免疫学

Gammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4⁺ T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4⁺ T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4⁺ lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4⁺ T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4⁺ T-cell regulation to prevent the development of virus-induced immunopathologies in the lung.

Key features

• High-resolution profiling of the functional properties of lung-infiltrating CD4⁺ T cells after viral infection using conventional multiparametric flow cytometry.

• Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining.

• Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4⁺ T cells.

• Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation.

Graphical overview

Graphical abstract depicting the interactions between immune cells infiltrating the alveolar niche and the lung during respiratory infection with a gammaherpesvirus (Murid herpesvirus 4, MuHV-4). Two distinct situations are represented: the inflammatory response developed during viral replication in the lung, either in the presence (WT mice) or absence of regulatory monocytes (CCR2KO mice). Sequential process of the experiment is represented, starting from intratracheal instillation of MuHV-4 virions to tissue dissociation and multicolor staining for flow cytometry analysis.

Type 1 regulatory T (Tr1) cells are an immunoregulatory CD4⁺ Foxp3^- IL-10^high T cell subset with therapeutic potential for various inflammatory diseases. Retroviral (RV) transduction has been a valuable tool in defining the signaling pathways and transcription factors that regulate Tr1 differentiation and suppressive function. This protocol describes a method for RV transduction of naïve CD4⁺ T cells differentiating under Tr1 conditions, without the use of reagents such as polybrene or RetroNectin. A major advantage of this protocol over others is that it allows for the role of genes of interest on both differentiation and function of Tr1 cells to be interrogated. This is due to the high efficiency of RV transduction combined with the use of an IL10^GFP/Foxp3^RFP dual reporter mouse model, which enables successfully transduced Tr1 cells to be identified and sorted for functional assays. In addition, this protocol may be utilized for dual/multiple transduction approaches and transduction of other lymphocyte populations, such as CD8⁺ T 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:

When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response.

Graphic abstract:

In-vitro simulation of restimulation-induced cell death in activated human T cells.

Blood endothelial cells (ECs) constitute the primary physical barrier to be crossed by circulating leukocytes, once attracted to a site of ongoing inflammation/infection. Upon a pro-inflammatory stimulus, such as tumor necrosis factor (TNF), ECs upregulate adhesion molecule expression to favor the adhesion and, subsequently, the transendothelial migration of the attracted lymphocytes. To address the ability of a cell to transmigrate through a monolayer of ECs, the classical transmigration assay is usually performed (Muller and Luscinskas, 2008). In the present protocol, adapted from Safuan et al. (2012), we describe an in vitro assay for assessing the functionality of the second step of the transendothelial migration, i.e., the firm adhesion of peripheral blood mononuclear cells (PBMCs) to ECs, under static conditions. By pre-incubating primary human umbilical cord ECs (HUVECs) with either innate lymphoid cell progenitors (ILCPs) or TNF, we were able to upregulate adhesion molecules on the EC surface. Then, by adding total PBMCs, we were able to both quantitatively and qualitatively analyze the cellular subtype and number of PBMCs that adhered to the pre-treated ECs. The important advantage of this technique is the possibility to perform functional studies on ECs biology since, differently from transwell-based strategies, it allows a good recovery of ECs at the end of the assay. Overall, this assay enables to interrogate how/if different stimulations/cell types can influence EC ability to retain PBMCs in vitro, under static conditions.

Graphic abstract:

The workflow of the Static Adhesion Assay.

Natural killer (NK) cells are large granular lymphocytes that keep in check the health of neighboring cells through a large array of intrinsically expressed germline-coded receptors. Most importantly, CD16 is a low affinity Fc receptor for IgG that mediates the antibody-dependent cellular cytotoxicity (ADCC) of NK cells, bridging the innate and adaptive immunities. There has been a significant interest in genetically engineering NK cells to enhance its ADCC, with the ultimate goal to produce off-the-shelf NK cell therapy products that can be combined with target-specific monoclonal antibodies to improve clinical outcomes. Previous protocols of ADCC assays use complex cell-based antigen-antibody models, which are both costly and time-consuming. This current protocol is devoid of target cells and uses plate-bound immobilized anti-CD16 antibodies as the trigger. It greatly shortens the experimental time, while faithfully evaluating NK cells ADCC.

Graphic abstract:

Workflow of stimulating NK cells via CD16 by plate-bound anti-CD16 mAb.

Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That’s why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.

Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability – together with the relatively easy quantification of phosphorylation – phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip et al. (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts (i.e., screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction.

During immune responses, B cells home to lymph nodes (LN), where they encounter antigens. Homing starts with capture and L-selectin-dependent rolling on the activated endothelium of high endothelial venules (HEV). After recognition of chemokines presented on HEV, activation of B cell integrins occurs mediating firm arrest. Subsequently, B cells crawl to the spot of extravasation to enter the LN. Extravasation can be visualized and quantified in vivo by intravital microscopy (IVM) of the inguinal LN. Here, we describe an established protocol that permits detailed in vivo analysis of B cell recruitment to LN under sterile inflammatory conditions. We describe data acquisition, exportation, quantification, and statistical analysis using specialized software. IVM of LN is a powerful technique that can provide a better understanding of B cell migratory behavior during inflammation in vivo.