Myoglobin expression boosts T-cell metabolism and antitumor effector function

The tumor microenvironment is frequently hypoxic and characterized by a scarcity of nutritional resources, including limited glucose availability. As effector T cells have high energy demands, tumor metabolism can contribute to T-cell dysfunction and exhaustion. In this study, we assessed hypoxia in spleen and tumor tissue from tumor-bearing C57BL/6J mice using RT-PCR, histology, and flow cytometry. Next, CD8⁺ T cells isolated from C57BL/6J or P14⁺ mice were transduced with a Thy1.1 (control) or Thy1.1-myoglobin (Mb) packaging retrovirus, and Mb expression was confirmed by RT-PCR and Western blot. The metabolism of these cells was analysed using flow cytometry, TEM, FIB-SEM, Seahorse assays, metabolomics, and luminescence-based approaches. Furthermore, effector function was measured in vitro by flow cytometry. P14⁺ CD45.1⁺ CD8⁺ Mb-transduced and control T cells were transferred into B16F10-gp33 or MC38-ova tumor-bearing mice and analysed by flow cytometry and histology. Finally, B16F10-gp33 tumor-bearing mice received additional treatment with an anti-PD-1 checkpoint inhibitor. Here, we demonstrate that expression of the oxygen-binding protein myoglobin in T cells can enhance their mitochondrial and glycolytic metabolic functions. Metabolites and TCA cycle intermediates were markedly increased in the presence of myoglobin and were associated with elevated ATP levels. Myoglobin-expressing T cells exhibited reduced HIF-1alpha expression after activation and during infiltration into the tumor microenvironment. Accordingly, myoglobin expression increased effector T-cell function against tumor cells in vitro, accompanied by reduced superoxide levels. Following adoptive transfer into tumor-bearing mice, myoglobin expression promoted greater infiltration into the tumor microenvironment. Although myoglobin-expressing T cells showed increased effector cytokine expression, PD-1 remained detectable and targetable by anti-PD-1 monoclonal antibodies, which-when combined with transfer of myoglobin-expressing T cells-achieved maximal efficacy in delaying tumor growth. Taken together, we show that expression of myoglobin in T cells enhances their metabolism, infiltration into tumor tissue, and effector function against cancer cells.