Acidity induces durable enhancement of T_reg cell suppressive functions for tumor immune evasion

The microenvironment within solid tumors often becomes acidic due to various factors associated with abnormal metabolism and cellular activities, including increased lactate production as a result of dysregulated tumor glycolysis. Recently, we have identified multiple tumor microenvironment (TME) factors that potentiate regulatory T (T_reg) cell function in evading anti-tumor immunosurveillance. Despite the strong correlation between lactate and acidity, the potential roles of acidity in intratumoral T_reg cell adaptation and underlying molecular mechanisms have gone largely unstudied. In this study, we demonstrate that acidity significantly enhances immunosuppressive functions of nT_reg cells, but not iT_reg cells, without altering the expression of either FoxP3 or the cell surface receptors CD25, CTLA4, or GITR in these cells. Surprisingly, the addition of lactate, often considered a major contributor to increased acidity of the TME, completely abolished the acidity-induced enhancement of nT_reg suppressive functions. Consistently, metabolic flux analyses showed elevated basal mitochondrial respiratory capacity and ATP-coupled respiration in acidity-treated nT_reg cells without altering glycolytic capacity. Genome-wide transcriptome and metabolomics analyses revealed alterations in multiple metabolic pathways, particularly the one-carbon folate metabolism pathway, with reduced SAM, folate, and glutathione, in nT_reg cells exposed to low pH conditions. Addition of a one-carbon metabolic contributor, formate, diminished the acidity-induced enhancement in nT_reg cell suppressive functions, but neither SAM nor glutathione could reverse the phenotype. Remarkably, in vitro transient treatment of nT_reg cells resulted in sustained enhancement of their functions, as evidenced by more vigorous tumor growth observed in mice adoptively receiving acidity-treated nT_reg cells. Further analysis of intratumoral infiltrated T cells confirmed a significant reduction in CD8+ T cell frequency and their granzyme B production. In summary, our study elucidates how acidity-mediated metabolic reprogramming leads to sustained Treg-mediated tumor immune evasion.