PROJECT SUMMARY The skin represents the first and outermost body’s line of defense, continuously dealing with potentially harmful environmental and physical stressors. To protect internal tissues, skin epithelial stem cells (SC) must exert diverse functions and form a barrier by proliferating, differentiating, secreting lipids and anti-microbial molecules. To allow for such a diverse functional output and specialization, SC rely on a high degree of heterogeneity within the skin. While this is mostly dictated at a developmental and anatomical level (such as the differences between the interfollicular and the hair follicle compartments), a considerable level of heterogeneity persists within the same compartment, as observed by recent single-cell RNA sequencing and clonal proliferation studies. However, how is this heterogeneity distributed across the tissue and what drives it remains unexplored. More importantly, what is the advantage and the consequence of establishing SC heterogeneity across the tissue remains an open question. In my preliminary studies, I observed that immune cells (and more specifically dendritic epidermal T cells, or DETC) in the skin fine tune and tailor their effector functions depending on the stem cells they are directly contacting (i.e. hair follicle of interfollicular epidermal SC). These findings provide a proof of concept that functional heterogeneity might arise from immune-SC crosstalk in the tissue. Therefore, here I plan to investigate whether the immune niche can drive SC heterogeneity within one SC unit (using the hair follicle as a model) and among distinct units across the tissue. More in detail, I aim to decipher: 1) how direct contact with DETC (which are uniformly distributed in every upper hair follicle) mediates functional SC specification within the hair follicle (Aim I); and 2) how proximity to lymphoid clusters (which are sparsely distributed across the skin and resemble tertiary lymphoid structures) dictates spatial and functional SC heterogeneity among different hair follicles across the tissue (Aim II). The results generated in this proposal will: 1) shed a new light in our understanding of SC-immune crosstalk in the skin and provide a novel perspective on the role of the immune system in the tissue and of SC heterogeneity; 2) allow to decode new molecular laws governing tissue homeostasis, which will be critical to comprehend what goes awry during disease; 3) establish a new paradigm and generate a toolkit to study cellular crosstalk within tissue microenvironments. Under the supervision of the world-renowned expert in stem cell biology, Dr. Elaine Fuchs, the co-mentorship of a leading immunologist and pioneer in genetic tools development for the study of cellular interaction, Dr. Gabriel Victora, I am ideally positioned to develop my technical skills, knowledge, and training. With my research background, training and career development, I will be able to establish a unique niche at the intersection...