Identification Of T Cell Receptor Chains Responsible For Alopecia Areata Pathogenesis Via Bulk And 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 has recently demonstrated that a subset of CD8+ NKG2D+ T cells, are both necessary and sufficient for development of AA in C3H/HeJ grafted mice. However, the antigens that are targeted by those CD8+ T cells are still 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 as well, pointing towards an antigenic drive for human AA. Here, we used single cell TCR sequencing of alpha and beta chains in CD8+ T cells isolated from lesional AA mouse skin to identify the TCRα chains that pair with the dominant TCRβ chains expressed by pathogenic CD8+ T cells. This approach allowed us to pinpoint two TCR α- and β-chain pairs that were markedly enriched in lesional skin. These two αβ chain pairs had similar amino acid motifs, and comprised 16% (13/81) and 18.5% (15/81) of the sequenced cells respectively. We are evaluating the pathogenicity of these 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 paves the way for both computational and experimental screening of potential autoantigens, as well as the identification of antigenic triggers for AA.