Immunology

This protocol provides a step-by-step approach for generating single-gene knockout in hard-to-transfect suspension immune cell lines like THP1, specifically demonstrated by knocking out the GSDMD gene. By employing CRISPR-Cas9 system delivered via lentivirus, this protocol enables precise gene disruption through targeted single-guide RNAs (sgRNAs). Key steps include designing specific sgRNAs, cloning them into a CRISPR vector, viral packaging, and transducing the target cells, followed by selection and validation. This optimized protocol is particularly useful for functional studies in immune cells, allowing researchers to reliably explore gene function in complex cellular pathways.

Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.

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.

Humanized immune system (HIS) mice are powerful tools for studying human immune system function and dysfunction and developing human-specific immunotherapeutics. The availability of sophisticated super immunodeficient mouse strains has allowed immune system humanization using transplants of human peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells. HIS mice are used extensively in immune-oncology, while there are fewer studies in autoimmunity, especially multiple sclerosis (MS). Using the protocol described here, we generated HIS mice that show key features of MS not represented in other widely used MS models [1]. Severely immunodeficient NOD.Cg-B2m^em1Tac Prkdc^scid Il2rg^tm1Sug/JicTac (B2m-NOG) mice, which lack murine B, T, and NK cells and murine major histocompatibility class I molecules and have defective innate immune responses, were transplanted with PBMC from HLA-DRB1-genotyped MS patients and healthy donors. Mice were successfully engrafted with hCD4 and hCD8 T and B lymphocytes and developed both spontaneous and experimental autoimmune encephalomyelitis (EAE)-enhanced T-cell lesions in the central nervous system. B-cell engraftment was highest in mice receiving cells from MS patients with serological evidence for Epstein–Barr virus (EBV) reactivation. This humanized MS model shows advantages over EAE, particularly spontaneous hCD8 T-cell lesions in the brain and spinal cord, mixed hCD8/hCD4 T-cell lesions in EAE-immunized mice, and more severe lesions in mice engrafted with PBMC from MS donors carrying the DRB1*15 MS susceptibility allele compared to DRB1*15-positive healthy and DRB1*13-positive MS donors. MS HIS mice represent simple and rapid tools for investigating human immunopathology and the efficacy of therapeutics at a personalized level.

In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCount^TM bead-based absolute counting technique to quantify IELs (CD45⁺ CD326/EpCAM- CD103⁺CD3⁺) and IECs (CD45- CD326/EpCAM⁺) in a C57BL/6 mouse model.

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.

Human immunodeficiency virus (HIV) remains a global health challenge with major research efforts being directed toward the unmet needs for a vaccine and a safe and scalable cure. Antiretroviral therapy (ART) suppresses viral replication but does not cure infection and so requires lifelong adherence. HIV-specific CD8+ T-cell responses play a crucial role in long-term HIV control as demonstrated in elite controllers, highlighting their potential in HIV cure strategies. Various HIV mouse models—including the human-hematopoietic stem cell (Hu-HSC) mouse, the bone marrow, liver, and thymus (BLT) mouse, and the human peripheral blood leukocyte (Hu-PBL) mouse—have deepened the understanding of HIV dynamics and facilitated the development of therapeutics. We developed the HIV participant-derived xenograft (HIV PDX) mouse model to enable long-term in vivo evaluation of bona fide autologous T-cell mechanisms of HIV control, including the antiviral activity of primary memory CD8+ (mCD8+) T cells taken directly from people with or without HIV, as well as testing potential immunotherapies. Additionally, this model faithfully recapitulates virus escape mutations in response to sustained CD8+ T-cell pressure, enabling the assessment of strategies to curb virus escape. In this model, NSG mice are engrafted with purified memory CD4+ (mCD4+) cells and infected with HIV; then, they receive autologous CD8+ T cells or T-cell products. Key advantages of this model include the minimization of graft-versus-host disease (GvHD), which severely limits peripheral blood mononuclear cell (PBMC) or total CD4-engrafted mice, the ability to evaluate long-term natural donor-specific T-cell responses in vivo, and the lack of use of human fetal tissues required for most humanized mouse models of HIV.

The endothelial barrier is a semipermeable cell layer covering the inside of blood vessels that regulates the flux of ions, macromolecules, and plasma from blood to tissues. Inflammation promotes an increase in vascular permeability, which can contribute to disease if not controlled properly. Thus, it is important to understand in detail the molecular mechanisms underlying inflammatory vascular hyperpermeability. While endothelial permeability can be measured in vitro, these assays do not recapitulate precisely the in vivo vasculature. Thus, in vivo assays are required to understand the full picture of vascular permeability regulation. Here, we describe an established assay that involves injection of Evans blue dye followed by intradermal injection of agents inducing vascular permeability. This assay is relatively easy to perform and provides reliable data on permeability regulation in vivo.

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.