RAG recombinase expression discriminates the development of natural killer cells

V(D)J recombination initiated by recombination-activating gene (RAG) endonucleases is a crucial process for the generation of diversified antigen receptors of T and B lymphocytes but regarded dispensable for the generation of innate natural killer (NK) lymphocytes that lack clonotypic receptors. However, a fraction of human and murine NK cells derives from RAG-expressing progenitors, which have non-productive rearrangements within their Ig and TCR loci. An impact of V(D)J rearrangements on maturation and function of NK cells has been suggested by mouse and human models. We studied the impact of RAG expression ontogeny on NK cell maturation and function by using RAG-fate mapping reporter permanently labeling NK progenitors and mature NK cells. To explore the impact of potential rearrangements on NK cell maturation, RAG-fate mapping reporter human induced pluripotent stem cell (iPSC) lines were generated by introduction of RSS-invEGFP constructs (an inverted EGFP cassette flanked by two recognition signal sequences (RSS)) into the AAVS1 locus using CRISPR/Cas9. Reporter iPSC lines were subsequently differentiated into hematopoietic stem progenitor cells (HSPC) and NK cells in vitro (protocol published by Euchner et al., Front Immunol, 2021). GFP+ and GFP- NK cell progenitors were sorted using FACS after week 1, week 2, and week 3 of NK cell differentiation. RNA-Seq analysis was performed on 3 replicates of each sample and time point.