Research ArticleImmunology

Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability

See allHide authors and affiliations

Science Signaling  17 May 2016:
Vol. 9, Issue 428, pp. ra51
DOI: 10.1126/scisignal.aad1576
  • Fig. 1 Themis1 enhances TCR signaling during thymic positive selection.

    (A) Contour plots represent the gating strategy to analyze GFP expression in preselection (TCRloCD69lo; red line in the GFP plot) and postselection (TCRloCD69hi or TCRintCD69hi; blue and green lines in the GFP plot, respectively) DP thymocytes from AND-Nur77-GFP transgenic mice. The abundance of the AND TCR was analyzed with anti-Vα11 antibodies. Histograms represent GFP expression in pre- and postselection DP thymocytes from AND-Nur77-GFP transgenic mice that either do or do not express the Bcl2 transgene (Bcl2-tg). Thymocytes from Themis1+/+ mice (Th+/+) were compared to those from Themis1−/− mice (Th−/−) or to Themis1 transgenic mice (Th-tg). Data are from one experiment and are representative of four independent experiments. (B) Left: Flow cytometric analysis of T cell development in Themis1+/+ and Themis1−/− mice that are either sufficient (Bim+/+) or deficient (Bim−/−) in the proapoptotic factor Bim. Contour plots represent either CD4 versus CD8 staining profiles of thymocytes (top) and splenocytes (bottom) or CD24 versus TCR staining profiles of CD4 SP thymocytes (middle). Numbers indicate the percentage of cells in the gated population. Right: Bar graphs present the mean percentages of CD4 and CD8 SP thymocytes (top), the mean ratio of mature CD24lo CD4 SP or CD24lo CD8 SP thymocytes to DP thymocytes (middle), and the mean percentages of splenic CD4+ and CD8+ T cells (bottom). Data are means ± SEM of three independent experiments containing one mouse of each genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test.

  • Fig. 2 Mass spectrometric analysis of the Themis1 interactome in thymocytes.

    (A) Table representing the main interactors of Themis1 in resting (unstimulated) thymocytes and in pervanadate-treated (stimulated) thymocytes from C57BL/6 mice. Proteins were filtered and sorted on the basis of the enrichment ratio (m.i., mean intensity) and the mean iBAQ metric as described in Materials and Methods. The complete list of proteins identified by proteomic analysis of Themis1-immunopurified samples is provided in table S1. WT, wild type; KO, knockout. (B) Schematic of those proteins that preferentially interact with Themis1 in stimulated thymocytes. Layouts indicate the classification of these proteins according to their signaling function (purple layout: adaptor proteins; green layout: effector proteins; gray layout: unknown function in T cell signaling). Keys indicate protein classification according to molecular function (phosphatase, GEF, and E3 ubiquitin ligase). Data are representative of three independent experiments.

  • Fig. 3 Themis1 enhances Vav1 activity in thymocytes.

    (A) Left: Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with low (3 μg/ml) or high (30 μg/ml) concentrations of preformed anti-CD3 and anti-CD4 antibody complexes for the indicated times. Total cytoplasmic extracts of the cells were then analyzed by Western blotting with antibodies against phosphorylated forms of ERK, SHP-1, Vav1, SLP-76, and p38 MAPK. Right: Graphs show the relative abundances of the indicated phosphorylated proteins as determined by calculating the ratios of the intensities of the bands corresponding to the phosphorylated proteins to those corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the loading control. The y axes represent means ± SD of the relative values calculated after normalization to the highest value in the low-dose condition. Data are from four independent experiments each including one mouse of the indicated genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. (B) Analysis of Vav1 phosphorylation by intracytoplasmic staining of thymocytes from AND-TCR transgenic mice that were either sufficient (Th+/+) or deficient (Th−/−) in Themis1. Histograms represent Vav1 phosphorylation on Tyr174 (Y174) in gated preselection (CD5lo) and postselection (CD5hi) DP thymocytes. Data are representative of three independent experiments each including one mouse of the indicated genotype. (C) Left: Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with high-dose anti-CD3 (α-CD3) and anti-CD4 (α-CD4) antibodies (30 μg/ml) for the indicated times. Cells were then lysed and subjected to Rac1-GTP pull-downs with a GST-PAK1-RBD fusion protein. Pull-downs and total cytoplasmic lysates were then analyzed by Western blotting (IB) with an anti-Rac1 antibody. Western blots are representative of three independent experiments each including one mouse of the indicated genotype. Right: Graphs show the ratios of Rac1-GTP to total Rac1. Data are representative of three independent experiments.

  • Fig. 4 Grb2 protein abundance is reduced in Themis1−/− thymocytes.

    (A and B) Thymocytes from Themis1+/+ and Themis1−/− mice were stimulated with premixed high-dose anti-CD3 (α-CD3) and anti-CD4 (α-CD4) antibodies (30 μg/ml) for the indicated times. Samples were then subjected to immunoprecipitation (IP) with antibodies specific for LAT (A) or Vav1 (B) and then analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are from one experiment and are representative of three independent experiments. (B) Total cytoplasmic extracts (TCE) from each sample were analyzed by Western blotting with antibodies against Vav1 or Grb2. (C) Left: Total cytoplasmic extracts of thymocytes from Themis1+/+ (Th+/+), Themis1−/− (Th−/−), and Grb2+/− (Gr+/−) mice were analyzed by Western blotting with anti-Grb2 and anti-GAPDH antibodies. Right: Densitometric analysis of the ratio of Grb2 band intensities to GADPH band intensities normalized to the ratio in WT control thymocytes, which was set at 1. Data are means ± SD of four independent experiments each including one mouse of the indicated genotype. **P < 0.01 by two-tailed, unpaired t test. (D) Flow cytometric analysis of Grb2 abundance in DP, CD4 SP, and CD8 SP thymocyte subsets from Themis1+/+ (Th+/+), Themis1−/− (Th−/−), and Grb2+/− (Gr+/−) mice. Data are representative of three independent experiments. (E) Left: Thymocytes from Grb2+/+ and Grb2+/− mice were stimulated with low (3 μg/ml) or high (30 μg/ml) concentrations of preformed anti-CD3 and anti-CD4 antibody complexes for the indicated times. Total cytoplasmic extracts of the cells were then analyzed by Western blotting with antibody against pVav1. Right: Graphs show the relative abundance of pVav1, as determined from a ratio of the intensity of the pVav1 bands to those of the bands corresponding to the GAPDH loading control. Western blots and densitometry are from a single experiment and are representative of three independent experiments.

  • Fig. 5 Grb2 protein turnover is increased in Themis1−/− thymocytes.

    (A) RT-PCR analysis of Grb2 mRNA abundance in total thymocytes from Themis1+/+, Themis1−/−, and Grb2+/− mice. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (B) Thymocytes from Themis1+/+ (Th+/+) and Themis1−/− (Th−/−) mice were left untreated (black bars) or were treated (white bars) for 16 to 18 hours with cycloheximide (10 μg/ml). Grb2 protein abundance was then analyzed by flow cytometry after intracytoplasmic staining of the cells with anti-Grb2 antibodies. Bar graphs show relative mean fluorescence intensities (MFIs) of Grb2 in treated cells as a percentage of the MFI of Grb2 in untreated cells, which was set at 100%. Data are means ± SD of three mice of each genotype. (C) Thymocytes from Themis1+/+ (black bars) and Themis1−/− mice (white bars) were left untreated or were treated with 1 mM MG132 for 16 to 18 hours. Grb2 protein abundance was analyzed by flow cytometry after intracytoplasmic staining of cells with anti-Grb2 antibodies. Bar graphs show the relative MFIs of Grb2 in treated cells as a percentage of the MFI of Grb2 in Themis1+/+ thymocytes, which was set at 100%. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (D) Thymocytes from Themis1+/+ and Themis1−/− mice were preincubated with MG132 and left untreated or treated with pervanadate (PV) for 5 min at 37°C. Grb2 was then immunoprecipitated from cellular extracts as described in Materials and Methods. Samples were analyzed by Western blotting with anti-ubiquitin and anti-Grb2 antibodies. Western blots are representative of three independent experiments each including one mouse of the indicated genotype. Data in (A) to (C) were analyzed by two-tailed unpaired t test. *P < 0.05, **P < 0.01.

  • Fig. 6 Transgenic expression of Themis1 in thymocytes increases Grb2 protein abundance and improves positive selection.

    (A) Left: Thymocytes from Themis1 WT (Themis1+/+) and Themis1 transgenic (Themis1-tg) mice were stimulated with preformed anti-CD3 and anti-CD4 antibody complexes (3 μg/ml) for the indicated times. Total cytoplasmic extracts were then analyzed by Western blotting with antibodies against pVav1 and pSLP76. GAPDH was used as a loading control. Right: Graphs show the relative abundances of the phosphorylated proteins as determined from a ratio of the intensity of the bands of phosphorylated proteins to those of GAPDH. The y axes represent means ± SD of the relative values calculated after normalization to the highest value in Themis1+/+ thymocytes. Data are from three independent experiments each including one mouse of the indicated genotype. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. Western blots are from one experiment and are representative of three independent experiments. (B) Flow cytometric analysis of Grb2 and Themis1 in total thymocytes from Themis1 transgenic mice (Tg) or littermate controls (WT). Data are representative of three independent experiments each including one mouse of the indicated genotype. (C) Left: Western blotting analysis of the relative amounts of Themis1, Grb2, and Vav1 in total thymocytes from Themis1 transgenic mice (Tg), WT littermate controls (T+/+), and Grb2+/− (Gr+/−) mice. Right: Bar graphs show the mean ratio ± SD of Grb2 band intensity values to Vav1 band intensity values, normalized to the ratio in T+/+ control thymocytes, which was set at 1. Data are means ± SD of three mice of each genotype. ‡P < 0.05 by two-tailed, unpaired t test. (D) Flow cytometric analysis of positive selection in AND Themis1 transgenic mice (Th-tg). Left: Contour plots represent the CD4 versus CD8 staining profiles of thymocytes from Themis1-tg mice and WT littermate controls (Th+/+) expressing the AND TCR. Histograms represent AND TCR surface staining with anti-Vα11 antibodies. Right: CD24 versus Vα11 staining profiles of CD4 SP thymocytes from Themis1-tg and littermate control mice expressing the AND TCR. Numbers indicate the percentage of cells in the gated population. Right: Bar graphs show the mean percentages of Vα11hi cells (top) and the mean ratio of mature CD4 SP (CD24lo) thymocytes to total DP thymocytes (bottom) in Themis1-tg and littermate control (WT) mice. Data are means ± SD of nine mice of each genotype. Five independent experiments were performed. *P < 0.05, **P < 0.01 by two-tailed, unpaired t test. Bottom: Cell surface staining of CD5 on gated DP and CD4 SP thymocyte subsets from Themis1-tg (Tg) and littermate control (Th+/+) mice. Data are representative of three independent experiments.

  • Fig. 7 Transgenic expression of Themis1 in Grb2+/− mice restores TCR signaling and positive selection.

    (A) Top: Total cytoplasmic extracts of thymocytes from Grb2+/+ and Grb2+/− mice that either expressed (+) or did not express (−) the Themis1 transgene (Themis1-tg) were analyzed by Western blotting with anti-Grb2 and anti-GAPDH antibodies. Bottom: Bar graphs show the mean ratio of Grb2 intensity values to GAPDH intensity values normalized to the ratio in WT control thymocytes, which was set at 1. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. (B) Thymocytes from Grb2+/+ and Grb2+/− mice that either expressed (+) or did not express (−) the Themis1 transgene (Themis1-tg) were stimulated with preformed anti-CD3 and anti-CD4 antibody complexes (30 μg/ml) for the indicated times. Total cytoplasmic extracts were analyzed by Western blotting with antibodies against the indicated phosphorylated proteins. Western blots are representative of three independent experiments. (C) Flow cytometric analysis of positive selection in AND-Grb2+/+ and in AND-Grb2+/− mice that did or did not express the Themis1 transgene (Themis1-tg). Contour plots represent CD4 versus CD8 staining profiles of thymocytes from mice of the indicated genotypes. Histograms represent the cell surface staining of the AND TCR on total thymocytes with anti-Vα11 antibodies. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of five mice (for Themis1+/+ and Grb2+/−) or three mice (for Themis1-tg and Themis1-tg/Grb2+/−). (D) Flow cytometric analysis of positive selection in AND ζ6Y/6Y and AND ζ6Y/6F mice that did or did not express the Themis1 transgene (Themis1-tg). Contour plots represent CD4 versus CD8 staining profiles of thymocytes from the indicated genotypes. Histograms represent AND TCR cell surface staining with anti-Vα11 antibodies on total thymocytes. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of three independent experiments each including one mouse of the indicated genotype. Data in (A), (C), and (D) were analyzed by two-tailed, unpaired t test. *P < 0.05, **P < 0.01.

  • Fig. 8 Themis1 hemideficiency exacerbates the defects in positive selection and TCR signaling in Grb2+/− mice.

    (A) Flow cytometric analysis of positive selection in AND-Grb2+/+ and in AND-Grb2+/− mice that were either WT (Themis1+/+) or heterozygous (Themis1+/−) for Themis. Contour plots represent CD4 versus CD8 staining profiles of thymocytes from mice of the indicated genotypes. Histograms show the cell surface staining of the AND TCR on thymocytes with anti-Vα11 antibody. Numbers indicate the percentages of cells in the gated populations. Right: Bar graphs show the mean percentages of CD4 SP thymocytes (top) and Vα11high thymocytes (bottom) from mice of the indicated genotypes. Data are means ± SD of five mice (for Grb2+/+Themis1+/+ and Grb2+/−Themis1+/+) or three mice (for Grb2+/+Themis1+/− and Grb2+/−Themis1+/−). *P < 0.05 by two-tailed, unpaired t test. (B) Histograms show the cell surface staining of CD5 and the relative amounts of Grb2 in CD4 SP thymocytes from mice of the indicated genotypes. Data are representative of three independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/428/ra51/DC1

    Materials and Methods

    Fig. S1. Themis1 enhances TCR signaling during thymic selection.

    Fig. S2. Analysis of the interaction between Themis1 and Vav1 in thymocytes and HEK 293T cells.

    Fig. S3. Themis1−/− thymocytes exhibit normal phosphorylation of Lck, ZAP-70, and LAT after cross-linking of the TCR with low or high concentrations of antibody complexes.

    Fig. S4. The amount of Grb2 that coimmunoprecipitates with c-Cbl and Sos1 is reduced in thymocytes from Themis1−/− mice.

    Fig. S5. Loss of c-Cbl does not affect Grb2 protein abundance in thymocytes.

    Fig. S6. Analysis of TCR signaling and Grb2 coimmunoprecipitation with Vav1 and LAT in Themis1 transgenic thymocytes.

    Fig. S7. Effect of Themis1 overexpression on T cell development.

    Fig. S8. Transgenic expression of Themis1 in Grb2+/− mice restores positive selection in TCR transgenic mice.

    Table S1. List of proteins identified by proteomic analysis of Themis1-immunopurified samples.

  • Supplementary Materials for:

    Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability

    Ekaterina Zvezdova, Judith Mikolajczak, Anne Garreau, Marlène Marcellin, Lise Rigal, Jan Lee, Seeyoung Choi, Gaëtan Blaize, Jérémy Argenty, Julien Familiades, Liqi Li, Anne Gonzalez de Peredo, Odile Burlet-Schiltz, Paul E. Love, Renaud Lesourne*

    *Corresponding author. Email: renaud.lesourne{at}inserm.fr

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Themis1 enhances TCR signaling during thymic selection.
    • Fig. S2. Analysis of the interaction between Themis1 and Vav1 in thymocytes and HEK 293T cells.
    • Fig. S3. Themis1−/− thymocytes exhibit normal phosphorylation of Lck, ZAP-70, and LAT after cross-linking of the TCR with low or high concentrations of antibody complexes.
    • Fig. S4. The amount of Grb2 that coimmunoprecipitates with c-Cbl and Sos1 is reduced in thymocytes from Themis1−/− mice.
    • Fig. S5. Loss of c-Cbl does not affect Grb2 protein abundance in thymocytes.
    • Fig. S6. Analysis of TCR signaling and Grb2 coimmunoprecipitation with Vav1 and LAT in Themis1 transgenic thymocytes.
    • Fig. S7. Effect of Themis1 overexpression on T cell development.
    • Fig. S8. Transgenic expression of Themis1 in Grb2+/− mice restores positive selection in TCR transgenic mice.
    • Legend for table S1

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.77 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of proteins identified by proteomic analysis of Themis1-immunopurified samples.

    Citation: E. Zvezdova, J. Mikolajczak, A. Garreau, M. Marcellin, L. Rigal, J. Lee, S. Choi, G. Blaize, J. Argenty, J. Familiades, L. Li, A. Gonzalez de Peredo, O. Burlet-Schiltz, P. E. Love, R. Lesourne, Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability. Sci. Signal. 9, ra51 (2016).

    © 2016 American Association for the Advancement of Science

Navigate This Article