Figure 2
Figure 2. TCR-dependent clustering and polarization of CD38-GFP from T cells at the T/APC contact zone. (A) Jurkat J77 cells transiently expressing CD38-GFP were allowed to conjugate with CMAC-labeled Raji cells pulsed or not pulsed with SEE. Then, cells were fixed, permeabilized, and stained for CD3-ζ as described in Figure 1 (red). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (Tokyo, Japan; 100×/1.3 NA oil objective). The white arrowheads point to the intracellular CD38-GFP or CD3-ζ. The arrows point to the plasma membrane CD38-GFP or CD3-ζ accumulated at the synapse. (B) Comparative analysis of the percentage of conjugates with CD38-GFP or endogenous CD3-ζ redistributed at the contact zone relative to the total number of CD38-GFP+ J77/Raji cell conjugates in the absence (□) or presence (■) of SEE. The data represent the mean plus SEM of 3 independent experiments. In each experiment, 50 to 70 conjugates were analyzed. (C) Jurkat J77 cells transiently transfected with CD38-GFP were seeded onto FN-coated coverslips. Raji cells labeled in red with the probe CM-TMR were incubated with SEE and added to the chambers. Cells were monitored by time-lapse confocal microscopy at 30-second intervals. Representative images of one T cell–SEE-pulsed APC conjugate are shown. Each time point shows the fluorescence images of Raji cells (red) and CD38-GFP fluorescence (green). (D) Endogenous CD38 distribution in untransfected permeabilized Jurkat J77 T cells (left panel) or untransfected permeabilized Raji B cells (third panel) was compared with CD38-GFP distribution in their respective transfected counterparts (second and fourth panels, respectively). The white arrowheads point to the intracellular CD38 or CD38-GFP. The arrows point to the plasma membrane CD38 or CD38-GFP. CD38-GFP distribution in transfected Jurkat cells differs greatly from the GFP distribution, which is largely cytosolic (second panel). Moreover, intracellular CD38 staining in untransfected Raji B cells does not colocalize with TO-PRO3, which is specific for nuclear staining (third panel). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (100×/1.3 NA oil objective).

TCR-dependent clustering and polarization of CD38-GFP from T cells at the T/APC contact zone. (A) Jurkat J77 cells transiently expressing CD38-GFP were allowed to conjugate with CMAC-labeled Raji cells pulsed or not pulsed with SEE. Then, cells were fixed, permeabilized, and stained for CD3-ζ as described in Figure 1 (red). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (Tokyo, Japan; 100×/1.3 NA oil objective). The white arrowheads point to the intracellular CD38-GFP or CD3-ζ. The arrows point to the plasma membrane CD38-GFP or CD3-ζ accumulated at the synapse. (B) Comparative analysis of the percentage of conjugates with CD38-GFP or endogenous CD3-ζ redistributed at the contact zone relative to the total number of CD38-GFP+ J77/Raji cell conjugates in the absence (□) or presence (■) of SEE. The data represent the mean plus SEM of 3 independent experiments. In each experiment, 50 to 70 conjugates were analyzed. (C) Jurkat J77 cells transiently transfected with CD38-GFP were seeded onto FN-coated coverslips. Raji cells labeled in red with the probe CM-TMR were incubated with SEE and added to the chambers. Cells were monitored by time-lapse confocal microscopy at 30-second intervals. Representative images of one T cell–SEE-pulsed APC conjugate are shown. Each time point shows the fluorescence images of Raji cells (red) and CD38-GFP fluorescence (green). (D) Endogenous CD38 distribution in untransfected permeabilized Jurkat J77 T cells (left panel) or untransfected permeabilized Raji B cells (third panel) was compared with CD38-GFP distribution in their respective transfected counterparts (second and fourth panels, respectively). The white arrowheads point to the intracellular CD38 or CD38-GFP. The arrows point to the plasma membrane CD38 or CD38-GFP. CD38-GFP distribution in transfected Jurkat cells differs greatly from the GFP distribution, which is largely cytosolic (second panel). Moreover, intracellular CD38 staining in untransfected Raji B cells does not colocalize with TO-PRO3, which is specific for nuclear staining (third panel). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (100×/1.3 NA oil objective).

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