© 2011 Nature America, Inc. All rights reserved. NATURE IMMUNOLOGY VOLUME 12 NUMBER 10 OCTOBER 2011 949 ARTICLES The innate lymphoid cell (ILC) family consists of lymphoid tissue– inducer cells (LTi cells), natural killer cells (NK cells) and cells that produce interleukin 17 (IL-17), IL-22, IL-5 and/or IL-13. ILC func- tions range from inducing the formation of lymphoid tissues to the innate immune response, stromal homeostasis and tissue remodeling 1 . ILC development is strictly dependent on expression of the transcrip- tion factor Id2 and the common γ-chain (γ c ) cytokine receptor 2–5 . ILCs differ from B cells and T cells, as they do not have recombined antigen receptors on their surface 2,6–9 . Among the ILC family, LTi cells, IL-17- and IL-22-producing cells all share the expression of the transcription factor RORγt 10 . IL-22-secreting cells mostly express the marker NKp46 and localize at mucosal sites 11–13 . NKp46 - RORγt + cells are found in the intestinal tract and the spleen and can secrete IL-17 and/or IL-22 (refs. 6,14,15). In the periphery of adult mice, CD3 - RORγt + ‘LTi-like’ cells have been found whose function could consist of either restoring damaged tissues or generating isolated lymphoid follicles 16–20 . CD45 + CD3 - CD4 + RORγt + LTi cells are present in the lymph nodes and Peyer’s patch anlagen 21–25 . In RORγt-deficient mice, the absence of LTi cells leads to loss of both lymph nodes and Peyer’s patches 25,26 . During development, LTi cells originate from CD4 - CD3 - Sca-1 + c-Kit int IL-7Rα + precursors in the fetal liver 27 , a phenotype typical of common lymphoid progenitors (CLPs). Bone marrow CLPs are able to generate B cells, T cells and conventional NK cells and have lost eryth- roid and myeloid potential 28 , whereas in the fetal liver, they still show residual myeloid potential 27 . Expression of the integrin α 4 β 7 by LTi progenitors is associated with loss of B cell potential 29 and requires a complex of Runx1 and the transcription factor CBFβ2 (ref. 30). CBFβ2-deficient mice lack LTi cells but not NK cells, which suggests a specific role for these transcription factors during the differentiation of LTi cells 30 . The differentiation program of ILCs remains unknown. Here we define the fetal and adult progenitor of RORγt + ILCs as a distinct fraction of CLPs and characterize the successive stages in the developmental pathway that led to RORγt + ILCs. Through analysis of the expression of α 4 β 7 and the chemokine receptor CXCR6, we define additional cell subsets that corresponded to the sequential steps of early ILC differentiation, before the acquisition of RORγt expression, which marks commitment to the ILC lineage. In addition, we show that bone marrow and fetal liver ILC progenitors differed in their final differentiation steps location and requirements. Fetal liver progeni- tor developed locally, whereas adult progenitors matured outside the bone marrow in a Notch2-dependent manner. RESULTS Fetal liver RORgt + ILCs differentiate from CLPs We analyzed RORγt expression in fetal liver progenitors. RORγt was expressed in 3% of CLPs (lineage-negative (Lin — ) Sca-1 lo c-Kit int IL-7Rα + ) but was never expressed in Lin - Sca-1 + c-Kit hi (LSK) IL-7Rα - cells (Fig. 1a and Supplementary Fig. 1). We cat- egorized CLPs according to their expression of the receptor tyrosine kinase Flt3, α 4 β 7 and CXCR6. RORγt was exclusively expressed in the α 4 β 7 + Flt3 - CXCR6 + subset, and RORγt + cells had higher IL-7Rα expression (Fig. 1a). We confirmed the absence of RORγt expres- sion in the α 4 β 7 - Flt3 - , α 4 β 7 - Flt3 + and CXCR6 - CLP subsets by quantifying transcripts of Rorc (which encodes RORγt). We call CLP populations that did not contain RORγt + cells ‘CLPγ - cells’ here. When cultured on OP9 mouse bone marrow stromal cells and OP9 stromal cells expressing the Notch ligand Delta-like 4 (OP9- DL4 stroma), fetal liver CLPγ - cells generated B cells, NK cells, T cells and RORγt + cells (Fig. 1b), which indicated that the CLPγ - 1 Institut Pasteur, Unité de Lymphopoièse, Paris, France. 2 L’Université Paris Diderot, Sorbonne Paris Cité (Cellule Pasteur), Paris, France. 3 Institut National de la Santé et de la Recherche Médicale U668, Paris, France. 4 Institut Pasteur, Plate Forme de Cytometrie, Paris, France. Correspondence should be addressed to R.G. (rachel.golub@pasteur.fr). Received 3 May; accepted 15 August; published online 11 September 2011; corrected after print 23 September 2011; doi:10.1038/ni.2105 Notch signaling is necessary for adult, but not fetal, development of RORgt + innate lymphoid cells Cécilie Possot 1–3 , Sandrine Schmutz 1,3 , Sylvestre Chea 1–3 , Laurent Boucontet 1,3 , Anne Louise 4 , Ana Cumano 1,3 & Rachel Golub 1–3 The transcription factor RORgt is required for the development of several innate lymphoid populations, such as lymphoid tissue– inducer cells (LTi cells) and cells that secrete interleukin 17 (IL-17) or IL-22. The progenitor cells as well as the developmental stages that lead to the emergence of RORgt + innate lymphoid cells (ILCs) remain undefined. Here we identify the chemokine receptor CXCR6 as an additional marker of the development of ILCs and show that common lymphoid progenitors lost B cell and T cell potential as they successively acquired expression of the integrin a 4 b 7 and CXCR6. Whereas fetal RORgt + cells matured in the fetal liver environment, adult bone marrow–derived RORgt + ILCs matured outside the bone marrow, in a Notch2-dependent manner. Therefore, fetal and adult environments influence the differentiation of RORgt + cells differently.