iTEC as a new experimental system for TEC biology

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Project Summary/Abstract The thymus is the key organ required for T cell generation and the formation of an adaptive immune response. One key cell type, endoderm-derived thymic epithelium, is required both for all thymus functions and to orchestrate the assembly and differentiation of all other cell types within the thymus. The genetic pathways underlying the specification and differentiation of these thymic epithelial cells (TECs) are still poorly understood. However, a single transcription factor, FOXN1, is known to control multiple key aspects of TEC proliferation, differentiation, and maintenance in both the fetal and postnatal thymus. Work from our lab and others has shown that Foxn1 acts differentially in TEC subsets and is incredibly dosage-sensitive, and that its expression in TEC progenitors is sufficient to drive most if not all of the TEC differentiation program. Because of this central role, Foxn1 is a key target in ongoing efforts to generate TEC by the directed differentiation of induced pluripotent stem cells (iPSCs). However, many questions remain about the precise FOXN1 functions in TEC differentiation and proliferation. While at least some of the FOXN1 targets required for TEC function are known, key questions remain unanswered, including the molecular pathways that establish TEC identity and initiate Foxn1 expression, and how diverse levels of FOXN1 in different TEC subsets differentially control TEC biology. We were part of a collaborative team that showed that enforced expression of FOXN1 in murine embryonic fibroblasts (MEFs) is sufficient to convert MEFs into functional TEC. These “induced TECs” (iTECs) can, upon transplantation, direct the assembly of a fully functional thymus organ that supports development of T cells in vivo. iTECs also show promise in promoting differentiation of immature thymocytes into single positive T cells in 2-dimensional culture. iTECs may thus provide a novel tool for long-term goals of generating autologous TEC in vitro that could be used to generate organoids for transplant, or for in vitro generation of T cells for therapeutic purposes. More broadly, the field of thymus biology lacks a viable in vitro culture system for studying the molecular requirements for TEC biology and differentiation, or TEC-thymocyte interactions that direct T cell development and selection. iTECs thus could provide a useful in vitro system for studying both FOXN1 function and the genetic pathways that control TEC differentiation and function. This proposal is designed to address key aspects of iTEC generation that limit its broader adoption as an experimental system. We propose three specific aims focused on improving the control of iTEC differentiation and proliferation that will allow us to develop this method for broad experimental applications: 1) Foxn1 dosage sensitivity during iTEC generation; 2) mechanisms to promote mTEC differentiation in iTEC cultures; and 3) MHCII expression and iTEC proliferation. Successful completion of the proposed experiments will substantially improve iTEC generation and function, with the goal of establishing iTECs as into a much-needed in vitro experimental system with broad utility for studying FOXN1 function and TEC biology, T cell differentiation, and TEC-thymocyte interactions.