SUMMARY Dendritic cells (DCs) are required to initiate and sustain T cell-dependent anti-cancer immunity. However, tumors often evade immune control by crippling normal DC function. The endoplasmic reticulum (ER) stress response factor XBP1 promotes intrinsic tumor growth directly, but whether it also regulates the host anti-tumor immune response is not known. Here we show that constitutive activation of XBP1 in tumor-associated DCs (tDCs) drives ovarian cancer (OvCa) progression by blunting anti-tumor immunity. XBP1 activation, fueled by lipid peroxidation byproducts, induced a triglyceride biosynthetic program in tDCs leading to abnormal lipid accumulation and subsequent inhibition of tDC capacity to support anti-tumor T cells. Accordingly, DC-specific XBP1 deletion or selective nanoparticle-mediated XBP1 silencing in tDCs restored their immunostimulatory activity in situ and extended survival by evoking protective type 1 anti-tumor responses. Targeting the ER stress response should concomitantly inhibit tumor growth and enhance anti-cancer immunity, thus offering a unique approach to cancer immunotherapy.

Summary Restoration of anti-tumor immunity by blocking PD-L1 signaling using antibodies has proven to be beneficial in cancer therapy. Here we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T cell dependent manner. Together, these data demonstrate a small molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression.

Dendritic cells are transformed to become immunosuppressive during ovarian cancer progression.

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extra-mucosal locations by increasing systemic IL-6, which drives mobilization of myeloid derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening anti-tumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently up-regulated in TLR5-unresponsive tumor-bearing mice, but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, anti-tumor immunity and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

Tumors evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1 – 4 . However, it remains unclear how intratumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer (OvCa), an aggressive malignancy refractory to standard treatments and current immunotherapies 5 – 8 , induces Endoplasmic Reticulum (ER) stress and activation of the IRE1α-XBP1 arm of the Unfolded Protein Response (UPR) 9 , 10 in T cells to control their mitochondrial respiration and anti-tumor function. XBP1 upregulation in T cells isolated from human OvCa specimens was associated with decreased intratumoral T cell infiltration and reduced IFNG mRNA expression. Malignant ascites fluid obtained from OvCa patients inhibited glucose uptake and caused N -linked protein glycosylation defects in T cells, leading to IRE1α/XBP1-driven suppression of mitochondrial activity and IFN-γ production. Mechanistically, XBP1 induction limited the influx of glutamine necessary to sustain T cell mitochondrial respiration under glucose-deprived conditions by regulating the abundance of glutamine carriers. Restoring N -linked protein glycosylation, abrogating IRE1α-XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to OvCa ascites. XBP1-deficient T cells in the metastatic OvCa milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, OvCa-bearing mice lacking XBP1 selectively in T cells demonstrated superior anti-tumor immunity, delayed malignant progression and increased overall survival. Therefore, controlling ER stress or targeting IRE1α-XBP1 signaling may help restore T cell metabolic fitness and anti-tumor capacity in cancer hosts.

The role of estrogens in anti-tumor immunity remains poorly understood. Here we show that estrogen signaling accelerates the progression of different estrogen insensitive tumor models by contributing to deregulated myelopoiesis by both driving the mobilization of myeloid-derived suppressor cells (MDSCs) and enhancing their intrinsic immunosuppressive activity in vivo. Differences in tumor growth are dependent on blunted anti-tumor immunity and, correspondingly, disappear in immunodeficient hosts and upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Therefore, estrogen signaling is a crucial mechanism underlying pathological myelopoiesis in cancer. Our work suggests that new anti-estrogen drugs that have no agonistic effects may have benefits in a wide range of cancers, independently of the expression of estrogen receptors in tumor cells, and may synergize with immunotherapies to significantly extend survival.

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Modulating the activity of microRNAs (miRNAs) provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DCs) to selectively supplement the immunomostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RISC. Endogenous processing of synthetic miR-155 favored Ago2- and, to a lesser extent, Ago4-loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent anti-tumor responses that abrogated the progression of established ovarian cancers. Our results demonstrate both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using non-viral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design and a mechanistic rationale for the clinical testing of non-viral miRNA mimetics.