Archive 4 checkpoints (1-4)

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+                                                                                               Article
+
+Systemic Human ILC Precursors Provide a Substrate
+for Tissue ILC Differentiation
+Graphical Abstract                                              Authors
+                                                                Ai Ing Lim, Yan Li, Silvia Lopez-Lastra, ...,
+                                                                Ido Amit, Hans Yssel, James P. Di Santo
+
+                                                                Correspondence
+                                                                james.di-santo@pasteur.fr
+
+                                                                In Brief
+                                                                Human innate lymphoid cell progenitors
+                                                                circulate systemically, differentiating into
+                                                                diverse subtypes in specific tissues in
+                                                                response to localized cues.
+
+
+
+
+Highlights                                                      Data Resources
+d   Discovery of circulating human ILC precursors (ILCPs)       GSE90834
+                                                                GSE90640
+d   Blood-borne ILCPs generate cytotoxic and helper ILCs
+    in vitro and in vivo
+
+d   ILCPs are present in lymphoid and non-lymphoid human
+    tissues
+
+d   Multi-potent ILCPs are present in RORC-deficient patients
+
+
+
+
+            Lim et al., 2017, Cell 168, 1086–1100
+            March 9, 2017 ª 2017 Elsevier Inc.
+            http://dx.doi.org/10.1016/j.cell.2017.02.021
+                                                                                                                         Article
+
+Systemic Human ILC Precursors Provide
+a Substrate for Tissue ILC Differentiation
+Ai Ing Lim,1,2,3 Yan Li,1,2 Silvia Lopez-Lastra,1,2,4 Ralph Stadhouders,5 Franziska Paul,6 Armanda Casrouge,1,2
+Nicolas Serafini,1,2 Anne Puel,7,8 Jacinta Bustamante,7,8 Laura Surace,1,2 Guillemette Masse-Ranson,1,2 Eyal David,6
+Helene Strick-Marchand,1,2 Lionel Le Bourhis,9 Roberto Cocchi,10 Davide Topazio,10 Paolo Graziano,10
+Lucia Anna Muscarella,10 Lars Rogge,11 Xavier Norel,12 Jean-Michel Sallenave,3,13 Matthieu Allez,9,14 Thomas Graf,5
+Rudi W. Hendriks,15 Jean-Laurent Casanova,7,8,16,17,18 Ido Amit,6 Hans Yssel,19 and James P. Di Santo1,2,20,*
+1Innate Immunity Unit, Institut Pasteur, 75724 Paris, France
+2Inserm U1223, 75015 Paris, France
+3Université Paris-Diderot, Sorbonne Paris Cité, 75205 Paris, France
+4Université Paris-Sud, Paris-Saclay, 91405 Orsay, France
+5Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain
+6Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel
+7Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Inserm U1163, 75015 Paris, France
+8Paris Descartes University, Imagine Institute, 75015 Paris, France
+9Inserm U1160, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, 75010 Paris, France
+10Scientific Institute for Research and Health Care ‘‘Casa Sollievo della Sofferenza,’’ 71013 San Giovanni Rotondo, Italy
+11Immunoregulation Unit, Institut Pasteur, 75724 Paris, France
+12Inserm U1148, Laboratory for Vascular Translational Science (LVTS), CHU X. Bichat, 75877 Paris, France
+13Inserm U1152, Faculté de Médicine site Bichat, Université Paris Diderot, Université Sorbonne Paris-Cité, 75018 Paris, France
+14Gastroenterology Department, Hôpital Saint-Louis, AP-HP, 75010 Paris, France
+15Department of Pulmonary Medicine, Erasmus MC, 3000 CA Rotterdam, Netherlands
+16St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York,
+
+NY 10065, USA
+17Howard Hughes Medical Institute, New York, NY 10065, USA
+18Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, 75015 Paris, France
+19Inserm U1135, Centre d’Immunologie et des Maladies Infectieuses, 75013 Paris, France
+20Lead Contact
+
+*Correspondence: james.di-santo@pasteur.fr
+ http://dx.doi.org/10.1016/j.cell.2017.02.021
+
+
+
+
+SUMMARY                                                                  INTRODUCTION
+
+Innate lymphoid cells (ILCs) represent innate ver-                       Innate lymphoid cells (ILCs) are a novel family of lymphoid
+sions of T helper and cytotoxic T cells that differen-                   effector cells that serve essential roles in the early immune
+tiate from committed ILC precursors (ILCPs). How                         response, comprising both ‘‘cytotoxic’’ ILCs (natural killer [NK]
+ILCPs give rise to mature tissue-resident ILCs                           cells) and ‘‘helper’’ ILCs. The latter are characterized by CD127
+remains unclear. Here, we identify circulating and tis-                  expression and categorized as three groups based on transcrip-
+                                                                         tion factors (TFs) and cytokine outputs. Group 1 innate lymphoid
+sue ILCPs in humans that fail to express the tran-
+                                                                         cells (ILC1s) express TBX21 (encoding T-BET) and produce
+scription factors and cytokine outputs of mature                         T helper (TH)1-associated cytokines interferon gamma (IFN-g)
+ILCs but have these signature loci in an epigeneti-                      and tumor necrosis factor alpha (TNF-a). Group 2 ILCs (ILC2s)
+cally poised configuration. Human ILCPs robustly                         express GATA-3 and related orphan receptor A (RORA) and
+generate all ILC subsets in vitro and in vivo. While                     secrete TH2-associated cytokines, interleukin (IL)-5, and IL-13.
+human ILCPs express low levels of retinoic acid re-                      Group 3 ILCs (ILC3s) utilize RORC (encoding RORgt) to drive
+ceptor (RAR)-related orphan receptor C (RORC) tran-                      production of the TH17-associated cytokines, IL-17 and/or
+scripts, these cells are found in RORC-deficient                         IL-22 (Serafini et al., 2015; Spits et al., 2013). These different
+patients and retain potential for EOMES+ natural killer                  ILC subsets are found in diverse lymphoid and non-lymphoid tis-
+(NK) cells, interferon gamma-positive (IFN-g+) ILC1s,                    sues and enriched at mucosal sites where they play essential
+interleukin (IL)-13+ ILC2s, and for IL-22+, but not for                  roles in barrier function and innate immune defense (Artis and
+                                                                         Spits, 2015; Eberl et al., 2015).
+IL-17A+ ILC3s. Our results support a model of tissue
+                                                                            Human ILC subsets were first identified in secondary lymphoid
+ILC differentiation (‘‘ILC-poiesis’’), whereby diverse
+                                                                         tissues and subsequently reported in intestine, lung, liver, and
+ILC subsets are generated in situ from systemically                      skin (Juelke and Romagnani, 2016). Two distinct populations
+distributed ILCPs in response to local environmental                     of IFN-g-producing ILC1s have been described. A T-BET+ cell
+signals.                                                                 expressing high levels of CD127 and CD161 has been identified
+
+
+1086 Cell 168, 1086–1100, March 9, 2017 ª 2017 Elsevier Inc.
+in tonsil and inflamed intestine (Bernink et al., 2013). In contrast,   and Spits, 2014; Munneke et al., 2014); Figures 1A and 1B).
+an intraepithelial ILC1 expressing NKp44 and CD103, but not             Further fractionation of PB ILCs into ILC1, ILC2, and ILC3 has
+CD127, resides at mucosal sites (Fuchs et al., 2013). Both              been achieved using phenotypic markers, including CD161,
+these ILC1s produce IFN-g in response to IL-12, but unlike NK           CRTh2, CD117, and NKp44, that distinguish ILC subsets in fetal
+cells, they show minimal EOMES expression. Human GATA-3+                and adult tissues (Spits et al., 2013). Circulating ILCs include pre-
+ILC2s express the chemoattractant receptor CRTh2 as well as             dominant CRTh2+ ILC2 and a CD117+ subset that lacks CRTh2
+IL-25R and IL-33R (Mjösberg et al., 2011), are systemically            expression (Figures 1A and 1B). While CD117+ cells are generally
+distributed, and produce IL-5 and IL-13 under physio- and               considered as circulating ILC3s (Hazenberg and Spits, 2014;
+pathological situations (Kim and Artis, 2015). Group 3 ILCs             Munneke et al., 2014), we found that PB CD117+ ILCs differ
+include RORgt+ fetal lymphoid tissue-inducer (LTi) cells in fetal       dramatically from gut CD117+ ILCs in that they lack protein
+lymph nodes and spleen (Cupedo et al., 2009) as well as adult           expression of NKp44 and RORgt that identifies ILC3 (Fig-
+lineage –CD127+CD117+ cells found in tonsils, intestine, spleen,        ure 1C and S1A). Accordingly, PB CD117+ ILCs do not produce
+skin, lung, endometrium, and decidua that produce IL-17A in             IL-17A or IL-22 after pharmacological stimulation, whereas gut
+response to IL-23 (Klose and Artis, 2016). A subset of ILC3s            CD117+ cells abundantly produce these ILC3-associated cyto-
+express natural cytotoxicity receptors (NCRs) and are enriched          kines (Figure 1D). Interestingly, circulating CD117+ ILCs express
+in IL-22-producing cells (Cella et al., 2009; Satoh-Takayama            high levels of IL-1R1 and CD45RA and are CD69 , whereas gut-
+et al., 2008).                                                          resident ILC3s are CD69+ but IL-1R1 and CD45RA (Figure 1C
+   Murine ILCs differentiate from hematopoietic stem cells (HSCs)       and S1B). These observations suggest that PB CD117+ ILCs
+via lymphoid progenitors to yield diverse ID2+TCF-1+PLZF+ ILC           differ from bona fide ILC3s.
+precursors (ILCPs) in fetal liver (FL) and adult bone marrow               PB CD117+ ILCs also do not express proteins for TF that
+(BM) (Constantinides et al., 2014; Yang et al., 2015). Several          characterize other known mature ILC subsets, including T-BET
+TFs and signaling pathways regulate this process in mice (Sera-         or EOMES (Figure 1C), although a fraction expressed PLZF
+fini et al., 2015); in contrast, human ILC development is less well     (Figure S1B). Accordingly, PB CD117+ ILCs failed to express
+characterized (Juelke and Romagnani, 2016). NK precursors               markers associated with NK cells, ILC1 and ILC2, such as
+(NKPs) have been identified in fetal liver, BM, cord blood (CB),        CD94, CD244, and CRTh2 (Figure S1B) and did not produce
+and adult tonsil (Renoux et al., 2015), whereas committed ILC3          IFN-g or IL-13 after stimulation with pharmacological activators
+precursors (ILC3P) are found in tonsil and intestinal lamina prop-      (Figure 1D). Taken together, these results suggest that PB
+ria but not peripheral blood (PB), thymus, or BM (Montaldo et al.,      CD117+ ILCs may represent an unusual ILC subset.
+2014). A recent study identified tonsillar human RORgt+ ILCPs
+that can develop into mature cytotoxic and helper ILCs (Scoville        Transcription and Chromatin Landscapes of CD117+
+et al., 2016). Interestingly, these human NKPs, ILC3Ps, and ILCPs       ILCs Reveal an ILCP Profile
+were CD34+ and enriched in secondary lymphoid tissues but               In order to further understand the identity of PB CD117+ ILCs,
+were rare or absent from the circulation. It was unclear how these      we profiled the transcriptomic and epigenetic landscapes of
+CD34+ hematopoietic precursors were developmentally related             highly purified circulating CD117+ ILCs compared to CD34+
+to mature tissue-resident ILC subsets.                                  HSCs (Figure 2A). We performed chromatin immunoprecipita-
+   In this report, we have extensively characterized the pheno-         tion (ChIP) followed by high-throughput sequencing using trans-
+typic, molecular, and functional attributes of PB and tissue            posase-mediated tagmentation (ChIPm-seq) (Schmidl et al.,
+CD117+ ILCs. This included an in vitro study of over 1,300 ILC          2015). To expose common and unique epigenetic features of
+clonal cultures as well as an in vivo analysis using humanized          CD34+ HSCs and CD117+ ILCs, we mapped histone H3 lysine
+mice. While ILCs with the CD117+ phenotype were previously              4 di-methyl modifications (H3K4Me2s), which marks both active
+proposed to represent human ILC3s (Hazenberg and Spits,                 and poised gene regulatory elements (GREs) (Koche et al.,
+2014), we find that PB CD117+ cells are remarkably enriched in          2011). We identified 18,000 and 35,000 GRE in CD117+ ILCs
+multi-potent and uni-potent ILCPs that can give rise in vitro           and CD34+ HSCs, respectively (Figure 2B; Table S1), the major-
+and in vivo to all known ILC subsets, including EOMES+ NK cells.        ity of which were located in introns and intergenic regions
+CD117+ ILCPs are found not only in the circulation, but also in tis-    (Figure S2A). A significant number of H3K4Me2+ GREs were
+sues where they retain ILC multi-potency. Our identification of         shared between the two cell types: 89% of GREs identified
+systemically distributed ILCPs suggests a model whereby circu-          in CD117+ ILCs showed similar enrichment in HSCs and
+lating and tissue ILCPs provide a cellular substrate for ILC differ-    were associated with 13,159 genes of which many encoded
+entiation in situ in response to local environmental signals.           housekeeping functions. Nevertheless, 11% of H3K4Me2+
+                                                                        GREs detected in CD117+ ILCs were absent in CD34+ HSCs,
+RESULTS                                                                 potentially regulating 2,283 genes. Pathway analysis of these
+                                                                        genes revealed a strong enrichment for immune system and
+Characterization of Human Peripheral Blood                              lymphocyte-related processes. Conversely, GREs only active
+CD117+ ILCs                                                             in CD34+ HSCs were located near genes involved in more gen-
+Circulating ILCs can be identified as a low-frequency population        eral pathways important for hematopoiesis, hemostasis, and
+(<0.2% of total CD45+ cells) within lineage CD7+CD56 CD127+             platelet activation (Figure 2C).
+peripheral blood (PB) cells of healthy individuals as well as pa-          We compared the transcriptome of CD117+ ILCs and CD34+
+tients suffering from diverse clinical syndromes ((Hazenberg            HSCs. Clear differences in gene expression profiles emerged,
+
+
+                                                                                                Cell 168, 1086–1100, March 9, 2017 1087
+Figure 1. Characterization of Peripheral Blood CD117+ILCs
+(A) Gating strategy for fluorescence-activated cell sorting (FACS) analysis of human PB ILCs. Total ILCs were gated on viable CD45+ Lin
+(CD3 CD4 CD5 TCRab TCRgd CD14 CD19 ) CD7+CD127+ cells (red). NK cells are identified by CD56dim (gray), ILC2s are marked by CRTh2+ cells (green,
+and CD117+ ILCs are gated on CRTh2 CD117+ population (blue).
+(B) Percentage of total ILCs from viable CD45+ and CD117+ ILCs from total ILCs of healthy adult donors in PB. Results from 27 healthy individuals
+(median).
+(C) Expression of surface phenotypes (NKp44, IL1R1, and CD69) and intracellular transcription factors (EOMES, T-BET, GATA-3, and RORgt) of PB CD117+ ILCs
+and gut CD117+NKp44+/ ILCs.
+(D) Functional profiles (IFN-g, IL-13, IL-22, and IL-17A) of PB CD117+ ILCs and gut CD117+NKp44+/ ILCs in response to 3 hr phorbol 12-myristate 13-acetate
+(PMA)/ionomycin stimulation. Data representative of at least three individuals analyzed from at least three independent experiments.
+See also Figure S1.
+
+
+
+with a large cluster of 1,540 genes expressed at substantially                involved in the broader development of diverse hematopoietic
+higher levels in CD117+ ILCs (Figure 2D; Table S2) including                  lineages, including ID1, GATA1, GATA2, and MYB (Figures 2D
+many genes linked to the lymphoid lineage (IKZF1, CD2, CD7,                   and S2B) as well as cytokine receptors for myeloid lineages
+and IL7R). In contrast, CD34+ HSCs highly expressed genes                     (CSF3R, CSF2RB, FLT3). Compared to HSCs, CD117+ ILCs
+
+
+1088 Cell 168, 1086–1100, March 9, 2017
+Figure 2. The Transcriptional Signature and Chromatin Landscape of CD117+ ILCs
+(A) Schematic for RNA-seq and ChIPm-seq analyses.
+(B) Heatmap depicting normalized ChIPm-seq signal showed H3K4me2 intensity of high-confidence regulatory region, of which 1,989 were unique to CD117+
+ILCs. Venn diagram shows the overlap between three categories of H3KMe2+ regions (‘‘CD34+ HSC-specific’’; ‘‘CD117+ ILC-specific’’ and ‘‘shared’’).
+                                                                                                                    (legend continued on next page)
+
+
+                                                                                                      Cell 168, 1086–1100, March 9, 2017 1089
+express high levels of TFs that have been shown to be essential               promoted the development of EOMES+CD94+ IFN-g-producing
+for murine ILC development, including ID2, GATA3, TOX, and                    NK cells and IL-23 was critical for IL-17A-producing ILC3s (Fig-
+TCF7. We did not detect transcripts characteristic of T and B                 ures 3A and S3D). These results not only define a cytokine milieu
+cells development, such as RAG1, RAG2, EBF1, CD3E,                            that supports multi-lineage ILC and NK cell generation (IL-2, IL-7,
+BCL11A, or LMO2 in CD117+ ILC, although some of these genes                   IL-1b, IL-23) but also suggest that the PB CD117+ ILCs harbor
+are expressed by CD34+ HSCs (Figure S2B).                                     multi-potent ILCPs.
+   As both transcriptomic and epigenetic analyses of CD117+                      We further characterized circulating CD117+ ILCPs using a
+ILCs identified strong lymphoid signatures, we intersected these              modified stromal cell-based culture system that is permissive
+datasets in order to gain insights into the developmental status              for B cell, T cell, and myeloid cell development (Figure 3C) (Moh-
+and potential of CD117+ ILCs. A substantial proportion (26%)                  tashami et al., 2010). Moreover, this system can extensively
+of the genes most highly expressed in CD117+ ILCs were located                expand human NK cells and ILC subsets at the clonal level
+in the direct vicinity of a CD117+ ILC-specific GREs (Figure 2E;              (Lim et al., 2016). We analyzed progeny of single PB CD117+
+Table S3). These included many transcription factors previously               ILCs cultured on OP9 and OP9-DL4 to identify EOMES+ NK cells
+implicated in mouse ILC development, including ID2, GATA3,                    and ILC subsets expressing signature TFs and producing IFN-g,
+ETS1, TOX, TCF7, RORA, ZBTB16, and NOTCH1 (Figures 2E                         IL-13, IL-17A, and/or IL-22 (Figures 3D and S3E). Our analysis of
+and S2A)—consistent with the commitment of CD117+ ILCs to                     over 340 clonal PB cultures allows several points to be made.
+the innate lymphoid fate. In contrast, we did not detect notable              First, PB CD117+ ILCs represent a heterogeneous population
+expression levels for TFs (EOMES, TBX21, RORC), cytokine re-                  of uni-potent and multi-potent ILCPs. Approximately half of the
+ceptors (CCR6, IL1RL1, IL23R), or signature cytokines (IFNG,                  cultures derived from single CD117+ ILCs generated a single
+IL13, IL5, IL22, IL17A) that are abundantly expressed by mature               ILC1, ILC2, or ILC3 subset and therefore represent lineage-
+ILCs (Koues et al., 2016). However, many of these mature ILC                  restricted ILCPs, whereas the remainder are multi-potent ILCPs
+identity genes were already marked with H3K4Me2, demon-                       that can give rise to two or more separate Lin CD7+ ILC lineages
+strating that they may reside in a poised state (Figures 2F and               (Figure 3E; Table S4). B cell and T cell potential was not
+S2B). Together, these analyses suggest that CD117+ ILC repre-                 observed. Second, within the multi-potent ILCP population, a
+sent lymphoid-biased progenitors reminiscent of multi-potent                  substantial fraction (between 9%–17%) are able to generate all
+ILCPs carrying a TF profile with key mature ILC signature genes               three ILC subsets and likely represent immature uncommitted
+in an epigenetically poised state.                                            ILCPs. Moreover, clonal IFN-g+ cultures also comprise EOMES+
+                                                                              NK cells demonstrating that some PB ILCPs have the potential to
+Peripheral Blood CD117+ ILCs Include Multi-                                   generate both ‘‘helper’’ and ‘‘cytotoxic’’ ILC lineages at the sin-
+potent ILCPs                                                                  gle-cell level. Third, a subset of Lin CD7+ ILC clones failed to
+In order to assess the hematopoietic potential of circulating                 produce any cytokine tested (Figure 3E; Table S4). As these
+CD117+ ILCs, we cultured these cells in the presence of various               clones maintained a high level of CD117, but lacked other ILC
+cytokines. As CD117+ ILCs express CD25, CD127, and IL-1R1                     markers, they may represent ILCPs that have not further differen-
+(Figures 1A, 1C, and S1B), we added IL-2, IL-7, and/or IL-1b to               tiated (Figure S3F). Fourth, Notch signals clearly influence the
+these cultures. While bulk cultures of CD117+ ILCs minimally                  cell fate potential of CD117+ ILCPs as multi-potentiality and
+expanded in the presence of IL-2 and IL-7, robust proliferation               development of ILC3-containing cells was enhanced on OP9-
+was observed when IL-1b was added to the cultures (Figure S3A).               DL4 (Figure 3E; Table S4). Together, these data identify PB
+The overall cell yield was reduced by more than 100-fold in the               CD117+ ILCs as a circulating pool of partially committed ILC pro-
+absence of any one of these cytokines (Figure S3A). Cultured                  genitors. The comparison of bulk and clonal assays clearly
+cells did not harbor B (CD19+) or T (CD3+CD5+) cells but                      demonstrate the importance of the single-cell approach to
+comprised a pure population of CD7+ cells that were CD161+                    define heterogeneity of CD117+ ILC cell fate potential and to
+and expressed variable levels of CD117 and CD25 (Figure 3A).                  establish functional multi-potency.
+Remarkably, expanded cells included some EOMES+CD94+
+NK cells as well as the three canonical ILC groups: IFN-g+ ILC1,              Circulating CD117+ ILCPs Have Multi-ILC Potential
+IL-13+ ILC2, and NKp44+IL-17A+IL-22+ ILC3 (Figures 3A and                     In Vivo in Humanized Mice
+S3B). ILC1 (NKp44 , CD25 ), ILC2 (NKp44 CD25+) and ILC3                       We next assessed the in vivo potential of PB CD117+ ILCPs.
+(NKp44+CD25 ) subsets expressed signature TFs (Figure S3C).                   Severely immunodeficient mouse strains engrafted with human
+While the additional presence of cytokines that can drive ILC1/               CD34+ HSC progenitors generate human lymphoid (B, T, NK)
+NK (IL-12, IL-18), ILC2 (IL-25, IL-33), or ILC3 (IL-23) development           and myeloid (DC, macrophage, neutrophils) cell subsets
+did not further increase cell yield over that obtained with IL-2,             (reviewed in Rongvaux et al., 2013). We used BALB/c
+IL-7, and IL-1b (Figures 3B and S3A), the addition of IL-12 clearly           Rag2 / Il2rg / SirpaNOD (BRGS) mice that are permissive for
+
+
+(C) Heatmap of significantly enriched molecular pathways associated with genes near GREs uniquely identified in CD117+ ILCs (blue) or CD34+ HSCs (orange).
+(D) Heatmap with clustering of differentially expressed genes between CD117+ ILCs (blue) and CD34+ HSCs (orange) as detected by RNA-seq.
+(E) Overlap of the 1,540 genes most highly expressed by CD117+ ILCs and 2283 genes associated with ‘‘CD117+ ILC-specific’’ H3KMe2+ GREs. Selected genes
+essential for ILC development are highlighted.
+(F) RNA-seq reads (n = 4; error bars denote SEM) and corresponding H3K4Me2 traces are shown for selected loci.
+See also Figure S2 and Tables S1, S2, and S3.
+
+
+
+1090 Cell 168, 1086–1100, March 9, 2017
+Figure 3. Cloning Reveals Multi-ILC Lineage Potential of CD117+ ILCs In Vitro
+(A) FACS analysis of bulk cultured CD117+ ILCs for surface phenotypes, intracellular EOMES, and cytokine expression after 3 hr PMA/ionomycin stimulation to
+identify NK cells (EOMES+ cells), ILC1s (IFN-g+ cells), ILC2s (IL-13+), and ILC3s (IL-22+ and/or IL-17A+).
+(B) Expansion of bulk cultured CD117+ ILCs (10 days) in stromal cell-free conditions with cytokines (20 ng/mL each). Results from four independent donors; not
+significant (ns), p > 0.05; **p < 0.01; ****p < 0.0001 using paired Student’s t test compared to IL-2, IL-7 only condition (median).
+
+                                                                                                                           (legend continued on next page)
+
+                                                                                                            Cell 168, 1086–1100, March 9, 2017 1091
+robust multi-lineage human hematopoietic cell engraftment,                        the mouse (Cherrier et al., 2012). Lin CD127+ ILCs within fetal
+including NK cells (Legrand et al., 2011). Human PB CD34+                         liver contain a predominant CD117+ subset (Figure S4A). Interest-
+HSCs and CD117+ ILCPs were transferred to newborn BRGS                            ingly, these cells express RORgt at levels exceeding their periph-
+mice; cytokine supplementation (human IL-2, IL-7, IL-1b, IL-23)                   eral blood counterparts (Figures 1C and S4A) and moreover
+was provided, and mice were analyzed 4 weeks later (Figure 4A).                   express CCR6, neuropilin-1 (NRP-1), but not NKp44 (Figures
+BRGS mice engrafted with human CD34+ HSCs developed                               S4A and S4B). Despite these differences, fetal liver CD117+
+CD19+ B cells and CD14/CD33+ myeloid cells in the bone marrow,                    ILCs did not produce significant amounts of IL-17A or IL-22 after
+while CD3/CD5+ T cells and Lin CD7+ cells were detected in                        stimulation (Figure S4C) suggesting that they were not fully
+the gut (Figure 4B). These include mature ILC3s as previously                     mature ILC3. Nevertheless, when fetal liver CD117+ ILCs were
+described (Zhang et al., 2015). In contrast, BRGS mice receiving                  expanded in vitro, IL-17A-producing ILC3s were abundantly
+PB CD117+ ILCPs developed Lin CD7+ cells but no myeloid cells,                    generated. Moreover, IL17A+ ILC3s developed on stromal cells
+B cells, or T cells. Human CD45+ progeny from transferred                         lacking the Notch ligand DL4 suggesting that Notch engagement
+CD117+ ILCPs were detected in multiple organs, including the                      is not necessary for this process (Figure 6A). Interestingly, bulk
+spleen, lung, gut, and liver (Figure 4C). At each of these tissue                 cultures of fetal liver CD117+ ILCs also contain IL-13- and IFN-
+sites, EOMES+ NK cells as well as diverse CD127+ ILC subsets                      g-producing cells, although at lower frequency. Clonal analysis
+could be identified that expressed RORgt and GATA-3 proteins                      revealed that fetal liver CD117+ ILCs harbor a high proportion of
+and produced IFN-g, IL-13, IL-17A, and/or IL-22 ex vivo upon                      ILC3-committed progenitors.Still, a substantial fraction of multi-
+stimulation (Figure 4C). These results demonstrate that PB                        potent ILCPs can be revealed in the presence of Notch ligands
+CD117+ cells comprise committed ILCPs having the capacity to                      (Figures 6B and 6C; Table S5).
+generate all known ILC subsets and NK cells in vivo.                                 We next characterized CD117+ ILCs from human CB. Like
+                                                                                  their PB counterparts, CB CD117+ ILCs lacked NKp44, CCR6,
+Human CD117+ ILCPs Develop from CD34+ HSCs In Vivo                                and NRP-1 and were CD45RA+ (Figures S4A and S4B). More-
+We next interrogated the developmental relationship between                       over, CB CD117+ ILCs failed to express RORgt and T-BET but
+CD34+ HSCs and CD117+ ILCPs in ‘‘humanized’’ BRGS mice                            were GATA-3lo, thus resembling PB ILCPs (Figures 1C and
+(Figure 5A). Mice engrafted with CD34+ HSCs harbored human                        S4A). Like PB CD117+ ILCs, CB CD117+ ILCs did not produce
+CD45+ cells in the BM, lung, liver, and spleen that included a va-                cytokines ex vivo after stimulation (Figure S4C). However, cul-
+riety of lineage+ T, B, and myeloid cells as well as mature NK                    ture of CB CD117+ ILCs generated IFN-g+ ILC1s, IL-13+ ILC2s,
+cells, ILC1 and ILC3 (Figure 5B, data not shown) as previously                    and IL-17A+ or IL-22+ ILC3s (Figure 6D). No T, B or myeloid cells
+described (Legrand et al., 2011; Zhang et al., 2015). Moreover,                   were detected in cultures of CB CD117+ ILCs (data not shown).
+within the subset of Lin CD7+ cells, a clearly defined subpopu-                   Further clonal analysis revealed that CB CD117+ ILCs harbored a
+lation of CD127+CD117+ cells could be discerned in multiple                       diverse mix of uni-potent and multi-potent ILCPs (Figures 6E and
+tissues that lacked T-BET and EOMES expression (Figures 5B                        6F; Table S5). Unlike fetal liver CD117+ ILCs, CB CD117+ ILCs
+and 5C). These included CD127+CD117+ cells that expressed                         were not biased toward ILC3-committed progenitors, but more
+low levels of GATA-3 and RORgt and were NKp44 (Figure 5D)                         closely resembled PB CD117+ ILCPs. As for ILCPs from PB or
+and therefore resembled PB CD117+ ILCPs. Ex vivo stimulation                      fetal liver, Notch stimulation resulted in an enhanced frequency
+failed to elicit cytokine production from CD127+CD117+ cells                      of multi-potent ILCPs (especially those having the potential
+(Figure 5E). We sorted these cells and cultured them in the pres-                 for IL-17A+ and IL-22+ ILC3s) and reduced the frequency of cy-
+ence of IL-2, IL-7, IL-1b, and IL-23. Expanded cells contained                    tokine ILC clones.
+subsets able to produce IFN-g, IL-13, IL-17A, and IL-22 (Fig-                        We also examined the phenotype and potential of CD117+
+ure 5E) thereby confirming the presence of human ILCPs. These                     ILCs from adult lung tissue. Lung CD117+ ILCs harbored
+results demonstrate that fetal liver CD34+ HSCs can give rise to                  discrete populations of NKp44+ and RORgt+ ILCs but were
+CD117+ ILCPs in vivo.                                                             largely CD45A (Figures S4A and S4B). Bulk cultures of lung
+                                                                                  CD117+ ILCs generated diverse cytokine-producing ILC sub-
+Human CD117+ ILCPs Are Present in Fetal Liver, Cord                               sets and EOMES+ NK cells (Figure 6G); further analysis using
+Blood, and Adult Lung                                                             clonal assays defined the NKp44 fraction of lung CD117+
+We next assessed the stage of development when human                              ILCs as a mixture of uni-potent and multi-potent ILCPs (Figures
+CD117+ ILCP arise. Fetal liver harbors several immature hemato-                   6H and 6I; Table S5). These results demonstrate that a variety of
+poietic precursor populations (Rollini et al., 2007) and is proposed              ILCPs, including multi-potent progenitors, are present in human
+as a site for the development of lymphoid tissue inducer cells in                 mucosal tissues.
+
+
+(C) Schematic diagram and morphology of CD117+ ILC-OP9 stromal co-culture system.
+(D) Single PB CD117+ ILCs were cultured on OP9 or OP9-DL4 stromal cells for 14–18 days. Cells were stimulated with PMA/ionomycin 3 hr before cytokine
+analysis. IL-17A and IL-22 producing cells were analyzed after gating on cells not expressing IFN-g or IL-13. Positive clones were considered when at least 100
+viable human CD45+ cells were detected by FACS. Presence of an ILC subset was scored when more than 5% of corresponding cytokine was detected in total
+viable CD45 cells.
+(E) Pie chart depicting all possible ILC combinations detected. Frequency of each single or multi ILC differentiation among total positive wells. Data summarized
+from four independent experiments with one donor each.
+See also Figure S3 and Table S4.
+
+
+
+1092 Cell 168, 1086–1100, March 9, 2017
+                     (legend on next page)
+
+
+
+
+Cell 168, 1086–1100, March 9, 2017 1093
+ILCPs Reside within Secondary Lymphoid Tissues                                     contrast, ILC1s were present and the percentage of ILC2s
+As human secondary lymphoid tissues (lymph nodes, tonsils)                         from total ILCs was significantly increased in the absence of
+harbor diverse ILC subsets and their precursors, we further char-                  RORC (Figure 7B). Sorted CD117+ ILCs from control and
+acterized tonsillar CD117+ ILCPs and assessed their cell fate po-                  RORC-deficient patients were cultured as described above.
+tential. CD117+ ILCs from pediatric tonsils harbor a predominant                   Robust growth of Lin CD7+ cells was observed with no signifi-
+NKp44+ ILC3 subset that can be stimulated to produce IL-17A                        cant difference between WT and RORC-deficient cells. We
+and IL-22 (Hoorweg et al., 2012). This population also appears                     identified diverse cytokine producing cells in these cultures,
+to have extensive functional plasticity as stimulation (using                      including those producing IFN-g, IL-13, or IL-22, however, there
+IL-1b, IL-12, IL-23) modifies their cytokine production (Bernink                   was a total absence of IL-17A-producing ILC3s (Figure 7C).
+et al., 2013, 2015; Cella et al., 2010). Within tonsillar CD117+                   Development of EOMES+IFN-g+ NK cells was not affected by
+ILCs, we found that NKp44 cells were CD45RA+ and NRP-1 ,                           the absence of RORC. These results demonstrate that RORC
+while NKp44+ cells were CD45RA and NRP-1+ (Figure S4B).                            is not required for the development of NK cells, ILC1, ILC2, or
+These findings suggest that NKp44+ ILC3s are more mature                           IL-22+ ILC3 but is essential for the generation of IL-17A+ ILC3s
+(Bernink et al., 2015). However, cytokine production profiles                      from ILCPs in humans.
+were different in bulk cultures from tonsillar NKp44 versus
+NKp44+ CD117+ ILCs (Figure 6J and 6M). In particular, IFN-g+                       DISCUSSION
+cells and IL-13+ cells were more obvious in cultures derived
+from NKp44 cells, especially on OP9 stroma (Figure 6J).                            In this report, we identify and characterize human ILCPs as a
+   In order to better understand the relationship between NKp44                    subset of Lin CD7+CD127+CD117+ cells in cord and adult blood
+and NKp44+ CD117+ ILCs, we generated clones from both                              as well as fetal liver and several adult tissues. Human ILCPs give
+subsets and analyzed their cytokine-production potential. Strik-                   rise to all mature ILC subsets that are capable of producing a
+ing differences were observed. Clones derived from NKp44+                          range of cytokines (IFN-g, IL-13, IL-17A, IL-22) after in vitro cul-
+CD117+ ILCs were highly enriched ILC3s producing IL-17A                            ture in an appropriate cytokine environment or after transfer
+and/or IL-22 (Figures 6N and 6O; Table S5). A fraction of clones                   in vivo to immunodeficient mice. Human ILCPs also generate
+co-expressed IFN-g (14%) that likely represent ‘‘plastic’’ ILC3s                   EOMES+ NK cells demonstrating their potential for both cyto-
+that may upregulate T-BET as previously shown (Bernink et al.,                     kine-producing and cytotoxic ILCs. This is the first evidence for
+2015). In contrast, clones derived from NKp44 CD117+ ILCs                          a circulating ILCP in any species and further demonstrates the
+were quite heterogeneous with cells producing not only IL-22                       broad systemic distribution of ILCPs within human lymphoid
+and/or IL-17A but also abundant single IFN-g+ clones as well                       and non-lymphoid tissues including mucosal sites (Figure S5).
+as single IL-13+ clones (Figures 6K and 6L; Table S5) that were                       The identification of human ILCP was possible thanks to a
+not detected from NKp44+ CD117+ ILCs (Figures 6N and 6O;                           robust OP9 stromal cell-based assay that could assess ILC po-
+Table S5). The presence of IFN-g+ ILC1 clones was unexpected                       tential at the single-cell level. With this approach, we analyzed
+given previous reports that tonsillar CD127+ ILC1s differentiate                   over 1,300 ILC clonal cultures and identified uni-potent ILCPs
+into IL-22-producing ILC3s in the presence of IL-2, IL-23, and                     that could give rise to IFN-g+ ILC1s, IL-13+ ILC2s, or IL-17A+
+IL-1b (Bernink et al., 2015). Lastly, multi-potent ILCPs giving                    and/or IL-22+ ILC3s as well as multi-potent human ILCPs
+rise to three ILC subsets were only found in NKp44 CD117+                          that could generate two or more ILC subsets. We further demon-
+ILCs. Taken together, these results suggest that tonsillar                         strate that human CD34+ HSCs can develop in vivo into CD117+
+CD117+s harbor NKp44 ILCPs as well as NKp44+ ILC3s.                                cells that harbored ILCPs with multi-lineage ILC potential. Both
+                                                                                   in vitro and in vivo ILCP differentiation to more mature ILC
+RORC-Deficient Patients Harbor ILCPs but Fail to                                   subsets required exogenous cytokines, especially IL-2, IL-7,
+Generate IL-17A+ ILC3s                                                             and IL-1b; as such, availability of these factors may condition
+A committed ILCP in human secondary lymphoid tissue with                           ILCP differentiation during immune responses. Our results sug-
+a CD34+CD45RA+CD117+ phenotype was shown to highly ex-                             gest a model for human ILCP development whereby plurip-
+press the TF RORC (Scoville et al., 2016). In order to address                     otent CD34+ HSCs progressively differentiate into multi-potent
+whether RORC is required for generation of human CD117+                            ILCPs (with the CD34 CD7+CD127+CD117+CD45RA+ pheno-
+ILCPs, we studied blood ILCs in RORC-deficient patients.                           type) with potential for ILC1, ILC2, ILC3, and EOMES+ NK
+RORC deficiency in humans is associated with mycobacterial                         cells. Both CD34+ HSCs and multi-potent ILCPs are present in
+disease and mucocutaneous candidiasis and a defect in Th1                          fetal liver suggesting that this tissue is permissive for this
+and Th17 differentiation (Okada et al., 2015). CD117+ ILCs are                     transition. While previously described human tonsillar CD34+
+reduced in patients with RORC deficiency, while CD56bright                         CD117+CD45RA+ ILCPs (Scoville et al., 2016) may represent
+and CD56dim NK cells were unaffected (Figures 7A and 7B). In                       an intermediate in this pathway, the absence of these cells in
+
+
+Figure 4. CD117+ ILCs Effectively Give Rise to Multi-ILC Lineage In Vivo
+(A) Schematic diagram of in vivo transfer experiment.
+(B and C) Newborn BRGS mice were injected with PB CD117+ ILCs or CD34+ HSCs and analyzed 4 weeks later. (B) FACS analysis for lymphocytes and myeloid
+surface markers gated on viable human CD45+ cells from bone marrow and gut. (C) ILC analysis within viable human CD45+ cell using CD127, NKp44, and CD117
+expression, intracellular TF (EOMES, GATA-3, and RORgt) and intracellular cytokines (IFN-g, IL-13, IL-22, and IL-17A) production in lung, gut, liver, and spleen of
+BRGS mice transferred with CD117+ ILCs. Representative data of at least four mice in each group from three independent experiments.
+
+
+
+1094 Cell 168, 1086–1100, March 9, 2017
+Figure 5. Human ILCPs Accumulate in Human Immune System Mice
+(A) Schematic diagram of generation of human immune system (HIS) mice. Fetal liver CD34+CD38 HSC were intrahepatically injected into newborn BRGS mice.
+Mice were analyzed 8–9 weeks later.
+(B) Representative FACS analysis of human ILCPs (Lin CD7+CD127+CD117+) in spleen, BM, lung, and liver of HIS mice.
+(C) Percentage of ILCPs from total human CD45+ in spleen, BM, lung, and liver of HIS mice (median).
+(D) FACS analysis of surface phenotypes and transcription factors profiles of ILCP and NK cells from spleen of HIS mice.
+(E) Cytokine production of splenic CD117+ ILCs pre- and post-culture on OP9-DL4 with IL-2, IL-7, IL-1b, and IL-23 for 10 days. Cytokine production was analyzed
+after 3 hr of PMA/ionomycin stimulation. Representative data of eight mice from at least three independent experiments.
+
+
+
+
+                                                                                                             Cell 168, 1086–1100, March 9, 2017 1095
+Figure 6. In Vitro Bulk and Clonal Assay of CD117+ ILCs from Lymphoid and Non-lymphoid Organs
+(A–O) Bulk (100–300 cells) or single CD117+ NKp44+/ CD117+ ILCs from different organs were sorted by FACS into 96-well round bottom plate pre-seeded with
+OP9 or OP9-DL4 and supplied with IL-2, IL-7, IL-1b, and IL-23 (20 ng/mL each). Intracellular cytokine production in respond to 3 hr PMA/ionomycin stimulation
+was performed to identify ILC1s (IFN-g+), ILC2s (IL-13+), and ILC3s (IL-22+ and/or IL-17A+) after 8–10 days bulk culture (A, D, G, J, and M) or 14–18 days single-cell
+culture (B, C, E, F, H, I, K, L, N, and O). Representative FACS analysis of bulk cultured CD117+NKp44 ILCs isolated from (A) fetal liver, (D) CB, (G) lung, (J) tonsil,
+                                                                                                                                  (legend continued on next page)
+
+1096 Cell 168, 1086–1100, March 9, 2017
+BM, as well as cord and adult blood (Scoville et al., 2016) sug-                driven survival (Marrack and Kappler, 2004). Taken together,
+gests that CD34+ ILCPs arise locally. The circulating and tissue                ILCPs appear to have some properties in common with naive
+CD34 ILCPs that we describe also harbor cells with more                         T cells, although a number of important differences exist that
+restricted uni-potent ILCs. While we have not identified a                      are consistent with their designation as immature progenitors.
+marker that allows distinction between multi-potent and uni-                       While uni-potent and multi-potent ILCPs were identified in
+potent ILCPs, we assume that they retain a precursor-product                    every human tissue sample tested, there were clearly differences
+relationship.                                                                   in the relative proportions of ILCPs that were uni- or multi-potent.
+   Transcriptomic and epigenomic analysis of circulating hu-                    It is therefore likely that each tissue harbors a unique ILCP
+man ILCPs revealed a signature consistent with a partial spec-                  ‘‘repertoire’’ conditioned by environmental signals. Notch sig-
+ification to the ILC lineage. TFs known to be critical for ILC                  nals appear to influence ILCP multi-potency and ILC3 fate,
+development in mice (including TCF7, TOX, ID2, ZBTB16, and                      particularly in the fetal liver. It is remarkable that other uni-potent
+GATA3) (Constantinides et al., 2014; Klose et al., 2014; Seehus                 ILCPs were rarely detected in this organ, suggesting that at this
+et al., 2015; Serafini et al., 2014; Yagi et al., 2014; Yang et al.,            stage of fetal development, the microenvironment may deliver
+2015) were clearly upregulated in ILCPs compared to circu-                      signals that strongly polarize ILCPs toward ILC3s. These results
+lating HSCs. In contrast, signatures of early B and T lymphopoi-                corroborate earlier findings in the mouse (Cherrier et al., 2012).
+esis were not obvious, consistent with the inability of ILCPs to                Notch-mediated signaling has been proposed to play a role in di-
+generate adaptive lymphocytes in vitro or in vivo. Previous                     recting lymphoid cell fate decisions, promoting the development
+studies have carefully analyzed the epigenomic and transcrip-                   of T-lineage primed precursors, as well as modifying ILCPs ho-
+tional landscape of human mucosal ILC subsets (Koues et al.,                    meostasis (Chea et al., 2016; Dallas et al., 2005). Soluble factors
+2016). These studies identified distinct transcriptional signa-                 are also likely to be involved in ILCPs ‘‘repertoires’’ as these cells
+tures for ILC1, ILC3, and conventional NK cells as well as                      express several cytokine receptors (IL-1R, IL-2R, IL-18R) that
+shared gene circuitry associated with ILC lineage differentiation               allow them to sense tissue inflammation and stress.
+under the control of defined ‘‘super enhancers’’ (Koues et al.,                    Regulation of TF expression dictates ILC fate as well as func-
+2016). In contrast, we find that the ILC group-defining TFs                     tion. Signature TFs have been identified for ILC subsets that
+(BCL11B, TBX21, EOMES, RORC) and effector molecules                             regulate their differentiation at the level of surface phenotype
+(IFNG, IL13, IL17A, IL22, IL26) identified by Koues et al.                      and effector outputs (Serafini et al., 2015). The TF RORC helps
+(2016) were not expressed by circulating ILCPs (Table S2),                      define the ILC3 subset and is required for ILC3 develop-
+suggesting commitment to ILC1, ILC2, or ILC3 had not yet                        ment and maintenance in mice (Satoh-Takayama et al., 2008;
+occurred. Interestingly, the loci encoding these factors were                   Sawa et al., 2010). RORC is expressed by human ILC3 and in
+still ‘‘poised’’ as evidenced by abundant H3K4Me2 epigenetic                    committed ILC3P (Montaldo et al., 2014). The recent report
+modifications consistent with establishing of ILC ‘‘regulomes’’                 that all human ILC subsets express RORC (Scoville et al.,
+at an early developmental stage (Shih et al., 2016). This chro-                 2016) suggested a broader role for this TF in human ILC differ-
+matin landscape likely facilitates rapid generation of differenti-              entiation. By analyzing blood from RORC-null patients, we
+ated ILC subsets following cytokine-driven expansion (Lara-                     could show that RORC was not required for global ILC differen-
+Astiaso et al., 2014; Zook et al., 2016).                                       tiation in humans, but rather was critical for IL-17 production
+   Mature ILCs and fully differentiated TH cells possess striking               by the ILC3 subset. ILCPs in RORC-deficient patients retained
+similarities (Spits et al., 2013). TH cells arise following antigen             the capacity to generate other cytokine-producing ILC and NK
+activation of naive T cells within specialized zones of secondary               cell subsets. It is noteworthy that IL-22+ ILC3s developed in
+lymphoid tissues. While the analogous sites and structures that                 a RORC-independent fashion, suggesting compensatory path-
+promote ILC differentiation are not known, it is interesting to                 ways for these cells in humans.
+consider circulating and tissue ILCPs as the functional innate                     We have previously shown that OP9 stroma minimizes human
+counterpart of naive T cells. Both naive T cells and ILCPs are                  ILC2 plasticity (Lim et al., 2016) and extend this observation by
+CD45RA+ and CD69 suggestive of a resting state. Human                           showing that in this culture system the vast majority of NKp44+
+and mouse ILCPs clearly demonstrate the potential for further                   ILC3 clones retain their functional attributes with little plasticity
+differentiation to all known ILC subsets and lack expression of                 toward the ILC1 phenotype. Moreover, previous reports pro-
+the signature TFs that drive mature ILC effector functions. As                  posed that ILC1 clones rapidly differentiate toward an ILC3
+such, ILCPs resemble naive T cells that can differentiate to                    fate in the presence of IL-1b (Bernink et al., 2015), whereas we
+diverse T helper subsets under appropriate environmental sig-                   found that ILC1 clones on OP9 stroma (containing IL-1b) re-
+nals. Nevertheless, human ILCPs have signature TFs in a poised                  tained their IFN-g signature. As such, our culture system ap-
+state that contrasts with the situation in naive T cells where these            pears useful to assess signals that promote ‘‘primary’’ ILC fate
+loci are actively repressed (Shih et al., 2016). Moreover, ILCPs                from ILCPs.
+expand extensively in the presence of cytokines, whereas naive                     Finally, our identification of circulating and tissue-resident
+T cell homeostasis is primarily maintained through cytokine-                    human ILCPs suggests a concept of ‘‘ILC-poiesis’’ in which
+
+
+and CD117+NKp44+ from (M) tonsil. Pie chart depicting all possible ILC combinations after clonal expansion of CD117+NKp44 ILCs from (B and C) fetal liver, (E
+and F) CB, (H and I) lung, (K and L) tonsil, and (N and O) CD117+ NKp44+ from tonsil on OP9 and OP9-DL4. Data summarized from at least two independent
+experiments with one donor each.
+See also Figure S4.
+
+
+
+                                                                                                            Cell 168, 1086–1100, March 9, 2017 1097
+Figure 7. Development Potential of ILCPs from RORC / Patients
+(A) FACS analysis of peripheral blood ILC subsets from healthy and RORC / patient samples.
+(B) Percentage of NKdim and NKbright from viable CD45+ cells, ILC1s, ILC2s, and ILCPs from total ILCs of PB of healthy and RORC / patients. Result from
+22 healthy donors and 2 RORC / patients. ns, p > 0.05; *p < 0.05; **p < 0.01 using unpaired Student’s t test (median).
+(C) ILCPs from healthy donor or RORC / patients were cultured on OP9-DL4 with IL-2, IL-7, IL-1b, and IL-23 for 8 days prior to analysis.
+See also Figure S5.
+
+
+
+
+1098 Cell 168, 1086–1100, March 9, 2017
+ILC differentiation can occur ‘‘on demand’’ in any tissue and at                    grant agreement HEALTH-F3-2012-305578 (PathCO) and 317057 (HOMIN).
+any age. A recent study using parabiosis in mice has proposed                       J.P.D. is a founder of AXENIS and a member of its advisory board.
+that ILCs are long-lived tissue-resident cells that do not recircu-
+                                                                                    Received: December 6, 2016
+late under steady state and some inflammatory conditions (Gas-
+                                                                                    Revised: January 17, 2017
+teiger et al., 2015). In contrast, the half-life of several mucosal                 Accepted: February 13, 2017
+ILC subsets in the mouse is on the order of weeks, suggesting                       Published: March 9, 2017
+that these cells must be renewed (Sawa et al., 2010). The discov-
+ery of a circulating ILCPs provides a mechanism to replenish tis-                   REFERENCES
+sue ILCs in response to steady-state losses and in the context of
+                                                                                    Artis, D., and Spits, H. (2015). The biology of innate lymphoid cells. Nature 517,
+infection and inflammation.                                                         293–301.
+                                                                                    Bernink, J.H., Peters, C.P., Munneke, M., te Velde, A.A., Meijer, S.L., Weijer,
+STAR+METHODS                                                                        K., Hreggvidsdottir, H.S., Heinsbroek, S.E., Legrand, N., Buskens, C.J.,
+                                                                                    et al. (2013). Human type 1 innate lymphoid cells accumulate in inflamed
+Detailed methods are provided in the online version of this paper                   mucosal tissues. Nat. Immunol. 14, 221–229.
+and include the following:                                                          Bernink, J.H., Krabbendam, L., Germar, K., de Jong, E., Gronke, K., Kofoed-
+                                                                                    Nielsen, M., Munneke, J.M., Hazenberg, M.D., Villaudy, J., Buskens, C.J.,
+    d KEY RESOURCES TABLE                                                           et al. (2015). Interleukin-12 and -23 control plasticity of CD127(+) group 1
+    d CONTACT FOR REAGENT AND RESOURCE SHARING                                      and group 3 innate lymphoid cells in the intestinal lamina propria. Immunity
+                                                                                    43, 146–160.
+    d EXPERIMENTAL MODEL AND SUBJECT DETAILS
+        B Human blood and tissues samples                                           Cella, M., Fuchs, A., Vermi, W., Facchetti, F., Otero, K., Lennerz, J.K., Doh-
+                                                                                    erty, J.M., Mills, J.C., and Colonna, M. (2009). A human natural killer cell sub-
+        B In vivo analysis using human immune system (HIS)
+                                                                                    set provides an innate source of IL-22 for mucosal immunity. Nature 457,
+            mice model                                                              722–725.
+    d METHOD DETAILS
+                                                                                    Cella, M., Otero, K., and Colonna, M. (2010). Expansion of human NK-22 cells
+        B Cell isolation from blood, tonsil, gut, fetal liver, and lung             with IL-7, IL-2, and IL-1beta reveals intrinsic functional plasticity. Proc. Natl.
+        B FACS analysis and cell sorting                                            Acad. Sci. USA 107, 10961–10966.
+        B Bulk RNA isolation, library construction, sequencing,                     Chea, S., Schmutz, S., Berthault, C., Perchet, T., Petit, M., Burlen-Defranoux,
+         and analysis                                                               O., Goldrath, A.W., Rodewald, H.R., Cumano, A., and Golub, R. (2016). Single-
+      B Chromatin immunoprecipitation and sequencing using                          cell gene expression analyses reveal heterogeneous responsiveness of fetal
+         ChipMentation                                                              innate lymphoid progenitors to Notch signaling. Cell Rep. 14, 1500–1516.
+
+      B ChIP-Seq data processing, analysis, and visualization                       Cherrier, M., Sawa, S., and Eberl, G. (2012). Notch, Id2, and RORgt sequen-
+                                                                                    tially orchestrate the fetal development of lymphoid tissue inducer cells.
+      B Bulk and single cell culture
+                                                                                    J. Exp. Med. 209, 729–740.
+    d QUANTIFICATION AND STATISTICAL ANALYSIS
+                                                                                    Constantinides, M.G., McDonald, B.D., Verhoef, P.A., and Bendelac, A. (2014).
+    d DATA AND SOFTWARE AVAILABILITY
+                                                                                    A committed precursor to innate lymphoid cells. Nature 508, 397–401.
+      B Data Resources
+                                                                                    Cupedo, T., Crellin, N.K., Papazian, N., Rombouts, E.J., Weijer, K., Grogan,
+    d ADDITIONAL RESOURCES
+                                                                                    J.L., Fibbe, W.E., Cornelissen, J.J., and Spits, H. (2009). Human fetal lymphoid
+      B Yssel’s Medium                                                              tissue-inducer cells are interleukin 17-producing precursors to RORC+
+                                                                                    CD127+ natural killer-like cells. Nat. Immunol. 10, 66–74.
+SUPPLEMENTAL INFORMATION                                                            Dallas, M.H., Varnum-Finney, B., Delaney, C., Kato, K., and Bernstein, I.D.
+                                                                                    (2005). Density of the Notch ligand Delta1 determines generation of B and
+Supplemental Information includes five figures and five tables and can be           T cell precursors from hematopoietic stem cells. J. Exp. Med. 201, 1361–1366.
+found with this article online at http://dx.doi.org/10.1016/j.cell.2017.02.021.     Eberl, G., Colonna, M., Di Santo, J.P., and McKenzie, A.N. (2015). Innate
+                                                                                    lymphoid cells. Innate lymphoid cells: a new paradigm in immunology. Science
+AUTHOR CONTRIBUTIONS                                                                348, aaa6566.
+                                                                                    Fuchs, A., Vermi, W., Lee, J.S., Lonardi, S., Gilfillan, S., Newberry, R.D., Cella,
+Conceptualization, A.I.L. and J.P.D.; Methodology, A.I.L., Y.L., R.S., F.P.,        M., and Colonna, M. (2013). Intraepithelial type 1 innate lymphoid cells are a
+H.S.-M., and H.Y.; Software, R.S., F.P., and E.D.; Formal Analysis, A.I.L.,         unique subset of IL-12- and IL-15-responsive IFN-g-producing cells. Immunity
+S.L.-L., R.S., F.P., and E.D.; Investigation, A.I.L., Y.L., S.L.-L., R.S., F.P.,    38, 769–781.
+A.C., N.S., L.S., and G.M.-R.; Resources, A.P., J.B., L.L.B, R.C., D.T., P.G.,
+                                                                                    Gasteiger, G., Fan, X., Dikiy, S., Lee, S.Y., and Rudensky, A.Y. (2015). Tissue
+L.A.M., L.R., X.N., J.-M.S., M.A., T.G., R.W.H., J.-L.C., and I.A.; Writing,
+                                                                                    residency of innate lymphoid cells in lymphoid and nonlymphoid organs. Sci-
+A.I.L., R.S., and J.P.D.; Funding Acquisition, J.P.D.; Supervision, J.P.D.
+                                                                                    ence 350, 981–985.
+                                                                                    Hazenberg, M.D., and Spits, H. (2014). Human innate lymphoid cells. Blood
+ACKNOWLEDGMENTS
+                                                                                    124, 700–709.
+We thank all the members of Innate Immunity Unit for helpful discussions, the       Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X.,
+Centre de Recherche Translationnelle, F. Pala and O. Fiquet (Institut Pasteur),     Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-
+and A. Sparaneo (Casa Sollievo della Sofferenza) for technical assistance. A.I.L.   determining transcription factors prime cis-regulatory elements required for
+is a scholar in Pasteur-Paris University (PPU) International PhD program and        macrophage and B cell identities. Mol. Cell 38, 576–589.
+supported by FP7 under grant agreement 317057 (HOMIN) and the Fondation             Hoorweg, K., Peters, C.P., Cornelissen, F., Aparicio-Domingo, P., Papazian,
+ARC. R.S. was supported by EMBO (ALTF 1201-2014) and Marie Curie (H2020-            N., Kazemier, G., Mjösberg, J.M., Spits, H., and Cupedo, T. (2012). Functional
+MSCA-IF-2014). Other support included grants from the Institut Pasteur,             differences between human NKp44(-) and NKp44(+) RORC(+) innate lymphoid
+Inserm, Laboratoire d’Excellence REVIVE (ANR-10-LBX-73), and FP7 under              cells. Front. Immunol. 3, 72.
+
+
+
+                                                                                                                  Cell 168, 1086–1100, March 9, 2017 1099
+Jaitin, D.A., Kenigsberg, E., Keren-Shaul, H., Elefant, N., Paul, F., Zaretsky, I.,    Quinlan, A.R., and Hall, I.M. (2010). BEDTools: a flexible suite of utilities for
+Mildner, A., Cohen, N., Jung, S., Tanay, A., and Amit, I. (2014). Massively par-       comparing genomic features. Bioinformatics 26, 841–842.
+allel single-cell RNA-seq for marker-free decomposition of tissues into cell           Renoux, V.M., Zriwil, A., Peitzsch, C., Michaëlsson, J., Friberg, D., Soneji, S.,
+types. Science 343, 776–779.                                                           and Sitnicka, E. (2015). Identification of a human natural killer cell lineage-
+Juelke, K., and Romagnani, C. (2016). Differentiation of human innate lymphoid         restricted progenitor in fetal and adult tissues. Immunity 43, 394–407.
+cells (ILCs). Curr. Opin. Immunol. 38, 75–85.                                          Rollini, P., Faes-Van’t Hull, E., Kaiser, S., Kapp, U., and Leyvraz, S. (2007).
+Kim, B.S., and Artis, D. (2015). Group 2 innate lymphoid cells in health and dis-      Phenotypic and functional analysis of human fetal liver hematopoietic stem
+ease. Cold Spring Harb. Perspect. Biol. 7, a016337.                                    cells in culture. Stem Cells Dev. 16, 281–296.
+Klose, C.S., and Artis, D. (2016). Innate lymphoid cells as regulators of immu-        Rongvaux, A., Takizawa, H., Strowig, T., Willinger, T., Eynon, E.E., Flavell, R.A.,
+nity, inflammation and tissue homeostasis. Nat. Immunol. 17, 765–774.                  and Manz, M.G. (2013). Human hemato-lymphoid system mice: current use
+                                                                                       and future potential for medicine. Annu. Rev. Immunol. 31, 635–674.
+Klose, C.S., Flach, M., Möhle, L., Rogell, L., Hoyler, T., Ebert, K., Fabiunke, C.,
+Pfeifer, D., Sexl, V., Fonseca-Pereira, D., et al. (2014). Differentiation of type 1   Saldanha, A.J. (2004). Java Treeview–extensible visualization of microarray
+ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.        data. Bioinformatics 20, 3246–3248.
+Cell 157, 340–356.                                                                     Satoh-Takayama, N., Vosshenrich, C.A., Lesjean-Pottier, S., Sawa, S., Loch-
+Koche, R.P., Smith, Z.D., Adli, M., Gu, H., Ku, M., Gnirke, A., Bernstein, B.E.,       ner, M., Rattis, F., Mention, J.J., Thiam, K., Cerf-Bensussan, N., Mandelboim,
+and Meissner, A. (2011). Reprogramming factor expression initiates wide-               O., et al. (2008). Microbial flora drives interleukin 22 production in intestinal
+spread targeted chromatin remodeling. Cell Stem Cell 8, 96–105.                        NKp46+ cells that provide innate mucosal immune defense. Immunity 29,
+                                                                                       958–970.
+Koues, O.I., Collins, P.L., Cella, M., Robinette, M.L., Porter, S.I., Pyfrom, S.C.,
+Payton, J.E., Colonna, M., and Oltz, E.M. (2016). Distinct gene regulatory path-       Sawa, S., Cherrier, M., Lochner, M., Satoh-Takayama, N., Fehling, H.J.,
+ways for human innate versus adaptive lymphoid cells. Cell 165, 1134–1146.             Langa, F., Di Santo, J.P., and Eberl, G. (2010). Lineage relationship analysis
+                                                                                       of RORgammat+ innate lymphoid cells. Science 330, 665–669.
+Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with
+                                                                                       Schmidl, C., Rendeiro, A.F., Sheffield, N.C., and Bock, C. (2015). ChIPmentation:
+Bowtie 2. Nat. Methods 9, 357–359.
+                                                                                       fast, robust, low-input ChIP-seq for histones and transcription factors. Nat.
+Lara-Astiaso, D., Weiner, A., Lorenzo-Vivas, E., Zaretsky, I., Jaitin, D.A., David,    Methods 12, 963–965.
+E., Keren-Shaul, H., Mildner, A., Winter, D., Jung, S., et al. (2014). Immunoge-
+                                                                                       Scoville, S.D., Mundy-Bosse, B.L., Zhang, M.H., Chen, L., Zhang, X., Keller,
+netics. Chromatin state dynamics during blood formation. Science 345,
+                                                                                       K.A., Hughes, T., Chen, L., Cheng, S., Bergin, S.M., et al. (2016). A progenitor
+943–949.
+                                                                                       cell expressing transcription factor RORgt generates all human innate
+Legrand, N., Huntington, N.D., Nagasawa, M., Bakker, A.Q., Schotte, R.,                lymphoid cell subsets. Immunity 44, 1140–1150.
+Strick-Marchand, H., de Geus, S.J., Pouw, S.M., Böhne, M., Voordouw, A.,
+                                                                                       Seehus, C.R., Aliahmad, P., de la Torre, B., Iliev, I.D., Spurka, L., Funari, V.A.,
+et al. (2011). Functional CD47/signal regulatory protein alpha (SIRP(alpha))
+                                                                                       and Kaye, J. (2015). The development of innate lymphoid cells requires TOX-
+interaction is required for optimal human T- and natural killer- (NK) cell homeo-
+                                                                                       dependent generation of a common innate lymphoid cell progenitor. Nat. Im-
+stasis in vivo. Proc. Natl. Acad. Sci. USA 108, 13224–13229.
+                                                                                       munol. 16, 599–608.
+Lim, A.I., Menegatti, S., Bustamante, J., Le Bourhis, L., Allez, M., Rogge, L.,
+                                                                                       Serafini, N., Klein Wolterink, R.G., Satoh-Takayama, N., Xu, W., Vosshenrich,
+Casanova, J.L., Yssel, H., and Di Santo, J.P. (2016). IL-12 drives functional
+                                                                                       C.A., Hendriks, R.W., and Di Santo, J.P. (2014). Gata3 drives development of
+plasticity of human group 2 innate lymphoid cells. J. Exp. Med. 213, 569–583.
+                                                                                       RORgt+ group 3 innate lymphoid cells. J. Exp. Med. 211, 199–208.
+Marrack, P., and Kappler, J. (2004). Control of T cell viability. Annu. Rev. Immu-     Serafini, N., Vosshenrich, C.A., and Di Santo, J.P. (2015). Transcriptional regu-
+nol. 22, 765–787.                                                                      lation of innate lymphoid cell fate. Nat. Rev. Immunol. 15, 415–428.
+McLean, C.Y., Bristor, D., Hiller, M., Clarke, S.L., Schaar, B.T., Lowe, C.B.,         Shih, H.Y., Sciumè, G., Mikami, Y., Guo, L., Sun, H.W., Brooks, S.R., Urban,
+Wenger, A.M., and Bejerano, G. (2010). GREAT improves functional interpre-             J.F., Jr., Davis, F.P., Kanno, Y., and O’Shea, J.J. (2016). Developmental acqui-
+tation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501.                        sition of regulomes underlies innate lymphoid cell functionality. Cell 165,
+Mjösberg, J.M., Trifari, S., Crellin, N.K., Peters, C.P., van Drunen, C.M., Piet,     1120–1133.
+B., Fokkens, W.J., Cupedo, T., and Spits, H. (2011). Human IL-25- and                  Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P., Eberl, G.,
+IL-33-responsive type 2 innate lymphoid cells are defined by expression of             Koyasu, S., Locksley, R.M., McKenzie, A.N., Mebius, R.E., et al. (2013). Innate
+CRTH2 and CD161. Nat. Immunol. 12, 1055–1062.                                          lymphoid cells–a proposal for uniform nomenclature. Nat. Rev. Immunol. 13,
+Mohtashami, M., Shah, D.K., Nakase, H., Kianizad, K., Petrie, H.T., and Zú-           145–149.
+ñiga-Pflücker, J.C. (2010). Direct comparison of Dll1- and Dll4-mediated Notch       Yagi, R., Zhong, C., Northrup, D.L., Yu, F., Bouladoux, N., Spencer, S., Hu, G.,
+activation levels shows differential lymphomyeloid lineage commitment out-             Barron, L., Sharma, S., Nakayama, T., et al. (2014). The transcription factor
+comes. J. Immunol. 185, 867–876.                                                       GATA3 is critical for the development of all IL-7Ra-expressing innate lymphoid
+Montaldo, E., Teixeira-Alves, L.G., Glatzer, T., Durek, P., Stervbo, U., Hamann,       cells. Immunity 40, 378–388.
+W., Babic, M., Paclik, D., Stölzel, K., Gröne, J., et al. (2014). Human RORgt(+)     Yang, Q., Li, F., Harly, C., Xing, S., Ye, L., Xia, X., Wang, H., Wang, X., Yu, S.,
+CD34(+) cells are lineage-specified progenitors of group 3 RORgt(+) innate             Zhou, X., et al. (2015). TCF-1 upregulation identifies early innate lymphoid pro-
+lymphoid cells. Immunity 41, 988–1000.                                                 genitors in the bone marrow. Nat. Immunol. 16, 1044–1050.
+Munneke, J.M., Björklund, A.T., Mjösberg, J.M., Garming-Legert, K., Bernink,         Zhang, Z., Cheng, L., Zhao, J., Li, G., Zhang, L., Chen, W., Nie, W., Reszka-
+J.H., Blom, B., Huisman, C., van Oers, M.H., Spits, H., Malmberg, K.J., and            Blanco, N.J., Wang, F.S., and Su, L. (2015). Plasmacytoid dendritic cells pro-
+Hazenberg, M.D. (2014). Activated innate lymphoid cells are associated with            mote HIV-1-induced group 3 innate lymphoid cell depletion. J. Clin. Invest.
+a reduced susceptibility to graft-versus-host disease. Blood 124, 812–821.             125, 3692–3703.
+Okada, S., Markle, J.G., Deenick, E.K., Mele, F., Averbuch, D., Lagos, M.,             Zook, E.C., Ramirez, K., Guo, X., van der Voort, G., Sigvardsson, M., Svens-
+Alzahrani, M., Al-Muhsen, S., Halwani, R., Ma, C.S., et al. (2015).                    son, E.C., Fu, Y.X., and Kee, B.L. (2016). The ETS1 transcription factor
+IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacte-                   is required for the development and cytokine-induced expansion of ILC2.
+rium in humans with bi-allelic RORC mutations. Science 349, 606–613.                   J. Exp. Med. 213, 687–696.
+
+
+
+
+1100 Cell 168, 1086–1100, March 9, 2017
+STAR+METHODS
+
+KEY RESOURCES TABLE
+
+
+
+REAGENT or RESOURCE                                      SOURCE                       IDENTIFIER
+Antibodies
+Anti-human CD3 FITC (Clone UCHT1)                        eBioscience                  Cat#11-0038
+Anti-human CD3 BUV737 (Clone UCHT1)                      BD Bioscience                Cat#564307
+Anti-human CD4 FITC (Clone OKT4)                         eBioscience                  Cat#11-0048
+Anti-human CD5 FITC (Clone UCHT2)                        eBioscience                  Cat#11-0059
+Anti-human alpha beta TCR FITC (Clone IP26)              eBioscience                  Cat#11-9986
+Anti-human gamma delta TCR FITC (Clone B1.1)             eBioscience                  Cat#11-9959
+Anti-human CD14 FITC (Clone TÜK4)                       Miltenyi Biotec              Cat#130-080-701
+Anti-human CD19 FITC (Clone LT19)                        Miltenyi Biotec              Cat#130-104-650
+Anti-human CD7 PE-CF594 (Clone M-T701)                   BD Bioscience                Cat#562541
+Anti-human CD56 AF700 (Clone B159)                       BD Bioscience                Cat#557919
+Anti-human CD127 PE-Cyanine 7 (Clone eBioRDR5)           eBioscience                  Cat#25-1278
+Anti-human CRTh2-PE (Clone BM16)                         Miltenyi Biotec              Cat#130-098-238
+Anti-human CD294 (CRTh2) Alexa Fluor 647 (Clone BM16)    BD Bioscience                Cat#558042
+Anti-human CD117 BV605 (Clone 104D2)                     Biolegend                    Cat#313218
+Anti-human NKp44 BB15/AF647 (Clone p44-8)                BD Bioscience                Cat#565099/558564
+Anti-human CD45RA VioBlue (Clone T6D11)                  Miltenyi Biotec              Cat#130-098-180
+Anti-human CD25 eFluor450 (Clone BC96)                   eBioscience                  Cat#48-0259
+Anti-human IL1R1 APC                                     R&D                          Cat#MAB2692
+Anti-human CD69 BV421 (Clone FN50)                       Biolegend                    Cat#310930
+Anti-human CD196 PerCP-Cy5-5 (Clone 11A9)                BD Bioscience                Cat#560467
+Anti-human CD304 BV421 (Clone 12C2)                      Biolegend                    Cat#354514
+Anti-human EOMES PE (Clone WD1928)                       eBioscience                  Cat#12-4877
+Anti-human T-BET BV421 (Clone 4B10)                      Biolegend                    Cat#644816
+Anti-human/mouse GATA-3 PerCP-eFluor 710 (CloneTWAJ)     eBioscience                  Cat#46-9966
+Anti-human/mouse RORgamma (t) APC / PE (Clone AFKJS-9)   eBioscience                  Cat#17-6988/12-6988
+Anti-human/mouse PLZF PE                                 eBioscience                  Cat#12-9320-82
+Anti-human CD94 APC-Vio770 (Clone REA113)                Miltenyi Biotec              Cat#130-101-146
+Anti-human IFNg FITC (Clone 45-15)                       Miltenyi Biotec              Cat#130-091-641
+Anti-human IL-13 BV421 (Clone JES10-5A2)                 BD Bioscience                Cat#563580
+Anti-human IL-22 PE-Cyanine7 (Clone 22URTI)              eBioscience                  Cat#25-7229
+Anti-human IL-17A APC (Clone CZ8-23G1)                   Miltenyi Biotec              Cat#130-094-519
+Anti-human CD45 BV711/AF700 (Clone HI30)                 BD Bioscience                Cat#564358/560566
+Anti-H3K4me2 (Clone Y47)                                 Abcam                        Cat#ab32356
+Anti-human CD3 biotin (Clone OKT3)                       eBioscience                  Cat#13-0037-82
+Anti-human CD4 biotin (Clone RPA-T4)                     eBioscience                  Cat#13-0049-82
+Anti-human CD19 biotin (Clone HIB19)                     eBioscience                  Cat#13-0199-82
+Anti-human CD123 biotin (Clone 6H6)                      eBioscience                  Cat#13-1239-82
+Anti-human CD14 biotin (Clone 61D3)                      eBioscience                  Cat#13-0149-82
+Anti-biotin MicroBeads                                   Miltenyi Biotec              Cat#130-090-485
+CD34 MicroBead Kit                                       Miltenyi Biotec              Cat#130-046-702
+Biological Samples
+Healthy human peripheral blood                           EFS Paris                    N/A
+RORC mutation patients peripheral blood                  Prof. JLC (Necker            N/A
+                                                         Medical School)
+                                                                                                    (Continued on next page)
+
+
+
+                                                                             Cell 168, 1086–1100.e1–e5, March 9, 2017 e1
+Continued
+REAGENT or RESOURCE                              SOURCE                          IDENTIFIER
+Human fetal liver                                Advanced Bioscience             N/A
+                                                 Resources
+Cord blood                                       EFS Paris                       N/A
+Human tonsil                                     Dr. RC & PG (Casa Sollievo      N/A
+                                                 della Sofferenza)
+Human Lung                                       Dr. JMS (Hôpital Bichat)       N/A
+Human Intestine                                  Dr. MA (Hôpital Saint Louis)   N/A
+Human AB Serum                                   EFS Paris                       N/A
+Chemicals, Peptides, and Recombinant Proteins
+Human IL-2 IS, premium grade                     Miltenyi Biotec                 Cat#130-097-744
+Human IL-7, premium grade                        Miltenyi Biotec                 Cat#130-095-362
+Human IL-12, premium grade                       Miltenyi Biotec                 Cat#130-096-704
+Human IL-18                                      MBL International               Cat#B001-5
+recombinant human IL-17E/IL-25                   R&D                             Cat#1258-IL
+recombinant human IL-33                          R&D                             Cat#3625-IL
+recombinant human IL-1b                          R&D                             Cat#201-IL
+recombinant human IL-23                          R&D                             Cat#1290-IL
+Phorbol 12-Myristate 13-acetate (PMA)            Sigma-Aldrich                   Cat#P-8139
+Ionomycin                                        Sigma-Aldrich                   Cat#I0634
+BD GolgiPlug Protein Transport Inhibitor         BD Bioscience                   Cat#555029
+Formaldehyde                                     Sigma-Aldrich                   Cat#F8775
+Bovine serum albumin                             Sigma-Aldrich                   Cat#A-3675
+2-amino ethanol                                  Sigma-Aldrich                   Cat#E-0135
+Apo-transferrin                                  Sigma-Aldrich                   Cat#T-2252
+Insulin                                          Sigma-Aldrich                   Cat#I-5500
+Iscove’s Modified Dulbeco’s Medium (Glutamax)    ThermoFisher                    Cat#31980
+Affinity Script                                  Agilent                         Cat#600107
+Exonuclease I                                    NEB                             Cat#M0293L
+mRNA Second Strand Synthesis                     NEB                             Cat#E6111L
+Turbo DNase I                                    Ambion                          Cat#AM2239
+RNA Fragmentation Reagent                        Ambion                          Cat#AM8740
+T4 RNA Ligase                                    NEB                             Cat#M0204L
+Kapa Hifi                                        Kapa Biosystems                 Cat#KK2601
+dNTP                                             Lavorah Gmbh                    Cat#NU-1005XS
+Agencourt AMPure XP                              Beckman Coulter                 Cat#A63881
+Critical Commercial Assays
+Foxp3/Transcription Factor Staining Buffer Set   eBioscience                     Cat# 00-5521
+BD Cytofix/Cytoperm Kit                          BD Bioscience                   Cat# 554714
+Dynabeads mRNA Direct Kit                        Life Technologies               Cat#61012
+Nextera DNA Library Prep Kit                     Illumina                        Cat#FC-121-1030
+T7 High Yield RNA Synthesis Kit                  NEB                             Cat#E2040S
+NextSeq 500 High Output v2 Kit                   Illumina                        Cat#FC-404-2005
+Deposited Data
+Bulk RNA Seq data                                NCBI GEO                        GEO: GSE90834
+Chip-Seq                                         NCBI GEO                        GEO: GSE90640
+Experimental Models: Cell Lines
+Mouse: OP9                                       Institut Pasteur                Cat#CRL-2749
+Mouse: OP9-DL4                                   Institut Pasteur                N/A
+                                                                                                (Continued on next page)
+
+
+
+e2 Cell 168, 1086–1100.e1–e5, March 9, 2017
+Continued
+REAGENT or RESOURCE                                            SOURCE                              IDENTIFIER
+
+Experimental Models: Organisms/Strains
+Mouse: BRGS (BALB/c Rag2 / IL-2Rgc / SIRPaNOD)                 Institut Pasteur                    N/A
+Software and Algorithms
+HOMER                                                          (Heinz et al., 2010)                http://homer.salk.edu/homer/
+BEDtools                                                       (Quinlan and Hall, 2010)            http://bedtools.readthedocs.io/
+                                                                                                   en/latest/
+Bowtie2                                                        (Langmead and Salzberg, 2012)       http://bowtie-bio.sourceforge.
+                                                                                                   net/bowtie2/manual.shtml
+GREAT                                                          (McLean et al., 2010)               http://bejerano.stanford.edu/
+                                                                                                   great/public/html/
+DESeq2                                                                                             https://bioconductor.org/packages/
+                                                                                                   release/bioc/html/DESeq2.html
+Flow Jo_v10                                                    FlowJo                              www.flowjo.com
+Prism 6                                                        Prism-Graphpad                      www.graphpad.com
+Other
+Yssel’s Medium                                                 In house                            N/A
+
+
+
+CONTACT FOR REAGENT AND RESOURCE SHARING
+
+Requests should be directed to and will be fulfilled by Lead Contact James Di Santo (james.di-santo@pasteur.fr).
+
+EXPERIMENTAL MODEL AND SUBJECT DETAILS
+
+Human blood and tissues samples
+Blood samples from healthy donors were obtained from Establissement Français du Sang (EFS, Paris). Blood samples from patients
+with RORC mutation have been previously reported (Okada et al., 2015) including P2 from Kindred A (homozygous for S38L/38L iso-
+form 1 or S17L/S17L isoform 2) and P6 from Kindred C (homozygous for Q441* isoform 1 or Q420* isoform 2). Fetal liver with gesta-
+tional age ranging from 14 to 20 weeks were obtained from Advanced Bioscience Resources Inc. following approval of an institutional
+medical ethical committee at Institut Pasteur and the French Ministry of Education and Research (IF-20080451). Umbilical cord blood
+was collected from normal deliveries. Tonsils were obtained from pediatric patients undergoing routine tonsillectomy (Scientific Insti-
+tute for Research and Health Care ‘‘Casa Sollievo della Sofferenza’’). Lung and colon samples were obtained from adenocarcinoma
+and colon cancer patients, respectively undergoing therapeutic resection (Dr. X. Norel and J.-M. Sallenave, Hôpital Bichat; Dr. M
+Allez, Hôpital Saint Louis). Informed consent was obtained from all patients and included protocols approved by the institutional re-
+view boards of Necker Medical School, Paris Descartes University, Hôpital Bichat, Hôpital Saint Louis, Assistance Publique - Hôpital
+de Paris.
+
+In vivo analysis using human immune system (HIS) mice model
+BALB/c Rag2—/—Il2rg—/—SirpaNOD (BRGS) mice have been described (Legrand et al., 2011) and were maintained in isolators at In-
+stitut Pasteur. CD34+ HSC or CD117+ ILC were sorted from peripheral blood of healthy donors using a FACS Aria. Fetal liver CD34+
+HSC were isolated using CD34 Microbead Kit (Miltenyi). For in vivo transfer experiment, 1-3 3 105 CD117+ ILC or CD34+ HSC were
+intrahepatically injected into sublethal irradiated (3Gy) new born (3-7 days-old) BRGS mice together with 0.3 mg of IL-2 and IL-7 (Mil-
+tenyi). Mice received IL-2, IL-7, IL-1b, and IL-23 (0.3 mg each) by intraperitoneal injection weekly and were analyzed four weeks post-
+transplantation. For generation of HIS mice, 2 3 105 fetal liver-derived CD34+ CD38- HSC were intrahepatically injected into sublethal
+irradiated (3Gy) new born (3-7 days-old) BRGS mice. Mice were sacrificed 8-9 weeks post-injection. Experiments were approved by
+ethical committee at Institut Pasteur and validated by French Ministry of Education and Research (MENESR#02162.02).
+
+METHOD DETAILS
+
+Cell isolation from blood, tonsil, gut, fetal liver, and lung
+Human peripheral blood mononuclear cells (PBMC) from CB and PB were isolated by Ficoll-Paque (GE Healthcare) density gradient
+centrifugation. Single cell suspension from fetal liver and tonsil was achieved by mechanical disruption through 70-mm filters. Lung
+and intestine samples were minced and digested with Liberase TL (25 mg/ml; Roche) and DNase I (50 mg/ml; Sigma-Aldrich) for 45min
+
+
+
+                                                                                          Cell 168, 1086–1100.e1–e5, March 9, 2017 e3
+in 37 C shaking incubator. Digested tissues were passed through 70-mm filters. Lymphocytes from liver, lung, and gut were isolated
+by Ficoll-Paque density gradient centrifugation.
+
+FACS analysis and cell sorting
+For FACS analysis, cells were first stained with Flexible Viability Dye eFluor 506 (eBioscience) for 10 min following by 20 min surface
+antibodies staining with Brilliant Stained Buffer (BD) on ice. For experiment involving intracellular TF staining, cells were fixed, per-
+meabilized, and stained using Foxp3/Transcription Factor Staining Buffer Kit (eBioscience). For intracellular cytokines staining, cells
+were stimulated with PMA (10ng/ml; Sigma) plus Ionomycin (1 mg/ml; Sigma) in the presence of Golgi Plug (BD) for 3 hr in 37 C incu-
+bator. Cells were fixed, permeabilized, and stained by Cytofix/Cytoperm Kit (BD). Samples were acquired on LSRFortessa (BD) and
+analyzed by FlowJ10 (Tree Star).
+   For cell sorting from healthy PB, PBMC were first depleted of T cell, B cell, pDC and monocytes by labeling with biotin-conjugated
+anti-CD3, anti-CD4, anti-CD19, anti-CD14, anti-CD123 followed by anti-biotin microbeads (Miltenyi) according to manufacturer’s in-
+structions. Sorting from CB and tissues were performed without lineage depletion. Bulk populations were sorted to a purity R 99% or
+as single cell index sorting (both using FACSAria II; BD).
+
+Bulk RNA isolation, library construction, sequencing, and analysis
+103 cells from each population were FACS sorted directly into 50 mL of lysis/binding buffer (Life Technologies). mRNA was captured
+with 15 mL Dynabeads oligo(dT) (Life Technologies), washed according to manufacturers’ instructions and eluted at 85 C with 6 mL of
+10mM Tris-HCl (pH7.5). We used a derivation of MARS-Seq as described (Jaitin et al., 2014), developed for single-cell RNA-seq to
+produce expression libraries of two replicates per population. Libraries were sequenced at an average depth of 5 million reads per
+library on the Ilumina NextSeq and aligned to the human reference genome (hg19). Reads were mapped using hisat (version 0.1.6);
+duplicate reads were filtered if they had identical UMIs. Expression levels were calculated and normalized to the total number of reads
+using HOMER software (http://homer.salk.edu).
+
+Chromatin immunoprecipitation and sequencing using ChipMentation
+FACS sorted cells (20-50K) were immediately crosslinked in PBS containing 1% formaldehyde (Sigma) for 10 min at room temper-
+ature for ChIP-seq analysis. Crosslinking was quenched by adding glycine (0.125M final concentration) followed by 5 min incubation
+at room temperature. Cells were placed on ice, washed with PBS and snap-frozen for storage at 80 C. Pellets were processed in
+parallel to minimize technical variation. Cells were resuspended in 100 mL sonication buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl
+pH8 and 1x EDTA-free complete protease inhibitors; Roche) and transferred to a 0.65ml Bioruptor sonication tube (Diagenode). After
+15 min incubation on ice, cells were sonicated for 30 cycles (30 s ON - 30 s OFF) using a Bioruptor Pico sonicator (Diagenode) to shear
+chromatin down to ± 250 bp fragments. Chromatin was equilibrated by adding 900 mL 10x ChIP dilution buffer (0.01% SDS, 1.1%
+Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH8, 167mM NaCl) and incubated overnight at 4 C with 1 mL of H3K4Me2-specific
+antibody (ab32356, Abcam) or normal rabbit IgG as a negative control (sc-2027, Santa Cruz). In addition, 20 mL of protein A Dyna-
+beads (Thermo Fisher Scientific) per IP were blocked in PBS containing 0.1% BSA (Sigma) by incubation overnight at 4 C. The next
+day, beads were resuspended in the original volume with ChIP dilution buffer and added to the chromatin extracts. After 2 hr of in-
+cubation at 4 C, beads were collected and washed with Low Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl
+pH8, 150mM NaCl), High Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH8, 500mM NaCl) and LiCl buffer
+(10mM Tris-HCl pH8, 1mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% deoxycholic acid). Chromatin-antibody immobilized on magnetic
+beads were then subjected to tagmentation as recently described (Schmidl et al., 2015). Eluted DNA was purified using MinElute spin
+columns (QIAGEN) and amplified for 8-12 cycles using Nextera PCR primers. Libraries were purified using dual (0.5x-2.0x) SPRI Am-
+pure XP beads (Beckman Coulter), pooled (up to 10 per sequencing run) and sequenced on a NextSeq500 (Illumina) running a single-
+read 75bp protocol.
+
+ChIP-Seq data processing, analysis, and visualization
+Reads were demultiplexed using BaseSpace (Illumina) and aligned to the human reference genome (hg19) using Bowtie (Langmead
+and Salzberg, 2012) with standard settings, removing reads that could not be uniquely mapped. Indexed and sorted bam files were
+parsed to HOMER (Heinz et al., 2010) for further analysis. Tag directories were generated for each sample with removal of duplicate
+reads (-tbp 1 option). BedGraph files displaying normalized counts (reads per million) were generated for direct visualization in the
+UCSC Genome Browser (https://genome.ucsc.edu/) using the makeUCSCfile HOMER script. H3K4Me2 enriched regions were iden-
+tified using HOMER findPeaks with -region -size 1000 -minDist 2500 options. Overlapping and non-overlapping regions between two
+samples were identified using the intersect function of BEDTools (Quinlan and Hall, 2010) or the HOMER mergePeaks script (-d given
+option) requiring a minimal overlap of 1bp. Sets of cell type-specific H3K4Me2+ regions were visualized as heatmaps with Java
+TreeView (Saldanha, 2004). Regions/peaks were assigned to putative target genes GREAT (McLean et al., 2010). GREAT was sub-
+sequently used to calculate enrichments of these genes for known pathway signatures using the whole genome as background.
+Fraction of reads in peaks (FRIP) and percentage of duplication metrics are included in Table S1.
+
+
+
+
+e4 Cell 168, 1086–1100.e1–e5, March 9, 2017
+Bulk and single cell culture
+All in vitro culture experiments were performed in Yssel’s medium supplemented with 2% human AB serum (EFS). 2-3 3 103 stromal
+cells were pre-seeded in 96-well round bottom plates one night before culture. For bulk culture, 100-300 FACS sorted cell were plated
+on the stromal cells. For cloning experiment, cells were index-sorted directly into the 96-well plates pre-seeded with stromal cells. Cy-
+tokines IL-2, IL-7 (20 ng/ml each, Miltenyi), IL-12, IL-18, IL-25, IL-33, IL-1b, IL-23 (20 ng/ml each, R&D) were provided in various com-
+binations as indicated. For bulk culture, fresh cytokines and medium were replenish every 5 days and analyzed after 10 days expansion.
+For cloning experiment, cytokines and medium were replenished every 7 days and analyzed after 14-18 days of culture.
+
+QUANTIFICATION AND STATISTICAL ANALYSIS
+
+Data are represented as Median unless specified. The sample size for each experiment and the replicate number of experiments are
+included in the figure legends.
+
+DATA AND SOFTWARE AVAILABILITY
+
+Data Resources
+The accession number for the RNA-Seq and ChIPm-Seq analysis have been deposited in the NCBI Gene Expression Omnibus under
+accession numbers GEO: GSE90834 and GEO: GSE90640.
+
+ADDITIONAL RESOURCES
+
+Yssel’s Medium
+Yssel’s Medium is prepared in house by using IMDM (Invitrogen) plus 0.25% (w/v) bovine serum albumin (Sigma), 1.8 mg/L 2-amino
+ethanol, 40 mg/L Apo-transferrin, 5 mg/L insulin and penicillin/Streptomycin.
+
+
+
+
+                                                                                          Cell 168, 1086–1100.e1–e5, March 9, 2017 e5
+Supplemental Figures
+
+
+
+
+Figure S1. CD117+ ILC Gating Strategy, Related to Figure 1
+(A) FACS analysis of ILCs from human peripheral blood and gut. CD117+ ILC were gated on lymphoid size, singlet and viable CD45+Lin—CD7+
+CD127+CRTh2—CD117+ population.
+(B) Protein staining for surface receptors and intracellular PLZF in blood CD117+ ILC (blue) and NK cells (gray).
+Figure S2. Transcriptional and Epigenetics Signature of PB CD117+ ILC, Related to Figure 2
+(A) Annotation of H3K4Me2+ regions as defined by their overlap with known genomic features.
+(B) Comparative transcriptional and epigenetics profiles of CD117+ ILC and CD34+ HSC for selective loci. Bar graphs show gene expression values identified by
+RNA-seq from CD117+ ILC (blue) and CD34+ HSC (orange) (n = 4, error bars denote SEM). Adjacent genome browser display normalized H3K4Me2 ChIPm-Seq
+signal in CD117+ ILC (blue) or CD34+ HSC (orange). Arrowheads mark regions of CD117+ ILC-specific H3K4Me2 enrichment.
+Figure S3. Cytokines Environment Dictate Progeny of CD117+ ILC, Related to Figure 3
+(A) Fold expansion of bulk cultured PB CD117+ ILC in cytokines (20 ng/ml each) for 10 days.
+(B) Cytokine production from in vitro expanded PB CD117+ ILC after stimulation.
+(C) Surface phenotype and TF expression in expanded PB CD117+ ILC.
+(D) Cytokines (IFN-g, IL-13, IL-22 and IL-17A) production in response to 3h PMA/ionomycin stimulation using different culture conditions.
+(E) Surface phenotype and TF expression of representative ILC1, ILC2 and ILC3 clones.
+(F) Surface phenotype, TF and cytokine production in PMA/ionomycin stimulated ‘cytokine negative’ clones.
+Figure S4. Gating Strategy and Characteristic of CD117+ ILC from Fetal Liver, Cord Blood, Lung, and Tonsil, Related to Figure 6
+(A) Gating strategy for FACS analysis to identify CD117+ ILC from fetal liver, cord blood, lung and tonsil. Surface expression for CRTh2 and NKp44, intracellular TF
+(RORgt, GATA-3 and T-BET) was compared between CD117+NKp44— ILC (solid blue), CD117+ NKp44+ (dashed blue), CRTh2+ ILC2 (green) and CD56+ NK
+cells (gray).
+(B) Surface phenotypes of CD117+NKp44— ILC (solid blue), CD117+NKp44+ (dashed blue), CRTh2+ ILC2 (green) and CD56+ NK cells (gray) from fetal liver, CB,
+lung and tonsil.
+(C) Functional profiles of CD117+ ILC (blue), and CD56+ NK cells (gray) from fetal liver, CB, lung and tonsil in response to 3 hr PMA/ionomycin stimulation.
+Figure S5. Model for Human ‘‘ILC-Poiesis,’’ Related to Figure 7
+Human ILCP (Lin—CD127+CD117+CD45RA+IL1R1+) within fetal and adult tissues as well as in the circulation can be detected that have potential for all ILC
+subsets and NK cells.
+