Assessment Of Specific T-Cell Receptor Engagement Without Necessity For Identification Of The Relevant Antigen

Individual cancers, even of the same cell type, express unique arrays of distinctive tumor antigens, requiring accurate laboratory measurement of induced immunity against them problematic. Fluorescently tagged reagents (dextramers) that selectively engage clonal T-cell receptors (TCR) can cytofluorographically quantify both frequency and avidity antigen-specific T-cells, but cannot be synthesized without prior identification of the relevant antigen. Since clinically evident tumors may contain as many as 300 unique point mutations capable of generating a large number of uniquely antigenic proteins, and since procurement of such information for each cancer is currently unrealistic, it is presently only possible to assess responses to anti-cancer immunotherapy by clinical determination of estimation of the tumor burden capacity. Therefore, there is a need to develop methodology that can quantify the collective anti-tumor T-cell response, without prior identification of the full array of expressed tumor antigens. We have developed a practical high-resolution method to measure antigen-specific CD8 T-cell responses, via T-cell proton extrusion, an immediate result of selective TCR engagement by antigen presenting cells. The fluorescent emission characteristics of hydroxypyrene trisulfonate (HPTS) correlate with solution-phase proton concentrations, manifesting as increased emission signals. We exploit this TCR characteristic within the context of T-cell activation and show that stimulation with anti-CD3 immunoglobulin stimulates measureable TCR to release protons to a significantly higher degree than unengaged TCR (