Results Skin Tregs generated early in life are crucial for adult skin homeostasis Inchul Cho 1,2 , Jessie Xu 1,2 , Niwa Ali 1,2 1. Centre for Stem Cells and Regenerative Medicine, UK; 2. The Francis Crick Institute, UK References 1. Ali, N., Zirak, B., Rodriguez, R., Pauli, M., Truong, H., Lai, K., Ahn, R., Corbin, K., Lowe, M., Scharschmidt, T., Taravati, K., Tan, M., Ricardo-Gonzalez, R., Nosbaum, A., Bertolini, M., Liao, W., Nestle, F., Paus, R., Cotsarelis, G., Abbas, A. and Rosenblum, M., 2017. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation. Cell, 169(6), pp.1119-1129.e11. 2. Mathur, A., Zirak, B., Boothby, I., Tan, M., Cohen, J., Mauro, T., Mehta, P., Lowe, M., Abbas, A., Ali, N. and Rosenblum, M., 2019. Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair. Immunity, 50(3), pp.655-667.e4. Introduction Regulatory T cells (Tregs) govern hair growth and wound healing by controlling the activity of stem cells in the mouse skin 1,2 . However, their functions in the neonatal skin during postnatal development is unknown. Methods We use Foxp3-EGFP-DTR transgenic mice to deplete Tregs during early stages of postnatal skin development. The skin from Treg-depleted mice are profiled by flow cytometric profiling, histology and whole skin RNA-sequencing 1. Neonatal Tregs are required for adult skin homeostasis Conclusion 2. RNA-seq analysis suggests neuronal defect in the skin immediately after Treg depletion 3. Tregs are required during early phase of postnatal skin development DT PBS Ventral DT PBS PBS DT 0 10 20 30 40 Follicular density ✱ PBS DT 0 2000 4000 6000 8000 10000 PIgment index ✱ Treg-depleted Treg-sufficient DGE Analysis GO Analysis Late DT DT PBS Harvest P6 P8 P10 P12 P28 PBS PBS Harvest Early DT DT PBS Harvest Full DT Harvest DT Phenotype PBS Late DT Early DT Full DT Lack of pigmentation No Not much Yes Yes Less hair No Not much Yes Yes Scaling skin No No Not much Yes PBS Late DT Early DT Full DT 4. Early DT and Full DT groups have high effector T cell (Teff), Langerin- myeloid numbers and LC activation on postnatal day 28. PBS Early DT Late DT Full DT 0 2000 4000 6000 Teffs (Abs No) Absolute number (per 6 SQCM) ✱✱ ns ✱ PBS Early DT Late DT Full DT 0 100 200 300 400 Langerin- (Abs No) Absolute number (per 6 SQCM) ✱✱✱ ✱✱ ns ✱✱ PBS Early DT Late DT Full DT 4000 5000 6000 7000 8000 9000 MHC2 (MFI) ✱✱ ✱ ns PBS Early DT Late DT Full DT 1. Neonatal Tregs are required for pigmentation and maintenance of hair follicles 2. Depletion of Tregs causes immediate transcriptomic changes associated with neurobiology. 3. Absence of Tregs during P6-P8, but not P10-P12, causes skin defects. 4. Elevated Teff and Langerin- myeloid number, as well was activated LCs, may contribute to skin defects. 5. We will assess how cytokines, secreted by Teffs, affect neuronal innervation of hair follicles. Dorsal P6 P8 P10 P12 P28 Harvest LCs