Transcriptional and immunohistological assessment of immune infiltration in pancreatic cancer.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes

Activating the Nucleic Acid-Sensing Machinery for Anticancer Immunity.

Nucleic acid sensing pathways have likely evolved as part of a broad pathogen sensing strategy intended to discriminate infectious agents and initiate appropriate innate and adaptive controls. However, in the absence of infectious agents, nucleic acid sensing pathways have been shown to play positive and negative roles in regulating tumorigenesis, tumor progression and metastatic spread. Understanding the normal biology behind these pathways and how they are regulated in malignant cells and in the tumor immune environment can help us devise strategies to exploit nucleic acid sensing to manipulate anti-cancer immunity

Myeloid MyD88 restricts CD8+ T cell response to radiation therapy in pancreatic cancer

Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD

Myeloid MyD88 restricts CD8

Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD8+ T cell responses from vaccination, similar to observations in Myd88-/- mice. Lyz2-specific loss of Myd88 in myeloid populations rendered tumors more susceptible to radiation therapy and elicited normal CD8+ T cell responses to vaccination. scRNAseq in Lyz2-Cre/Myd88fl/fl mice revealed gene signatures in macrophages and monocytes indicative of enhanced type I and II interferon responses, and improved responses to RT were dependent on CD8+ T cells and IFNAR1. Together, these data implicate MyD88 signaling in myeloid cells as a critical source of immunosuppression that hinders adaptive immune tumor control following radiation therapy

α-1 Antitrypsin Inhibits Caspase-3 Activity, Preventing Lung Endothelial Cell Apoptosis

α-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema

Abstract 6410: Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors

The balance of innate signaling through adaptor proteins such as MyD88 and TRIF is critical in directing the pattern of inflammatory responses following exposure to endogenous adjuvants. While innate stimuli can cause inflammation that supports adaptive immunity, cancer myeloid cells can be pre-polarized such that the same inflammatory signals cause myeloid cells to suppress adaptive immune responses. We aim to investigate the signals regulating type I IFN secretion by innate immune cells in tumors in order to improve type I IFN secretion, activation of innate immune cells, direct macrophage polarization and improve CD8+ T cell mediated anti-tumor immunity. To better understand the role of MyD88-mediated signaling in driving response to innate adjuvants released following RT in solid tumors, we developed MyD88 conditional knockout mouse models. MyD88 was deleted specifically in CD11c expressing dendritic cells (DC), Lck expressing T cells, or Lyz2 expressing myeloid cells that include macrophages, monocytes and granulocytes. Mice bearing pancreatic tumors (Panc02-SIY or PK5L1940) were randomized to no treatment or 16 Gy CT-guided RT and followed for treatment responses. Lyz2-Cre/MyD88fl/fl mice demonstrated improved responses to RT in pancreatic tumors as compared to control MyD88fl/fl mice, or Lck-Cre mice. The improved responses in Lyz2-Cre/MyD88fl/fl mice were shown to be dependent on CD8+ T cells as well as on type I IFN signaling. To analyze mechanisms of response, tumors were digested into a single cell suspension and CD45+ cells were isolated for single cell sequencing three days post-RT. Lyz2-Cre/MyD88fl/fl mice showed diminished Th1 and Th2-type T cells and had a higher M1/TAM ratio compared to MyD88fl/fl mice. Loss of MyD88 in myeloid cells resulted in increased activation of IFN-dependent transcriptional responses in multiple immune cell populations in the tumor. To model responses ex vivo, bone marrow-derived macrophages (BMDMs) were activated with lipopolysaccharide (LPS) or a synthetic cyclic dinucleotide (CDA) for analysis of cytokine secretion in either monolayer or 3D spheroid culture conditions. BMDM derived from Lyz2-Cre/MyD88fl/fl cultured as a monolayer showed significantly altered secretion of TNF-α, IL-10 and IL-6 on activation with LPS as compared to controls. Preliminary data obtained from 3D spheroid culture systems demonstrated that 3D interactions between cancer cells and macrophages resulted in a significant increase in BMDM co-stimulatory phenotype following activation with innate stimuli compared to 2D cultures. These data suggest that conventional innate signaling through MyD88 in myeloid populations suppresses IFN production and adaptive immune control of irradiated tumors. 3D cultures are an effective tool to study cellular interactions ex vivo and screen novel interventions prior to in vivo translation