Curtis J. Perry

Hepatic Acetyl CoA Links Adipose Tissue Inflammation to Hepatic Insulin Resistance and Type 2 Diabetes

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulins ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.

Prostaglandin E2 and programmed cell death 1 signaling coordinately impair CTL function and survival during chronic viral infection

More than 10% of the worlds population is chronically infected with HIV, hepatitis C virus (HCV) or hepatitis B virus (HBV), all of which can cause severe disease and death. These viruses persist in part because continuous antigenic stimulation causes the deterioration of virus-specific cytotoxic T lymphocyte (CTL) function and survival. Additionally, antiviral CTLs autonomously suppress their responses to limit immunopathology by upregulating inhibitory receptors such as programmed cell death 1 (PD-1). Identification and blockade of the pathways that induce CTL dysfunction may facilitate the clearance of chronic viral infections. We found that the prostaglandin E2 (PGE2) receptors EP2 and EP4 were upregulated on virus-specific CTLs during chronic lymphocytic choriomeningitis virus (LCMV) infection and suppressed CTL survival and function. We show that the combined blockade of PGE2 and PD-1 signaling was therapeutic in terms of improving viral control and augmenting the numbers of functional virus-specific CTLs. Thus, PGE2 inhibition is both an independent candidate therapeutic target and a promising adjunct therapy to PD-1 blockade for the treatment of HIV and other chronic viral infections.

Chronic viral infection promotes sustained Th1-derived immunoregulatory IL-10 via BLIMP-1

During the course of many chronic viral infections, the antiviral T cell response becomes attenuated through a process that is regulated in part by the host. While elevated expression of the immunosuppressive cytokine IL-10 is involved in the suppression of viral-specific T cell responses, the relevant cellular sources of IL-10, as well as the pathways responsible for IL-10 induction, remain unclear. In this study, we traced IL-10 production over the course of chronic lymphocytic choriomeningitis virus (LCMV) infection in an IL-10 reporter mouse line. Using this model, we demonstrated that virus-specific T cells with reduced inflammatory function, particularly Th1 cells, display elevated and sustained IL-10 expression during chronic LCMV infection. Furthermore, ablation of IL-10 from the T cell compartment partially restored T cell function and reduced viral loads in LCMV-infected animals. We found that viral persistence is needed for sustained IL-10 production by Th1 cells and that the transcription factor BLIMP-1 is required for IL-10 expression by Th1 cells. Restimulation of Th1 cells from LCMV-infected mice promoted BLIMP-1 and subsequent IL-10 expression, suggesting that constant antigen exposure likely induces the BLIMP-1/IL-10 pathway during chronic viral infection. Together, these data indicate that effector T cells self-limit their responsiveness during persistent viral infection via an IL-10-dependent negative feedback loop.

CCR7 expression alters memory CD8 T-cell homeostasis by regulating occupancy in IL-7- and IL-15-dependent niches.

Significance IL-7 and IL-15 have been shown to play crucial roles in maintaining the population of memory T cells, which are important in fighting against secondary infections, but how effector and memory T cells receive these cytokines is incompletely understood. We investigated the role of C-C receptor 7 (CCR7) in the development of memory T cells using CCR7-deficient mice. CCR7-deficient memory T cells showed an abnormal migratory pattern and increased survival, especially in the lungs and bone marrow, in an IL-15–dependent fashion compared with wild type memory cells. We conclude that the localization of memory T cells is important to “see” these cytokines for homeostasis. C-C receptor 7 (CCR7) is important to allow T cells and dendritic cells to migrate toward CCL19- and CCL21-producing cells in the T-cell zone of the spleen and lymph nodes. The role of this chemokine receptor in regulating the homeostasis of effector and memory T cells during acute viral infection is poorly defined, however. In this study, we show that CCR7 expression alters memory CD8 T-cell homeostasis following lymphocytic choriomeningitis virus infection. Greater numbers of CCR7-deficient memory T cells were formed and maintained compared with CCR7-sufficient memory T cells, especially in the lung and bone marrow. The CCR7-deficient memory T cells also displayed enhanced rates of homeostatic turnover, which may stem from increased exposure to IL-15 as a consequence of reduced exposure to IL-7, because removal of IL-15, but not of IL-7, normalized the numbers of CCR7-sufficient and CCR7-deficient memory CD8 T cells. This result suggests that IL-15 is the predominant cytokine supporting augmentation of the CCR7−/− memory CD8 T-cell pool. Taken together, these data suggest that CCR7 biases memory CD8 T cells toward IL-7–dependent niches over IL-15–dependent niches, which provides insight into the homeostatic regulation of different memory T-cell subsets.

Myeloid-targeted immunotherapies act in synergy to induce inflammation and antitumor immunity

Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-&agr;, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-&agr; and IFN-&ggr;. Together, this study demonstrates the potential for targeting TAMs to convert a “cold” into an “inflamed” tumor microenvironment capable of eliciting protective T cell responses.

Uv-Induced Somatic Mutations Elicit A Functional T Cell Response In The Yummer1.7 Mouse Melanoma Model

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single‐cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1−/− mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti‐CTLA‐4 and anti‐PD‐1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses.

IL-7 plays a critical role for the homeostasis of allergen-specific memory CD4 T cells in the lung and airways

Memory T cells respond rapidly to repeated antigen exposure and can maintain their population for extended periods through self-renewal. These characteristics of memory T cells have mainly been studied during viral infections, whereas their existence and functions in allergic diseases have been studied incompletely. Since allergic patients can suffer repeated relapses caused by intermittent allergen exposure, we hypothesized that allergen- specific memory Th2 cells are present and the factors necessary for the maintenance of these cells are provided by the lung and airways. Using a murine model of airway inflammation, we found that allergen-specific CD4 T cells survived longer than 70 days in the lung and airways in an IL-7 dependent fashion. These T cells showing homeostatic proliferation were largely found in the mediastinal lymph node (mLN), rather than the airways; however, cells residing in the lung and airways developed recall responses successfully. We also found that CD4 T cells exhibited differential phenotypes in the mLN and in the lung. Altogether, we believe that allergen-specific memory T cells reside and function in the lung and airways, while their numbers are replenished through homeostatic turnover in the mLNs. Furthermore, we determined that IL-7 signaling is important for the homeostasis of these cells.

Tumor regression mediated by oncogene withdrawal or erlotinib stimulates infiltration of inflammatory immune cells in EGFR mutant lung tumors

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells. Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment. We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. Erlotinib treatment also led to increased maturation of myeloid cells and an increase in CD40+ dendritic cells. Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlights potential avenues for investigation of targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer.

Abstract A083: Modulation of the immune system as a strategy to treat EGFR mutant lung adenocarcinoma

Fifteen percent of lung adenocarcinomas harbor mutations in the Epidermal Growth Factor Receptor (EGFR). Although EGFR mutant tumors show sensitivity to specific tyrosine kinase inhibitors (TKIs) like erlotinib, resistance develops after an average of 12 months and there are currently no approved therapeutic strategies to overcome resistance. Therefore, novel approaches to delay resistance and improve patient survival are crucial. Recent discoveries in tumor immunology have revealed the potential of immune-based therapies to treat lung malignancies. In particular, immune checkpoint inhibitors such as anti-PD-1 have shown efficacy in a subset of patients with metastatic lung adenocarcinoma with minimal toxicity. These promising data led us to investigate the benefit of immune modulatory agents in EGFR mutant lung cancer. As a first step, we set out to explore the role of the immune system in this lung adenocarcinoma subset using a tetracycline-inducible mouse model of EGFR mutant lung cancer. We found, in these models, that lung-specific expression of mutant EGFR and consequent tumor formation leads to the establishment of an immunosuppressive tumor microenvironment characterized by an overall decrease in CD4 T cells, infiltration of Tregs, up-regulation of PD-1 expression on both helper and cytotoxic T cells and a diminished cytokine production by these T cells. Additionally, by flow cytometry and quantitative PCR of FACS-sorted cells, we have observed increased expression of PD-L1 on alveolar macrophages, which represent over 60% of the CD45+ cells in these tumors, suggesting an engagement of the adaptive and innate arms of the immune system to mediate immunosuppression via the PD-1/PD-L1 axis. Furthermore, when the tumor suppressor gene p53 was deleted, we observed a marked decrease in tumor infiltrating CD8 T cells when compared to p53 wild-type tumors. Currently, we are evaluating the functional role of elements of the immune system in these models to delineate their effects on tumor progression and therapeutic response. Moreover, we are investigating whether the observed immunosuppression in these tumors can be altered by immunomodulatory agents such as the anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors alone or in combination with EGFR TKIs. Citation Format: Deborah Ayeni, Ping-Chih Ho, Curtis Perry, Susan Kaech, Katerina Politi. Modulation of the immune system as a strategy to treat EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A083.