Smart Luminescent Sensors for Molecular Super-resolution Imaging of Protein Kinase Cascades in Regulatory T-cells
Abstract: Health disorders caused by abnormal immune responses represent one of the major biomedical challenges nowadays. These disorders range from autoimmune diseases to the failure of the immune system to control tumour growth, occurring in cancer. For all these problems, T cell-based therapies are identified as promising treatments that may move personalised medicine to the next level. Regulatory T cells (Treg) play a critical role in maintaining peripheral and central tolerance and their number, stability and function are tightly regulated. However, the inability to select Treg based on their functional activity has represented to date a major hurdle in the successful implementation of Treg-therapies.
Recently, the importance of intracellular phosphorylation and kinase cascades in regulating the immunosuppressive activity of Treg has been highlighted. In an interdisciplinary approach, in this project we will develop a family of smart luminescent biosensors, specially designed to provide quantitative information using novel super-resolution microscopy techniques and flow cytometry, capable of simultaneously monitoring in real-time the activity of kinases critical for T cell function and activation (AKT1, AMPK and ERK1/2).
These sensors will be first tested in vitro, and then in cellulo using cell lines to optimize their application in microscopy and flow cytometry. In parallel, the sensors will be used to characterize the heterogeneity in kinase activity of ex vivo expanded primary Treg isolated from mouse spleens and subsequently test their stability and immunosuppressive properties.
This project will represent a proof-of-concept of the applicability of luminescent kinase biosensors to study live Treg activation and sort specific Treg subsets, which has the potential to majorly improve novel Treg-based immunotherapies.
The project PID2020-114256RB-I00 is funded by the Spanish Ministerio de Ciencia e Innovación and the Agencia Estatal de Investigación (MCIN/AEI/ 10.13039/501100011033).