Lineage-Specific Metabolic Properties and Vulnerabilities of T Cells in the Demyelinating Central Nervous System.

Multiple sclerosis (MS) is a disease that is characterized by immune-mediated destruction of CNS myelin. Current MS therapies aim to block peripheral immune cells from entering the CNS. Although these treatments limit new inflammatory activity in the CNS, no treatment effectively prevents long-term disease progression and disability accumulation in MS patients. One explanation for this paradox is that current therapies are ineffective at targeting immune responses already present in the CNS. To this end, we sought to understand the metabolic properties of T cells that mediate ongoing inflammation in the demyelinating CNS. Using experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a well-studied model of MS, we showed that the CD4+ and CD8+ T cells that invade the EAE CNS are highly glycolytic. Elevated glycolytic rates in T cells isolated from the EAE CNS correlate with upregulated expression of glycolytic machinery and is essential for inflammatory responses to myelin. Surprisingly, we found that an inhibitor of GAPDH, 3-bromopyruvic acid (3-BrPa), blocks IFN-γ, but not IL-17A, production in immune cells isolated from the EAE CNS. Indeed, in vitro studies confirmed that the production of IFN-γ by differentiated Th1 cells is more sensitive to 3-BrPa than is the production of IL-17A by Th17 cells. Finally, in transfer models of EAE, 3-BrPa robustly attenuates the encephalitogenic potential of EAE-driving immune cells. To our knowledge, these data are among the first to demonstrate the metabolic properties of T cells in the demyelinating CNS in vivo.

Modulation of PKM activity affects the differentiation of TH17 cells.

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-ß1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function.

Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells.

In the context of solid tumors, there is a positive correlation between the accumulation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) and favorable clinical outcomes. However, CD8+ TILs often exhibit a state of functional exhaustion, limiting their activity, and the underlying molecular basis of this dysfunction is not fully understood. Here, we show that TILs found in human and murine CD8+ melanomas are metabolically compromised with deficits in both glycolytic and oxidative metabolism. Although several studies have shown that tumors can outcompete T cells for glucose, thus limiting T cell metabolic activity, we report that a down-regulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+ TILs. Provision of pyruvate, a downstream product of ENOLASE 1, bypasses this inactivity and promotes both glycolysis and oxidative phosphorylation, resulting in improved effector function of CD8+ TILs. We found high expression of both enolase 1 mRNA and protein in CD8+ TILs, indicating that the enzymatic activity of ENOLASE 1 is regulated posttranslationally. These studies provide a critical insight into the biochemical basis of CD8+ TIL dysfunction.

Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma.

Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T cells with hyperpolarized mitochondria were also visible in the blood of ccRCC patients. Importantly, provision of pyruvate to bypass glycolytic defects or scavengers to neutralize mitochondrial ROS could partially restore TIL activation. Thus, strategies to improve metabolic function of ccRCC CD8 TIL may promote the immune response to ccRCC.

Effector lymphocyte-induced lymph node-like vasculature enables naive T-cell entry into tumours and enhanced anti-tumour immunity.

The presence of lymph node (LN)-like vasculature in tumours, characterized by expression of peripheral node addressin and chemokine CCL21, is correlated with T-cell infiltration and positive prognosis in breast cancer and melanoma patients. However, mechanisms controlling the development of LN-like vasculature and how it might contribute to a beneficial outcome for cancer patients are unknown. Here we demonstrate that LN-like vasculature is present in murine models of melanoma and lung carcinoma. It enables infiltration by naive T cells that significantly delay tumour outgrowth after intratumoral activation. Development of this vasculature is controlled by a mechanism involving effector CD8 T cells and NK cells that secrete LTα3 and IFNγ. LN-like vasculature is also associated with organized aggregates of B lymphocytes and gp38(+) fibroblasts, which resemble tertiary lymphoid organs that develop in models of chronic inflammation. These results establish LN-like vasculature as both a consequence of and key contributor to anti-tumour immunity.

Tyrosine kinase inhibition in leukemia induces an altered metabolic state sensitive to mitochondrial perturbations.

PURPOSE: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL(+) leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph(+) leukemia and AML upon TK inhibition. EXPERIMENTAL DESIGN: Ph(+) cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. RESULTS: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL(+) leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. CONCLUSIONS: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL(+) and FLT3(ITD) leukemias.