Immunology

Chemically induced murine colitis models are widely used to understand intestinal homeostasis and inflammatory responses during acute and chronic gut inflammation, such as inflammatory bowel disease (IBD). Resident populations of immune cells, together with those recruited during an inflammatory response, maintain intestinal immunity by mounting an effective immune response to enteropathogenic microbes while at the same time maintaining tolerance against commensals. To better understand the disease mechanism, studying different immune cell populations and their dynamic changes during infection and inflammation is essential. However, isolating healthy and viable immune populations, particularly hyperactivated neutrophils and macrophages from the inflamed gut (i.e., active disease site), is challenging as tissues are usually subjected to rigorous enzymatic digestion for an extended period. Here, we describe a method that uses a cell dissociator (Medimachine II from Syntec International) to separate intestinal tissue after short enzymatic digestion to obtain a single-cell suspension. This technique facilitates the isolation of immune cells from mouse intestinal tissues in high quantity and with superior viability in a very short time frame. This protocol delivers 80%–90% cell viability, which is 1.5 to 2-fold higher than conventional methods of isolating cells from inflamed mouse colons. The composition, phenotype, activation state, and gene expression profile of cells isolated using this protocol can be assessed by using multiple methods, including, but not limited to, flow cytometry, quantitative PCR, immunoblotting, mass spectrometry, single-cell RNA sequencing, and functional readouts such as reactive oxygen species (ROS) production.

In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS Express^TM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS Express^TM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies.

The growing demand for advanced analytical techniques to explore complex cellular targets of nanotherapeutics has driven the development of innovative methodologies. This protocol presents a refined approach for fluorescent labeling and flow cytometric analysis of colonic cells following oral lipid nanoparticle (LNP) treatment, focusing on LNP uptake in colonic cell subpopulations in a DSS-induced colitis mouse model. By integrating optimized fluorochrome selection and gating strategies with advanced t-distributed stochastic neighbor embedding (t-SNE) analysis, this method enables precise identification and multidimensional visualization of LNP-targeted epithelial and macrophage populations under the complex conditions of inflamed colon tissue. Building on our previous studies demonstrating the effectiveness of nanoparticles in targeted drug delivery, this approach highlights the utility of flow cytometry for assessing uptake efficiency and cellular targeting. Unlike conventional protocols, it incorporates t-SNE for enhanced multidimensional analysis, allowing for the detection of subtle cellular patterns and the delineation of intricate clusters. By addressing gaps in traditional methodologies, this protocol provides a robust and reproducible framework for investigating in vivo cellular targets and optimizing drug delivery strategies for nanomedicines.

γδ T cells play a critical role in homeostasis and diseases such as infectious diseases and tumors in both mice and humans. They can be categorized into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells. While CD27 expression segregates these two subsets in mice, little is known about human γδT17 cell differentiation and expansion. Previous studies have identified γδT17 cells in human skin and mucosal tissues, including the oral cavity and colon. However, human γδ T cells from peripheral blood mononuclear cells (PBMCs) primarily produce IFN-γ. In this protocol, we describe a method for in vitro expansion and polarization of human γδT17 cells from PBMCs.

Key Features

• Expansion of γδ T cells from peripheral blood mononuclear cells.

• Human IL-17A-producing γδ T-cell differentiation and expansion using IL-7 and anti-γδTCR.

• Analysis of IL-17A production post γδ T-cell expansion.

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3⁺ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4⁺ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4⁺ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4⁺ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4⁺ T cells in an antigen-specific context.

Key features

• This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022).

• This protocol requires transgenic mice, such as OTIIxRag2^-/- mice and the cognate peptide OVA_323–339, to assess mTEC antigen presentation to CD4⁺ T cells.

• This requires specific equipment such as a Miltenyi Biotec AutoMACS^® Pro Separator, a BD FACSAria^TM III cell sorter, and a BD^® LSR II flow cytometer.

Graphical overview

Dendritic cells have been investigated for cell-based immunotherapy for various applications. The low abundance of dendritic cells in blood hampers their clinical application, resulting in the use of monocyte-derived dendritic cells as an alternative cell type. Limited knowledge is available regarding blood-circulating human dendritic cells, which can be divided into three subsets: type 2 conventional dendritic cells, type 1 conventional dendritic cells, and plasmacytoid dendritic cells. These subsets exhibit unique and desirable features for dendritic cell-based therapies. To enable efficient and reliable human research on dendritic cell subsets, we developed an efficient isolation protocol for the three human dendritic cell subsets, resulting in pure populations. The sequential steps include peripheral blood mononuclear cell isolation, magnetic-microbead lineage depletion (CD14, CD56, CD3, and CD19), and individual magnetic-microbead isolation of the three human dendritic cell subsets.

Graphical overview

Scheme of the dendritic cell (DC) isolation protocol. Starting material for this process is human blood (buffy coat or aphaeresis). From that, peripheral blood mononuclear cells (PBMCs) are isolated by using ficoll gradient centrifugation. Then, an enrichment for DCs is performed using semi-automated equipment. From the enriched fraction, DC subsets are obtained by magnetic cell sorting.

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection.

Key features

• Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice.

• Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry.

• Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice.

• Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.

Graphical overview

Overview of the methods to trace the migration of Leishmania and monocytes from the skin to lymphatic tissues by flow cytometry. Infective metacyclic promastigotes (from axenic culture) and monocytes (isolated from the bone marrow of donor mice) are labeled with fluorescent cell tracers. After intradermal injection into the test mouse (1, 2), migratory cells and infected cells are isolated from the skin and lymphoid tissues of the test mouse. These cells are then labeled with fluorescent antibodies against myeloid cells and recognized according to the differential excitation/emission wavelengths of the fluorochromes by flow cytometry.

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.

Key features

• Ex vivo co-culture model of the alveolar niche.

• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.

Graphical overview
This protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).

This co-culture experiment is composed of three steps (Graphical overview):
1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively.
2. Culture of these ECs and bone marrow MOs for three days.
3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.

Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow–derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.

The nematode Haemonchus placei is a pathogenic parasite, the most seriously affecting ruminant’s health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) from H. placei transitory infective larvae (xL₃). ESP from xL₃ were obtained from the in vitro infective larvae (L₃) maintained in Hank’s medium at 37 °C with 5% CO₂ for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays.

Graphical overview