PROJECT SUMMARY/ ABSTRACT: Vitiligo is an autoimmune skin disease characterized by the progressive destruction of melanocytes by autoreactive CD8+ T cells, resulting in disfiguring patches of white depigmented skin that cause significant psychological distress among patients. The gold standard treatment involves narrow band ultraviolet B (NBUVB) therapy, which induces the migration of hair follicle melanocyte precursors, in conjunction with topical steroids to suppress the local immune. Unfortunately, treatment is time consuming and re-pigmentation is often uneven. It is unknown why some depigmented areas persist and others re-pigment within the same lesion. Our recent work using non-invasive multiphoton microscopy (MPM) and single-cell RNA sequencing (scRNA-seq) has demonstrated that a subpopulation of keratinocytes enriched in vitiligo skin is important in disease persistence. We termed these cells ‘stress keratinocytes’ as they upregulate molecules associated with inflammation, wounding and other injuries. In this K08 application, we propose to study how local signals from keratinocytes interact with T cells and melanocytes to drive vitiligo persistence and repigmentation after treatment. Using MPM imaging, scRNA-seq and spatial transcriptomics, we will define if stress keratinocytes signals to T cells and paucity of melanocyte recruiting factors are enriched in treatment resistant areas (specific aim 1). We will also compare test to see whether adding these factors locally is sufficient to promote repigmentation (specific aim 2). Completion of this work will define how local tissue factors shape immune responses and melanocyte homeostasis in the skin has implications beyond vitiligo. This K08 application is designed to provide Dr. Jessica Shiu, MD PhD, the scientific training and professional development necessary to become an independent R01-funded investigator in the field of cutaneous biology. She will be mentored by Dr. Anand Ganesan, a physician scientist and a pigment cell biology expert with expertise on single cell genomics and skin imaging. Her secondary mentors, Drs. Bogi Andersen and Qing Nie, have extensive experience in keratinocyte biology and single-cell RNA sequencing analyses. Additional scientists, Drs. Mihaela Balu and Tinoco will provide further expertise in noninvasive imaging and cutaneous immunity. The work takes place within the outstanding scientific environment at UCI in the Department of Dermatology and Skin Biology Resource Center and has the support of multiple state-of-the-art centers including the NSF-Simons Center for Multiscale Cell Fate Research and Genomics High Throughput Facility. This training plan will help her develop technical skills on noninvasive imaging and spatial transcriptomics as well as quantitative methods needed for the analysis of dynamic populations of keratinocytes so that she will be positioned as a leading physician scientist in the field of cutaneous biology.