Immunology

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:

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.

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.

The skin plays an important role in protecting the body from pathogens and chemicals in the external environment. Upon injury, a healing program is rapidly initiated and involves extensive intercellular communication to restore tissue homeostasis. The deregulation of this crosstalk can lead to abnormal healing processes and is the foundation of many skin diseases. A relatively overlooked cell type that nevertheless plays critical roles in skin homeostasis, wound repair, and disease is the dendritic epidermal T cells (DETCs), which are also called γδT-cells. Given their varied roles in both physiological and pathological scenarios, interest in the regulation and function of DETCs has substantially increased. Moreover, their ability to regulate other immune cells has garnered substantial attention for their potential role as immunomodulators and in immunotherapies. In this article, we describe a protocol to isolate and culture DETCs and analyse them in vivo within the skin. These approaches will facilitate the investigation of their crosstalk with other cutaneous cells and the mechanisms by which they influence the status of the skin.

Graphic abstract:

Overall workflow to analyse DETCs in vitro and in vivo.

Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format. The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields.

Graphic abstract:

One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.

Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4⁺ T cells, CD8⁺ T cells, γδT cells, and regulatory T cells.

Functional and mechanistic studies of CD4⁺ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (T_H17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting cells (APC) for TCR activation. The protocol described here employs plate-bound antibodies and a T_H17-polarizing cytokine cocktail to activate and differentiate naïve CD4⁺ T (T_nai) cells, reflecting a simple and robust protocol for in vitro T_H17n differentiation. Using T cells that are genetically engineered with an IL-17 reporter, this protocol may enable the rapid production of a pure population of IL17-expressing CD4⁺ T cells for system biology studies and high-throughput functional screening.

Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). In this protocol, a robust method to determine low intracellular ROS production using H₂DCFDA was validated by several ROS-specific as well as NOX-2-specific inhibitors. Cells were treated with inhibitors or control substances prior to treatment with the ROS-inducer of interest. H₂DCFDA was added only for the last 30 min of the treatment schedule. To terminate its conversion, we used a ROS-specific inhibitor until analysis by flow cytometry within the FITC-channel (Ex: 488 nm/Em: 519 nm). In summary, this protocol allows the detection of signaling-relevant intracellular ROS production in cell lines and primary immune cells (e.g., Mono Mac 6 cells and Bone marrow-derived dendritic cells, respectively). Using this method in combination with specific inhibitors, we were able to validate even exceptionally low amounts of ROS produced by NOX-2 and relevant for immune-regulatory signaling.

Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol infection model, and flow cytometry to track Mtb-infected cells in the lung immediately following aerosol infection and fluorescence-activated cell sorting (FACS) to isolate naïve, bystander, and Mtb-infected cells for downstream applications, including RNA-sequencing. This protocol provides the ability to monitor Mtb-infection and cell-specific responses within the context of the lung environment, which is known to modulate the function of both resident and recruited populations. Using this protocol, we discovered that alveolar macrophages respond to Mtb infection in vivo by up-regulating a cell protective transcriptional response that is regulated by the transcription factor Nrf2 and is detrimental to early control of the bacteria.