Increasing Foxp3+ regulatory T-cell (Treg) numbers and/or their function are innovative approaches to immunosuppression post-transplantation. T-effector (TE) and Treg cells have differing metabolic needs, but little is known of the contribution of mitochondrial functions to T cells during alloresponses. In studies of Treg and TE cells, we noted that CD3Ε/CD28 stimulation led to activation of oxidative phosphorylation (OXPHOS) regulators, including Sirt3 (Sirtuin-3), PGC1Α (Peroxisome proliferator-activated receptor-Γ coactivator-1Α) and phosphorylated AMPKΑ (AMP-activated protein kinase-Α). While either cell type could be inhibited by ≥0.1 ΜM rotenone, an inhibitor of mitochondrial complex-I, at 0.05 ΜM, TE proliferation was unaffected, but Tregs had impaired suppressive function (41±0.11% reduction, p<0.001 n=4). These data suggest that OXPHOS is involved in both TE and Treg metabolism, but disproportionally more so for Tregs. Genetic and pharmacologic targeting of histone/protein deacetylase-9 (HDAC-9) improves Treg suppressive function and promotes allograft survival. We now show that deletion of HDAC9 increased mitochondrial respiration (p=0.03 n=5) and ROS production (p=0.001 n=3) in Treg but not TE. HDAC9 is an inhibitor of myocyte enhancer factor-2D (MEF-2D), which promotes PGC1Α expression, and PGC1Α is important to mitochondrial biogenesis and OXPHOS. Indeed, HDAC9-/- Tregs had increased PGC1Α and Sirt3 expression (mRNA/protein), and 20-fold more AMPKΑ phosphorylation (p<0.001). Moreover, deletion of Sirt3 in mice led to decreased Treg suppressive function in vitro (p=0.02 n=4), and conditional deletion of PGC1Α in Tregs led to impaired allograft survival. In summary, key regulators of energy metabolism in Tregs and TE were identified and shown to be required for allograft acceptance. These pathways can be regulated using pharmacologic HDAC inhibition. These studies provide a novel approach to improve Treg function and promote allograft survival, and give new insights into the fundamental mechanisms by which immune cells function during alloresponses.

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