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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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PDK1-mTOR signaling regulates IL-15–induced E4BP4 expression in vitro. (A) Quantitative RT-PCR analysis of NK cell–related genes in sorted wild-type CD3−NK1.1+ cells before and after stimulation with IL-15–IL-15R complexes; the results were normalized to those of β-actin and are presented relative to those of untreated cells, set as 1. Data were pooled from three independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (B) Intracellular staining was used to analyze the expression of E4BP4, Eomes, and T-bet on gated CD3−NK1.1+ cells by flow cytometry before and after stimulation with IL-15–IL-15R complexes; the results were normalized and are presented relative to MFI of untreated cells, set as 1. Data were pooled from four independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (C) Quantitative RT-PCR analysis of E4bp4 in sorted wild-type CD3−NK1.1+ cells after stimulation with IL-15–IL-15R complexes in the presence of the indicated inhibitors or of DMSO (control); the results were normalized to those of β-actin and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (D) Intracellular staining was used to analyze the expression of E4BP4 and Eomes on gated CD3−NK1.1+ cells by flow cytometry after stimulation with IL-15–IL-15R complexes plus indicated inhibitors; the results were normalized and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (E) Intracellular staining was used to analyze the expression of E4BP4 in CD3−CD122high NK1.1+ NK cells by flow cytometry from indicated mice before and after stimulated with IL-15–IL-15R complexes. Representative overlaid histogram is shown (left). Red lines, unstimulated; Blue lines, stimulated. Results are presented relative to MFI values before treatment. Data were pooled from two independent experiments (right, n = 4). ***, P < 0.0005.
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fig1: PDK1-mTOR signaling regulates IL-15–induced E4BP4 expression in vitro. (A) Quantitative RT-PCR analysis of NK cell–related genes in sorted wild-type CD3−NK1.1+ cells before and after stimulation with IL-15–IL-15R complexes; the results were normalized to those of β-actin and are presented relative to those of untreated cells, set as 1. Data were pooled from three independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (B) Intracellular staining was used to analyze the expression of E4BP4, Eomes, and T-bet on gated CD3−NK1.1+ cells by flow cytometry before and after stimulation with IL-15–IL-15R complexes; the results were normalized and are presented relative to MFI of untreated cells, set as 1. Data were pooled from four independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (C) Quantitative RT-PCR analysis of E4bp4 in sorted wild-type CD3−NK1.1+ cells after stimulation with IL-15–IL-15R complexes in the presence of the indicated inhibitors or of DMSO (control); the results were normalized to those of β-actin and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (D) Intracellular staining was used to analyze the expression of E4BP4 and Eomes on gated CD3−NK1.1+ cells by flow cytometry after stimulation with IL-15–IL-15R complexes plus indicated inhibitors; the results were normalized and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (E) Intracellular staining was used to analyze the expression of E4BP4 in CD3−CD122high NK1.1+ NK cells by flow cytometry from indicated mice before and after stimulated with IL-15–IL-15R complexes. Representative overlaid histogram is shown (left). Red lines, unstimulated; Blue lines, stimulated. Results are presented relative to MFI values before treatment. Data were pooled from two independent experiments (right, n = 4). ***, P < 0.0005.

Mentions: E4BP4 was reported to be the most specific transcription factor required for the NK cell lineage development (Gascoyne et al., 2009; Kamizono et al., 2009), but how it is regulated remains unknown. A real-time PCR assay revealed that IL-15 could preferentially and significantly up-regulate mRNAs encoding E4BP4 and Eomes (Fig. 1 A). To evaluate the E4BP4 protein level, we developed an intracellular staining assay to quantify its dynamic changes before and after IL-15 stimulation. To our surprise, E4BP4 was rarely detectable in resting NK cells. Upon IL-15 triggering, E4BP4 exhibited a more than fivefold increase in NK cells, and Eomes, which is considered to be involved downstream of E4BP4 (Male et al., 2014), exhibited slight up-regulation (Fig. 1 B). However, T-bet expression was not enhanced by IL-15 stimulation (Fig. 1 B).


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)

PDK1-mTOR signaling regulates IL-15–induced E4BP4 expression in vitro. (A) Quantitative RT-PCR analysis of NK cell–related genes in sorted wild-type CD3−NK1.1+ cells before and after stimulation with IL-15–IL-15R complexes; the results were normalized to those of β-actin and are presented relative to those of untreated cells, set as 1. Data were pooled from three independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (B) Intracellular staining was used to analyze the expression of E4BP4, Eomes, and T-bet on gated CD3−NK1.1+ cells by flow cytometry before and after stimulation with IL-15–IL-15R complexes; the results were normalized and are presented relative to MFI of untreated cells, set as 1. Data were pooled from four independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (C) Quantitative RT-PCR analysis of E4bp4 in sorted wild-type CD3−NK1.1+ cells after stimulation with IL-15–IL-15R complexes in the presence of the indicated inhibitors or of DMSO (control); the results were normalized to those of β-actin and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (D) Intracellular staining was used to analyze the expression of E4BP4 and Eomes on gated CD3−NK1.1+ cells by flow cytometry after stimulation with IL-15–IL-15R complexes plus indicated inhibitors; the results were normalized and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (E) Intracellular staining was used to analyze the expression of E4BP4 in CD3−CD122high NK1.1+ NK cells by flow cytometry from indicated mice before and after stimulated with IL-15–IL-15R complexes. Representative overlaid histogram is shown (left). Red lines, unstimulated; Blue lines, stimulated. Results are presented relative to MFI values before treatment. Data were pooled from two independent experiments (right, n = 4). ***, P < 0.0005.
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fig1: PDK1-mTOR signaling regulates IL-15–induced E4BP4 expression in vitro. (A) Quantitative RT-PCR analysis of NK cell–related genes in sorted wild-type CD3−NK1.1+ cells before and after stimulation with IL-15–IL-15R complexes; the results were normalized to those of β-actin and are presented relative to those of untreated cells, set as 1. Data were pooled from three independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (B) Intracellular staining was used to analyze the expression of E4BP4, Eomes, and T-bet on gated CD3−NK1.1+ cells by flow cytometry before and after stimulation with IL-15–IL-15R complexes; the results were normalized and are presented relative to MFI of untreated cells, set as 1. Data were pooled from four independent experiments. **, P < 0.005; ***, P < 0.0005 versus control. (C) Quantitative RT-PCR analysis of E4bp4 in sorted wild-type CD3−NK1.1+ cells after stimulation with IL-15–IL-15R complexes in the presence of the indicated inhibitors or of DMSO (control); the results were normalized to those of β-actin and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (D) Intracellular staining was used to analyze the expression of E4BP4 and Eomes on gated CD3−NK1.1+ cells by flow cytometry after stimulation with IL-15–IL-15R complexes plus indicated inhibitors; the results were normalized and are presented relative to those of DMSO-treated cells, set as 1. Data represent the mean ± SEM (3 repeats). **, P < 0.005 versus DMSO. (E) Intracellular staining was used to analyze the expression of E4BP4 in CD3−CD122high NK1.1+ NK cells by flow cytometry from indicated mice before and after stimulated with IL-15–IL-15R complexes. Representative overlaid histogram is shown (left). Red lines, unstimulated; Blue lines, stimulated. Results are presented relative to MFI values before treatment. Data were pooled from two independent experiments (right, n = 4). ***, P < 0.0005.
Mentions: E4BP4 was reported to be the most specific transcription factor required for the NK cell lineage development (Gascoyne et al., 2009; Kamizono et al., 2009), but how it is regulated remains unknown. A real-time PCR assay revealed that IL-15 could preferentially and significantly up-regulate mRNAs encoding E4BP4 and Eomes (Fig. 1 A). To evaluate the E4BP4 protein level, we developed an intracellular staining assay to quantify its dynamic changes before and after IL-15 stimulation. To our surprise, E4BP4 was rarely detectable in resting NK cells. Upon IL-15 triggering, E4BP4 exhibited a more than fivefold increase in NK cells, and Eomes, which is considered to be involved downstream of E4BP4 (Male et al., 2014), exhibited slight up-regulation (Fig. 1 B). However, T-bet expression was not enhanced by IL-15 stimulation (Fig. 1 B).

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