The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway

Abstract

Innate lymphoid cells (ILCs) including lymphoid tissue-inducer (LTi) cells, IL-22-producing NKp46 + innate cells and IL-13-producing nuocytes play important roles in regulating intestinal microbiota, defence against pathogens and formation of lymphoid tissue [1][2][3][4] . Their development is dependent on Id2 and Rorγt or Rorα 5-7 . Lineage tracing experiments have shown that the common lymphoid precursor gives rise to nuocytes, LTi cells and NKp46 + ILCs 6,8,9 , but these studies have not deciphered the discrete steps and transcription factors that specify ILC subset development, activation and maintenance. Whether NKp46 + ILCs arise directly from LTi cells, or rather represent a separate lineage that diverges earlier in development, remains controversial [10][11][12] . We investigated the requirement for the transcriptional master regulator T-bet (encoded by Tbx21) which is critical for the development of both T cells and NK cells 13,14 , in driving differentiation of ILC populations. Here we report that T-bet played an essential role for the development of NKp46 + ILCs, but was dispensable for LTi cells or nuocytes. Tbx21 +/+ LTi cells adopted an NKp46 + phenotype in vitro and in vivo but not in the absence of Tbx21. Decrease of T-bet expression coordinately reduced Notch1 and Notch2 and we show Notch signaling is necessary for the transition of LTi cells into NKp46 + ILCs. In addition, Tbx21 −/− mice have an accumulation in CD4 − LTi cells and differentiation into NKp46 + ILCs came solely from this population. Our results pinpoint T-bet as the critical regulator of NKp46 + ILC differentiation by regulation of Notch2 signaling. NKp46 + cells are an important element of the protective intestinal mucosal cellular arsenal, and here, we uncover the distinct molecular pathways that guide the development of NKp46 + ILCs. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author InformationThe authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Immunol. Author manuscript; available in PMC 2014 July 01. Supplementary Fig. 2).Analysis of T-bet expression in innate lymphocyte populations by intracellular staining and mRNA levels revealed high expression of T-bet in both NKp46 + ILCs and NK (lin − Rorγt − NKp46 + NK1.1 + ) cells isolated from the intestine, while T-bet was not expressed in LTi cells (Fig. 1c). The related T-box factor Eomes, that performs similar functions to Tbet in CD8 + T cells and NK cells, was not expressed in NKp46 + ILCs or LTi cells ( Supplementary Fig. 3a). Thus, T-bet appears to play a crucial role in the development of NKp46 + ILCs. Similar to T-bet, Blimp1 (encoded by Prdm1) was strongly differentially expressed between LTi and NKp46 + ILC populations ( Supplementary Fig. 3b). However, mice homozygously deficient for Blimp1 (Blimp1 gfp/gfp ) did not show any apparent defect in the development of ILC subsets ( Supplementary Fig. 3c). Thus, Blimp1 is dispensable for ILC differentiation while T-b...

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets

“…However, the overall number of Rorγt + ILC3 was similar suggesting that loss of NKp46 did not adversely affect the development of total ILC3 at steady-state. Nevertheless, analyses of T-bet expression revealed that it was upregulated within the NCR + subset in C57BL/6 mice as previously reported, 25,26 while NCR − ILC3 from Ly5.1 C14R mice did not show elevated levels of T-bet when compared with the NCR − population in C57BL/6 mice suggesting that NCR + cells fail to develop (Figure 3C).

10.1080/2162402X.2018.1475875-F0003Figure 3.Ly5.1 C14R mice have abnormal numbers of ILC3.

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“…NKp46 is also a key marker of one of the three subsets of ILC3. 18 In Ly5.1 C14R mice, as expected NCR + ILC3 were not detectable via NKp46 expression but we also did not see enrichment of expression of T-bet which is normally associated with induction of this subset, 25,26 although ILC3 development itself was not affected implying that activation of NKp46 is important for the transcriptional program of these cells. This is supported by cell fate mapping experiments that suggest that instability of NKp46 expression in ILC3s found in the intestine reflects a major role of this receptor in tuning the very dynamic environmental signals encountered that drive ILC3 plasticity.…”

Functional and phenotypic heterogeneity of group 3 innate lymphoid cells

“…Vitamin A deprivation results in a loss of intestinal ILC3 in adult mice, which can be reversed upon supplementation of diet with the vitamin A‐derived metabolite retinoic acid 44, 45. Similarly, Ahr acts to sense soluble aromatic hydrocarbons – present in the diet and produced by commensal bacteria – and is essential for the development of both LTi‐like and NKp46 + ILC3, as well as IL‐22 production 14, 27, 28, 46, 47, 48, 49, 50, 51. Together these signals tune ILC3 numbers and responses and establish bidirectional interactions between ILC3 and the commensal microbiota during the initial colonization of barrier tissue sites.…”

“…2). NKp46 + ILC3 are characterized by subset‐restricted expression of multiple genes required for their development and maturation, such as Il12rb2 , Tbx21 and Notch1 ,51, 61 and are defined by expression of eponymous NK cell‐associated receptors. Recent evidence suggests that NKp46 expression may act as a novel pattern recognition molecule to facilitate responses to fungal pathogens,62 whereas engagement of NKp44 on human ILC3 results in pro‐inflammatory cytokine production 63.…”

“…In addition to CCR6 + LTi‐like ILC3 and NKp46 + ILC3, the small intestine contains a population of ‘double‐negative’ ROR γ t + CCR6 − NKp46 − ILC3 61, 71. ‘Double‐negative’ ILC3 represent intestinal precursors of the NKp46 + ILC3 subset, and acquire NKp46 and IFN‐ γ expression in a process driven by up‐regulation of T‐bet and dependent upon IL‐23, Notch signalling and the microbiota 51, 61, 71. In addition, fate‐mapping studies revealed the presence of NKp46 + T‐bet + ‘ex‐ILC3’ that had lost ROR γ t‐expression in response to pro‐inflammatory cytokine stimulation and that functionally and phenotypically resemble ILC1 5, 61, 71 (Fig.…”