Innate lymphoid cells (ILCs) and conventional natural killer (cNK) cells develop from bone marrow lymphoid progenitors through a transcription factor cascade whose core is now established: NFIL3 specifies NFIL3+ lymphoid precursors, TOX drives their progression to early innate lymphoid progenitors, and TCF-1 enforces lineage commitment. What remains unresolved is the topmost layer of this cascade: which signals induce Batf and Nfil3 in all-lymphoid and alpha-lymphoid progenitors — in particular whether interleukin-7 receptor engagement directly activates STAT3 and STAT6 to turn on Batf transcription, whether BATF pioneer activity feeds into NFIL3-dependent specification in vivo, what upstream signals induce Nfil3 itself, and how the pre-existing accessible chromatin landscape in progenitors, on which NFIL3 and TCF-1 binding depends, is established by factors such as RUNX and PU.1.

Background

Innate lymphoid cells (ILCs) are a family of lymphocytes that lack antigen-specific receptors yet produce the same cytokines as T helper cells; they comprise group 1 ILCs (including natural killer, NK, cells), group 2 ILCs, and group 3 ILCs, and they govern barrier immunity and tissue homeostasis. In adult mouse bone marrow, ILC and NK development proceeds through a sequence of increasingly restricted progenitors: common lymphoid progenitors (CLPs), all-lymphoid progenitors (ALPs), alpha-lymphoid progenitors (αLPs, defined by α4β7 integrin expression), NFIL3+ lymphoid precursors (recently named NLPs), early innate lymphoid progenitors (EILPs), and common innate lymphoid progenitors (CHILPs) or ILC precursors (ILCPs). A series of transcription factors (TFs) — proteins that bind DNA and control gene-expression programs — controls these transitions. NFIL3 (also called E4BP4, a basic leucine zipper TF) is required for NK cell development (Kamizono et al. 2009) and for all ILC subsets (Seillet et al. 2014; Geiger et al. 2014). TOX, an HMG-box TF, is required for generation of a common ILC progenitor (Seehus et al. 2015; Aliahmad et al. 2010). TCF-1 (encoded by Tcf7, an HMG-box TF related to LEF1) is upregulated at the EILP stage, is required for efficient generation of all adult ILC subsets, and enforces ILC lineage commitment (Yang et al. 2015; Harly et al. 2019). Single-cell analyses showed that Nfil3 expression is transient at the earliest progenitor stages while Tox persists, consistent with Nfil3 acting first (Ishizuka et al. 2016; Yu Y et al. 2016).

Several mechanistic links were long unresolved. First, it was unclear how NFIL3 relates to TOX — whether NFIL3 induces TOX or the two act in parallel (Yu X et al. 2014 reported NFIL3 required for TOX induction in adult bone marrow, but the absence of lymph nodes in NFIL3-deficient mice confounded interpretation; Seehus et al. 2015 stated the exact relationship remained to be elucidated). Second, the identity of direct NFIL3 target genes in the NK lineage was open; Male et al. 2014 resolved the key ones, showing NFIL3 directly binds regulatory regions of Eomes and Id2. Third, Harly et al. 2019 showed that TCF-1 binds a pre-existing, DNase-accessible chromatin landscape established in upstream ALPs, but which factors build that landscape remained undefined; conditional deletion of Cbfb (the cofactor for RUNX TFs) from hematopoietic progenitors eliminates ALPs and downstream ILC progenitors (Harly et al. 2018), and a shared 1-kb cis-regulatory element that initiates Tcf7 expression in both T cells and ILCs was identified, with an essential Notch-binding site in T cells but not in ILCs (Harly et al. 2020) — leaving the trans-acting input that initiates Tcf7 in ILCs unknown. Fourth, Liu et al. 2020 reported that BATF (a basic leucine zipper TF with pioneer activity, i.e., the ability to bind closed chromatin and open it) is induced at the αLP stage by interleukin-7 (IL-7), binds and activates the Nfil3 gene, and is required for normal ILC progenitor numbers; however, STAT3 and STAT6 (signal transducers and activators of transcription downstream of cytokine receptors) were implicated only by inhibitor experiments, and whether they are directly activated by IL-7 receptor (IL-7R) signaling in these progenitors, as well as BATF's chromatin-remodeling targets, remained open.

The central architecture has now been resolved: Léger et al. 2025, using Il7r-lineage tracing, identified NFIL3+ NLP intermediates, showed that Nfil3-null mice lack all bone marrow ILC-specified precursors, that forced NFIL3 expression in ALPs is sufficient to generate Tcf7+ cells producing all ILC subsets and cNK cells, and that NFIL3 initiates specification by inducing Tox, Id2, Gata3, Tcf7 and Zbtb16, with genome-wide NFIL3 binding mapped by CUT&RUN (a targeted antibody-based method for profiling protein-DNA binding) in ILC precursors. The 2026 review by Das, Ding, Harly and Bhandoola codifies the cascade: RUNX factors are required for ALP development onward, NFIL3 for ALP-to-NLP specification, TOX for NLP-to-EILP progression, and TCF-1 at the EILP-to-ILCP commitment checkpoint. Notably, that review does not discuss BATF at all, and Léger et al. 2025 found that Batf is not detectably upregulated during early ILC specification and that neither Batf nor Batf3 ectopic expression induces ILC specification — so BATF's relationship to the NFIL3 apex is actively contested. The unresolved residue of the original question therefore concentrates at the very top of the cascade: the receptor-to-transcription-factor wiring that turns on Batf and Nfil3, and the establishment of the permissive chromatin substrate on which the now-mapped NFIL3→TOX→TCF-1 program acts.

Problem Statement

In adult mouse bone marrow lymphoid progenitors, determine the molecular mechanisms that constitute the most upstream, still-unresolved layer of the ILC/NK specification cascade — the layer that activates the now-established NFIL3 → TOX → TCF-1 program. Specifically:

  1. By what molecular mechanism does interleukin-7 receptor (IL-7R) engagement upregulate the transcription factor Batf at the alpha-lymphoid progenitor (αLP) stage: are STAT3 and STAT6 directly phosphorylated by IL-7R-associated JAK kinases in these progenitors and recruited to the Batf promoter, or do they act indirectly (for example, licensed by other cytokines) to permit IL-7-dependent Batf induction?

  2. Does BATF participate in NFIL3-dependent ILC specification in vivo? In particular, is BATF binding to the Nfil3 locus required for Nfil3 induction in the progenitors that seed ILC development, or is BATF's documented requirement for ILC progenitor numbers exerted through other targets? If BATF participates, identify the chromatin-remodeling steps and direct target loci through which BATF pioneer activity acts at these stages; if it does not, provide evidence reconciling BATF's necessity for progenitor numbers with its failure to induce specification.

  3. Which upstream signal(s) induce Nfil3 expression at the all-lymphoid-progenitor (ALP)-to-NFIL3+ lymphoid precursor (NLP) transition?

  4. How is the pre-existing accessible chromatin landscape in ALPs — the substrate on which NFIL3 and TCF-1 binding depends — established, including the specific contributions of RUNX factors and PU.1 to opening these elements, and which trans-acting factor(s) initiate Tcf7 expression in the ILC lineage through the shared 1-kb Tcf7 regulatory element?

All four components are jointly required, and they are to be integrated into a consistent model of the cascade's input layer.

References

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