Limits...
Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent.

Westernberg L, Conche C, Huang YH, Rigaud S, Deng Y, Siegemund S, Mukherjee S, Nosaka L, Das J, Sauer K - Elife (2016)

Bottom Line: Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb).This is reversed by inhibition of Akt, mTOR or glucose metabolism.Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, United States.

ABSTRACT
β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb(-/-) thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4(+)CD8(+) cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

No MeSH data available.


Related in: MedlinePlus

Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.(A,B) Sorted DN3 cells from 6.5 week old Itpkb+/+ or Itpkb-/- mice were seeded onto Delta-like 1 Notch ligand-expressing OP9DL1 or Notch ligand-free OP9 stroma cells and analyzed for CD4/CD8 expression 4 days later. (A) Representative FACS data from input (day 0) or day 4 cultures. The numbers indicate % cells in the DP or DN gates, respectively. Representative of 5 independent experiments. (B) Bar-graphs showing mean ± SEM Itpkb+/+ (black bars) or Itpkb-/- (open bars) % DP cells after 4-day culture on OP9DL1 or OP9 cells, averaged from 4 independent experiments. Significance for genotype differences was analyzed as in Figure 2 (n = 4). (C,D) Fetal thymic lobes from Itpkb+/+ or Itpkb-/- embryos harvested on day 15.5 of embryogenesis (E15.5) from the same mother were cultured in the presence of ethanol (vehicle) or 20 μM rapamycin for 4 days, harvested and analyzed. (C) Representative FACS plots of CD4/CD8 expression on total thymocytes. Numbers denote % cells in the respective gate. (D) Bar graph of mean ± SEM % DP cells for each condition and genotype from 3 independent experiments. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 5).DOI:http://dx.doi.org/10.7554/eLife.10786.010
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764578&req=5

fig7: Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.(A,B) Sorted DN3 cells from 6.5 week old Itpkb+/+ or Itpkb-/- mice were seeded onto Delta-like 1 Notch ligand-expressing OP9DL1 or Notch ligand-free OP9 stroma cells and analyzed for CD4/CD8 expression 4 days later. (A) Representative FACS data from input (day 0) or day 4 cultures. The numbers indicate % cells in the DP or DN gates, respectively. Representative of 5 independent experiments. (B) Bar-graphs showing mean ± SEM Itpkb+/+ (black bars) or Itpkb-/- (open bars) % DP cells after 4-day culture on OP9DL1 or OP9 cells, averaged from 4 independent experiments. Significance for genotype differences was analyzed as in Figure 2 (n = 4). (C,D) Fetal thymic lobes from Itpkb+/+ or Itpkb-/- embryos harvested on day 15.5 of embryogenesis (E15.5) from the same mother were cultured in the presence of ethanol (vehicle) or 20 μM rapamycin for 4 days, harvested and analyzed. (C) Representative FACS plots of CD4/CD8 expression on total thymocytes. Numbers denote % cells in the respective gate. (D) Bar graph of mean ± SEM % DP cells for each condition and genotype from 3 independent experiments. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 5).DOI:http://dx.doi.org/10.7554/eLife.10786.010

Mentions: Further supporting accelerated development, Itpkb-/- sorted DN3 cells generated larger proportions of DP cells than WT DN3 cells after 4-day co-culture on OP9DL1 stroma cells (Ciofani and Zuniga-Pflucker, 2005; Ciofani et al., 2004) (Figure 7A,B, Figure 9—figure supplement 1). Finally, Itpkb-/- E15.5 fetal thymic organ cultures (FTOC) produced more DP cells than WT controls (Figure 7C,D).10.7554/eLife.10786.010Figure 7.Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.


Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent.

Westernberg L, Conche C, Huang YH, Rigaud S, Deng Y, Siegemund S, Mukherjee S, Nosaka L, Das J, Sauer K - Elife (2016)

Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.(A,B) Sorted DN3 cells from 6.5 week old Itpkb+/+ or Itpkb-/- mice were seeded onto Delta-like 1 Notch ligand-expressing OP9DL1 or Notch ligand-free OP9 stroma cells and analyzed for CD4/CD8 expression 4 days later. (A) Representative FACS data from input (day 0) or day 4 cultures. The numbers indicate % cells in the DP or DN gates, respectively. Representative of 5 independent experiments. (B) Bar-graphs showing mean ± SEM Itpkb+/+ (black bars) or Itpkb-/- (open bars) % DP cells after 4-day culture on OP9DL1 or OP9 cells, averaged from 4 independent experiments. Significance for genotype differences was analyzed as in Figure 2 (n = 4). (C,D) Fetal thymic lobes from Itpkb+/+ or Itpkb-/- embryos harvested on day 15.5 of embryogenesis (E15.5) from the same mother were cultured in the presence of ethanol (vehicle) or 20 μM rapamycin for 4 days, harvested and analyzed. (C) Representative FACS plots of CD4/CD8 expression on total thymocytes. Numbers denote % cells in the respective gate. (D) Bar graph of mean ± SEM % DP cells for each condition and genotype from 3 independent experiments. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 5).DOI:http://dx.doi.org/10.7554/eLife.10786.010
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764578&req=5

fig7: Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.(A,B) Sorted DN3 cells from 6.5 week old Itpkb+/+ or Itpkb-/- mice were seeded onto Delta-like 1 Notch ligand-expressing OP9DL1 or Notch ligand-free OP9 stroma cells and analyzed for CD4/CD8 expression 4 days later. (A) Representative FACS data from input (day 0) or day 4 cultures. The numbers indicate % cells in the DP or DN gates, respectively. Representative of 5 independent experiments. (B) Bar-graphs showing mean ± SEM Itpkb+/+ (black bars) or Itpkb-/- (open bars) % DP cells after 4-day culture on OP9DL1 or OP9 cells, averaged from 4 independent experiments. Significance for genotype differences was analyzed as in Figure 2 (n = 4). (C,D) Fetal thymic lobes from Itpkb+/+ or Itpkb-/- embryos harvested on day 15.5 of embryogenesis (E15.5) from the same mother were cultured in the presence of ethanol (vehicle) or 20 μM rapamycin for 4 days, harvested and analyzed. (C) Representative FACS plots of CD4/CD8 expression on total thymocytes. Numbers denote % cells in the respective gate. (D) Bar graph of mean ± SEM % DP cells for each condition and genotype from 3 independent experiments. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 5).DOI:http://dx.doi.org/10.7554/eLife.10786.010
Mentions: Further supporting accelerated development, Itpkb-/- sorted DN3 cells generated larger proportions of DP cells than WT DN3 cells after 4-day co-culture on OP9DL1 stroma cells (Ciofani and Zuniga-Pflucker, 2005; Ciofani et al., 2004) (Figure 7A,B, Figure 9—figure supplement 1). Finally, Itpkb-/- E15.5 fetal thymic organ cultures (FTOC) produced more DP cells than WT controls (Figure 7C,D).10.7554/eLife.10786.010Figure 7.Itpkb-loss in DN3 cells causes accelerated, Notch-independent development to the DP stage.

Bottom Line: Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb).This is reversed by inhibition of Akt, mTOR or glucose metabolism.Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, United States.

ABSTRACT
β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb(-/-) thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4(+)CD8(+) cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

No MeSH data available.


Related in: MedlinePlus