Transcriptional Regulation of Natural Killer Cell Development and Functions

Abstract

Natural killer (NK) cells are the major lymphocyte subset of the innate immune system. Their ability to mediate anti-tumor cytotoxicity and produce cytokines is well-established. However, the molecular mechanisms associated with the development of human or murine NK cells are not fully understood. Knowledge is being gained about the environmental cues, the receptors that sense the cues, signaling pathways, and the transcriptional programs responsible for the development of NK cells. Specifically, a complex network of transcription factors (TFs) following microenvironmental stimuli coordinate the development and maturation of NK cells. Multiple TFs are involved in the development of NK cells in a stage-specific manner. In this review, we summarize the recent advances in the understandings of TFs involved in the regulation of NK cell development, maturation, and effector function, in the aspects of their mechanisms, potential targets, and functions.

Keywords: IL-12; IL-15; IL-2; IL-21; NK cell; development; transcription factors.

Figure 1

Early programs that commit progenitors to natural killer (NK) cell lineage. (A) General schema of hematopoietic stem cells (HSCs)-lymphoid-primed multipotential progenitors (LMPPs)-common lymphoid progenitors (CLPs) to NK cell progenitors (NKPs). (B) Stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt3L) interact with their tyrosine kinase receptors c-Kit and Flt3L, respectively. The signaling pathways initiated through these receptors, primarily mediated by Jak2-Stats, PI(3)K-mTORC1-S6K1/4EBP1, or Jak2-PLC-γ2-NF-κB, promote cell survival, proliferation, and migration between niches. This activation also leads to essential gene transcriptions including IL-2Rβ. (C) IL-7-mediated activation via CD127 results in the maintenance of cell-fate integrity, survival, and proliferation.

Figure 2

Transcription Factors (TFs) involved in distinct murine NK cell developmental stages. Notch and Ikaros promote Lineage negative (Lin⁻) CD34⁺Sca-1⁺CD117⁺ hematopoietic stem cells (HSCs) committing to common lymphoid progenitors (CLPs, Lin⁻CD34⁺Sca-1^LowCD117^LowFlt3⁺), while FoxO1 suppresses this commitment to keep the HSCs in a quiescent state. Runx3, E4bp4, PU.1, and Id2 enhance NK cell lineage transition to CD27⁺CD244⁺CD127⁺CD122⁺ NK cell precursors (NKPs). FoxO1 suppresses NK lineage transition. Committed immature NK cells (iNKs) that are marked by the expression of NKG2D are regulated by E4bp4 and Ets1. Runx3, Ets1, Id2, IRF2, Gata3, Eomes, and Tox1/2 promote the iNK into mature NK cells (mNKs), which are defined by the expression of NCR1, NK1.1, Ly49, CD49b, and CD51. Aiolos and T-bet further enhance mNK to terminal NK cells (tNK) with the expression of CD11b, Ly49s, and KLRG1 and loss of CD27.

Figure 3

TFs involved in distinct human NK cell developmental stages. Notch proteins facilitate transition from Lin⁻CD34⁺ HSCs to CLPs (defined by Lin⁻CD34⁺CD244⁺). Commitment to NK cell lineage (named NKPs), marked by CD117⁺CD127⁺CD122⁺IL1R1⁺, is activated by Id2 and RUNX3. The transition to iNK is defined by higher expression of IL1R1 and the expression of NKG2D, CD335, CD337, and CD161, which is enhanced by NFIL3 and ETS1. GATA2 and EOMES promote commitment to transitional NK cells (TransNKs) between the immature and the mature stage, which are defined by NKG2D⁺CD335⁺CD337⁺CD161⁺CD56^bright population. T-bet promotes them to NKp80⁺CD56^dimCD16⁺KIR^−/+ mNKs. The commitment of terminally mature NK cells (TermNKs) is marked by CD56^dimCD16⁺CD57⁺ KIR⁺.

Figure 4

Potential transcriptional networks in murine NK cell development. Potential downstream and upstream regulators of individual transcription factors involved in murine NK cell development were identified using String database (https://string-db.org/). Gene of interest is shown in red font. ‘—’ represent data from curated databases; ‘—’ experimentally determined; ‘—’ gene neighborhood; ‘—’ gene fusions; ‘—’ gene cooccurrence; ‘—’ text mining; ‘—’ co-expression; and ‘—’ protein homology. (A) The transition from HSCs to CLPs is regulated by Notch1, Ikaros (encoded by Ikzf1), and Foxo1. (B) Committed to NK lineage is mainly governed by Id2, Nfil3, and Runx3. (C) NKPs transit to immature NK cells under the control of PU.1 (encoded by Spi1), Ets-1, and Irf2. (D) The maturation of NK cells is activated by Gata3, Eomes, and T-bet (encoded by Tbx21).

Figure 5

Potential transcriptional networks in human NK cell development. Potential downstream and upstream regulators of individual transcription factors involved in human NK cell development were identified using String database String database (https://string-db.org/). Gene of interest is shown in red font. ‘—’ represent data from curated databases; ‘—’ experimentally determined; ‘—’ gene neighborhood; ‘—’ gene fusions; ‘—’ gene cooccurrence; ‘—’ text mining; ‘—’ co-expression; and ‘—’ protein homology. (A) Human HSCs transit to CLPs under the help of NOTCH1, IKAROS, and FOXO1. (B) The commitment to NK lineage is mainly regulated by ID2, NFIL3, and RUNX3. (C) Transition from NKPs to iNK is potentially governed by PU.1, ETS-1, and IRF2. (D) The maturation of human NK cells is regulated by GATA3, EOMES, and T-bet.

Figure 6

TFs involved in the effector functions of NK cells. The 3-D protein structural motifs of TFs involved in the development and functions of NK cells are shown. Most TFs enhance both cytotoxicity and IFN-γ production of NK cells (black font). However, FoxO1 suppresses both effector functions as a balance (red). Current evidence shows that Ets-1 is involved only in cytotoxicity and IRF-2 and Gata3 are only involved in IFN-γ production. Tox2 and PU.1 may contribute to the IFN-γ production, though no direct evidence exists. Notch, Runx3, and Aiolos are not involved in NK cell effector functions. The ‘?’ in Figure 6 represents no direct evidence showing the effect on IFN-γ production.

Figure 7

Targets of TFs in murine NK cells. (A) Notch proteins regulate NK cell development partially through upregulated expression of Ets1 and Ikaros. Ikaros is indispensable due to its role in the expression of Flt3 and activating the transcription of cKit by directly binding to its promoter region. Interaction with Runx1 is required for PU.1 to bind the DNA motif. PU.1 promotes the expression of cKit and Klf4 to contribute to NK cell development. PU.1 upregulates the expression levels of Ly49A, Ly49D, and DAP12. PU.1 suppresses the expression of Ets-1 which directly binds the promoter region of Id2, Tbx21, and Jak3. The augmented level of Id2, T-bet, and Jak3 regulate NK cell development in different ways. Ets-1 promotes the expression of NCR1, Ly49D, and Ly49H. The line between PU.1 and Ets1 represents that PU.1 directly binds to Ets-1 promoter region and inhibit Ets-1 transcription. (B) During NK cell development, Nfil3 is repressed by Ets-1, and promotes the transcription of Id2 and Eomes. Nfil3 upregulates NCR1, NKG2D, and DX5 and downregulates Ly49C, Ly49I, Ly49F, and Klrg1. Tox2 directly binds to the promoter region of Tbx21 and activates its transcription. Tox2 augments the expression of NKR1 to fulfil the NK cell effector function. Runx3 cooperates with Ets-1 to promote NK cell proliferation. Eomes maintains the expression of NK1.1 and NCR1 during NK cell development. Eomes augments the expression of DX5 and Ly49s and represses the expression of TRAIL. Eomes can bind to the Ifng promoter region and activate its transcription. T-bet maintains the expression of CD11b, DX5, and Klrg1 but suppresses the expression of c-Kit. T-bet activates transcription of Gzmb and Prf1. T-bet and Eomes cooperatively bind to the Il2rb promoter region. The dashed line between Ets1 and Nfil3 represents that the Ets1 inhibits the transcription of Nfil3 in an indirect way.