Lactate regulation of activation in CD8+ T cells

CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in tissues can result in extended exposure of cytotoxic T cells to the metabolite lactate. Lactate can be immunosuppressive, at least in part due to its association with tissue acidosis. We show here that the lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and is capable of displacing glucose as a carbon source. Activation in the presence of a pH neutral form of lactate significantly alters the CD8+ T cell transcriptome, including the expression of key effector differentiation markers such as granzyme B and interferon-gamma. Our studies reveal the novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.

TAMI-47. MIR-542-3P CONTRIBUTES TO THE HK2-MEDIATED HIGH GLYCOLYTIC PHENOTYPE IN HUMAN GLIOMA CELLS

BACKGROUND The elevation of glucose metabolism is linked to high-grade gliomas such as glioblastoma multiforme (GBM). The high glycolytic phenotype is associated with cellular proliferation and resistance to treatment with chemotherapeutic agents in GBM. MicroRNA-542-3p (miR-542-3p) has been implicated in several tumors including gliomas. However, the role of miR-542-3p in glucose metabolism in human gliomas remains unclear. METHODS We measured the levels of cellular proliferation in human glioma cells. We measured the glycolytic activity in miR-542-3p knockdown and over-expressed human glioma cells. We measured the levels of miR-542-3p and HK2 in glioma tissues from patients with low- and high-grade gliomas using imaging analysis. RESULTS We show that knockdown of miR-542-3p significantly suppressed cellular proliferation in human glioma cells. Knockdown of miR-542-3p suppressed HK2-induced glycolytic activity in human glioma cells. Consistently, over-expression of miR-542-3p increased HK2-induced glycolytic activity in human glioma cells. The levels of miR-542-3p and HK2 were significantly elevated in glioma tissues of patients with high-grade gliomas relative to that in low-grade gliomas. The elevation of HK2 levels in patients with high-grade gliomas were positively correlated with the high levels of miR-542-3p in GBM and low-grade gliomas (LGG) based on the datasets from the Cancer Genome Atlas (TCGA) database. Moreover, the high levels of miR-542-3p were associated with poor survival rate in the TCGA database. CONCLUSIONS miR-542-3p contributes to the HK2-mediated high glycolytic phenotype in human glioma cells.

NIMG-41. PH-WEIGHTED MOLECULAR MRI AS AN EARLY BIOMARKER OF METABOLIC RESPONSE TO IDH INHIBITION IN IDH MUTANT GLIOMAS

The current study tested the hypotheses that (1) pH-weighted MRI measurements of tumor acidity reflect glycolytic activity in human gliomas, (2) that tumor acidity, and thus glycolytic activity, differs between human IDH mutant (mIDH) and wild type gliomas, and (3) that an increase in tumor acidity, suggestive of increased glycolytic activity, occurs after inhibition of mutant IDH enzyme activity and reduction in 2HG, an oncometabolite. To test these hypotheses, we employed a custom pH-weighted amine chemical exchange saturation transfer echoplanar (CEST-EPI) technique in 152 patients with IDH mutant or wildtype glioma prior to surgery and 11 patients before and after treatment with AG120 or AG881 enrolled at our institution in a phase 1 perioperative study in patients with recurrent, non-enhancing, IDH mutant low-grade gliomas (NCT03343197). Results from image-guided biopsies in more than 100 patients demonstrated a significant correlation between MTRasym at 3ppm, a measure of tumor acidity from pH-weighted amine CEST-EPI, and expression of key glycolytic proteins including GLUT3 (R2=0.2188,P=0.0105), HK2 (R2=0.1788,P=0.0314), LDHA (R2=0.1111,P=0.0071), and MCT1 (R2=0.1228,P=0.0039) as well as ex vivo extracellular flux analysis estimates of ATP consumption from glycolysis (R2=0.6684,P=0.0021). Data suggests a significantly lower acidity (MTRasym@3ppm) within non-enhancing tumor in IDH mutant gliomas when compared to IDH wild type gliomas (P< 0.0001). Patients in a phase 1 perioperative study showed a shift toward higher tumor acidity (i.e. higher glycolytic activity) following inhibition of IDH based on 2HG suppression in resected tumors, but at levels below that of IDH wild type gliomas. Levels of 2HG within the tumor after IDH inhibition were inversely correlated with post-treatment tumor acidity (R2=0.6342, P=0.0180). Overall, results suggest mIDH gliomas have low levels of glycolytic activity, and successful inhibition of the mutant IDH enzyme results in reduction in 2HG and a measurable metabolic shift toward elevated glycolysis as evidenced using pH-weighted molecular MRI.

[18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma

The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.

High-resolution positron emission microscopy of patient-derived tumor organoids

Tumor organoids offer new opportunities for translational cancer research, but unlike animal models, their broader use is hindered by the lack of clinically relevant imaging endpoints. Here, we present a positron-emission microscopy method for imaging clinical radiotracers in patient-derived tumor organoids with spatial resolution 100-fold better than clinical positron emission tomography (PET). Using this method, we quantify 18F-fluorodeoxyglucose influx to show that patient-derived tumor organoids recapitulate the glycolytic activity of the tumor of origin, and thus, could be used to predict therapeutic response in vitro. Similarly, we measure sodium-iodine symporter activity using 99mTc- pertechnetate and find that the iodine uptake pathway is functionally conserved in organoids derived from thyroid carcinomas. In conclusion, organoids can be imaged using clinical radiotracers, which opens new possibilities for identifying promising drug candidates and radiotracers, personalizing treatment regimens, and incorporating clinical imaging biomarkers in organoid-based co-clinical trials.

Elevated glycolysis imparts functional ability to CD8+ T cells in HIV infection

The mechanisms inducing exhaustion of HIV-specific CD8+ T cells are not fully understood. Metabolic programming directly influences T-cell differentiation, effector function, and memory. We evaluated metabolic profiles of ex vivo CD8+ T cells in HIV-infected individuals. The baseline oxygen consumption rate of CD8+ T cells was elevated in all infected individuals and CD8+ T cells were working at maximal respiratory capacity. The baseline glycolysis rate was enhanced only during early untreated HIV and in viral controllers, but glycolytic capacity was conserved at all stages of infection. CD8+ T-cell mTOR activity was found to be reduced. Enhanced glycolysis was crucial for HIV-specific killing of CD8+ T cells. CD8+ T-cell cytoplasmic GAPDH content was reduced in HIV, but less in early infection and viral controllers. Thus, CD8+ T-cell exhaustion in HIV is characterized by reduced glycolytic activity, enhanced OXPHOS demands, dysregulated mTOR, and reduced cytoplasmic GAPDH. These data provide potential metabolic strategies to reverse CD8+ T-cell dysfunction in HIV.

Trained Immunity Confers Prolonged Protection From Listeriosis

Trained immunity refers to the ability of the innate immune system exposed to a first challenge to provide an enhanced response to a secondary homologous or heterologous challenge. We reported that training induced with β-glucan one week before infection confers protection against a broad-spectrum of lethal bacterial infections. Whether this protection persists over time is unknown. To tackle this question, we analyzed the immune status and the response to Listeria monocytogenes (L. monocytogenes) of mice trained 9 weeks before analysis. The induction of trained immunity increased bone marrow myelopoiesis and blood counts of Ly6Chigh inflammatory monocytes and polymorphonuclear neutrophils (PMNs). Ex vivo, whole blood, PMNs and monocytes from trained mice produced increased levels of cytokines in response to microbial products and limited the growth of L. monocytogenes. In vivo, following challenge with L. monocytogenes, peripheral blood leukocytes were massively depleted in control mice but largely preserved in trained mice. PMNs were reduced also in the spleen from control mice, and increased in the spleen of trained mice. In transwell experiments, PMNs from trained mice showed increased spontaneous migration and CXCL2/MIP2α-induced chemotaxis, suggesting that training promotes the migration of PMNs in peripheral organs targeted by L. monocytogenes. Trained PMNs and monocytes had higher glycolytic activity and mitochondrial respiration than control cells when exposed to L. monocytogenes. Bacterial burden and dissemination in blood, spleen and liver as well as systemic cytokines and inflammation (multiplex bead assay and bioluminescence imaging) were reduced in trained mice. In full agreement with these results, mice trained 9 weeks before infection were powerfully protected from lethal listeriosis. Altogether, these data suggest that training increases the generation and the antimicrobial activity of PMNs and monocytes, which may confer prolonged protection from lethal bacterial infection.