Research ArticleT CELL DEVELOPMENT

RORγt limits the amount of the cytokine receptor γc through the prosurvival factor Bcl-xL in developing thymocytes

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Science Signaling  28 Aug 2018:
Vol. 11, Issue 545, eaam8939
DOI: 10.1126/scisignal.aam8939
  • Fig. 1 RORγt deficiency increases the abundance of surface γc on preselection thymocytes.

    (A) Surface γc abundance and mean fluorescence intensity (MFI) were assessed in CD4, CD8 DN, DP, and TCRβhi mature CD4 and CD8 SP cells by flow cytometry. Bar graphs show means ± SEM of three independent experiments with a total of six mice. (B) Surface γc abundance (MFI) was determined on TCRβlow preselection DP cells and TCRβhi mature SP cells from WT and RORγtKO mice. Data are means ± SEM of eight independent experiments. (C) Sorted TCRβlow DP and TCRβhi SP cells from WT and RORγtKO thymocytes were assessed for Il2rg mRNA abundance. We performed quantitative real-time polymerase chain reaction (qRT-PCR) analysis with primers specific for the mRNA encoding γc (25), and signals were normalized to that corresponding to Actb. Results show means ± SEM of nine independent experiments. (D) Analysis of the change in surface γc abundance during WT and RORγtKO thymocyte development, as defined by staining for CD24 and TCRβ, followed by flow cytometry. Top and middle: Representative histograms and contour plots. Bottom: Data are means ± SEM of four independent experiments. (E) Surface γc abundance on preselection DP thymocytes as defined by staining for CD69 and CCR7. Top: Representative contour plots. Bottom left: Representative histogram showing γc staining in population 1 cells from the indicated mice. Data are representative of three independent experiments. Bottom right: Data are means ± SEM of three experiments showing the MFI of γc staining on CD4+CD8+ DP cells among population 1 (CD69CCR7) cells. Ab, antibody. (F) Flow cytometric analysis of IL-4Rα abundance on the surface of population 1 (CD69CCR7) cells from the indicated mice. Left: Representative histograms. Right: Data are means ± SEM of two experiments showing the MFI of IL-4Rα staining on DP cells among population 1 cells. N.S., not significant; KO, knockout.

  • Fig. 2 RORγt overexpression does not reduce γc abundance.

    (A) Thymocyte development in RORγtTg mice. Total thymocyte numbers were determined, and CD4, CD8 profiles of TCRβhi cells were assessed for WT and RORγtTg mice by flow cytometry. Cell numbers are means ± SEM, whereas histograms and contour plots are representative of five independent experiments. (B) Intracellular RORγt abundance and surface γc abundance were assessed on TCRβlow preselection DP and TCRβhi mature SP thymocytes of WT and RORγtTg mice by flow cytometry. Histograms are representative of five independent experiments. (C) Total lymph node (LN) cell numbers and CD4, CD8 profiles of TCRβ+ LN cells were determined for WT and RORγtTg mice. Cell numbers are means ± SEM, whereas histograms and contour plots are representative of five independent experiments. (D) Intracellular RORγt and surface γc abundance were assessed on WT, RORγtTg, and RORγtKO LN T cells by flow cytometry. Histograms are representative of five independent experiments. (E) Left: Ex vivo interferon-γ (IFN-γ) and IL-17 production in WT and RORγtTg CD4 LN T cells. Right: Data are means ± SEM of four independent experiments with a total of four mice for each genotype. (F) In vitro differentiation of naïve WT and RORγtTg CD4+ T cells into T helper 17 (TH17) cells. Sorted naïve CD4+ T cells from the indicated mice were cultured for 5 days under TH17-skewing conditions. IFN-γ and IL-17 production were assessed by intracellular staining. Top: Dot plots are representative of two independent experiments. Bottom: Data are means ± SEM of two independent experiments with a total of four mice for each genotype.

  • Fig. 3 Transgenic RORγt restores T cell development in RORγtKO mice.

    (A and B) Thymocyte development in RORγtKO/Tg mice. Surface TCRβ abundance (A) and total numbers of thymocytes (B) were determined for WT, RORγtKO, and RORγtKO/Tg mice. Data in (A) are representative of three experiments. Data in (B) are means ± SEM of three independent experiments. (C) Quantitation of RORγt protein abundance in TCRβlow preselection DP thymocytes from WT, RORγtKO, and RORγtKO/Tg mice. Data are means ± SEM of four independent experiments. (D) Surface γc abundance on TCRβlow DP cells of WT, RORγtKO, and RORγtKO/Tg mice was assessed by flow cytometry. Left: Histograms are representative of four independent experiments. Right: Data are means ± SEM of four independent experiments with a total of five mice for each genotype.

  • Fig. 4 Transgenic Bcl-xL reduces γc abundance in RORγtKO mice.

    (A) Thymocytes of the indicated mice were incubated overnight at 37°C in cell culture medium. Cell viability was determined the next day by assessing propidium iodide (PI) exclusion by flow cytometry. Data are means ± SEM of five independent experiments with a total of five mice for each genotype. (B) TCRβ abundance on CD69CCR7 (population I) DP thymocytes of RORγtKO and RORγtKOBcl-xLTg mice was assessed by flow cytometry. Histograms are representative of three independent experiments. (C) Surface γc abundance on TCRβlow DP cells of WT, RORγtKO, and RORγtKOBcl-xLTg mice was assessed by flow cytometry. Left: Histograms are representative of five independent experiments. Right: Data are means ± SEM of five independent experiments with a total of eight mice for each genotype. (D) Surface γc abundance on TCRβlow DP thymocytes of the indicated mice was assessed by flow cytometry. Data are means ± SEM of three independent experiments with five WT, four RORγtKO, three Mcl-1Tg, four BimKO, and three NoxaKO mice. (E) Surface γc abundance on Mcl-1–deficient TCRβlow DP thymocytes was assessed by gating on human CD4 reporter protein (hCD4)–positive cells in Mcl1fl/flCD2-Cre mice. Data are means ± SEM of three independent experiments with five WT, four RORγtKO, and three Mcl1fl/flCD2-Cre mice. (F) Forward scatter (FSC) signals were determined in thymocytes of the indicated mice as a measure of cell size. Data are means ± SEM of four independent experiments with four mice for each genotype.

  • Fig. 5 RORγt is required to suppress metabolic activity in immature DP thymocytes.

    (A) Electron microscopy of sorted TCRβlow DP thymocytes from the indicated mice. Images are representative of at least five analyses per genotype. (B) Cytoplasmic areas were determined from electron microscopy images of TCRβlow DP thymocytes from the indicated mice using the National Institutes of Health (NIH) ImageJ software. Data are means ± SEM of five to nine cells for each genotype. (C) Mitochondrial content was determined as the ratio of mtDNA to nDNA upon total DNA extraction and PCR analysis of sorted TCRβlow DP thymocytes from the indicated mice. Data are means ± SEM of two independent experiments. (D) Histogram shows representative MitoTracker Green staining of TCRβlow DP thymocytes from the indicated mice. Data are representative of four independent experiments. (E) Analysis of the amount of intracellular phosphorylated mammalian target of rapamycin (p-mTOR) in TCRβlow DP thymocytes from the indicated mice. Data are means ± SEM of four independent experiments. (F) Flow cytometric analysis of cell surface CD71 abundance on TCRβlow DP (left) and mature TCRβhi CD4 SP (right) thymocytes from the indicated mice. Histograms are representative of three independent experiments. (G) FSC signals were used to determine cell size for WT CD4 SP thymocytes stimulated with plate-bound antibodies against TCR and CD28 (αTCRβ/CD28; each at 1 μg/ml) in the presence or absence of 10 mM 2-deoxy-d-glucose (2-DG). Data are means ± SEM of three independent experiments. (H) Cell surface γc (left) and CD69 (right) abundance was determined on WT CD4 SP thymocytes stimulated with plate-bound antibodies against TCR and CD28 (each at 1 μg/ml) in the presence or absence of 10 mM 2-DG. Data are means ± SEM of three independent experiments.

  • Fig. 6 Enforced Bcl-xL expression reduces γc abundance.

    (A) Intracellular Bcl-xL and cell surface abundance of γc on thymocytes from RORγtKO, Bcl-xLTg, and WT mice were determined by flow cytometry. Left: Histograms are representative of three independent experiments. Right: Bar graphs are means ± SEM of three independent experiments with a total of three mice for each genotype. (B) Intracellular Bcl-xL and cell surface abundance of γc on thymocytes from RORγtKO, RORγtKO/Tg, and WT mice were determined by flow cytometry. Left: Histograms are representative of three independent experiments. Right: Bar graphs are means ± SEM of three independent experiments with a total of three mice for each genotype.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/545/eaam8939/DC1

    Fig. S1. Surface abundance of γc on RORγtKO thymocytes.

    Fig. S2. Analysis of γc surface abundance on RORγtKO thymocytes.

    Fig. S3. Thymocyte development in RORγtTg mice.

    Fig. S4. Thymocyte development in RORγtKO and RORγtKO/Tg mice.

    Fig. S5. Phenotypic characterization of RORγtKOBcl-xLTg thymocytes.

    Fig. S6. Thymocyte development in Mcl1fl/flCD2-Cre mice.

    Fig. S7. Surface γc abundance on Bcl-xLTg thymocytes.

  • This PDF file includes:

    • Fig. S1. Surface abundance of γc on RORγtKO thymocytes.
    • Fig. S2. Analysis of γc surface abundance on RORγtKO thymocytes.
    • Fig. S3. Thymocyte development in RORγtTg mice.
    • Fig. S4. Thymocyte development in RORγtKO and RORγtKO/Tg mice.
    • Fig. S5. Phenotypic characterization of RORγtKOBcl-xLTg thymocytes.
    • Fig. S6. Thymocyte development in Mcl1fl/flCD2-Cre mice.
    • Fig. S7. Surface γc abundance on Bcl-xLTg thymocytes.

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