Abstract Establishment of an immunosuppressive microenvironment is one mechanism of tumor immune escape. Elevated levels of 5’-deoxy-5’-methylthioadenosine (MTA) were detected in various tumor entities and spurred interest as putative immuno-inhibitory metabolic dysregulation. Loss of MTA phosphorylase (MTAP) activity in tumor cells was identified as major cause of MTA accumulation. Lack of MTAP expression is associated with an inferior response towards adjuvant interferon-α therapy and a higher risk for metastatic disease in malignant melanoma. Here we examined the role of tumor-secreted MTA in suppression of anti-tumor T cell responses. The effect of MTA was investigated in human polyclonal or antigen-specific T cells. We evaluated proliferation, viability, activation, differentiation, clonal expansion and effector function of the T cells as well as the phosphorylation of major signaling pathways. In addition, co-culture experiments of T cells with an MTA-secreting tumor cell line were performed. Finally, we used retroviral transduction to generate MTAP-overexpressing T cells as a strategy to overcome MTA-mediated inhibitory effects. MTA strongly reduced proliferation, expression of activation markers, viability, induction and expansion as well as differentiation and effector functions of antigen-specific T cells in a dose dependent manner. Moreover, MTA-mediated suppression could be confirmed using an MTA-secreting tumor cell line, which impaired T cell proliferation in mixed lymphocyte-tumor cell cultures. Mechanistically, we found MTA to interfere with different T cell signaling pathways, with the most noteworthy influence on AKT phosphorylation, thereby explaining the reduction in proliferation and viability. Moreover, we found that MTA also influences intracellular protein methylation, also a critical factor for proper T cell function. To address these broad inhibitory functions of MTA as tumor metabolite we aimed to enhance T cell resistance to MTA by equipping T cells with higher levels of the MTA catabolizing enzyme MTAP. We thus successfully generated stable MTAP-overexpressing T cells, which revealed less sensitivity against MTA mediated effects such as inhibition of proliferation, cytotoxicity, or viability upon co-culture with MTA when compared to mock controls. These results suggest a promising approach to reconstitute T cell function in presence of a normally inhibitory tumor microenvironment. Our data emphasizes the importance of tumor metabolites such as MTA in the tumor microenvironment for tumor immune escape. Furthermore, we were able to identify potential molecular mechanisms for MTA induced T cell inhibition, which offer the opportunity to study pharmacological approaches to overcome tumor induced immune inhibition in T cells and will help to develop more effective immune-based therapies against MTAP-deficient tumors. Citation Format: Carolin Strobl, Frederik Henrich, Marina Kreutz, Anja-Kathrin Bosserhoff, Andreas Mackensen, Michael Aigner. 5’-deoxy-5’-methylthioadenosine (MTA) impairs human T-cell functions and constitutes a novel immuno-suppressing tumor metabolite. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5118.