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Runx3 specifies lineage commitment of innate lymphoid cells

Abstract

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells.

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Figure 1: ILC1 cells and all ILC3 subsets express Runx3 mainly from the distal promoter.
Figure 2: Cbfbf/fNKp46-Cre mice lack NKp46+ ILC3 and ILC1 cells in the intestine.
Figure 3: Defects in ILC3 differentiation in Runx3f/fNKp46-Cre and Runx3f/fVav1-Cre mice.
Figure 4: Runx3 regulates the expression of RORγt and subsequent expression of AHR in ILC3 cells.
Figure 5: Runx3 is essential for the survival of ILC1 cells but not that of ILC3 cells.
Figure 6: Cell-intrinsic requirement for Runx3 in the development of ILC1 and ILC3 cells.
Figure 7: Runx3 in ILC cells is critical for the control of acute infection with C. rodentium.

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Acknowledgements

We thank E. Vivier (Centre d'Immunologie de Marseille-Luminy) for NKp46-Cre mice; D.R. Littman (New York University) for Cbfbf/f, Runx3f/f and Runx3d+/YFP mice; S. Taffner for technical assistance; H. Miyoshi and A. Fuchs for technical advice; and D.K. Sojka, and S. Demehri for discussions. Supported by the Howard Hughes Medical Institute (W.M.Y.) and the US National Institutes of Health (R01AI097244 to T.Eg.; and T32-GM007200 for M.D.B.).

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T.Eb. and W.M.Y. designed experiments and analyzed data; T.Eb., C.S., S.H.R., B.P.-D., L.Y., M.D.B. and T.S.S. performed the experiments; D.L. and Y.G. supplied reagents; T.Eg. analyzed data obtained by chromatin immunoprecipitation followed by deep sequencing; and T.Eb., Y.G., T.S.S., M.C., T.Eg. and W.M.Y. wrote the paper.

Corresponding author

Correspondence to Wayne M Yokoyama.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Absence of NKp46+ ILC3 cells in PPs and reduction in the number of NK cells in tissues of Cbfbf/fNKp46-Cre mice.

(a) Flow cytometry analyzing NK cells (CD3 CD19 NK1.1+ NKp46+) in liver, skin and salivary gland of Runx3d+/YFP mice for DX5, CD49a and distal Runx3-YFP. Shaded histograms indicate wild-type mice. Upper plots were gated on Live CD45+ CD3 CD19 cells. Arrows in the two upper panels indicate gated cells shown in the lower histogram. Numbers in quadrants and outlined area indicate percent cells in each. (b) Flow cytometry of PP lymphocytes showing ILC3 subsets in Cbfb+/fNKp46-Cre and Cbfbf/fNKp46-Cre mice. Upper plots were gated on Live CD45+ CD3 CD19 cells. (c) Absolute cell numbers of ILC3 subsets in PPs of Cbfb+/fNKp46-Cre and Cbfbf/fNKp46-Cre mice. (d, e) Frequency (d) and absolute cell numbers (e) of indicated NK cells (CD3 CD19 NK1.1+ NKp46+) and their DX5 and CD49a expression (d) in spleen, liver, salivary gland and skin of Cbfb+/fNKp46-Cre and Cbfbf/fNKp46-Cre mice. Upper plots were gated on Live CD45+ CD3 CD19 cells. Arrows in the upper panels indicate gated cells shown in the lower plots (d). Numbers in outlined areas or quadrants indicate percent cells in each (a, b, d). Data are representative of more than three experiments (a, b, d) or from two independent experiments (b and d; mean and s.d. of three mice). *p< 0.05 and **p< 0.01 by Student’s t-test. NS, not significant.

Supplementary Figure 2 Normal differentiation of ILC progenitor cells in Runx3f/fVav1-Cre mice.

Frequency (a, b, c) and absolute number (d) of common lymphoid progenitor cells (CLP: Live CD45+ Lin cKitlo CD127+ sca1lo Flt3+ α4β7), α4β7 integrin-expressing CLP (αLP: Live CD45+ Lin cKitlo CD127+ sca1lo Flt3 α4β7+), common progenitor to all helper-like innate lymphoid cell lineages (CHILP: Live CD45+ Lin CD127+ α4β7+ Flt3 CD25), ILC2 precursor (ILC2P: Live CD45+ Lin CD127+ α4β7+ Flt3 CD25+) and common precursor to ILC (ILCP: Live CD45+ Lin cKit+ CD127+ α4β7+ PLZF+) in bone marrow of Runx3f/f and Runx3f/fVav1-Cre mice. Plots are gated on Live CD45+ Lin CD127+ sca1lo cells in a, Live CD45+ Lin CD127+ cells in b (left plot of each pair), Live CD45+ Lin CD127+ cells in c. Arrows in the left panels indicate gated cells shown in the right plots. Numbers in quadrants and outlined area indicate percent cells in each. Data are representative of at least two independent experiments (mean and s.d. of three mice in d). NS, not significant.

Supplementary Figure 3 Runx3 is necessary for NK cells and ILC1 cells in tissues.

(a) Flow cytometry of Live CD45+ CD3 Cd19 cells showing NK and ILC1 cells in spleen, intraepithelial lymphocytes (IELs) of small intestine, salivary gland and skin of Runx3+/fNKp46-Cre and Runx3f/fNKp46-Cre mice. (b) Flow cytometry showing frequency of liver NK cells and their DX5 and CD49a expression in Runx3+/fNKp46-Cre and Runx3f/fNKp46-Cre mice. Plots are gated on Live CD45+ CD3 Cd19 cells in a and b (upper). Arrows in the two upper panels indicate gated cells shown in the lower plots in b. (c) Absolute cell numbers of indicated NK cells (CD3 CD19 NK1.1+ NKp46+) and ILC1 cells (CD45+ CD3 CD19 NK1.1+ NKp46+ CD49a+) in Runx3+/fNKp46-Cre and Runx3f/fNKp46-Cre mice. Numbers in outlined areas and quadrants indicate percent cells in each. Data are representative of three experiments (a, b) or from two independent experiments (c; mean and s.d. of three mice). *p < 0.05 and **p < 0.01 by Student’s t-test.

Supplementary Figure 4 CD127+ ILC lineage-negative (ILCLN) cells in RORγt-deficient mice and RORγt expression in Runx3-deficient LTi cells

(a) Flow cytometry of CD45+ CD3 CD19 CD127+ NK1.1 LPLs from wild-type (WT) and Rorc(gt)GFP/GFP mice for GATA-3 and RORγt expression as detected by anti-RORγt staining. (Anti-RORγt was necessary for simultaneous staining of GATA-3 in permeabilized cells.) Numbers in outlined areas or quadrants indicate percent cells in each. (b) Flow cytometry (left) for enteric fetal LTi0 (CD45+ CD3 CD11c CD127+ CD4) and LTi4 (CD45+ CD3 CD11c CD127+ CD4+) cells and their RORγt expression (right) in Runx3f/f and Runx3f/fVav1-Cre mice (E17.5). Plots were gated on CD45+ CD3 CD11c LPLs of intestine. Arrows in the two left panels indicate gated cells shown in the right histogram. (c) Absolute numbers of LTi0 and LTi4 cells in the indicated mice. Data are representative of more than three independent experiments (mean and s.d of 3-5 mice in c). NS, not significant by Student’s t-test.

Supplementary Figure 5 T-bet expression in ILCs is not affected by the absence of Runx3.

(a) Flow cytometry of CD45+ CD3 CD19 (Lin) CD127+ NK1.1 cells for GATA-3 and RORγt (upper panels) and CD45+ Lin CD127+ NK1.1 GATA-3int cells for CD4 and NKp46 (lower panels) from large intestinal LPLs of Cbfb+/fNKp46-Cre and Cbfbf/fNKp46-Cre mice. Arrows in the two upper panels indicate gated cells shown in the lower plots. Numbers indicate percent cells in outlined areas or quadrants. (b) Flow cytometry showing T-bet expression in CD45+ Lin CD127+ NK1.1 GATA-3int NKp46+ or CD4+ cells and CD45+ Lin NK1.1+ NKp46+ CD127+ (ILC1) or CD127 (NK) cells among LPLs of Cbfb+/fNKp46-Cre and C bfbf/fNKp46-Cre mice. Data are representative of three experiments (a, b).

Supplementary Figure 6 AHR is downstream of RORγt in ILC3 cells.

(a) Expression of indicated genes in CD45+ CD3 CD19 NK1.1 CD127+ LPLs of wild-type (WT) and Rorc(gt)+/GFP mice by qRT-PCR, relative to Actb expression. Data are representative of two independent experiments (mean and s.d. of triplicates). *p < 0.01 by Student’s t-test. (b) Tracks of Runx3 (SRR95607, green) and H3K4me1 (SRR955609, blue) ChIP-seq traces in splenic NK cells, and H3K4me2 (GSM1004811, dark green), p300 (GSM1004849, pink) and RORγt (GSM1004855, green) ChIP-seq traces in Th17 cells.

Supplementary Figure 7 Effect of deficiency in CBF-β or Runx3 on the survival of NK cells in tissues.

(a) Flow cytometry of CD3+ CD19+ cells and indicated NK cells (CD3 CD19 NK1.1+ NKp46+) of Cbfb+/fNKp46-Cre, Cbfbf/fNKp46-Cre, Runx3+/fNKp46-Cre and Runx3f/fNKp46-Cre mice for apoptosis markers. (b) Histograms depicting Ki-67 expression in indicated NK cells of Cbfb+/fNKp46-Cre, Cbfbf/fNKp46-Cre, Runx3+/fNKp46-Cre and Runx3f/fNKp46-Cre mice. Numbers in quadrants indicate percent cells in each. Data are representative of three experiments.

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Ebihara, T., Song, C., Ryu, S. et al. Runx3 specifies lineage commitment of innate lymphoid cells. Nat Immunol 16, 1124–1133 (2015). https://doi.org/10.1038/ni.3272

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