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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.

View Article: PubMed Central - HTML - PubMed

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

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IL-15 augments NK cell metabolic activation and proliferation via PI3K–PDK1–mTOR signaling. Splenocytes from PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were stimulated with recombinant IL-15–IL-15R complexes overnight. (A) Expression of CD71 and CD98 was detected by flow cytometry (left) and was quantified as mean fluorescence intensity. The results are presented relative to unstimulated cells, set as 1 (right). Data represent the mean ± SEM of 5 mice per group. **, P < 0.005. (B). Intracellular phosphorylated S6, AKT T308, and AKT S473 were detected by flow cytometry (left), and the MFI was calculated. The protein expression levels in PDK1 fl/fl NK cells are presented relative to PDK1fl/fl/Vav1-Cre+ mice, set as 1 (right). Data represent the mean ± SEM of 3 mice per group. Data are representative of two independent experiments. **, P < 0.005. (C) WT bone marrow cells were stimulated with recombinant IL-15–IL-15R complexes overnight in the presence of the indicated pharmacological inhibitors or DMSO, as a negative control. Flow cytometry was used to detect CD71 and CD98, and quantification is presented as the MFI. Data represent the mean ± SEM of 3 independent experiments. **, P < 0.005, ***, P < 0.0005. (D) WT mice were injected with IL-15–IL-15R complexes every 3 d together with the mTOR inhibitor Torin1 or DMSO. The absolute number of peripheral blood CD3−NKp46+NK cells was quantified on the indicated days. Fold change was calculated as describe in Fig. 3 D. Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05.
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fig5: IL-15 augments NK cell metabolic activation and proliferation via PI3K–PDK1–mTOR signaling. Splenocytes from PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were stimulated with recombinant IL-15–IL-15R complexes overnight. (A) Expression of CD71 and CD98 was detected by flow cytometry (left) and was quantified as mean fluorescence intensity. The results are presented relative to unstimulated cells, set as 1 (right). Data represent the mean ± SEM of 5 mice per group. **, P < 0.005. (B). Intracellular phosphorylated S6, AKT T308, and AKT S473 were detected by flow cytometry (left), and the MFI was calculated. The protein expression levels in PDK1 fl/fl NK cells are presented relative to PDK1fl/fl/Vav1-Cre+ mice, set as 1 (right). Data represent the mean ± SEM of 3 mice per group. Data are representative of two independent experiments. **, P < 0.005. (C) WT bone marrow cells were stimulated with recombinant IL-15–IL-15R complexes overnight in the presence of the indicated pharmacological inhibitors or DMSO, as a negative control. Flow cytometry was used to detect CD71 and CD98, and quantification is presented as the MFI. Data represent the mean ± SEM of 3 independent experiments. **, P < 0.005, ***, P < 0.0005. (D) WT mice were injected with IL-15–IL-15R complexes every 3 d together with the mTOR inhibitor Torin1 or DMSO. The absolute number of peripheral blood CD3−NKp46+NK cells was quantified on the indicated days. Fold change was calculated as describe in Fig. 3 D. Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05.

Mentions: PDK1 is a master molecule linking the PI3K pathway with mTOR activation via Akt. To explore whether mTOR-dependent PDK1 signaling is involved in the early NK cell development promoted by IL-15, we stimulated PDK1−/− splenocyte with recombinant IL-15–IL-15R complexes and detected the expression of nutritional receptors and mTOR signaling in CD3−CD122highNK1.1+ cells, which could eliminate the developmental discrepancy in CD122 levels between the two genotypes. We found that after overnight IL-15 stimulation, PDK1-sufficient NK cells displayed a two-to fivefold increase in CD71 and CD98, two nutritional receptors, whereas PDK1−/− NK cells nearly lost the ability to up-regulate these receptors (Fig. 5 A), and these cells consistently displayed impaired activation of AKT-mTOR1 signaling upon IL-15 exposure (Fig. 5 B). To further exclude the possibility that the impaired metabolic activation by IL-15 is caused by the variation in CD122 levels between the genotypes, several pharmacological inhibitors were chosen. Blockage of PI3K–PDK1–mTOR activation largely prevented the IL-15–triggered up-regulation of CD71 and CD98 on bone marrow and splenic NK cells (Fig. 5 C and not depicted), further demonstrating that IL-15 is able to trigger NK cell metabolic activation, which requires mTOR-dependent PI3K signaling. To directly test mTOR signaling in NK cell development, wild-type mice were injected with Torin1 to suppress mTOR activity. As expected, the proproliferative role of IL-15 was notably diminished by mTOR inhibition (Fig. 5 D). Together, these data demonstrate that PDK1 signaling is required for NK cell metabolic activation and proliferation downstream of the IL-15 receptor via activating mTOR.


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)

IL-15 augments NK cell metabolic activation and proliferation via PI3K–PDK1–mTOR signaling. Splenocytes from PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were stimulated with recombinant IL-15–IL-15R complexes overnight. (A) Expression of CD71 and CD98 was detected by flow cytometry (left) and was quantified as mean fluorescence intensity. The results are presented relative to unstimulated cells, set as 1 (right). Data represent the mean ± SEM of 5 mice per group. **, P < 0.005. (B). Intracellular phosphorylated S6, AKT T308, and AKT S473 were detected by flow cytometry (left), and the MFI was calculated. The protein expression levels in PDK1 fl/fl NK cells are presented relative to PDK1fl/fl/Vav1-Cre+ mice, set as 1 (right). Data represent the mean ± SEM of 3 mice per group. Data are representative of two independent experiments. **, P < 0.005. (C) WT bone marrow cells were stimulated with recombinant IL-15–IL-15R complexes overnight in the presence of the indicated pharmacological inhibitors or DMSO, as a negative control. Flow cytometry was used to detect CD71 and CD98, and quantification is presented as the MFI. Data represent the mean ± SEM of 3 independent experiments. **, P < 0.005, ***, P < 0.0005. (D) WT mice were injected with IL-15–IL-15R complexes every 3 d together with the mTOR inhibitor Torin1 or DMSO. The absolute number of peripheral blood CD3−NKp46+NK cells was quantified on the indicated days. Fold change was calculated as describe in Fig. 3 D. Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05.
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fig5: IL-15 augments NK cell metabolic activation and proliferation via PI3K–PDK1–mTOR signaling. Splenocytes from PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were stimulated with recombinant IL-15–IL-15R complexes overnight. (A) Expression of CD71 and CD98 was detected by flow cytometry (left) and was quantified as mean fluorescence intensity. The results are presented relative to unstimulated cells, set as 1 (right). Data represent the mean ± SEM of 5 mice per group. **, P < 0.005. (B). Intracellular phosphorylated S6, AKT T308, and AKT S473 were detected by flow cytometry (left), and the MFI was calculated. The protein expression levels in PDK1 fl/fl NK cells are presented relative to PDK1fl/fl/Vav1-Cre+ mice, set as 1 (right). Data represent the mean ± SEM of 3 mice per group. Data are representative of two independent experiments. **, P < 0.005. (C) WT bone marrow cells were stimulated with recombinant IL-15–IL-15R complexes overnight in the presence of the indicated pharmacological inhibitors or DMSO, as a negative control. Flow cytometry was used to detect CD71 and CD98, and quantification is presented as the MFI. Data represent the mean ± SEM of 3 independent experiments. **, P < 0.005, ***, P < 0.0005. (D) WT mice were injected with IL-15–IL-15R complexes every 3 d together with the mTOR inhibitor Torin1 or DMSO. The absolute number of peripheral blood CD3−NKp46+NK cells was quantified on the indicated days. Fold change was calculated as describe in Fig. 3 D. Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05.
Mentions: PDK1 is a master molecule linking the PI3K pathway with mTOR activation via Akt. To explore whether mTOR-dependent PDK1 signaling is involved in the early NK cell development promoted by IL-15, we stimulated PDK1−/− splenocyte with recombinant IL-15–IL-15R complexes and detected the expression of nutritional receptors and mTOR signaling in CD3−CD122highNK1.1+ cells, which could eliminate the developmental discrepancy in CD122 levels between the two genotypes. We found that after overnight IL-15 stimulation, PDK1-sufficient NK cells displayed a two-to fivefold increase in CD71 and CD98, two nutritional receptors, whereas PDK1−/− NK cells nearly lost the ability to up-regulate these receptors (Fig. 5 A), and these cells consistently displayed impaired activation of AKT-mTOR1 signaling upon IL-15 exposure (Fig. 5 B). To further exclude the possibility that the impaired metabolic activation by IL-15 is caused by the variation in CD122 levels between the genotypes, several pharmacological inhibitors were chosen. Blockage of PI3K–PDK1–mTOR activation largely prevented the IL-15–triggered up-regulation of CD71 and CD98 on bone marrow and splenic NK cells (Fig. 5 C and not depicted), further demonstrating that IL-15 is able to trigger NK cell metabolic activation, which requires mTOR-dependent PI3K signaling. To directly test mTOR signaling in NK cell development, wild-type mice were injected with Torin1 to suppress mTOR activity. As expected, the proproliferative role of IL-15 was notably diminished by mTOR inhibition (Fig. 5 D). Together, these data demonstrate that PDK1 signaling is required for NK cell metabolic activation and proliferation downstream of the IL-15 receptor via activating mTOR.

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