1. Volume 36, Issue 6, Pages 921-932 (June 2012)
Gene Deregulation and Chronic Activation in Natural Killer Cells Deficient in the Transcription Factor ETS1 
Kevin Ramirez, Katherine J. Chandler, Christina Spaulding, Sasan Zandi, Mikael Sigvardsson, Barbara J. Graves, Barbara L. Kee 
Immunity 
Volume 36, Issue 6, Pages (June 2012)
DOI: /j.immuni
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Immunity 2012 36, 921-932DOI: (10.1016/j.immuni.2012.04.006)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 ETS1 Functions at the Earliest Stages of NK Cell Development
(A and B) Flow cytometry analysis of Ets1+/+ and Ets1−/− BM using (A) NK1.1 and DX5 to identify NKP (NK1.1−DX5−), iNK (NK1.1+DX5−), and mNK (NK1.1+DX5+) cells in CD122+ Lineage (CD19−CD3−TCR-β−CD4−CD8−TER119−)− cells or (B) FLT3 and CD122 to identify CLP (FLT3+CD122−), pre-NKP (FLT3−CD122−), and rNKP (FLT3−CD122+) in Lin−CD27+CD244+CD127+ cells. The frequency of cells in the gated areas is indicated.
(C and D) The total number of (C) BM NKP, iNK, and mNK and splenic mNK (sNK) or (D) CLP, pre-NKP, and rNKP is presented. Each circle represents one mouse, horizontal bar indicates average. Closed circles, Ets1+/+ mice; open circles, Ets1−/− mice. n = 4–7 for each genotype. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by a Student's t test, #p < 0.05 by a paired t test but not significant with a standard t test.
(E) Relative contribution of Ets1+/+ and Ets1−/− CD45.2+ cells in HSCs, MPPs, LMPPs, CLPs, NKP, iNK, and mNK from mixed BM chimeras with WT CD45.1+ competitors. Squares, Ets1+/+; diamonds, Ets1−/−. n = 9 except CLPs where n = 7.
See Figure S1 for analysis of splenic mNK cells, pre-pro-NK cells, and flow cytometery analysis of BM chimeras.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 2 Identification of ETS1-Dependent Genes in NK Cells
(A) Microarray analysis of mRNA from Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells. Probe sets with differential expression in mNK cells are shown as is their mRNA expression across multiple hematopoietic cell populations.
(B) Analysis of ETS1 binding to ETS1-dependent NK cell genes in a CD4+ T cell line by ChIP-seq. Genes associated with ETS1-bound regions were determined by the closest TSS. Pie graphs show the percent of ETS1-dependent NK genes that had human orthologs (left) and the number of genes that were bound by ETS1 (right).
(C) MEME analysis of sequences bound by ETS1 in ETS1-dependent NK cell genes.
(D) KEGG analysis for functional pathways regulated by NK cell genes with a 1.5× threshold of differential expression.
See Table S1 for ETS1-dependent NK cell genes.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3 Deregulation of Multiple NK Cell Genes in the Absence of ETS1
(A) Heat maps for a subset of ETS1-dependent genes in Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells (duplicates combined). Asterisk indicates that ETS1 was bound near this gene by ChIP-seq in CD4 T cells.
(B) qPCR analysis of Tbx21, Ncr1, Cd122, Idb2, Ltb, and Lair1 mRNA in Ets1+/+ (black) and Ets1−/− (white) pro-NK cells (Lin−CD122+DX5−) and mNK cells. At least three independent experiments were performed.
(C) qPCR analysis of Tbx21 and Ltb mRNA in NKPs. Error bars indicate SD of triplicate measurements. Two independent experiments were performed.
(D) Anti-ETS1 ChIP for EBS in Tbx21 and Cd122. qPCR SP5, Hbb, and Ebf1 are at regions that lack an EBS.
See Table S2 for ETS1 binding sites in CD4+ T cells.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4 Idb2 Is Regulated by ETS1 in NK Cells and Their Precursors
(A) Promoter-luciferase reporter assay via regions 5′ of the Idb2 TSS, including the endogenous EBS site (GGA) or a mutated (m) EBS (GGT). One representative experiment from at least four is shown.
(B) EMSA of proteins in PTL cell extracts binding to the Idb2 EBS in the presence (+) or absence (−) of cold competitor or α-ETS1, α-MEF1, or α-ELF1.
(C) ChIP was performed on sorted splenic mNK cells via α-ETS1 followed by qPCR with primers flanking the EBS in Idb2 or for sequences lacking an EBS (SP5, Hbb, and Ebf1).
(D) qPCR analysis for Idb2, Ets1, Elf1, and Gabpa mRNA relative to Hprt mRNA in sorted NK cell and NK cell progenitor populations.
Error bars represent standard error of triplicate measurements and one of at least two to three replicate experiments is shown. See Figure S2 for Ets1 mRNA expression in NK cell lines and activated NK cells.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5
Ets1−/− mNK Cells Had Decreased Activating NKR Expression and Activating NKR-Triggered Degranulation
(A) Flow cytometry analysis for the activating NKRs NKp46, Ly49D, and Ly49H in BM and spleen of Ets1+/+ (dark line) and Ets1−/− (gray line) mice. Shaded histogram shows isotype control.
(B) Summary of the percent of mNK cells expressing NKp46, Ly49D, and Ly49H. Ets1+/+ (filled), Ets1−/− (open); horizontal bar indicates average. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4; ns, p > 0.05.
(C) Flow cytometry analysis for the activating NKRs NK1.1 and NKG2D on Ets1+/+ and Ets1−/− mNK cells. Histograms are as indicated in (A).
(D and E) Summary of percent (D) CD107a+ or (E) IFN-γ+ after a 5 hr stimulation with PMA plus ionomycin, α-NK1.1, α-NKG2D, or IgG.
See Figure S2 for activating NKR expression in mixed BM chimeras and representative flow cytometery staining for CD107a and IFN-γ.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6
Ets1−/− mNK Cells Have Characteristics of Chronic Cytokine Stimulation
(A) Heat maps for a subset of ETS1-repressed genes in Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells. Asterisk indicates that ETS1 was bound near this gene in CD4+ T cells as determined by ChIP-seq.
(B–D) qPCR analysis for (B) Gzmb, (C) Nfil3, and (D) Ikzf2 mRNA in Ets1+/+ (black) and Ets1−/− (white) mNK cells. Expression is plotted relative to transcripts from Hprt. Error bars represent standard error of triplicate measurements and one of at least two to three replicate experiments are shown.
(E and F) Flow cytometry analysis for (E) SSC and CD69 or (F) the inhibitory NKRs Ly49A, Ly49G2, and Ly49E on BM and splenic mNK cells from Ets1+/+ (black) and Ets1−/− (gray).
(G) Summary of the percent of mNK cells expressing Ly49A, Ly49G2, and Ly49E. Ets1+/+ (filled), Ets1−/− (open), horizontal bar indicates average. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4; ns, p > 0.05.
Cells in (E) were isolated from CD45.1 (WT):CD45.2 (WT or Ets1−/−) chimeric mice. See Figure S3 for Ly49A, Ly49G2, and Ly49E staining on mNK cells from mixed BM chimeras.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions

9. Figure 7 Ets1−/− mNK Cells Are Hyperresponsive to IL-15
(A) Percent of single Ets1+/+ (filled) and Ets1−/− (open) pro-NK and mNK cells giving rise to visible colonies after 10 days in cultures supplemented with IL-2.
(B) FSC versus SSC on typical mNK cell progeny from (A).
(C) Flow cytometry analysis for GZMB and BrdU incorporation in splenic mNK cells 24 hr after initiation of culture in varying concentrations of IL-15. The percent of Granzyme B+ BrdU+ cells is indicated. One of three experiments is shown.
(D) Average ± SD of percent GZMB+ BrdU+ cells from three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < The splenic mNK cells were isolated from BM chimeras.
Immunity  , DOI: ( /j.immuni )
Copyright © 2012 Elsevier Inc. Terms and Conditions