Figure 5
Figure 5. TCR-dependent clustering of CD38 from B cells and CD31 from T cells at the T/APC contact zone. (A) Raji cells transiently transfected with the CD38-GFP construct were pulsed or not with SEE and allowed to conjugate with Jurkat J77 T cells. Then, cells were fixed, permeabilized, and stained for CD3-ζ (red). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope. CD38-GFP (green; 100×/1.3 NA oil objective). Bars represent 5 μm for all panels. CD3-ζ (red), green fluorescence merged with red fluorescence images, and the corresponding DIC images superimposed on blue fluorescence images from CMAC-loaded Raji cells are shown. The white arrowheads point to the intracellular CD38-GFP or CD3-ζ. The arrows point to the plasma membrane CD3-ζ accumulated at the synapse. (B) CD38-GFP–transfected Raji B cells were treated as in panel A except that after permeabilization, cells were stained for CD31 (red). CD38-GFP (green), CD31 (red), green fluorescence merged with red fluorescence images, and the corresponding DIC images superimposed on blue fluorescence images from CMAC-loaded Raji cells are shown. The white arrowheads point to the intracellular CD38-GFP. The arrows point to the plasma membrane CD38-GFP or CD31 accumulated at the synapse. (C) Comparative analysis of the percentage of conjugates with CD38-GFP (in B cells), CD31 (in T cells), or CD3-ζ (in T cells) redistributed at the contact zone relative to the total number of J77/CD38-GFP+ Raji cell conjugates in the absence (□) or presence (■) of SEE. The data represent the mean plus SEM of 3 independent experiments. In each experiment, 40 to 70 conjugates were analyzed. (D) FACS analysis of Jurkat J77 cells (top) or Raji B cells (bottom). Cells were stained with specific antibodies against CD38 (left) or anti-CD31 (right). (E) Quantification of the number of CD38, CD3, or CD31 molecules in Jurkat J77 T cells by using the Cellquant Calibrator kit. (F) Quantification of the number of CD38, CD19, and CD31 molecules in Raji B cells using the same kit as in panel E. Data in panels E and F represent the mean plus or minus SEM of 5 independent experiments.

TCR-dependent clustering of CD38 from B cells and CD31 from T cells at the T/APC contact zone. (A) Raji cells transiently transfected with the CD38-GFP construct were pulsed or not with SEE and allowed to conjugate with Jurkat J77 T cells. Then, cells were fixed, permeabilized, and stained for CD3-ζ (red). Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope. CD38-GFP (green; 100×/1.3 NA oil objective). Bars represent 5 μm for all panels. CD3-ζ (red), green fluorescence merged with red fluorescence images, and the corresponding DIC images superimposed on blue fluorescence images from CMAC-loaded Raji cells are shown. The white arrowheads point to the intracellular CD38-GFP or CD3-ζ. The arrows point to the plasma membrane CD3-ζ accumulated at the synapse. (B) CD38-GFP–transfected Raji B cells were treated as in panel A except that after permeabilization, cells were stained for CD31 (red). CD38-GFP (green), CD31 (red), green fluorescence merged with red fluorescence images, and the corresponding DIC images superimposed on blue fluorescence images from CMAC-loaded Raji cells are shown. The white arrowheads point to the intracellular CD38-GFP. The arrows point to the plasma membrane CD38-GFP or CD31 accumulated at the synapse. (C) Comparative analysis of the percentage of conjugates with CD38-GFP (in B cells), CD31 (in T cells), or CD3-ζ (in T cells) redistributed at the contact zone relative to the total number of J77/CD38-GFP+ Raji cell conjugates in the absence (□) or presence (■) of SEE. The data represent the mean plus SEM of 3 independent experiments. In each experiment, 40 to 70 conjugates were analyzed. (D) FACS analysis of Jurkat J77 cells (top) or Raji B cells (bottom). Cells were stained with specific antibodies against CD38 (left) or anti-CD31 (right). (E) Quantification of the number of CD38, CD3, or CD31 molecules in Jurkat J77 T cells by using the Cellquant Calibrator kit. (F) Quantification of the number of CD38, CD19, and CD31 molecules in Raji B cells using the same kit as in panel E. Data in panels E and F represent the mean plus or minus SEM of 5 independent experiments.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal