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. 2012 Jan 27;36(1):55-67.
doi: 10.1016/j.immuni.2011.11.016. Epub 2012 Jan 18.

The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation

Scott M Gordon  1 , Julie Chaix, Levi J Rupp, Junmin Wu, Sharline Madera, Joseph C Sun, Tullia Lindsten, Steven L Reiner
Affiliations

The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation

Scott M Gordon et al. Immunity. .

"V体育2025版" Abstract

Natural killer (NK) cells play critical roles defending against tumors and pathogens VSports手机版. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis. .

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"V体育安卓版" Conflict of interest statement

The authors declare no competing financial interests or other conflicts of interest.

Figures

Figure 1
Figure 1. Eomes required for maturation of NK cells
(A) NK immaturity marker TRAIL correlates with absence of Eomes, and maturity marker DX5 correlates with presence of Eomes in wild-type (WT) splenic and hepatic NK cells. Plots represent CD3ε−CD19−Gr-1− NK1.1+NKp46+CD122+ cells (NK cells). Data are representative of at least 5 experiments. (B) Integrin αv, CXCR3, and CXCR6 are expressed predominantly by DX5− NK cells. Plots represent hepatic NK cells. Integrin αv+CXCR3+CXCR6+ TRAIL+ NK cells were also present at low frequency in spleen and bone marrow (data not shown). “CXCR6” represents detection of CXCL16-Fc, which binds to surface CXCR6 (Matloubian et al., 2000). Data are representative of 3–5 experiments. (C) TRAIL+ NK cells express low amounts of S1pr1 (encoding S1P1) and S1pr5 (encoding S1P5) compared to DX5+ NK cells as assessed by qRT-PCR. Data are representative of 2 experiments. (D) EomesFlox/Flox, Vav-Cre+ mice are deficient in percentage and absolute numbers of NK cells. Data are representative of at least 5 independent experiments. “Lin” denotes staining with anti-CD3, -CD19, -Gr-1, and -Ter119. Error bars indicate standard deviation. *, p<0.05; **, p<0.001. (E and F) EomesFlox/Flox, Vav-Cre+ and wild-type Eomes− NK cells express markers of immaturity, while Eomes+ NK cells express markers of maturity. Analysis of maturation markers TRAIL and DX5 (E) and Ly49 receptors (F) in EomesFlox/Flox, Vav-Cre+, wild-type Eomes−, and wild-type Eomes+ hepatic NK cells. Data are representative of 3–5 experiments. (G) Expression of maturity markers CD27 and CD11b on hepatic EomesFlox/Flox, Vav-Cre+ and wild-type Eomes− and Eomes+ NK cells. Data are representative of 3 experiments. (H) Kinetic analysis of NK cell maturation (Eomes expression) in mice of indicated ages. Day 0 (d0) denotes the day of birth. Data are representative of 2 experiments. (I) Analysis of maturity markers TRAIL and DX5 in adult medullary NK cells of the indicated states of maturity. Data are representative of 3 experiments. See also Figure S1.
Figure 2
Figure 2. Eomes− NK cells can give rise to Eomes+ NK cells
(A) Highly purified, wild-type TRAIL+ or DX5+ donor NK cells were adoptively transferred into Il2rg−/−Rag2−/− recipients. One week post-transfer, indicated organs of recipient mice were harvested and donor NK cells were analyzed for expression of Eomes, Ly49 receptors, and maturation markers TRAIL and DX5. This assay reveals TRAIL+Eomes+ NK cells, which may represent a transient, intermediate stage that is ordinarily out-populated by TRAIL−DX5+Eomes+ cells in intact adults. (B) Two weeks post-transfer of wild-type TRAIL+ NK cells, donor NK cells were analyzed for indicated markers. (C) Hepatic wild-type or EomesFlox/Flox, Vav-Cre+ TRAIL+ donor NK cells were adoptively transferred into Il2rg−/−Rag2−/− recipients. Bone marrow of recipient mice was harvested after 1w and donor NK cells were analyzed for expression of indicated markers. (D) Medullary TRAIL+ or DX5+ NK cells of indicated sub-stages of maturity were adoptively transferred into Il2rg−/−Rag2−/− recipients. Indicated organs of recipient mice were harvested after 1w, and donor NK cells were analyzed for expression of Eomes and maturation markers TRAIL, CD27, and CD11b. Data are representative of 3 experiments. See also Figure S2.
Figure 3
Figure 3. Eomes required to maintain some aspects of mature NK cells
(A and B) Splenic NK cells with floxed alleles of Eomes were purified and either treated with TAT-Cre or sham-treated directly ex vivo. Donor cells were transferred into Il2rg−/−Rag2−/− recipients and analyzed 7–10d later. Expression of Ly49A, Ly49G2, Ly49H (A) and Ly49D (B) was stable in mature NK cells from which Eomes was temporally deleted. Data are representative of at least 2 separate experiments. (B) Derepression of TRAIL and downregulation of CD49b by mature NK cells after temporal deletion of Eomes. Ly49D+ NK cells were analyzed for maturation markers TRAIL and DX5 (bottom rows). Data are representative of 5 experiments. See also Figure S3.
Figure 4
Figure 4. T-bet stabilizes immature, Eomes− NK cells and either T-bet or Eomes required for NK lineage marker stability
(A and B) Expression of maturation markers TRAIL and DX5 (A), along with expression of T-bet and Eomes (B), in wild-type, EomesFlox/Flox, Vav-Cre+, and Tbx21−/− hepatic NK cells. Data representative of at least 5 experiments. (C and D) T-bet-deficient neonates are deficient in NK cells. (C) Developmental analysis of hepatic and splenic NK cell compartments in wild-type and Tbx21−/− mice of indicated ages. Day 0 (d0) denotes the day of birth. Data are representative of at least 5 experiments. (D) Quantification of absolute number of NK cells in wild-type and Tbx21−/− neonatal mice, aged d0 to d2. Error bars indicate standard deviation. **, p<0.001; ***, p<0.0001. (E) Acute loss of T-bet destabilizes TRAIL+ NK cells. Hepatic NK cells with floxed alleles of Tbx21 were purified and either treated with TAT-Cre or sham-treated directly ex vivo. Donor cells were transferred into Il2rg−/−Rag2−/− recipients and analyzed 7–10d later. NK cells from livers of recipient mice were analyzed for expression of Eomes and Ly49 receptors. Data are representative of 2 experiments. (F) Absence of NK cells in indicated organs of mice doubly deficient in T-bet and Eomes. Data are representative of 2 experiments. (G) Loss of NK lineage antigens after acute loss of Eomes in the setting of T-bet deficiency. Purified, Tbx21−/−EomesFlox/Flox NK cells transferred to Il2rg−/−Rag2−/− recipient mice in vivo (top row) or cultured in 2000U/mL rIL-2 in vitro (bottom row) after TAT-Cre or sham-treatment. Organs of recipient mice were analyzed 1w after adoptive transfer. Cells cultured in IL-2 were analyzed after 5d. Plots represent presently or formerly mature NK cells, based on purified starting population and gating of CD3− (Ly49D+ and/or LyG2+) cells (gating shown in Figure S4M). CD3−Ly49+ cells were analyzed for expression of Eomes and the NK lineage antigens NK1.1 and NKp46. Data are representative of 3 in vitro and 2 in vivo experiments. (H) Loss of DX5 and TRAIL staining in T-bet-deficient NK cells following temporal deletion of Eomes. CD3−Ly49D+ cells as depicted in upper row of panel G were further analyzed for staining of CD49b (DX5) and TRAIL. See also Figure S4.
Figure 5
Figure 5. Functional analyses of NK cells lacking T-bet and Eomes
(A–C) Expression of effector genes and proteins by TRAIL+Eomes− and DX5+Eomes+ NK cells. (A) Levels of mRNA for genes encoding Eomes, Perforin, and Granzyme C were analyzed by qRT-PCR. Error bars indicate standard error of the mean (SEM). (B) Expression of Granzyme B (Gzmb) by wild-type, unstimulated, hepatic Eomes− and Eomes+ NK cells (far left panel), degranulation by wild-type hepatic Eomes− and Eomes+ NK cells after stimulation with PMA and Iono for 4h in vitro (left middle panel), expression of IFN-γ by wild-type hepatic Eomes− and Eomes+ NK cells after stimulation with rIL-12 and rIL-18 for 4h in vitro (right middle panel), and expression of TNF-α by wild-type hepatic Eomes− and Eomes+ NK cells after stimulation with PMA and Iono for 4h in vitro (far right panel). (C) Expression of Granzyme B (Gzmb) by wild-type, EomesFlox/Flox, Vav-Cre+, and Tbx21−/−, unstimulated, hepatic NK cells (top row) and expression of IFN-γ by wild-type, EomesFlox/Flox, Vav-Cre+, and Tbx21−/− hepatic Eomes− and Eomes+ NK cells after stimulation with recombinant IL-12 and IL-18 for 4h in vitro (bottom rows). (D–F) Response by wild-type, Tbx21−/−, and EomesFlox/Flox, Vav-Cre+ NK cells 7 days after MCMV infection. (D) Analysis of Ly49D and Ly49H expression by splenic (top) and hepatic (bottom) NK cells from MCMV-infected mice of indicated genotypes. (E) Expression of maturation and activation markers KLRG1, CD11b, TRAIL, and DX5 by hepatic Ly49H+ and Ly49H− NK cells of indicated genotypes. (F) More highly activated, hepatic NK cells from MCMV-infected EomesFlox/Flox, Vav-Cre+ mice can induce CD49b and repress TRAIL. All data representative of at least 2 experiments. See also Figure S5.
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