The intimate relationship between the epithelium and the immune system is crucial for maintaining tissue homeostasis, with perturbations in epithelial-immune interactions linked to autoimmune disease and cancer. Whereas stem cell-derived organoids are powerful models of tissue-specific epithelial function, these structures 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 tissueresident lymphocytes (TRMs), a portion of which integrate within the IIO epithelium and survey the barrier. IIO formation was driven by TRM migration and interaction with epithelial cells, as orchestrated by TRM- enriched transcriptomic programs governing cell motility and epithelial inspection. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients, and found that the system recapitulates clinical outcomes and the underlying cellular mechanisms. Inflammation was associated with the emergence of an activated population of CD8+ T cells, which progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and likely mediated by a Th1- like CD4+ population, which initially displayed a cytokine producing character and subsequently became cytotoxic itself. A system amenable to direct perturbation and interrogation, IIOs allowed us to identify the Rho pathway as a novel target for mitigating immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and the underlying inter-lineage immune interactions, IIOs can be used to broadly study tissue-resident immune responses in the context of tumorigenesis, infectious and autoimmune diseases.

Human intestinal organoids with an autologous tissue-resident immune compartment