The Application of Organoids in the Study of Antiviral Innate Immunity.

Antiviral innate immunity plays a critical role in the defense against viral infections, yet its complex interactions with viruses have been challenging to study using traditional models. Organoids, three-dimensional (3D) tissue-like structures derived from stem cells, have emerged as powerful tools for modeling human tissues and studying the complex interactions between viruses and the host innate immune system. This chapter summarizes relevant applications of organoids in antiviral innate immunity studies and provides detailed information and experimental procedures for using organoids to study antiviral innate immunity.

Anisakis extracellular vesicles elicit immunomodulatory and potentially tumorigenic outcomes on human intestinal organoids.

BACKGROUND: Anisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear. METHODS: Here, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA. RESULTS: Anisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5-20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed. CONCLUSIONS: The results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host's anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited.

The use of organoids in creating immune microenvironments and treating gynecological tumors.

Owing to patient-derived tumor tissues and cells, significant advances have been made in personalized cancer treatment and precision medicine, with cancer stem cell-derived three-dimensional tumor organoids serving as crucial in vitro models that accurately replicate the structural, phenotypic, and genetic characteristics of tumors. However, despite their extensive use in drug testing, genome editing, and transplantation for facilitating personalized treatment approaches in clinical practice, the inadequate capacity of these organoids to effectively model immune cells and stromal components within the tumor microenvironment limits their potential. Additionally, effective clinical immunotherapy has led the tumor immune microenvironment to garner considerable attention, increasing the demand for simulating patient-specific tumor-immune interactions. Consequently, co-culture techniques integrating tumor organoids with immune cells and tumor microenvironment constituents have been developed to expand the possibilities for personalized drug response investigations, with recent advancements enhancing the understanding of the strengths, limitations, and applicability of the co-culture approach. Herein, the recent advancements in the field of tumor organoids have been comprehensively reviewed, specifically highlighting the tumor organoid co-culture-related developments with various immune cell models and their implications for clinical research. Furthermore, this review delineates the current state of research and application of organoid models regarding the therapeutic approaches and related challenges for gynecological tumors. This study may provide a theoretical basis for further research on the use of patient-derived organoids in tumor immunity, drug development, and precision medicine.

Unveiling the functional roles of patient-derived tumour organoids in assessing the tumour microenvironment and immunotherapy.

Recent studies have established the pivotal roles of patient-derived tumour organoids (PDTOs), innovative three-dimensional (3D) culture systems, in various biological and medical applications. PDTOs, as promising tools, have been established and extensively used for drug screening, prediction of immune response and assessment of immunotherapeutic effectiveness in various cancer types, including glioma, ovarian cancer and so on. The overarching goal is to facilitate the translation of new therapeutic modalities to guide personalised immunotherapy. Notably, there has been a recent surge of interest in the co-culture of PDTOs with immune cells to investigate the dynamic interactions between tumour cells and immune microenvironment. A comprehensive and in-depth investigation is necessary to enhance our understanding of PDTOs as promising testing platforms for cancer immunotherapy. This review mainly focuses on the latest updates on the applications and challenges of PDTO-based methods in anti-cancer immune responses. We strive to provide a comprehensive understanding of the potential and prospects of PDTO-based technologies as next-generation strategies for advancing immunotherapy approaches.

Epithelial organoid supports resident memory CD8 T cell differentiation.

Resident memory T cells (TRMs) play a vital role in regional immune defense. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency and low cell survival rates have limited the implementation of TRM-focused high-throughput assays. Here, we engineer a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation. These in-vitro-generated TRMs are phenotypically and transcriptionally similar to in vivo TRMs. Pharmacological and genetic approaches showed that transforming growth factor ß (TGF-ß) signaling plays a crucial role in their differentiation. The VEOs in our model are susceptible to viral infections and the CD8 T cells are amenable to genetic manipulation, both of which will allow a detailed interrogation of antiviral CD8 T cell biology. Altogether we have established a robust in vitro TRM differentiation system that is scalable and can be subjected to high-throughput assays that will rapidly add to our understanding of TRMs.

Human organoids with an autologous tissue-resident immune compartment.

The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.

Organoids: new frontiers in tumor immune microenvironment research.

The tumor microenvironment (TME) contains cells that regulate medication response and cancer growth in a major way. Tumor immunology research has been rejuvenated and cancer treatment has been changed by immunotherapy, a rapidly developing therapeutic approach. The growth patterns of tumor cells in vivo and the heterogeneity, complexity, and individuality of tumors produced from patients are not reflected in traditional two-dimensional tumor cell profiles. On the other hand, an in vitro three-dimensional (3D) model called the organoid model is gaining popularity. It can replicate the physiological and pathological properties of the original tissues in vivo. Tumor cells are the source of immune organoids. The TME characteristics can be preserved while preserving the variety of tumors by cultivating epithelial tumor cells with various stromal and immunological components. In addition to having genetic and physical similarities to human diseases and the ability to partially reconstruct the complex structure of tumors, these models are now widely used in research fields including cancer, developmental biology, regenerative mechanisms, drug development, disease modeling, and organ transplantation. This study reviews the function of organoids in immunotherapy and the tumor immune milieu. We also discuss current developments and suggest translational uses of tumor organoids in immuno-oncology research, immunotherapy modeling, and precision medicine.

Quantitatively Evaluating Interactions between Patient-Derived Organoids and Autologous Immune Cells by Microfluidic Chip.

The coculture of patient-derived tumor organoids (PDOs) and autologous immune cells has been considered as a useful ex vivo surrogate of in vivo tumor-immune environment. However, the immune interactions between PDOs and autologous immune cells, including immune-mediated killing behaviors and immune-related cytokine variations, have yet to be quantitatively evaluated. This study presents a microfluidic chip for quantifying interactions between PDOs and autologous immune cells (IOI-Chip). A baffle-well structure is designed to ensure efficient trapping, long-term coculturing, and in situ fluorescent observation of a limited amount of precious PDOS and autologous immune cells, while a microbeads-based immunofluorescence assay is designed to simultaneously quantify multiple kinds of immune-related cytokines in situ. The PDO apoptosis and 2 main immune-related cytokines, TNF-α and IFN-γ, are simultaneously quantified using samples from a lung cancer patient. This study provides, for the first time, a capability to quantify interactions between PDOs and autologous immune cells at 2 levels, the immune-mediated killing behavior, and multiple immune-related cytokines, laying the technical foundation of ex vivo assessment of patient immune response.

Using Ex Vivo Tonsil Organoids to Study Memory B Cells.

Tools to study memory B cell (MBC) development and function are needed to understand their role in supporting sustained protection against recurrent infections. While human MBCs are traditionally measured using blood, there is a growing interest in elucidating their behavior within lymphoid tissues, which are the main sites where adaptive immune responses are orchestrated. In this chapter, we introduce a high-throughput organoid system that is derived from primary human lymphoid tissues. The approach can recapitulate many hallmarks of successful adaptive immune responses and capture inter-individual variation in response to a variety of stimuli. Lymphoid tissue organoids enable characterization of pre-existing antigen-specific MBCs within an entirely human system and can provide valuable insights into MBC dynamics.

Cancer organoids 2.0: modelling the complexity of the tumour immune microenvironment.

The development of neoplasia involves a complex and continuous interplay between malignantly transformed cells and the tumour microenvironment (TME). Cancer immunotherapies targeting the immune TME have been increasingly validated in clinical trials but response rates vary substantially between tumour histologies and are often transient, idiosyncratic and confounded by resistance. Faithful experimental models of the patient-specific tumour immune microenvironment, capable of recapitulating tumour biology and immunotherapy effects, would greatly improve patient selection, target identification and definition of resistance mechanisms for immuno-oncology therapeutics. In this Review, we discuss currently available and rapidly evolving 3D tumour organoid models that capture important immune features of the TME. We highlight diverse opportunities for organoid-based investigations of tumour immunity, drug development and precision medicine.

A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

Identification and validation of tumor-specific T cell receptors from tumor infiltrating lymphocytes using tumor organoid co-cultures.

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy.

Patient derived cancer organoids model the response to HER2-CD3 bispecific antibody (BsAbHER2) generated from hydroxyapatite gene delivery system.

HER2-positive cancer is a prevalent subtype of malignancy with poor prognosis, yet current targeted therapies, like Trastuzumab and pyrotinib, have resulted in remission in patients with HER2-positive cancer. This study provides a novel approach for immunotherapy based on a hydroxyapatite (HA) gene delivery system producing a bispecific antibody for HER2-positive cancer treatment. An HA nanocarrier has been synthesized by the classical hydrothermal method. Particularly, the HA-nanoneedle system was able to mediate stable gene expression of minicircle DNA (MC) encoding a humanized anti-CD3/anti-HER2 bispecific antibody (BsAbHER2) in vivo. The produced BsAbs exhibited a potent killing effect not only in HER2-positive cancer cells but also in patient-derived organoids in vitro. This HA-nanoneedle gene delivery system features simple large-scale preparation and clinical applicability. Hence, the HA-nanoneedle gene delivery system combined with minicircle DNA vector encoding BsAbHER2 reported here provides a potential immunotherapy strategy for HER2-positive tumors.

In vitro immunity: an overview of immunocompetent organ-on-chip models.

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of microphysiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summarise the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilise microfluidic devices that are either commercially available or custom-made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field.

Patient-Derived Tumor Organoids Combined with Function-Associated ScRNA-Seq for Dissecting the Local Immune Response of Lung Cancer.

In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient-derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function-associated single-cell RNA sequencing (FascRNA-seq) platform allowing both phenotypic evaluation and scRNA-seq at single-organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA-seq data of individual pLCOs with their responses to anti-PD-1 (αPD-1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti-tumor immunity, to identify potential tumor-reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA-seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.

Virus neutralization assays for human respiratory syncytial virus using airway organoids.

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

In silico study of heterogeneous tumour-derived organoid response to CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for treating cancers. This method consists in modifying the patients' T-cells to directly target antigen-presenting cancer cells. One of the barriers to the development of this type of therapies, is target antigen heterogeneity. It is thought that intratumour heterogeneity is one of the leading determinants of therapeutic resistance and treatment failure. While understanding antigen heterogeneity is important for effective therapeutics, a good therapy strategy could enhance the therapy efficiency. In this work we introduce an agent-based model (ABM), built upon a previous ABM, to rationalise the outcomes of different CAR T-cells therapies strategies over heterogeneous tumour-derived organoids. We found that one dose of CAR T-cell therapy should be expected to reduce the tumour size as well as its growth rate, however it may not be enough to completely eliminate it. Moreover, the amount of free CAR T-cells (i.e. CAR T-cells that did not kill any cancer cell) increases as we increase the dosage, and so does the risk of side effects. We tested different strategies to enhance smaller dosages, such as enhancing the CAR T-cells long-term persistence and multiple dosing. For both approaches an appropriate dosimetry strategy is necessary to produce "effective yet safe" therapeutic results. Moreover, an interesting emergent phenomenon results from the simulations, namely the formation of a shield-like structure of cells with low antigen expression. This shield turns out to protect cells with high antigen expression. Finally we tested a multi-antigen recognition therapy to overcome antigen escape and heterogeneity. Our studies suggest that larger dosages can completely eliminate the organoid, however the multi-antigen recognition increases the risk of side effects. Therefore, an appropriate small dosages dosimetry strategy is necessary to improve the outcomes. Based on our results, it is clear that a proper therapeutic strategy could enhance the therapies outcomes. In that direction, our computational approach provides a framework to model treatment combinations in different scenarios and to explore the characteristics of successful and unsuccessful treatments.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.