096 Identification of T cell receptor α and β chains responsible for AA pathogenesis via single cell TCR sequencing

Alopecia Areata (AA) is an autoimmune form of hair loss affecting more than 6 million people in the USA. Our laboratory recently demonstrated that a cell subset, CD8+ NKG2D+ T cells, are both necessary and sufficient for development of AA in C3H/HeJ grafted mice. The antigens that are targeted by those CD8+ T cells are however unknown. Previously, we performed next generation T cell receptor (TCR) β-chain sequencing on CD8+ T cell subsets isolated from lesional skin and skin-draining lymph nodes in the C3H/HeJ mouse model of AA. This revealed T cell clonal expansion in lesional skin around the time of hair loss, as well as TCR CDR3β motifs shared between independent lesional skin samples, which supports the notion that the disease is antigen-driven. Moreover, clonal expansion also occurred in lesional skin of human AA patients, pointing towards antigenic drive underlying human AA as well. Here, we used single cell TCR sequencing of alpha and beta chain pairs in CD8+ T cells isolated from lesional AA mouse skin to identify TCR pairs expressed by pathogenic CD8+ T cells. Unexpectedly, we found that one β-chain CDR3 amino acid motif, CASSXGLGGRX*QYF, was markedly enriched in lesional skin. This motif was present in one or several isoforms in the AA lesional skin of all analyzed mice. Interestingly, the paired TCRα chain CDR3 sequence varied from mouse to mouse, suggesting that the specificity is more strongly linked to the TCR β-chain. We are evaluating the pathogenicity of the identified TCR pairs by introducing them into retrogenic C3H/HeJ mice and asking whether they are sufficient to induce AA. The identification and validation of pathogenic TCR pairs in AA facilitates both computational and experimental screening of potential autoantigens, as well as the identification of antigenic triggers for AA.