Aerobic glycolysis but not GLS1-dependent glutamine metabolism is critical for anti-tumor immunity and response to checkpoint inhibition.

Tumor cells undergo uncontrolled proliferation driven by enhanced anabolic metabolism including glycolysis and glutaminolysis. Targeting these pathways to inhibit cancer growth is a strategy for cancer treatment. Critically, however, tumor-responsive T cells share metabolic features with cancer cells, making them susceptible to these treatments as well. Here, we assess the impact on anti-tumor T cell immunity and T cell exhaustion by genetic ablation of lactate dehydrogenase A (LDHA) and glutaminase1 (GLS1), key enzymes in aerobic glycolysis and glutaminolysis. Loss of LDHA severely impairs expansion of T cells in response to tumors and chronic infection. In contrast, T cells lacking GLS1 can compensate for impaired glutaminolysis by engaging alternative pathways, including upregulation of asparagine synthetase, and thus efficiently respond to tumor challenge and chronic infection as well as immune checkpoint blockade. Targeting GLS1-dependent glutaminolysis, but not aerobic glycolysis, may therefore be a successful strategy in cancer treatment, particularly in combination with immunotherapy.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Transforming growth factor-ß-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection.

Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-ß repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.

Methio "mine"! Cancer cells steal methionine and impair CD8 T-cell function.

Tumor cells steal methionine from CD8 T cells. High expression of the methionine transporters SLC7A5 and SLC43A2 allows tumor cells to outcompete CD8 T cells for methionine uptake. Lower methionine concentrations in CD8 T cells lead to reduced levels of dimethylated H3K79, an active epigenetic mark, which in turn results in reduced STAT5 expression and activity.

Early precursor T cells establish and propagate T cell exhaustion in chronic infection.

CD8+ T cells responding to chronic infections or tumors acquire an 'exhausted' state associated with elevated expression of inhibitory receptors, including PD-1, and impaired cytokine production. Exhausted T cells are continuously replenished by T cells with precursor characteristics that self-renew and depend on the transcription factor TCF1; however, their developmental requirements are poorly understood. In the present study, we demonstrate that high antigen load promoted the differentiation of precursor T cells, which acquired hallmarks of exhaustion within days of infection, whereas early effector cells retained polyfunctional features. Early precursor T cells showed epigenetic imprinting characteristic of T cell receptor-dependent transcription factor binding and were restricted to the generation of cells displaying exhaustion characteristics. Transcription factors BACH2 and BATF were key regulators with opposing functions in the generation of early precursor T cells. Overall, we demonstrate that exhaustion manifests first in TCF1+ precursor T cells and is propagated subsequently to the pool of antigen-specific T cells.

Reference Genes for Expression Studies in Human CD8+ Naïve and Effector Memory T Cells under Resting and Activating Conditions.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells.

Tumor-derived TGF-ß inhibits mitochondrial respiration to suppress IFN-γ production by human CD4+ T cells.

Transforming growth factor-ß (TGF-ß) is produced by tumors, and increased amounts of this cytokine in the tumor microenvironment and serum are associated with poor patient survival. TGF-ß-mediated suppression of antitumor T cell responses contributes to tumor growth and survival. However, TGF-ß also has tumor-suppressive activity; thus, dissecting cell type-specific molecular effects may inform therapeutic strategies targeting this cytokine. Here, using human peripheral and tumor-associated lymphocytes, we investigated how tumor-derived TGF-ß suppresses a key antitumor function of CD4+ T cells, interferon-γ (IFN-γ) production. Suppression required the expression and phosphorylation of Smad proteins in the TGF-ß signaling pathway, but not their nuclear translocation, and depended on oxygen availability, suggesting a metabolic basis for these effects. Smad proteins were detected in the mitochondria of CD4+ T cells, where they were phosphorylated upon treatment with TGF-ß. Phosphorylated Smad proteins were also detected in the mitochondria of isolated tumor-associated lymphocytes. TGF-ß substantially impaired the ATP-coupled respiration of CD4+ T cells and specifically inhibited mitochondrial complex V (ATP synthase) activity. Last, inhibition of ATP synthase alone was sufficient to impair IFN-γ production by CD4+ T cells. These results, which have implications for human antitumor immunity, suggest that TGF-ß targets T cell metabolism directly, thus diminishing T cell function through metabolic paralysis.

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis.

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8+ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8+ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8+ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter-linked and important for CD8+ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL-2 production - as well as subsequent IL-2 dependent TNF, IFN-γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8+ T cells.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Human regulatory T cells lack the cyclophosphamide-extruding transporter ABCB1 and are more susceptible to cyclophosphamide-induced apoptosis.

ATP-binding cassette (ABC) transporters, including ABC-transporter B1 (ABCB1), extrude drugs, metabolites, and other compounds (such as mitotracker green (MTG)) from cells. Susceptibility of CD4(+) regulatory T (Treg) cells to the ABCB1-substrate cyclophosphamide (CPA) has been reported. Here, we characterized ABCB1 expression and function in human CD4(+) T-cell subsets. Naïve, central memory, and effector-memory CD4(+) T cells, but not Treg cells, effluxed MTG in an ABCB1-dependent manner. In line with this, ABCB1 mRNA and protein was expressed by nonregulatory CD4(+) T-cell subsets, but not Treg cells. In vitro, the ABCB1-substrate CPA was cytotoxic for Treg cells at a 100-fold lower dose than for nonregulatory counterparts, and, inversely, verapamil, an inhibitor of ABC transporters, increased CPA-toxicity in nonregulatory CD4(+) T cells but not Treg cells. Thus, Treg cells lack expression of ABCB1, rendering them selectively susceptible to CPA. Our findings provide mechanistic support for therapeutic strategies using CPA to boost anti-tumor immunity by selectively depleting Treg cells.

Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch.

Antigen-experienced memory T cells acquire effector function with innate-like kinetics; however, the metabolic requirements of these cells are unknown. Here we show that rapid interferon-γ (IFN-γ) production of effector memory (EM) CD8(+) T cells, activated through stimulation mediated by the T cell antigen receptor (TCR) and the costimulatory receptor CD28 or through cognate interactions, was linked to increased glycolytic flux. EM CD8(+) T cells exhibited more glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity at early time points, before proliferation commenced, than did naive cells activated under similar conditions. CD28 signaling via the serine-threonine kinase Akt and the metabolic-checkpoint kinase mTORC2 was needed to sustain TCR-mediated immediate-early glycolysis. Unlike glycolysis in proliferating cells, immediate-early glycolysis in memory CD8(+) T cells was rapamycin insensitive. Thus, CD8(+) memory T cells have an Akt-dependent 'imprinted' glycolytic potential that is required for efficient immediate-early IFN-γ recall responses.

Epstein-Barr virus negativity among individuals older than 60 years is associated with HLA-C and HLA-Bw4 variants and tonsillectomy.

Epstein-Barr virus (EBV) infects ≈ 95% of the adult population. The factors that confer protection in the remaining ≈ 5% remain unknown. In an exploratory study, we assessed immunogenetic factors and tonsillectomy in a cohort of 17 EBV-negative and 39 EBV-positive healthy individuals aged >60 years. Analyses of HLA genotypes revealed an association between EBV negativity and the presence of HLA-C-35T/T and/or HLA-Bw4 alleles. In addition, EBV-negative donors presented with a history of tonsillectomy more often than EBV-positive donors.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.