The project started with an initial evaluation on the role of succinate in CD4+ T cells, in particular the generation of different CD4+ T cell populations. The research activity focused on the pro-inflammatory CD4+ subsets named T helper 1 (Th1), Th17 and Th2 cells, which are important players in autoimmune and atopic disease. In parallel, the importance of succinate on the generation of the CD4+ anti-inflammatory subset named regulatory T cells (Tregs) was evaluated. To this purpose, two different approaches were used: CD4+ T cells were treated with extracellular succinate or with cell-permeable inhibitors of succinate dehydrogenase (SDH), the enzyme that degrades succinate into the cell. The latter approach was used to induce intracellular succinate accumulation in T cells. It was first observed that, despite the fact that CD4+ T cell express on their surface the cellular receptor for succinate, extracellular succinate did not affect the activation or functionality of T cells. In particular, succinate did not significantly modulate the in vitro generation of pro-inflammatory Th1, Th17 and Th2 cells or anti-inflammatory Tregs. Pharmacological inhibition of SDH moderately increased the pro-inflammatory potential of Th1 cells, even though this effect was mild. Overall, the results indicate a minor role for extracellular or intracellular succinate in the modulation of CD4+ T cell activity.
In parallel with the above experiments, the expression of different isoforms of the enzyme PK was evaluated in CD4+ T cells. The results indicate that, upon in vitro activation, two different isoforms of PK (PKM1 and PKM2) are upregulated in CD4+ T cells. In particular, the PKM2 isoform undergoes a complex regulation upon activation, which involves its phosphorylation at multiple sites and accumulation in the nucleus of activated CD4+ T cells. Of note, at all phases of activation, this enzyme is present in equilibrium between a monomeric/dimeric form (i.e. one/two enzyme subunits joined together) and a tetrameric form (i.e. four enzyme subunits joined together). Treatment of CD4+ T cells with PKM2 pharmacological activators, which induce the tetramerisation of the PKM2 enzyme, limited PKM2 nuclear translocation and strongly inhibited T cell activation, proliferation and cytokine production. These activators prevented the engagement of glycolysis, a metabolic pathway that was previously shown to be essential for T cell activation. Importantly, this is associated with a block in the generation of both pro-inflammatory Th1 and Th17 cells and parallel induction of anti-inflammatory Tregs in vitro. Finally, treatment with PKM2 activators ameliorated experimental autoimmune encephalomyelitis, the mouse model of human multiple sclerosis, by inhibiting Th1- and Th17-mediated inflammation in the central nervous system. Overall, these results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.
