The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5 0 -deoxy-5 0 -methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTAcatabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAPcoding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.
Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.
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.
Metabolic changes of malignant cells lead to the secretion of tumor metabolites which contribute to the shaping of a favorable milieu for tumor immune escape facilitating cancer development and resistance to anti-tumor immunotherapy. Recently, disrupted methylthioadenosine metabolism drew interest as further putative immunoinhibitory metabolic dysregulation. A broad spectrum of tumor entities was reported to lack methylthioadenosine phosphorylase (MTAP) expression leading to elevated levels of its substrate 5’-deoxy-5’-methylthioadenosine (MTA). MTAP deficiency is reported to correlate with worse response to adjuvant interferon-alpha therapy and a higher risk for metastatic disease in malignant melanoma providing evidence for immunobiological relevance. We therefore examined the effect of the tumor metabolite MTA on human T cell responses. We could show that MTA has a strong inhibitory impact on human T cell function regarding proliferation, viability, activation, differentiation, tumor antigen-specific expansion and cytotoxicity. We therefore aimed to overexpress MTAP in human T cells by retroviral transduction as a putative strategy to overcome MTA-mediated inhibition of T cells. In addition, we investigated the effect of MTA on T cell metabolism and maturation of human monocyte derived mDC. We successfully generated stable MTAP-overexpressing primary human T cells. First studies were performed to examine the resistance to MTA after equipping the T cells with higher levels of its metabolizing enzyme MTAP. MTAPhigh T cells revealed less sensitivity against inhibitory effects of MTA on proliferation, viability or cytotoxicity compared to mock transduced controls suggesting a promising approach to strengthen the performance of T cells facing the MTA-rich milieu of MTAP-deficient tumors. Mechanistically, we found MTA to interfere with several signaling pathways. Of most importance, MTA impaired Akt phosphorylation as well as intracellular protein methylation, both crucial processes for proper T cell function. In addition, we studied the combined effect of MTA and PRMT5 inhibition on T cells. Several metabolic studies focusing on glycolysis and fatty acid metabolism could confirm our findings that MTA keeps T cells in a rather inactive, naïve state. Finally, we found MTA to interfere with DC maturation and their potential to induce cytokine secretion of T cells. Our data emphasizes the crucial role of tumor metabolites such as MTA in the tumor milieu for tumor immune escape. Additionally, the identification of potential molecular mechanisms of MTA-induced immunosuppression offer the opportunity to pharmacologically tackle MTA-mediated immunoinhibition and will help to develop more effective immune-based therapies against MTAP-deficient tumors. Citation Format: Carolin D. Strobl, Frederik Henrich, Katrin Singer, Katrin Peter, Marina Kreutz, Anita Kremer, Andreas Mackensen, Michael Aigner. MTA-mediated inhibition of human T cells: Mechanism and MTAP overexpression as putative overcoming strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2017-3971
An immunosuppressive tumor microenvironment is considered one of the main problems in the treatment of different malignancies with immunotherapies like adoptive T cell transfer. Metabolites like lactate - which is produced by tumor cells even in the presence of sufficient oxygen ("Warburg-effect") - contribute to this setting. Another metabolic alteration found in many malignancies is a high level of 5‘-deoxy-5‘methylthioadenosine (MTA), due to a reduction of the catabolizing enzyme methylthioadenosine phosphorylase (MTAP). In case of malignant melanoma, MTAP-deficiency has been demonstrated to result in a higher invasive potential as well as in a worse response of melanoma cells to an interferon-α therapy. Additionally, studies showed an anti-inflammatory activity on macrophages and lymphocytes, emphasizing a tumor-promoting effect of MTA. We hypothesized that MTA has a direct suppressive effect on the induction of adaptive immunity. Indeed, polyclonally stimulated CD8+ and CD4+ T cells showed a strongly diminished proliferation in the presence of MTA (50μM). MTA pre-treated T cells remain in the G0 Phase of the cell cycle. Furthermore, in a co-culture-system of antigenic peptide-pulsed DCs with autologous CD8+ T cells, addition of 25μM MTA led to an inhibition of proliferation, activation and consequently to lower antigen-specificity. A potential toxic effect of MTA on resting T cells could be excluded as apoptosis was not observed until day seven of culture. In contrast, antigen-stimulated, activated CD8+ T cells that were incubated in co-culture with DCs in presence of 25μM MTA showed high apoptosis levels and decreasing cell numbers. Since MTA has been described as a protein methylation inhibitor, we conducted western blot analyses of polyclonally stimulated CD8+ T cells and found that presence of MTA lead to decreased protein methylation in CD8+ T cells. To prove the hypothesis that MTA exerts the effects on T cells by protein methylation inhibition, we compared MTA to another known inhibitor of protein methylation (adenosine-2,3-dialdehyde, AdOx) and found that AdOx as well inhibited antigen-specificity, proliferation and activation of T cells at comparable concentrations. In addition to direct effects of MTA on T cells, monocytes cultured in the presence of higher doses MTA (150μM) differentiated to DCs that showed a smaller phenotype as well as impaired expression of co-stimulatory and maturation markers when compared to DCs generated without MTA. MTA-DCs were still capable of activating T cells and inducing antigen-specificity in co-cultures, however, cytokine secretion of MTA-DCs stimulated CD8+ T cell was strongly impaired. In summary we provide in vitro evidence of a potential new immune evasion mechanism via secretion of MTA from tumor cells as well as an interesting new mechanism of non-toxic suppression of T cells via interfering with protein methylation. Citation Format: Frederik C. Henrich, Katrin Singer, Kerstin Förster-Poller, Katrin Peter, Bernadette Neueder, Dimitrios Mougiakakos, Marina Kreutz, Andreas Mackensen, Michael Aigner. Suppression of human adaptive immunity by the tumor metabolite 5′-deoxy-5′-methylthioadenosine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1898. doi:10.1158/1538-7445.AM2013-1898
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