Differentiation of cord blood-derived Innate Lymphoid Cells 1  into KIR+NKG2A- natural killer cells in a human stem cell niche.

KIR+NKG2A- natural killer (NK) cells can detect and eliminate malignant and infected cells that have downregulated single HLA class I molecules to escape T cell recognition. So far, these KIRonly NK cells cannot be efficiently expanded in vitro without concomitant co-expression of NKG2A, which modulates their specificity. In this context, we recently demonstrated that circulating innate lymphoid cells 1 (cILC1s) have NK cell progenitor potential and can be differentiated into KIRonly NK cells using murine feeder cells. Here, we established an animal-free culture system enabling the generation and expansion of NK cells from cord blood (CB)-derived cILC1s using human mesenchymal stem cells (MSCs) as feeder cells. Compared to the murine niche provided by the OP9-DL1 cell line, human MSCs generally enabled a much more efficient generation of NK cells, resulting in significantly higher yields of KIR+ NK cells. The frequency of KIRonly NK cells could be further increased by addition of the soluble NOTCH ligand DLL1. Furthermore, we utilized the cILC1/MSC platform to study education of KIRonly NK cells by HLA-C-encoded ligands in a human stem cell niche. This effect was strongest for homozygous (C1/C1) compared to heterozygous (C1/C2) donors, suggesting that cognate KIR/KIR ligand interaction mediates a gene-dosage-dependent education effect. Altogether, this optimized culture protocol overcomes previous limitations by enabling efficient generation of KIR-expressing NK cells in an animal-free, GMP-compatible system. The presented approach may facilitate the clinical translation of NK cell-based strategies for cellular immunotherapy and in addition provides a platform for mechanistic studies of NK cell education.