Giacomo Lazzaroni

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. We combined human intestinal immuno-organoids and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics, which was associated with an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, particularly for liver-targeted treatments. However, predicting human-specific outcomes, such as transduction efficiency and hepatotoxicity, remains challenging. Reliable <i>in vitro</i> models are urgently needed to bridge the gap between preclinical studies and clinical applications. This study presents the first comparative evaluation of AAV transduction across multiple induced pluripotent stem cell (iPSC)-derived hepatocyte organoid donors, offering a novel platform for assessing vector performance in human liver models. The transduction efficiency and hepatotoxicity of eight AAV serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, and AAV9) were tested in iPSC-derived liver organoids and hepatic cell lines (HepG2 and HepaRG). AAV6 and AAV8 exhibited the highest transduction efficiency in organoids, while AAV4 and AAV5 were the least effective. Transduction variability was observed across different donors and cell lines. Notably, no significant hepatotoxicity, measured by AST (aspartate aminotransferase) release and viability measurements, was observed, indicating that AAVs do not induce immediate liver damage <i>in vitro</i>. This study introduces iPSC-derived hepatocyte organoids as a novel and effective tool for predicting AAV transduction efficiency and safety, with potential to enhance the translation of gene therapies to clinical applications.

Abstract Lung-resident immune cells, spanning both innate and adaptive compartments, preserve the integrity of the respiratory barrier, but become pathogenic if dysregulated 1 . Current in vitro organoid models aim to replicate interactions between the alveolar epithelium and immune cells but have not yet incorporated lung-specific immune cells critical for tissue residency 2 . Here we address this shortcoming by describing human lung alveolar immuno-organoids (LIO) that contain an autologous tissue-resident lymphoid compartment, primarily composed of tissue-resident memory T cells (TRMs). Additionally, we introduce lung alveolar immuno-organoids with myeloid cells (LIOM), which include both TRMs and a macrophage-rich alveolar myeloid compartment. The resident immune cells formed a stable immune-epithelial system, frequently interacting with the epithelium and promoting a regenerative alveolar transcriptomic profile. To understand how dysregulated inflammation perturbed the respiratory barrier, we simulated T-cell-mediated inflammation in LIOs and LIOMs and used single-cell transcriptomic analyses to uncover the molecular mechanisms driving immune responses. The presence of innate cells induced a shift in T cell identity from cytotoxic to immunosuppressive, reducing epithelial cell killing and inflammation. Based on insights obtained with bulk RNA-seq data from the phase 3 IMpower150 trial, we tested whether LIOM cultures could model clinically-relevant but poorly understood pulmonary side effects caused by immunotherapies such as the checkpoint inhibitor atezolizumab 3 . We observed a decrease in immunosuppressive T cells and identified gene signatures that matched the transcriptomic profile of patients with drug-induced pneumonitis. Given its effectiveness in capturing outcomes and mechanisms associated with a prevalent pulmonary disease, this system unlocks opportunities for studying a wide range of immune-related pathologies in the lung.