLR4 in human skeletal muscle from chronic kidney disease patients, by immunohistochemistry and image analysis. Unlike the commonly-used Western blot method, immunohistochemistry allows for the observation of protein distribution in the intact tissue while, image analysis, its quantification. In fact, our data highlighted our hypothesis that an enhanced TLR4 skeletal muscle cell expression contributes to the activation of the downward inflammatory pathway in uremic sarcopenia. In this protocol, we describe the procedure for immunostaining TLR4 in human skeletal muscle and for quantifying it by image analysis.

Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular localization of internalized dsRNA via flow cytometry and confocal microscopy respectively.

In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step. Under a microscope, these reactions appear as punctate fluorescent spots, indicating successful PLA reaction and suggesting protein-protein interaction between the two antigens.

In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of immune cells on fungi and the cell wall, making them promising tools for exploring host-pathogen interactions during fungal infection.

The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.