Regulatory T cells keep the immune system in check and limit collateral damage during pathogen invasion. These cells are required to prevent the development of autoimmune and autoinflammatory disorders, protect the host against allergic reactions, and control the immune response during acute and chronic infections. Now, over a decade since FoxP3 was revealed as the defining transcription factor of the majority of regulatory T cells (known as FoxP3+ Tregs), we are beginning to have a good understanding of the transcriptional profiles of these cells and their dependence on signals emanating from IL-2 and/or T cell receptors. In contrast, little is known about how these cells are regulated by ubiquitin cascades that promote signaling, endocytosis or degradation of the many proteins expressed by regulatory T cells. We hypothesize that Ndfip1 and Ndfip2 promote the activation of Itch, and related E3 ubiquitin ligases to regulate Treg homeostasis and function. Since this grant was initially funded in 2011, we have defined key biologic processes that are regulated by Ndfip1, a small membrane bound protein that activates Itch and related Nedd4-family E3 ubiquitin ligases. However, due to a lack of techniques that would allow us to define physiologically relevant substrates of these ubiquitin pathways, our studies were mostly descriptive. We have spent the past year trying to overcome these obstacles, designing strategies that could be used to screen for substrates. These efforts have been extremely successful and we are now poised to reveal key aspects defining how these ubiquitin complexes function. We are one of the first labs to define substrates that are ubiquitylated in T cells and the first to do so in primary cells rather than in cell lines. To accomplish this, we hav needed to 1) optimize the anti-K-e-GG enrichment strategy (as it requires a very high number of cells and to date has thus only been accomplished using transformed cell lines), 2) devise a new strategy using tandem ubiquitin binding entities (TUBEs) to enrich for ubiquitylated substrates, and 3) found ways to pair these approaches with additional approaches to increase our confidence as we select potential substrates for validation. Having these approaches in hand, we are now ready to expand our studies to explore other E3s and adaptors that are expressed in Tregs. Our aims are to 1) determine whether and how Ndfip1 limits TCR signaling and the frequency of eTregs, 2) determine whether and how Ndfip1 promotes Treg function and FoxP3 stability, and 3) to determine the relative contributions of Ndfip1 and Ndfip2 and their cognate E3s in Treg homeostasis and function. To accomplish these aims, I have assembled a team of investigators with expertise in Treg biology and ubiquitin pathway analysis (Oliver), mass spectrometry (Seeholzer), FoxP3 methylation (Wells), and the generation of mice using CRISPR/Cas9 (Henao-Majia). Together, we are poised to make significant contributions towards understanding how ubiquitin regulates key aspects of Treg biology.