Kinase activity rewiring in T cell trans-differentiation - SUMMARY The research project focuses on understanding and modulating the plasticity of Th17 cells, specifically their ability to transdifferentiate into Treg cells (TregexTh17), a process with potential implications for autoimmune diseases, organ transplants, and malignancies. New insights indicate that AMP-activated protein kinase (AMPK) activity is highly involved in T cell plasticity. Yet, uncovering the biochemical mechanism employed by AMPK to drive TregExTh17 generation remains a significant challenge in the field, primarily due to the lack of appropriate technical approaches. We have developed a set of fluorescent kinase sensors capable of measuring the activity signatures of various kinases. These sensors employ diverse chemical approaches, offering a broad spectrum of colors ranging from UV to NIR. These sensors (i) are based on the specific substrate peptide sequence and are thus highly selective for the kinase/substrate pair of interest, an approach well-established in biosensing, (ii) are non toxic and thus suitable for live cell use, (iii) display rapid reactivity, and (iv) minimally interfere with the native cellular environment, preserving T cell functions. In this application, we propose a combinatorial approach using these cutting-edge, highly sensitive, and selective fluorescent biosensors to decipher the AMPK activity signatures responsible for driving Th17-to-Treg plasticity (TregExTh17) (Aim 1), and (ii) harnessing this plasticity through developing Phosphorylation Targeting Chimeras (PHICS), molecules designed to bring AMPK in proximity to key substrates involved in this plasticity, and phosphorylate/activate them as necessary to promote TregExTh17cells (Aim 2). After the R21 project, we will have developed a comprehensive fluorescent toolbox for monitoring AMPK kinase activity signatures in T cells and their relationship with function. By establishing connections between these signatures and their impact on Th17-to-Treg plasticity, we aim to unveil the translational potential of this innovative approach in deciphering the mechanisms governing this plasticity. Furthermore, we anticipate that developing a novel class of small molecules capable of selectively modulating substrate phosphorylation will strongly impact all contexts in which modulating immune responses is desired. These achievements will generate crucial data for R01 applications. 1
