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PDK1 orchestrates early NK cell development through induction of E4BP4 expression and maintenance of IL-15 responsiveness.

Yang M, Li D, Chang Z, Yang Z, Tian Z, Dong Z - J. Exp. Med. (2015)

Bottom Line: It remains largely unknown which signal is required to induce E4BP4 expression and what effects it has during NK cell differentiation.Thus, we identify a role for PDK1 signaling as a key mediator in regulating E4BP4 expression during early NK cell development.Our findings underscore the importance of IL-15 self-responsiveness through a positive feedback loop that involves PDK1-mTOR-E4BP4-CD122 signaling.

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Affiliation: School of Medicine, and Center of Animal Facility, Tsinghua University, Beijing 100086, China.

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Severely reduced NK cellularity and loss of NK cell activity in PDK1-deficient mice. (A) CD3−NKp46+ NK cells were isolated from spleen and bone marrow, and percentages are indicated in representative flow cytometric plots. (B) Absolute numbers of CD3−NKp46+ NK cells are also indicated in tissues and organs from PDK1fl/fl and PDK1fl/fl/Vav1-Cre+ mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data were pooled from two individual experiments (n = 4 or more). **, P < 0.005; ***, P < 0.0005. (C) Mice were treated with 5-FU before bone marrow was isolated and depleted of B220+ and CD3+ and NK1.1+ cells. Depleted bone marrow from CD45.1 WT mice was mixed with either WT or PDK1−/− bone marrow cells expressing CD45.2 at a 1:1 ratio. Cells were then injected into sublethally irradiated RAG1−/−γc− recipient mice. CD45.1 versus CD45.2 expression on gated CD3−CD122+NKp46+ was detected by flow cytometry. The numbers show the percentages in relevant quadrant. (D, left) Representative flow cytometry of CFSE+ cells obtained from spleen, LN, or blood of the indicated recipient mice 18 h after injection with an equal number of WT or β2-microglobulin (β2M)–deficient splenocytes labeled with various concentrations of the cytosolic dye CFSE. R1, CFSElow splenocytes from WT mice; R2, CFSEhigh splenocytes from β2M-deficient mice. (D, right) Quantification of the percent rejection of β2M-deficient splenocytes. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 6 mice per group). (E) Representative flow cytometry plot (left) and quantification of the percentages (right, percent rejection) of RMA-S cells in the peritoneal cavity on 18 h after intraperitoneal injection in the indicated mice. A mixture of NK cell–sensitive RMA-S cells expressing green fluorescent protein (GFP; outlined in red) together with NK cell-resistant RMA cells expressing the fluorescent protein DsRed (outlined in black) were injected. The numbers near square boxes represent percentages of RMA-S or RMA relative to the total number of injected cells. Immunocompromised RAG1−/−γc− mice were used as a control. Injected cells are shown. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 4 mice per group). Data are representative of two independent experiments. ***, P < 0.0005.
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fig2: Severely reduced NK cellularity and loss of NK cell activity in PDK1-deficient mice. (A) CD3−NKp46+ NK cells were isolated from spleen and bone marrow, and percentages are indicated in representative flow cytometric plots. (B) Absolute numbers of CD3−NKp46+ NK cells are also indicated in tissues and organs from PDK1fl/fl and PDK1fl/fl/Vav1-Cre+ mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data were pooled from two individual experiments (n = 4 or more). **, P < 0.005; ***, P < 0.0005. (C) Mice were treated with 5-FU before bone marrow was isolated and depleted of B220+ and CD3+ and NK1.1+ cells. Depleted bone marrow from CD45.1 WT mice was mixed with either WT or PDK1−/− bone marrow cells expressing CD45.2 at a 1:1 ratio. Cells were then injected into sublethally irradiated RAG1−/−γc− recipient mice. CD45.1 versus CD45.2 expression on gated CD3−CD122+NKp46+ was detected by flow cytometry. The numbers show the percentages in relevant quadrant. (D, left) Representative flow cytometry of CFSE+ cells obtained from spleen, LN, or blood of the indicated recipient mice 18 h after injection with an equal number of WT or β2-microglobulin (β2M)–deficient splenocytes labeled with various concentrations of the cytosolic dye CFSE. R1, CFSElow splenocytes from WT mice; R2, CFSEhigh splenocytes from β2M-deficient mice. (D, right) Quantification of the percent rejection of β2M-deficient splenocytes. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 6 mice per group). (E) Representative flow cytometry plot (left) and quantification of the percentages (right, percent rejection) of RMA-S cells in the peritoneal cavity on 18 h after intraperitoneal injection in the indicated mice. A mixture of NK cell–sensitive RMA-S cells expressing green fluorescent protein (GFP; outlined in red) together with NK cell-resistant RMA cells expressing the fluorescent protein DsRed (outlined in black) were injected. The numbers near square boxes represent percentages of RMA-S or RMA relative to the total number of injected cells. Immunocompromised RAG1−/−γc− mice were used as a control. Injected cells are shown. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 4 mice per group). Data are representative of two independent experiments. ***, P < 0.0005.

Mentions: To identify a potential role for PDK1-medated signaling in NK cell physiology, we extensively analyzed NK cell development and function in PDK1−/− mice. Compared with PDK1-sufficient mice, PDK1−/− mice exhibited a nearly 95% reduction in the number of NK cells in the spleen and bone marrow (Fig. 2, A and B). The remarkable reduction in NK cell pool could also be found in other lymphoid organs, including the lymph nodes, liver, and lungs (Fig. 2 B). To investigate the cell-intrinsic effect of PDK1 for NK cell development, bone marrow mixtures were adoptively transferred into sublethally irradiated immunodeficient RAG1−/−γc− mice. In contrast to CD45.2+ WT, PDK1−/− bone marrow cells failed to reconstitute NK cell pool efficiently (Fig. 2 C), suggesting the critical requirement for PDK1 in NK cell development is cell intrinsic.


PDK1 orchestrates early NK cell development through induction of E4BP4 expression and maintenance of IL-15 responsiveness.

Yang M, Li D, Chang Z, Yang Z, Tian Z, Dong Z - J. Exp. Med. (2015)

Severely reduced NK cellularity and loss of NK cell activity in PDK1-deficient mice. (A) CD3−NKp46+ NK cells were isolated from spleen and bone marrow, and percentages are indicated in representative flow cytometric plots. (B) Absolute numbers of CD3−NKp46+ NK cells are also indicated in tissues and organs from PDK1fl/fl and PDK1fl/fl/Vav1-Cre+ mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data were pooled from two individual experiments (n = 4 or more). **, P < 0.005; ***, P < 0.0005. (C) Mice were treated with 5-FU before bone marrow was isolated and depleted of B220+ and CD3+ and NK1.1+ cells. Depleted bone marrow from CD45.1 WT mice was mixed with either WT or PDK1−/− bone marrow cells expressing CD45.2 at a 1:1 ratio. Cells were then injected into sublethally irradiated RAG1−/−γc− recipient mice. CD45.1 versus CD45.2 expression on gated CD3−CD122+NKp46+ was detected by flow cytometry. The numbers show the percentages in relevant quadrant. (D, left) Representative flow cytometry of CFSE+ cells obtained from spleen, LN, or blood of the indicated recipient mice 18 h after injection with an equal number of WT or β2-microglobulin (β2M)–deficient splenocytes labeled with various concentrations of the cytosolic dye CFSE. R1, CFSElow splenocytes from WT mice; R2, CFSEhigh splenocytes from β2M-deficient mice. (D, right) Quantification of the percent rejection of β2M-deficient splenocytes. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 6 mice per group). (E) Representative flow cytometry plot (left) and quantification of the percentages (right, percent rejection) of RMA-S cells in the peritoneal cavity on 18 h after intraperitoneal injection in the indicated mice. A mixture of NK cell–sensitive RMA-S cells expressing green fluorescent protein (GFP; outlined in red) together with NK cell-resistant RMA cells expressing the fluorescent protein DsRed (outlined in black) were injected. The numbers near square boxes represent percentages of RMA-S or RMA relative to the total number of injected cells. Immunocompromised RAG1−/−γc− mice were used as a control. Injected cells are shown. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 4 mice per group). Data are representative of two independent experiments. ***, P < 0.0005.
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fig2: Severely reduced NK cellularity and loss of NK cell activity in PDK1-deficient mice. (A) CD3−NKp46+ NK cells were isolated from spleen and bone marrow, and percentages are indicated in representative flow cytometric plots. (B) Absolute numbers of CD3−NKp46+ NK cells are also indicated in tissues and organs from PDK1fl/fl and PDK1fl/fl/Vav1-Cre+ mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data were pooled from two individual experiments (n = 4 or more). **, P < 0.005; ***, P < 0.0005. (C) Mice were treated with 5-FU before bone marrow was isolated and depleted of B220+ and CD3+ and NK1.1+ cells. Depleted bone marrow from CD45.1 WT mice was mixed with either WT or PDK1−/− bone marrow cells expressing CD45.2 at a 1:1 ratio. Cells were then injected into sublethally irradiated RAG1−/−γc− recipient mice. CD45.1 versus CD45.2 expression on gated CD3−CD122+NKp46+ was detected by flow cytometry. The numbers show the percentages in relevant quadrant. (D, left) Representative flow cytometry of CFSE+ cells obtained from spleen, LN, or blood of the indicated recipient mice 18 h after injection with an equal number of WT or β2-microglobulin (β2M)–deficient splenocytes labeled with various concentrations of the cytosolic dye CFSE. R1, CFSElow splenocytes from WT mice; R2, CFSEhigh splenocytes from β2M-deficient mice. (D, right) Quantification of the percent rejection of β2M-deficient splenocytes. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 6 mice per group). (E) Representative flow cytometry plot (left) and quantification of the percentages (right, percent rejection) of RMA-S cells in the peritoneal cavity on 18 h after intraperitoneal injection in the indicated mice. A mixture of NK cell–sensitive RMA-S cells expressing green fluorescent protein (GFP; outlined in red) together with NK cell-resistant RMA cells expressing the fluorescent protein DsRed (outlined in black) were injected. The numbers near square boxes represent percentages of RMA-S or RMA relative to the total number of injected cells. Immunocompromised RAG1−/−γc− mice were used as a control. Injected cells are shown. Each symbol represents an individual mouse; small horizontal lines indicate the mean (n = 4 mice per group). Data are representative of two independent experiments. ***, P < 0.0005.
Mentions: To identify a potential role for PDK1-medated signaling in NK cell physiology, we extensively analyzed NK cell development and function in PDK1−/− mice. Compared with PDK1-sufficient mice, PDK1−/− mice exhibited a nearly 95% reduction in the number of NK cells in the spleen and bone marrow (Fig. 2, A and B). The remarkable reduction in NK cell pool could also be found in other lymphoid organs, including the lymph nodes, liver, and lungs (Fig. 2 B). To investigate the cell-intrinsic effect of PDK1 for NK cell development, bone marrow mixtures were adoptively transferred into sublethally irradiated immunodeficient RAG1−/−γc− mice. In contrast to CD45.2+ WT, PDK1−/− bone marrow cells failed to reconstitute NK cell pool efficiently (Fig. 2 C), suggesting the critical requirement for PDK1 in NK cell development is cell intrinsic.

Bottom Line: It remains largely unknown which signal is required to induce E4BP4 expression and what effects it has during NK cell differentiation.Thus, we identify a role for PDK1 signaling as a key mediator in regulating E4BP4 expression during early NK cell development.Our findings underscore the importance of IL-15 self-responsiveness through a positive feedback loop that involves PDK1-mTOR-E4BP4-CD122 signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Medicine, and Center of Animal Facility, Tsinghua University, Beijing 100086, China.

Show MeSH
Related in: MedlinePlus