Prostaglandin E2 Alters the Differentiation and Function of Antigen-Specific T Cells By Targeting the Metabolic Gene Regulatory Network Downstream of mTORC1

Abstract Umbilical cord blood transplantation (UCBT) has extended the availability of hematopoietic stem cell transplantation to patients without compatible adult donors. Studies in zebrafish and mouse models have shown that the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (PGE2), increases HSC number and homing. In a human clinical trial of double UCBT, PGE2 decreased time to engraftment, promoted early T cell chimerism, favored generation of long-lived memory CD8+ cells and reduced the incidence of CMV viremia. To obtain mechanistic insight of how PGE2 affects the differentiation program of antigen-specific T cells, we used a well-established model of T effector (TEFF) vs. T memory (TM) differentiation of TCR-transgenic OTI T cells. OTI cells were stimulated with Ova257-264 plus APC with or without PGE2, followed by incubation with either IL-2 or IL-7, which are critical for the development of CD8+ TEFF and TM, respectively. Antigen-specific stimulation with or without PGE2 followed by IL-2 resulted in differentiation to CD44+CD62L- TEFF cells. In contrast, antigen-specific stimulation followed by IL-7 resulted in generation of CD44+CD62L+ central memory T cells. Under these conditions, PGE2 treatment gave rise to a phenotype of CD44-CD62L+Bcl2+Sca1+ cells, consistent with T stem cell memory. Two key pathways regulated by AMPK and mTOR have a decisive role on TM differentiation. AMPK can promote the generation of TM cells. However, we found that PGE2 inhibited AMPK activation indicating that differentiation of TM by PGE2 is not mediated by AMPK. Because the mTORC1-specific inhibitor rapamycin can promote the generation of TM, we focused our studies on the effects of PGE2 on mTORC1. Two classes of direct downstream targets of mTORC1 have been well characterized. mTOR phosphorylates the ribosomal protein S6 kinases (S6K1/2) and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP1/2), both of which control specific steps in the initiation of cap-dependent translation. mTORC1 activation can stimulate glycolysis as well as lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia inducible factor 1a (HIF1a) and sterol regulatory element-binding protein (SREBP1/2). HIF1a is regulated downstream of 4E-BP whereas SREBP is regulated downstream of S6K1. Although the mTORC1 inhibitor rapamycin can promote differentiation of TM and Treg cells, recent studies revealed that mTORC1, via its effects on lipid metabolism has a mandatory role on Treg differentiation. We found that PGE2 treatment during antigen-specific stimulation of OTI cells with Ova257-264 resulted in activation of mTOR as determined by phosphorylation of mTOR, 4E-BP and S6K and phosphorylation of Akt on the mTOR-specific site. Surprisingly, when antigen-specific stimulation was followed by IL-7, PGE2 treatment inhibited expression of the 4E-BP downstream targets Myc, HIF1a, the glycolysis genes Glut1, HK2, LDH-A, and the uptake of glucose. Phosphorylation of S6K was also impaired and lipid biosynthesis was suppressed as determined by decreased expression of fatty acid synthase FASN. In contrast, a metabolic program of lipid utilization was activated, characterized by increase of CPT1a, which promotes fatty acid transport in the mitochondria for b-oxidation, and the lipid oxidase Acox1. Mitochondria biogenesis analyzed by Mitotracker staining and expression of mitochondrial genes MTOC-1, TIMM50 and COX5a were also enhanced. In bioenergetics studies control-treated antigen-specific T cells had a glycolytic phenotype with elevated extracellular acidification rate (ECAR) whereas PGE2-treated cells had elevated oxygen consumption rate (OCR) and increased OCR/ECAR ratio, indicating preferential use of oxidative phosphorylation to generate energy. Thus, mTORC1 might regulate differentiation of antigen-specific T cells to TM by promoting lipid biosynthesis upon engaging distinct downstream targets in response to extracellular cues, thereby providing the required fuel for the bioenergetic demands of TM cells. Our studies reveal an unexpected mechanism by which PGE2 regulates the functional fate of T cells by modifying mTORC1 downstream signals and altering T cell metabolic imprints. These findings have implications for harnessing immune memory in the context of tumor-specific and pathogen-specific immunity. Disclosures No relevant conflicts of interest to declare.