Investigating the Development of Tregs from iPSCs by Manipulating Exogenous and Endogenous FOXP3 Expression

About This Grant

PROJECT SUMMARY/ABSTRACT Tregs play a crucial role in maintaining immunologic tolerance and preventing autoimmune diseases. Current treatments for these conditions often involve immunosuppressive medications, which can have harmful side effects and limited effectiveness. Our research aims to unlock new possibilities in stem cell science by manipulating the expression of the transcription factor FOXP3, the master regulator of Treg development, during T cell differentiation of induced pluripotent stem cells (iPSCs). This work seeks to understand how FOXP3 expression can be most effectively regulated during iPSC differentiation and the impact of specific approaches on T cell differentiation. I propose two specific aims to achieve this goal: Aim 1 explores the effects of introducing an exogenous source of FOXP3 on iPSC differentiation. We will examine how different levels, timing, and isoforms of exogenous FOXP3 expression influence Treg development and functionality. Aim 2 focuses on identifying and manipulating Notch signaling effectors to direct Treg lineage commitment. We will create a comprehensive gene regulatory network and employ machine learning through the Python library CellOracle to model transcription factor perturbations for candidate genes in silico. To achieve these aims, I have applied new strategies to an in vitro model of T cell development, the artificial thymic organoid (aka ATO), developed by our group. The ATO platform is currently the only in vitro system that robustly supports mature CD4+ T cell production through the developmental stages that mirror conventional thymopoiesis. I have effectively increased FOXP3 expression during iPSC differentiation in the ATO model using the following methods: constitutive overexpression via lentiviral transduction, CRISPR- Cas9 knock-in for stage-specific expression, and small molecule modulation. This multi-faceted approach allows for the mechanistic investigation of Treg development from iPSCs and will provide foundational knowledge for generating iPSC-derived Tregs as adoptive cell therapy for autoimmunity. Expected outcomes of this work include a detailed understanding of how FOXP3 expression levels and timing affect Treg development. We will also define the regulatory role of Notch signaling on FOXP3 expression for this process. This knowledge will facilitate the development of future Treg therapies, offering new hope for patients with autoimmune diseases. Our work will enhance the mechanistic understanding of iPSC differentiation into the Treg lineage and propel research in stem cell-based therapies for autoimmunity. By developing a robust platform for Treg generation from iPSCs, our project holds the potential to transform autoimmune disease treatment and advance the field of stem cell-based therapies.