Figure 3.
Dynamitin-disrupted T cells form altered LFA-1 and CD3 clusters at the contact area with the APC. (A) LFA-1 and CD3 cluster at the IS in dynamitin-disrupted cells. Conjugates between J77 Jurkat control (GFP) or dynamitin-disrupted cells were formed, fixed as in Fig. 1, and stained for LFA-1 or CD3, followed by rhodamine red X–labeled anti–mouse secondary antibody (red in merged images). GFP, green; R, Raji APC (CMAC-labeled, blue). (B) LFA-1 and CD3 show a correct SMAC architecture in control cells (GFP) but not in dynamitin-disrupted cells. A confocal z slice of conjugates formed and processed as in A is shown (3D). LFA-1 is shown in red (Alexa 568–labeled anti–mouse secondary antibody) and CD3 was detected with 448 antibody, followed by an Alexa 647–labeled anti–rabbit secondary antibody (blue). GFP, green. (C) In dynamitin-disrupted clones, CD3 clusters occupy a larger area and appear scattered at the contact area with the APC. A confocal z section of conjugates formed and processed as in A is shown (3D). CD3 was detected with T3b monoclonal antibody followed by rhodamine red X–labeled anti–mouse secondary antibody (red). Asterisks indicate SEE-Raji APC (B and C). (C, right) Quantitative analysis of the CD3-stained area at the IS. Data are means ± SEM. 20 cells were analyzed per condition. *, P < 0.05 versus GFP (Student's t test). Bars, 10 μm.