1. Volume 20, Issue 4, Pages 417-428 (April 2004)
HDAC6 Deacetylase Activity Links the Tubulin Cytoskeleton with Immune Synapse Organization 
Juan M Serrador, Jose Román Cabrero, David Sancho, Marı́a Mittelbrunn, Ana Urzainqui, Francisco Sánchez-Madrid 
Immunity 
Volume 20, Issue 4, Pages (April 2004)
DOI: /S (04)

2. Figure 1
Acetylated Microtubules Are Concentrated at the T Cell/APC Contact Site Surrounding Clusters of CD3 and LFA-1
Raji cells were allowed to conjugate with J77 cells on FN-80-coated coverslips. Then, cells were fixed, permeabilized, and stained with the indicated antibodies.
(A) Cell conjugates in the absence (upper panel) or presence of 1 μg/ml SEE (lower panel) were double stained for α-tubulin (green) and acetylated α-tubulin (red) and analyzed by confocal microscopy. Merged and DIC pictures are shown at the right. Asterisks indicate the localization of Raji cells in cell conjugates.
(B) SEE-pulsed J77/Raji cell conjugates were double stained for acetylated α-tubulin (red) and either CD3 (green, upper panel) or LFA-1 (green, lower panel). At the right, the fluorescent staining of Raji cells (blue), acetylated tubulin (red), and CD3 or LFA-1 (green) were superimposed on the DIC images.
Immunity  , DOI: ( /S (04) )

3. Figure 2
Acetylated Tubulin Increases in T Cells upon Specific Interaction with APCs
(A) Raji cells (1.5 × 106) were pulsed or not with 1 μg/ml of either SEB or SEE and allowed to interact with J77 cells (4.5 × 106) for 40 min at 37°C. Then, acetylated α-tubulin and α-tubulin were detected in cell lysates by immunoblot under nonreducing conditions, as stated in the Experimental Procedures.
(B) J77 cells (5 × 106) were stimulated for 40 min at 37°C with the indicated Abs. Then, acetylated and total α-tubulin were detected in cell lysates by immunoblot under nonreducing conditions.
(C) Kinetics of tubulin acetylation and CD3ζ phosphorylation in J77/Raji cell conjugates. J77 cells, pretreated or not for 30 min with 3 μM TSA, were allowed to interact with Raji cells in the presence of SEE or SEB for the indicated times and then acetylated, and total α-tubulin was detected by immunoblot under nonreducing conditions. Densitometry values are shown at the middle of panels. To detect phospho-CD3ζ, CD3ζ was immunoprecipitated with the anti-CD3ζ chain 488 pAb, resolved on SDS-PAGE under reducing conditions, and immunoblotted with the anti-phosphotyrosine 4G10 mAb.
(D) J77 cells were pretreated or not with TSA and allowed to interact with Raji cells as in (C). Then, cell conjugates were lysed and immunoprecipitated for α-tubulin. Proteins in immunoprecipitates were immunoblotted with anti-α-tubulin-FITC, anti-CD3ζ, anti-phospho-tyrosine (4G10), and anti-ZAP-70 antibodies. Molecular weights are indicated to the left.
Immunity  , DOI: ( /S (04) )

4. Figure 3 Characterization of Exogenous HDAC6 Expressed in T Cells
(A) HDAC6 is concentrated at the contact site of T cells with APCs. CMAC-labeled Raji cells (blue) were allowed to interact with either purified CD4+ peripheral blood lymphocytes (PBLCD4+) or J77 cells in the presence or absence of SEE. Then, cell conjugates were fixed, permeabilized, and stained for HDAC6 and acetylated α-tubulin or Vβ8 TcR. Arrows point to MTOCs.
(B) Purified CD4+ PBLs and J77 cells were pretreated for 30 min with either 3 μM TSA or 5 mM sodium butyrate and allowed to interact with CMAC-labeled Raji cells (blue) in presence of SEE. T/APC conjugates were stained for acetylated microtubules (red). Fluorescence and DIC superimposed images are also shown. Arrows point to acetylated tubulin in T cells.
(C) Lysates from untransfected J77 cells or cells transfected with wild-type HDAC6-GFP, dead deacetylase HDAC6-GFP, or GFP were immunoblotted with an anti-GFP pAb. Molecular weights are indicated to the left.
(D) Levels of acetylated tubulin in HDAC6-transfected J77 cells. J77 cells were transfected with GFP−, HDAC6-GFP, or dead deacetylase HDAC6-GFP. After 24 hr, cells were treated or not with either 3 μM TSA or 5 mM sodium butyrate, and the level of acetylated tubulin was determined by flow cytometry as stated in the Experimental Procedures. A representative experiment from three is shown.
(E) Exogenous HDAC6 are concentrated at the contact site of J77 cells with Raji cells. SEE-pulsed Raji cells were allowed to interact with either wild-type HDAC6- or dead deacetylase HDAC6-transfected J77 cells. Then, cell conjugates were fixed, permeabilized, and stained for acetylated tubulin. Arrowheads point to the APCs contact sites of T cells.
Immunity  , DOI: ( /S (04) )

5. Figure 4
Dynamics of HDAC6 during the Specific Interaction of T Cells and APCs
Raji cells were pulsed with 1 μg/ml of SEE and adhered to FN-80-coated coverslips. Then, HDAC6-transfected J77 cells were added, and cell images were recorded at 30 s intervals. Raji cells were labeled with the blue probe CMAC. Each time point shows the DIC image superimposed on the fluorescent image of HDAC6-GFP. Time from the initial cell contact (min) is indicated at the upper left corner of each image. Asterisks indicate Raji cells.
Immunity  , DOI: ( /S (04) )

6. Figure 5
Overexpression of HDAC6 Disrupts the Clustering of CD3 at the c-SMAC
(A) J77 cells transfected with GFP, wild-type, or dead deacetylase HDAC6-GFP were incubated for 30 min at 37°C with SEE-pulsed Raji cells and stained for CD3ζ. At the right, the corresponding XZ projections of the interface area of contact for each conjugates are represented. DIC images and the superimposed staining of CD3ζ and HDAC6-GFP are also shown. Asterisks indicate the localization of APCs in cell conjugates.
(B) Percentage of Vβ8 CD4+ PBL and J77 cells transfected with GFP, wild-type, or dead deacetylase HDAC6-GFP treated or not with either TSA, trapoxin B, or sodium butyrate, displaying accumulation of CD3 at the contact sites with Raji cells. Data correspond to the arithmetic mean ± SEM of four independent experiments.
(C) J77 cells pretreated with 3 μM TSA (■), 100 nM trapoxin B (▴), or 10 mM sodium butyrate (•) were mixed with Raji cells pulsed with SEE at 1 μg/ml, and the clustering of CD3 was analyzed at different times. A representative experiment out of three is shown.
(D) J77 cells were transfected with a mock (control) or with HDAC6 siRNA; protein levels of HDAC6, acetylated α-tubulin, and total tubulin were determined by immunoblotting with specific antibodies.
(E) CD3 clustering at the c-SMAC is enhanced in HDAC6 knockdown J77 cells. The accumulation of CD3 at the contact site with Raji cells in control and HDAC6 knockdown cells was assessed as in (B), except for a shorter interaction with SEE-pulsed Raji cells (10 min).
Immunity  , DOI: ( /S (04) )

7. Figure 6
Overexpression of HDAC6 Disrupts the Organization of LFA-1 at the p-SMAC
J77 cells transfected with plasmids encoding wild-type or dead deacetylase HDAC6-GFP were incubated for 30 min at 37°C with SEE-pulsed Raji cells. Then, cell conjugates were stained for LFA-1 and analyzed by laser confocal microscopy. Representative confocal sections and three-dimensional reconstruction of T cell/APC conjugates which J77 cells overexpressed either wild-type HDAC6-GFP (upper panel) or the dead deacetylase HDAC6-GFP mutant (lower panel) are shown. Twenty cell conjugates for three independent experiments were analyzed for each transfectant. LFA-1 was detected at the p-SMAC of 9 (GFP), 2 (HDAC6-GFP), and 11 (H216A/H611A-GFP) cells. At the right, the corresponding XZ projections of the junction area of each transfectant with the APC are represented. DIC images and the superimposed staining of LFA-1 and either wild-type or the dead deacetylase mutant HDAC6-GFP are also shown. Asterisks indicate the APCs.
Immunity  , DOI: ( /S (04) )

8. Figure 7
Functional Role of HDAC6 in MTOC Translocation and T Cell Activation
(A) J77 cells transfected with wild-type or dead deacetylase HDAC6-GFP were incubated for 30 min at 37°C in the presence of SEE-pulsed Raji cells. Then, cells were fixed, permeabilized, and stained for α-tubulin. Representative images of wild-type HDAC6-GFP-expressing cell conjugates in the absence or presence of 3 μM TSA (upper and middle panels, respectively) and the dead deacetylase HDAC6 mutant (lower panel) are shown. DIC images, the fluorescent staining of Raji cells (blue), and superimposed HDAC6 and α-tubulin staining are also shown.
(B) Percentage of Vβ8 CD4+ PBL and transfected J77 cells mixed with SEE-pulsed Raji cells in presence or absence of TSA, trapoxin B, or sodium butyrate with their MTOC in close proximity to the cell plasma membrane between the nucleus and the Raji contact site. Data correspond to the arithmetic mean ± SEM of four independent experiments. At least 200 conjugates/experiment were scored.
(C) J77 cells pretreated with 3 μM TSA (■), 100 nM trapoxin B (▴), or 10 mM sodium butyrate (•) were mixed with SEE-pulsed Raji cells and the translocation of the MTOC was analyzed at different times. A representative experiment out of three is shown.
(D) IL-2 was determined in the supernatants from HDAC6-GFP, H216A/H611A-GFP, and GFP stably transfected J77 cells and Raji B cell conjugates in the presence (+SEE) or absence (−SEE) of SEE (0.5 μg/ml). In other experiments, phorbol-12-myristate-13-acetate (PMA, 20 ng/ml) and ionomycin (1μg/ml) were added to the medium, and the cells were cultured for 6 hr. Results show the arithmetic mean ± SD of IL-2 production (pg/ml) from three independent experiments.
(E) CD69-positive cells were determined in Vβ8-positive J77 cells in conjugates, as in (D). Results are expressed as arithmetic mean ± SD of percentage of CD69+ cells in the Vβ8+ subset from three independent experiments.
Immunity  , DOI: ( /S (04) )