Immunology

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry–based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers.

Key features

• This protocol describes a flow cytometry–based method to analyze the myeloid cell response in retinopathy mouse models.

• The protocol can distinguish between microglia- and monocyte-derived macrophages.

• It can be modified to incorporate markers of interest.

We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.

Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4⁺ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify Lineage⁻Thy1.2⁺ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology.

Key features

• In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33.

• Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.

• During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels of Gata-3 and Ki-67 and produce greater amounts of IL-5 and IL-13.

Graphical overview

The sirtuin 6 has emerged as a regulator of acute and chronic immune responses. Recent findings show that SIRT6 is necessary for mounting an active inflammatory response in macrophages. In vitro studies revealed that SIRT6 is stabilized in the cytoplasm to promote tumor necrosis factor (TNFα) secretion. Notably, SIRT6 also promotes TNFα secretion by resident peritoneal macrophages upon lipopolysaccharide (LPS) stimulation in vivo. Although many studies have investigated SIRT6 function in the immune response through different genetic and pharmacological approaches, direct measurements of in vivo SIRT6 expression in immune cells by flow cytometry have not yet been performed. Here, we describe a step-by-step protocol for peritoneal fluid extraction, isolation, and preparation of peritoneal cavity cells, intracellular SIRT6 staining, and flow cytometry analysis to measure SIRT6 levels in mice peritoneal macrophages. By providing a robust method to quantify SIRT6 levels in different populations of macrophages, this method will contribute to deepening our understanding of the role of SIRT6 in immunity, as well as in other cellular processes regulated by SIRT6.

Graphical abstract:

Employing a novel mouse model of immune related adverse events (irAEs) induced by combination of anti-PD1 and anti-CTLA-4 antibodies, we visualized immune infiltration into the liver, lung, pancreas, and colon. Here, we describe the avidin-biotin conjugate (ABC) method used to stain T cells (CD4 and CD8), B cells (CD19), macrophages (F4/80), and cells bound by the in vivo administered rat anti-mouse antibodies for chromogenic immunohistochemistry (IHC). Using a biotinylated goat anti-rat antibody, we detected the localization of cells bound to the in vivo antibodies for PD-1 and CTLA-4. IHC has advantages over other techniques, namely antibody availability, resistance to photobleaching, and greater sensitivity. Additionally, detection and localization of in vivo antibodies can be used in mice models to infer their therapeutic efficacy, stability, and function.

Graphical abstract:

During adaptive immune responses, germinal centers (GC) appear as transient microstructures, in which antigen-specific B and T cells interact with each other. Because only the antigen-activated B and T cells, such as Plasmablasts or follicular T helper (Tfh) cells, are present in GC, the in depth-analysis of GC is of great interest. To identify the cells that reside within GC, the majority of studies use the expression of specific surface molecules for analysis by flow cytometry. To do so, the tissue has to be disrupted for the preparation of single-cell suspensions. Thereby, the local information regarding neighborhoods of B cells and T cells and their potential interaction is lost. To study GC in vivo within their original microenvironment, we established a protocol for the isolation of GC by laser microdissection. To enable the identification of GC for subsequent transcriptomic analysis, the degradation of mRNA was diminished by using frozen tissues and by establishing a rapid staining protocol. This procedure enables histological and transcriptomic analysis of individual GC even within one lymphoid organ.

Macrophages are important for host defense against intracellular pathogens like Salmonella and can be differentiated into two major subtypes. M1 macrophages, which are pro-inflammatory and induce antimicrobial immune effector mechanisms, including the expression of inducible nitric oxide synthase (iNOS), and M2 macrophages, which exert anti-inflammatory functions and express arginase 1 (ARG1). Through the process of phagocytosis, macrophages contain, engulf, and eliminate bacteria. Therefore, they are one of the first lines of defense against Salmonella. Infection with Salmonella leads to gastrointestinal disorders and systemic infection, termed typhoid fever. For further characterization of infection pathways, we established an in vitro model where macrophages are infected with the mouse Salmonella typhi correlate Salmonella enterica serovar Typhimurium (S.tm), which additionally expresses red fluorescent protein (RFP). This allows us to clearly characterize macrophages that phagocytosed the bacteria, using multi-color flow cytometry.

In this protocol, we focus on the in vitro characterization of pro- and anti-inflammatory macrophages displaying red fluorescent protein-expressing Salmonella enterica serovar Typhimurium, by multi-color flow cytometry.

Autoreactive T cells in autoantibody-mediated autoimmune diseases can be divided into two major subsets: (i) follicular T helper cells (Tfh) that provide T cell help in germinal centers (GC) and (ii) effector T (Teff) cells that immigrate into peripheral tissue sites such as the skin and mediate local inflammation. To study the sequence of events leading to the loss of tolerance in autoantibody-mediated autoimmune diseases it is required to investigate both T cell subsets simultaneously. This approach is hampered mainly because the appearance of skin inflammation in mouse models is a random process, which makes it difficult to define the location of inflammation at the right time point. To overcome this problem, we developed a scratching technique for ear skins that leads to the establishment of chronic autoimmune wounds in the mouse model for the pemphigoid-like disease epidermolysis bullosa acquisita. By defining the exact place where the skin wounds should form, this protocol enables a detailed analysis of skin-immigrating Teff cells. Of note, this protocol induces GC in draining lymph nodes in parallel so that Tfh cells in GC can be investigated concurrently. This protocol is not restricted to T cells and can be used for any other skin-immigrating inflammatory cells.

The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8⁺ T cell-mediated inhibition of HIV replication in CD4⁺ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures [isolated CD4⁺ T cells or CD8⁺ depleted peripheral blood mononuclear cells (PBMCs)], HIV strains (laboratory strains, isolates, reporter viruses) and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA) have been reported.

Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24⁺ cells in replicate cultures and the corresponding 95% confidence intervals. We identify methodological and analysis changes that could be incorporated into other protocols to improve assay reproducibility. We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8 T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions.

Graphic abstract:

Here, we describe a combinatorial approach in reverse vaccinology to identify immunogenic class I major histocompatibility complex (MHC) displayed epitopes derived from a morbillivirus named pestes des petits ruminants (PPRV). The protocol describes an in silico prediction of immunogenic epitopes using an IEDB tool. The predicted peptides were further analysed by molecular docking with mouse class I MHC (H-2K^b), to assess their binding affinity, and their immunogenicity was validated, using acellular and cellular assays. Finally, an enumeration of the expanded PPRV-specific CD8⁺ T cells in infected or immunized mice against the immunogenic peptides was performed ex vivo. Synthetic peptide derivatives from different structural and non-structural proteins of PPRV were used to measure the extent of stabilized H2-K^b, using an ELISA based acellular assay and TAP deficient RMA/s cells. Fluorescently labelled H2-K^b-tetramers were generated by displacing a UV photocleavable conditional ligand with the PPRV-peptides. The resulting reagents were used to identify and enumerate virus-specific CD8⁺ T cells in immunized or PPRV-infected mice. The combinatorial approach described here could be used to identify immunogenic epitopes of any pathogen, autoantigens, as well as cancer antigens.

Graphic abstract:

Figure 1. General schematic to identify immunogenic peptides and their stabilization on MHC I molecule.

Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie et al., 1994), has enabled convenient visualization of gene expression in vivo, this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences. Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frøkjær-Jensen, 2019). However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure.

Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing. Here, we present a reliable protocol in which C. elegans nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye. Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined. We have successfully used this protocol (O’Hagan et al., 2011 and 2017; Power et al., 2020) to preserve and detect post-translational modifications of tubulin in C. elegans ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).