Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor


Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage–restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: TOX is expressed in ILC progenitors and mature ILC lineages.
Figure 2: TOX is required for the development of ILC progenitors.
Figure 3: TOX is required for the development of mature ILCs.
Figure 4: TOX regulates the development of ILCs by a cell-intrinsic mechanism.
Figure 5: Whole-transcriptome analysis of α4β7+ progenitor cells reveals a block in the induction of the ILC gene program in Tox−/− mice.
Figure 6: In vitro ILC specification is blocked early in the absence of TOX.
Figure 7: In the absence of TOX, there is a cell-intrinsic differentiation defect in the generation of ILC1 cells and functional ILC2 cells.

Accession codes

Primary accessions

Gene Expression Omnibus


  1. 1

    Artis, D. & Spits, H. The biology of innate lymphoid cells. Nature 517, 293–301 (2015).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Klose, C.S. et al. Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157, 340–356 (2014).

    CAS  Article  Google Scholar 

  4. 4

    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  PubMed  PubMed Central  Google Scholar 

  5. 5

    Yagi, R. et al. The transcription factor GATA3 is critical for the development of all IL-7Rα-expressing innate lymphoid cells. Immunity 40, 378–388 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

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

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

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

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Nussbaum, J.C. et al. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature 502, 245–248 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Molofsky, A.B. et al. Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J. Exp. Med. 210, 535–549 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Halim, T.Y. et al. Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation. Immunity 40, 425–435 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Sun, Z. et al. Requirement for RORγ in thymocyte survival and lymphoid organ development. Science 288, 2369–2373 (2000).

    CAS  Google Scholar 

  12. 12

    Kim, M.Y. et al. Heterogeneity of lymphoid tissue inducer cell populations present in embryonic and adult mouse lymphoid tissues. Immunology 124, 166–174 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

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

    CAS  Google Scholar 

  14. 14

    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  PubMed  Google Scholar 

  15. 15

    Mortha, A. et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. Science 343, 1249288 (2014).

    PubMed  PubMed Central  Google Scholar 

  16. 16

    Sonnenberg, G.F., Monticelli, L.A., Elloso, M.M., Fouser, L.A. & Artis, D. CD4+ lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 34, 122–134 (2011).

    CAS  Google Scholar 

  17. 17

    Magri, G. et al. Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells. Nat. Immunol. 15, 354–364 (2014).

    CAS  Article  Google Scholar 

  18. 18

    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  PubMed  PubMed Central  Google Scholar 

  19. 19

    Yang, Q., Saenz, S.A., Zlotoff, D.A., Artis, D. & Bhandoola, A. Cutting edge: natural helper cells derive from lymphoid progenitors. J. Immunol. 187, 5505–5509 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Rankin, L.C. et al. The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway. Nat. Immunol. 14, 389–395 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Yokota, Y. et al. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397, 702–706 (1999).

    CAS  Google Scholar 

  22. 22

    Satoh-Takayama, N. et al. IL-7 and IL-15 independently program the differentiation of intestinal CD3NKp46+ cell subsets from Id2-dependent precursors. J. Exp. Med. 207, 273–280 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

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

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Constantinides, M.G., McDonald, B.D., Verhoef, P.A. & Bendelac, A. A committed precursor to innate lymphoid cells. Nature 508, 397–401 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Seillet, C. et al. Nfil3 is required for the development of all innate lymphoid cell subsets. J. Exp. Med. 211, 1733–1740 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Geiger, T.L. et al. Nfil3 is crucial for development of innate lymphoid cells and host protection against intestinal pathogens. J. Exp. Med. 211, 1723–1731 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Yu, X. et al. The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor. eLife 3, e04406 (2014).

  28. 28

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

    CAS  Google Scholar 

  29. 29

    Wilkinson, B. et al. TOX: an HMG box protein implicated in the regulation of thymocyte selection. Nat. Immunol. 3, 272–280 (2002).

    CAS  Google Scholar 

  30. 30

    O'Flaherty, E. & Kaye, J. TOX defines a conserved subfamily of HMG-box proteins. BMC Genomics 4, 13 (2003).

    PubMed  PubMed Central  Google Scholar 

  31. 31

    Aliahmad, P. & Kaye, J. Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245–256 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    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. Nat. Immunol. 11, 945–952 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Aliahmad, P., Kadavallore, A., de la Torre, B., Kappes, D. & Kaye, J. TOX is required for development of the CD4 T cell lineage gene program. J. Immunol. 187, 5931–5940 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Rawlins, E.L., Clark, C.P., Xue, Y. & Hogan, B.L. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development 136, 3741–3745 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Zhang, D.H., Cohn, L., Ray, P., Bottomly, K. & Ray, A. Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272, 21597–21603 (1997).

    CAS  PubMed  Google Scholar 

  36. 36

    Klose, C.S. et al. A T-bet gradient controls the fate and function of CCR6RORγt+ innate lymphoid cells. Nature 494, 261–265 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Gat. Aliahmad, P., Seksenyan, A. & Kaye, J. The many roles of TOX in the immune system. Curr. Opin. Immunol. 24, 173–177 (2012).

    Google Scholar 

  38. 38

    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  PubMed  PubMed Central  Google Scholar 

  39. 39

    Spencer, S.P. et al. Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier immunity. Science 343, 432–437 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    van de Pavert, S.A. et al. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 508, 123–127 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Iwata, M. et al. Retinoic acid imprints gut-homing specificity on T cells. Immunity 21, 527–538 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Gascoyne, D.M. et al. The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development. Nat. Immunol. 10, 1118–1124 (2009).

    CAS  Google Scholar 

  43. 43

    Xu, W. et al. NFIL3 orchestrates the emergence of common helper innate lymphoid cell precursors. Cell Rep. 10, 2043–2054 (2015).

    CAS  Google Scholar 

  44. 44

    Tachibana, M. et al. Runx1/Cbfbeta2 complexes are required for lymphoid tissue inducer cell differentiation at two developmental stages. J. Immunol. 186, 1450–1457 (2011).

    CAS  PubMed  Google Scholar 

  45. 45

    Artegiani, B. et al. Tox: a multifunctional transcription factor and novel regulator of mammalian corticogenesis. EMBO J. 34, 896–910 (2015).

    CAS  PubMed  Google Scholar 

  46. 46

    Ho, I.C., Hodge, M.R., Rooney, J.W. & Glimcher, L.H. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85, 973–983 (1996).

    CAS  PubMed  Google Scholar 

  47. 47

    Robinette, M.L. et al. Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat. Immunol. 16, 306–317 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Yang, X.O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 29–39 (2008).

    CAS  Google Scholar 

Download references


We thank members of the Cedars-Sinai Medical Center Flow Cytometry Core, especially G. Hultin and L. Dieu, for assistance with cell isolation; G. Martins for assistance with the generation of TH2 cells; A. Seksenyan and A. Kadavallore for discussions; the Cedars-Sinai Medical Center Biomedical Sciences and Translational Medicine Graduate Program; the NIH Tetramer Core Facility (contract HHSN272201300006C) for CD1d tetramers; and J.C. Zuniga-Pflucker (University of Toronto) for OP9-DL1 cells. Supported by the US National Institutes of Health (DK098310 to I.D.I., and 5R01AI054977 to J.K.).

Author information




C.R.S. and J.K. were responsible for overall design and execution of experiments and data analysis; C.R.S. performed the bulk of the experiments; P.A. designed and performed the characterization of the TOX reporter mice; B.d.l.T. provided technical assistance for animal experiments; I.D.I. aided in the isolation and analysis of LP cells; L.S. and V.A.F. performed RNA-seq and data analysis; and C.R.S. and J.K. wrote the manuscript, with input from all other authors.

Corresponding author

Correspondence to Jonathan Kaye.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of lung-resident ILC2 cells and NKT cells.

(a) Lung ILC2s were identified from WT, Rag1−/− and nu/nu mice. (b) TOM expression in gated ILC2s or NKT, with staining of control cells derived from WT mice shown for comparison in filled histogram. (c) Quantitation of low, intermediate, and high TOM populations in ILC2s (d) Lung ILC2s (LinCD45+Thy-1+ST2+), NKT (Lin+CD45+Thy-1+ST2+), and other (Lin+CD45+Thy-1+ST2) cells were identified from WT animals. Identity of cell populations was confirmed by specific binding of PBS-57/CD1d tetramer to NKT but not ILC2s, or other Lin+ cells (histograms). Staining with unloaded-CD1d tetramer is also shown as a control. ***P <0.001, NS=not significant, P >0.05 (Student’s t-test). Data are representative of one experiment (a), 15 experiments (b), or three experiments (d), or are compiled from 15 experiments (c).

Supplementary Figure 2 α4β7+ progenitor cells and BM ILC2p cells are decreased in the absence of TOX.

(a) BM from WT and Tox−/− mice was analyzed for presence of Linα4β7+CD127+Flt3 cells. (b,c) Compiled data of frequency (b) and number (c) of α4β7+ progenitors. (d) Staining for BM ILC2p from WT or Tox−/− treated mice as indicated. (e,f) Frequency (e) and absolute numbers (f) of BM ILC2p cells from WT and Tox−/− mice treated as indicated. *P <0.05, **P <0.01, ***P <0.001 (Student’s t-test). Data are representative of four experiments (a) or five experiments (d) or compiled from four (b,c) or five (e,f) experiments using one (b,c) or two pooled (e,f) animal(s) per experiment.

Supplementary Figure 3 The NK1.1+NKp46+ population consists of ILC1 cells and a minor population of ILC3 cells in the gut LP.

(a,b) Flow cytometry of colon (a) and small intestine (b). Data are representative of three mice analyzed independently.

Supplementary Figure 4 ILC3 cells are present in the small intestine LP of TOX-deficient mice.

(a) LinCD45+ cells from WT and Tox−/− spleen and small intestine LP. ILC3s were defined as Lin (CD3CD8αCD19Gr-1) CD45+RORγt+. Data in columns are derived from the same anima. (b) Absolute numbers of splenic ILC3 subtypes from indicated genotype. (c) Flow cytometry of small intestine LP ILC2s and ILC3s. (d) ILC3s from small intestine LP isolated from WT and Tox−/− mice analyzed for expression of NKp46 and T-bet. Histograms are gated on the NKp46T-bet ILC3 populations as shown. (e) Frequency (Top) and absolute numbers (Bottom) of indicated cell populations as gated in (a). (f) Cells derived from the thymus (left) and small intestine LP (right) from a Tox−/− animal were analyzed as shown. *P <0.05, **P <0.01, NS=not significant, P >0.05 (Student’s t-test). Data shown are from two animals (a), or are representative of three experiments (c,d), or two experiments (f), or compiled from three (b,e) experiments using one animal per experiment.

Supplementary Figure 5 Unsupervised hierarchical clustering of RNA-seq data.

Data are derived from four independent progenitor cell isolations for each genotype, using 2-3 pooled animals per experiment.

Supplementary Figure 6 Tox−/− CLPs have normal CD127 expression and B cell lineage potential.

(a) CD127 expression on WT and Tox−/−CLP. As control, staining of LinCD127+Sca-1Flt3 cells is shown. (b) CLP were isolated from WT and Tox−/− animals and cultured with OP9 cells and appropriate cytokines. Production of B cells (B220+CD19+) was analyzed after six and 12 days in culture. Data are representative of two experiments (a,b).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 3 (PDF 4499 kb)

Supplementary Table 2

Table of well-annotated genes differentially expressed between WT and Tox−/− progenitors. (XLSX 102 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Seehus, C., Aliahmad, P., de la Torre, B. et al. The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat Immunol 16, 599–608 (2015).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing