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

  1. Luise Westernberg
  2. Claire Conche
  3. Yina Hsing Huang
  4. Stephanie Rigaud
  5. Yisong Deng
  6. Sabine Siegemund
  7. Sayak Mukherjee
  8. Lyn'Al Nosaka
  9. Jayajit Das
  10. Karsten Sauer  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Geisel School of Medicine, United States
  3. The Ohio State University, United States
Research Article
Cite this article as: eLife 2016;5:e10786 doi: 10.7554/eLife.10786
11 figures and 1 table

Figures

Itpkb protein is expressed in DN3 cells from Itpkb+/+ but not Itpkb-/- mice.

Shown are immunoblots (IB) of Itpkb immunoprecipitates (IP, top) or whole cell lysates (WCL, bottom) from Itpkb+/+ or Itpkb-/- DN3 cell-enriched Rag2-/- thymocytes (left) or sorted CD53- DP thymocytes (right), resolved via SDS-PAGE and probed with antibodies against Itpkb (top) or PLCγ1 (bottom, loading control) as in (Miller et al., 2007; Huang et al., 2007).

https://doi.org/10.7554/eLife.10786.003
Altered β-selection in Itpkb-/- mice.

(A) Flow cytometric profiles of thymocytes from Itpkb+/+ and Itpkb-/- littermate mice. Top, CD4 and CD8 expression. Upper center, HSA and mature lineage marker (Lin = CD11b, CD11c, CD19, B220, CD49b, Gr-1, Ter119, TCRγ) expression on CD4-CD8- (DN) cells. Lower center, CD44 and CD25 expression on HSAhighLin- DN cells. The bottom gates denote DN3, transitional DN3-4 and DN4 cells from right to left. Bottom two panels, TCRβ expression on DN4 cells, and HSA and TCRβ expression on CD4-CD8+ cells with ISP (HSAhighTCRβlow) and mature CD8 T cell (HSAlowTCRβhigh) gates. Numbers denote % cells per gate. Representative of at least 7 independent experiments. (B) Mean ± SEM %DN3 cells, %DN4 cells or %DN3:%DN4 cell ratio in Itpkb+/+ or Itpkb-/- 5–7 week old littermates. Statistical significance of genotype differences was analyzed by unpaired two-tailed Student's t-tests (n = 7). (C) Total numbers of thymocytes, Lin-HSAhigh CD44-CD25+ DN3, CD44-CD25int DN3-4, TCRβ-CD44-CD25- DN4, CD8α+HSAhighTCRβlow ISP or CD4+CD8+ DP cells in individual Itpkb+/+ or Itpkb-/- mice. Horizontal lines denote means ± SEM. Significance of genotype differences was analyzed as in (B). nWT = 9, nItpkb-/- = 11. ns, no significant difference. (D) Histograms of surface or total cellular TCRβ or CD3 levels on the indicated thymocyte populations from Itpkb+/+ (black) or Itpkb-/- (gray) mice. Representative of ≥3 independent experiments.

https://doi.org/10.7554/eLife.10786.004
Surface marker expression, steady-state proliferation and viability of Itpkb+/+ and Itpkb-/- thymocytes.

(A) Surface levels of the indicated markers for activation or β-selection on thymocyte subpopulations from Itpkb+/+ (solid) or Itpkb-/- (hatched) mice. (B) Nr4a1/Nur77-GFP expression in Itpkb+/+ or Itpkb-/- Nr4a1/Nur77-GFP transgenic (solid or hatched black, respectively) or non-transgenic (gray) mice. Representative of ≥3 independent experiments. (C) Steady-state proliferative status of the indicated thymocyte subpopulations in Itpkb+/+ (black) or Itpkb-/- (gray) mice was analyzed by Ki67 stain (top, representative of 3 independent experiments) or BrdU incorporation assay (bottom, representative of 2 independent experiments). Thin open histograms, Itpkb+/+ isotype or BrdU-uninjected, respectively, negative control. TCRβlow DP cells were analyzed as they represent the majority of DP cells and Itpkb-/- mice lack TCRβhigh DP cells (Wen et al., 2004). (D) Steady-state viability of the indicated thymocyte subpopulations in Itpkb+/+ (black) or Itpkb-/- (gray) mice was analyzed by AnnexinV (AnnV) stain. Representative of 4 independent experiments.

https://doi.org/10.7554/eLife.10786.005
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.

https://doi.org/10.7554/eLife.10786.006
Figure 5 with 1 supplement
Accelerated differentiation of Itpkb-/- DN3 thymocytes.

(A) In silico analysis of β-selection kinetics in Itpkb-/- and Itpkb+/+ mice. Scheme of intrathymic DP cell development from progenitors. The velocities of relevant developmental transitions are characterized by rate constants K (identical between genotypes) and K1, Kand K3 (development from DN3 to DP cells, set to over two-fold higher in Itpkb-/- vs. WT mice, Table 1). Rate constants Kd1-Kd4 for subset turnover via proliferation and death were considered identical between genotypes. (B) Predicted steady-state numbers of the indicated thymocyte populations in Itpkb+/+ (black) or Itpkb-/- (red) mice. (C) Left, CD4/CD8 expression on embryogenesis day (E) 16.5 or 17.5 fetal thymocytes. Center, CD44/CD25 expression on CD4-CD8-HSAhighLin-TCRγ- cells (Figure 5—figure supplement 1A). Numbers denote % cells per gate. CD4-CD8+ fetal thymocytes are ≥92% ISP (Figure 5—figure supplement 1B). Right, mean ± SEM DP cell number (#) or % in E16.5 or E17.5 Itpkb+/+ or Itpkb-/- thymi. Significance of genotype differences was analyzed as in Figure 2 (nE16.5 = 2, nE17.5 = 3). E16.5 data with t-test from another experiment in Figure 5—figure supplement 1C,D.

https://doi.org/10.7554/eLife.10786.007
Figure 5—figure supplement 1
Raw and replicate data related to Figure 5C.

(A,B) HSA and combined Lin/TCRγ expression on the DN (A) and CD4-CD8+ (B) cells in Figure 5C, left panel. The HSAhighLin-TCRγ- gates in (A) were analyzed for DN cell subsets in Figure 5C, center panel. The HSAhighLin-TCRγ- gates in (B) denote ISP. ISP comprise ≥92% of CD4-CD8+ cells in E16.5 and E17.5 fetal thymi in both Itpkb+/+ and Itpkb-/- mice. (C) CD4 and CD8 expression on Itpkb+/+ or Itpkb-/- fetal thymocytes from embryos harvested on day 16.5 of embryogenesis (E16.5) from the same mother. Numbers denote % cells per gate. (D) Mean ± SEM numbers or % of DP cells in E16.5 Itpkb+/+ or Itpkb-/- fetal thymi. Significance of the indicated comparisons was analyzed as in Figure 2 (nWT = 3, nKO = 5).

https://doi.org/10.7554/eLife.10786.008
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 = 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.

https://doi.org/10.7554/eLife.10786.009
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).

https://doi.org/10.7554/eLife.10786.010
Increased pre-TCR signaling via PI3K/Akt/mTOR in Itpkb-/-DN3 cells.

(A,B) We analyzed (A) cellular content of T308-phosphorylated active Akt (pAktT308), S2481-phosphorylated mTOR (pmTORS2481), S235/S236-phosphorylated ribosomal protein S6 (pS6S235/S236), Glut1 protein, T202/Y204-phosphorylated Erk (pErkT202/Y204) and Akt protein, and (B) cell size via side/forward-scatter analysis (SSC-A/FSC-A) in the indicated thymocyte populations of Itpkb+/+ (black histograms) or Itpkb-/- (gray histograms) mice by FACS. Thin open histograms, Itpkb+/+ isotype or second antibody stained negative controls. Bold open histograms, Calyculin A-treated positive controls. Arrowheads show gate positions. In (B), numbers indicate % cells per large cell gate. Representative of at least 2 (pS6S235/S236), 3 (total Akt) or 8 (else) independent experiments.

https://doi.org/10.7554/eLife.10786.011
Figure 9 with 1 supplement
Itpkb renders β-selection Notch-dependent.

(A,B) Addition of inhibitors of Akt, mTOR or glucose metabolism reverses the accelerated development of Itpkb-/- DN3 cells and re-establishes Notch-dependence. Shown are mean ± SEM Itpkb+/+ (solid black bars) or Itpkb-/- (pen bars) % DP cells after 4-day culture on OP9DL1 (A) or OP9 (B) cells without or with addition of carrier (solid gray bars; DMSO, ethanol or PBS, respectively), 500 nM Akt-inhibitor VIII in DMSO (Akt-I, added once on day 0), 4 μM rapamycin in ethanol (Rapa, added once on day 0) or 500 μM 2-deoxy-D-glucose in PBS (2DG, added once daily), averaged from 3 (Akt-I), 4 (rapamycin) or 2 (2DG) independent experiments. Significance of the indicated comparisons was analyzed as in Figure 2. Replicate numbers are indicated above each bar. Representative FACS-data in Figure 9—figure supplement 1.

https://doi.org/10.7554/eLife.10786.012
Figure 9—figure supplement 1
Raw FACS data from one representative experiment included in the averaged data in Figure 9.

Itpkb+/+ or Itpkb-/- sorted DN3 cells were cultured for 4 days on OP9DL1 (A,C,E) or OP9 (B,D,F) stroma cells without (left) or with once on day 0 (Akt-I, rapamycin) or once-daily (2DG) addition of carrier (center of each panel), 500 nM Akt-inhibitor VIII in DMSO (Akt-I), 4 μM rapamycin in ethanol or 500 μM 2-deoxy-D-glucose in PBS (2DG). Numbers denote % cells in the respective DP or DN gates. Representative of 3 (A,B), 4 (C,D) or 2 (E,F) independent experiments.

https://doi.org/10.7554/eLife.10786.013
Itpkb-loss reduces the Notch-dependence of DN thymocyte development to DP cells in vivo.

Shown are (A) CD4/CD8 expression on total thymocytes and (B) HSA/TCRβ expression on CD4-CD8+ thymocytes from Rag2-/- and Rag2-/-Itpkb-/- mice two days post α-CD3 antibody injection. Starting 3–4 hr before α-CD3 injection, the mice were treated once daily with orally administered γ-secretase inhibitor LY-411,575 or vehicle (Wong et al., 2004). Numbers indicate % cells per respective gate. The gates in (B) denote CD8+HSAhigh ISP (Petrie and Zuniga-Pflucker, 2007; Xiong et al., 2011). Representative of two independent experiments (n = 3).

https://doi.org/10.7554/eLife.10786.014
Antagonistic signaling by PI3K and Itpkb controls the kinetics and Notch-dependence of β-selection.

(A) We propose a model in which pre-TCR and Notch signaling both activate PI3K to produce PIP3 in DN3/DN3-4 cells. PIP3 then recruits and activates Akt to increase glucose metabolism via the Akt/mTOR pathway. This is required for DN3-to-DP cell differentiation. However, pre-TCR signaling also activates Itpkb to produce IP4, which competes with PIP3 for Akt PH domain binding and limits Akt recruitment, Akt and mTOR activation in pre-TCR expressing DN3/DN3-4 cells. IP4 may have additional effectors, indicated by the question mark. By limiting downstream glucose metabolism, this "IP4 brake" delays the kinetics of β-selection and renders this process dependent on Notch costimulation. (B) Without Itpkb, IP4 no more dampens Akt activation and pre-TCR signaling alone sufficiently activates Akt/mTOR signaling to trigger DP cell development in the absence of Notch engagement. (C) In the presence of Notch-signals, Akt is now hyperactivated and causes an accelerated DN3-to-DP cell differentiation.

https://doi.org/10.7554/eLife.10786.015

Tables

Table 1

Parameters used in our mathematical models.

https://doi.org/10.7554/eLife.10786.016
GenotypeK
(cells/day)
K1
(days-1)
Kd1
(days-1)
K2
(days-1)
K3
(days-1)
Kd4
(days-1)
WT15.4 × 1040.10.10.1620.070.00058
Itpkb-/-15.4 × 1040.20.10.4860.210.00058
Rag2-/-Itpkb+/+00.10.10.1620.070.00058
Rag2-/-Itpkb-/-00.20.10.4860.210.00058

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