A committed precursor to innate lymphoid cells



Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers1,2,3,4,5,6,7,8,9. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties10, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development11,12, by using lineage tracing and transfer studies. PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+DX5 ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages13. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

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Figure 1: ILC lineage tracing in PLZFGFPcre reporter mice.
Figure 2: PLZFhigh cells in fetal liver and bone marrow.
Figure 3: PLZFhigh cells are ILC progenitors.
Figure 4: PLZF is required for ILC development.


  1. 1

    Fuchs, A. et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells. Immunity 38, 769–781 (2013)

    CAS  Article  Google Scholar 

  2. 2

    Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464, 1367–1370 (2010)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463, 540–544 (2010)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Price, A. E. et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc. Natl Acad. Sci. USA 107, 11489–11494 (2010)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Monticelli, L. A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nature Immunol. 12, 1045–1054 (2011)

    CAS  Article  Google Scholar 

  6. 6

    Satoh-Takayama, N. et al. Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 29, 958–970 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Sanos, S. L. et al. RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nature Immunol. 10, 83–91 (2009)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Luci, C. et al. Influence of the transcription factor RORγt on the development of NKp46+ cell populations in gut and skin. Nature Immunol. 10, 75–82 (2009)

    CAS  Article  Google Scholar 

  9. 9

    Cella, M. et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature 457, 722–725 (2009)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Spits, H. et al. Innate lymphoid cells–a proposal for uniform nomenclature. Nature Rev. Immunol. 13, 145–149 (2013)

    CAS  Article  Google Scholar 

  11. 11

    Savage, A. K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008)

    CAS  Article  Google Scholar 

  12. 12

    Kovalovsky, D. et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nature Immunol. 9, 1055–1064 (2008)

    CAS  Article  Google Scholar 

  13. 13

    Aliahmad, P., de la Torre, B. & Kaye, J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nature Immunol. 11, 945–952 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Savage, A. K., Constantinides, M. G. & Bendelac, A. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J. Immunol. 186, 5801–5806 (2011)

    CAS  Article  Google Scholar 

  15. 15

    Constantinides, M. G., Picard, D., Savage, A. K. & Bendelac, A. A naive-like population of human CD1d-restricted T cells expressing intermediate levels of promyelocytic leukemia zinc finger. J. Immunol. 187, 309–315 (2011)

    CAS  Article  Google Scholar 

  16. 16

    Peng, H. et al. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123, 1444–1456 (2013)

    CAS  Article  Google Scholar 

  17. 17

    Yoshida, H. et al. Expression of α4β7 integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells. J. Immunol. 167, 2511–2521 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Sawa, S. et al. Lineage relationship analysis of RORγt+ innate lymphoid cells. Science 330, 665–669 (2010)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Possot, C. et al. Notch signaling is necessary for adult, but not fetal, development of RORγt+ innate lymphoid cells. Nature Immunol. 12, 949–958 (2011)

    CAS  Article  Google Scholar 

  20. 20

    Cherrier, M., Sawa, S. & Eberl, G. Notch, Id2, and RORγt sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J. Exp. Med. 209, 729–740 (2012)

    CAS  Article  Google Scholar 

  21. 21

    Gleimer, M., von Boehmer, H. & Kreslavsky, T. PLZF controls the expression of a limited number of genes essential for NKT cell function. Front. Immunol. 3, 374 (2012)

    CAS  Article  Google Scholar 

  22. 22

    Hoyler, T. et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity 37, 634–648 (2012)

    CAS  Article  Google Scholar 

  23. 23

    Mjösberg, J. et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 37, 649–659 (2012)

    Article  Google Scholar 

  24. 24

    Wong, S. H. et al. Transcription factor RORα is critical for nuocyte development. Nature Immunol. 13, 229–236 (2012)

    CAS  Article  Google Scholar 

  25. 25

    Halim, T. Y. et al. Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Immunity 37, 463–474 (2012)

    CAS  Article  Google Scholar 

  26. 26

    Yang, Q. et al. T cell factor 1 is required for group 2 innate lymphoid cell generation. Immunity 38, 694–704 (2013)

    CAS  Article  Google Scholar 

  27. 27

    Mielke, L. A. et al. TCF-1 controls ILC2 and NKp46+RORγt+ innate lymphocyte differentiation and protection in intestinal inflammation. J. Immunol. 191, 4383–4391 (2013)

    CAS  Article  Google Scholar 

  28. 28

    Constantinides, M. G. & Bendelac, A. Transcriptional regulation of the NKT cell lineage. Curr. Opin. Immunol. 25, 161–167 (2013)

    CAS  Article  Google Scholar 

  29. 29

    Lee, E. C. et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65 (2001)

    CAS  Article  Google Scholar 

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We thank W. Yokoyama for discussion; F. Gounari and R. de Pooter for advice on OP9 cultures; H. Gudjonson for statistical advice; D. Leclerc, J. Cao, M. Olsen and R. Duggan for help with cell sorting; V. Bindokas and R. Mathew for help with fluorescence microscopy. This work was supported by NIH grants R01HL118092, R01AI038339 and P30DK42086 and by The Howard Hughes Medical Institute (A.B.).

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M.G.C., B.D.M. and P.A.V. designed research, performed experiments and analysed data. M.G.C. and A.B. wrote the paper. A.B. supervised the research.

Corresponding author

Correspondence to Albert Bendelac.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 PLZF expression and lineage tracing in PLZFGFPcre mice.

a, A sequence encoding an IRES and a GFP-cre fusion protein was inserted immediately after the Zbtb16 stop codon in C57BL/6J ES cells and knock-in mice were bred to ACTB-FLPe mice to excise the neomycin resistance cassette and generate the PLZFGFPcre allele. b, FACS analysis of the indicated populations from PLZFGFPcre+/− ROSA26-YFP mice. c, Summary of data (mean ± s.e.m.) from 2–5 mice analysed in 2 or more independent experiments.

Extended Data Figure 2 Gating strategy for analysis of ILC and LTi among LPL.

ILC2 cells were identified as IL-7Rα+KLRG1+ among CD3εCD19 LPL (top left), and then gated Thy1.2+ (not shown). CD3εCD19 LPL were gated as IL-7Rα+KLRG1 (top left) and then subsetted into CCR6+CD4+ (CD4+ LTi cells) and CCR6+CD4 (CD4 LTi cells) (bottom left). NCR+ ILC3 were identified as CD3εCD19 LPL that expressed NKp46 but not NK1.1 (top right).

Extended Data Figure 3 Transcription factor expression by PLZFhigh bone marrow precursors.

Quantitative PCR with reverse transcription analysis for Tbx21 and Rora as indicated. NKP are LinCD27+IL-7RαFlt3CD122+ BM cells. Mean ± s.e.m. of data from 2–3 independent experiments.

Extended Data Figure 4 PLZFhigh-derived NK1.1+ cells are distinct from CLP-derived NK1.1+ cells.

CD45.2 Rag2−/−Il2rg−/− mice were injected with equivalent numbers of CD45.2 PLZFhigh cells and CD45.1 CLP (800 of each) and the resulting NK1.1+CD3εTCRβ cells present in the spleen were analysed 5–7 weeks later by FACS, as indicated. Note that PLZFhigh-derived cells expressed higher amount of surface NKp46, whether they were identified as CD45.2+ or as CD45.1 in reciprocal staining experiments. Similar results were obtained for lung NK1.1+ cells. Data representative of 5 chimaeras from 2 independent experiments.

Extended Data Figure 5 FTOC of PLZFhigh cells.

FACS analysis of PLZFhigh and CLP cells (100 of each) co-cultured for 15 days in FTOC (a). The percentages of PLZFhigh- or CLP-derived cells that are CD3ε+ are summarized in the bar graph (b). Data representative of 7 independent cultures.

Extended Data Figure 6 Additional characterization of PLZFhigh cells after culture on OP9 cells.

a, FACS analysis of PLZFhigh or CLP cells from adult BM cultured on OP9 for 4 days showing expression of T1/ST2 on ICOShigh cells. Data representative of 4 replicate cultures from 2 independent experiments. b, FACS analysis of fetal liver PLZFhigh cells after culture on OP9 for 7 days, showing expression of GATA3 by ICOShigh cells and RORγt by ICOSint cells. Data representative of 2 independent experiments.

Extended Data Figure 7 Proposed model of ILC development.

A CLP-derived IL-7Rα+α4β7+ population bifurcates into RORγthigh LTi precursors (LTiP) and PLZFhigh ILCP, the latter of which gives rise to all ILC lineages. Whether NKP cells develop directly from CLPs or progress through an IL-7Rα+α4β7+ stage has yet to be determined.

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Constantinides, M., McDonald, B., Verhoef, P. et al. A committed precursor to innate lymphoid cells. Nature 508, 397–401 (2014). https://doi.org/10.1038/nature13047

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