065 Longitudinal analysis of T cell dynamics in alopecia areata at single-cell resolution

Abstract

Alopecia areata (AA) is defined by autoreactive T cells that attack hair follicles and lead to non-scarring hair loss. We previously demonstrated that AA is driven by cytotoxic CD8+ T cells that undergo clonal expansion. However, the relationship between CD8+ T cell pathogenicity and clonality in AA, as well as their temporal changes throughout disease course, remain poorly understood. Here, we conducted a time-course study in which we performed concomitant single-cell RNA and T cell receptor (TCR) sequencing analysis of T cells in both the skin and skin-draining lymph nodes (SDLNs) prior to, during, and after AA onset in the graft-induced C3H/HeJ mouse model. We observed a striking increase in CD8+ T cell clonality in both the skin and SDLNs during AA onset, with little to no clonal expansion in other T cell subsets. Clonality increased throughout AA progression, but later decreased in chronic AA, when the mice exhibited total body hair loss. Although we identified shared TCR sequences between AA skin and SDLNs, the degree of T cell clonality was increased in the skin, suggesting a two-step mechanism in which activated CD8+ T cells undergo an initial round of expansion in the SDLNs, followed by subsequent round(s) upon antigen exposure in the skin. We functionally investigated the causal role of specific TCR αβ pairs by generating retrogenic TCR mice expressing an expanded TCR sequence identified in our single-cell studies. Mice expressing expanded TCRs developed AA-like hair loss, whereas control mice expressing unexpanded TCR sequences did not. Our results demonstrate that individual expanded CD8+ T cell clones are sufficient to cause disease induction. These findings have broad implications for the development of novel therapeutics in AA aimed at the depletion of expanded pathogenic T cells, as well as clinical immunomonitoring in AA patients by following the dynamics of T cell clonality.