Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers1,2,3,4,5,6,7,8,9. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties10, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development11,12, by using lineage tracing and transfer studies. PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+DX5− ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages13. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.
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We thank W. Yokoyama for discussion; F. Gounari and R. de Pooter for advice on OP9 cultures; H. Gudjonson for statistical advice; D. Leclerc, J. Cao, M. Olsen and R. Duggan for help with cell sorting; V. Bindokas and R. Mathew for help with fluorescence microscopy. This work was supported by NIH grants R01HL118092, R01AI038339 and P30DK42086 and by The Howard Hughes Medical Institute (A.B.).
The authors declare no competing financial interests.
Extended data figures and tables
a, A sequence encoding an IRES and a GFP-cre fusion protein was inserted immediately after the Zbtb16 stop codon in C57BL/6J ES cells and knock-in mice were bred to ACTB-FLPe mice to excise the neomycin resistance cassette and generate the PLZFGFPcre allele. b, FACS analysis of the indicated populations from PLZFGFPcre+/− ROSA26-YFP mice. c, Summary of data (mean ± s.e.m.) from 2–5 mice analysed in 2 or more independent experiments.
ILC2 cells were identified as IL-7Rα+KLRG1+ among CD3ε−CD19− LPL (top left), and then gated Thy1.2+ (not shown). CD3ε−CD19− LPL were gated as IL-7Rα+KLRG1− (top left) and then subsetted into CCR6+CD4+ (CD4+ LTi cells) and CCR6+CD4− (CD4− LTi cells) (bottom left). NCR+ ILC3 were identified as CD3ε−CD19− LPL that expressed NKp46 but not NK1.1 (top right).
Quantitative PCR with reverse transcription analysis for Tbx21 and Rora as indicated. NKP are Lin−CD27+IL-7Rα−Flt3−CD122+ BM cells. Mean ± s.e.m. of data from 2–3 independent experiments.
CD45.2 Rag2−/−Il2rg−/− mice were injected with equivalent numbers of CD45.2 PLZFhigh cells and CD45.1 CLP (800 of each) and the resulting NK1.1+CD3ε−TCRβ− cells present in the spleen were analysed 5–7 weeks later by FACS, as indicated. Note that PLZFhigh-derived cells expressed higher amount of surface NKp46, whether they were identified as CD45.2+ or as CD45.1− in reciprocal staining experiments. Similar results were obtained for lung NK1.1+ cells. Data representative of 5 chimaeras from 2 independent experiments.
FACS analysis of PLZFhigh and CLP cells (100 of each) co-cultured for 15 days in FTOC (a). The percentages of PLZFhigh- or CLP-derived cells that are CD3ε+ are summarized in the bar graph (b). Data representative of 7 independent cultures.
a, FACS analysis of PLZFhigh or CLP cells from adult BM cultured on OP9 for 4 days showing expression of T1/ST2 on ICOShigh cells. Data representative of 4 replicate cultures from 2 independent experiments. b, FACS analysis of fetal liver PLZFhigh cells after culture on OP9 for 7 days, showing expression of GATA3 by ICOShigh cells and RORγt by ICOSint cells. Data representative of 2 independent experiments.
A CLP-derived IL-7Rα+α4β7+ population bifurcates into RORγthigh LTi precursors (LTiP) and PLZFhigh ILCP, the latter of which gives rise to all ILC lineages. Whether NKP cells develop directly from CLPs or progress through an IL-7Rα+α4β7+ stage has yet to be determined.
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Constantinides, M., McDonald, B., Verhoef, P. et al. A committed precursor to innate lymphoid cells. Nature 508, 397–401 (2014). https://doi.org/10.1038/nature13047
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