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TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow

Nature Immunology volume 16, pages 1044–1050 (2015)Cite this article

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Abstract

The cellular and molecular events that drive the early development of innate lymphoid cells (ILCs) remain poorly understood. We show that the transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILPs) expressing high amounts of TCF-1. EILPs lacked efficient T and B lymphocyte potential but efficiently gave rise to NK cells and all known adult helper ILC lineages, indicating that they are the earliest ILC-committed progenitors identified so far. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILPs provides a basis for deciphering additional signals that specify ILC fate.

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Figure 1: TCF-1 is required for the efficient generation of all known adult ILCs and their progenitors.
Figure 2: Identification of EILPs, a novel TCF-1–expressing BM cell population.
Figure 3: Comparison of EILPs with BM lymphoid progenitors.
Figure 4: EILPs efficiently give rise to all four ILC lineages in vivo.
Figure 5: EILPs efficiently give rise to all four ILC lineages at the clonal level.
Figure 6: EILPs persist in the absence of Id2.

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Acknowledgements

We thank M. Greene and G. Massey for essential support. We thank the laboratories of A. Haczku, Y. Belkaid, R. Bosselut and D. Artis for technical and scientific advice. OP9-DL1 stromal cell lines were obtained from J.C. Zuniga-Pflucker (University of Toronto, Toronto, Ontario, Canada). Reagents for recombineering were kindly provided by D.L. Court (National Cancer Institute, Frederick, Maryland, USA) and N.G. Copeland (Houston Methodist Research Institute, Houston, Texas, USA). We thank T.L. Saunders at the University of Michigan for blastocyst injection of Tcf7-GFP+ embryonic stem cells. We also thank Y. Yokota (University of Fukui, Fukui, Japan) for Id2−/− mice. This work was supported by the American Cancer Society (RSG-11-161-01-MPC to H.-H.X.) and the National Institutes of Health (AI105351, AI112579, AI115149 and AI119160 to H.-H.X.; AI059621, AI098428 and HL11074103 to A.B.). A.B. is supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, and the Center for Cancer Research.

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Authors and Affiliations

  1. T-Cell Biology and Development Unit, Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

    Qi Yang, Christelle Harly, Longyun Ye & Avinash Bhandoola

  2. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Qi Yang, Xuefeng Xia, Haikun Wang & Xinxin Wang

  3. Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Fengyin Li, Shaojun Xing, Shuyang Yu, Xinyuan Zhou & Hai-Hui Xue

  4. CCR Collaborative Bioinformatics Resource, National Cancer Institute, Bethesda, Maryland, USA

    Maggie Cam

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Contributions

Q.Y. performed most of the experiments with the help of C.H., L.Y., X.X., H.W. and X.W.; Tcf7GFP mice were generated by S.Y. and X.Z. and characterized by F.L. and S.X. M.C. analyzed the microarray data; Q.Y., H.-H.X. and A.B. designed the study, analyzed the data and wrote the manuscript.

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Correspondence to Hai-Hui Xue or Avinash Bhandoola.

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Integrated supplementary information

Supplementary Figure 1 TCF-1 is required for the efficient generation of all known adult ILC subsets.

(a) The frequencies of previously described pre-NKPs, refined-purity NKPs (rNKPs) and pre-pro-NKPs were examined and compared in Tcf7+/+ and Tcf7−/− mice. (b) Gating strategies to identify different mature ILC subsets in BM LSK chimeric mice. (c) The number of cells of each mature ILC subset was measured and compared in Tcf7+/+ versus Tcf7−/− mice. (d) 5,000 Tcf7−/− LSK cells or control Tcf7+/+ LSK cells (Cd45.2) were mixed with 5,000 wild-type competitor LSK cells (CD45.1) and injected intravenously into irradiated (950 rad) recipient mice (CD45.1). Mice were analyzed at 12–16 weeks post-transplant. Representative profiles of donor chimerism are shown. (e) Donor chimerism of competitive LSK chimeras was quantified. Data are from 3–4 mice per group and are representative of two (a) or three independent experiments (b,d) or are pooled from three independent experiments (c,e). Data are presented as mean and s.e.m. *P < 0.05, **P < 0.01.

Supplementary Figure 2 Generation of the Tcf7-EGFP reporter allele.

(a) Targeting strategy. Depicted on top are a partial structure of the Tcf7 gene, transcription start sites for TCF-1 isoforms, and the conservation track from the UCSC genome browser. The resulting targeted Tcf7 allele is shown in the bottom panel, with the locations of the EGFP expression cassette, NEO cassette, two Frt sites and two LoxP sites shown. (b) Details of the targeting approach. Shown on the top is a partial structure of the Tcf7 gene, with filled bars denoting exons (all numbered), key enzyme sites, and relative locations of 5‘ and 3‘ probes used in Southern blotting. Shown at the bottom is the structure of the resulting targeted Tcf7 allele, highlighting the targeting arms in the targeting construct. Note that an extra BamHI site was inserted immediately after the downstream LoxP site, and an extra ApaLI was embedded in the EGFP expression cassette. These two extra enzyme sites were used to facilitate detection of the targeted allele by Southern blotting. (c) Identification of ES clones with expected homologous recombination. Genomic DNA was extracted from ES cell clones generated after targeting construct electroporation and selection, digested with ApaL1, and blotted with the 5‘ probe, which detects the wild-type allele at approximately 9.4 kb and the targeted allele at approximately 6.6 kb (left panel). Another portion of genomic DNA was digested with BamHI and blotted with the 3‘ probe, which detects the WT allele at about 9.8 kb and the targeted allele at about 7 kb (right panel). The primers used in Southern blotting were as follows: 5‘ probe, 5‘-gcaccatgatcctgtttgat and 5‘-gcgcgtttttaaacatgtgag; 3‘ probe, 5‘-ccctagcagctcatcagacc and 5‘-ctctggcccacagtagaagc. (d) Confirmation of GFP expression in T cells of Tcf7GFP mice. Representative flow cytometric plots depict the GFP expression of thymus DP cells and spleen T and B cells in Tcf7GFP (green) or control mice (gray). (e) Representative flow cytometric plots depicting GFP expression in the indicated susbets of Tcf7GFP (green) or control mice (open histogram). Data are representative of three independent experiments (c–e).

Supplementary Figure 3 EILPs developed into ILCs in vitro.

(a,b) 100 EILPs or CLPs were plated on OP9 (a) or OP9-DL1 (b) stroma in the presence of FL (5 ng/ml) and IL-7 (1 ng/ml or 10 ng/ml). The emergence of progeny was examined after 10 days of culture. (c) 100 EILPs were cultured with OP9 stroma in the presence of SCF (30 ng/ml), IL-7 (30 ng/ml), and IL-2 (30 ng/ml). Cultures were examined after 10 days. (d) EILPs and the other indicated BM and thymus early progenitors were sorted by flow cytometry cell sorting, and genome-wide microarray analysis was performed. The heat map shows the expression levels of canonical genes for validation of the microarray analysis. Data are representative of three independent experiments (a–c).

Supplementary Figure 4 Functional EILPs are not detected in the thymus.

(a) 10 EILPs were cultured on OP9 stroma with the indicated cytokines for 10 days. Expansion rates were examined as the number of CD45+ progeny that emerged per progenitor input. (b) Single EILPs were plated on OP9 stroma at one EILP per well in 96-well plates in the presence of SCF, IL-2 and IL-7 for 10 days. The bar graph indicates the percentage of positive wells that contained more than 10 CD45+ daughter cells. (c) 100 EILPs, LMPPs, CLPs and ETPs were cultured on OP9 stroma for 7 days in the presence of SCF, IL-2, and IL-7. The number of progeny per cell input was examined by flow cytometry. (d) Representative flow profiles of thymocytes of Tcf7GFP mice. Plots were gated on thymus Lin− cells. A few cells with the EILP phenotype (Lin−TCF-1+Thy-1−IL-7Ralo-neg) were identified, among which some expressed α4β7. (e) Thymus α4β7+ and α4β7− EILP-like cells (Lin−TCF-1+Thy-1.2−IL-7Ralo-neg) were sorted and cultured on OP9 stroma for 7 days in the presence of SCF, IL-2, and IL-7. The number of progeny was examined by flow cytometry. BM EILPs were used as a positive control. The bar graph shows the number of progeny per progenitor cell input. Data are from three wells per group (a), five independent experiments (b) or three independent experiments (c,e) or are representative of three independent experiments (d). Data are presented as mean and s.e.m. *P < 0.05, **P < 0.01.

Supplementary Figure 5 Id2−/− EILP failed to give rise to B and T lymphocytes in vitro

(a) 200 CLP or EILP from Id2+/+ or Id2−/− mice were sorted and cultured on OP9 in the presence of FL (5ng/ml) and IL-7 (10ng/ml). The emergence of progeny was examined after 7 days of culture. (b) 200 CLP or EILP from Id2+/+ or Id2-/- mice were sorted and cultured on OP9-DL1 in the presence of FL (5ng/ml) and IL-7 (10ng/ml). The emergence of progeny was examined after 7 days of culture. Data are representative of three independent experiments.

Supplementary Figure 6 Model of early ILC development

Diagram depicts a model for early ILC development.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 902 kb)

Supplementary Table 1

List of antibodies used for flow cytometry analysis (XLS 30 kb)

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Yang, Q., Li, F., Harly, C. et al. TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow. Nat Immunol 16, 1044–1050 (2015). https://doi.org/10.1038/ni.3248

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