Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell activation

Abstract

Natural killer cells belong to the family of innate lymphoid cells comprising the frontline defense against infected and transformed cells. Development and activation of natural killer cells is highly dependent on interleukin-15 signaling. However, very little is known about the transcription program driving this process. The transcription factor Runx3 is highly expressed in natural killer cells, but its function in these cells is largely unknown. We show that loss of Runx3 impaired interleukin-15-dependent accumulation of mature natural killer cells in vivo and under culture conditions and pregnant Runx3(-/-) mice completely lack the unique population of interleukin-15-dependent uterine natural killer cells. Combined chromatin immunoprecipitation sequencing and differential gene expression analysis of wild-type versus Runx3-deficient in vivo activated splenic natural killer cells revealed that Runx3 cooperates with ETS and T-box transcription factors to drive the interleukin-15-mediated transcription program during activation of these cells. Runx3 functions as a nuclear regulator during interleukin-15-dependent activation of natural killer cells by regulating the expression of genes involved in proliferation, maturation, and migration. Similar studies with additional transcription factors will allow the construction of a more detailed transcriptional network that controls natural killer cell development and function.

FIG 1

Runx3 is expressed in NKC from the NKP stage, and its loss affects their maturation. (A) Western blot analysis of FACS-sorted mouse spleen NKC (NK1.1⁺ CD3⁻), IL-2-cultured mouse spleen NKC, and IL-2-cultured human blood NKC reveals expression of Runx3. (B) NKP cells express Runx3. BM of compound Runx3^{P1-AFP/+/P2-EGFP/+} mice was analyzed for GFP expression in Lin⁻ (Lin = Ter119, B220, Gr1, CD3, CD8, and CD11b) NK1.1⁻ NKG2D⁺ CD122⁺ NKPs. (C) Most NK1.1⁺ NKC express Runx3. Lin⁻ NK1.1⁺ cells of compound Runx3^{P1-AFP/+/P2-EGFP/+} mice were analyzed for GFP expression. (D) Analysis of Runx3^P1-AFP/+ and Runx3^P2-EGFP/+ expression in BM. CD122⁺ CD3⁻ BM lymphocytes were analyzed for coexpression of NKp46 and either P1- or P2-derived GFP expression. (E) Percentages of NKp46⁺ NKC in BM and spleen of WT and Runx3^−/− mice. (F) Bar graphs showing the frequency of WT and Runx3^−/− NKC subsets out of total NKp46⁺ spleen cells under resting conditions. Mean values are shown for the four maturation stages of NKC (n = 5). Significance, WT versus Runx3^−/− NKC: ∗, P < 0.05. CD11b, CD11b⁺ CD27⁻; DP, CD11b⁺ CD27⁺; CD27, CD11b⁻ CD27⁺; DN, CD11b⁻ CD27⁻.

FIG 2

Impaired IL-2- and IL-15-induced proliferation of cultured primary Runx3^−/− NKC. (A) Numbers of WT and Runx3^−/− NKC (DX5⁺ or NKp46⁺) accumulating following 7 days of culture in the presence of IL-2 (n = 22) or IL-15 (n = 8). Significance: ∗∗, P < 0.01; ∗, P < 0.05. (B) CFSE dilution assay of WT and Runx3^−/− NKC on day 6 of culture in the presence of IL-2 or IL-15. The red line indicates CFSE intensity before culturing, immediately after cell labeling. (C) Analysis of apoptotic (annexin V-positive) cells in WT and Runx3^−/− NKC at day 6 of culture with IL-2 or IL-15.

FIG 3

Impaired accumulation of mature Runx3^−/− splenic NKC following IL-15/Rα or IL-2 activation in vivo. (A) Percentages of NKp46⁺ NKC in WT and Runx3^−/− BM and spleen under resting conditions (T0) (n = 8 and n = 12, respectively) and following injection of IL-15/Rα (n = 13 and n = 16, respectively) or IL-2 (n = 5). Significance: ∗∗, P < 0.001. (B) BrdU incorporation in BM (upper panels) and spleen (lower panels) NKC (NKp46⁺ cells) 24 h after the second IL-15/Rα injection. (C) Bar graphs showing the frequency of WT and Runx3^−/− NKC subsets in spleen following IL-15/Rα activation. Mean values are shown for the four populations (n = 5). Significance, WT versus Runx3^−/− NKC: ∗∗, P < 0.001; ∗, P < 0.005. (D) Impaired maturation of Runx3^−/− NKC under resting and activated conditions. Percentages of iNK (CD27⁻ CD11b⁻ and CD27⁺ CD11b⁻) cells in WT and Runx3^−/− spleen under resting conditions (T0) and following administration of IL-15/Rα. Significance: WT versus Runx3^−/− at T0 and IL-15, P < 0.01. (E) IFN-γ production by WT and Runx3^−/− spleen NKC following IL-15 activation in vivo.

FIG 4

Runx3^−/− pregnant mice lack uNKC. (A) DBA and anti-Runx3 immunostaining show Runx3 expression in uNKC. (B) WT and Runx3^−/− implantation site sections stained with PAS (a to d, E11; panels c and d represent enlarged boxed regions in panels a and b, respectively; arrowheads in panel c mark uNKC) or DBA (e and f, E6.5; g to j, E11; panels i and j represent enlarged regions in panels g and h, respectively). (C) IHC of Runx3 (upper panels) and DBA (lower panels) on sections of E10.5 implantation sites of pregnant Rag^−/− γc^−/− mice to which WT (left panels) or Runx3^−/− (right panels) fetal liver cells were transferred ∼2 months earlier. Bars, 1 mm (Ba and b), 50 μm (Bc, d, i, and j), 250 μm (Be to h), and 100 μm (C).

FIG 5

Runx3 promoter usage in NKC. (A) E11 implantation site sections of Runx3^P1-AFP/+ or Runx3^P2-EGFP/+ pregnant mice stained with anti-GFP. (B) E11 implantation sites of Runx3^{P1-AFP/P1-AFP} mice analyzed by DBA IHC (upper panel) or Runx3 (lower panel). Bars (A and B), 50 μm. (C) FACS analysis of GFP expression in IL-2-cultured NKC of Runx3^P1-AFP/+ and Runx3^P2-EGFP/+ mice. (D) RT-PCR analysis of Runx3 common and P1- and P2-specific transcripts in RNA prepared from WT and Runx3^{P1-AFP/P1-AFP} NKC on day 7 of culture with IL-2. (E) Western blot documenting Runx3 expression in IL-2-cultured WT and Runx3^{P1-AFP/P1-AFP} NKC. The 46-kDa Runx3 P1 isoform is clearly detected in the WT lane, while in the Runx3^{P1-AFP/P1-AFP} lane, the two typical P2 Runx3 45- and 43-kDa isoforms (76) are detected. (F) Analysis of WT and Runx3^{P1-AFP/P1-AFP} NKC numbers following 7 days in culture with IL-2.

FIG 6

Integrated analysis of Runx3 ChIP-seq and gene expression data of in vivo IL-15-activated NKC. (A) Intersection of differentially expressed genes (Runx3^−/−/WT ≥ 1.5-fold; P < 0.05) in the three spleen NKC subpopulations of WT and Runx3^−/− mice (CD27, CD27⁺ CD11b⁻; DP, CD27⁺ CD11b⁺; CD11b, CD11b⁺ CD27⁻). (B) Pie chart showing the genomic distribution of Runx3 peaks in in vivo IL-15/Rα-activated NKC. (C) Analysis of Runx3 peaks in NKC in vivo. Runx3 peak numbers, corresponding genes, and percentages of peaks with RUNX motif (RCCRCA) in resting NK cells (68) and in in vivo IL-15-activated NKC. (D) De novo-discovered enriched TF motifs in Runx3 peaks in in vivo IL-15/Rα-activated NKC. (E) In vivo IL-15-activated Runx3-regulated genes: intersection between the list of genes containing Runx3 peaks with a RUNX motif and the list of differentially expressed genes (Runx3^−/−/WT ≥ 1.5-fold; P < 0.05) in the three spleen NKC populations.

FIG 7

Runx3-regulated genes in NKC. (A) Heat map showing Runx3^−/−/WT fold expression (log 2 ratio) for a subset of Runx3-regulated genes in in vivo IL-15-activated iNKC (CD27, CD27⁺ CD11b⁻) and mNKC (CD11b, CD11b⁺ CD27⁻). (B) qRT-PCR analysis of Runx3-regulated genes in in vivo IL-15-activated spleen iNK (CD27⁺ CD11b⁻) and mNK (CD11b⁺ CD27⁻) cell populations. (C) FACS analysis of Runx3-regulated genes: Kit expression in iNKC (CD27⁺ CD11b⁻) 24 h after the second IL-15/Rα injection and CD226, CD81, Cxcr3, and Itgαx in spleen NKC 48 h after the second IL-15/Rα injection.