Kelly L. Singel

<h3>Background</h3> We evaluated the overall and site-specific incidence of cancer in subjects with primary immunodeficiency diseases (PIDD) enrolled in the United States Immune Deficiency Network (USIDNET) registry compared with age-adjusted cancer incidence in the Surveillance, Epidemiology and End Results Program (SEER) database. <h3>Objective</h3> We hypothesized that subjects with PIDD would have an increased incidence of cancer due to impaired immune function. <h3>Methods</h3> Overall and site-specific cancer incidence rates were evaluated in subjects with PIDD (<i>n</i> = 3658) enrolled in the USIDNET registry from 2003 to 2015 and compared with age-adjusted incidence rates in the SEER database. <h3>Results</h3> We observed a 1.42-fold excess relative risk of cancer in subjects with PIDD compared with the age-adjusted SEER population (<i>P</i> < .001). Men with PIDD had a 1.91-fold excess relative risk of cancer compared with the age-adjusted male population (<i>P</i> < .001), while women with PIDD had similar overall cancer rates compared with the age-adjusted female population. Of the 4 most common malignancies in men and women in SEER (lung, colon, breast, and prostate cancers), we found no significant increase in these diagnoses in subjects with PIDD. Significant increases in lymphoma in both men (10-fold increase, <i>P</i> < .001) and women (8.34-fold increase, <i>P</i> < .001) with PIDD were observed. <h3>Conclusions</h3> Excess incidence of cancer occurred in subjects with PIDD. An excess of lymphoma in specific PIDD populations principally drove this increased incidence, while no increased risk of the most common solid tumor malignancies was observed. These data point to a restricted role of the immune system in protecting from specific cancers.

NADPH oxidase (NOX) isoforms together have multiple functions that are important for normal physiology and have been implicated in the pathogenesis of a broad range of diseases, including atherosclerosis, cancer and neurodegenerative diseases. The phagocyte NADPH oxidase (NOX2) is critical for antimicrobial host defence. Chronic granulomatous disease (CGD) is an inherited disorder of NOX2 characterized by severe life-threatening bacterial and fungal infections and by excessive inflammation, including Crohn's-like inflammatory bowel disease (IBD). NOX2 defends against microbes through the direct antimicrobial activity of reactive oxidants and through activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the breakdown of cell membranes and extracellular release of chromatin and neutrophil granular constituents that target extracellular pathogens. Although the immediate effects of oxidant generation and NETosis are predicted to be injurious, NOX2, in several contexts, limits inflammation and injury by modulation of key signalling pathways that affect neutrophil accumulation and clearance. NOX2 also plays a role in antigen presentation and regulation of adaptive immunity. Specific NOX2-activated pathways such as nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional factor that induces antioxidative and cytoprotective responses, may be important therapeutic targets for CGD and, more broadly, diseases associated with excessive inflammation and injury.

Neutrophils are the first responders to infection and injury and are critical for antimicrobial host defense. Through the generation of reactive oxidants, activation of granular constituents and neutrophil extracellular traps, neutrophils target microbes and prevent their dissemination. While these pathways are beneficial in the context of trauma and infection, their off-target effects in the context of tumor are variable. Tumor-derived factors have been shown to reprogram the marrow, skewing toward the expansion of myelopoiesis. This can result in stimulation of both neutrophilic leukocytosis and the release of immature granulocytic populations that accumulate in circulation and in the tumor microenvironment. While activated neutrophils have been shown to kill tumor cells, there is growing evidence for neutrophil activation driving tumor progression and metastasis through a number of pathways, including stimulation of thrombosis and angiogenesis, stromal remodeling, and impairment of T cell-dependent anti-tumor immunity. There is also growing appreciation of neutrophil heterogeneity in cancer, with distinct neutrophil populations promoting cancer control or progression. In addition to the effects of tumor on neutrophil responses, anti-neoplastic treatment, including surgery, chemotherapy, and growth factors, can influence neutrophil responses. Future directions for research are expected to result in more mechanistic knowledge of neutrophil biology in the tumor microenvironment that may be exploited as prognostic biomarkers and therapeutic targets.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate β-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.

Epithelial ovarian cancer (EOC) typically presents with advanced disease. Even with optimal debulking and response to adjuvant chemotherapy, the majority of patients will have disease relapse. We evaluated cytokine and chemokine profiles in ascites at primary surgery as biomarkers for progression-free survival (PFS) and overall survival (OS) in patients with advanced EOC.Retrospective analysis of patients (n =70) who underwent surgery at Roswell Park Cancer Institute between 2002 and 2012, followed by platinum-based chemotherapy.The mean age at diagnosis was 61.8 years, 85.3% had serous EOC, and 95.7% had stage IIIB, IIIC, or IV disease. Univariate analysis showed that ascites levels of tumor necrosis factor (TNF)-α were associated with reduced PFS after primary surgery. Although the ascites concentration of interleukin (IL)-6 was not by itself predictive of PFS, we found that stratifying patients by high TNF-α and high IL-6 levels identified a sub-group of patients at high risk for rapid disease relapse. This effect was largely independent of clinical prognostic variables.The combination of high TNF-α and high IL-6 ascites levels at primary surgery predicts worse PFS in patients with advanced EOC. These results suggest an interaction between ascites TNF-α and IL-6 in driving tumor progression and resistance to chemotherapy in advanced EOC, and raise the potential for pre-treatment ascites levels of these cytokines as prognostic biomarkers. This study involved a small sample of patients and was an exploratory analysis; therefore, findings require validation in a larger independent cohort.

Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes.Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE.The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02).In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

Objectives To investigate the impact of ascites volume on ovarian cancer outcomes. Methods Clinicopathologic features of a cohort of patients with ovarian cancer were obtained from a curated database at a single institution. Progression free survival (PFS) and overall survival (OS) were recorded. Ascites volume at primary surgery was dichotomized at 2000 mL and comparisons for high and low volume ascites were made. Additionally, to elucidate interactions between ascites and ovarian tumor progression, we evaluated the effect of intraperitoneal administrations of murine cell-free ascites versus saline in a syngeneic mouse model of epithelial ovarian cancer. Results Out of 685 patients identified, 58% had ascites present at the time of initial surgery. Considering the volume of ascites continuously, each liter of ascites was associated with shorter PFS (HR = 1.12, 95% CI: 1.07–1.17) and OS (HR = 1.12, 95%CI: 1.07–1.17). Patients with ascites greater than the median of 2000 mL had significantly shorter PFS (14.5 months vs. 22.7 months; p < 0.001) and OS (27.7 months vs. 42.9 months; p < 0.001). After adjusting for stage, presence of ascites was inversely associated with ability to achieve optimal cytoreductive surgery. Consistent with these correlative results in patients, intraperitoneal administrations of murine cell-free ascites accelerated ovarian cancer progression in mice. Conclusions The volume of ascites at initial diagnosis of ovarian cancer correlated with worse PFS and OS. The effect of large volume on prognosis is likely to be in part related to reduced likelihood for complete resection of tumor (R0). If these findings are confirmed in independent studies, consideration should be made to add the presence of large volume ascites at diagnosis to the staging criteria for ovarian cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells.

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1β and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages.

Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47 (phox-/-)) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47 (phox-/-) mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

Cancer immunotherapies have generated remarkable clinical responses for some patients with advanced/metastatic disease, prompting exploration of rational combination therapies to bolster anti-tumor immunity in patients with limited response or those who experience tumor progression following an initial response to immunotherapy. In contrast to other tumor indications, objective response rates to single-agent PD-1/PD-L1 blockade in ovarian cancer are limited, suggesting a need to identify combinatorial approaches that lead to tumor regression in a setting where checkpoint blockade alone is ineffective. Using a pre-clinical model of aggressive intraperitoneal ovarian cancer, we have previously reported on a heterologous prime/boost cancer vaccine that elicits robust anti-tumor immunity, prolongs survival of tumor-bearing mice, and which is further improved when combined with checkpoint blockade. As tumor control in this model is CD8 + T cell dependent, we reasoned that the prime/boost vaccine platform could be used to explore additional treatment combinations intended to bolster the effects of CD8 + T cells. Using whole tumor transcriptomic data, we identified candidate therapeutic targets anticipated to rationally combine with prime/boost vaccination. In the context of a highly effective cancer vaccine, CD27 agonism or antibody-mediated depletion of granulocytic cells each modestly increased tumor control following vaccination, with anti-PD-1 therapy further improving treatment efficacy. These findings support the use of immunotherapies with well-defined mechanisms(s) of action as a valuable platform for identifying candidate combination approaches for further therapeutic testing in ovarian cancer.

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8+ T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity.

Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites.Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival.Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups.These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

Objective: Standard therapy for metastatic EOC involves surgical debulking and chemotherapy. Surgery in metastatic disease is noncurative, and recurrence of disease is driven by residual tumor. However, little is known about the effect of surgery on residual tumor. Our objective is to evaluate acute changes induced by surgery on the residual tumor microenvironment in syngeneic murine epithelial ovarian cancer (EOC).

Abstract Ovarian cancer (OC) is often diagnosed at advanced stages and presents with ascites. Necrosis is a hallmark of advanced cancer and releases DAMPs that activate innate immune responses (e.g., neutrophils). While neutrophils are critical in host defense, their role in the context of tumor is less understood. CTLs correlate with better outcomes in advanced OC, and immunotherapy for cancer is based on augmentation of CTL responses. Our prior studies showed that granulocytes from ascites of patients with newly diagnosed OC suppressed donor T cell proliferation ex vivo. The current work probes mechanisms for this suppression. In ascites from patients with newly diagnosed OC, ~90% of cells were inflammatory (CD45+). Neutrophils comprised ~15% of CD45+ cells and the neutrophil:CD8+ T cell ratio was 1.5:1. In ex vivo studies, cell-free ascites (ASC; 500g) attracted normal donor neutrophils (NDN). While neither ASC nor NDN alone impaired CD3/CD28-stimulated normal donor T cell proliferation, ASC (10/15 patients) when combined with NDN (1:1) completely suppressed T cell proliferation. Suppression by ASC + NDN required cell contact and was lost if NDN were added after overnight CD3/CD28 stimulation of T cells. Finally, heat-inactivating ASC (56C, 1h) abrogated the ability of NDN to suppress T cell proliferation. Here we provide evidence that ascites recruits neutrophils to the tumor microenvironment and induces a suppressive phenotype of neutrophils that requires cell contact and heat-labile cell-free constituents. Ongoing studies aim to identify the putative protein(s) in ascites and the signaling effects on neutrophils. Our long-term goal is to develop novel approaches to overcome this suppression and improve durable anti-tumor immunity.

&lt;div&gt;Abstract&lt;p&gt;T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related Spotlight by Cassatella, p. 725.&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123&lt;sup&gt;high&lt;/sup&gt; expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related Spotlight by Cassatella, p. 725.&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123&lt;sup&gt;high&lt;/sup&gt; expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;CD33+CD11b+CD14-CD15- fraction of OC ascites cells contains variable proportions of HLA-DR+CD123mid dendritic cells.&lt;/p&gt;

&lt;p&gt;Anti-human mAb used in flow cytometry analysis and cell sorting for this study.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4: Structure, binding affinity and in vitro potency of FB inhibitor.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S1: Gating strategies for flow cytometry and Image-stream.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6: Evaluation of NFAT and NF-kB translocation, glucose uptake and phosphorylation of p70S6 kinase in CD4+ T cells.&lt;/p&gt;

&lt;p&gt;Supplementary Tables 1-2&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2: OC ascites fluid exposure increases neutrophil lifespan, increases the proportion of low-density neutrophils, increases LOX-1 expression, and induces neutrophil nuclear hypersegmentation&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3: OC ascites fluid activates the classical and alternative pathways.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5: Evaluation of neutrophil subpopulations after exposure to suppressor ASC with and without C3 inhibition or non-suppressor ASC.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5: Evaluation of neutrophil subpopulations after exposure to suppressor ASC with and without C3 inhibition or non-suppressor ASC.&lt;/p&gt;

&lt;p&gt;CD33+CD11b+CD14-CD15- fraction of OC ascites cells contains variable proportions of HLA-DR+CD123mid dendritic cells.&lt;/p&gt;

&lt;p&gt;Anti-human mAb used in flow cytometry analysis and cell sorting for this study.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3: OC ascites fluid activates the classical and alternative pathways.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2: OC ascites fluid exposure increases neutrophil lifespan, increases the proportion of low-density neutrophils, increases LOX-1 expression, and induces neutrophil nuclear hypersegmentation&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4: Structure, binding affinity and in vitro potency of FB inhibitor.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6: Evaluation of NFAT and NF-kB translocation, glucose uptake and phosphorylation of p70S6 kinase in CD4+ T cells.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S1: Gating strategies for flow cytometry and Image-stream.&lt;/p&gt;

&lt;p&gt;Supplementary Tables 1-2&lt;/p&gt;

1520 Background: We reported the overall and site-specific incidence of cancer in the United States Immune Deficiency Network (USIDNET) Registry.We hypothesized that patients with Primary Immunodeficiency Diseases will have an increased incidence of cancer due to impaired immune function. Methods: Data were abstracted from the USIDNET Registry patients (n = 3,665) from 2003 to 2015. Site-specific cancer incidence rates were generated for patients within the registry. Age adjusted incidence rates for were generated using the SEER database for comparison to the patients within the USIDNET registry. Results: We observed a 1.34-fold excess relative risk of cancer (p < .001) in patients with PIDD compared to age-adjusted SEER population. In men we found this excess relative risk to increase to 1.8-fold excess relative risk of cancer (p < .001), however in women the increase risk of cancer was not significant. When analyzing the four most common malignancies diagnosed in men and women in the United States (lung, colon, breast, and prostate cancers) we found no statistically significant increase in these diagnoses in the USIDNET Registry patients. We observed statistically significant increases in lymphoma in both men (10-fold excess relative risk p < .001) and women (8-fold excess relative risk p < .001) in the registry. In men within the registry we observed a statistically significant increase in skin cancer (4.45 fold excess relative risk p < .001) and thyroid cancer (4-fold excess relative risk p = .002), and in women we observed a statistically significant increase in skin (3.19 fold excess relative risk p < .001) and stomach cancer (3-fold excess relative risk p = .015). Conclusions: Excess incidence of cancer occurred in patients within the USIDNET Registry. This increased incidence was principally driven by an excess of lymphoma in specific PIDD populations, while no increased risk of the most common solid tumor malignancies was observed. These data point to a restricted role of the immune system in protecting from specific cancers and question the role of immunosurveillance in incident risk of common solid tumor cancers.

Abstract CD8 T cell activation prompts extensive transcriptome remodeling underlying effector differentiation and function. Regulation of transcriptome composition by the mitogen-inducible nuclear cap-binding complex (CBC) adaptor protein ARS2 has critical cell type-specific consequences, including thymic T cell survival. Here we show that ARS2 was upregulated by CD28 during activation of peripheral T cells, was essential for anti-tumor immunity, and facilitated T cell activation-induced alternative splicing. The novel splicing function of ARS2 was mediated at least in part by recruitment of splicing factors to nascent transcripts including the M2 isoform of pyruvate kinase ( Pkm2 ), a key determinant of CD8 T cell effector properties. Notably, ARS2-directed Pkm2 splicing occurred days after stimulation of PI3K-indepdendent CD28 signaling and increased glycolysis beyond levels determined by PI3K signaling during T cell priming. Thus, ARS2-directed Pkm2 splicing represents a mechanism by which CD28 drives glycolytic metabolism, allowing for optimal effector cytokine production and T cell anti-tumor immunity.

Objectives: Ovarian cancer vaccine development is hampered by the availability of powerful immune adjuvants that can induce sustained innate immunity and augment adaptive cellular responses in the tumor-bearing host. Accumulation of myeloid cells in the tumor microenvironment is a known obstacle to durable antitumor immunity. We evaluated the strategy of vaccination with the novel TLR9/NOD2 ligand (MIS-416) followed by myeloid cell depletion in the murine model of epithelial ovarian cancer (EOC).

5545 Background: Mitochondrial damage-associated molecular patterns, comprised of mtDNA and formylated peptides, are released after cellular injury and activate innate immune responses. Extracellular mtDNA activates TLR-9 and other pathogen recognition receptors. While mitochondrial metabolism and DNA mutations have been widely studied in tumor cells, this study analyzes mtDNA at an extracellular level as a marker of injury. Since OC is associated with cellular necrosis, we reason that extracellular mtDNA may be a molecular prognostic marker. Methods: Serum collected in OC patients prior to primary surgery (n=41) and from age-matched cancer free women (n=3) was analyzed for mtDNA. Q-PCR with cytochrome B primers specific for mtDNA was done. The high group was pre-specified as patients with serum mtDNA > 5-fold above the mean control level, and the remainder was defined as the low group. Primary endpoint was PFS. Results: 41% had high levels of mtDNA. 76% in the high mtDNA and 74% in the low groups were Stage 3. Histology and grade were similar. The mean age at diagnosis was 69. Median PFS in the high and low groups was 228 vs. 489 days, respectively (p = 0.17). The high mtDNA group had quicker relapse (HR=2.7; p = 0.0278) and a reduced OS trend (HR=3.5; p = 0.0593) within the first year of surgery. Consistent with this effect, additional exploratory analysis showed that patients with serum mtDNA in the highest 15% had markedly reduced PFS (5 vs. 16 mo; p = 0.0015) and OS (8 vs. 50 mo; p = 0.00022) compared to the remaining patients. The relationship between pre-treatment serum mtDNA levels and PFS and OS involve a threshold effect rather than a direct linear relationship. Comparing pre-treatment serum mtDNA levels to published data on serum CA-125 levels from a Gynecology Oncology Group study that analyzed 1,299 patients with OC, we observed significantly greater effect sizes with serum mtDNA in predicting prognosis, with the greatest effect in the top 15% serum mtDNA group. Conclusions: Increased levels of circulating mtDNA correlated with significantly reduced PFS and OS. If validated in an independent cohort, pre-treatment serum mtDNA may be of value as a prognostic marker for OC.

e22087 Background: Mitochondrial damage-associated molecular patterns, made of mtDNA and formylated peptides, are released after cellular injury and recruit/activate neutrophils. NETs made of stretches of extracellular DNA, histones, and granular constituents, trap and kill invading pathogens, but also cause tissue injury. In mice, NETs bind tumor cells and accelerate invasion and metastasis. Mitochondrial extract and mtDNA activate NETs in human neutrophils. In addition, we observed that increased pretreatment mtDNA levels in serum and ascites significantly correlate with shorter PFS and OS in OC patients. Based on these findings, we aimed to evaluate: a) The effects of cell-free ascites from OC patients on normal donor neutrophils; b) Whether tumor-infiltrating neutrophils undergo NETosis. Methods: Purified neutrophils were cultured with media, mtDNA, or cell-free ascites from patients with OC collected at surgery. Arginase 1 (ARG1) expression in neutrophils (CD15+), which in tumor-bearing mice is associated with an immunosuppressive phenotype, was assessed by flow cytometry. NETs were identified by immunofluorescence microscopy showing co-localization of extracellular DNA, histone, and neutrophil elastase. In separate studies, we evaluated NETosis in fixed, paraffin-embedded cytokeratin-7 OC tumor, and NETs were identified by co-localization of the same immunofluorescence markers. Results: Cell-free ascites from different patients had variable effects on ARG1 expression, ranging from no effect to a reduction in ARG1 levels, while purified mtDNA decreased ARG1 expression. In addition, ascites and purified mtDNA induced NET generation in normal neutrophils. Finally, we observed NETs in resected OC tumor (n = 3). Conclusions: Pretreatment serum and ascites mtDNA levels are promising prognostic biomarkers in patients with OC. Our findings support a model in which mtDNA released as products of necrosis during OC, accelerate tumor progression by recruitment/activation of neutrophils and stimulation of NETosis. Further work will delineate the relationship between quantitative markers of NETosis and PFS in patients with OC.

Abstract Ovarian cancer (OC) is often diagnosed at advanced stages. Necrosis is a hallmark of advanced cancer and releases DAMPs that activate innate immune responses. It is well known that CD8+ T cells mediate antitumor immunity and immunosuppressive myeloid cells can impair T cell responses. In contrast, there is limited knowledge on the role of activated neutrophils in the tumor microenvironment. In patients with newly diagnosed OC, ~90% of ascites cells were inflammatory (CD45+) with varying proportions of neutrophils, monocytes and lymphocytes. Although patient variability existed, neutrophils comprised ~15% of CD45+ cells and the neutrophil:CD8+ T cell ratio was 1:1. In ex vivo studies, cell-free ascites (CFA) attracted normal donor neutrophils (NDN). Neutrophil extracellular traps (NETs) are a distinct mode of death, and characterized by extracellular release of DNA, histones, and granular products. We found that CFA induced NDN to generate NETs ex vivo. In co-culture studies, CD3/CD28-stimulated normal donor T cells were incubated with media; purified NDN 1:1; CFA; or NDN + CFA. After 72h, T cell proliferation was measured by [3H] thymidine incorporation. While CFA or NDN alone did not suppress T cell proliferation, CFA-activated NDN completely suppressed proliferation. Suppression did not affect T cell viability or induce apoptosis, and was not reversed by the addition of arginine, N-acetyl-L-cysteine or IL-2. Suppression required T cell contact with NDN. These results support a model in which ascites-activated neutrophils suppress T cell responses, thereby abrogating antitumor immunity. Further studies will identify ascites constituents that activate neutrophils and mechanisms for neutrophil-mediated T cell suppression.

Abstract While spectacular responses to cancer immunotherapy have been observed in some patients, the majority of responses are short-lived with ultimate tumor relapse. Therefore, it is crucial to identify strategies that will effectively synergize with immunotherapy to improve treatment outcome. Using a pre-clinical mouse model, we explored the use of a potent heterologous prime/boost vaccine strategy for the treatment of metastatic intraperitoneal ovarian cancer. Priming with an adjuvant based vaccine followed by boosting with a novel oncolytic Maraba viral vector elicited robust tumor-specific CD8+ T cell responses, with high numbers of therapy-induced CD8+ T cells effectively trafficking to the tumor microenvironment. While this approach greatly improved tumor control and long-term survival compared to treatment with the priming vaccine alone, the combination therapy was not curative and cellular analysis suggested that T cells within the tumor microenvironment were functionally suppressed/exhausted. Transcriptional profiling of tumors following therapy revealed that prime/boost vaccination led to induction of numerous inflammatory processes, with gene signatures consistent with not only CD8+ T cell infiltration, but also upregulation of multiple co-stimulatory and immune checkpoint receptors, induction of chemokine networks associated with both lymphoid and myeloid cell trafficking, as well as infiltration of immunosuppressive myeloid cell populations. We reasoned that these gene signatures could be used to design rational combination therapies that would have the potential to further enhance tumor attack following prime/boost vaccination. Using this strategy, we observed that checkpoint blockade using αPD-1 in combination with prime/boost vaccination resulted in a dramatic improvement in tumor control, as did transient depletion of granulocytic myeloid cells (but not monocytes/macrophages) following treatment. Current studies are underway that combine prime/boost vaccination with relevant co-stimulatory agonist antibodies, as well as inhibitors of candidate chemokine networks identified through tumor profiling. These findings underscore the importance of designing treatment strategies that not only elicit robust anti-tumor T cell responses, but also improve the duration and/or magnitude of immune attack within the tumor microenvironment. Additionally, our data suggest that interrogating the tumor microenvironment during treatment can identify unique therapeutic targets that have the potential to further improve therapeutic impact. Citation Format: AJ Robert Mcgray, Cheryl Eppolito, Anthony Miliotto, Raya Huang, Kelly Singel, Jonathan Pol, Kyle Stephenson, Brahm H. Segal, Brian Lichty, Kunle Odunsi. Tumor immune profiling identifies multiple unique therapeutic targets that improve vaccination + oncolytic virotherapy against metastatic ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4557. doi:10.1158/1538-7445.AM2017-4557

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 μM and 45 μM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 μM), soluble VWF (KD = 0.58 μM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.

Objective: Intravital microscopy (IVM) is a dynamic imaging modality that allows for real-time observation of tumor vasculature and immune cell trafficking. A recent clinical trial in melanoma patients was the first application of high-magnification, real-time IVM to patients at the time of surgery. To date there are no reports of IVM in deep-seated tumors including ovarian cancer. We tested the feasibility of using IVM during ovarian cancer cytoreductive surgery in order to characterize tumor vasculature and expand the ability of IVM from subcutaneous sites to the peritoneal cavity.

Abstract Metastatic epithelial ovarian cancer (EOC) is associated with ascites and accumulation of immunosuppressive myeloid cells. Granulocytic myeloid-derived suppressor cells are an immature population that impairs anti-tumor immunity. We previously found that granulocytes from the ascites of patients with newly diagnosed EOC were morphologically mature and suppressed ex vivo anti-CD3/CD28-stimulated T cell proliferation. Circulating neutrophils (PMN) from EOC patients were not suppressive, but acquired a suppressor phenotype (defined as ≥ 1 log10 reduction of T cell proliferation) after ascites supernatant exposure. Healthy donor PMN exposed to ascites supernatants acquired the same suppressor phenotype and also inhibited T cell activation and cytokine production. Our goal was to delineate the mechanisms for this PMN suppressor phenotype. Treatment of ascites supernatants with compstatin, an inhibitor of C3 activation, fully abrogated the PMN suppressor phenotype. Inhibition of C5 activation by anti-C5 or OmCI partially abrogated T cell suppression. Neutralizing Ab against C7, a required component of the membrane attack complex had no effect. Malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype, showing generalizability of the phenotype. NADPH oxidase-deficient PMN from patients with X-linked chronic granulomatous disease did not acquire a suppressor phenotype following ascites supernatant exposure, indicating that the suppressor phenotype is NADPH oxidase-dependent. Our results point to PMN impairing T cell expansion and activation in the tumor microenvironment and the potential for targeting C3 and NADPH oxidase as therapeutic approaches.

Abstract Pro-inflammatory (M1) macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C&gt;U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here, we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections of normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C&gt;U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of deleterious C&gt;U RNA editing occurred in THOC5 , encoding a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces pro-inflammatory M1 markers including IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1β and IL-6 cytokine secretion, and increases glycolysis and glycolytic capacity. Thus, APOBEC3A cytidine deaminase plays an important role in transcriptomic and functional polarization of M1 macrophages.