Hegewisch-Solloa, E.; Melsen, J. E.; Nalin, A.; Ravichandran, H.; Rendeiro, A.; Mundy-Bosse, B.; Melms, J. C.; Eisman, S.; Izar, B.; Grunstein, E.; Connors, T.; Elemento, O.; Freud, A.; horowitz, a.; Mace, E.

Secondary lymphoid tissue, including tonsil, supports human NK cell development, but the spatial organization and tissue niches that drive this differentiation remain undefined. Here, we used single cell analysis of cyclic immunofluorescence to generate a comprehensive atlas of human NK cell development in tissue. By integrating regional localization, chemokine signaling, cytokine availability, and cell phenotype, we show that NK cell differentiation follows a reproducible spatial trajectory defined by stage-specific cell-cell interactions. Notably, CD34+ NK cell progenitors are found in the interfollicular domain in proximity to high endothelial venules and preferentially interact with lymphatic endothelial cells, suggesting their route of progenitor entry into tissue. Mature NK cells are primarily found in the T-cell rich parafollicular domain, where they interact with other NK cells and T cell subsets. Local inflammation increases NK cell frequency in tissue through both proliferation of NK progenitors and recruitment of circulating mature NK cells. Finally, we identify a subset of tonsil stromal cells that support differentiation of NK cells in vitro and proliferation of NK precursors in situ. Together, these findings demonstrate that spatial localization defines human NK cell development and provide an in situ definition of niches that support human NK cell differentiation in tonsil.