Immunology

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.

Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8⁺ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens.

This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs), HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection, especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs, known for their high production of humoral factors such as Type I interferons (IFNs), the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways, especially innate immunity, allows for species-independent antiviral activity, reducing the likelihood of viral escape mutations. Additionally, the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens, including emerging viruses, positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs, offering a novel avenue for HTA discovery.

Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the Sleeping Beauty system to generate stably transfected cells. The system uses a “resurrected” transposase gene named Sleeping Beauty found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the Sleeping Beauty transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the Sleeping Beauty transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-bisphosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression.

The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system–associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry.

The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures. An important aspect in this respect was the experimental validation of TCR-mediated anti-tumor reactivity by means of an in vitro functional assay, which is the subject of the present protocol. This assay involves the transient transfection of mRNA gene constructs encoding TCRα/β pairs into a well-defined human T-cell line, followed by co-cultivation with the tumor cells of interest and detection of T-cell activation by flow cytometry. Due to the high transfectability and the low background reactivity of the mock-transfected T-cell line to a wide variety of tumor cells, this assay offers a highly robust and versatile platform for the functional screening of large numbers of TCR clonotypes as identified in scRNAseq data sets. Whereas the assay was initially developed to test TCRs of human origin, it was more recently also applied successfully for the screening of TCRs of murine origin.

Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials’ development due to the lack of underlying theory governing cell response to materials’ chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials’ HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions.

Key features

• Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O₂ plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating.

• Preparations of 7 mm × 7 mm polymers employing pin printing system.

• Provision of confined area for each polymer using ProPlate^® multi-well chambers.

• Compatibility of this platform was validated using adherent cells [primary human monocyte–derived macrophages (MDMs)) and non-adherent cells (primary human monocyte–derived dendritic cells (moDCs)].

• Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet^®).

Graphical overview

Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium (S.tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl₃) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe²⁺ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe²⁺ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress.

Key features

• Easy quantification of Fe²⁺ in cultured macrophages with a fluorescent probe.

• Analysis of iron in living cells without the need for fixation.

• Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria.

Graphical overview

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Graphical overview

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8⁺ and CD4⁺ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes.

Key features

• This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I.

• Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.