ABSTRACT Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we

examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor–product relationships that link the subsets identified. This analysis identified two

successive stages of ILC development within T cell factor 1-positive (TCF-1+) early innate lymphoid progenitors (EILPs), which we named ‘specified EILPs’ and ‘committed EILPs’. Specified

EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation

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DIFFERENTIATION DOWNSTREAM OF LINEAGE COMMITMENT IN ILC1S IS DRIVEN BY HOBIT ACROSS TISSUES Article 30 August 2021 STAGE-SPECIFIC GATA3 INDUCTION PROMOTES ILC2 DEVELOPMENT AFTER LINEAGE

COMMITMENT Article Open access 05 July 2024 RECIPROCAL TRANSCRIPTION FACTOR NETWORKS GOVERN TISSUE-RESIDENT ILC3 SUBSET FUNCTION AND IDENTITY Article 23 September 2021 DATA AVAILABILITY The

accession number for the raw data of the RNA-Seq is GSE113767. The accession number for the raw data of the DNase-Seq and ChIC-Seq is GSE128483. All other relevant data are available from

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influenza “OMICs” data defines a role for UBR4 in virus budding. _Cell Host Microbe_ 18, 723–735 (2015). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank H.-R.

Rodewald for sharing _Il7r-iCre_ mice and J. Richa from the Transgenic and Chimeric Mouse Facility, University of Pennsylvania, for injection of _Tcf7_YFP embryonic stem cells into mouse

blastocysts. We thank T. Ciucci, R. Bosselut, J. Chen, the CCR Sequencing Facility, the CCR Flow Cytometry Core Facility, and the DNA Sequencing Facility of the University of Pennsylvania

for technical support. This work utilized the computational resources of the NIH high-performance computing Biowulf cluster (http://hpc.nih.gov). This research was supported by the

Intramural Research Program of the NIH, National Cancer Institute and Center for Cancer Research, and by grants from the NIH (AI121080 and AI139874 to H.-H.X.), Veteran Affairs BLR&D

Merit Review Program (BX002903A to H.-H.X.), and Foundation pour la Recherche Médicale (DEQ20170839118 to C.H.) and National Research Agency Investissements d’Avenir via the program LabEX

IGO (ANR-11-LABX-0016-01 to C.H.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute, National Institutes of

Health, Bethesda, MD, USA Christelle Harly, Devin Kenney & Avinash Bhandoola * CRCINA, INSERM, CNRS, Université d’Angers, Université de Nantes, Nantes, France Christelle Harly * LabEx

IGO ‘Immunotherapy, Graft, Oncology’, Nantes, France Christelle Harly * Systems Biology Center, National Heart, Lung, and Blood Institute, National Iinstitutes of Health, Bethesda, MD, USA

Gang Ren, Binbin Lai & Keji Zhao * Department of Medicine, Division of Translational Medicine and Human Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia,

PL, USA Tobias Raabe * Department of Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA Qi Yang * Office of Science and Technology Resources, Office of the Director,

Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Margaret C. Cam * Department of Microbiology, Interdisciplinary Immunology Graduate

Program, Carver College of Medicine, University of Iowa, Iowa City, IA, USA Hai-Hui Xue * Iowa City Veterans Affairs Health Care System, Iowa City, IA, USA Hai-Hui Xue Authors * Christelle

Harly View author publications You can also search for this author inPubMed Google Scholar * Devin Kenney View author publications You can also search for this author inPubMed Google Scholar

* Gang Ren View author publications You can also search for this author inPubMed Google Scholar * Binbin Lai View author publications You can also search for this author inPubMed Google

Scholar * Tobias Raabe View author publications You can also search for this author inPubMed Google Scholar * Qi Yang View author publications You can also search for this author inPubMed 

Google Scholar * Margaret C. Cam View author publications You can also search for this author inPubMed Google Scholar * Hai-Hui Xue View author publications You can also search for this

author inPubMed Google Scholar * Keji Zhao View author publications You can also search for this author inPubMed Google Scholar * Avinash Bhandoola View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS C.H. designed the research and performed most of the experiments, alongside D.K. and G.R. C.H., B.L., M.C.C. and A.B. analyzed

the data. C.H., M.C.C., T.R. and A.B. produced the figures. C.H., T.R., Q.Y. and H.-H.X. designed and generated the new mouse models. C.H., K.Z. and A.B. directed and oversaw the

experiments. C.H. and A.B. wrote the paper. All authors helped to design the research, and read and commented on the manuscript. CORRESPONDING AUTHOR Correspondence to Avinash Bhandoola.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION: Ioana Visan was the primary editor on this article and

managed its editorial process and peer review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 REPRESENTATIVE GATING STRATEGY USED FOR FLOW CYTOMETRIC ANALYSIS. (A) Single cell

suspensions made from bone marrow are depleted of RBCs using osmotic lysis, then depleted for LinILC+ cells (see Methods), stained using DAPI, and analyzed by flow cytometry. (B) For

visualization of ILC precursors, an additional LinILC- Kit+ gate is applied before gating shown in Fig. 1a. SUPPLEMENTARY FIGURE 2 DIFFERENTIATION POTENTIAL OF EILPS _IN VITRO_. Flow

cytometric analysis of cultures from single EILPs sorted into 96 well plates and cultured for 10 days in either SF7-GM3 or SF7-GM3-MG6 conditions. (A-B) Examples of single EILP-derived

colonies showing the lineages identified. Arrows show successive gating. Numbers indicate the percentage of cells in each gate. (C) Composition of the ILC+ wells. Each column represents one

well, positive wells are indicated in black for individual lineage. (D) Quantification of absolute numbers of Mac-1+ cells per Mac-1+ colony in SF7-GM3 condition (n=50 colonies) and

SF7-GM3-MG6 condition (n=191 colonies). Data are presented as mean + SD. A two-tailed unpaired Student’s t-test was performed to determine significance, and showed that difference is not

significant. (E) Representative profile of Mac-1+ colonies gated on Mac-1+ cells. (F) Percentage of Mac-1+ wells containing cDC1, cDC2, or both lineages as shown in e. All data are

representative of three independent experiments. SUPPLEMENTARY FIGURE 3 GENERATION OF A _TCF7__YFP_ REPORTER MOUSE. (A) Strategy for the generation of a _Tcf7__YFP_ allele by insertion of a

_P2A-YFP_ sequence downstream of the _Tcf7_ C-terminus, followed by deletion of the floxed neo cassette using a _CMV-Cre_ mouse strain. The P2A ribosomal skipping peptide allows bicistronic

generation of separate _Tcf7_ and YFP molecules, but both driven by the _Tcf7_ promoter at equimolar amounts. _Tcf7_ exons are numbered (E3-E9), and the scale and the location of the MfeI

restriction sites and Southern blot probe used to screen the targeted ES clones are indicated. (B) Identification by Southern blot of an ES clone with the expected homologous recombination

(+/-) compared to a wild-type clone (-/-). Genomic DNA was extracted, digested with MfeI, and blotted with the probe shown in a. (C) Flow cytometric analysis of LinILC- Kit+ CD122low α4β7+

BM cells of the indicated mouse strains. Data are representative of three independent experiments. SUPPLEMENTARY FIGURE 4 CHARACTERIZATION OF EILP SUBSETS. (A) RNA-seq analysis of ALPs,

sEILPs, cEILPs and ILCPs. Hierarchical clustering using complete linkage calculated from Euclidian distances. (B-C) Flow cytometric analysis of cultures from single sEILPs and cEILPs sorted

into 96 well plates and cultured for 10 days in either SF7-GM3 or SF7-GM3-MG6 conditions. Data are pooled from 2 out of 3 representative experiments. (B) Frequency of wells containing ILC

progenitors as shown in Supplementary Fig. 2a, in SF7-GM3 condition. Data are presented as % of ILC positive wells ± SEP for n=78 sEILP wells and n=143 cEILP wells. (C) Frequency of ILC

positive colonies containing the indicated combination of mature ILCs, identified as shown in Supplementary Fig. 2a. (D) Flow cytometric analysis of sEILPs and cEILPs. (E) Flow cytometric

analysis of intracellular DAPI staining on sEILPs and cEILPs. (F) Flow cytometric analysis of cultures from sEILPs after two days in SF7 condition. Based on Mac-1, TCF-1, Thy1, and PLZF

expression (left), the derived populations were separated into DC (Mac-1+, grey), sEILP (TCF-1+ PLZF- Thy1-, black), cEILP (TCF-1+ PLZF+ Thy1-, orange), and ILCP (TCF-1+ PLZF+ Thy1+,

purple), and analyzed for expression of transcription factors (right). Arrows show successive gating. (G-H) Flow cytometric analysis of sEILP1s (green), sEILP2s (blue), cEILPs (orange) and

pre-DCs (grey) in _Tcf7__YFP_ mice (G) or wild-type mice (H). (I) Flow cytometric analysis of cultures from _Tcf7-_YFP+ sEILP1s and sEILP2s after two days in SF7 condition, gated on

_Tcf7-_YFP- cells. (J-K) Flow cytometric analysis of cultures from single sEILP1 and sEILP2 cells sorted into 96 well plates after 10 days in SF7-GM3 or SF7-GM3-MG6 conditions. (J)

Quantification of wells containing cDC1s, cDC2s, or both lineages as defined in Supplementary Fig. 2e. (K) Quantification of absolute numbers of DCs per DC positive colony in n=159 colonies

derived from sEILP1s and n=6 colonies derived from sEILP2s. Data are presented as mean + SD. A two-tailed unpaired Student’s t-test with Welsh correction was performed to determine

significance. ***_p_<0.005. (E,F,I) Numbers indicate the percentage of cells in each gate. All data are representative of three independent experiments. SUPPLEMENTARY FIGURE 5 DC

POTENTIAL OF EILPS _IN VIVO_. (A-B) Flow cytometric analysis of BM cells from wild-type mouse defining the indicated populations. Arrows show successive gating. (C) Flow cytometric analysis

of pre-DCs defined in a (grey), pre-cDC1s defined in b (blue), sEILPs (black) and CD11c+ EILPs defined in Fig. 4d (red). (D) Flow cytometric analysis of the indicated BM precursors (black)

compared to Lin Kit BM cells (grey). (E) Flow cytometric analysis of _Il7r-Cre R26-stop-YFP Tcf7__EGFP/+_ DC subsets defined in b. (A-E) Data are representative of three independent

experiments. (F) Flow cytometric analysis of BM cells from CD45.1+ mice that were lethally irradiated (850 rads), injected with _Tox__-/-_ or wild-type littermate CD45.2+LinILC-KithighSca-1+

BM cells mixed with CD45.1+LinILC-KithighSca-1+ BM cells, and reconstituted for 10–12 weeks. Profiles of LinILC-KithighSca-1+ cells and granulocytes from BM, and cDC1s (CD8α+Mac-1lo) and

cDC2s (CD8α-Mac-1hi) from spleen are shown. Data are pooled from two independent experiments that gave similar results, and presented as mean ± SEM for n=7 mice per group. A two-tailed

unpaired Student’s t-test was performed to determine significance. ns, not significant. (A,B,E,F) Numbers indicate the percentage of cells in each gate. SUPPLEMENTARY FIGURE 6 GENERATION OF

A _TCF7__EGFPNULL_ MOUSE AND ANALYSIS OF EILPS IN _TCF7__NULL_ MICE. (A) Strategy of generation of a _Tcf7__EGFPnull_ allele by breeding the _Tcf7__EGFP_ mouse with the _CMV-Cre_ mouse

strain. (B) Flow cytometric analysis of CD3ε+ splenocytes from _Tcf7__-/-_ (grey shaded histogram), _Tcf7__EGFP/+_ (black histogram), and _Tcf7__EGFPnull/-_ (red histogram) mice for GFP

expression on unfixed samples, or TCF-1 expression detected by intracellular staining with antibodies targeting either the N-terminal (C63D9) or C-terminal (C46C7) domains of TCF-1. (C) Flow

cytometric analysis of thymus from mice of the indicated genotype. (D) Numbers of thymocytes for _Tcf7__-/-_ (n=4), _Tcf7__EGFP/+_ (n=6), and _Tcf7__EGFPnull/-_ (n=6) mice pooled from three

independent experiments. Data are presented as mean ± SEM. (E) Flow cytometric analysis of LinILC- Kit+ CD122low BM cells from _Tcf7__EGFPnull/+_ and _Tcf7__EGFPnull/-_ littermate mice. (F)

Flow cytometric analysis of EILPs from e. (G) Flow cytometric analysis of LinILC-depleted BM cells from mice of the indicated genotype. Arrows show successive gating. (H) Flow cytometric

analysis and quantification of LinILC- Kit+ BM cells of _Tcf7__EGFPnull/+_ and _Tcf7__EGFPnull/-_ littermate mouse. Data are presented as mean ± SEM for n=3 mice per group. (C,E,G) Numbers

indicate the percentage of cells in each gate. (D,H) A two-tailed unpaired Student’s t-test was performed to determine significance. ns, not significant; **_p_<0.01, ***_p_<0.005.

(B-C,E-H) Data are representative of three independent experiments. (i) RNA-seq analysis averaged from 3 ALP samples, 2 _Tcf7__EGFPnull/-_ EILP samples, and 2 _Tcf7__EGFPnull/+_ sEILP

samples. Significance was calculated using a linear model (anova), applying the empirical Bayes method for estimating variance. Volcano plots show significantly upregulated or downregulated

genes by more than two-fold from ALPs to wild-type sEILPs colored in red and green respectively. SUPPLEMENTARY FIGURE 7 TCF-1 MECHANISM OF ACTION. (A,B) Flow cytometric analysis of cultures

from single sEILP sorted from _Tcf7__EGFPnull/+_ and _Tcf7__EGFPnull/-_ littermate mice into 96 well plates, after 10 days in SF7-GM3 or SF7-GM3-MG6 conditions. (A) Quantification of wells

containing ILCs, DCs, or both lineages. (B) Quantification of absolute numbers of Mac-1+ DCs per DC positive colony. Data are pooled from both cytokine conditions and presented as mean + SD.

Statistics are calculated using n=12 DC positive colonies derived from _Tcf7__EGFPnull/+_ sEILPs and n=34 DC positive colonies derived from _Tcf7__EGFPnull/-_ sEILPs. A two-tailed unpaired

Student’s t-test with Welsh correction was performed to determine significance. **_p_<0.01. (C-E) scRNA-seq analysis of _Tcf7_-GFP_+_ BM progenitors isolated from _Tcf7__EGFPnull/-_ mice

and compared to wild-type ALPs and _Tcf7_-GFP_+_ BM progenitors. (C) _t_-SNE plots showing pseudo-time ordering scores of individual ALP (n=786 cells), _Tcf7_-GFP_+_ _Tcf7__EGFP/+_ cells

(n=1799) and _Tcf7_-GFP_+_ _Tcf7__EGFPnull/-_ cells (n=594) shown in Fig. 6b along the two progressions from Fig. 1e. The ordering score of individual cells is represented in colors going

from light grey to violet for a given progression. Cells that are not part of the progression are dark grey. (D) Quantification of TCF-1 deficient and sufficient sEILP1s in each progression

from b, calculated as percentage of sEILP1. The size of the circle is relative to the percentage of cells. (E) Expression of the indicated genes of individual TCF-1 deficient (black) and

sufficient (pink) sEILP1s in each progression (main or alt. for alternative) as shown in c and d. Statistics are calculated on n=224 main sEILP1s and n=75 alt. sEILP1s that are TCF-1

deficient, and n=585 main sEILP1s and n=84 alt. sEILP1s that are TCF-1 sufficient. (F) LEAP analysis modelling the transcriptional network underlying ILC development. Interactions between

transcription factors. Only interactions with the 10 most connected controllers are represented. Controllers downregulated during ILC development are shown in green, controllers upregulated

are in red. The size of each controller is proportional to its network connectivity. See Supplementary Table 6 for the whole network. (G) TCF-1 ChIC-seq analysis in EILPs and DNase-seq

analysis in ALPs, EILPs, ILCPs. Heat map centered on TCF-1 binding sites in EILPs (± 2kb) that are not located in region of DNase I hypersensitivity in ALPs (left), and quantification of

DNase I hypersensitivity enrichment (right). (H) scRNA-seq analysis as in c-e. Expression of the indicated genes of individual TCF-1 deficient (n=224, black) and sufficient (n=585, pink)

sEILP1s from the Main developmental progression as shown in c and d. (E,H) A two-sided Wilcoxon rank-sum test was used to determine the significance of gene expression differences between

TCF-1 deficient and sufficient cells for a given subset. **_p_<0.01, ***_p_<0.005. See also Supplementary Table 4. (I) Scheme of early ILC development showing the progenitor-successor

relationships between EILP subsets, and their developmental fate. The transcription factors required for the described developmental progression are indicated. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figs. 1–7 REPORTING SUMMARY SUPPLEMENTARY TABLE 1: GENES DYNAMICALLY EXPRESSED DURING WILD-TYPE ILC DEVELOPMENT IN SCRNA-SEQ Cell clusters separated

by pseudotime were compared with each other. Clusters as defined in Fig. 1c are compared along the main (ALP (_n_ = 752), sEILP1 (_n_ = 585), cEILP (_n_ = 409), ILCP (_n_ = 414), ILC2P (_n_ 

= 111)) or alternative pseudotimes (ALP (_n_ = 751), sEILP1 (_n_ = 84), sEILP2 (_n_ = 187)), as defined in Fig. 1e. Log-transformed average expressions and fold changes are shown. A

two-sided Wilcoxon rank-sum test was used to determine significance. SUPPLEMENTARY TABLE 2: GENES DIFFERENTIALLY EXPRESSED BETWEEN TCF7EGFPNULL/− EILPS AND WILD-TYPE SEILPS IN BULK RNA-SEQ

Gene expression is shown as the log-transformed average for each sample. Fold changes in expression between TCF-1-deficient EILPs and wild-type sEILPs are indicated. SUPPLEMENTARY TABLE 3:

GENES DIFFERENTIALLY EXPRESSED BETWEEN TCF7EGFPNULL/− AND WILD-TYPE SEILP1S AND SEILP2S IN SCRNA-SEQ The gene expression of TCF-1-deficient and sufficient cells separated by clusters, as

shown in Fig. 6c, was compared. Statistics were calculated on _n_ = 270 sEILP1s and _n_ = 276 sEILP2s that were TCF-1 deficient, and _n_ = 615 sEILP1s and _n_ = 187 sEILP2s that were TCF-1

sufficient. Log-transformed average expressions and fold changes are shown. A two-sided Wilcoxon rank-sum test was used to determine significance. SUPPLEMENTARY TABLE 4: GENES DIFFERENTIALLY

EXPRESSED BETWEEN TCF7EGFPNULL/− AND WILD-TYPE SEILP1S SEPARATED BY PSEUDOTIMES IN SCRNA-SEQ The gene expression of TCF-1-deficient and sufficient sEILP1s separated by pseudotimes, as shown

in Supplementary Fig. 7c,d, was compared. Statistics were calculated on _n_ = 224 main sEILP1s and _n_ = 75 alternative sEILP1s that were TCF-1 deficient, and _n_ = 585 main sEILP1s and _n_

 = 84 alternative sEILP1s that were TCF-1 sufficient. Log-transformed average expressions and fold changes are shown. A two-sided Wilcoxon rank-sum test was used to determine significance.

SUPPLEMENTARY TABLE 5: CORRELATION NETWORK Putative regulatory relationships between controllers and targets are indicated, along with their corresponding maximum absolute correlation

coefficient. All cells in the main trajectory of differentiation (_n_ = 2,633) were used to calculate Pearson’s correlations. Only maximum absolute correlations of ≥0.2 were considered,

which corresponded to an FDR < 5 × 10–5. The pseudotime lag of expression between a controller and its targets is indicated. SUPPLEMENTARY TABLE 6: TCF-1 CHIC-SEQ PEAKS The coordinates

for individual TCF-1 binding peaks, as identified by ChIC-Seq, are indicated and mapped to the nearest gene. SUPPLEMENTARY TABLE 7: TCF-1 GENE TARGETS TCF-1 gene targets were identified

using the correlation network (Supplementary Table 5), TCF-1 binding (Supplementary Table 6) and _Tcf7_EGFPnull/− RNA-Seq data (Supplementary Tables 3 and 4). RIGHTS AND PERMISSIONS Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Harly, C., Kenney, D., Ren, G. _et al._ The transcription factor TCF-1 enforces commitment to the innate lymphoid cell lineage. _Nat

Immunol_ 20, 1150–1160 (2019). https://doi.org/10.1038/s41590-019-0445-7 Download citation * Received: 08 August 2018 * Accepted: 12 June 2019 * Published: 29 July 2019 * Issue Date:

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