A single-cell transcriptional reference for the functional and developmental diversity of neonatal innate lymphoid cells

Circulating innate lymphoid cells (cILCs) comprise a complex mixture of subsets with effector functions and progenitor potential toward mature ILCs and natural killer (NK) cells. Here, we dissected cord blood (CB) cILC complexity using single-cell RNA sequencing (RNA-seq) combined with developmental and functional analyses. cILC1s comprise six different subsets, with four showing different maturation degrees and two resembling NK cell progenitors. Despite previously described transcriptional similarity to T cells, the developmental potential of cILC1s was restricted to NK cells using an artificial thymic organoid (ATO) model. cILC2s could be divided into four main subsets: CD161⁺, CD117⁺, activated cILC2s, and cytotoxic cILC2s. Finally, a CD161⁺CD28⁺CD117^low ILC3 subset was identified that secreted IFNγ upon co-stimulation with a CD28 superagonist, suggesting an alternative activation stimulus for cILC3s. Altogether, this in-depth analysis provides a detailed picture of cILC diversity in immunologically naive CB and constitutes a versatile resource for further exploration of their translational potential.