Letter | Published:

Single-cell RNA-seq identifies a PD-1hi ILC progenitor and defines its development pathway

Nature volume 539, pages 102106 (03 November 2016) | Download Citation


Innate lymphoid cells (ILCs) functionally resemble T lymphocytes in cytotoxicity and cytokine production but lack antigen-specific receptors, and they are important regulators of immune responses and tissue homeostasis1,2. ILCs are generated from common lymphoid progenitors, which are subsequently committed to innate lymphoid lineages in the α-lymphoid progenitor, early innate lymphoid progenitor, common helper innate lymphoid progenitor and innate lymphoid cell progenitor compartments3,4,5,6,7,8. ILCs consist of conventional natural killer cells and helper-like cells (ILC1, ILC2 and ILC3)9. Despite recent advances1,2,10, the cellular heterogeneity, developmental trajectory and signalling dependence of ILC progenitors are not fully understood. Here, using single-cell RNA-sequencing (scRNA-seq) of mouse bone marrow progenitors, we reveal ILC precursor subsets, delineate distinct ILC development stages and pathways, and report that high expression of programmed death 1 (PD-1hi) marked a committed ILC progenitor that was essentially identical to an innate lymphoid cell progenitor. Our data defined PD-1hiIL-25Rhi as an early checkpoint in ILC2 development, which was abolished by deficiency in the zinc-finger protein Bcl11b but restored by IL-25R overexpression. Similar to T lymphocytes, PD-1 was upregulated on activated ILCs. Administration of a PD-1 antibody depleted PD-1hi ILCs and reduced cytokine levels in an influenza infection model in mice, and blocked papain-induced acute lung inflammation. These results provide a perspective for exploring PD-1 and its ligand (PD-L1) in immunotherapy, and allow effective manipulation of the immune system for disease prevention and therapy.

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We thank F. Colucci and J. Di Santo for providing Rag2−/−Il2rg−/− mice. We thank the Sanger Institute RSF (J. Bussell, D. Key, A. Kirton, L. Bulman, S. Kemp, P. Green, P. Zielezinski, R. Lacey, C. Rogerson, A. Logan and G. Notley), Flow Cytometry Core Facility (B. L. Ng, J. Graham and C. Hall), Single Cell Genomic Core Facility (S. Loren and I. Bronner) and DNA sequencing pipeline (N. Smerdon) for technical assistances. We thank K. Chen, J. Pramanik and R. Miragaia for technical help. C.W. is supported by the Plan of Youth Growth from Shanghai Municipal Agricultural Committee (Hunongqingzi (2015. No. A-35)). L.L. is funded by National Natural Science Foundation of China (31370904, 81671579). G.T.B. is supported by the Australian Research Council (Future Fellowship FT110100283) and the National Health and Medical Research Council (Fellowship 10402092). A.N.J.M. is supported by the Medical Research Council (U105178805) and Wellcome Trust (100963/Z/13/Z). This work is supported by Wellcome Trust (grant number 098051) (P.L).

Author information

Author notes

    • Yong Yu
    • , Jason C. H. Tsang
    •  & Cui Wang

    These authors contributed equally to this work


  1. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK

    • Yong Yu
    • , Jason C. H. Tsang
    • , Cui Wang
    • , Simon Clare
    • , Juexuan Wang
    • , Xi Chen
    • , Cordelia Brandt
    • , Leanne Kane
    • , Lia S. Campos
    • , Sarah A. Teichmann
    • , Gordon Dougan
    •  & Pentao Liu
  2. Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China

    • Jason C. H. Tsang
  3. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

    • Jason C. H. Tsang
  4. Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China

    • Cui Wang
  5. Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China

    • Liming Lu
  6. The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia

    • Gabrielle T. Belz
  7. Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3010, Australia

    • Gabrielle T. Belz
  8. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK

    • Andrew N. J. McKenzie
  9. European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

    • Sarah A. Teichmann
  10. Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0SP, UK

    • Gordon Dougan


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Y.Y. designed research, performed experiments and analysed data. J.C.H.T. performed all of the bioinformatics analyses, C.W. performed experiments. S.C. and C.B. performed influenza infection. J.W. generated the Bcl11btdTomato conditional knockout reporter mice. X.C. performed ChIP–PCR. L.K. did the histologic section staining. L.S.C. analysed the histologic data. L.L. contributed intellectually to the PD-1 experiments. G.T.B. and A.N.J.M. provided Id2GFP and Il13+/tdTomato reporter mice, respectively. S.A.T. and G.D. provided intellectual input for the experiments performed in their laboratories. Y.Y., J.C.H.T. and P. L. wrote the paper. Y. Y. and P. L. conceived the PD-1 as an ILC marker concept. P.L. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Pentao Liu.

Reviewer Information

Nature thanks I. Amit and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This file contains clustering information of bone marrow progenitors.

  2. 2.

    Supplementary Table 2

    This file contains clustering information of PD-1hi cells.

  3. 3.

    Supplementary Table 3

    This file contains differential gene analysis of WT C6 vs C8.

  4. 4.

    Supplementary Table 4

    This file contains ILC gene set in GESA

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