Research ArticleTHYMUS

Metabolic signaling directs the reciprocal lineage decisions of αβ and γδ T cells

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Science Immunology  06 Jul 2018:
Vol. 3, Issue 25, eaas9818
DOI: 10.1126/sciimmunol.aas9818
  • Fig. 1 Dynamic regulation of cell metabolism and mTORC1 activity and the requirement of RAPTOR in thymocyte development.

    (A and B) OCR in thymocyte subsets under basal condition (A) or in response to the indicated mitochondrial inhibitors (B). Oligo, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Rot, rotenone. SRC of DN3 cells is shown. (C and D) Measurement of ECAR (C) and glycolytic activity (D) in freshly isolated thymocyte subsets. DPM, disintegrations per minute. (E) Ratio of OCR to ECAR in thymocyte subsets. (F) Relative phosphorylation of S6 [mean fluorescence intensity (MFI) in DN3 cells is set to 1] in thymocyte subsets. (G) Total thymocyte cellularity of WT and Rptor−/− mice. Each symbol represents an individual mouse (n = 8 each group). (H) Left: Flow cytometry of thymocyte subsets in WT and Rptor−/− mice. Right: Cellularity of the indicated thymocyte subsets (DN, CD4CD8; DP, CD4+CD8+; CD8SP, CD4CD8+TCRβ+; and CD4SP, CD4+CD8TCRβ+). Each symbol represents an individual mouse (WT, n = 7; Rptor−/−, n = 6). Data are means ± SEM. ns, not significant; *P < 0.05, **P < 0.001, and ***P < 0.0001. One-way analysis of variance (ANOVA) with Tukey’s test (A, C, and E), two-way ANOVA with Bonferroni’s test (B), or two-tailed unpaired t test (G and H). Data are combination of three independent experiments (A to C, and E) or representative of three (D) or at least 10 independent experiments (F to H). Numbers indicate percentage of cells in gates.

  • Fig. 2 RAPTOR deficiency impairs the DN-to-DP transition in αβ T cell development.

    (A) Flow cytometry of DN1 to DN4 subsets (gated on lineage-negative DN thymocytes) in WT and Rptor−/− mice (left). Frequencies (middle) and cellularity (right) of DN subsets. Each symbol represents an individual mouse (WT, n = 6; Rptor−/−, n = 5). (B) Left: Gating strategy for ISP cells (CD4CD8+TCRβ). Right: Flow cytometry (top) and frequency and cellularity (bottom) of ISP cells in WT and Rptor−/− mice. Each symbol represents an individual mouse (WT, n = 9; Rptor−/−, n = 8). (C and D) Sublethally irradiated Rag1−/− mice were reconstituted with a 1:1 mixture of CD45.1+ BM and either CD45.2+ WT or Rptor−/− BM cells to generate mixed BM chimeras. Flow cytometry of thymocyte subsets (C, top) or ISP cells (D, top) in CD45.2+ WT and Rptor−/− donor-derived cells or in CD45.1+ spike-derived cells (C and D, bottom). (E) Expression of CD4 and CD8 on WT and Rptor−/− DN3 cells cocultured with OP9-DL1 cells for 3 or 8 days. (F) Left: Flow cytometry of thymocyte subsets in WT OTII and Rptor−/− OTII mice (n = 4 each group). Right: Cellularity of total thymocytes. Data are means ± SEM. *P < 0.0005 and **P < 0.0001. One-way ANOVA with Tukey’s test (A), two-tailed Mann-Whitney test (B, frequency), or two-tailed unpaired t test (B, cellularity). Data are representative of at least three independent experiments (A to F). Numbers indicate percentage of cells in quadrants or gates.

  • Fig. 3 Loss of RAPTOR promotes γδ T cell development.

    (A) Flow cytometry (left) of TCRγδ+ cells in WT and Rptor−/− thymocytes. Frequency (middle) and cellularity (right) of TCRγδ+ cells. Each symbol represents an individual mouse (WT, n = 6; Rptor−/−, n = 5). (B) Flow cytometry of TCRγδ+ cells derived from WT and Rptor−/− donor (CD45.2+, left) or spike (CD45.1+, right) cells in the reconstituted BM chimeras as described in Fig. 2C. (C) Distribution of DP (left) and γδ T cells (right) between CD45.1+- and CD45.2+-derived cells in the mixed BM chimeras generated as described in Fig. 2C, after normalization against nondeleting DN1 thymocytes. (D) Expression of CD73 and CD24 on CD4CD8− TCRγδ+ thymocytes from WT and Rptor−/− mice. (E) Frequency (left) and cellularity (right) of CD73+ TCRγδ+ cells in (D). Each symbol represents an individual mouse (n = 4 each group). (F) Expression of CD73 on TCRγδ+ cells in the thymus and spleen of WT and Rptor−/− mice, with MFI plotted above graphs. Data are means ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005. Two-tailed Mann-Whitney test (A and E for frequency analyses), one-tailed unpaired t test (C), or two-tailed unpaired t test (A and E for cellularity). Data are representative of at least three independent experiments (A to F). Numbers indicate percentage of cells in quadrants or gates.

  • Fig. 4 Interplay between mTORC1 and MYC in developing thymocytes.

    (A) Flow cytometry of TCRβ+ and TCRγδ+ cells in WT and Myc−/− thymocytes. (B) Frequency of TCRγδ+ cells (left) and the ratio of TCRγδ+ to TCRβ+ cells (right) in WT and Myc−/− thymocytes. Each symbol represents an individual mouse (n = 4 each group). (C) Expression of CD73 and CD24 on CD4CD8−  TCRγδ+ thymocytes from WT and Myc−/− mice (left). Frequency (middle) and cellularity (right) of CD73+ TCRγδ+ cells. Each symbol represents an individual mouse (WT, n = 5; Myc−/−, n = 4). (D) Expression of CD73 on TCRγδ+ cells in the thymus and spleen of WT and Myc−/− mice, with MFI plotted above graphs. (E) Expression of GFP-MYC in thymocyte subsets of WT and Rptor−/− mice, with MFI plotted above graphs. (F and G) Plots of p-S6 (F) and p–4E-BP1 (G) in thymocyte subsets of WT and Myc−/− mice, with MFI plotted above graphs. Data are means ± SEM. *P < 0.05. Two-tailed Mann-Whitney test (B and C for frequency analyses) or two-tailed unpaired t test (B for ratio analyses and C for cellularity). Data are representative of at least three independent experiments (A to E) or two independent experiments (F and G). Numbers indicate percentage of cells in quadrants or gates.

  • Fig. 5 RAPTOR controls metabolic balance between glycolysis and OXPHOS and production of ROS in fate determination of DN3 cells.

    (A) OCR in WT and Rptor−/− DN3 cells under basal conditions or in response to the indicated mitochondrial inhibitors (SRC of WT and Rptor−/− cells is shown). (B) Measurement of ECAR in WT and Rptor−/− DN3 cells. (C) Ratio of OCR to ECAR in WT and Rptor−/− DN3 cells. (D and E) Analysis of ROS production in DN3 cells (D) and TCRγδ+ cells (E) from WT and Rptor−/− mice. (F and G) Left: WT DN3a cells were cocultured with OP9-DL1 cells in the absence or presence of NAC or GSH for 3 days, followed by analysis of CD4 and TCRγδ expression (F) and CD4 and CD8 expression (G). Right: Numbers of TCRγδ+ (F) and CD4+CD8+ DP cells (G) in the culture (n = 3 each group). (H) Left: WT and Rptor−/− DN3a cells were cocultured with OP9-DL1 cells in the absence or presence of NAC for 5 days, followed by analysis of CD4 and TCRγδ expression. Right: Fold change of the ratio of TCRγδ+ to CD4+ cells (WT without NAC treatment is set to 1), and frequencies of TCRγδ+ and CD4+ cells (WT without NAC treatment is set to 1, n = 3 each group). (I) Left: WT and Rptor−/− DN3a cells were cocultured with OP9-DL1 cells in the absence or presence of NAC for 5 days, followed by analysis of CD44 and CD25 expression. Right: Fold change of the ratio of CD25+CD44 (DN3) to CD25CD44 (DN4) populations of WT and Rptor−/− cells without or with NAC treatment (WT without NAC treatment is set to 1). (J) Cellularity of total cells in (H). Data are means ± SEM. *P < 0.05 and **P < 0.01. Two-way ANOVA with Bonferroni’s test (A), one-way ANOVA with Tukey’s test (F to I), or two-tailed unpaired t test (J). Data are the combination of three independent experiments (A to C) or representative of at least three (D to J) independent experiments. Numbers indicate percentage of cells in quadrants or gates.

  • Fig. 6 scRNA-seq reveals that RAPTOR coordinates immune signaling and anabolic metabolism in thymocyte fate decisions.

    (A) scRNA-seq of WT and RAPTOR-deficient DN cells, followed by a tSNE visualization of the 21,332 single cells analyzed. (B) tSNE visualization of eight clusters partitioned by unsupervised clustering. (C) tSNE visualization of DN3a gene signature expressed by individual cells. (D) Association of different gene signatures with the eight single-cell clusters, with the relative overrepresentation of gene signatures for a specific cluster determined using one-tailed Mann-Whitney test. Clusters 1, 5, and 8 contained DN3a cells, whereas the remaining clusters contained post-selection thymocytes. (E) Frequencies of WT and Rptor−/− cells in the different clusters. Clusters significantly enriched for presence of WT or Rptor−/− cells were marked. (F) Violin plots of signatures of pre-TCR and ERK signaling among the eight clusters. A violin plot combines the box plot and the local density estimation into a single display. The black bars and thin lines within the violin plots indicate the interquartile range (first quantile to third quantile) and the entire range of the data (up to 1.5-fold of interquartile range from first/third quantile), respectively, and the white dots in the center indicate the median values. (G) Violin plots of signatures of mTORC1 signaling, MYC targets, and glycolysis. (H and I) tSNE (H) and violin (I) plots of γδ T cell gene signature. (J) Pseudotime densities for the eight clusters. Data are means ± SEM. ***P < 0.0001. One-tailed Mann-Whitney test (D), logistic regression (E), and two-tailed Mann-Whitney test (F, G, and I). Data are from one experiment (WT, n = 2; Rptor−/−, n = 3; A to J).

  • Fig. 7 RAPTOR tunes the strength of the ERK/EGR1/ID3 signaling axis in fate choices.

    (A) Immunoblot analysis of p-ERK, EGR1, ID3, and β-ACTIN in WT and Rptor−/− DN3 cells. (B and C) Plots of p-ERK (B) and expression of ID3 (C) in WT and Rptor−/− DN3 cells, with MFI plotted in graphs. (D) Cellularity of CD4+CD8+ DP cells from the indicated mice. Each symbol represents an individual mouse (WT, n = 5; Rptor−/−, n = 5; Erk2−/−, n = 4; and Rptor−/−Erk2−/−, n = 4). (E) Left: Flow cytometry of CD73 and CD24 expression on TCRγδ+ thymocytes in the indicated mice. Right: Frequency and cellularity of CD73+ TCRγδ+ cells. Each symbol represents an individual mouse (WT, n = 5; Rptor−/−, n = 5; Erk2−/−, n = 4; and Rptor−/−Erk2−/−, n = 4). (F) Expression of CD73 on TCRγδ+ cells in (E) shown as the fold change (MFI on WT cells is set to 1). Data are means ± SEM. *P < 0.05 and ***P < 0.001. One-way ANOVA with Tukey’s test (D to F). Data are representative of at least three independent experiments (A to F).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/25/eaas9818/DC1

    Materials and Methods

    Fig. S1. Dynamic regulation of nutrient transporters and cell growth in developing thymocytes.

    Fig. S2. mTOR signaling is required for thymocyte development.

    Fig. S3. RAPTOR deficiency diminishes cell growth, proliferation, and nutrient transporter expression of ISP cells, but not cell survival.

    Fig. S4. RAPTOR controls metabolic gene expression programs in thymocytes.

    Fig. S5. RAPTOR deficiency affects γδ T cell development.

    Fig. S6. Loss of RHEB or RICTOR does not affect the development of γδ T cells.

    Fig. S7. SCAP and HIF1α are dispensable for T cell development.

    Fig. S8. MYC is implicated in αβ and γδ T cell development.

    Fig. S9. RAPTOR-deficient DN3 cells exhibit increased ROS but normal mitochondrial mass and membrane potential.

    Fig. S10. Modulation of ROS production alters fate choices of DN3 cells.

    Fig. S11. mTORC1 activation integrates signals from pre-TCR and NOTCH.

    Fig. S12. RAPTOR deficiency alters transcriptional programs and signaling and metabolic pathways.

    Fig. S13. Monocle pseudotime trajectory of single-cell transcriptomics data.

    Fig. S14. Loss of RAPTOR or MYC enhances signal strength.

    Fig. S15. Schematics of mTORC1 and metabolic control of redox homeostasis and signal strength in T cell lineage choices.

    Table S1. Raw data sets.

    Reference (54)

  • Supplementary Materials

  • The PDF file includes:
    • Materials and Methods
    • Fig. S1. Dynamic regulation of nutrient transporters and cell growth in developing thymocytes.
    • Fig. S2. mTOR signaling is required for thymocyte development.
    • Fig. S3. RAPTOR deficiency diminishes cell growth, proliferation, and nutrient transporter expression of ISP cells, but not cell survival.
    • Fig. S4. RAPTOR controls metabolic gene expression programs in thymocytes.
    • Fig. S5. RAPTOR deficiency affects γδ T cell development.
    • Fig. S6. Loss of RHEB or RICTOR does not affect the development of γδ T cells.
    • Fig. S7. SCAP and HIF1α are dispensable for T cell development.
    • Fig. S8. MYC is implicated in αβ and γδ T cell development.
    • Fig. S9. RAPTOR-deficient DN3 cells exhibit increased ROS but normal mitochondrial mass and membrane potential.
    • Fig. S10. Modulation of ROS production alters fate choices of DN3 cells.
    • Fig. S11. mTORC1 activation integrates signals from pre-TCR and NOTCH.
    • Fig. S12. RAPTOR deficiency alters transcriptional programs and signaling and metabolic pathways.
    • Fig. S13. Monocle pseudotime trajectory of single-cell transcriptomics data.
    • Fig. S14. Loss of RAPTOR or MYC enhances signal strength.
    • Fig. S15. Schematics of mTORC1 and metabolic control of redox homeostasis and signal strength in T cell lineage choices.
    • Reference ( 54)

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  • Other Supplementary Material for this manuscript includes the following:
    • Table S1 (Microsoft Excel format). Raw data sets.

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