Interleukin (IL)-21 in Combination with IL-15 Induces the Differentiation of Human Umbilical Cord Blood CD34−Lineage− Cells into Pseudomature Lytic NK Cells.
Authors: Bonanno Giuseppina , Procoli Annabella , Mariotti Andrea , Corallo Maria , Scambia Giovanni , Pierelli Luca , Leone Giuseppe , Rutella Sergio 
Published: October 2019

Abstract
CD34−lineage− cells encompass an average 0.16% of human umbilical cord blood (UCB) mononuclear cells. We have previously shown that IL-15 promotes the stromal cell-dependent differentiation of UCB CD34−lineage− cells into bipotent T/NK progenitor cells. IL-21, a CD4+ T-cell-derived IL-2 receptor γ-chain signalling cytokine, is a crucial regulator of NK cell development and function. IL-21 increases the generation of mature NK cells from CD34+ haematopoietic stem cells (HSC) in combination with IL-15, IL-7, SCF and Flt3 ligand. To date, it has not been established whether IL-21 might replace the stromal cell requirements and synergise with IL-15 in the induction of NK cell differentiation of CD34−lineage− cells. In the present study, highly purified CD34−lineage− cells from human UCB were cultured in medium containing IL-15, SCF and Flt3 ligand, either in the absence or presence of IL-21. Cells were analysed at different time intervals for surface expression of NK cell-associated antigens (CD56, CD16, NKG2A/CD94 inhibitory receptor, CD244 and NKG2D activating receptors, killer-cell immunoglobulin-like receptors, NKp46 triggering receptor), mRNA signals for NK-specific transcription factors (inhibitor of DNA binding protein 2 or ID2) and bcl-2 family members, T-cell-receptor (TCR)γ rearrangement status, IFN-γ release, and lytic activity against NK-sensitive tumour targets. On day +30 of culture in the presence of IL-15 and IL-21, CD34−lineage− cells were expanded by 35-fold on average, compared with 21-fold in cultures nurtured with IL-15 alone. IL-21 per se was incapable of activating the proliferation of CD34−lineage− cells or promote the emergence of NK cell-associated surface markers (see below). After 30 days of culture in the presence of IL-21, 80–90% of the developing NK cells exhibited a CD56brightCD16neg phenotype. Consistent with the antigen expression profile previously assigned to pseudomature NK cells, IL-21-differentiated NK cells (IL-21NKs) stained negatively for killer-cell immunoglobulin-like receptors (CD158a and CD158b). Whereas NKG2A/CD94 expression on IL-21NKs mirrored that on IL-15-differentiated NK cells, NKG2D could be detected on an average 65% of IL-21NKs compared with 10% of cells maintained with IL-15. In sharp contrast to IL-15 alone, the combination of IL-21 and IL-15 markedly up-regulated mRNA signals for bcl-2, bcl-xL, TGF-β, GATA-3 and, perhaps more importantly, ID2, a master switch required for NK-cell development. At variance with IL-15-differentiated NK cells, IL-21NKs harboured unrearranged TCRγ genes, secreted copious amounts of IFN-γ in culture supernatants and were capable of lysing NK-sensitive tumour cell targets after pre-activation with exogenous IL-2.
Figure Figure
The present study establishes a novel role for IL-21 in the expansion and differentiation of pseudomature lytic NK cells, primarily in a synergistic context with IL-15. IL-21 might represent a valuable strategy to expand lytic NK cells from UCB CD34−lineage− cells for immunotherapy purposes.