Kinetics of ligand binding and drug response in a whole cell system using flow injection analysis

T-cell stimulation through the T-cell receptor (TCR) complex is one of the initiating factors of an immune response. While the mechanism by which TCR binding to its ligand, the major histocompatibility complex (MHC) protein-peptide complex, is perceived as a signal is not known, evidence suggests that the kinetics rather then the affinity of the interaction controls the outcome. To understand the T-cell signaling mechanism it is necessary to measure both the kinetics of MHC-TCR binding and cellular activation.; Current techniques for measuring receptor-ligand binding do not allow determination of kinetic parameters using living cells. The first part of my thesis project has entailed developing a new fluorescence technique, based on flow injection analysis, which uses live cells to measure receptor-ligand binding kinetics and affinities. The kinetic rate constants and affinities measured with this new FIA method compare favorably with kinetics determined by surface plasmon resonance.; The ultimate goal of this project is to be able to correlate receptor ligand binding to cellular activation. Ideally these measurements would be conducted concurrently. Since T-cells are non-adherent cell and the system used to determine kinetic parameters relies on attaching cells to microcarrier beads, the system needed to be reconfigured. The second part of my thesis describes a FIA method that overcomes this limitation by immobilizing the cells in a soft agarose matrix within the perfusion detector chamber. The system was validated by determining the [Ca2+] flux EC50 for carbachol activation of the M1 acetylcholine receptor expressed in Chinese hamster ovary (CHO) cells.; In a final set of experiments environmentally sensitive fluorescent dyes responsive to mitochondrial membrane potential, intracellular pH, or intracellular calcium ion concentration were used to monitor the response of either the MBP specific T-cell clone B10A-E3 or freshly isolated B10-PL splenocytes triggered by a variety of stimuli and it was found that the calcium signaling was the most robust and easily detected of these signals.