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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 regulates CD122 expression and IL-15 responsiveness in vivo. (A) Representative flow cytometry profile of CD122 versus NK1.1 expression on CD3− bone marrow cells. Three populations, CD122highNK1.1+ (R1), CD122intNK1.1int (R2), and CD122−NK1.1int (R3), are outlined (left). Absolute numbers of indicated NK cell populations from spleen and bone marrow are also shown (right). Data were pooled from two independent experiments (n = 6). **, P < 0.005, ***, P < 0.0005. (B). Representative overlaid histograms demonstrating CD122 expression on gated R1 NK cells in the spleens and BM of PDK1fl/fl (WT) and PDK1fl/fl/Vav1-Cre+ (KO) mice (left); the absolute MFI (ΔMFI) were quantified (right). Data were pooled from two independent experiments (n = 6). *, P < 0.05. (C). Profiling developmental markers on BM CD3−CD122+NK1.1+ cells from PDK1fl/fl mice and BM CD3−CD122−NK1.1int NK cells in PDK1fl/fl/ Vav1-Cre+ mice. n = 5 per group. ***, P < 0.0005. (D). PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were injected with IL-15–IL-15R complexes every 3 d. The absolute number of peripheral blood CD3−NKp46+NK cells was monitored on the indicated days. Fold change was calculated simply as the ratio of the NK cell number at each time point after IL-15–IL-15R treatment to the initial NK cell number in untreated mice (day 0). Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05. (E) Representative flow cytometry plot showing Annexin V staining and Caspase activity in naive NK cells from the indicated mice (left). Numbers adjacent to the outlined areas (left) indicate the percentage of Annexin V– or caspase-positive cells. Quantifications were performed from two independent experiments (right, n = 5). *, P < 0.05. (F). Intracellular staining of Bcl2 and Bim in naive NK cells from the indicated mice (left). Quantifications were performed from two independent experiments (right, n = 5). **, P < 0.005.
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fig3: PDK1 regulates CD122 expression and IL-15 responsiveness in vivo. (A) Representative flow cytometry profile of CD122 versus NK1.1 expression on CD3− bone marrow cells. Three populations, CD122highNK1.1+ (R1), CD122intNK1.1int (R2), and CD122−NK1.1int (R3), are outlined (left). Absolute numbers of indicated NK cell populations from spleen and bone marrow are also shown (right). Data were pooled from two independent experiments (n = 6). **, P < 0.005, ***, P < 0.0005. (B). Representative overlaid histograms demonstrating CD122 expression on gated R1 NK cells in the spleens and BM of PDK1fl/fl (WT) and PDK1fl/fl/Vav1-Cre+ (KO) mice (left); the absolute MFI (ΔMFI) were quantified (right). Data were pooled from two independent experiments (n = 6). *, P < 0.05. (C). Profiling developmental markers on BM CD3−CD122+NK1.1+ cells from PDK1fl/fl mice and BM CD3−CD122−NK1.1int NK cells in PDK1fl/fl/ Vav1-Cre+ mice. n = 5 per group. ***, P < 0.0005. (D). PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were injected with IL-15–IL-15R complexes every 3 d. The absolute number of peripheral blood CD3−NKp46+NK cells was monitored on the indicated days. Fold change was calculated simply as the ratio of the NK cell number at each time point after IL-15–IL-15R treatment to the initial NK cell number in untreated mice (day 0). Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05. (E) Representative flow cytometry plot showing Annexin V staining and Caspase activity in naive NK cells from the indicated mice (left). Numbers adjacent to the outlined areas (left) indicate the percentage of Annexin V– or caspase-positive cells. Quantifications were performed from two independent experiments (right, n = 5). *, P < 0.05. (F). Intracellular staining of Bcl2 and Bim in naive NK cells from the indicated mice (left). Quantifications were performed from two independent experiments (right, n = 5). **, P < 0.005.

Mentions: Because PDK1-deficient NK cells were resistant to IL-15–induced E4BP4 up-regulation, the impaired NK cell development probably resulted from the compromised IL-15 signaling. We initially detected IL-15–dependent lineage cells in PDK1−/− mice. The absolute numbers of NK cells (CD3−NK1.1+), NK-T cells (CD3+NK1.1int), and memory CD8+ T cells (CD8+CD122+) were remarkably diminished in PDK1−/− mice (unpublished data). To further clarify this effect, we analyzed CD122 expression on CD3−NK1.1+ cells. PDK1−/− mice exhibited dramatically fewer CD3− CD122 high NK1.1+ cells (R1) in the detected lymphoid organs, like bone marrow, spleen, lung, and liver (Fig. 3 A and not depicted). Additionally, the CD122 intensity on PDK1−/− CD3−NK1.1+ cells was significantly lower and had a tendency to be gradually attenuated (Fig. 3, A and B). Surprisingly, there was a noticeable population of CD122-negative NK cells, which exhibited the distinct phenotype of intermediate expression of NK1.1, called NK1.1int. These cells accumulated mainly in the bone marrow rather than other organs (data not shown), of PDK1−/− mice, whereas the population was rarely seen in WT mice (Fig. 3 A). To further characterize this population, we confirmed that the cells expressed high levels of CD117 and CD127, the two earliest markers for NK cells (Fig. 3 C), and were not reactive for CD1d tetramer binding (unpublished data); this excluded the possibility of the cells being NK-T cells, which also exhibit NK1.1int. The population was negative for NKp46, a marker for type I group of innate lymphoid cell (ILC1; Fig. 3 C). Collectively, these data indicate that PDK1 is essential for preserving a CD122high state during early NK cell development.


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 regulates CD122 expression and IL-15 responsiveness in vivo. (A) Representative flow cytometry profile of CD122 versus NK1.1 expression on CD3− bone marrow cells. Three populations, CD122highNK1.1+ (R1), CD122intNK1.1int (R2), and CD122−NK1.1int (R3), are outlined (left). Absolute numbers of indicated NK cell populations from spleen and bone marrow are also shown (right). Data were pooled from two independent experiments (n = 6). **, P < 0.005, ***, P < 0.0005. (B). Representative overlaid histograms demonstrating CD122 expression on gated R1 NK cells in the spleens and BM of PDK1fl/fl (WT) and PDK1fl/fl/Vav1-Cre+ (KO) mice (left); the absolute MFI (ΔMFI) were quantified (right). Data were pooled from two independent experiments (n = 6). *, P < 0.05. (C). Profiling developmental markers on BM CD3−CD122+NK1.1+ cells from PDK1fl/fl mice and BM CD3−CD122−NK1.1int NK cells in PDK1fl/fl/ Vav1-Cre+ mice. n = 5 per group. ***, P < 0.0005. (D). PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were injected with IL-15–IL-15R complexes every 3 d. The absolute number of peripheral blood CD3−NKp46+NK cells was monitored on the indicated days. Fold change was calculated simply as the ratio of the NK cell number at each time point after IL-15–IL-15R treatment to the initial NK cell number in untreated mice (day 0). Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05. (E) Representative flow cytometry plot showing Annexin V staining and Caspase activity in naive NK cells from the indicated mice (left). Numbers adjacent to the outlined areas (left) indicate the percentage of Annexin V– or caspase-positive cells. Quantifications were performed from two independent experiments (right, n = 5). *, P < 0.05. (F). Intracellular staining of Bcl2 and Bim in naive NK cells from the indicated mice (left). Quantifications were performed from two independent experiments (right, n = 5). **, P < 0.005.
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fig3: PDK1 regulates CD122 expression and IL-15 responsiveness in vivo. (A) Representative flow cytometry profile of CD122 versus NK1.1 expression on CD3− bone marrow cells. Three populations, CD122highNK1.1+ (R1), CD122intNK1.1int (R2), and CD122−NK1.1int (R3), are outlined (left). Absolute numbers of indicated NK cell populations from spleen and bone marrow are also shown (right). Data were pooled from two independent experiments (n = 6). **, P < 0.005, ***, P < 0.0005. (B). Representative overlaid histograms demonstrating CD122 expression on gated R1 NK cells in the spleens and BM of PDK1fl/fl (WT) and PDK1fl/fl/Vav1-Cre+ (KO) mice (left); the absolute MFI (ΔMFI) were quantified (right). Data were pooled from two independent experiments (n = 6). *, P < 0.05. (C). Profiling developmental markers on BM CD3−CD122+NK1.1+ cells from PDK1fl/fl mice and BM CD3−CD122−NK1.1int NK cells in PDK1fl/fl/ Vav1-Cre+ mice. n = 5 per group. ***, P < 0.0005. (D). PDK1fl/fl or PDK1fl/fl/Vav1-Cre+ mice were injected with IL-15–IL-15R complexes every 3 d. The absolute number of peripheral blood CD3−NKp46+NK cells was monitored on the indicated days. Fold change was calculated simply as the ratio of the NK cell number at each time point after IL-15–IL-15R treatment to the initial NK cell number in untreated mice (day 0). Data represent the mean ± SEM of 3 mice per time point and are representative of two independent experiments. *, P < 0.05. (E) Representative flow cytometry plot showing Annexin V staining and Caspase activity in naive NK cells from the indicated mice (left). Numbers adjacent to the outlined areas (left) indicate the percentage of Annexin V– or caspase-positive cells. Quantifications were performed from two independent experiments (right, n = 5). *, P < 0.05. (F). Intracellular staining of Bcl2 and Bim in naive NK cells from the indicated mice (left). Quantifications were performed from two independent experiments (right, n = 5). **, P < 0.005.
Mentions: Because PDK1-deficient NK cells were resistant to IL-15–induced E4BP4 up-regulation, the impaired NK cell development probably resulted from the compromised IL-15 signaling. We initially detected IL-15–dependent lineage cells in PDK1−/− mice. The absolute numbers of NK cells (CD3−NK1.1+), NK-T cells (CD3+NK1.1int), and memory CD8+ T cells (CD8+CD122+) were remarkably diminished in PDK1−/− mice (unpublished data). To further clarify this effect, we analyzed CD122 expression on CD3−NK1.1+ cells. PDK1−/− mice exhibited dramatically fewer CD3− CD122 high NK1.1+ cells (R1) in the detected lymphoid organs, like bone marrow, spleen, lung, and liver (Fig. 3 A and not depicted). Additionally, the CD122 intensity on PDK1−/− CD3−NK1.1+ cells was significantly lower and had a tendency to be gradually attenuated (Fig. 3, A and B). Surprisingly, there was a noticeable population of CD122-negative NK cells, which exhibited the distinct phenotype of intermediate expression of NK1.1, called NK1.1int. These cells accumulated mainly in the bone marrow rather than other organs (data not shown), of PDK1−/− mice, whereas the population was rarely seen in WT mice (Fig. 3 A). To further characterize this population, we confirmed that the cells expressed high levels of CD117 and CD127, the two earliest markers for NK cells (Fig. 3 C), and were not reactive for CD1d tetramer binding (unpublished data); this excluded the possibility of the cells being NK-T cells, which also exhibit NK1.1int. The population was negative for NKp46, a marker for type I group of innate lymphoid cell (ILC1; Fig. 3 C). Collectively, these data indicate that PDK1 is essential for preserving a CD122high state during early NK cell development.

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