Figure 3.
Figure 3. Lck instructs LAT and TCRζ vesicular release at the immunological synapse. (A–D) Primary CD4 T cells were transfected with a siRNA control (siCtr; A and B) or a siRNA MAL (siMAL; C and D) and allowed to form immunological synapses with superantigen-loaded Raji cells for 30 min. Cells were stained for Lck and LAT (A and C) or Lck and TCRζ (B and D), and then analyzed by 3D confocal microscopy. (E–G) Population analysis of at least 20 conjugates per group quantifying the 3D fluorescence at the synapse relative to total cell fluorescence in cells processed in the same way as cells in A–D in the presence (sAg+) or in the absence (sAg−) of superantigen. (H) Primary CD4 T cells were transfected with siRNA control (siCtr), siRNA MAL (siMAL), or siRNA Syt7 (siSyt7) and cell lysates were analyzed for MAL, Syt7, and actin expression by Western blot. (I–L) Primary CD4 T cells were transfected with a siRNA control (siCtr; I and J) or a siRNA Syt7 (siSyt7; K and L) and allowed to form immunological synapses with superantigen-loaded Raji cells for 30 min. Cells were stained for Lck and LAT (I and K) or LAT and TCRζ (J and L). (M–O) Population analysis of at least 20 conjugates per group, quantifying the 3D fluorescence at the cell junction relative to total cell fluorescence in cells processed in the same way as cells in I–L and activated in the presence (sAg+) or in absence (sAg−) of superantigen and analyzed by 3D confocal microscopy. Confocal images were post-treated by deconvolution and 1 µm-thick medial stack is shown. Synaptic clustering and intracellular compartments are highlighted by arrows and arrowheads, respectively. Each dot in plots represents one conjugate. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.

Lck instructs LAT and TCRζ vesicular release at the immunological synapse. (A–D) Primary CD4 T cells were transfected with a siRNA control (siCtr; A and B) or a siRNA MAL (siMAL; C and D) and allowed to form immunological synapses with superantigen-loaded Raji cells for 30 min. Cells were stained for Lck and LAT (A and C) or Lck and TCRζ (B and D), and then analyzed by 3D confocal microscopy. (E–G) Population analysis of at least 20 conjugates per group quantifying the 3D fluorescence at the synapse relative to total cell fluorescence in cells processed in the same way as cells in A–D in the presence (sAg+) or in the absence (sAg−) of superantigen. (H) Primary CD4 T cells were transfected with siRNA control (siCtr), siRNA MAL (siMAL), or siRNA Syt7 (siSyt7) and cell lysates were analyzed for MAL, Syt7, and actin expression by Western blot. (I–L) Primary CD4 T cells were transfected with a siRNA control (siCtr; I and J) or a siRNA Syt7 (siSyt7; K and L) and allowed to form immunological synapses with superantigen-loaded Raji cells for 30 min. Cells were stained for Lck and LAT (I and K) or LAT and TCRζ (J and L). (M–O) Population analysis of at least 20 conjugates per group, quantifying the 3D fluorescence at the cell junction relative to total cell fluorescence in cells processed in the same way as cells in I–L and activated in the presence (sAg+) or in absence (sAg−) of superantigen and analyzed by 3D confocal microscopy. Confocal images were post-treated by deconvolution and 1 µm-thick medial stack is shown. Synaptic clustering and intracellular compartments are highlighted by arrows and arrowheads, respectively. Each dot in plots represents one conjugate. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.

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