Mass cytometry reveals innate lymphoid cell differentiation pathways in the human fetal intestine.

Innate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional heterogeneity exists, and their differentiation pathways remain largely unclear. We applied mass cytometry to analyze ILCs in the human fetal intestine and distinguished 34 distinct clusters through a t-SNE-based analysis. A lineage (Lin)CD7CD127CD45ROCD56 population clustered between the CD127 ILC and natural killer (NK) cell subsets, and expressed diverse levels of Eomes, T-bet, GATA3, and RORγt. By visualizing the dynamics of the t-SNE computation, we identified smooth phenotypic transitions from cells within the LinCD7CD127CD45ROCD56 cluster to both the NK cells and CD127 ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the LinCD7CD127CD45ROCD56CD8a cells could develop into CD45RA NK cells and CD127RORγt ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells.