Regulatory T cells (Tregs) are a promising therapeutic tool for inducing transplantation tolerance. Tregs can be divided into those of thymic origin (tTreg) and those which arise in the periphery or induced in vitro (pTregs and iTregs, respectively). tTreg and iTreg share many key features such as their reliance on Foxp3 expression, but they differ in the repertoire of their TCRs and the epigenetic regulation in Foxp3 locus. Exciting new studies in the field of immunometabolism have shown that cellular metabolism of different types of immune cells including T cells can affect their fate and function. Although growing insights in this field suggest the manipulation of Treg metabolic traits as a therapeutic strategy, there is actually a surprising paucity of information regarding the metabolism of distinct Treg subsets and human Tregs. Here, we performed a detailed comparative analyses of human tTregs and iTregs subsets to understand their metabolic signatures.

We activated ex vivo Tregs (tTregs) from healthy donor PBMCs in the presence of polyclonal stimulation and IL-2, and generated iTregs by naïve CD4+ T conventional cells in iTreg skewing conditions that include IL-2, TGFb and ATRA. Using seahorse analyses, we found that by day 3 post activation both human Treg subsets similarly engage glycolysis. By day 7, iTregs showed increased propensity to favor glycolytic metabolism unlike tTregs. This correlated with a decrease in FOXP3 expression in iTregs, a feature associated with their instability. In contrast, tTregs maintain FOXP3 expression exhibiting reduced glycolysis. These suggested that each of Treg subsets have distinct requirements for glycolysis in temporally manner. To understand this further, we inhibited glycolysis by 2-deoxy-D-glucose (2DG) at the onset as well as at day 3 post activation. Notably, addition of 2DG at the onset dramatically diminished FOXP3 expression in iTregs. In contrast, inhibiting glycolysis showed at best a modest effect on FOXP3 expression on tTregs. Furthermore, once FOXP3 expression was upregulated, 2DG treatment did not affect the maintenance of FOXP3 expression for either Treg subset.

Unlike murine system, our data show that glycolysis is indispensable for FOXP3 expression and induction in both Treg subsets during the initial stages of activation. Moreover, timing and duration of manipulation in each of Treg subsets are critical components to effectively control Tregs by glycolysis.

CITATION INFORMATION: Tanimine N., Priyadharshini B., Germana S., Turka L. Differential Effects of Inhibiting Glycolysis on Human Treg Subsets Am J Transplant. 2017;17 (suppl 3).

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