Figure 4
Figure 4. Defective TCR-induced CD38 clustering at the immunologic synapse in a Lck-deficient T-cell line. (A) JCaM1.6 T cells were transiently transfected with the CD38-GFP construct and allowed to conjugate with CMAC-labeled Raji cells pulsed or not pulsed with SEE. Then, cells were fixed, permeabilized, and stained for CD3-ζ (red) as described in Figure 1. Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (100×/1.3 NA oil objective). CD38-GFP (green), CD3-ζ (red), and the green fluorescence merged with red fluorescence 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 CD38-GFP or CD3-ζ. (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+ JCaM 1.6/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 T cells transiently transfected with the CD38-GFP construct, untreated or treated with PP2 (10 μM), were stimulated with CMAC-labeled Raji B cells pulsed or not pulsed with SEE, stained with anti–CD3-ζ (red), and analyzed as in panel A. (D) 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/SEE-pulsed Raji conjugates in the absence (■) or presence of PP2 (▨). In each experiment, more than 40 conjugates were analyzed. (E) Jurkat J77 T cells treated or untreated with PP2 (10 μM) were stimulated with CMAC-labeled Raji B cells pulsed or not pulsed with SEE, stained with anti-CD38 (green) and anti-CD3-ζ (red), and analyzed by a Leica TCS-SP5 confocal scanning laser microscope (63×/1.4 NA oil objective); scale bar equals 4 μm. In this panel, intact cells were incubated with the anti-CD38 mAb HB136 followed by an Alexa Fluor 488–goat anti-mouse IgG, saturated with mouse serum. CD3-ζ was detected by permeabilizing cells (0.5% Triton X-100) prior staining with the anti–CD3-ζ 488 rabbit antibody, followed by the incubation with a Rhodamine Red X–labeled goat anti-rabbit IgG (H + L) highly cross-adsorbed. (F) Comparative analysis of the percentage of conjugates with endogenous CD38 or CD3-ζ redistributed at the contact zone relative to the total number of J77/SEE-pulsed Raji cell conjugates in the absence (■) or presence of PP2 (▨). Data representative of 2 independent experiments. In each experiment, more than 100 conjugates were analyzed.

Defective TCR-induced CD38 clustering at the immunologic synapse in a Lck-deficient T-cell line. (A) JCaM1.6 T cells were transiently transfected with the CD38-GFP construct and allowed to conjugate with CMAC-labeled Raji cells pulsed or not pulsed with SEE. Then, cells were fixed, permeabilized, and stained for CD3-ζ (red) as described in Figure 1. Cells were examined using an Olympus Cell R IX81 motorized system inverted microscope (100×/1.3 NA oil objective). CD38-GFP (green), CD3-ζ (red), and the green fluorescence merged with red fluorescence 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 CD38-GFP or CD3-ζ. (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+ JCaM 1.6/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 T cells transiently transfected with the CD38-GFP construct, untreated or treated with PP2 (10 μM), were stimulated with CMAC-labeled Raji B cells pulsed or not pulsed with SEE, stained with anti–CD3-ζ (red), and analyzed as in panel A. (D) 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/SEE-pulsed Raji conjugates in the absence (■) or presence of PP2 (▨). In each experiment, more than 40 conjugates were analyzed. (E) Jurkat J77 T cells treated or untreated with PP2 (10 μM) were stimulated with CMAC-labeled Raji B cells pulsed or not pulsed with SEE, stained with anti-CD38 (green) and anti-CD3-ζ (red), and analyzed by a Leica TCS-SP5 confocal scanning laser microscope (63×/1.4 NA oil objective); scale bar equals 4 μm. In this panel, intact cells were incubated with the anti-CD38 mAb HB136 followed by an Alexa Fluor 488–goat anti-mouse IgG, saturated with mouse serum. CD3-ζ was detected by permeabilizing cells (0.5% Triton X-100) prior staining with the anti–CD3-ζ 488 rabbit antibody, followed by the incubation with a Rhodamine Red X–labeled goat anti-rabbit IgG (H + L) highly cross-adsorbed. (F) Comparative analysis of the percentage of conjugates with endogenous CD38 or CD3-ζ redistributed at the contact zone relative to the total number of J77/SEE-pulsed Raji cell conjugates in the absence (■) or presence of PP2 (▨). Data representative of 2 independent experiments. In each experiment, more than 100 conjugates were analyzed.

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