Massively parallel interrogation and mining of natively paired human TCRαβ repertoires

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCR alpha beta clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCR alpha beta-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCR alpha beta clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing. T cell receptors are identified in large-scale screening of T cell repertoires cloned from primary human T cells.