Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine


Fetal lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch (PP) organogenesis, but where these specialized group 3 innate lymphoid cells (ILC3s) develop remains unclear. Here, we identify extrahepatic arginase-1+ Id2+ fetal ILC precursors that express a transitional developmental phenotype (ftILCPs) and differentiate into ILC1s, ILC2s and ILC3s in vitro. These cells populate the intestine by embryonic day (E) 13.5 and, before PP organogenesis (E14.5–15), are broadly dispersed in the proximal gut, correlating with regions where PPs first develop. At E16.5, after PP development begins, ftILCPs accumulate at PP anlagen in a lymphotoxin-α–dependent manner. Thus, ftILCPs reside in the intestine during PP development, where they aggregate at PP anlagen after stromal cell activation and become a localized source of ILC populations.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Adult LTi-like cells express Arg1.
Figure 2: Innate lymphoid cells express Arg1 in the fetal gut.
Figure 3: Fetal Arg1-YFP+RNT cells aggregate at the developing PP anlage.
Figure 4: Characterization of Arg1-YFP+RNT cells.
Figure 5: Arg1-YFP+RNT cells differentiate into mature ILCs.
Figure 6: Arg1-YFP expression by LinId2+IL-7Rα+α4β7+Flt3 cells in fetal liver and adult bone marrow.


  1. 1

    Eberl, G. et al. An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells. Nat. Immunol. 5, 64–73 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Yoshida, H. et al. IL-7 receptor α+ CD3– cells in the embryonic intestine induces the organizing center of Peyer's patches. Int. Immunol. 11, 643–655 (1999).

    CAS  Article  Google Scholar 

  3. 3

    De Togni, P. et al. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264, 703–707 (1994).

    CAS  Article  Google Scholar 

  4. 4

    Fütterer, A., Mink, K., Luz, A., Kosco-Vilbois, M.H. & Pfeffer, K. The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9, 59–70 (1998).

    Article  Google Scholar 

  5. 5

    Honda, K. et al. Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer's patch organogenesis. J. Exp. Med. 193, 621–630 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Koni, P.A. et al. Distinct roles in lymphoid organogenesis for lymphotoxins α and β revealed in lymphotoxin β–deficient mice. Immunity 6, 491–500 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Okuda, M., Togawa, A., Wada, H. & Nishikawa, S. Distinct activities of stromal cells involved in the organogenesis of lymph nodes and Peyer's patches. J. Immunol. 179, 804–811 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Spits, H. & Cupedo, T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. Annu. Rev. Immunol. 30, 647–675 (2012).

    CAS  Article  Google Scholar 

  9. 9

    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  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Satoh-Takayama, N. et al. IL-7 and IL-15 independently program the differentiation of intestinal CD3-NKp46+ cell subsets from Id2-dependent precursors. J. Exp. Med. 207, 273–280 (2010).

    CAS  Article  Google Scholar 

  12. 12

    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 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    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 

  15. 15

    Sandler, N.G., Mentink-Kane, M.M., Cheever, A.W. & Wynn, T.A. Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair. J. Immunol. 171, 3655–3667 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Herbert, D.R. et al. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20, 623–635 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Hesse, M. et al. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J. Immunol. 167, 6533–6544 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Munder, M., Eichmann, K. & Modolell, M. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype. J. Immunol. 160, 5347–5354 (1998).

    CAS  PubMed  Google Scholar 

  19. 19

    Bando, J.K., Nussbaum, J.C., Liang, H.E. & Locksley, R.M. Type 2 innate lymphoid cells constitutively express arginase-I in the naive and inflamed lung. J. Leukoc. Biol. 94, 877–884 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Reese, T.A. et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 447, 92–96 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Kanamori, Y. et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J. Exp. Med. 184, 1449–1459 (1996).

    CAS  Article  Google Scholar 

  22. 22

    Eberl, G. & Littman, D.R. Thymic origin of intestinal αβ T cells revealed by fate mapping of RORγt+ cells. Science 305, 248–251 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Eberl, G. Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway? Nat. Rev. Immunol. 5, 413–420 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Adachi, S., Yoshida, H., Kataoka, H. & Nishikawa, S. Three distinctive steps in Peyer's patch formation of murine embryo. Int. Immunol. 9, 507–514 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Klose, C.S. et al. A T-bet gradient controls the fate and function of CCR6-RORγt+ innate lymphoid cells. Nature 494, 261–265 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Vonarbourg, C. et al. Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt+ innate lymphocytes. Immunity 33, 736–751 (2010).

    CAS  Article  Google Scholar 

  27. 27

    El Kasmi, K.C. et al. Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens. Nat. Immunol. 9, 1399–1406 (2008).

    CAS  Article  Google Scholar 

  28. 28

    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  Article  Google Scholar 

  29. 29

    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  Article  Google Scholar 

  30. 30

    de Boer, J. et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314–325 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H.J. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur. J. Immunol. 37, 43–53 (2007).

    CAS  Article  Google Scholar 

Download references


The authors thank V. Nguyen (UCSF Flow Cytometry Core) and Z. Wang (Sabre Sorting Facility) for cell sorting, and D. Sheppard, D. Kioussis (MRC National Institute for Medical Research), D. Littman (New York University), E. Robey (University of California, Berkeley), H. Luche and H. Fehling (University Clinics Ulm, Germany) for providing mice for these studies. We also thank J. Cyster and L. Lanier for critical reading of the manuscript. Supported by the Howard Hughes Medical Institute (R.M.L.), National Institutes of Health (AI026918, AI030663 and AI119944 to R.M.L.), the Sandler Asthma Basic Research Center at UCSF (R.M.L.) and the UCSF Biomedical Sciences (BMS) Graduate Program (J.K.B.).

Author information




J.K.B. and R.M.L. designed experiments and wrote the manuscript. J.K.B. conducted experiments and H.-E.L. provided reagents.

Corresponding author

Correspondence to Richard M Locksley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of Arg1-YFP+NK1.1+ cells.

(a) YFP expression in NK1.1+CD3- cells from the spleen and liver of Arg1YFP mice at different ages post-birth. (b) RORγ(t) protein expression in YFP+NK1.1+ cells sorted from spleens of 20-day-old Arg1YFP mice. The gray shaded area indicates CD4+ T cells. (c) YFP expression in NK1.1+ cells from Arg1YFPRag2−/− spleens of 20-day-old mice. (d) Eomesodermin expression in YFP+NK1.1+ cells sorted from spleens of 20-day-old Arg1YFP mice (solid line). The gray shaded area represents an isotype control and the dotted line represents sorted YFPNK1.1+CD5 NK cells. (e) Expression of IL-7Rα, NKp46, and NKG2D in adult spleen and liver. Shaded areas indicate isotype controls. Data are representative of 2 independent experiments (a-e).

Supplementary Figure 2 Dissection strategy for determining YFP+ cell localization before PP development.

Division of the fetal small intestine into three regions of equal length for flow cytometry experiments in Fig. 3a.

Supplementary Figure 3 Arg1 is not required for PP development.

PP numbers per intestine (left) and follicles per PP (right) in VavcreArg1fl/fl mice and VavcreArg1fl/+ controls (n = 7 mice per group or n = 54-56 PP). P>0.05 (unpaired Student’s t-test). Data are pooled from two independent experiments.

Supplementary Figure 4 Specificity of RORγ(t) antibodies to RORγt in fetal Arg1+ ILCs.

Rorc(γt)GFP/+ or Rorc(γt)GFP/GFP (knockout) cells from E16.5 intestines were stained with RORγ(t) antibodies. Cells were pre-gated on CD45+CD11bIL-7Rα+ cells, and then further gated on CD4+NK1.1 cells for LTis, ST2+NK1.1 cells for ILC2s, or CD4NK1.1ST2 cells to enrich for RNT cells. Data are representative of two independent experiments.

Supplementary Figure 5 A subset of Arg1+RNT cells expresses CD25.

(a) CD25 staining in Arg1YFP+RORγt(fm)NK1.1 cells isolated from E15.5 fetal intestines. CD25 expressing RNT cells are shown in the top left quadrant. (b) T-bet and RORγ(t) expression in Arg1YFP+RNTCD25 (left quadrants) and Arg1YFP+RNTCD25+ cells (right quadrants) isolated from E16.5 fetal intestines. (c) Histograms for T-bet and RORγ(t) expression in Arg1YFP+RNTCD25 cells. Data are representative of two independent experiments.

Supplementary Figure 6 Transcription factor expression in GATA-3+ fetal liver ILC precursors.

Transcription factor staining in E14.5 LinIL-7Rα+α4β7+Flt3CD25ST2 fetal liver cells. Data are representative of three independent experiments.

Supplementary Figure 7 Functional assessment of ILCs derived from Arg1-YFP+RNT cells.

(a) Cytokine expression by 3 h PMA-and Ionomycin-stimulated ILCs at day 10 of culture. (b) LTα1β2 expression by ILCs at day 10 of culture. Data are representative of two independent experiments (a-b).

Supplementary Figure 8 Variation between experiments pooled for Figure 5h.

Each pie represents an independent experiment in which 96 single Arg1YFP+RNT cells were sorted and cultured for 6 days.

Supplementary Figure 9 Arg1+RNT cells in adult intestinal tissue.

CD45+, lymphocyte-sized Arg1+ cells in adult lamina propria and Peyer’s patches include NK1.1RORγt(fm)KLRG1CD25 cells. Data are representative of two independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 1789 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bando, J., Liang, HE. & Locksley, R. Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine. Nat Immunol 16, 153–160 (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