Changes in metabolic phenotype and cellular ATP production during CD4+ T cell activation.

Abstract

Abstract Activation of CD4+ T cells is followed by rapid proliferation and differentiation into specific subsets (Treg or Teff). These transitions are accompanied by tight regulated changes in energetic demand and cellular metabolic reprogramming. Using Agilent Seahorse extracellular flux analysis, we have developed a cell-based assay for simultaneous measurement of the two-main cellular ATP-producing pathways, i.e. glycolysis and oxidative phosphorylation. The assay allows for quantification of real time changes in total ATP production rate, and the fractional contribution of the individual pathways to support bioenergetic demands. We found that naïve CD4+ T cells obtain most of its ATP from mitochondrial oxidative phosphorylation. After activation with anti CD3/CD28 conjugated beads, total ATP production rate significantly increases, and that rise is sustained by an increase in glycolytic ATP production, but also in mitochondrial ATP production. When glucose is replaced by galactose in the assay medium, T cell activation is inhibited despite partial compensation of total ATP production through mitochondrial respiration. Time-course of bioenergetic phenotype during T-cell expansion shows a deep increase in ATP production rate up to day 6–7 with glycolysis contributing up to 70% of total ATP production. When glucose is replaced by galactose 72 hr after activation, total ATP production rate was fully compensated by mitochondrial ATP production but still the inhibition of glycolytic activity by galactose reduces cell proliferation. The results obtained highlight the role of glycolytic activity beyond energetic supply and its importance for biosynthetic and redox requirements or cell activation and proliferation.