Abstract IA20: Sugar high: Glycolytic flux wrecks stem-cell like T cells.

Adoptive cell transfer (ACT) – the ex vivo expansion and re-infusion of antigen (Ag)-specific T cells – represents a highly effective and potentially curative systemic therapy for patients with advanced solid and hematologic cancers. Patients that fail to obtain objective clinical responses receive T cells that have shorter telomeres and that express lower levels of CD27, indications that ineffective T cells are ‘senescent’ or ‘exhausted.’ Patient responses are correlated with the persistence of anti-tumor T cells, but very little are known in mouse or in man about the metabolism the fuels the complete and durable destruction of tumor masses. Preclinical models using human and mouse T cells have begun to reveal the qualities of highly effective tumor-specific T cells. CD8+ T cells have been described as being naive (TN) or one of four antigen-experienced subtypes representing a continuum of differentiation and maturation: stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminally differentiated effector T cells (TEFF). ACT of less differentiated TN, TSCM and TCM subsets have consistently demonstrated superior in vivo expansion, persistence, and antitumor capacities relative to the more differentiated TEM and TEFF cells, consistent with retrospective analyses from human ACT trials. Studies of the dynamics of the metabolism of anti-tumor T cells started with the analysis of key metabolites in naive T cells, which rely on the oxidation of fatty acids and amino acids as a primary source of energy. Following antigen encounter, naive CD8+ T cells shifted to a glycolytic metabolism to sustain the rapid proliferation and biosynthetic demands of effector T cell functions. However, it remained unclear whether these changes in glucose metabolism ultimately influence effector versus memory T cell fate or the tumor destructive capacity of T cells. Using a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells, we found that CD8+ T cells exhibiting limited glucose incorporation had a gene expression profile characteristic of memory precursors expressing genes like Bcl6, Tcf7 and Lef1 and an increased capacity to persist after transfer. All of these qualities are associated with the stem cell-like qualities of self-renewal and multipotency. T cells that took up high amounts of labeled glucose, reflecting a high dependence on glycolytic pathways of energy production, expressed high levels of Prdm1, Gzmb and Prf1 and were destined to be short-lived. Consistent with these observations, activation of CD8+ T cells in the presence of a glycolytic inhibitor, 2-deoxyglucose (2DG) enhanced the generation of memory cells as assessed by gene expression, phenotype and function. Furthermore, 2DG-treated T cells established long-term memory in both lymphoid and non-lymphoid organs and mediated potent regression of large, vascularized tumors. Importantly, enforcing glycolytic flux by ectopically over-expressing the glycolytic enzyme PGAM-1 severely impaired the ability of CD8+ T cells to form long-term memory. These findings indicate that glycolytic flux limits the generation of memory CD8+ T cells and provides novel pharmacological and genetic approaches to enhance T-cell memory and improve T cell-based immunotherapy by targeting glucose dependent metabolism. It is critical to understand the metabolic requirements of anti-tumor T cells, which themselves exist in a microenvironment where tumor cells are also avidly competing for glucose and glutamine to fuel their own ‘Warburg’ metabolism which is dependent on glycolysis. Pharmacologic and genetic control of critical metabolic pathways may represent an important aspect of future tumor immunotherapies. Additional Reading: 1. NP Restifo, ME Dudley and SA Rosenberg. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol. Mar 22;12(4):269-81, 2012. 2. A Wang, S Chandran, SA Shah, Y Chiu, BC Paria, T Aghamolla, MM Alvarez-Downing, CC Lee, S Singh, T Li, ME Dudley, NP Restifo, SA Rosenberg and US Kammula. The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans. Sci Transl Med. Aug 29;4(149):149ra120, 2012. 3. L Gattinoni, CA Klebanoff and NP Restifo, Paths to stemness: building the ultimate anti-tumour T cell. Nat Rev Cancer. Oct;12(10):671-84, 2012. Citation Format: Nicholas P. Restifo. Sugar high: Glycolytic flux wrecks stem-cell like T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA20.