Regulation of CD4⁺ T cell polarisation and function by pyruvate kinase M2 (PKM2)

Abstract

Background: Autoimmune diseases, such as autoimmune hepatitis, are driven by a functional imbalance between effector T cells and T regulatory cells. Therapeutic options in many cases still lack specificity and employ general immune suppression with glucocorticoids or purine analogs. When activated in the course of autoimmune disease, CD4+ T cells switch to a pro-proliferative metabolism characterized by aerobic glycolysis – a process also known as Warburg effect. Pyruvate kinase M2, which is expressed in proliferative tissue, plays a key role in this glycolytic switch as it catalyzed the last and rate limiting step of glycolysis. It changes from a catalytically active tetramer toward a less active dimer amplifying glycolysis and acting as a transcription factor for glycolytic enzymes as well as inflammatory cytokines. By diverging intermediates from glycolysis into biosynthetic pathways for lipid, nucleotide and amino acid synthesis, this change provides substrates for cell growth, proliferation, and cytokine production. To the contrary, T regulatory cells are believed to rely on oxidative phosphorylation for fueling.

Methods: CD4+ T cells with a cre-mediated PKM2 knockout were isolated from mouse spleens and polarised into Th1 and Th17 CD4+ T cells as well as T regulatory cells. Using seahorse metabolic rate assay, restimulation and suppression assay these cells were characterized regarding their glycolytic metabolism, proliferation and function and compared with regular CD4+ T cells. Additionally, the effects of a small molecule activator of PKM2 (TEPP-46) were tested regarding its influence on proliferation and cytokine production.

Results: Here we show that a knockout of PKM2 in CD4+ T cells results in altered glycolytic metabolism despite compensatory overexpression of PKM1. Subsequently, reduced production of IL-2 as well as IFNγ could be measured. In Th17 cells particularly, a strongly pronounced reduction of glycolytic activity was observed. Th17 proliferation and polarisation were affected with a reduced expression of RORγT and an altered expression of the ectonucleotidases CD39 and CD73. Contrarily to what was observed in the effector T cells, T regulatory cells showed enhanced expression of their signature transcription factor FoxP3 upon PKM2 knock out. Treatment of cultured CD4+ T cells with the small molecule activator TEPP-46 resulted in impairment of activation and proliferation, again with the most pronounced effect in the Th17 cell subset. Upon TEPP-46 application, IL-2 production was reduced in Th1 cells as was IL-17 expression in Th17 cells. Yet, in vitro results may differ from in vivo effects of a PKM2 knockout as liver damage in a Concanavalin A model of autoimmune hepatitis persisted in mice with a CD4+ T cells specific PKM2 knockout.

Conclusion: This work demonstrated the relevance of PKM2 to CD4+ T cell proliferation and function in vitro. A strongly pronounced effect was observed in Th17 CD4+ T cells, possibly explained by the relevance of the HIF1α signature to their metabolic and inflammatory profile. In vivo effects of a knockout of PKM2 in Th17 cells in organ inflammation still remain to be better examined in different disease models.