Investigating the Role of PPAR Delta in Alloreactive T Cells

Cellular metabolism is a key determinant in T cell fate and function. We have previously demonstrated that murine alloreactive CD8 T cells upregulate fatty acid oxidation (FAO) during graft-versus-host disease (GVHD), concomitant with elevated levels of peroxisome proliferator-activated receptor delta (PPARδ), a ligand-triggered transcription factor that regulates FAO. Notably, PPARδ is not activated in T cells from syngeneic bone marrow transplants or following dendritic cell immunization. To investigate the impact of PPARδ activation more closely, we stimulated naïve B6 T cells with splenocytes from a B6xDBA F1 donor in a mixed leukocyte reaction (MLR) with the PPARδ specific agonist, GW501516. T cells treated with GW501516 increased acetyl-Coenzyme A acyltransferase 2 and perilipin-2 levels in CD8 T cells 2 to 4-fold and 6 to 7-fold in CD4 T cells, respectively. Significant increases were also observed in carnitine palmitoyltransferase 1A (CPT1a) and carnitine-acylcarnitine translocase, both components of the carnitine shuttle pathway responsible for transporting long-chain fatty acids (LCFAs) across the mitochondrial matrix. Culturing agonist-treated cells with 3H-palmitate demonstrated an increase in 3H2O production that was inhibitable by etomoxir, confirming a GW501516-driven increase in FAO in allogenically-stimulated cells. Consistent with this data, the deletion of PPARδ in alloreactive donor cells impaired the ability of CD8 T cells to perform FAO post-transplant. Together, these data indicate that PPARδ drives FAO gene transcription and subsequent FAO engagement in allogenically-stimulated T cells.

Transplantation of PPARd KO T cells into allogeneic recipients modestly increased survival but did not fully ameliorate GVHD. This led us to hypothesize that compensatory metabolic pathways were being engaged in PPARδ KO T cells. To test this idea, wildtype (WT) and KO donor CD8 T cells were harvested from allogeneic recipient spleens on day 7 post-transplant, followed by targeted and untargeted metabolomic analyses on cell lysates. Targeted metabolite analysis revealed elevated levels of glutamine and succinate in PPARδ KO T cells, suggesting an uptick in glutamine metabolism in these cells. Furthermore, mass spectrometry discovered and validated two unique metabolites during untargeted analysis. O-phosphorylethanolamine levels were 0.64-fold lower, whereas pantothenic acid levels (vitamin B5) were 2.93-fold higher (p<0.01) in KO CD8 T cells. Pantothenic acid can be readily catabolized to replenish the cell’s coenzyme-A (CoA) pools, which influences T cell homeostasis and polarization, as well as being acetylated into acetyl-CoA to serve as a TCA fuel source. Thus, alloreactive CD8 donor T cells appear to adapt to loss of PPARδ through the upregulation of glutamine metabolism and an increased reliance on metabolid fuels provide through the panthothenate pathway. Future work will interrogate the vitamin B5/CoA axis in PPARδ KO alloreactive CD8 T and examine potential upstream influence by AMPK and mTOR, the two primary energy regulators of T cells, with the long-term goal of pinpointing therapeutic targets in reducing GVHD.