Glu-MTs are rapidly induced after T cell stimulation. (A and B) Jurkat cells (A and B), CH7C17 Jurkat cells (B), or peripheral blood lymphocytes (PBL; B) were conjugated to APCs loaded or not with SEE, SEB, or hemagglutinin (HA) peptide as indicated. The distribution of Glu-, Tyr-, and acetyl-MTs was analyzed with specific antibodies (A). The histogram represents the percentage of T cells with Glu- or acetyl-MTs (B). (C) Conjugates formed by Jurkat cells with SEE-loaded Raji cells were stained for Glu- and Tyr-MTs. The images were analyzed under a confocal microscope and the resulting stacks were deconvolved and subsequently reconstructed in 3D. Views of the cell-to-cell contact rotated 0°, 20°, 40°, and 60° are shown for Glu-MT–stained cells in one conjugate. The arrow indicates the position of the MTOC and the arrowheads point to Glu-MTs that bend away from the IS following the T cell curvature. (D) Equal numbers of Jurkat and Raji cells or Jurkat cells conjugated to Raji cells loaded or not loaded with SEE as indicated were processed for immunoblotting for Glu-, acetyl-, and Tyr-tubulin, or for GAPDH as a loading control. The histogram represents Glu-MT levels relative to control Jurkat cells. (E) Jurkat cells were conjugated to latex beads coated with anti-TCR antibodies for the indicated times in the presence or absence of 2.5 µM PP2. Cells were analyzed by immunoblotting for Glu-, acetyl-, and Tyr-tubulin or for GAPDH. The graphics represents the levels of Glu-, Tyr-, and acetyl-tubulin at different times of conjugation relative to those at zero time. (F) Distribution of Glu- and Tyr-MTs in Jurkat cells conjugated to SEE-loaded APCs for 15 min and then treated with 2 µM nocodazole for 30 min. At least 40 T cells were analyzed in B. Quantitative data in B, D, and E are summarized as means ± SEM from three independent experiments (error bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars, 5 µm.