Abstract B49: Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells
Authors: Zhang Ying , Zhou Xiangyang , Kurupati Raj , Filisio Flavia , Hudaihed Abeer , Giles-Davis Wynetta , Ertl Hildegund CJ 
Published: October 2015

Abstract
The 5-year survival rate of late-stage melanoma patients is less than 5%. Adoptive immune transfer has demonstrated the potency of melanoma-associated antigen (MAA)-specific T cells in inducing clinical responses. Nevertheless, cancer vaccines against melanoma that aim to induce such T cells have largely been ineffective in patients with advanced disease. One major barrier of cancer vaccine efficacy is immunoinhibition within the tumor microenvironment (TME). Vaccine-induced MAA-specific tumor infiltrating T cells (TILs) commonly show signs of T cell exhaustion within TME by upregulating co-inhibitory molecules combined with loss of functions. This has traditionally been viewed as the result of chronic antigen stimulation. However, our data show that TILs directed to an antigen that is not expressed by the tumor also become exhausted, which led us to hypothesize that exhaustion of TILs is linked to metabolic stress within the TME.
Upon activation the energy production of T cells switches from oxidative phosphorylation (OXPHOS) to glycolysis, which requires enhanced uptake of glucose for ATP production. Glycolysis is also used by tumor cells, which may lead to glucose deprivation within the TME, forcing MAA-specific TILs to rely on OXPHOS. OXPHOS requires oxygen, which due to lack of blood supply can also be limiting within TME. TILs therefore face dual metabolic jeopardy, i.e., lack of glucose and oxygen, which may affect their functions and thereby impair the efficacy of cancer vaccines. Using a B16 mouse melanoma model and an adenoviral vector based polyepitope melanoma vaccine, we tested if vaccine-induced TILs experience metabolic stress within the TME and whether this contributes to T cell exhaustion. Our results indicate that tumors recruit vaccine-induced tyrosinase-related protein (Trp)-1-specific CD8+ T cells, which proliferate within TME, upregulate co-inhibitory markers and become functionally impaired. Activated CD8+ T cells directed to an unrelated antigen expressed by another vaccine were also recruited to the tumors; although they fail to proliferation they also increased co-inhibitors programmed death (PD)-1 and lymphocyte activation gene (LAG)-3 expression with downregulation of T-bet, a critical transcription factor regulating T cell effector functions. Comparing markers of mitochondrial functions and oxidative deficiencies between TILs and CD8+ T cells stimulated in vitro under conditions producing energy through OXPHOS or glycolysis and under normal or reduced oxygen levels, we showed that activated CD8+ TILs undergo oxidative stress evidenced by loss of mitochondrial membrane potential (MMP), increases of mitochondrial reactive oxygen species (MROS) and enhanced levels of hypoxia inducible factor 1 subunit α (HIF-1α) and its downstream target glucose transporter (Glut)1. This metabolic profile was largely mirrored by T cells activated in vitro under hypoxia. Furthermore, our data demonstrated links between the T cells metabolic stress and their exhaustion status. Enhanced expression of PD-1 was linked to lack of glucose as cells cultured in galactose significantly increased PD-1 compared to cells with access to glucose. Increased expression of LAG-3 was most pronounced on cells cultured under hypoxia or in medium with drug that stabilizes HIF-1α. Knocking down HIF-1α in activated CD8+ T cells cultured under hypoxia significantly reduced their LAG-3 expression and increased their T-bet levels, implying a direct impact of hypoxia-induced HIF-1α on the expression of co-inhibitor LAG-3 and overall exhaustion status of T cells.
In summary, our data indicate that within the TME metabolic stress due to lack of glucose and oxygen rather than sustained activation causes T cell exhaustion. These findings suggest that lifting the metabolic stress of MAA-specific TILs may improve the therapeutic efficacy of cancer vaccines.
Citation Format: Ying Zhang, Xiangyang Zhou, Raj Kurupati, Flavia Filisio, Abeer Hudaihed, Wynetta Giles-Davis, Hildegund CJ Ertl. Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B49.