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Abstract

Intestinal epithelial cells function as a protective barrier towards potentially harmful luminal content. They release immune regulators to attract basally residing immune cells, which interact to sample luminal antigens initiating immune responses and control inflammation. Alterations in either the intestinal epithelial barrier integrity or intestinal immune cell function are correlated with gut dysbiosis, and inflammatory processes such as in autoimmune diseases. Most knowledge regarding the interaction between intestinal immune cells and epithelial cells has been acquired from mouse studies or in vitro systems using cell lines or blood-derived immune cells. However, these approaches are not able to mimic the human IEB with tissue-residing immune cells which are characterized by their antigen experienced phenotype. With the recent advances in organoid technology the development brings us closer to developing a near-physiological human co-culture model. One of the unresolved challenges is finding a dependable source of human intestinal tissue and establish methods to store, preserve, and extract a sufficient number of viable cells from biopsies. Here, a novel human autologous co-culture model was developed using stem cell-derived organoids and tissue-resident immune cells. Organoids are a powerful tool to closely mimic the IEB composition in a 3D culture. The establishment of a co-culture system with mouse cells laid the foundation for this achievement, using the mouse model for proof-of-principle analysis. This analysis underpinned the relevance of the interaction, demonstrating broad transcriptomic effects on colon ECs due to CX3CR1 deficiency, which is crucial for transepithelial protrusions. MPs paracrinally influence the response of mouse intestinal organoids, with effects varying based on the integrity of the IEB and the MPs inflammatory state'. This response is further modulated by the health status of the MPs, be it in a steady-state or in an autoimmune condition. Finally, the reunion of tissue-resident immune cells with organoids in vitro enabled the combined analysis of paracrine and physical interactions in a near-physiologic, but tailored and controllable environment. A protocol was established that defined not only isolation and cultivation conditions, but also identified relevant readouts and peak of interaction using live-cell imaging, providing the critical fundamental knowledge for the transition to a human co-culture system. To develop a co-culture from human autologous tissue, extra preparatory steps were needed. This involved creating a set of protocols to process an entire human intestine. Additionally, a cryopreservation method for a tissue biobank was established. This was essential to separate the time and location of sampling from the later isolation of immune cells and crypts necessary for organoid generation, ensuring the creation of viable co-cultures. After successfully creating a consistent human intestinal co-culture involving epithelial and resident immune cells, I focused my research to validating and characterizing this system. Initially, using a custom-made image analysis tool, the peak interaction time was identified as 48 h with 6.6 cells in direct physical contact with the organoids. Next, the IX sensitivity and functionality of the co-culture was confirmed using a bacterial stimulant, which resulted in twice the number of immune cells closely associating with the organoids. Third, to discern the specific cells interacting and to uncover the effects of direct interaction at the individual cell level, I employed spatial multiplexing and scRNA-seq techniques. After refining protocols and image analysis for spatial multiplexing, I validated 13 markers in fixed co-culture sections. The data showed 7% of immune cells, mainly CD20+ B cells and CD3+ T cells, in contact with organoids, with smaller populations of DCs and macrophages present. ScRNA-seq indicated significant changes in B and T cell cluster sizes and expression profiles in co-culture compared to those without organoids. Epithelial cells, particularly early colonocytes, also displayed altered expression profiles when co-cultured with immune cells. Integrating spatial multiplexing data with single-cell transcriptomic analysis, initial results hint at both physical and paracrine interactions in the co-culture of LP immune cells and organoids, causing shifts in both epithelial and immune cell populations. The primary interacting immune cells appear to be B and T cells, likely driving the observed changes. This advanced human co-culture model offers unparalleled insights into the intricate crosstalk between immune and epithelial cells, enhancing our understanding of intestinal mucosal immunology. Moreover, its potential extends beyond mere understanding; it sets the stage for groundbreaking drug discovery methods.