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)^-CD7⁺CD127^-CD45RO⁺CD56⁺ 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 Lin^-CD7⁺CD127^-CD45RO⁺CD56⁺ cluster to both the NK cells and CD127⁺ ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the Lin^-CD7⁺CD127^-CD45RO⁺CD56⁺CD8a^- cells could develop into CD45RA⁺ NK cells and CD127⁺RORγt⁺ ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells.