Immunology

This protocol describes the preparation, administration, and analysis of a nanoparticle-based therapeutic strategy (nanoPDLIM2) in combination with PD-1 immune checkpoint blockade immunotherapy and chemotherapy for the treatment of lung cancer in mouse preclinical studies. NanoPDLIM2 uses a polyethyleneimine (PEI)-based delivery system that encapsulates PDLIM2 expression plasmids for reconstituting PDLIM2 that is repressed in tumors. This approach induces tumor immunogenicity, suppresses drug resistance, and improves treatment efficacy when used in combination with carboplatin, paclitaxel, and anti-PD-1 antibodies. The protocol describes steps for mouse lung tumor induction, nanoPDLIM2 and other therapeutic reagents’ preparation and administration, and subsequent analysis of tumor burden, immune response, and toxicity, providing a reproducible approach for investigators.

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.

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 adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available. Our protocol describes the ex vivo expansion of potentially long-lived human T memory stem cells (T_SCM), starting from a limited number of peripheral blood mononuclear cells (PBMCs). Using the perforin gene as an example, we provide detailed instructions for precise gene repair of human T cells and the expansion of T_SCM. The efficiency of precise gene repair can be increased by suppressing unintended non-homologous end-joining (NHEJ) events. Our protocol yields edited T-cell populations that are ready for phenotyping, genome-wide off-target analysis, and functional characterization.

PD-1 is an immune checkpoint on T cells. Antibodies to PD-1 or its ligand PD-L1 are gaining popularity as a leading immunotherapy approach. In the US, 40% of all cancer patients will be treated with anti-PD-1 or anti-PD-L1 antibodies but, unfortunately, only 30% will respond, and many will develop immune-related adverse events. There are nine FDA-approved anti-PD-1/PD-L1 antibodies, and approximately 100 are in different stages of clinical development. It is a clinical challenge to choose the correct antibody for a given patient, and this is critical in advanced malignancies, which often do not permit a second-line intervention. To resolve that, an in vitro assay to compare the performance of the different anti-PD-1/PD-L1 antibodies is not only a critical tool for research purposes but also a possible tool for personalized medicine. There are some assays describing the binding affinity and function of anti-PD-1/PD-L1 antibodies. However, a significant limitation of existing assays is that they need to consider the location of PD-1 in the immune synapse, the interface between the T cell and tumor cells, and, therefore, ignore a critical component in its biology. To address this, we developed and validated an imaging-based assay to quantify and compare the ability of different anti-PD-1/PD-L1 antibodies to remove PD-1 from the immune synapse. We correlated that with the same antibodies' ability to increase cytokine secretion from the targeted cells. The strong correlation between PD-1 location and its function in vitro and in vivo within the antibody treatment setting validates this assay's usability, which is easily recordable and straightforward.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector–target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality.

Exosomes are lipid bilayer–enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell–derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.