ABSTRACT This proposal will enable the applicant to acquire mentored research training and develop into a productive, independent physician-scientist in the field of dermatology. Alopecia areata (AA) is an autoimmune skin disease, characterized by patchy non-scarring hair loss, with no cure. AA is caused by inappropriate activation of autoreactive T cells that target and damage keratinocytes of anagen hair follicles (HFs) due to the collapse of HF immune privilege (IP). Why anagen HFs undergo IP collapse is not well-understood. Recently, Janus kinase inhibitors (JAKi) have shown clinical efficacy in AA patients by suppressing immune-cell activity at the HF, however, JAKi do not work for all AA patients. A better mechanistic understanding of JAK-STAT regulation can lead to more effective AA treatments. Our preliminary data indicates that we have a novel AA-like mouse model that exhibits spontaneous alopecia in mice genetically null for autoimmune regulator (Aire-/-). Aire is a transcriptional regulator expressed in medullary thymic epithelial cells that eliminates autoreactive T cells. We and others have recently shown that Aire is also expressed in epidermal and follicular keratinocytes. Increasing clinical evidence also supports the importance of Aire in AA, as patients with loss-of-function mutations in AIRE have an increased risk of developing AA. We observe adult female C57BL/6 Aire-/- (germline) mice spontaneously develop AA-like lesions (n=35/73). Skin biopsies from alopecic Aire-/- mice resemble human AA lesions on macroscopic, histopathologic, and molecular levels. Additionally, we observed upregulation in JAK-STAT signaling in Aire-/- skin lesions and AIRE-deficient cultured keratinocytes alongside downregulated expression of PIAS1, a STAT1 inhibitor. These findings suggest that 1) Aire is a critical regulator of HF IP in AA and 2) AIRE is a key suppressor of JAK-STAT signaling in keratinocytes. This proposal will address current gaps in our understanding of HF IP and JAK-STAT signaling in AA. In Aim 1, we hypothesize that the loss of either thymic or keratinocyte Aire contribute to HF IP collapse. To test this, we will harvest CD8+NKG2D+ T cells from alopecic Aire-/- mice, subcutaneously inject them into Aire+/+ mice and monitor them for AA onset. Cytolytic T cell activity will be assessed via keratinocyte co-culture experiments. The results of these studies will reveal whether thymic Aire loss is sufficient to trigger HF IP collapse. In parallel, we will test whether skin-specific deletion of Aire is sufficient to trigger HF IP collapse by utilizing tamoxifen-treated Airefl/flK5-CreERT2 mice. In Aim 2, we hypothesize that AIRE contributes to the regulation of IFNγ-JAK-STAT signaling in cultured keratinocytes. To test this, we will determine how AIRE influences the PIAS1, STAT1, and the PIAS1-STAT1 complex, assess which AIRE domains are required for JAK-STAT signaling, and identify the AA cytokines and chemokines expressed by AIRE-/- ke...