The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells
before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.