How the strength ofT cell receptor signalling controls cytotoxic T cell polarisation for targeted killing (360G-Wellcome-099789_Z_12_A)

The proposed project aims to investigate how cytotoxic T lymphocytes (CTL) respond to variations of T Cell Receptor (TCR) signal strength at an intracellular level.Altered peptide ligands (APL) have been characterised to change the TCR: peptide-major histocompatibility complex interaction and subsequently alter TCR signal strength. Using APL provided by Ed Palmer, his biophysical measurements of these interactions, particularly dwelltime, can be correlated to physiological measurements. These will aim to identify any killing defects imposed by the APL and consequently any intracellular causes of defects will be investigated. This includes investigating strength of TCR signalling modules, such as calcium flux and extracellular signal-related kinase phosphorylation. The project will progress to investigating defects in conjugation and organisation of the immune synapse by immunofluorescence techniques. Next, defects in centrosome and granule polarisation will be investigated via live cell imaging to identify variations in velocity, granule clustering dynamics and timing post signalling and synapse formation, in order to elucidate how the order of these events are impacted by dwell time. This will provide insights into the determinate stages for target killing. Further work will involve following organelles with roles in TCR signalling but poorly defined roles in CTL killing, such as recycling endosomes.