Myoglobin expression boosts T-cell metabolism and antitumor effector function

Introduction/Rationale: The tumor microenvironment is often highly hypoxic and typically suffers from a marked lack of essential nutrients, including limited glucose availability. Because effector T cells have substantial energy requirements, the altered metabolic conditions created by the tumor can significantly contribute to impaired T-cell function and the development of T-cell exhaustion.

Methods: In this study, we assessed hypoxia in spleen and tumor tissue of tumor-bearing C57BL/6J mice using RT-PCR, histology, and flow cytometry. CD8⁺ T cells from C57BL/6J or P14⁺ mice were transduced with Thy1.1 (control) or Thy1.1–myoglobin (Mb) retrovirus, and Mb expression was verified by RT-PCR and western blot. Cellular metabolism was analyzed using flow cytometry, electron microscopy, Seahorse assays, metabolomics and luminescence-based methods. Mb-expressing or control P14⁺ or OT-I⁺ T cells were transferred into B16F10-gp33 or MC38-ova tumor-bearing mice and examined by flow cytometry and histology. Additionally, B16F10-gp33 tumor-bearing mice received anti–PD-1 treatment.

Results: We show that expression of the oxygen-binding protein myoglobin (Mb) in T cells enhances their mitochondrial and glycolytic metabolism, increasing metabolites, tricarboxylic acid cycle intermediates and ATP. Mb-expressing T cells showed reduced HIF-1α after activation and during tumor infiltration. Consequently, Mb boosted effector T-cell function against tumor cells in vitro while lowering superoxide. After adoptive transfer into tumor-bearing mice, Mb promoted greater T-cell infiltration into the tumor microenvironment. Although Mb-expressing T cells produced more effector cytokines, they still expressed PD-1 and responded to anti–PD-1 treatment, which combined with Mb-T-cell transfer most effectively delayed tumor growth.

Conclusion: Taken together, our findings show that Mb expression in T cells enhances their metabolic activity, promotes their infiltration into tumor tissue, and improves their effector function against cancer cells.