Immunology

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro.

Anti-CD19 chimeric antigen receptor (CAR)-natural killer (NK) cells are expected to demonstrate anti-CD19 CAR-T-cell-like efficacy against relapsed and refractory B-cell malignancies and autoimmune diseases, with fewer adverse events and the added advantage of permitting the use of allogeneic cells. However, the methodology for generating CAR-NK cells remains under development. Although various cell sources and expansion methods are available, feeder cells derived from cancerous tissue have been most commonly employed to promote ex vivo expansion of NK cells. In the protocol described herein, NK cells are expanded from adult peripheral blood mononuclear cells using CD2- and NKp46-specific stimulating antibodies in combination with multiple cytokines. The activated NK cells can be genetically modified using a retroviral vector. Subsequent culture of these cells yields large numbers of anti-CD19 CAR-NK cells. The current method, which enables feeder-free, large-scale generation of anti-CD19 CAR-NK cells, eliminates the risk of tumor cell contamination and may facilitate safer clinical application.

Natural killer (NK) cells are crucial innate immune effectors, mediating cytotoxicity against cancer and infected cells through receptors such as NKG2D. Reliable quantification of NK cell subsets is essential for evaluating NK cell-based immune responses in cancer research. Unlike other assays, including traditional flow cytometry used in assessing NK cells, imaging flow cytometry (IFC) is a simple and direct method for quantitative analysis of NK cells. This protocol describes the necessary procedures, including harvesting splenocytes, acquiring these cells labeled with NKG2D antibodies, and analyzing IFC data with IDEAS^® software. We applied this protocol to quantitatively assess the number of splenic NKG2D⁺ NK cells in mice injected with SVTneg2 cancer cells (which carry the p53 G242A missense mutation) and compared them to mice injected with EMT6 cancer cells (which have wild-type p53) or normal fibroblasts. We found that the SVTneg2 cancer cells significantly decreased the number of NKG2D⁺ NK cells in mice by approximately 2-fold (933 cells vs. 2360 cells, p < 0.001) compared with mice injected with EMT6 cancer cells. This IFC protocol can be applied to directly quantify NK cells in vivo. This quantitative protocol allows novices to quickly handle the analysis of cytotoxic NK cells with a single NKG2D marker. Further multicolor flow cytometry and cytokine assay may be required to precisely define the subtypes and effects of NK cells in anticancer immunity.

We established a step-by-step approach for generating a single-nucleotide mutation in the promoter region of an immune regulatory gene in human monocyte THP-1 cells by employing a plasmid-based CRISPR-Cas9 system delivered via transfection with a homology-directed repair template DNA (HDR). Key steps include designing a single-guide RNA (sgRNA), cloning it into a CRISPR plasmid encoding the Cas9 protein, transfection of the plasmid constructs along with single-stranded oligonucleotide repair template (ssODNs) into THP-1 cells, followed by selection and validation. This approach provides a precise and relevant model to investigate the role of single polymorphisms in the regulation of inflammatory gene expression in human monocytes. In addition to the rs1024611 single-nucleotide polymorphism (SNP), this CRISPR/Cas9-based strategy is broadly applicable to functional studies of noncoding and coding variants in innate immune genes.

Nanoparticle vaccines can provide advantages over traditional vaccine methodologies, including adjuvant delivery to enhance the effectiveness of recombinant antigens. Many approaches exist to formulate different vaccine nanoparticles, which are designed for different biomolecular cargos, adjuvant compositions, and disease targets. Here, a protocol is described to produce nanoliposomes whose surface is decorated with recombinant protein influenza antigens with monophosphoryl lipid A and QS-21 adjuvants incorporated into the lipid bilayer for protection against influenza infection. This protocol includes methods for producing adjuvanted liposomes and coupling with His-tagged antigens for surface decoration of the particle. This allows for a rapid methodology of producing immunogenic antigen-presenting liposomes that can be tailored to display a combination of influenza surface antigens.

Obesity is a risk factor for many diseases. The 3T3-L1 cell line is often used to obtain mature adipocytes, but these lack the inflammatory phenotype observed in obesity. Using a cocktail of cytokines that mimics the secretome of macrophages found in the inflammatory adipose tissue, we developed a protocol for obtaining mature inflammatory adipocytes. This model was validated at gene (RT-qPCR) and protein levels (multiplex adipokine array) as we found a decrease of adipogenic markers (C/EBPα, PPARУ, adiponectin, and CD36) and an increase of pro-inflammatory cytokines (IL-6, IL-1β, CXCL1, CXCL10, TNF-α, ICAM-1, and lipocalin-2). We provide a relevant in vitro model for studying the impact of low-grade chronic inflammation caused by obesity and its downstream effects on metabolic disorders and tumor microenvironments.

Experimental autoimmune encephalomyelitis (EAE) is a widely used rodent model of multiple sclerosis (MS), typically induced with pertussis toxin (PTX) to achieve robust disease onset. However, PTX has been shown to exert broad immunomodulatory effects that include disruption of G protein-coupled receptor (GPCR) signaling, altered T-cell response, and exogenous suppression of regulatory T cells, all of which are not present in human MS pathophysiology. Moreover, PTX also obscures the sex differences observed in MS, limiting the translational value of EAE models that rely on it. Given EAE’s widespread use in preclinical therapeutic testing, there is a critical need for a model that better recapitulates both clinical and immunological features of MS without PTX-induced confounds. Here, we demonstrate a non-pertussis toxin (non-PTX) EAE model in C57BL/6 mice, using optimized concentrations of complete Freund’s adjuvant (CFA), Mycobacterium tuberculosis, and myelin oligodendrocyte glycoprotein (MOG_35-55) peptide. This model recapitulates hallmark features of MS that include demyelination, neuroinflammation, motor deficits, and neuropathic pain. Importantly, it retains sex-specific differences in disease onset and pathology, providing a more physiologically and clinically relevant platform for mechanistic and translational MS research.

It is common practice for laboratories to discard clotted blood or freeze it for future DNA extraction after extracting serum from a serum-separating tube. If freezing for DNA extraction, the blood clot is not usually cryopreserved, which leads to cell membrane fragility. In this protocol, we describe steps to isolate high-quality nuclei from leukocytes derived from whole blood samples frozen without a cryoprotective medium. Nuclei isolated from this protocol were able to undergo ATAC (assay for transposase-accessible chromatin) sequencing to obtain chromatin accessibility data. We successfully characterized and isolated B cells and T cells from leukocytes isolated from previously frozen blood clot using Miltenyi’s gentleMACS Octo Dissociator coupled with flow sorting. Nuclei showed round, intact nuclear envelopes suitable for downstream applications, including bulk sequencing of nuclei or single-cell nuclei sequencing. We validated this protocol by performing bulk ATAC-seq.

Congenital renal disorders, such as the Potter sequence, result from renal dysgenesis. To explore a prenatal therapeutic approach for fetuses with kidney insufficiency, we established an in utero transplantation protocol using donor fetal kidneys. Although numerous rodent studies have reported cellular injections into fetal recipients, no protocol to date has described whole-organ transplantation during gestation. Here, we present a step-by-step method for grafting donor fetal kidneys (embryonic day 14.0–16.5) into allogeneic rat fetuses at embryonic day 18.0–18.5, resulting in term neonates that retain the grafts postnatally. A 15–16 G needle preloaded with the donor kidney is inserted transuterinely, depositing the organ into the subcutaneous space of the fetus. Four days later, the term pups are delivered naturally and evaluated for graft development. This protocol enables organ-level transplantation and longitudinal assessment of graft maturation within the unique fetal environment, which differs markedly from adult settings in terms of growth factor availability and immune reactivity. To our knowledge, this is the first protocol to successfully achieve whole-organ transplantation directly into fetuses in utero. Therefore, the model provides a valuable platform for studying developmental organogenesis, fetal immunology, and regenerative strategies that leverage embryonic cues.

The tissue explant culture (histoculture) is a method that involves maintaining small pieces taken from an organ ex vivo or post mortem in a controlled laboratory setting. Such a technique has a number of advantages: unlike the 2D, organoid, or on-chip cultures, tissue explants preserve the whole complexity of the original tissue in vivo, its structure, extracellular matrix, and the diverse cell populations, including resident immune cells. The explant culture method can be applied to human tissue specimens obtained from biopsies or autopsies, provided that proper ethical protocols are followed. This avoids the difficulties that may arise in translating results obtained on animal models into biomedical research for humans. This advantage makes histocultures especially desirable for studying human pathogenesis in the course of infectious diseases. The disadvantage of the method is the limited lifespan of the cultured tissues; however, a number of approaches allow extending tissue viability to a period sufficient for observing the infection onset and development. Here, we provide a protocol for lung explant maintenance that allows tracing the local effects of infection with SARS-CoV-2 in humans. Further applications of the lung tissues cultured according to this protocol include, but are not limited to, histochemical and immunohistochemical studies and microscopy, FACS, qPCR, and ELISA-based analysis of the conditioned culture media.