Kueh, Hao YYirdaw, Hailemikael K2024-09-092024-09-092024-09-092024Yirdaw_washington_0250O_27143.pdfhttps://hdl.handle.net/1773/51803Thesis (Master's)--University of Washington, 2024T-cells can discriminate between foreign and self-peptides through a process known as kinetic proofreading, where time lag in signaling enables the cell to selectively respond to stably binding foreign peptides. Kinetic proofreading is believed to arise in the T-cell receptor (TCR) signaling pathway; however, the exact biochemical reactions in this pathway that underlie proofreading have remained unclear. A recent experimental modeling study in our lab attributed proofreading to the phosphorylation, diffusion, and condensation of a key signaling protein, LAT, following TCR and peptide major histocompatibility complex (pMHC) binding. This model uses time delays in the nucleation of LAT clusters and condensates for kinetic proofreading, allowing very selective and persistent responses to high-affinity pMHC ligands. Moreover, the nucleation and growth of LAT condensates also amplify low signals from foreign peptides. Our model predicts that LAT condensation and clustering occur optimally at intermediate pMHC densities. Here, to test this key prediction, we used a protein scaffold to present pMHCs at defined intervals and measured resultant signaling through LAT and other associated components through immunofluorescence staining and imaging. We further used live imaging to test the model. Here in these experiments, we cloned reporter proteins for key TCR signaling pathway components both upstream and downstream of LAT (TCRz, LAT, ZAP70, Grb2, and PLCg), and transfected them into a Jurkat T-cell line. We then measured phosphorylation and activation responses in T-cells using live imaging. These tools will allow us to test model predictions and broadly investigate the dynamics and mechanisms of antigen discrimination through T-cell signaling.application/pdfen-USnoneBioengineeringBioengineeringThesis