PROJECT SUMMARY/ABSTRACT Enteric pathogens infect their human host via the gastrointestinal tract causing significant morbidity, mortality, and economic hardship worldwide. The intestine is a complex organ comprised of a layer of epithelial cells, the lamina propria containing immune cells, and a layer of smooth muscle cells organized into villi and crypts. Currently available in vitro models of the human intestine have major limitations, e.g., lack of immune cells or microbiota, and therefore, understanding how human enteric pathogens behave in their native host environment has presented significant challenges. Systems more closely mimicking the human intestine are needed to gain a better understanding of intestinal diseases, host - pathogen interactions and to facilitate the development of more effective therapies. Recent studies revealed that primary human pluripotent stem cells can be differentiated in vitro into three-dimensional human intestinal organoids (HIOs) with a pseudo-lumen and polarized intestinal epithelium similar to the organization found in the intestine in vivo. Importantly, HIOs contain the major secretory and absorptive cell types of the intestinal epithelium. However, HIOs lack microfold (M) cells and immune cells, which are critical for induction of mucosal immunity and balancing of appropriate tolerogenic and/or inflammatory responses to food antigens, commensal bacteria and pathogens. Imbalances in intestinal homeostasis result in devastating diseases such as inflammatory bowel disease (IBD), and M cells are important targets for improving oral vaccine efficacy. Therefore, the goal of Project 2 is to develop immune HIOs containing M cells and immune cells. We hypothesize that immune HIOs, i.e., HIOs co-cultured with immune cells, will more faithfully mimic the human intestine than current models and thus will be superior for studying host-pathogen interactions, drug development and disease pathogenesis. To develop complex human intestinal organoids with immune cells, we will: 1) establish and benchmark a complex organoid model by co-culturing immune cells with HIOs (immune HIOs); 2) determine the response of the complex organoid model to infection with Salmonella enterica serovar Typhimurium, Clostridium difficile and human norovirus; and 3) determine the suitability of immune HIOs with IBD-associated mutations to model aspects of disease pathogenesis. The effects of microbiota-derived products on the function of immune HIOs will also be evaluated. Our studies will synergize with Project 1, which will elucidate the interactions of HIOs with commensal or pathogenic bacteria in the absence of immune cells, and with Project 3, which will engineer a platform for the HIOs to allow intra- and extra-luminal flow and sampling. Our proposed research is significant because it is expected to provide a novel system to facilitate studies of intestinal dysfunction. The immune HIO model has the potential to fast-track development of effective vaccines, anti-infectives and treatment options for intestinal diseases like IBD.