Figure 3
TCR arrested GFP-FoxP3-CD4–positive T cells are relatively resistant to the arrest reversal effects of CTLA-4. (A) Left panel: Tracing patterns of GFP-FoxP3-CD4–positive T cells and GFP-FoxP3-negative-CD4–positive T cells. T cells were initially activated for CTLA-4 surface expression and then rested for 24 hours before use for experiments. Cells were monitored over 20 minutes for random movement on glass slides coated with 2 μg/mL of ICAM-1-Fc in the presence of soluble anti-CD3 alone, or in combination with various anti–CTLA-4 concentrations. Stimulation with soluble antibody isotype served as a negative control. Rabbit anti–hamster antibody was used for crosslinking. Top panels: GFP-FoxP3–negative T cells; bottom panels: GFP-FoxP3–positive T cells. (B) CTLA-4 and LFA-1 (CD11a) are expressed at similar levels of Tconvs and Tregs. Left panel: CTLA-4–expression in GFP-FoxP3–positive and GFP-FoxP3–negative T cells. Cells were stained with CTLA-4-PE and analyzed by FACS. Right panel: Mean fluorescence intensity (MFI) of LFA-1 expression in FoxP3-positive and -negative T cells after activation. (C) Measurements of the velocities of GFP-FoxP3-CD4–positive T cells and GFP-FoxP3–negative CD4-positive T cells. Left panel: Velocity of cells were monitored on glass slides coated with 2 μg /mL of ICAM-1-Fc in the presence of soluble anti-CD3 alone, or in combination with various anti–CTLA-4 concentrations. Rabbit anti–hamster antibody was used for crosslinking. Right panel: Histogram showing mean velocity of the 2 populations in response to different anti–CTLA-4 concentrations. Bottom left panel: Measurements of displacement of GFP-FoxP3-CD4–positive T cells and GFP-Foxp3-CD4–negative T cells. Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant; *P < .05; **P < .01; ***P < .001. Data are representative of at least 3 separate experiments.

TCR arrested GFP-FoxP3-CD4–positive T cells are relatively resistant to the arrest reversal effects of CTLA-4. (A) Left panel: Tracing patterns of GFP-FoxP3-CD4–positive T cells and GFP-FoxP3-negative-CD4–positive T cells. T cells were initially activated for CTLA-4 surface expression and then rested for 24 hours before use for experiments. Cells were monitored over 20 minutes for random movement on glass slides coated with 2 μg/mL of ICAM-1-Fc in the presence of soluble anti-CD3 alone, or in combination with various anti–CTLA-4 concentrations. Stimulation with soluble antibody isotype served as a negative control. Rabbit anti–hamster antibody was used for crosslinking. Top panels: GFP-FoxP3–negative T cells; bottom panels: GFP-FoxP3–positive T cells. (B) CTLA-4 and LFA-1 (CD11a) are expressed at similar levels of Tconvs and Tregs. Left panel: CTLA-4–expression in GFP-FoxP3–positive and GFP-FoxP3–negative T cells. Cells were stained with CTLA-4-PE and analyzed by FACS. Right panel: Mean fluorescence intensity (MFI) of LFA-1 expression in FoxP3-positive and -negative T cells after activation. (C) Measurements of the velocities of GFP-FoxP3-CD4–positive T cells and GFP-FoxP3–negative CD4-positive T cells. Left panel: Velocity of cells were monitored on glass slides coated with 2 μg /mL of ICAM-1-Fc in the presence of soluble anti-CD3 alone, or in combination with various anti–CTLA-4 concentrations. Rabbit anti–hamster antibody was used for crosslinking. Right panel: Histogram showing mean velocity of the 2 populations in response to different anti–CTLA-4 concentrations. Bottom left panel: Measurements of displacement of GFP-FoxP3-CD4–positive T cells and GFP-Foxp3-CD4–negative T cells. Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant; *P < .05; **P < .01; ***P < .001. Data are representative of at least 3 separate experiments.

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