Research ArticleIMMUNOGENOMICS

Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

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Science Immunology  08 Dec 2017:
Vol. 2, Issue 18, eaan3796
DOI: 10.1126/sciimmunol.aan3796
  • Fig. 1 STAT4 directly targets Runx transcription factors in activated NK cells.

    Splenic NK cells (TCRβCD19CD3εLy6GTER119TCRγδNK1.1+) were sorted from WT mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated). STAT4 ChIP was performed, followed by high-throughput DNA sequencing. (A) Proportions of STAT4 genome-wide occupancy at promoter (2 kb upstream and 0.5 kb downstream from TSS), intronic, exonic, or distal intergenic regions in cytokine-stimulated NK cells are shown. (B) RNA-seq was performed on splenic Ly49H+ WT NK cells and Stat4−/− NK cells sorted from mixed chimeras 2 days after MCMV infection. Venn diagram of overlap between differentially expressed genes (top; Padj < 0.05) identified through RNA-seq and reproducible STAT4-bound regions identified through ChIP-seq (bottom; IDR < 0.05). RNA-seq data were performed on n = 3 per condition. (C) Bar graphs depict the top 20 genes with greatest fold enrichment of STAT4 binding over input calculated by MACS2 in transcription factors that show differential expression in RNA-seq data. (D) Representative gene tracks for indicated core-binding factors from STAT4 ChIP-seq. ChIP-seq data are representative of three independent experiments with n = 15 to 20 pooled mice per group per experiment.

  • Fig. 2 STAT4 facilitates a permissive epigenetic landscape (H3K4me3) in activated NK cells.

    Splenic NK cells (TCRβCD19CD3εLy6GTER119TCRγδNK1.1+) were isolated from WT and Stat4−/− mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated; unstim). H3K4me3 ChIP was performed, followed by high-throughput DNA sequencing. (A) Global proportions of H3K4me3 permissive marks at promoter, intronic, exonic, or distal intergenic regions in cytokine-stimulated NK cells are shown. (B) Bar plots depict number of peaks that change on the basis of fold change (FC) of stimulated versus unstimulated NK cells. FC was calculated by taking the difference between log2-transformed normalized counts for each condition. Only peaks that showed a log2 FC greater than a magnitude of 1 were counted. (C) Meta-peak of all H3K4me3 promoter regions. Overlap of midpoints of ChIP fragments (defined as regions between properly paired sequence reads) for each TSS region was counted for each base pair ± 1 kb from the transcriptional start site. Line plot depicts average signal for all regions for each base pair. (D) Heat map of all H3K4me3 binding regions, with each row representing a single-peak region, row-clustered by normalized peak counts. Signal is displayed as normalized read counts over 5 kb centered at the peak region and is binned at 100-bp windows. (E) H3K4me3 signals from Cbfb, Runx1, Runx2, and Runx3 loci plotted as normalized fragment counts binned at 200 bp across a 10-kb window centered on the transcriptional start site. (F) Zoomed-in histograms of STAT4 ChIP and H3K4me3 ChIP reads mapped to Runx1 and Runx3 loci. Dashed box within boxed tracks indicate STAT4 ChIP called peak region. Data are representative of two independent experiments with n = 15 to 20 pooled mice per group per experiment.

  • Fig. 3 Runx1 and Runx3 are up-regulated during MCMV infection.

    (A) Graph shows mRNA expression of highest induced lymphocyte-specific transcription factors in splenic Ly49H+ NK cells sorted from uninfected and MCMV-infected animals on day 2 PI, as assessed by microarray [data provided by the Immunological Genome Consortium (41)]. Data are shown as fold change in microarray signal intensity for the infected versus uninfected samples (n = 3 biological replicates per group and representative of three separate experiments). Solid black bars denote significant up-regulation or down-regulation as compared with uninfected controls (P < 0.05, two-tailed unpaired Student’s t test). (B) Normalized counts of Runx1, Runx2, Runx3, and Cbfb in splenic Ly49H+ NK cells sorted from MCMV-infected mice on day 2 PI and uninfected mice (top) or in unstimulated (US) or IL-12 plus IL-18–treated (12 + 18; 16-hour stimulation) splenic NK cells (bottom), as assessed by RNA-seq (n = 2 to 3 biological replicates per group). (C) RNA-seq was performed on purified Ly49H+ WT NK cells and Stat4−/− NK cells from uninfected and MCMV-infected mixed BMC mice (day 2 PI). Normalized counts of Runx family members are shown (n = 2 to 3 biological replicates per group). (D) RNA-seq was performed on purified Ly49H+ and Ly49H WT NK cells from uninfected and MCMV-infected mice (day 2 PI). Normalized counts of Runx1 and Runx3 are shown (n = 2 to 3 biological replicates per group). Data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

  • Fig. 4 CBF-β is required for NK cell development and host protection against MCMV infection.

    (A) Percentage and absolute numbers of NK cells (LinNK1.1+) in the bone marrow (BM) and in peripheral organs of Cbfbfl/fl × NKp46iCre mice and Cbfbfl/fl littermate controls. (B) Representative flow cytometric plots show CD27 and CD11b expression of Lin NK1.1+ NK cells in the blood of Cbfbfl/fl × NKp46iCre mice and Cbfbfl/fl littermate controls. Lin refers to TCRβCD19CD3εLy6GTER119TCRγδ cells. Data are representative of four independent experiments, with n = 3 to 5 mice. Samples were compared using two-tailed unpaired Student’s t test, and data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Top graph shows survival of Cbfbfl/fl × NKp46iCre and Cbfbfl/fl littermate control mice infected with a lethal dose of MCMV. Bottom graph shows viral titers in the blood at 4 days PI. Data are representative of two to four independent experiments, with n = 3 to 5 mice. Samples were compared using two-tailed unpaired Student’s t test, and data are presented as means ± SEM (*P < 0.05).

  • Fig. 5 Inducible deletion of CBF-β in NK cells results in defective expansion and generation of memory cells after viral infection.

    (A) Schematic of experiment: WT:Cbfbfl/fl × UbcERT2-Cre–mixed bone marrow chimeras were treated with 1 mg of tamoxifen or oil daily for 5 days before adoptive transfer of Ly49H+ NK cells into Ly49H-deficient hosts. Recipient mice were infected with MCMV 1 day later. (B) Graphs depict the relative and absolute percentage of transferred WT and Cbfbfl/fl × UbcERT2-Cre NK cells in the blood at various time points after MCMV infection. (C) Percentage of transferred WT and Cbfbfl/fl × UbcERT2-Cre NK cells in indicated organs at day 28 PI. (D) CD27 versus CD11b expression of WT and Cbfbfl/fl × UbcERT2-Cre Ly49H+ NK cells at day 14 PI. Error bars show SEM, and graphs are representative of three independent experiments, with n = 4 to 6 mice per group (**P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test).

  • Fig. 6 Runx1 and Runx3 are critical and nonredundant in promoting NK cell expansion and survival during MCMV infection.

    WT and Runx1fl/fl × Nkp46iCre, or WT and Runx3fl/fl × Nkp46iCre Ly49H+ NK cells were cotransferred into Ly49H-deficient hosts and infected with MCMV. (A) Relative and absolute percentage of WT and Runx1fl/fl × Nkp46iCre Ly49H+ NK cells. (B) Percentage of transferred WT and Runx1fl/fl × Nkp46iCre NK cells in the spleen and liver at day 32 PI. (C) CD27 versus CD11b and KLRG1 expression of WT and Runx1fl/fl × Nkp46iCre transferred NK cells in the blood at day 7 PI. (D) Memory NK cells isolated at day 28 PI were stimulated with IL-12 plus IL-18 (IL-12+18) or PMA plus ionomycin (PMA+iono) for 4 hours or were untreated. Percentages of memory WT and Runx1fl/fl × Nkp46iCre NK cells producing IFN-γ (left) or degranulating (right) are shown. (E) Graphs show percentage of WT and Runx3fl/fl × Nkp46iCre transferred NK cells in the blood at depicted time points as well as (F) in the spleen and liver at day 32 PI. (G) CD27 versus CD11b and KLRG1 expression of transferred NK cells in the blood for each group at day 7 PI. (H) Memory NK cells isolated at day 28 PI were stimulated as described in (D) and percentages of IFN-γ (left) or degranulating (right) memory NK cells are shown. Data are representative of two independent experiments, with n = 3 to 4 mice. Samples were compared using two-tailed unpaired Student’s t test, and data are presented as means ± SEM (**P < 0.01, ***P < 0.001).

  • Fig. 7 Runx1 promotes a cell cycle program during MCMV infection.

    Splenic NK cells harvested from WT or Runx1fl/fl × Nkp46iCre mice were adoptively transferred into Ly49H-deficient recipients and infected with MCMV. RNA-seq was performed on WT and Runx1-deficient Ly49H+ NK cells harvested from the spleen 3 days PI. (A) Heat map depicting row-normalized blind rlog counts calculated by DESeq2. Genes that show differential expression at a cutoff of Padj < 0.05 are shown. (B) Top five enriched pathways as calculated by GAGE. Bar plots depict –log10 P values calculated by GAGE. (C) Cumulative distribution function plot of moderated log2 fold changes calculated by DESeq2 comparing cell cycle genes (red) and all genes (black). P value was calculated by one-sided Kolmogorov-Smirnov test. (D) WT and Runx1-deficient NK cells were labeled with CTV before adoptive transfer and MCMV infection. Representative histograms of CTV expression in Ly49H+ NK cells of indicated genotypes at 3.5 days PI are shown, and data are representative of three independent experiments, with n = 3 to 4 mice.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/18/eaan3796/DC1

    Fig. S1. Epigenetic regulation by STAT4 in cytokine-stimulated NK cells.

    Fig. S2. Cell-intrinsic requirement of CBF-β for the development of mature NK cells.

    Fig. S3. CBF-β is critical for NK cell survival after homeostatic proliferation.

    Fig. S4. Runx1- or Runx3-deficient mice have normal NK cell numbers.

    Table S1. Raw data for Figs. 3 to 6 and figs. S3 and S4.

  • Supplemental Materials

    Supplementary Material for:

    Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

    Moritz Rapp, Colleen M. Lau, Nicholas M. Adams, Orr-El Weizman, Timothy E. O?Sullivan, Clair D. Geary, Joseph C. Sun*

    *Corresponding author. Email: sunj{at}mskcc.org

    Published 8 December 2017, Sci. Immunol. 2, eaan3796 (2017)
    DOI: 10.1126/sciimmunol.aan3796

    This PDF file includes:

    • Fig. S1. Epigenetic regulation by STAT4 in cytokine-stimulated NK cells.
    • Fig. S2. Cell-intrinsic requirement of CBF-β for the development of mature NK cells.
    • Fig. S3. CBF-β is critical for NK cell survival after homeostatic proliferation.
    • Fig. S4. Runx1- or Runx3-deficient mice have normal NK cell numbers.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data for Figs. 3 to 6 and figs. S3 and S4.

    Files in this Data Supplement:

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