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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 controls β-selection cell-autonomously.B/T cell-depleted BM from CD45.1 Itpkb+/+ and CD45.2 Itpkb-/- mice was mixed at a 1:1 ratio and injected into CD45.1/CD45.2 lethally irradiated hosts. 7 weeks later, thymocytes were analyzed by FACS. (A) Top, thymocyte expression of CD45.1 and CD45.2. The other panels show expression of the indicated markers on CD45.1+CD45.2-Itpkb+/+ or CD45.1-CD45.2+Itpkb-/- donor-derived thymocytes, using the gating strategy in Figure 2A. Numbers denote % cells per gate. (B) Chimerism of the indicated thymocyte subpopulations, expressed as mean ± SEM ratio of CD45.1-CD45.2+Itpkb-/- to CD45.1+CD45.2-Itpkb+/+ donor-derived thymocytes. (C) Mean ± SEM ratio of total DN3 cell numbers to TCRβ- DN4 cell numbers in Itpkb+/+ or Itpkb-/- donor-derived thymocytes. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 3). (D) CD2 and CXCR4 expression on Itpkb+/+ (solid) and Itpkb-/- (hatched) thymocyte subsets in mixed BM chimeras. Representative of 3 independent hosts. (E) Ki67 expression in Itpkb+/+ (black) and Itpkb-/- (gray) DN3, DN3-4 and DN4 cells in mixed BM chimeras. Open histogram, Itpkb+/+ isotype staining negative control. Representative of 3 independent hosts.DOI:http://dx.doi.org/10.7554/eLife.10786.006
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fig4: Itpkb controls β-selection cell-autonomously.B/T cell-depleted BM from CD45.1 Itpkb+/+ and CD45.2 Itpkb-/- mice was mixed at a 1:1 ratio and injected into CD45.1/CD45.2 lethally irradiated hosts. 7 weeks later, thymocytes were analyzed by FACS. (A) Top, thymocyte expression of CD45.1 and CD45.2. The other panels show expression of the indicated markers on CD45.1+CD45.2-Itpkb+/+ or CD45.1-CD45.2+Itpkb-/- donor-derived thymocytes, using the gating strategy in Figure 2A. Numbers denote % cells per gate. (B) Chimerism of the indicated thymocyte subpopulations, expressed as mean ± SEM ratio of CD45.1-CD45.2+Itpkb-/- to CD45.1+CD45.2-Itpkb+/+ donor-derived thymocytes. (C) Mean ± SEM ratio of total DN3 cell numbers to TCRβ- DN4 cell numbers in Itpkb+/+ or Itpkb-/- donor-derived thymocytes. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 3). (D) CD2 and CXCR4 expression on Itpkb+/+ (solid) and Itpkb-/- (hatched) thymocyte subsets in mixed BM chimeras. Representative of 3 independent hosts. (E) Ki67 expression in Itpkb+/+ (black) and Itpkb-/- (gray) DN3, DN3-4 and DN4 cells in mixed BM chimeras. Open histogram, Itpkb+/+ isotype staining negative control. Representative of 3 independent hosts.DOI:http://dx.doi.org/10.7554/eLife.10786.006

Mentions: To explore if the altered β-selection is caused by thymocyte-intrinsic Itpkb-loss, we injected a 1:1 mix of mature T/B cell-depleted CD45.1 WT and CD45.2 Itpkb-/- BM into lethally irradiated CD45.1/CD45.2-congenic hosts and analyzed reconstituted thymocyte subsets 6–8 weeks later. We distinguished WT vs. Itpkb-/- donor-derived cells by CD45 allelic expression. Compared to WT controls, Itpkb-/- donor-derived thymocytes reproduced the published cell-intrinsic block at the DP stage (Wen et al., 2004) and the increased DN3 and reduced DN4 cell proportions, partial loss of TCRβ- DN4 and ISP cells, and increased (DN3 + DN3-4):TCRβ- DN4 cell ratio of Itpkb-/- mice (Figure 4A–C). Moreover, Itpkb-/- vs. WT donor-derived DN3-4 and DN4 cells overexpressed CD2 and tended to upregulate CXCR4 (Figure 4D). Ki67 staining was again similar between genotypes (Figure 4E). Thus, Itpkb-/- thymocytes show a cell-intrinsically altered β-selection not rescued by a WT environment and the presence of WT thymocytes.10.7554/eLife.10786.006Figure 4.Itpkb controls β-selection cell-autonomously.


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 controls β-selection cell-autonomously.B/T cell-depleted BM from CD45.1 Itpkb+/+ and CD45.2 Itpkb-/- mice was mixed at a 1:1 ratio and injected into CD45.1/CD45.2 lethally irradiated hosts. 7 weeks later, thymocytes were analyzed by FACS. (A) Top, thymocyte expression of CD45.1 and CD45.2. The other panels show expression of the indicated markers on CD45.1+CD45.2-Itpkb+/+ or CD45.1-CD45.2+Itpkb-/- donor-derived thymocytes, using the gating strategy in Figure 2A. Numbers denote % cells per gate. (B) Chimerism of the indicated thymocyte subpopulations, expressed as mean ± SEM ratio of CD45.1-CD45.2+Itpkb-/- to CD45.1+CD45.2-Itpkb+/+ donor-derived thymocytes. (C) Mean ± SEM ratio of total DN3 cell numbers to TCRβ- DN4 cell numbers in Itpkb+/+ or Itpkb-/- donor-derived thymocytes. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 3). (D) CD2 and CXCR4 expression on Itpkb+/+ (solid) and Itpkb-/- (hatched) thymocyte subsets in mixed BM chimeras. Representative of 3 independent hosts. (E) Ki67 expression in Itpkb+/+ (black) and Itpkb-/- (gray) DN3, DN3-4 and DN4 cells in mixed BM chimeras. Open histogram, Itpkb+/+ isotype staining negative control. Representative of 3 independent hosts.DOI:http://dx.doi.org/10.7554/eLife.10786.006
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Related In: Results  -  Collection

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fig4: Itpkb controls β-selection cell-autonomously.B/T cell-depleted BM from CD45.1 Itpkb+/+ and CD45.2 Itpkb-/- mice was mixed at a 1:1 ratio and injected into CD45.1/CD45.2 lethally irradiated hosts. 7 weeks later, thymocytes were analyzed by FACS. (A) Top, thymocyte expression of CD45.1 and CD45.2. The other panels show expression of the indicated markers on CD45.1+CD45.2-Itpkb+/+ or CD45.1-CD45.2+Itpkb-/- donor-derived thymocytes, using the gating strategy in Figure 2A. Numbers denote % cells per gate. (B) Chimerism of the indicated thymocyte subpopulations, expressed as mean ± SEM ratio of CD45.1-CD45.2+Itpkb-/- to CD45.1+CD45.2-Itpkb+/+ donor-derived thymocytes. (C) Mean ± SEM ratio of total DN3 cell numbers to TCRβ- DN4 cell numbers in Itpkb+/+ or Itpkb-/- donor-derived thymocytes. Significance of the indicated comparisons was analyzed as in Figure 2 (n = 3). (D) CD2 and CXCR4 expression on Itpkb+/+ (solid) and Itpkb-/- (hatched) thymocyte subsets in mixed BM chimeras. Representative of 3 independent hosts. (E) Ki67 expression in Itpkb+/+ (black) and Itpkb-/- (gray) DN3, DN3-4 and DN4 cells in mixed BM chimeras. Open histogram, Itpkb+/+ isotype staining negative control. Representative of 3 independent hosts.DOI:http://dx.doi.org/10.7554/eLife.10786.006
Mentions: To explore if the altered β-selection is caused by thymocyte-intrinsic Itpkb-loss, we injected a 1:1 mix of mature T/B cell-depleted CD45.1 WT and CD45.2 Itpkb-/- BM into lethally irradiated CD45.1/CD45.2-congenic hosts and analyzed reconstituted thymocyte subsets 6–8 weeks later. We distinguished WT vs. Itpkb-/- donor-derived cells by CD45 allelic expression. Compared to WT controls, Itpkb-/- donor-derived thymocytes reproduced the published cell-intrinsic block at the DP stage (Wen et al., 2004) and the increased DN3 and reduced DN4 cell proportions, partial loss of TCRβ- DN4 and ISP cells, and increased (DN3 + DN3-4):TCRβ- DN4 cell ratio of Itpkb-/- mice (Figure 4A–C). Moreover, Itpkb-/- vs. WT donor-derived DN3-4 and DN4 cells overexpressed CD2 and tended to upregulate CXCR4 (Figure 4D). Ki67 staining was again similar between genotypes (Figure 4E). Thus, Itpkb-/- thymocytes show a cell-intrinsically altered β-selection not rescued by a WT environment and the presence of WT thymocytes.10.7554/eLife.10786.006Figure 4.Itpkb controls β-selection cell-autonomously.

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