669 Lactate uptake through MCT11, a novel monocarboxylate transporter, enforces dysfunction in terminally exhausted T cells

BackgroundUpon infiltration into tumors, T cells experiencing persistent antigen stimulation progressively differentiate into a state of dysfunction, known as exhaustion. Exhausted T cells are characterized by the sustained upregulation of co-inhibitory molecules and reduced effector cytokine production. Additionally, exhausted T cells exist in a state of metabolic dysfunction in the tumor microenvironment (TME), due to disrupted mitochondrial biogenesis, hypoxia and lack of metabolites. Highly glycolytic tumor and stromal cells outcompete T cells for glucose, and secrete lactate into the TME, acidifying the extracellular space. Recent studies have shown lactate can be metabolized by tumor infiltrating Tregs and macrophages. We hypothesized that CD8+ tumor-infiltrating lymphocytes (TIL) may also take up lactate as an alternative carbon source to meet their metabolic demands.MethodsFor lactate uptake experiments, B16 melanoma single cell suspensions from B6 mice were loaded with the pH sensitive dye pHrodo, then pulsed with 5µM lactic acid. MCT11 KO OT-I T cells were generated via transfection of Slc16a11 sgRNA-Cas9 ribonucleoprotein complexes, and adoptively transferred into B16-OVA bearing mice.ResultsRNA sequencing and flow cytometry data from CD8+ T cell subsets in the TME revealed MCT11 (encoded by Slc16a11), a monocarboxylate transporter (MCT) only recently discovered, to be highly and uniquely expressed in terminally exhausted T cells (Tex). As lactate is an abundant monocarboxylate in tumors, we asked whether MCT11 supports lactate uptake into Tex cells. Antibody blockade of MCT11 resulted in reduced lactic acid uptake, but whether lactic acid promoted or inhibited effector function. Intriguingly, overexpression of MCT11 in OT-I T cells adoptively transferred into B16-OVA bearing mice resulted in accelerated exhaustion: increased co-inhibitory marker expression and decreased TNFa and IFN production. Conversely, knockdown of MCT11 in the same model resulted in decreased co-inhibitory marker expression and increased TNFa and IFN production. Further, MCT11 KO OT-I T cells used therapeutically had decreased tumor burden over mice treated with control OT-I T cells. As MCT11's uptake function was blocked with an antibody, we also used the antibody therapeutically, revealing that single-agent MCT11 antibody treatment reduced tumor burden and increased survival in B16 melanoma bearing mice.ConclusionsOur data support a model where exhausted CD8+ T cells upregulate MCT11, which renders them sensitive to toxic lactic acid in the TME. Our data suggest MCT11 could be deleted on therapeutic T cells or blocked using an antibody on endogenous T cells to render exhausted T cells impervious to lactic acid such and promote tumor eradication.

Basigin drives intracellular accumulation of l-lactate by harvesting protons and substrate anions

Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant.

Universal Glia to Neurone Lactate Transfer in the Nervous System: Physiological Functions and Pathological Consequences

Whilst it is universally accepted that the energy support of the brain is glucose, the form in which the glucose is taken up by neurones is the topic of intense debate. In the last few decades, the concept of lactate shuttling between glial elements and neural elements has emerged in which the glial cells glycolytically metabolise glucose/glycogen to lactate, which is shuttled to the neural elements via the extracellular fluid. The process occurs during periods of compromised glucose availability where glycogen stored in astrocytes provides lactate to the neurones, and is an integral part of the formation of learning and memory where the energy intensive process of learning requires neuronal lactate uptake provided by astrocytes. More recently sleep, myelination and motor end plate integrity have been shown to involve lactate shuttling. The sequential aspect of lactate production in the astrocyte followed by transport to the neurones is vulnerable to interruption and it is reported that such disparate pathological conditions as Alzheimer’s disease, amyotrophic lateral sclerosis, depression and schizophrenia show disrupted lactate signalling between glial cells and neurones.

Elucidating the Role and Regulation of a Lactate Permease as Lactate Transporter in Bacillus coagulans DSM1

ABSTRACT A key feature of Bacillus coagulans is its ability to produce l-lactate via homofermentative metabolism. A putative lactate permease-encoding gene (lutP) and the gene encoding its regulator (lutR) were identified in one operon in B. coagulans strains. LutP orthologs are highly conserved and located adjacent to the gene cluster related to lactate utilization in most lactate-utilizing microorganisms. However, no lactate utilization genes were found adjacent to lutP in all sequenced B. coagulans strains. The stand-alone presence of lutP in l-lactate producers indicates that it may have functions in lactate production. In this study, B. coagulans DSM1 was used as a representative strain, and the critical roles of LutP and its regulation were described. Transport property assays showed that LutP was essential for lactate uptake. Its regulator LutR directly interacted with the lutP-lutR intergenic region, and lutP transcription was activated by l-lactate via regulation by LutR. A biolayer interferometry assay further confirmed that LutR bound to an 11-bp inverted repeat in the intergenic region, and lutP transcription began when the binding of LutR to the lutP upstream sequence was inhibited. We conclusively showed that lutP encodes a functional lactate permease in B. coagulans. IMPORTANCE Lactate-utilizing strains require lactate permease (LutP) to transport lactate into cells. Bacillus coagulans LutP is a previously uncharacterized lactate permease with no lactate utilization genes situated either adjacent to or remotely from it. In this study, an active lactate permease in an l-lactate producer, B. coagulans DSM1, was identified. Lactate supplementation regulated the expression of lactate permease. This study presents physiological evidence of the presence of a lactate transporter in B. coagulans. Our findings indicate a potential target for the engineering of strains in order to improve their fermentation characteristics.

Recurrent glucose deprivation leads to the preferential use of lactate by neurons in the ventromedial hypothalamus

Increased GABAergic output in the ventromedial hypothalamus (VMH) contributes to counterregulatory failure in recurrently hypoglycemic (RH) rats, and lactate, an alternate fuel source in the brain, contributes to this phenomenon. The current study assessed whether recurring bouts of glucose deprivation enhanced neuronal lactate uptake and, if so, whether this influenced γ-aminobutyric acid (GABA) output and the counterregulatory responses. Glucose deprivation was induced using 5-thioglucose (5TG). Control rats received an infusion of artificial extracellular fluid. These groups were compared with RH animals. Subsequently, the rats underwent a hypoglycemic clamp with microdialysis. To test whether 5TG affected neuronal lactate utilization, a subgroup of 5TG-treated rats was microinjected with a lactate transporter inhibitor [cyano-4-hydroxycinnamate (4CIN)] just before the start of the clamp. Both RH and 5TG raised VMH GABA levels, and this was associated with impaired counterregulatory responses. 4CIN reduced VMH GABA levels and restored the hormone responses in the 5TG group. We then evaluated [14C]lactate uptake in hypothalamic neuronal cultures. Recurring exposure to low glucose increased monocarboxylate transporter-2 mRNA expression and augmented lactate uptake. Taken together, our data suggest that glucose deprivation, per se, enhances lactate utilization in hypothalamic neurons, and this may contribute to suppression of the counterregulatory responses to hypoglycemia.

MCT4 is a high affinity transporter capable of exporting lactate in high-lactate environments

MCT4 is an H+-coupled transporter expressed in metastatic cancer cells, macrophages, and other highly glycolytic cells, where it extrudes excess lactate generated by the Warburg phenomenon or by hypoxia. Intriguingly, its reported Kmfor lactate, obtained with pH-sensitive probes, is more than an order of magnitude higher than physiological lactate. Here we examined MCT4-rich MDA-MB-231 cells using the FRET sensor Laconic and found a median Kmfor lactate uptake of only 1.7 mM, while parallel estimation in the same cells with a pH probe gave a Kmof 27 mM. The median Kmof MCT4 for lactate was 0.7 mM in MCT4-expressing HEK293 cells and 1.2 mM in human macrophages, suggesting that high substrate affinity is a robust property of the transporter. Probed with the FRET sensor Pyronic, MCT4 showed a Kmfor pyruvate of only 4.2 mM in MDA-MB-231 cells, as opposed to > 150 mM reported previously. We conclude that prior estimates of MCT4 affinity based on pH probes were severely biased by the confounding action of pH regulatory mechanisms. Numerical simulation showed that MCT4, but not MCT1 or MCT2, endows cells with the capability of lactate extrusion in high lactate environments. The revised kinetic properties and novel transport assays may help in developing small-molecule MCT4 blockers for research and therapy.