Cathepsin-B-dependent apoptosis triggered by antithymocyte globulins: a novel mechanism of T-cell depletion by Marie-Cécile Michallet, Frederic Saltel, Xavier Preville, Monique Flacher, Jean-Pierre Revillard, and Laurent Genestier Blood Volume 102(10):3719-3726 November 15, 2003 ©2003 by American Society of Hematology

High concentrations of ATG induce apoptosis of PBL High concentrations of ATG induce apoptosis of PBL. (A-B) PBLs were incubated with rabbit (▪) or horse (•) ATGs, F(ab′)2 fragments of ATG (□), or nonmitogenic ATG number 5 (○) over a range of concentrations. High concentrations of ATG induce apoptosis of PBL. (A-B) PBLs were incubated with rabbit (▪) or horse (•) ATGs, F(ab′)2 fragments of ATG (□), or nonmitogenic ATG number 5 (○) over a range of concentrations. In panel A, [3H]TdR uptake was measured after 72 hours of culture. In panel B, apoptosis was measured by surface binding of annexin V after 24 hours of culture. (C) Kinetics of ATG-induced apoptosis. PBLs were incubated with NRS alone (○), rabbit ATGs (250 μg/mL; ▪), or horse ATGs (500 μg/mL; •), and apoptosis was measured by surface binding of annexin V. (A-C) Results are expressed as the mean ± SEM from triplicate measurements of one experiment representative of several independent experiments. (D) PBLs were incubated for 24 hours in the presence of NRS (control) or ATG (250 μg/mL). Anti-CD95 (7C11; 1 μg/mL) was added to 3-day-activated lymphoblasts during 24 hours. DNA fragmentation was analyzed by flow cytometry staining using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. Results shown are representative of 4 independent experiments. (E) Morphologic features of cells after treatment with NRS or ATG were observed using transmission electron microscopy. Original magnification, × 10 000. Marie-Cécile Michallet et al. Blood 2003;102:3719-3726 ©2003 by American Society of Hematology

Apoptosis of T lymphocytes at high concentrations of ATG is not the consequence of an AICD process. Apoptosis of T lymphocytes at high concentrations of ATG is not the consequence of an AICD process. (A) PBLs were activated by rabbit ATG at the indicated concentration in the presence or absence of ZB4 (2.5 μg/mL) and TNF-R-Ig (20 μg/mL), and apoptosis was measured at 24 hours. (B) Activated lymphoblasts were treated for 24 hours with either medium, CD178 (100 ng/mL) in the presence or absence of ZB4 (2.5 μg/mL), or TNF-α (50 ng/mL) in the presence or absence of TNF-R-Ig (20 μg/mL). (A-B) Apoptosis was measured by surface binding of annexin V. (C) Analysis of caspase-8 processing in the presence of rabbit ATG. PBLs were treated with ATG for 24 hours. As positive control, activated lymphoblasts were treated with anti-CD95 (7C11; 1 μg/mL) for 6 hours. (D) Kinetics of FLIPs expression in PBLs treated with rabbit ATG (50 or 250 μg/mL). (C-D) Cells were lysed after a wash with PBS, and proteins were loaded and separated on 12% SDS-PAGE followed by Western blotting with the anti-caspase-8 mAb (C) or the anti-FLIP mAb (D). Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-β-actin mAb. Results from one experiment representative of 2 independent experiments showing similar results. (E) PBLs were incubated with NRS or ATG (250 μg/mL) for 24 hours in the presence of CsA (1 μg/mL), RPM (500 nM), or dexamethasone (1 μM). Apoptosis was measured by surface binding of annexin V, and results shown are representative of 2 independent experiments. Marie-Cécile Michallet et al. Blood 2003;102:3719-3726 ©2003 by American Society of Hematology

Kinetics of cytochrome c release during ATG-induced apoptosis of T cells. Kinetics of cytochrome c release during ATG-induced apoptosis of T cells. PBLs were incubated with NRS, rabbit ATG (250 μg/mL), or horse ATG (250 μg/mL). (A) At the indicated times, cytosolic extracts were prepared and proteins were separated on 15% SDS-PAGE followed by Western blotting with the anti-cytochrome c mAb or the anti-cytochrome oxidase mAb. Anti-cytochrome oxidase served as a marker of mitochondrial contamination of the extracts. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-β-actin mAb. Apoptosis (%) was measured by surface binding of annexin V at the indicated times. (B) After 24-hour treatment, cytochrome c release was visualized by immunofluorescence staining. Results are from one representative experiment of 2 independent experiments showing similar results. Original magnification, × 63. Marie-Cécile Michallet et al. Blood 2003;102:3719-3726 ©2003 by American Society of Hematology

Role of caspases in apoptosis induced by high concentrations of ATG Role of caspases in apoptosis induced by high concentrations of ATG. (A) Effect of zVAD-fmk on DNA fragmentation induced by ATG. The caspase inhibitor zVAD-fmk (100 μM) was added to PBLs for 1 hour, and then cells were incubated with NRS or ATG (250 μg/mL). Role of caspases in apoptosis induced by high concentrations of ATG. (A) Effect of zVAD-fmk on DNA fragmentation induced by ATG. The caspase inhibitor zVAD-fmk (100 μM) was added to PBLs for 1 hour, and then cells were incubated with NRS or ATG (250 μg/mL). DNA fragmentation was analyzed at the indicated time by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. (B) Caspase-3 and -7 processing. PBLs were treated with rabbit ATG at indicated concentrations and lysed at 24 hours, and proteins were separated on SDS-PAGE. (C) Analysis of PARP cleavage. PBLs were incubated with NRS or ATG (250 μg/mL) and preactivated T cells with anti-CD95 (7C11 1 μg/mL). At 24 hours, cells were lysed and proteins were separated on SDS-PAGE. The p85 band corresponds to the caspase-3 cleavage product of PARP. (D) Caspase-9 cleavage. Cells were treated with rabbit ATG and lysed at 24 hours, and proteins were separated on SDS-PAGE and analyzed by Western blotting with an anti-caspase-9 antibody that recognizes the proform (p46) and the cleaved forms (p37/p34) of caspase-9. As positive control, 3-day-activated PBLs were treated with STS (0.5 μM) for 12 hours. Amounts of loaded proteins were controlled for homogeneity by probing membranes with anti-β-actin mAb. All data are from one representative experiment of 2 independent experiments showing similar results. Marie-Cécile Michallet et al. Blood 2003;102:3719-3726 ©2003 by American Society of Hematology

Cathepsin B is translocated from lysosomes into the cytosol during ATG-induced apoptosis. Cathepsin B is translocated from lysosomes into the cytosol during ATG-induced apoptosis. (A-B) PBLs were treated by NRS alone or ATG (250 μg/mL) in the presence or absence of indicated protease inhibitors. (A) The percentage of apoptotic cells was measured 24 hours later by surface binding of annexin V. Results are mean ± SEM from a triplicate experiment and are representative of 3 independent experiments. (B) DNA fragmentation was analyzed at 24 hours by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. (C) Visualization of cathepsin B release from lysosomes into cytosol in PBLs treated by horse ATG (50 or 250 μg/mL). Staining with lysotracker red (acidic organelle-selective probe) was used to characterize lysosomal membrane alterations. (D) Cells were treated with rabbit or horse ATG (250 μg/mL), and cytosolic extracts were prepared at indicated times. Equal amounts of proteins were analyzed for cathepsin B activity using the fluorogenic protease substrate zRR-AMC. Results are mean ± SEM from a triplicate experiment. Marie-Cécile Michallet et al. Blood 2003;102:3719-3726 ©2003 by American Society of Hematology