Immune-epithelial interactions orchestrate stem-cell niche formation and morphogenesis requiring TGF-β signaling

Epithelial morphogenesis is generally attributed to mesenchymal and extracellular matrix interactions, whereas the role of immune cells in tissue assembly remains poorly defined. Here, we established a patient-derived, single-cell 3D hydrogel organoid system to examine immune-epithelial interactions during human mammary morphogenesis. CD45⁺ immune cells integrate into developing breast epithelial structures and persist throughout organoid formation, recapitulating their spatial organization in vivo. Functionally, immune cells act as instructive regulators in a donor-dependent manner, enhancing organoid number, branching, and proliferation. Reciprocal mixing experiments demonstrate that this phenotype is determined by the immune compartment, identifying distinct "inducer" and "non-inducer" states. Live imaging reveals that inducer immune cells undergo directed migration and establish stable early interactions with nascent epithelial clusters, whereas non-inducer cells fail to engage. Although inducer co-cultures produce a pro-morphogenic cytokine environment, soluble factors alone are insufficient to reproduce the phenotype. Instead, TGF-β signaling is required under these conditions, as pathway inhibition attenuates immune-driven morphogenesis whereas exogenous ligand is insufficient. These findings identify immune cells as essential, context-dependent regulators of enhanced epithelial morphogenesis in this organoid system.