Integrin αV-Mediated Spatially Restricted Activation of Autocrine TGF-β1 Programs Cytotoxic Tissue-Resident NK Cell Differentiation 2310290

Abstract Introduction Tissue-resident natural killer (trNK) cells differentiation requires TGF-β1 for tissue residency. As TGF-β1 often suppresses NK function, the mechanism by which human NK cells acquire residency without compromising cytotoxicity remains unclear. We hypothesized that Integrin αV-mediated spatially restricted activation of autocrine TGF-β1 programs cytotoxic trNK cells in the presence of IL-15, thereby preserving effector function. Methods Conventional PBNK cells were cultured on integrin αVβ8- coated surfaces supplemented with IL-15. Phenotype, signaling, cytotoxicity (against SCC cells ), and transcriptional profiles were characterized using flow cytometry, functional assays, SMAD2/3 phosphorylation analysis, and bulk RNA-seq. Human NSCLC CosMx SMI datasets were used for spatial transcriptomics to map trNK cells and integrin αV expressing populations. Results Integrin αVβ8 exposure elicited a trNK-like phenotype in IL-15-stimulated PBNK cells while retaining strong tumor cell killing. Mechanistically, IL-15 induced latent TGF-β1 secretion, which accumulated on the NK cell surface and was locally activated by integrin αVβ8. This resulted in spatially confined, autocrine TGF-β1 signaling, triggered low-level SMAD2/3 phosporylation. Transcriptomic analysis revealed enrichment of cytotoxic, metabolic, and integrin-associated pathways, distinct from NK cells differentiated with high-dosage soluble TGF-β1 treatment. Spatial transcriptomics of a human NSCLC showed that CD49a+CD103+ trNK cells preferentially localized within stromal regions, in close proximity to epithelial cells expressing integrin αV. Conclusion We uncover a novel integrin αV-dependent mechanism for the programming of highly cytotoxic CD49a+CD103+ tissue-resident NK cells. This involves PBNK cell-secreted latent TGF-β1, followed by its localized activation via epithelial integrin αV (αVβ6/αVβ8). This spatially confined signaling drives low-level SMAD2/3 phosphorylation to establish tissue residency while preserving effector function. Funding Source NIH (1R35DE030054-01) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)