Induction of apoptosis of peripheral blood mononuclear cells by antithymocyte globulin (ATG) in aplastic anemia: an in vivo and in vitro study

Antithymocyte globulin (ATG) is the standard therapy for aplastic anemia (AA) patients who are not eligible for allogeneic bone marrow transplantation (BMT). The exact mechanism of action of ATG is still not known. Profound lymphopenia is observed throughout the treatment period with ATG and appears to contribute to its immunomodulatory effect. One of the possible mechanisms, which could account for ATG-mediated lymphopenia, is by induction of apoptosis of peripheral blood mononuclear cells (PBMCs). However, there is no conclusive evidence to support this mechanism. We investigated whether ATG could induce an in vivo apoptosis in PBMCs of 12 AA patients undergoing ATG therapy by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay using flow cytometry. A significant increase in the percentage of apoptosis was observed in six patients. The median percentage prior to therapy was 3% (range: 1-10%), which increased to a peak median value of 27% (range 17-66%) with therapy. ATG also induced an in vitro apoptosis of normal PBMCs as demonstrated by Annexin V and TUNEL staining using flow cytometry. Induction of apoptosis was dose dependent: 52% of the PBMCs exhibited Annexin V positivity after incubation with ATG at a dose of 500 mug/ml for 6 h, and 37% of the PBMCs exhibited DNA fragmentation after incubation with ATG at a dose of 1000 mug/ml for 24 h as demonstrated by TUNEL assay. Thus, ATG induces in vivo apoptosis in PBMCs of AA patients undergoing therapy as well as an in vitro apoptosis in normal PBMCs, and apoptosis may be an important mechanism for ATG-induced lymphopenia.