A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

Abstract

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

Figure 1. DOCK8 interacts constitutively and colocalizes with WASp and WIP in T cells.

(A and B) Co-IP of WIP and WASp with DOCK8, but not MALT1, in human peripheral blood T cells (A) and mouse splenic T cells (B). Aliquots from the lysates used for IP were probed with DOCK8, WASp, WIP, and MALT1 to ensure equal loading. (C) Lack of a detectable effect of TCR ligation with anti-CD3 mAb on the association of DOCK8 with WIP and WASp in human blood T cells. Lysates were probed with a phospho-specific ERK1/ERK2 (p-ERK1/2) Ab to verify TCR signaling and with GAPDH as a loading control. IP with an IgG isotype Ab was used as a negative control. (D) Co-IP of WIP and WASp with DOCK8, but not MALT1, in the human DND41 T cell line. (E) Colocalization of WIP and WASp with DOCK8. Representative DIC images (left), confocal immunofluorescence microscopic images (middle), and merged images (right) of DND41 T cells permeabilized and stained with Abs against DOCK8, WIP, and WASp, followed by fluorochrome-labeled secondary Abs. Original magnification, ×63. Scale bars: 2 μm. Data in A–D represent 3 independent experiments each. Images in E are representative of 50 cells that were examined in 2 independent experiments.

Figure 2. WIP bridges DOCK8 to WASp.

(A and B) Representative immunoblot and quantitative analysis of co-IP of WIP and DOCK8 from peripheral blood T cell lysates from an HC and a WASp^null patient (A) and of mouse splenic T cells from WT and Was^–/– mice (B). T cells were examined 0 and 10 minutes after (+) anti-CD3 stimulation. Using densitometric scanning, quantification of the results was performed by calculating the ratio of WIP/DOCK8 bands in the DOCK8 immunoprecipitates to that in the lysates relative to controls. The results in A represent 2 WAS patients and 2 controls examined in 2 independent experiments, and in B, 3 WT and 3 Was^–/– mice were examined in 3 independent experiments. Aliquots from the lysates used for IP were probed with MALT1 in A and with GAPDH in B to ensure equal loading. Error bars in A and B represent the mean ± SEM. Student’s t test. (C) Co-IP of rWIP-EGFP, but not EGFP, with rMyc-tagged DOCK8 (DOCK8-Myc) proteins (top panel) and of rDOCK8-Myc with rWASp-Flag, but not rMALT1-Flag, in the presence, but not in the absence, of rWIP-EGFP (bottom panel). (D) Map of the WIPΔWBD protein. (E) Representative immunoblot of the co-IP of WIP-EGFP and WIPΔWBD-EGFP with Myc-tagged DOCK8 in 293T cell transfectants. LRRC8A-Myc and EGFP transfectants were used as negative controls, and lysate aliquots were probed for Myc and EGFP to ensure equal loading. The * denotes a non-specific band. (F) Co-IP of DOCK8 and WASp with WIP in human T cells, using the 3D10 and D12C5 anti-WIP mAbs directed against distinct epitopes in the N-terminus and C-terminus of WIP, respectively. Data in C, E, and F represent 4 independent experiments.

Figure 3. WIP bridges DOCK8 to actin.

(A) Map of the WIPΔABD protein. (B) Representative immunoblot of the co-IP of WIP-EGFP and WIPΔABD-EGFP with Myc-tagged DOCK8 in 293T cell transfectants. LRRC8A-Myc and EGFP transfectants were used as negative controls, and an aliquot of the lysates used for the coprecipitation was probed for Myc and EGFP to ensure equal loading. The * denotes a non-specific band. (C and D) Representative immunoblot (C) and quantitative analysis (D) of the co-IP of WIP, WASp, and actin with DOCK8 in splenic T cells from WT and WIPΔABD knockin mice. An aliquot of the lysates used for the co-IP was probed for DOCK8, WIP, WASp, and actin as a loading control. Quantification of the results was performed by calculating the relative ratio of actin/DOCK8, WIP/DOCK8, and WASp/DOCK8 in DOCK8 immunoprecipitates relative to lysates and normalizing the values to those obtained in WT T cells by setting the WT ratio to 1. Ctrl, control. Data are representative of 3 independent experiments in B and 2 independent experiments in C and D. Error bars in D represent the mean ± SEM.

Figure 4. DOCK8 GEF activity mediates TCR-driven WASp activation.

(A) Representative (left) and pooled (right) results of the generation of active WASp following TCR ligation in human T cells from an HC and a DOCK8^null patient as determined by immunoblotting WASp and N-WASp CSAb immunoprecipitates with anti–WASp F-8 mAb. Aliquots from total lysates were probed for WASp to ensure equal loading and for p-ERK1/2 to verify TCR signaling. Results are representative of 2 experiments involving 2 patients and 2 controls. Quantification was performed by calculating the ratio of activated WASp in the IP to total WASp in the lysates, relative to controls. (B) Ribbon diagram of the DOCK8-CDC42 complex. Residue S1827 is in red, and the region affected by the S1827P mutation is in magenta. (C) Interaction of DOCK8 residue S1827 with CDC42 (top) and its disruption by the S1827P DOCK8^pri mutation (bottom). (D) GEF activity for CDC42 of WT DOCK8 and DOCK8^pri DHR2 domains. Relative fluorescence units (RFU) of MANT-GTP over time are shown. Results are representative of 3 experiments. (E) Representative immunoblot and quantitation of DOCK8 expression in Dock8^pri/pri and WT mice. Quantification was performed by calculating the DOCK8/GAPDH ratio relative to that in WT controls. (F and G) Representative immunoblot (F) and quantitation (G) of the association of DOCK8 with WIP and WASp in T cells from DOCK8-mutant mice. Quantification was performed by calculating the WIP/DOCK8 ratio in DOCK8 immunoprecipitates relative to that in WT controls. (H and I) Representative (H) and pooled (I) results of the generation of active WASp following TCR ligation in splenic T cells from Dock8^–/–, Dock8^pri/pri, and WT mice. The experiment was performed and the results expressed as described in A. Results in E–I are representative of 3 experiments using 3 mice from each strain. Error bars in A, E, G, and I represent the mean± SEM. *P < 0.05, by Student’s t test.

Figure 5. Defective actin cytoskeleton structure and function in DOCK8-deficient T cells.

(A) Representative EM images of the apical membrane of T cells stimulated with anti-CD3 mAb. Shown are the cytoskeletal actin fibers associated with the cytoplasmic side of the adherent plasma membranes. Results are representative of 2 experiments with T cells from 1 mouse of each strain. More than 50 cells were examined in each experiment. Scale bar: 200 nm. (B) Actin filament length in T cell membranes from the Dock8^–/–, Dock8^pri/pri, and WT mice represented in A; 114 WT T cells, 139 Dock8^–/– T cells, and 120 Dock8^pri/pri T cells were measured in 2 independent experiments. (C) Representative FACS analysis of FITC-phalloidin staining for F-actin in resting T cells from Dock8^–/–, Dock8^pri/pri, and WT mice and quantitative analysis of the results as a percentage of F-actin content in T cells from WT controls. Results are representative of 3 independent experiments using 3 mice from each strain. (D) Increase in the F-actin content of T cells from Dock8^–/–, Dock8^pri/pri, and WT mice following stimulation with anti-CD3 mAb. Results are expressed as the increase in the mean fluorescence intensity (MFI) of F-actin from the baseline (time 0). Results are representative of 3 independent experiments using 3 mice from each strain. Error bars in B and C and symbols and bars in D represent the mean ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, by Student’s t test.

Figure 6. DOCK8 is essential for TCR-driven actin foci formation and mechanotransduction.

(A and B) Representative images of splenic CD4⁺ T cells from WT, Dock8^–/–, and Dock8^pri/pri mice plated on anti-CD3– and ICAM-1–coated glass chambers (A) or a superantigen-treated endothelial cell monolayer (B) and stained for F-actin (phalloidin, pseudocolored green) and pCasL (pseudocolored red). Orange indicates merging. The extracellular striated pattern observed in B (phalloidin panel on the left) is due to the F-actin fluorescence contributed by the endothelial cell cytoskeleton. Original magnification, ×100. Scale bar: 5 μm. (C–G) Quantitative analysis of actin foci (C and E), pCasL intensity (D and F), and synaptic area (G) in T cells. Each data point represents a value obtained for a single cell. Similar results (A–G) were obtained in 2 independent experiments. Error bars in C–G represent the mean ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, by Student’s t test.

Figure 7. Defective spreading and in vitro migration of Dock8^–/– and Dock8^pri/pri T cells.

(A) FACS analysis of CD44, PSGL-1, LFA-1, and VLA-4 surface expression by CD4⁺ Th1 cells from Dock8^–/–, Dock8^pri/pri, and WT control mice. Results are representative of 3 independent experiments using 2 mice each per strain. (B) Accumulation of CD4⁺ Th1 cells over E-selectin–, ICAM-1 plus 250 ng/ml SDF-1α–, and VCAM-1–coated surfaces under a range of shear stress conditions. (C) Representative photomicrographs and pooled results of spreading of CD4⁺ Th1 cells over ICAM-1. Arrows indicate fully spread cells scored with 2 points, and solid triangles indicate partially spread cells scored with 1 point. Round, unspread cells were scored with no points to calculate the mean spreading score of cells adherent to ICAM-1 in 5 fields of view at 0.5 dyn/cm². Original magnification, ×20. Scale bar: 50 μm. Duplicate coverslips were assessed in 3 independent studies. (D) Th1 cell transmigration across a TNF-α–treated MHEC monolayer under 0.8 dyn/cm² shear stress normalized to the percentage of WT Th1 cells. Pooled results in B–D represent 3 independent experiments each using CD4⁺ Th1 cells from 1 mouse per strain. ***P < 0.001, **P < 0.01, and *P <0.05, by Student’s t test.

Figure 8. Defective in vivo homing of DOCK8-deficient T cells.

(A) Representative FACS analysis of a mixture of equal numbers of Alexa Fluor 555–labeled WT T cells (designated by red lettering) and Alexa Fluor 488–labeled WT, Dock8^–/–, and Dock8^pri/pri T cells (designated by green lettering) used for injection into genetically matched WT recipients. (B) Representative FACS plots from the blood and LNs of WT recipients 1 hour after i.v. administration of 1:1 mixtures of Alexa Fluor 555–labeled WT T cells and Alexa Fluor 488–labeled WT, Dock8^–/–, or Dock8^pri/pri T cells. (C) Quantitative analysis of cells from the blood and LNs of WT recipients obtained 1 hour after i.v. administration of 1:1 mixtures of equal numbers of Alexa Fluor 555–labeled WT T cells and Alexa Fluor 488–labeled WT, Dock8^–/–, or Dock8^pri/pri T cells. Graph shows the homing index of T cells from Dock8^–/–, Dock8^pri/pri, and WT mice relative to the mean homing index of WT T cells set at 1.0. (D) Percentages of annexin V⁺ eFluor-780^– apoptotic cells and eFluor-780⁺ dead cells among Alexa Fluor 488–labeled WT, Dock8^–/–, and Dock8^pri/pri donor T cells in pooled LN cells from WT recipients. Results in A–D are representative of 2 independent experiments each using 5 recipients per donor strain. Error bars represent the mean ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, by Student’s t test.

Figure 9. A DOCK8-WIP-WASp complex links the TCR to the actin cytoskeleton reorganization.

DOCK8, WIP, and WASp exist in a complex in T cells. TCR ligation activates DOCK8, promoting its GEF activity for CDC42. CDC42-GTP generated by DOCK8 binds to WASp, which is closely associated with DOCK8, causing it to assume an open conformation that allows its C-terminal verproliin, central, acidic (VCA) domain to bind to the ARP2/3 complex and initiate actin polymerization. The generated F-actin is stabilized by binding to WIP and is essential for actin cytoskeleton–dependent T cell functions. These include actin foci formation at the immune synapse, mechanotransduction, and TEM into LNs and tissues, both of which are critical for T cell–mediated immune surveillance.