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.
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Gene Expression Omnibus
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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.).
The authors declare no competing financial interests.
Integrated supplementary information
(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 (Lin−CD45+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 Lin−α4β7+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.
(a) Lin−CD45+ cells from WT and Tox−/− spleen and small intestine LP. ILC3s were defined as Lin− (CD3−CD8α−CD19−Gr-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 NKp46−T-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.
Data are derived from four independent progenitor cell isolations for each genotype, using 2-3 pooled animals per experiment.
(a) CD127 expression on WT and Tox−/−CLP. As control, staining of Lin−CD127+Sca-1−Flt3− 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).
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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). https://doi.org/10.1038/ni.3168
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