PROJECT SUMMARY/ABSTRACT T-cells are among the most potent and precise immune cells in the body?s defensive arsenal. T-cell receptors (TCRs), that are unique to each T-cell clone, provide antigen recognition capability to cytolytic T cells (CTLs), which allows for targeted killing of infected or cancerous cells. Healthy cells are recognized as ?self? and do not trigger a killing response from CTLs. It is the repertoire of all T-cells and their respective TCRs that collectively provides immune protection against threats posed by foreign pathogens or cancerous cells in the body. TCRs are encoded by highly variable genes expressed in T-cells that result in an abundant diversity of TCRs in humans, which can now be routinely decoded by DNA sequence analysis. However, there is still no tractable method to determine exactly which antigens are being recognized by the TCR expressed by a given T-cell of interest. This is important because it is the knowledge of antigens that is necessary to understand cellular immunology at its most fundamental level. Further, in a practical sense, the identity of T- cell antigens is necessary to design new immune interventions, including vaccines, biopharmaceuticals, or cell-based therapies. Immune-based therapies for complex pathogens such as HIV, malaria and tuberculosis require optimal T-cell antigens for specificity, as do many therapies for cancer, where our knowledge of antigens remains rudimentary. In the current proposal, we aim to leverage DNA sequencing technology to develop a method to discover DNA sequences that encode antigens recognized by T-cells of interest. This will be accomplished by making DNA libraries encoding large sets of possible antigens, transferring the DNA libraries into laboratory cell lines that will express the encoded antigens at their cell surfaces, screening these cells against a T-cell population of interest, isolating the specific target cells that were recognized by T-cells, and then identifying the antigens carried within the recovered target cells using DNA sequencing. This new technology will be reduced to practice using known, model T-cells and antigens and, once optimized, it will be mobilized toward the comprehensive mapping of T-cell/antigen interactions in health and disease.