SUMMARY

T cells of the immune system recognize small antigen peptide fragments loaded onto Major Histocompatibility Complex (pMHC) molecules through their T Cell Receptor (TCR). The recognition of antigenic pMHC by the TCR is an extremely sensitive and specific process, discriminating as few as a single antigenic pMHC from the self majority while remaining tolerant to uninfected cells. This unique sensitivity and specificity have been intensely studied, but much is still unknown regarding the antigen recognition process. A horizontal atomic force microscope was developed to assist in parsing this unique behavior. Utilizing this system, periods of upregulated adhesion, called TCR ligand memory, were investigated between 1E6 TCR and a panel of pMHC of varying potency. The strength of these periods of upregulated adhesion, indicative of an upregulated sensitivity to antigen, positively correlated with antigen potency. Pharmacological treatments suggested that this mechanism is controlled by proximal signaling and membrane organization. This behavior was simulated to extract estimates of kinetic parameters and showed that TCRs quickly upregulate their kinetics several magnitudes upon initial antigen recognition. Additionally, OT-1 double positive thymocytes were probed by pMHC using a biomembrane force probe with different ligands under the presence of CD8, a coreceptor which also binds MHC independently of TCR. Negatively selecting ligands resulted in catch-bonds, and positively selecting ligands resulted in slip bonds. Simulation-based analysis on these data sets indicated that this mechanism was not the result of passive processes. Force induced formation of long-lived bonds, indicating that mechanical forces are priming formation of a larger complex which enhances lifetime. Simulations of the BFP assay suggest that mechanotransduction by the TCR resulted in active heterodimerization of CD8 and TCR via interactions between intracellular tails of CD3(TCR) and Lck(CD8).