Regulatory T-cells are more resistant to genotoxic agents compared to effector T-cells

Background: Both host and graft regulatory T cells (Treg) may provide protection from graft-versus-host disease (GVHD) in the mouse system. Findings from a murine model of syngeneic GVHD suggest that host Treg preferentially survive a lethal dose of irradiation and confer protection from disease. In humans, various combinations of genotoxic agents are actually used for myeloablative conditioning. The sensitivity of human Treg to DNA damage induced apoptosis has so far not been investigated. Objective:To evaluate the resistance of Treg to apoptosis induced by genotoxic agents compared to effector T cells (Teff) in vitro. Materials and methods: PBMC of healthy donors were exposed to either ionizing irradiation or etoposide. After incubation, samples were labelled with annexin V FITC or anti-activated caspase 3 FITC combined with monoclonal antibody conjugates to CD4, CD25 and CD127 or to surface antigen CD4 and transcription factor FoxP3. Propidium iodide or fixable infrared viability dye were included in the staining protocol as dead cell markers. The frequency of apoptotic cells among Treg and effector CD4+ T lymphocytes was determined by flow cytometric analysis at different time points (up to 72h). CD45RA staining was used to analyse the role of differentiation for resistance to apoptosis. Expression of anti-apoptotic proteins bcl-2 and Bcl-xL by Treg and Teff was compared. Results: Both activation of caspase 3 and exposure of phosphatidylserine following DNA damage in vitro was observed with significantly higher frequency in Treg compared to effector CD4 lymphocytes. This effect was observed in both etoposide treated cultures and those exposed to ionizing irradiation. Similar results were obtained by identifying Treg as CD25highCD127- or as FoxP3+ events. The resistance to DNA damage by Treg could not be explained by their differentiation to a CD45RA negative memory phenotype. When measured as mean fluorescence intensity, anti-apoptotic proteins Bcl-2 and bcl-xL were not more expressed by Treg compared to Teff. Conclusion: Our observations suggest a higher resistance to DNA damage of regulatory T cells compared to effector T cells in vitro. The higher resistance cannot be explained by differentiation to a memory phenotype or differential expression of anti-apopotic proteins bcl-2 and bcl-xL. The higher resistance to genotoxic agents of Treg could affect the relative size ratio of Treg to effector T cells following conditioning.