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

Abstract

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.

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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.

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Acknowledgements

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.).

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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.

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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.

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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). https://doi.org/10.1038/ni.3057

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