Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism.

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Novel SIRPα Antibodies That Induce Single-Agent Phagocytosis of Tumor Cells while Preserving T Cells.

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Retinoic acid signaling acts as a rheostat to balance Treg function.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

Basis of the intrinsic flexibility of the Cε3 domain of IgE.

Allergic reactions are triggered by the interaction between IgE and its high-affinity receptor, FcεRI. Various studies have mapped the interaction surface between IgE and its cellular receptors to the third constant domain of IgE (Cε3). The isolated Cε3 domain has been shown to exist as a molten globule, and the domain retains significant flexibility within the context of the IgE protein. Here we have analyzed the structural basis of the intrinsic flexibility of this domain. We have compared the sequence of the Cε3 domain to the sequences of other members of the C1 subset of the immunoglobulin superfamily and observed that Cε3 has an unusually high electrostatic charge and an unusually low content of hydrophobic residues. Mutations restoring Cε3 to a more canonical sequence were introduced in an attempt to derive a more structured domain, and several mutants display decreased levels of disorder. Engineered domains of Cε3 with a range of structural rigidities could serve as important tools for the elucidation of the role of flexibility of the Cε3 domain in IgE's biological functions.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

Targeting PIM Kinase with PD1 Inhibition Improves Immunotherapeutic Antitumor T-cell Response.

PURPOSE: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor. EXPERIMENTAL DESIGN: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1-/-PIM2-/-PIM3-/-) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both. RESULTS: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination. CONCLUSIONS: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Similarities and Distinctions of Cancer and Immune Metabolism in Inflammation and Tumors.

It has been appreciated for nearly 100 years that cancer cells are metabolically distinct from resting tissues. More recently understood is that this metabolic phenotype is not unique to cancer cells but instead reflects characteristics of proliferating cells. Similar metabolic transitions also occur in the immune system as cells transition from resting state to stimulated effectors. A key finding in immune metabolism is that the metabolic programs of different cell subsets are distinctly associated with immunological function. Further, interruption of those metabolic pathways can shift immune cell fate to modulate immunity. These studies have identified numerous metabolic similarities between cancer and immune cells but also critical differences that may be exploited and that affect treatment of cancer and immunological diseases.

MYC and MCL1 Cooperatively Promote Chemotherapy-Resistant Breast Cancer Stem Cells via Regulation of Mitochondrial Oxidative Phosphorylation.

Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation in vivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC.

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.

Reduced Warburg effect in cancer cells undergoing autophagy: steady- state 1H-MRS and real-time hyperpolarized 13C-MRS studies.

Autophagy is a highly regulated, energy dependent cellular process where proteins, organelles and cytoplasm are sequestered in autophagosomes and digested to sustain cellular homeostasis. We hypothesized that during autophagy induced in cancer cells by i) starvation through serum and amino acid deprivation or ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism would be affected, reducing flux to lactate, and that this effect may be reversible. We probed metabolism during autophagy in colorectal HT29 and HCT116 Bax knock-out cells using hyperpolarized (13)C-magnetic resonance spectroscopy (MRS) and steady-state (1)H-MRS. 24 hr PI103-treatment or starvation caused significant reduction in the apparent forward rate constant (k(PL)) for pyruvate to lactate exchange compared with controls in HT29 (100 µM PI-103: 82%, p = 0.05) and HCT116 Bax-ko cells (10 µM PI-103: 53%, p = 0.05; 20 µM PI-103: 42%, p<0.0001; starvation: 52%, p<0.001), associated with reduced lactate excretion and intracellular lactate in all cases, and unchanged lactate dehydrogenase (LDH) activity and increased NAD+/NADH ratio following PI103 treatment or decreased LDH activity and unchanged NAD+/NADH ratio following starvation. After 48 hr recovery from PI103 treatment, k(PL) remained below control levels in HT29 cells (74%, p = 0.02), and increased above treated values, but remained below 24 hr vehicle-treated control levels in HCT116 Bax-ko cells (65%, p = 0.004) both were accompanied by sustained reduction in lactate excretion, recovery of NAD+/NADH ratio and intracellular lactate. Following recovery from starvation, k(PL) was significantly higher than 24 hr vehicle-treated controls (140%, p = 0.05), associated with increased LDH activity and total cellular NAD(H). Changes in k(PL) and cellular and excreted lactate provided measureable indicators of the major metabolic processes accompanying starvation- and drug-induced autophagy. The changes are reversible, returning towards and exceeding control values on cellular recovery, which potentially identifies resistance. k(PL) (hyperpolarized (13)C-MRS) and lactate ((1)H-MRS) provide useful biomarkers for the autophagic process, enabling non-invasive monitoring of the Warburg effect.