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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.

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.

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.