Studying the mechanisms of initiation and progression of beta cell death in T1D - the progress of type 1 diabetes research has been limited by the accessibility of the target organ, the pancreas

The recent progress in the design and manufacturing of microfluidic platform has allowed the development of vascularized micro-endocrine pancreas on a chip. However, neither primary islets, nor islet-like structures produced from embryonic stem cells or induced pluripotent stem cells (ipsc) have allowed to build a fully syngeneic system, yet. However, full histocompatibility is necessary to introduce in those devices immune cells and study the mechanisms of immune attack to the b cells. Thus, the primary goal of the UG3 phase of the proposal is to build the elements that will allow this histocompatibility and the introduction of immune cells from patients into the vascularized micro-organ. Towards this end, we will use a series of ipsc cell lines from 3 T1D patients and 3 control individuals to produce endocrine cells, blood vessels, tissue-resident macrophages, and fibroblasts. While large quantities of pbmcs are available from each donor, clonal populations of CD4 and CD8 t cells of known antigen specificity will be produced for each individual using crispr/CAS9 editing and TCR viral transfer. The micro-organ and each cell type will be tested in vitro for functionality as well as their capacity at responding to inflammatory mediators. This analysis will combine functional tests such as gsis for b cells and permeability for blood vessels, and phenotypic analysis by immunofluorescence microscopy (confocal and 2- photon confocal) and single cell transcriptome. The UH3 phase will integrate this platform into the understanding of b cell death and its mechanisms. The immunological interrogation will proceed in three steps: 1- what cells are presenting antigens to CD4 and CD8 t cells? 2- what are the target cells and effectors molecules of CD4 and CD8 t cells? 3- can we show that CD4 are cytotoxic by bystander effects, and CD8 directly cytotoxic? To complement these mechanistic studies, a genome wide screen of b cell resistance to death will be performed to identify/confirm the pathways of killing. In addition, we will take advantage of our unique system to screen candidate compounds capable of protecting b cells from death. Finally, we will attempt to examine the behavior of the exocrine pancreas by testing organoids in the same inflammatory context than the one used for islets and t cells. Our path to success is built on the complementarity and congruency of the three experts leading this effort: jeff millman, a force in the world of ipsc differentiation and type 1 diabetes, chris hughes who designed and engineered the original microfluidic system and is a world leader in vascular biology, and LUC teyton, an immunologist of type 1 diabetes.