Activated protein C modulates T-cell metabolism and epigenetic FOXP3 induction via α-Ketoglutarate.

A direct regulation of adaptive immunity by the coagulation protease activated protein C (aPC) has recently been established. T-cell pre-incubation with aPC for 1 hour prior to transplantation increases FOXP3+ Tregs and reduces acute graft versus host disease (aGvHD) in mice, but the underlying mechanism remains unknown. As cellular metabolism modulates epigenetic gene regulation and plasticity in T-cells, we hypothesized that aPC promotes FOXP3+ expression by altering T-cell metabolism. To this end, T-cell differentiation was assessed in vitro using mixed lymphocyte reaction or plate-bound α-CD3/CD28 stimulation and ex vivo using T-cells isolated from aGvHD mice without and with aPC preincubation or analyses of mice with high plasma aPC levels. In stimulated CD4+CD25- cells, aPC induces FOXP3 expression while reducing expression of Th1-cell markers. Increased FOXP3 expression is associated with altered epigenetic markers (reduced 5-methylcytosine and H3K27me3) and reduced Foxp3 promoter methylation and activity These changes are linked to metabolic quiescence, decreased glucose and glutamine uptake, decreased mitochondrial metabolism (reduced TCA metabolites and mitochondrial membrane potential), and decreased intracellular glutamine and α-ketoglutarate levels. In mice with high aPC plasma levels, T-cell subpopulations in the thymus are not altered, reflecting normal T-cell development, while FOXP3 expression in splenic T-cells is reduced. Glutamine and α-ketoglutarate substitution reverse aPC-mediated FOXP3+ induction and abolish aPC-mediated suppression of allogeneic T-cell stimulation. These findings show that aPC modulates cellular metabolism in T-cells, reducing glutamine and α-ketoglutarate levels, which results in altered epigenetic marks, Foxp3 promoter demethylation and induction of FOXP3 expression, thus favoring a Treg-like phenotype.