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In cancers with tumor-infiltrating lymphocytes (TILs), monoclonal antibodies (mAbs) that block immune checkpoints such as CTLA-4 and PD-1/PD-L1 promote antitumor T-cell immunity. Unfortunately, most cancers fail to respond to single-agent immunotherapies. T regulatory cells, myeloid derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) dampen antitumor immune responses by preventing T-cell infiltration and/or activation. Few studies have explored combinations of immune-checkpoint antibodies that target multiple suppressive cell populations within the TME, and fewer have studied the combinations of both agonist and antagonist mAbs on changes within the TME. Here, we test the hypothesis that combining a T-cell–inducing vaccine with both a PD-1 antagonist and CD40 agonist mAbs (triple therapy) will induce T-cell priming and TIL activation in mouse models of nonimmunogenic solid malignancies. In an orthotopic breast cancer model and both subcutaneous and metastatic pancreatic cancer mouse models, only triple therapy was able to eradicate most tumors. The survival benefit was accompanied by significant tumor infiltration of IFNγ-, Granzyme B-, and TNFα-secreting effector T cells. Further characterization of immune populations was carried out by high-dimensional flow-cytometric clustering analysis and visualized by t-distributed stochastic neighbor embedding (t-SNE). Triple therapy also resulted in increased infiltration of dendritic cells, maturation of antigen-presenting cells, and a significant decrease in granulocytic MDSCs. These studies reveal that combination CD40 agonist and PD-1 antagonist mAbs reprogram immune resistant tumors in favor of antitumor immunity.

Supplementary Table S1, Supplementary Figures S1-7

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor–deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.

In cancers with tumor-infiltrating lymphocytes (TILs), monoclonal antibodies (mAbs) that block immune checkpoints such as CTLA-4 and PD-1/PD-L1 promote antitumor T-cell immunity. Unfortunately, most cancers fail to respond to single-agent immunotherapies. T regulatory cells, myeloid derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) dampen antitumor immune responses by preventing T-cell infiltration and/or activation. Few studies have explored combinations of immune-checkpoint antibodies that target multiple suppressive cell populations within the TME, and fewer have studied the combinations of both agonist and antagonist mAbs on changes within the TME. Here, we test the hypothesis that combining a T-cell–inducing vaccine with both a PD-1 antagonist and CD40 agonist mAbs (triple therapy) will induce T-cell priming and TIL activation in mouse models of nonimmunogenic solid malignancies. In an orthotopic breast cancer model and both subcutaneous and metastatic pancreatic cancer mouse models, only triple therapy was able to eradicate most tumors. The survival benefit was accompanied by significant tumor infiltration of IFNγ-, Granzyme B-, and TNFα-secreting effector T cells. Further characterization of immune populations was carried out by high-dimensional flow-cytometric clustering analysis and visualized by t-distributed stochastic neighbor embedding (t-SNE). Triple therapy also resulted in increased infiltration of dendritic cells, maturation of antigen-presenting cells, and a significant decrease in granulocytic MDSCs. These studies reveal that combination CD40 agonist and PD-1 antagonist mAbs reprogram immune resistant tumors in favor of antitumor immunity.

The prime function of the arterial smooth muscle cell (SMC) in adult individuals is to contract and relax, thereby regulating blood flow to target tissues. However, in several vascular diseases, arterial SMCs in the adult vessel undergo major changes in structure and function. For example, SMCs can take on properties that allow them to proliferate and migrate, to promote calcification, to undergo apoptosis, and to orchestrate other cell types to take on different properties. These altered SMCs are often viewed as vascular troublemakers. However, these responses can also be seen as an attempt of the SMC to mend a diseased blood vessel. The plasticity and diversity of SMC populations and the presence of SMC progenitor cells contribute to the growing complexity of SMC biology. In the past couple of years, several new mechanisms that allow SMCs to proliferate, migrate, promote calcification, and communicate with and respond to other cell types have been discovered. Much of this work has been published in ATVB , and some of that body of work is highlighted in this article. We emphasize areas that have generated substantial interest, including novel mechanisms governing SMC phenotypic switching, SMC progenitor cells, noncoding RNAs as regulators of SMC function and phenotype, novel mechanisms of cross talk between SMCs and other cells and differences in regulation of SMCs and other vascular cells. The arterial SMC exhibits an impressive potential for plasticity. SMCs have been known to exist in different phenotypic states for decades, and the switch from a quiescent contractile phenotype to a synthetic proliferative phenotype is thought to play an important role in cardiovascular disease (for a historical perspective1). Thus, switching to a synthetic phenotype is generally thought to enable SMCs to migrate and proliferate in atherosclerotic lesions,2 thereby forming a fibrous cap covering a core of …

Summary: Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets. : Diabetes is associated with cardiovascular complications due to atherosclerosis, but the role of elevated glucose in this process is still uncertain. Nishizawa et al. now test the long-standing hypothesis that increased glucose in myeloid cells, important contributors to atherosclerosis, stimulates inflammatory activation and atherosclerosis. Although the glucose transporter GLUT1 was required for inflammatory activation, findings in mice with myeloid-cell-specific GLUT1 overexpression suggest that if increased glucose flux promotes atherosclerosis, myeloid cells are not sufficient for this effect.

The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in β-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on β-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.

Dopamine-producing neurons fire with both basal level tonic patterns and phasic bursts. Varying affinities of the five dopamine receptors have led to a hypothesis that higher affinity receptors are primarily activated by basal level tonic dopamine, while lower affinity receptors may be tuned to be sensitive to higher levels caused by phasic bursts. Genetically modified mice provide a method to begin to probe this hypothesis. Here we discuss three mouse models. Dopamine-deficient mice were used to determine which behaviors require dopamine. These behaviors were then analyzed in mice lacking D1 receptors and in mice with reduced phasic dopamine release. Comparison of the latter two mouse models revealed a similar failure to learn about and respond normally to cues that indicate either a positive or negative outcome, giving support to the hypothesis that phasic dopamine release and the D1 receptor act in the same pathway. However, the D1 receptor likely has additional roles beyond those of phasic dopamine detection, because D1 receptor knockout mice have deficits in addition to what has been observed in mice with reduced phasic dopamine release.

A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Bibhav Poudel, Tara Robinson, Brian Christmas, Kayla Cruz, Skylar Woolman, Christine Rafie, Blake Scott, Valerie Wall, Todd Armstrong, Elizabeth Jaffee. Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4936.

More from mTOR Leigh syndrome is a rare, untreatable, inherited neurodegenerative disease in children that is caused by functional disruption of mitochondria, the cell's energy-producing organelles. Johnson et al. (p. 1524 , published online 14 November; see Perspective by Vafai and Mootha ) show that rapamycin, a drug used clinically as an immunosuppressant and for treatment of certain cancers, delayed the onset and progression of neurological symptoms in a mouse model of Leigh syndrome and significantly extended survival of the animals. Rapamycin inhibits the so-called “mTOR” signaling pathway, which is currently under intense study because it plays a contributory role in many common diseases.

Chronological aging has been studied extensively in laboratory yeast by culturing cells into stationary phase in synthetic complete medium with 2% glucose as the carbon source. During this process, acidification of the culture medium occurs due to secretion of organic acids, including acetic acid, which limits survival of yeast cells. Dietary restriction or buffering the medium to pH 6 prevents acidification and increases chronological life span. Here we set out to determine whether these effects are specific to laboratory-derived yeast by testing the chronological aging properties of the vineyard yeast strain RM11. Similar to the laboratory strain BY4743 and its haploid derivatives, RM11 and its haploid derivatives displayed increased chronological life span from dietary restriction, buffering the pH of the culture medium, or aging in rich medium. RM11 and BY4743 also displayed generally similar aging and growth characteristics when cultured in a variety of different carbon sources. These data support the idea that mechanisms of chronological aging are similar in both the laboratory and vineyard strains.