Rabbit Anti T-Lymphocyte Globulin Induces Apoptosis in Peripheral Blood Mononuclear Cell Compartments and Leukemia Cells, While Hematopoetic Stem Cells Are Apoptosis Resistant

Binding of ATG to PBMNC compartments. Unseparated PBMNC were incubated with the indicated concentrations of ATG (X-axis is depicted in 2 sectors, left sector range: 0 to 3 μg/mL, right sector range: 10 to 250 μg/mL) for 20 minutes, stained with FITC-labeled antirabbit IgG, and analyzed by fluorocytometry. Binding is expressed as mean fluorescence intensity (A). CD4-, CD8-, CD20-, CD14-, and CD56-positive selected cells (purity ≥90%) were incubated with ATG 20 μg/mL for 20 minutes and stained with FITC antirabbit IgG (filled histograms) versus incubation with rabbit IgG isotype control (open histograms) (B). Double staining with PE-labeled anti-CD antibodies and FITC antirabbit IgG after incubation of unseparated PBMNC with ATG 20 μg/mL confirms the complete ATG binding of PBMNC compartments (C).

ATG induces apoptosis in PBMNC compartments. PBMNC incubated with ATG 250 μg/mL for 6 h and 24 h were analyzed by triple-color fluorescence for the lineage specific marker (PE anti CD) and Annexin V/PI staining for apoptosis detection. AnnexinV/PI plots represent the gate (R1) of the indicated CD marker.

Time course of ATG-induced apoptosis in PBMNC compartments. Time-dependent apoptosis rates as analyzed by AnnexinV/PI positivity of PBMNC cell compartments incubated with ATG 250 μg/mL from 3 hours to 24 hours. Values represent mean ± SEM from 2 experiments.

ATG binding to HSC and apoptosis induction. Incubation with ATG 20 μg/mL for 20 minutes results in complete binding to CD34-positive HSC (A). Annexin V/PI detection of apoptosis induced by incubation with ATG 500 μg/mL for 24h in the HSC gate (CD34⁺) (B). Percentage of nonapoptotic CD34⁺ HSC following incubation with the indicated concentrations of ATG for 6 hours and 24 hours defined by AnnexinV/PI negativity (mean ± SEM from 2 experiments) (C).

Colony growth of HSC in the presence of ATG. Unseparated leukapheresis products of 9 healthy HSC donors were plated at 1.5 × 10⁵ cells/mL in methylcellulose as described. ATG was added at 20 μg/mL and 250 μg/mL. Significantly (P < .0013) more colonies (CFU) could be observed at 14 days in the culture with ATG 20 μg/mL, whereas ATG 250 μg/mL abrogated colony growth (mean: solid bars) (A). In HSC positively selected for CD34 no growth stimulation by ATG could be observed. CD34 selected HSC of 2 healthy donors were plated at 5 × 10³ cells/mL (open symbols; mean: dotted bars) and 15 × 10³ cells/mL (filled symbols; mean: solid bars) in the presence of the indicated ATG concentrations. At 14 days colony growth was reduced with both ATG concentrations (B).

ATG induces apoptosis in leukemia cell lines. HL-60 cells incubated with ATG 250 μg/mL for 6 hours show high rates for Annexin V/PI positivity (A) and comparable MMP as measured by loss of ΔΨ (reduced DIOC6 uptake) (B). Concentration-dependent induction of apoptosis by ATG in myeloblastic cell lines KG1, K562, HL-60, and U937 (C) and lymphoblastic cell lines Jurkat, Daudi, DG 75, and YT (D) after incubation with the indicated concentrations of ATG for 6 hours by Annexin V/PI staining (mean ± SEM from 2 independent experiments).

ATG induces apoptosis in primary leukemia cells. Specific Apoptosis rates for primary leukemia cells from 10 patients with CLL (A), 6 patients with ALL (B), and 8 patients with AML (C) incubated with ATG 250 μg/mL for 24 hours measured by triple fluorescence for leukemia lineage marker and Annexin V/PI (mean from 2 experiments) (∗apoptosis rate slightly below control).