Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the and genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither nor knockout strongly reduced lactate secretion. In contrast, double knockout (-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, -DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates and compared with their WT counterparts. Under hypoxia (1% oxygen), however, suppression completely abolished growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both and need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by / genetic disruptions is responsible for the tumors' escape and growth.

No abstract

Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.

Heat-inactivation of sera is used to reduce possible disturbing effects of complement factors in cell-culture experiments, but it is controversially discussed whether this procedure is appropriate or could be neglected. Here, we report a strong impact of heat-inactivation of human sera on the activation and effector functions of human CD4+ T cells. While T cells cultured with native sera were characterized by a higher proliferation rate and higher expression of CD28, heat-inactivated sera shaped T cells towards on-blast formation, higher cytokine secretion (interferon γ, tumor necrosis factor, and interleukin-17), stronger CD69 and PD-1 expression, and increased metabolic activity. Heat-inactivated sera contained reduced amounts of complement factors and regulators like C1 inhibitor, but increased concentrations of circulating immune complexes. Substitution of C1 inhibitor reduced the beneficial effect of heat-inactivation in terms of cytokine release, whereas surface-molecule expression was affected by the addition of complex forming anti-C1q antibody. Our data clearly demonstrate a beneficial effect of heat-inactivation of human sera for T cell experiments but indicate that beside complement regulators and immune complexes other components might be relevant. Beyond that, this study further underpins the strong impact of the complement system on T cell function.