Research ArticleGENETICS

Discrete roles and bifurcation of PTEN signaling and mTORC1-mediated anabolic metabolism underlie IL-7–driven B lymphopoiesis

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Science Advances  31 Jan 2018:
Vol. 4, no. 1, eaar5701
DOI: 10.1126/sciadv.aar5701
  • Fig. 1 Regulation of metabolic activity in developing B cells and IL-7–mediated proteome landscape in pro-B cells.

    (A) OCR of LinB220+CD43+IgM cells, CD25+ pre-B cells (LinB220+CD43IgMCD25+), immature B cells (B220+IgM+), and circulating mature B cells (B220hiIgM+) measured using a Seahorse XF24 analyzer. (B) RC7 from microarray data of common lymphoid progenitors (CLPs) and each developing B cell subset from ImmGen (gene expression relative to CLP). The central rectangle represents the first to third quartile, with notch in the plot corresponding to the median expression of all genes in the sample. End of the whiskers represents minimum and maximum level of expression of the genes in the cluster. (C) Functional annotations of RCs by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Hallmark databases (FDR < 0.2). (D) Cluster analysis of DE proteins in temporal proteomic profiling of pro-B cells stimulated with 0, 1, 4, and 16 hours of IL-7. (E) Five WPCs during pro-B cell activation were defined by WGCNA. Each line indicates the relative abundance of each protein and is color-coded by the cluster membership determined by correlation analysis between individual protein and the consensus of the WPC (see the color bar). (F) Functional annotations of WPCs by GO, KEGG, and Hallmark databases (FDR < 0.2). (G and H) IL-7–mediated temporal expression of mTORC1 signaling (G) and Myc targets (H). (I) Four interconnected modules of organelles and ribosome components were derived from WPC2. The number and names of proteins and the representative functional term for each module are shown. See also fig. S1 and data S1 and S2.

  • Fig. 2 mTORC1, but not mTORC2, is required for pro-B cell development and pro-B to pre-B cell transition.

    (A to D) Representative flow cytometry plots of BM cells from wild-type (WT) and Cd2icreMtorfl/fl mice. (A) Expression of B220 and CD43 on BM lymphocytes, with the percentages of B220+CD43+IgM cells indicated (after further gating IgM+ cells out of B220+CD43+ cells; see fig. S1A for gating scheme). Right: Number of designated populations. (B) Expression of BP-1 and CD24 on B220+CD43+IgM cells, with the percentages of fraction A (CD24BP-1), fraction B (CD24+BP-1), and fraction C/C′ (CD24+BP-1+) cells indicated. Right: Number of indicated subset cells. (C) Expression of B220 and CD25 on BM lymphocytes, with the percentage of B220+CD25+ pre-B cells indicated. Right: Number of B220+CD25+ pre-B cells. (D) Expression of B220 and IgM on BM lymphocytes, with the percentages of pro-B/pre-B cells (B220+IgM), immature B cells (B220+IgM+), and circulating mature B cells (B220hiIgM+) indicated. Right: Number of immature and mature B cells. (E to H) Representative flow cytometry plots of BM cells of WT, Cd2icreRptorfl/fl, Cd2icreRictorfl/fl, and Cd2icreRptorfl/flRictorfl/fl mice and the number of indicated populations. (E) Expression of B220 and CD43 on BM lymphocytes, with the percentage of B220+CD43+IgM cells indicated. (F) Hardy classification of pro-B cell subsets in the B220+CD43+IgM gate [from (E)], with the percentages of fraction A (CD24BP-1), fraction B (CD24+BP-1), and fraction C/C′ (CD24+BP-1+) cells indicated. (G) Expression of B220 and CD25 on BM lymphocytes, with the percentage of B220+CD25+ pre-B cells indicated. (H) Expression of B220 and IgM on BM lymphocytes, with the percentages of pro-B/pre-B cells (B220+IgM), immature B cells (B220+IgM+; statistics are on the right), and circulating mature B cells (B220hiIgM+) indicated. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with Tukey’s test). Results represent at least four independent experiments. Data are means ± SEM. Numbers indicate percentage of cells in gates. See also fig. S2.

  • Fig. 3 Raptor-mTORC1 is essential to mediate IL-7 signaling and Myc induction but not Stat5 activation.

    (A) Expression of CD71 on fraction A, B, and C/C′ cells from WT and Cd2icreRptorfl/fl mice. Numbers indicate the MFI of CD71 from a representative experiment. (B and C) EdU incorporation (at 1 hour after intravenous injection of EdU) (B) and active caspase-3 staining (C) in fraction A, B, and C/C′ cells from WT and Cd2icreRptorfl/fl mice, with the percentages of EdU+ and active caspase-3+ cells indicated from a representative experiment plotted in the graphs. (D) Cellularity of BM cells after IL-7 stimulation in vitro. Total BM cells were cultured in the presence of IL-7 (5 ng/ml) for 1 week, and cell numbers were counted on days 0, 4, and 7. (E) Immunoblot analysis of p-Stat5, p-S6, and p-4EBP1 in freshly sorted pro-B cells with or without IL-7 stimulation. (F) Immunoblot analysis of p-Stat5, p-S6K, and p-S6 in the in vitro cultured B cells. Lin BM cells were cultured with OP9 stromal cells with IL-7 for 5 days, and cells were washed and rested overnight before they were restimulated with IL-7 for the indicated time points in the presence or absence of rapamycin. DMSO, dimethyl sulfoxide. (G) Expression of BP-1 and CD24 on BM LinB220+CD43+IgM cells from mice injected with vehicle control, recombinant IL-7 (250 μg/kg), rapamycin (15 mg/kg), or IL-7 plus rapamycin, with the percentages of fraction A, B, C, and C′ cells indicated. Right: Number of fraction C and C′ cells. (H) Relative CD71 expression on fraction C cells from mice treated in (G). The MFI of CD71 from mice treated with vehicle control was set as 1. (I) Human BM pro-B cells (CD3CD19+CD10+CD34+CD127+) were sorted from healthy donors. Cells were stimulated with recombinant human IL-7 (10 ng/ml) for 24 hours. Expression of CD71 (left) and cell size [measured by forward light scatter (FSC); right] were analyzed by flow cytometry. Numbers in plots indicate the MFI. (J) Frequency of EGFP-Myc+ cells in fraction A, B, C, and C′ cells from WT/MycEGFP and Cd2icreRptorfl/fl/MycEGFP mice cultured for 4 hours in medium (left) or with IL-7 (right). (K) Five RWPCs during pro-B cell activation were defined by WGCNA. The central rectangle represents the first to third quartile, with notch in the plot corresponding to the median expression of all proteins in the sample. End of the whiskers represents minimum and maximum level of expression of the proteins in the cluster. (L) Functional annotations of RWPCs by Hallmark and KEGG databases (FDR < 0.1). (M and N) Rapamycin-dependent IL-7–induced temporal expression of proteins in mTORC1 signaling (M) and Myc targets (N). **P < 0.01, ***P < 0.001 [Mann-Whitney test (J) and one-way ANOVA with Tukey’s test (G and H)]. Results represent three (A to C, E, and F) or two (D, G, and I) independent experiments or are pooled from four (J) or two (G and H) independent experiments. Data are means ± SEM. Numbers indicate percentage of cells in gates. See also figs. S3 to S5 and data S3.

  • Fig. 4 Loss of Raptor prevents Myc-induced B cell malignancy.

    (A) Survival curve of Eμ-Myc, Cd2icreRptorfl/+Eμ-Myc, and Cd2icreRptorfl/flEμ-Myc mice (P = 0.0005, log-rank test). (B and C) Representative flow cytometry plots of BM cells of WT, Eμ-Myc, Cd2icreRptorfl/fl, and Cd2icreRptorfl/flEμ-Myc mice. (B) Expression of B220 and CD43 on BM lymphocytes, with the percentages of B220+CD43 and B220+CD43+ cells indicated. (C) Expression of B220 and IgM on BM lymphocytes, with the percentages of pro-B/pre-B cells (B220+IgM), immature B cells (B220+IgM+), and circulating mature B cells (B220hiIgM+) indicated. (D) Analysis of CD71 and CD98 expression and cell size (via FSC) of pro-B cells. Numbers indicate the MFI. (E) Frequency of CD19+ B cells in the blood of WT, Eμ-Myc, Cd2icreRptorfl/fl, and Cd2icreRptorfl/flEμ-Myc mice. (F) Immunoblot analysis of p-S6 and p-4EBP1 in pro-B cells from WT, Eμ-Myc, Cd2icreRptorfl/fl, and Cd2icreRptorfl/flEμ-Myc mice. (G) Immunoblot analysis of Myc expression and phosphorylation of eIF4G and eIF4E in pro-B cells from WT, Eμ-Myc, Cd2icreRptorfl/fl, and Cd2icreRptorfl/flEμ-Myc mice. (H) 3D rendering of STORM imaging of polyribosomes (red) and Myc (green) in pro-B cells treated with IL-7. Left: Representative images of unoccupied (top) and Myc-occupied polyribosome (bottom). Right: The percentage of Myc-occupied polyribosomes in each imaged cell was enumerated. Data were pooled from three independent experiments, with a total of 35 cells from WT and 45 cells from Cd2icreRptorfl/fl mice examined. ***P < 0.001, ****P < 0.0001 [one-way ANOVA with Tukey’s test (E) and Mann-Whitney test (H)]. Results represent four (B and C), three (D, F, and H), or two (E and G) independent experiments. Data are means ± SEM. Numbers indicate percentage of cells in gates. See also fig. S6.

  • Fig. 5 Deletion of PTEN inhibits B cell development independent of mTORC1.

    (A) Immunoblot analysis of PTEN in fraction A cells, pro-B cells, pre-B cells, and immature B cells with or without IL-7 stimulation for 4 hours. (B to D) Left: Representative flow cytometry plots of BM cells of WT and Cd2icrePtenfl/fl mice. Right: Absolute number of indicated populations. (B) Expression of B220 and CD43 on BM lymphocytes, with the percentage of B220+CD43+ cells indicated. Right: Number of B220+CD43+IgM B cell precursors. (C) Expression of BP-1 and CD24 on B cell precursors, with the percentages of fraction A (CD24BP-1), fraction B (CD24+BP-1), and fraction C/C′ (CD24+BP-1+) cells indicated. Right: Number of each subset. (D) Expression of B220 and CD25 on BM lymphocytes, with the percentage of B220+CD25+ pre-B cells indicated. Right: Number of B220+CD25+ pre-B cells. (E to H) Representative flow cytometry plots of BM cells of WT, Cd2icrePtenfl/fl, Cd2icreRptorfl/fl, and Cd2icrePtenfl/flRptorfl/fl mice. (E) Expression of B220 and CD43 on BM lymphocytes, with the percentage of B220+CD43+IgM cells indicated. Right: Number of B220+CD43+IgM cells. (F) Hardy classification of pro-B cell subsets in the B220+CD43+IgM gate [from (B)], with the percentages of fraction A (CD24BP-1), fraction B (CD24+BP-1), and fraction C/C′ (CD24+BP-1+) cells indicated. Bottom: Numbers of fraction A, B, and C/C′ cells. (G) Expression of B220 and CD25 on BM lymphocytes, with the percentage of B220+CD25+ pre-B cells indicated. Right: Number of B220+CD25+ pre-B cells. (H) Expression of B220 and IgM on BM lymphocytes, with the percentage of pro-B/pre-B cells (B220+IgM), immature B cells (B220+IgM+), and mature B cells (B220hiIgM+) indicated. Right: Number of immature B cells. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA with Tukey’s test). Results represent at least four independent experiments. Data are means ± SEM. Numbers indicate percentage of cells in gates. See also fig. S7.

  • Fig. 6 PTEN promotes early B cell development through Foxo1-dependent and Foxo1-independent mechanisms.

    (A) Expression of IL-7Rα on fraction A, B, and C/C′ cells of WT and Cd2icrePtenfl/fl mice. Numbers indicate the MFI. (B to E) Representative flow cytometry plots of BM cells from WT, Cd2icrePtenfl/fl, Cd2icrePtenfl/+Foxo1-CA, and Cd2icrePtenfl/flFoxo1-CA mice. (B) Expression of IL-7Rα in pro-B cells. Numbers indicate the MFI. (C) Expression of B220 and CD43 on BM lymphocytes, with the percentage of B220+CD43+ and B220+CD43 cells indicated. (D) Hardy classification of pro-B cell subsets in the B220+CD43+IgM gate (from C), with the percentages of fraction A (CD24BP-1), fraction B (CD24+BP-1), and fraction C/C′ (CD24+BP-1+) cells indicated. (E) Expression of B220 and CD25 on BM lymphocytes, with the percentage of B220+CD25+ pre-B cells indicated. (F) Analysis of apoptotic cell death via annexin V staining on fraction A, B, and C/C′ cells. (G) Expression of Bim in fraction A, B, and C/C′ cells of WT and Cd2icrePtenfl/fl mice. Numbers indicate the MFI. (H) EdU incorporation in fraction A, B, and C/C′ cells of WT and Cd2icrePtenfl/fl mice. (I) Reverse transcription polymerase chain reaction (RT-PCR) analysis of Myc and Myb expression in pro-B cells. (J) Immunoblot analysis of p-Stat5 in pro-B cells of WT and Cd2icrePtenfl/fl mice. (K) RT-PCR analysis of Pax5, Ebf1, and Tcf3 expression in fraction B and C/C′ cells of WT and Cd2icrePtenfl/fl mice. *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney test for cell frequencies and unpaired Student’s t test for cell numbers). Data represent four (A and F), three (J), or two (B to E, G to I, and K) independent experiments. Data are means ± SEM. Numbers indicate percentage of cells in gates. See also fig. S8.

  • Fig. 7 Loss of mTORC1 and PI3K overactivation exert differential effects on immunoglobulin gene rearrangement and expression.

    (A and B) Intracellular (i.c.) expression of Igμ chains in pro-B cells from Cd2icreMtorfl/fl (A) or Cd2icrePtenfl/fl mice (B). (C and D) Semiquantitative RT-PCR analysis of the expression of the rearranged VH7183-DJCμ and VHJ558-DJμ transcripts in fivefold serial dilutions of complementary DNA prepared from sorted fraction B and C/C′ cells from WT and Cd2icreRptorfl/fl (C) or Cd2icrePtenfl/fl mice (D). (E to G) Representative flow cytometry plots of BM cells (E and F) and splenocytes (G) of WT, IgHEL, and Cd2icrePik3ca*IgHEL mice. (E and F) Expression of B220 and HEL (E) and B220 and IgM (F) on BM lymphocytes. (G) Expression of T cell receptor β (TCRβ) and B220 on splenocytes. Results represent three (A and B) or two (C to G) independent experiments. Numbers indicate percentage of cells in quadrants or gates. See also fig. S9.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/1/eaar5701/DC1

    fig. S1. IL-7Rα mediates dynamic metabolic changes during B cell development.

    fig. S2. mTORC1, but not mTORC2, is required for B cell development.

    fig. S3. mTORC1 promotes B cell development independent of Stat5.

    fig. S4. mTORC1 promotes Myc protein expression to support B cell development.

    fig. S5. mTORC1-mediated transcriptional and translational programs in pro-B cells.

    fig. S6. An mTORC1-Myc circuitry controls B cell malignancy.

    fig. S7. Deficiency of PTEN or overactivation of PI3K blocks early B cell development.

    fig. S8. Impaired differentiation and survival in PTEN-deficient pro-B cells.

    fig. S9. Expression of Rag1 and Rag2 in Raptor- and PTEN-deficient B cells.

    data S1. Bioinformatics analysis of gene expression in developing B cell subsets.

    data S2. Whole proteome analysis of IL-7–stimulated pro-B cells.

    data S3. Whole proteome analysis of IL-7– and rapamycin-treated pro-B cells.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. IL-7Rα mediates dynamic metabolic changes during B cell development.
    • fig. S2. mTORC1, but not mTORC2, is required for B cell development.
    • fig. S3. mTORC1 promotes B cell development independent of Stat5.
    • fig. S4. mTORC1 promotes Myc protein expression to support B cell development.
    • fig. S5. mTORC1-mediated transcriptional and translational programs in pro-B cells.
    • fig. S6. An mTORC1-Myc circuitry controls B cell malignancy.
    • fig. S7. Deficiency of PTEN or overactivation of PI3K blocks early B cell development.
    • fig. S8. Impaired differentiation and survival in PTEN-deficient pro-B cells.
    • fig. S9. Expression of Rag1 and Rag2 in Raptor- and PTEN-deficient B cells.
    • Legends for data S1 to S3

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • data S1 (Microsoft Excel format). Bioinformatics analysis of gene expression in developing B cell subsets.
    • data S2 (Microsoft Excel format). Whole proteome analysis of IL-7–stimulated pro-B cells.
    • data S3 (Microsoft Excel format). Whole proteome analysis of IL-7– and rapamycin-treated pro-B cells.

    Files in this Data Supplement:

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