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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

Accelerated differentiation of Itpkb-/- DN3 thymocytes.(A,B) Mathematically predicted numbers of DN4 (A) and DP cells (B) in Rag2-/-Itpkb+/+ (black) or Rag2-/-Itpkb-/- (red) mice on the indicated days post α-CD3 antibody injection. >98% of thymocytes in Rag2-/- mice are DN3 cells (C,D), and progenitor influx within 3 days is negligible. Thus, we set K = 0 in our model for (A,B). (C,D) CD4/CD8 expression on thymocytes (C) and CD44/CD25 expression on DN cells (D) from Itpkb+/+Rag2-/- or Itpkb-/-Rag2-/- mice before (day 0), or 2 or 3 days post α-CD3 antibody injection. Gates in (D) denote DN1, DN2, DN3, DN3-4 and DN4 cells in clock-wise order, numbers % cells in the DN3 and DN4 gates. Representative of 7 independent experiments. (E) Measured mean ± SEM DN4 cell (upper panel) and ISP (lower panel) numbers in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 injection. Significance of the indicated comparisons was analyzed as in Figure 2. For DN4 cell numbers, n = 4, 4, 6 or 6 Rag2-/- and 4, 4, 5 or 5 Itpkb-/-Rag2-/- mice, respectively. For ISP numbers, n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (F) Mean ± SEM DP cell % (upper panel) or number (lower panel) in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 antibody injection. Significance of genotype differences per day was analyzed as in Figure 2. n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (G) Annexin V (AnnV) staining of DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from uninjected Rag2-/- (open histograms) or α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/-(gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 3 independent experiments and 3–4 mice per genotype. Uninjected Rag2-/- mice contain dying cells in the DN4, CD8-ISP and DP cell gates due to failed β-selection at the DN3 stage. These serve as positive controls for the Annexin V stain. (H) Ki67 expression in DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/- (gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 2 independent experiments and 3 mice per genotype. Open histograms, day 0 WT isotype control. (I) CD2, CD5, CD71 and CXCR4 surface-levels on, and transgenic Nr4a1/Nur77-GFP expression in DN3 or DN4 thymocytes from uninjected Rag2-/- (open histograms) or α-CD3 injected Itpkb+/+Rag2-/- (black) or Itpkb-/-Rag2-/- (gray) mice 2 days post injection. The <1% CD44-CD25- negative control cells in uninjected Rag2-/- mice are non-T cells. Representative of ≥3 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.10786.009
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fig6: Accelerated differentiation of Itpkb-/- DN3 thymocytes.(A,B) Mathematically predicted numbers of DN4 (A) and DP cells (B) in Rag2-/-Itpkb+/+ (black) or Rag2-/-Itpkb-/- (red) mice on the indicated days post α-CD3 antibody injection. >98% of thymocytes in Rag2-/- mice are DN3 cells (C,D), and progenitor influx within 3 days is negligible. Thus, we set K = 0 in our model for (A,B). (C,D) CD4/CD8 expression on thymocytes (C) and CD44/CD25 expression on DN cells (D) from Itpkb+/+Rag2-/- or Itpkb-/-Rag2-/- mice before (day 0), or 2 or 3 days post α-CD3 antibody injection. Gates in (D) denote DN1, DN2, DN3, DN3-4 and DN4 cells in clock-wise order, numbers % cells in the DN3 and DN4 gates. Representative of 7 independent experiments. (E) Measured mean ± SEM DN4 cell (upper panel) and ISP (lower panel) numbers in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 injection. Significance of the indicated comparisons was analyzed as in Figure 2. For DN4 cell numbers, n = 4, 4, 6 or 6 Rag2-/- and 4, 4, 5 or 5 Itpkb-/-Rag2-/- mice, respectively. For ISP numbers, n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (F) Mean ± SEM DP cell % (upper panel) or number (lower panel) in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 antibody injection. Significance of genotype differences per day was analyzed as in Figure 2. n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (G) Annexin V (AnnV) staining of DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from uninjected Rag2-/- (open histograms) or α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/-(gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 3 independent experiments and 3–4 mice per genotype. Uninjected Rag2-/- mice contain dying cells in the DN4, CD8-ISP and DP cell gates due to failed β-selection at the DN3 stage. These serve as positive controls for the Annexin V stain. (H) Ki67 expression in DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/- (gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 2 independent experiments and 3 mice per genotype. Open histograms, day 0 WT isotype control. (I) CD2, CD5, CD71 and CXCR4 surface-levels on, and transgenic Nr4a1/Nur77-GFP expression in DN3 or DN4 thymocytes from uninjected Rag2-/- (open histograms) or α-CD3 injected Itpkb+/+Rag2-/- (black) or Itpkb-/-Rag2-/- (gray) mice 2 days post injection. The <1% CD44-CD25- negative control cells in uninjected Rag2-/- mice are non-T cells. Representative of ≥3 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.10786.009

Mentions: To corroborate these findings in an in vivo system that allows one to kinetically study β-selection of a synchronized DN3 cell population, we generated Itpkb-/-Rag2-/-mice. Rag2-loss causes a developmental arrest at the DN3 stage due to blocked TCRβ expression. Injected anti-CD3 antibodies (α-CD3) can crosslink CD3ε on Rag-/-DN3 cells and trigger their differentiation to DP cells (Campese et al., 2006). Our mathematical model predicted immediately increasing DN4 cell numbers, and DP cell accumulation at ≥2 days post α-CD3 injection in Rag2-/- mice (Figure 6A,B). Simulating over two fold faster DN3-to-DP development in Rag2-/-Itpkb-/- mice predicted transiently increased DN4 cell accumulation resulting in earlier DP cell accumulation. Experiments confirmed the predictions: Thymocyte development was blocked at the DN3 stage in Itpkb-/-Rag2-/-and Rag2-/- control mice (Figure 6C,D). α-CD3 injection triggered progressive accumulation of DN4, ISP and DP cells 2 and 3 days later in both genotypes (Figure 6C–F). However, Itpkb-/-Rag2-/- mice accumulated larger proportions of DN4 (33% vs. 18%), ISP (3% vs. 1%) and DP cells (20% vs. 1%) than Rag2-/- mice on day 2 when Rag2-/- mice barely had any DP cells. Itpkb-/- DN4 and ISP cell numbers tended to be increased on day 2 but this was not statistically significant (Figure 6E). Itpkb-/-Rag2-/- mice continued to accumulate more DP cells towards an ~four fold excess over Rag2-/- controls on day 3 (Figure 6F). This resulted mostly from faster development, as both genotypes showed similar thymocyte subset viability and proliferation post α-CD3 injection (Figure 6G,H).10.7554/eLife.10786.009Figure 6.Accelerated differentiation of Itpkb-/- DN3 thymocytes.


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)

Accelerated differentiation of Itpkb-/- DN3 thymocytes.(A,B) Mathematically predicted numbers of DN4 (A) and DP cells (B) in Rag2-/-Itpkb+/+ (black) or Rag2-/-Itpkb-/- (red) mice on the indicated days post α-CD3 antibody injection. >98% of thymocytes in Rag2-/- mice are DN3 cells (C,D), and progenitor influx within 3 days is negligible. Thus, we set K = 0 in our model for (A,B). (C,D) CD4/CD8 expression on thymocytes (C) and CD44/CD25 expression on DN cells (D) from Itpkb+/+Rag2-/- or Itpkb-/-Rag2-/- mice before (day 0), or 2 or 3 days post α-CD3 antibody injection. Gates in (D) denote DN1, DN2, DN3, DN3-4 and DN4 cells in clock-wise order, numbers % cells in the DN3 and DN4 gates. Representative of 7 independent experiments. (E) Measured mean ± SEM DN4 cell (upper panel) and ISP (lower panel) numbers in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 injection. Significance of the indicated comparisons was analyzed as in Figure 2. For DN4 cell numbers, n = 4, 4, 6 or 6 Rag2-/- and 4, 4, 5 or 5 Itpkb-/-Rag2-/- mice, respectively. For ISP numbers, n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (F) Mean ± SEM DP cell % (upper panel) or number (lower panel) in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 antibody injection. Significance of genotype differences per day was analyzed as in Figure 2. n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (G) Annexin V (AnnV) staining of DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from uninjected Rag2-/- (open histograms) or α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/-(gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 3 independent experiments and 3–4 mice per genotype. Uninjected Rag2-/- mice contain dying cells in the DN4, CD8-ISP and DP cell gates due to failed β-selection at the DN3 stage. These serve as positive controls for the Annexin V stain. (H) Ki67 expression in DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/- (gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 2 independent experiments and 3 mice per genotype. Open histograms, day 0 WT isotype control. (I) CD2, CD5, CD71 and CXCR4 surface-levels on, and transgenic Nr4a1/Nur77-GFP expression in DN3 or DN4 thymocytes from uninjected Rag2-/- (open histograms) or α-CD3 injected Itpkb+/+Rag2-/- (black) or Itpkb-/-Rag2-/- (gray) mice 2 days post injection. The <1% CD44-CD25- negative control cells in uninjected Rag2-/- mice are non-T cells. Representative of ≥3 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.10786.009
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fig6: Accelerated differentiation of Itpkb-/- DN3 thymocytes.(A,B) Mathematically predicted numbers of DN4 (A) and DP cells (B) in Rag2-/-Itpkb+/+ (black) or Rag2-/-Itpkb-/- (red) mice on the indicated days post α-CD3 antibody injection. >98% of thymocytes in Rag2-/- mice are DN3 cells (C,D), and progenitor influx within 3 days is negligible. Thus, we set K = 0 in our model for (A,B). (C,D) CD4/CD8 expression on thymocytes (C) and CD44/CD25 expression on DN cells (D) from Itpkb+/+Rag2-/- or Itpkb-/-Rag2-/- mice before (day 0), or 2 or 3 days post α-CD3 antibody injection. Gates in (D) denote DN1, DN2, DN3, DN3-4 and DN4 cells in clock-wise order, numbers % cells in the DN3 and DN4 gates. Representative of 7 independent experiments. (E) Measured mean ± SEM DN4 cell (upper panel) and ISP (lower panel) numbers in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 injection. Significance of the indicated comparisons was analyzed as in Figure 2. For DN4 cell numbers, n = 4, 4, 6 or 6 Rag2-/- and 4, 4, 5 or 5 Itpkb-/-Rag2-/- mice, respectively. For ISP numbers, n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (F) Mean ± SEM DP cell % (upper panel) or number (lower panel) in Itpkb+/+Rag2-/- (solid line) or Itpkb-/-Rag2-/- (hatched) mice before (day 0) or 1, 2 or 3 days after α-CD3 antibody injection. Significance of genotype differences per day was analyzed as in Figure 2. n = 6, 5, 7 or 7 Rag2-/- and 4, 4, 6 or 5 Itpkb-/-Rag2-/- mice, respectively. (G) Annexin V (AnnV) staining of DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from uninjected Rag2-/- (open histograms) or α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/-(gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 3 independent experiments and 3–4 mice per genotype. Uninjected Rag2-/- mice contain dying cells in the DN4, CD8-ISP and DP cell gates due to failed β-selection at the DN3 stage. These serve as positive controls for the Annexin V stain. (H) Ki67 expression in DN3, DN3-4, DN4, HSAhiCD8+ ISP or DP cells from α-CD3 antibody injected Itpkb+/+Rag2-/- (black filled histograms) or Itpkb-/-Rag2-/- (gray filled histograms) mice two (left) or three (right) days post antibody injection. Representative of 2 independent experiments and 3 mice per genotype. Open histograms, day 0 WT isotype control. (I) CD2, CD5, CD71 and CXCR4 surface-levels on, and transgenic Nr4a1/Nur77-GFP expression in DN3 or DN4 thymocytes from uninjected Rag2-/- (open histograms) or α-CD3 injected Itpkb+/+Rag2-/- (black) or Itpkb-/-Rag2-/- (gray) mice 2 days post injection. The <1% CD44-CD25- negative control cells in uninjected Rag2-/- mice are non-T cells. Representative of ≥3 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.10786.009
Mentions: To corroborate these findings in an in vivo system that allows one to kinetically study β-selection of a synchronized DN3 cell population, we generated Itpkb-/-Rag2-/-mice. Rag2-loss causes a developmental arrest at the DN3 stage due to blocked TCRβ expression. Injected anti-CD3 antibodies (α-CD3) can crosslink CD3ε on Rag-/-DN3 cells and trigger their differentiation to DP cells (Campese et al., 2006). Our mathematical model predicted immediately increasing DN4 cell numbers, and DP cell accumulation at ≥2 days post α-CD3 injection in Rag2-/- mice (Figure 6A,B). Simulating over two fold faster DN3-to-DP development in Rag2-/-Itpkb-/- mice predicted transiently increased DN4 cell accumulation resulting in earlier DP cell accumulation. Experiments confirmed the predictions: Thymocyte development was blocked at the DN3 stage in Itpkb-/-Rag2-/-and Rag2-/- control mice (Figure 6C,D). α-CD3 injection triggered progressive accumulation of DN4, ISP and DP cells 2 and 3 days later in both genotypes (Figure 6C–F). However, Itpkb-/-Rag2-/- mice accumulated larger proportions of DN4 (33% vs. 18%), ISP (3% vs. 1%) and DP cells (20% vs. 1%) than Rag2-/- mice on day 2 when Rag2-/- mice barely had any DP cells. Itpkb-/- DN4 and ISP cell numbers tended to be increased on day 2 but this was not statistically significant (Figure 6E). Itpkb-/-Rag2-/- mice continued to accumulate more DP cells towards an ~four fold excess over Rag2-/- controls on day 3 (Figure 6F). This resulted mostly from faster development, as both genotypes showed similar thymocyte subset viability and proliferation post α-CD3 injection (Figure 6G,H).10.7554/eLife.10786.009Figure 6.Accelerated differentiation of Itpkb-/- DN3 thymocytes.

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