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 O 2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O 2 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 O 2 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 O 2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O 2 or conventional conditions, and MDCs from low O 2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O 2 and conventional culture conditions were similarly potent allostimulatory APCs. The O 2 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.