Figure 2.
Figure 2. Effect of CD40 activation on DC apoptosis. Cells were activated for 6 hours with the indicated mAbs or isotype controls, before annexin V FITC and 7-AAD staining and flow cytometry. This staining distinguishes between viable (7-AAD-/annexin V-), early apoptotic (annexin V+/7-AADdim), and late apoptotic (annexin V+/7-AAD+) DCs. (A-B) Apoptotic effect of the isotype control (A) or anti-CD40 clone B-B20 (B) on immature/LPS-matured DCs, B lymphocytes, and CD8+ T cells. Similar results were obtained in 10 independent experiments. (C) Effect of anti-CD40 (clone B-B20) and anti-CD80 antibodies on LPS-matured DC apoptosis. Similar results were obtained in 4 independent experiments. Numbers in the quadrants indicate the percentages of positive cells. (D) Effect of agonistic anti-CD40 (clones BB20, G28-5, and mAb89; gray bars), agonistic anti-CD80 (black bars), and isotype control (white bars) on apoptosis of immature, LPS-matured, or TNF-α-matured DCs. The percentage of apoptotic cells represents the sum of early and late apoptotic cells. Results are the means ± SEM of 4 independent experiments. (E) LPS-matured DCs were treated for 6 hours with increasing concentrations of anti-CD40 (B-B20) or the isotype control before staining with annexin V FITC and 7-AAD. Results are the means ± SEM of 3 independent experiments. (F) Immature and LPS-matured DCs were treated with 5 μg/mL anti-CD40 (B-B20) or the isotype control and were stained at various times with annexin V FITC and 7-AAD. Ten thousand events were acquired in the FS/SS gate. Results are expressed as anti-CD40-induced specific apoptosis (% apoptotic DCs following treatment with anti-CD40) - (% apoptotic DCs following treatment with the isotype control). DCs undergo apoptosis and form apoptotic bodies that are no longer detected in the FS/SS gate. This explains the fall in the percentage of apoptotic cells after 12 hours of activation. Results are means ± SEM of 5 independent experiments. (G) DCs were matured with LPS (1 μg/mL) or anti-CD40 B-B20 (5 μg/mL) for 24 hours, then activated for 6 hours with B-B20 or isotype control (5 μg/mL) and stained with annexin V FITC and 7-AAD. Results represent the mean ± SEM of 3 independent experiments.

Effect of CD40 activation on DC apoptosis. Cells were activated for 6 hours with the indicated mAbs or isotype controls, before annexin V FITC and 7-AAD staining and flow cytometry. This staining distinguishes between viable (7-AAD-/annexin V-), early apoptotic (annexin V+/7-AADdim), and late apoptotic (annexin V+/7-AAD+) DCs. (A-B) Apoptotic effect of the isotype control (A) or anti-CD40 clone B-B20 (B) on immature/LPS-matured DCs, B lymphocytes, and CD8+ T cells. Similar results were obtained in 10 independent experiments. (C) Effect of anti-CD40 (clone B-B20) and anti-CD80 antibodies on LPS-matured DC apoptosis. Similar results were obtained in 4 independent experiments. Numbers in the quadrants indicate the percentages of positive cells. (D) Effect of agonistic anti-CD40 (clones BB20, G28-5, and mAb89; gray bars), agonistic anti-CD80 (black bars), and isotype control (white bars) on apoptosis of immature, LPS-matured, or TNF-α-matured DCs. The percentage of apoptotic cells represents the sum of early and late apoptotic cells. Results are the means ± SEM of 4 independent experiments. (E) LPS-matured DCs were treated for 6 hours with increasing concentrations of anti-CD40 (B-B20) or the isotype control before staining with annexin V FITC and 7-AAD. Results are the means ± SEM of 3 independent experiments. (F) Immature and LPS-matured DCs were treated with 5 μg/mL anti-CD40 (B-B20) or the isotype control and were stained at various times with annexin V FITC and 7-AAD. Ten thousand events were acquired in the FS/SS gate. Results are expressed as anti-CD40-induced specific apoptosis (% apoptotic DCs following treatment with anti-CD40) - (% apoptotic DCs following treatment with the isotype control). DCs undergo apoptosis and form apoptotic bodies that are no longer detected in the FS/SS gate. This explains the fall in the percentage of apoptotic cells after 12 hours of activation. Results are means ± SEM of 5 independent experiments. (G) DCs were matured with LPS (1 μg/mL) or anti-CD40 B-B20 (5 μg/mL) for 24 hours, then activated for 6 hours with B-B20 or isotype control (5 μg/mL) and stained with annexin V FITC and 7-AAD. Results represent the mean ± SEM of 3 independent experiments.

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