Co-agonists in human T cell activation

The proportion of MHC molecules on an antigen-presenting cell that present antigenic peptide to a responding T-cell is small compared to those presenting non-stimulatory self peptides – the T-cell must find the antigenic “needle in the haystack” of seemingly irrelevant MHC molecules. T-cell responses are enhanced by the presence of seemingly irrelevant endogenous pMHC, a phenomenon termed co-agonism. Non-stimulatory pMHC complexes have not yet been studied in clinically relevant settings. Here we determine the requirements of human hepatitis B virus (HBV)-specific T-cells for recognition of co-agonists. TCR-like monoclonal antibodies, recognizing the MHC-peptide complex, will enable determination of the amount of antigen displayed on the surface of the APC in relation to other non-stimulatory MHC class-I molecules. T-cells from a patient can be “engineered” to express a TCR recognizing HBV, which can then be transferred back into the patient for therapy. We will determine the role of co-agonists in activation of such engineered T-cells. We will use single chain pMHCI molecules to determine the molecular requirements for human T cell recognition of co-agonists, separating CD8 binding to agonist and co-agonist, determining the strength of interaction between CD8 and non-stimulatory MHCI required for effective co-agonism. We will analyze the role of co-agonists in MHCI-restricted CD4+ T-cells (particularly useful in T-cell therapy) which may have very different co-agonist requirements from normal MHCI-restricted T-cells. We will use advanced imaging techniques (TIRF and FRET microscopy) to investigate signalling parameters during co-agonist-aided T-cell activation at the immunological synapse. We will investigate the recruitment of Lck, and its activation, along with the phosphorylation of TCR/CD3 ITAMS. Combinations of transduced fluorescent molecules for live imaging, and of antibodies for fixed cell imaging will be used to identify the role of co-agonists in the formation of TCR signaling microclusters during formation of the immunological synapse.