T lymphocytes expressing a chimeric antigen receptor (CAR) targeting antigens on tumor cells have demonstrated tremendous promise in combating cancer, with two therapies receiving FDA-approval in the last year. In addition to immune activation, CARs can suppress immune responses in an antigen-dependent manner when expressed on regulatory T lymphocytes (Tregs). CAR Tregs have shown promise in treating autoimmune disease, graft vs. host disease, and transplant rejection in preclinical studies with improved activity and fewer off-target effects than current treatments. As such, advances in the safety and specificity of CAR Treg therapy could have a huge beneficial impact to millions of patients worldwide. An obstacle facing CAR Treg therapy is that the current CAR designs found in the clinic can only target a single antigen with limited specificity, a major concern in Treg therapy. Additionally, these current designs are too rigid to facilitate optimization. To address these issues, our lab has recently developed a split, universal and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical upgrades. These features can mitigate over-activation, and enhance specificity of the engineered T cells. As CAR Treg therapies rapidly progress through clinical trials, understanding the mechanism of action of CARs in Tregs is urgently needed to facilitate CAR Treg therapy advancement. However, our current mechanistic understanding of how CARs work, especially in Tregs, is limited. Given that protein phosphorylation plays a critical role in CAR signaling, a global phosphoproteomic analysis on CAR Treg activation will reduce our knowledge gap by uncovering novel signaling pathways and identifying potential targets for optimization. In preparation for this proposal, we have demonstrated that our SUPRA CAR system is fully functional in human primary Tregs. We have also developed a quantitative mass spectrometry workflow to interrogate the phosphoproteomics of human T cell signaling. We will address our hypothesis that through a better understanding of CAR signaling and a programmable and inducible CAR system, we can achieve a higher level of control over Treg immune response, which will ultimately lead to a more effective and safer therapy for a wide range of autoimmune diseases. We propose to combine synthetic biology and Treg biology to expand the capability of our SUPRA CAR system in primary Tregs, and subsequently use proteomics to develop a mechanistic understanding on how the SUPRA CAR system controls Treg signaling. Specifically, we aim to: Aim 1: Expand the signaling domain repertoire in SUPRA CARs to control Treg responses. Aim 2: Establish combinatorial AND logic antigen detection in human Tregs. Aim 3: Define global phosphoproteomics signatures of SUPRA CARs signaling in Tregs. This work on CAR design could significantly improve the safety and specificity of Treg therapies and complement existing efforts that emphasize the optimization of Treg activity.