Research ArticleImmunology

Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity

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Sci. Signal.  12 Jun 2018:
Vol. 11, Issue 534, eaan8799
DOI: 10.1126/scisignal.aan8799
  • Fig. 1 GAP activity is not intrinsic to thymocyte plexin-D1.

    (A) Sema3E induces activated Cdc42 Rho GTPase in the DP-257-20-109 model DP thymocyte cell line. Left: The cells were treated with the indicated concentrations of sema3E for 10 min. Right: The cells were treated with sema3E (3 μg/ml) for the indicated times. These parameters were selected as optimal for all subsequent assays. Relative activation was determined by measuring band intensities from GST-PAK1-CRIB coprecipitation assay. Data are representative of three experiments. (B) Plexin-D1 surface expression on parental DP257-20-109 cells and transfected DP257-20-109 cells stably expressing full-length plexin-D1 was assessed by flow cytometry. Data are representative of four experiments. FITC, fluorescein isothiocyanate; plexin-D1hi, plexin-D1 overexpression in DP257-20-109 cells. (C) Top: Measurement of active Cdc42 in DP257-20-109 cells (parental), DP257-20-109 cells overexpressing full-length plexin-D1 (plexin-D1), and DP257-20-109 cells expressing myristoylated plexin-D1 cytoplasmic tail (myr–plexin-D1–cyto) after treatment with sema3E (3 μg/ml) for 10 min. Bottom: Measurement of active Cdc42 in the indicated cell lines after treatment with CXCL12 (100 ng/ml) for 3 min. The responses of cells overexpressing full-length plexin-D1 represent the mean of three independent transfected clones, whereas those of cells expressing the myr–plexin-D1–cyto construct are the mean of six independent clones. Data are means ± SEM and are representative of two independent experiments. (D) Assay of monomeric plexin-D1 cytoplasmic tail GAP activity for Cdc42, R-Ras, Rac1, and Rnd3 (control). We used p50-RhoGAP as a positive control. Data are means ± SEM of four replicates and are representative of three experiments.

  • Fig. 2 Thymocyte GAP proteins interact with the cytoplasmic tail of plexin-D1.

    (A) List of the mouse thymocyte GAP proteins that were selected for analysis. (B) Western blotting analysis of FLAG-GAP proteins immunoprecipitated from 5 × 105 seeded cells to detect coimmunoprecipitated plexin-D1 (top). Whole-cell lysates were also analyzed by Western blotting to detect full-length plexin-D1 (middle) and the indicated GAPs (bottom). Data are representative of six experiments. (C) Analysis of cytoskeletal collapse and detachment in 5 × 105 seeded cells expressing the indicated GAPs alone or together with plexin-D1. (D) Summary analysis of the percentage of cells expressing the indicated GAPs that exhibited cell rounding or detachment from the experiments shown in (C). Data are means ± SEM of five experiments. The dashed red line marks two SDs above the control. For all conditions, 3109 ± 209 cells were counted. (E) Transcriptional profiles of thymocytes developing from the early thymocyte progenitor (ETP) stage to the SP stages for genes encoding the indicated GAPs, Plxnd1, and Sema3e (21). SP profiles represent the mean of pooled CD4SP and CD8SP profiles for simplicity. ISP, immature single-positive CD8 thymocyte. (F) TAGAP locus single-nucleotide polymorphisms (SNPs) associated with human autoimmune disease. Shading intensity for each pathology represents exponent of probability of association from B (borderline) to −7 (P < 10−7). See table S2 for details. (G) Direct coimmunoprecipitation of recombinant plexin-D1–cyto by FLAG-Tagap. Plexin-D1–cyto–myc-His6 (1.25 μg) was incubated with beads coated with the anti-FLAG antibody M2 (lane 2) or with similar beads saturated with recombinant FLAG-Tagap (lane 3). Lane 1 shows recombinant FLAG-Tagap beads alone. After three washes in lysis buffer, coimmunoprecipitated plexin-D1 cytoplasmic domain was eluted with SDS–polyacrylamide gel electrophoresis (PAGE) buffer and detected by Western blotting with anti–plexin-D1 antibody. Bottom: Detection of recombinant Tagap in the samples of the same eluates by Western blotting with an anti-FLAG antibody. Data are representative of three experiments.

  • Fig. 3 Inducible suppression of Tagap expression by lentiviral shRNA transgenes.

    (A) Left: RNAi-induced repression of Tagap mRNA in the thymus of WT (n = 3), T8 (n = 8), and T12 (n = 3) inducible KD transgenic (tg) strains treated with doxycycline for 5 to 8 weeks. Right: Loss of FLAG-Tagap expression after doxycycline (dox)–induced expression of Tagap-specific short hairpin RNA (shRNA) was assessed by Western blotting analysis. Bar graph shows pooled densitometry data from three experiments. The unshaded arrow indicates a cluster of nonspecific bands that served as a loading control. (B and C) Thymocyte subset representation [CD4CD8 DN (double negative), CD4+CD8+ DP, CD4+ CD4SP, and CD8+ CD8SP] as total cell numbers (B) and FACS-determined population distribution (C) in WT mice and the T8 and T12 Tagap KD strains. Data are means ± SEM of four mice per group and are representative of five experiments. (D) Assessment of thymocyte maturation as determined by measurement of increasing TCRβ abundance and postselection signaling as determined by cell surface expression of CD69 in WT mice and the T8 and T12 Tagap KD mice strains. Data are means ± SEM of four mice per group and are representative of five experiments. (E) Plexin-D1 abundance, as assessed by measurement of sema3E-Fcγ2c binding, was determined by flow cytometric analysis of thymocytes from the indicated mice. Blue, plexin-D1; gray, isotype control immunoglobulin G2c (IgG2c); thymocytes from three mice per sample. (F and G) Thymocytes isolated from control (top) or Tagap KD (bottom) mice were allowed to adhere to VCAM-1–coated migration chambers. (F) Left: Adherence before the introduction of sema3E (5 μg/ml) under flow (2.5 μl/min). Right: Adherence after 40 min of exposure to sema3E and an increase in flow to 15 μl/min. Yellow circles outline Tagap KD adherent cells that detached in the presence of sema3E. Data are representative of the three experiments shown in (G). (G) Summary of the percentage of thymocytes released by sema3E. Data are means ± SEM of three experiments. P < 0.005.

  • Fig. 4 Tagap KD thymocytes show impaired cortical-medullary translocation and ectopic medullary formations.

    (A) Top: Postselection CD69+ cells (white) in WT and Tagap KD thymus. CD4 cells are in green, CD8 cells are in red, and CD4+CD8+ DP thymocytes are in yellow-orange. CD69+ signal alone is shown in the monochrome panels. The dashed yellow line shows the junction between the cortex (c) and the medulla (m). The solid yellow line shows the capsule. For both WT and Tagap KD, 22 confocal images (0.25 mm × 0.25 mm) were analyzed. Scale bars (confocal images), 25 μm. Ectopic medullary structures formed in the Tagap KD thymus (lower enlarged panel on right; white arrow indicates example of sub-capsular cortical medullary formation). The intracellular Foxp3 signal is shown in white. The corticomedullary junction is shown by yellow lines (dashed in insets). White boxes outline the areas enlarged in the insets. Eighty confocal images (0.1 mm × 0.1 mm) for WT mice and 61 confocal images (0.1 mm × 0.1 mm) for Tagap KD were analyzed. (B) Top: The numbers of CD69+ cells in WT (n = 80 confocal sections) and Tagap KD mice (n = 61 confocal sections). Data are means ± SEM. Middle: Medullary density for CD4SP and CD8SP thymocytes in WT and Tagap KD mice. Data are means ± SEM. Bottom: Numbers of Foxp3+ CD4SP cells in WT and Tagap KD mice. Data are means ± SEM. n.s., not significant. (C) Flow cytometric analysis of plexin-D1 (left) and neuropilin-1 (right) expression on WT B6 mouse thymic CD4+ subsets. The median fluorescence intensity (MFI) indicated. Data are representative of three experiments. Middle and right: Total (Foxp3+ CD4SP) and mature (CD25+Foxp3+ CD4SP) thymic Treg cells in WT and Tagap KD thymus. Data are means ± SEM of three or four mice per group and are representative of 11 experiments. (D) Flow cytometric analysis of CD5 cell surface expression on DP, CD4SP, and CD8SP cells from the indicated mice. Data are means ± SEM of four mice per group and are representative of five experiments. (E) Genotype representation of WT [green fluorescent protein (GFP)] and T12 Tagap KD (GFP+) thymic subsets in mixed bone marrow chimeras. Data are means ± SEM of nine mice per group and are representative of three experiments.

  • Fig. 5 Tagap functions downstream of plexin-D1 as a RhoGAP.

    (A) Assessment of the relative Cdc42 activation after sema3E binding to plexin-D1 on thymocytes from WT and Tagap KD mice. Results are normalized to control for each condition and are means ± SEM of four experiments. (B) Cdc42 activation by sema3E in WT and Tagap KD thymocytes was detected by specific binding to GST-PAK1 protein-binding domain CRIB motif fusion protein. Data are representative of four experiments. The active Cdc42 signal represents total recovered active Cdc42, whereas the total Cdc42 signal (inactive and active) represents 5% of the sample. (C) Full-length mouse Tagap was expressed as an N-terminal FLAG fusion protein (left; silver stain) and then incubated at concentrations from 0 to 80 nM with the indicated Rho GTPases or Ras p21 loaded with GTP (right). GTP hydrolysis was assayed by measuring the release of Pi. Data are means ± SEM of four samples and are representative of three experiments. (D) Regulation of active RhoA abundance by sema3E in WT and Tagap KD thymocytes. Data are means ± SEM of four samples and are representative of three experiments; for WT cells, P < 0.0001 between control and sema3E-treated; for Tagap KD cells, there was no significant difference. Top: Western blotting analysis of the relative amounts of total RhoA in each sample. (E) Proposed model for how Tagap regulates sema3e/plexin-D1 function in thymocytes. Strong expression of full-length plexin-D1 in cortex-localized DP thymocytes in the absence of sema3E leads to very low basal Cdc42 activation, with no increase in response to CXCL12 (Fig. 1C) and minimal CXCL12-directed migration (2) due to sequestration of a Cdc42 GAP, possibly Arhgap9 or Arhgap26 (Fig. 2A). Upon binding to sema3E, plexin-D1 forms homodimers, generating an altered cytoplasmic domain-binding interface, releasing Cdc42GAP and recruiting tagap together with other GTPase regulatory proteins. Tagap facilitates the cycling of RhoA between active and inactive states, which leads to cytoskeletal reorganization and the release of activated β1 integrins and loss of adhesion. The separation of plexin-D1 from Cdc42 GAP further enables increases in active Cdc42 abundance, which accelerates chemokine-directed migration (2).

  • Table 1 Cortical and medullary distributions of Foxp3+ Treg cells in WT, Tagap KD, and Plxnd1 CKO mice.

    Plxnd1 WT and CKO thymi (three mice per genotype; B6 background) and WT and Tagap KD thymi [from two T8 mice and one T12 mouse for each condition; nonobese diabetic (NOD) background, all doxycycline-treated] were analyzed by confocal staining. Data are means ± SEM. Medulla refers to the normal medullary formations and not to the irregular, subcapsullary ectopic structures. Data are based on the analysis of 90 confocal images (0.2 mm × 0.15 mm) for B6 WT and Plxnd1 CKO thymi, 80 confocal images (0.2 mm × 0.15 mm) for NOD WT thymi, and 61 confocal images (0.2 mm × 0.15 mm) for Tagap KD thymi. Cells were only counted as being Foxp3+ if a distinctive ring of membrane CD4 staining could be distinguished around the intracellular white Foxp3 signal.

    GenotypeB6 WTB6 Plxnd1
    CKO
    NOD WTNOD Tagap
    KD
    Foxp3+ Treg/mm2
    Medulla108.5 ± 12.3320.6 ± 37.3558.9 ± 46.1931.3 ± 68.3
    Cortex2.6 ± 0.716.9 ± 3.148.6 ± 11.5131.0 ± 20.9
    Medulla/
    cortical
    ratio
    41.719.011.57.1
    Change
    relative
    to WT
    medulla*
    ×2.95×1.67
    Change
    relative to
    WT cortex*
    ×6.50×2.70

    *P < 0.0001.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/534/eaan8799/DC1

      Fig. S1. Characterization of the DP257-20-109 DP thymocyte-like cell line.

      Fig. S2. Binding of GTPases to the plexin-D1 cytoplasmic tail in a mammalian two-hybrid assay.

      Fig. S3. Morphologies of cells coexpressing plexin-D1 and selected GAPs.

      Fig. S4. Thymic morphology in WT and Plxnd1 CKO mice.

      Fig. S5. Treg cell representation and TCR repertoire development in Tagap KD mice.

      Fig. S6. Gating strategy for distinct thymocyte subsets.

      Fig. S7. Thymocyte development in WT and Tagap KD hematopoietic chimeras.

      Fig. S8. Calibration of GAP assay with p50-RhoGAP.

      Table S1. Bioinformatically predicted mammalian RhoGAPs and protocol for target selection.

      Table S2. Association of human TAGAP SNPs with autoimmunity.

      Movie S1. Sema3E-mediated release of β1 integrin from VCAM-1 for WT thymocytes.

      Movie S2. Sema3E-mediated release of β1 integrin from VCAM-1 for Tagap KD thymocytes.

      References (4357)

    • Supplementary Materials for:

      Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity

      Jonathan S. Duke-Cohan*, Yuki Ishikawa, Akihiro Yoshizawa, Young-Il Choi, Chin-Nien Lee, Oreste Acuto, Stephan Kissler*, Ellis L. Reinherz*

      *Corresponding author. Email: ellis_reinherz{at}dfci.harvard.edu (E.L.R.); jonathan_duke-cohan{at}dfci.harvard.edu (J.S.D.-C.); stephan.kissler{at}joslin.harvard.edu (S.K.)

      This PDF file includes:

      • Fig. S1. Characterization of the DP257-20-109 DP thymocyte-like cell line.
      • Fig. S2. Binding of GTPases to the plexin-D1 cytoplasmic tail in a mammalian two-hybrid assay.
      • Fig. S3. Morphologies of cells coexpressing plexin-D1 and selected GAPs.
      • Fig. S4. Thymic morphology in WT and Plxnd1 CKO mice.
      • Fig. S5. Treg cell representation and TCR repertoire development in Tagap KD mice.
      • Fig. S6. Gating strategy for distinct thymocyte subsets.
      • Fig. S7. Thymocyte development in WT and Tagap KD hematopoietic chimeras.
      • Fig. S8. Calibration of GAP assay with p50-RhoGAP.
      • Table S1. Bioinformatically predicted mammalian RhoGAPs and protocol for target selection.
      • Table S2. Association of human TAGAP SNPs with autoimmunity.
      • Legends for movies S1 and S2
      • References (4357)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mp4 format). Sema3E-mediated release of β1 integrin from VCAM-1 for WT thymocytes.
      • Movie S2 (.mp4 format). Sema3E-mediated release of β1 integrin from VCAM-1 for Tagap KD thymocytes.

      © 2018 American Association for the Advancement of Science