Figure 3

Anti-human CCR7 mAb induces cell death in xenografted Granta-519 cells from the subcutaneous tumors. The Annexin-V-FITC/7-AAD assay was used to quantitatively determine the percentage of non-viable cells following exposure to the anti-CCR7 mAb. (A) Granta-519 cells maintained in culture medium supplemented with 10% FCS (upper dot plot). Xenografted Granta-519 cells harvested from subcutaneous tumors in the control group (middle dot plot). Xenografted Granta-519 cells recovered from subcutaneous tumors in the CCR7 mAb-treated group (bottom dot plot). The Annexin V/7-AAD double staining made it possible to distinguish among viable cells (Annexin-V-/7-AAD-), early apoptotic cells (Annexin-V+/7-AAD-), late apoptotic cells (Annexin-V+/7-AAD+) and dead cells (Annexin-V-/7-AAD+). A representative experiment is shown (n = 5). (B) Percentage of dead cells (Annexin-V-/7-AAD+) in tumors from the control group (white bar, n = 5) or the treated group (black bar, n = 5). The mean ± SEM is shown. P-value denotes significant differences between the percentage of dead cells in the subcutaneous tumors from PBS- and anti-CCR7 mAb-treated mice. (C) Splenocytes from NOD/SCID mice but not from NSG mice mediate anti-CCR7 mAb-dependent cellular cytotoxicity. Calcein-UV-labeled Granta-519 target cells previously incubated either with IC or anti-CCR7 mAb (100 μg/ml) were plated with mice splenocytes at effector to target ratio of 10:1 for 24 hours. The mean percentage of specific lysis +/- SEM is shown from seven independent experiments with NOD/SCID mice and three independent experiments with NSG mice. P-values refer to the differences between the specific death mediated by the IC and the anti-CCR7 mAb in the corresponding strain of mice.