Antigens, CD8; Genes, MHC Class I; Molecular Imaging; Receptors, Antigen, T-Cell, alpha-beta
Cytotoxic T-cells (CTLs) are of paramount importance in immune defense against tumors and viruses. They are exquisitely sensitive towards antigen as they can detect with their T-cell antigen receptors (TCRs) the presence of even a single antigenic peptide-loaded MHC molecule I (pMHCI) among thousands of structurally related yet non-stimulatory pMHCs (Marco Purbhoo, 2004). Antigen recognition takes place within the special constraints of the immunological synapse between a T-cell and an antigen presenting cell. Since biochemical experimentation invariably requires the destruction of at least one of the synaptic membranes it does not account for the specific microenvironment in which T-cell antigen recognition occurs. We are therefore employing a molecular imaging approach in which we confront cytotoxic TCR transgenic T-cells with a glass-supported lipid bilayer functionalized with accessory molecules and a wild-type pMHCI or CD8 deficient pMHCI. In this defined system we can set the concentration and composition of the molecules in order to determine the influence of CD8 and accessory molecules on antigen-recognition in the case of high and low -, agonistic and antagonistic antigen and correlate TCR-pMHCI engagement with downstream signaling events. Furthermore we want to determine the 2D Kd and lifetimes of TCR-pMHC interaction.
Techniques, methods & infrastructure
- Molecular cloning
- Protein engineering (protein expression, purification (FPLC), protein refold, protein labeling)
- Molecular Imaging (TIRF, FRET, single molecule microscopy)