Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression

Abstract

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c+ MHC II(NEG), CD11c+ MHC II(INT), CD11c+ MHC II(HI) expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O2 or conventional conditions, and MDCs from low O2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O2 and conventional culture conditions were similarly potent allostimulatory APCs. The O2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.