Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high-risk leukemias. Other innate lymphoid cells (ILCs Keywords: G-CSF · HSCT · ILC · ILC development · NK cells · PlerixaforAdditional supporting information may be found in the online version of this article at the publisher's web-site In the context of HSCT, NK cells play a central role in Tcell depleted HLA-haploidentical HSCT for the cure of high-risk leukemias. Indeed, alloreactive NK cells mediate graft-versusleukemia activity, contributing to the elimination of leukemia blast surviving the preparative regimen, while sparing normal tissues [6]. Moreover, before the adaptive immune response becomes fully competent, NK cells can provide protection against viral reactivation and/or primary infections [7]. Given the involvement of helper-ILCs in anti-microbial responses and in tissue remodeling, it is conceivable that, after HSCT, they may exert a protective role against chemo/radiotherapy-induced tissue damage (mucositis), development of graft-versus-host disease (GvHD), and infections. It has recently been shown that the early appearance after HSCT of donor natural cytotoxicity receptor (NCR) + ILC3 correlates with a decreased incidence of GvHD [8]. Thus, it would be important to define which conditions and factors may influence ILC differentiation. Indeed, conditioning regimen, presence of residual leukemia blasts, and infections could modulate ILC development. In this context, it has been shown that leukemia cells releasing the pro-inflammatory cytokine IL-1β influence ILC3 and NK-cell differentiation [9]. Moreover, in patients with acute myeloid leukemia, the frequency of ILC subsets is altered at diagnosis with a marked reduction of NCR + ILC3s [10]. It is conceivable that also the HSC source and the graft manipulation could influence post-transplant ILC recovery. Thus, we investigated the ILC differentiation potential of HSC isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). Results and discussion HSCs isolated from different sources display differences in the generation of NK cells and ILC3sCells isolated from BM, PB, TCR-α/β, and CD19 depleted PB (PB-α/β − 19 − ; α/βT cell and CD19 cell depleted mobilized peripheral blood), and UCB were analyzed for their phenotype after gating on CD45 + CD34 + cells (hereinafter referred to as HSC), as shown in Fig. 1A Fig. 2A-B). Notably, although the percentages of ILCs were significantly higher in BM cultures, BM HSCs displayed the lowest expansion rate (Fig. 2C). Thus, when considering the absolute numbers of CD56 + CD161 + ILCs, UCB cultures gave the highest recovery (Fig. 2D) Fig. S2A), although increasing at later time points, particularly in BM cultures (not shown). Upon stimulation, CD56 Supporting Information+ CD161 + cells produced mainly the ILC3-associated cytokines IL-22 and IL-8, while the percentages of IFN-γ-producing cells, i.e. NK cells, were low (Fig. 2H). Thus, BM and UCB HSCs display a better capability of in vitro differentiati...