Crossreactive αβ T Cell Receptors Are the Predominant Targets of Thymocyte Negative Selection

The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4^-CD8β^- intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation.