Involvement of Fas and TNF pathways in the induction of apoptosis of T cells by antithymocyte globulin

Abstract

Antithymocyte globulin (ATG) is the treatment of choice for those aplastic anemia patients who are not suitable for bone marrow transplantation (BMT). ATG is also used for the treatment of rejections in organ transplantation and as a conditioning regimen in BMT. Despite the proven efficacy of ATG in these areas, its mechanism of action is not known. Profound T-cell lymphopenia observed in vivo with ATG treatment is supposed to contribute to its therapeutic effect. We have previously shown that apoptosis is one of the mechanisms responsible for ATG-induced lymphopenia. Our next objective was to investigate the effect of ATG on modulation of Fas and TNF pathways, the two main pathways of T-cell apoptosis. Maximum surface expression of Fas on T cells was observed after 24 h at an ATG dose of 100 µg/ml; at this dose 88% of cells expressed Fas as compared to 26% of untreated cells. Surface expression of FasL was found to peak after 24 h at an ATG dose of 1000 µg/ml when 34% of cells were positive for FasL as compared to 1.5% of untreated T cells. Tumor necrosis factor (TNF)-a production was found to be maximum after 6 h at 1000 µg/ml dose (20%) as measured by intracellular cytokine staining of T cells. TNF-a production was also measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of lymphocytes treated with ATG for 6 h. A dose-dependent increase in TNF-a production was found in these supernatants with a plateau being achieved at an ATG dose of 1000 µg/ml. We conclude that ATG-induced apoptosis in T cells involves both Fas and TNF pathways and TNF-a is produced much earlier than Fas and FasL expression.