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Tumor cells release nucleic acid-containing proinflammatory complexes, termed nucleic acid-containing damage-associated molecular patterns (NA DAMPs), passively upon death and actively during stress. NA DAMPs activate pattern recognition receptors on cells in the tumor microenvironment leading to prolonged and intensified inflammation that potentiates metastasis. No strategy exists to control endogenous or therapy-induced inflammation in cancer patients. We discovered that the generation 3.0 polyamidoamine dendrimer (PAMAM-G3) scavenges NA DAMPs and mitigates their proinflammatory effects. In this study, we tested if the nucleic acid scavenger (NAS) PAMAM-G3 reduces lung metastasis in murine models of breast cancer. Our data indicate that PAMAM-G3 treatment decreases cell-free DNA levels and reduces lung metastasis in the experimental intravenous tumor-injection model and the postsurgical tumor-resection model of 4T1 breast cancer. Reduction in lung metastasis is associated with reduction in inflammatory immune cell subsets and proinflammatory cytokine levels in the tumor and the periphery. This study is the first example of NAS-mediated inhibition of metastasis to the lung. The study results provide a strong rationale for inclusion of NAS therapy in women with breast cancer undergoing standard-of-care surgery.

IntroductionB cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients.MethodsFlow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs.ResultsB cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival.ConclusionThese data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Objective To perform an exploratory, descriptive pilot study of the systemic and local immune environment in patients with vesicoureteral reflux (VUR) and bladder-bowel dysfunction (BBD). Methods Consecutive children with VUR undergoing intravesical ureteral reimplantation were enrolled. Patients were assessed for presence of BBD by reported patient history and validated questionnaire. Fresh blood and bladder tissue, collected at the time of surgery, were immediately processed for analysis. Immune cell compositions were determined via flow cytometry. Immune cell activation was also defined at the time of analysis. LegendPlex assay analysis was utilized to define levels of circulating chemokines and cytokines. Results A total of 7 patients were enrolled. Although percentages of circulating immune cells in the blood of those with VUR/BBD and VUR alone were similar, within bladder tissue, VUR/BBD demonstrated increased immune infiltrates compared to VUR alone. Bladder sample analysis showed that B cells, and Effector Memory and Naive T cell percentages were significantly increased in VUR/BBD patients compared to VUR patients. T cell expression of PD1 was increased in bladder tissues of BBD/VUR. Additionally, analysis of circulating neutrophils displayed significantly increased upregulation of PDL-1 in patients with VUR/BBD vs those with VUR only. Conclusion These pilot data suggest an immune-rich microenvironment is present within VUR. Severity of inflammation appeared to correlate with presence of BBD. This implies that targeting pelvic inflammation may be a novel therapy for children with VUR- or non-VUR-related BBD. Follow-up studies are currently underway.

BACKGROUND: In melanoma patients, microscopic tumor in the sentinel lymph node biopsy (SLN) increases the risk of distant metastases but the transition from tumor in the SLN to metastatic disease remains poorly understood. METHODS: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g. tumor) or by fluorescent cell subset markers (e.g. CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune related proteins were collected and normalized to account for system variation and ROI area. RESULTS: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years. CONCLUSION: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

BackgroundWe previously reported results from a phase 1 study testing intratumoral recombinant poliovirus, lerapolturev, in 12 melanoma patients. All 12 patients received anti-PD-1 systemic therapy before lerapolturev, and 11 of these 12 patients also received anti-PD-1 after lerapolturev. In preclinical models lerapolturev induces intratumoral innate inflammation that engages antitumor T cells. In the current study, prelerapolturev and postlerapolturev tumor biopsies and blood were evaluated for biomarkers of response.MethodsThe following analyses were performed on tumor tissue (n=11): (1) flow cytometric assessment of immune cell density, (2) NanoString Digital Spatial profiling of protein and the transcriptome, and (3) bulk RNA sequencing. Immune cell phenotypes and responsiveness to in vitro stimulation, including in vitro lerapolturev challenge, were measured in peripheral blood (n=12).ResultsThree patients who received anti-PD-1 therapy within 30 days of lerapolturev have a current median progression-free survival (PFS) of 2.3 years and had higher CD8+T cell infiltrates in prelerapolturev tumor biopsies relative to that of 7 patients with median PFS of 1.6 months and lower CD8+T cell infiltrates in prelerapolturev tumor biopsies. In peripheral blood, four patients with PFS 2.3 years (including three that received anti-PD-1 therapy within 30 days before lerapolturev and had higher pretreatment tumor CD8+T cell infiltrates) had significantly higher effector memory (CD8+, CCR7-, CD45RA-) but lower CD8+PD-1+ and CD4+PD-1+ cells compared with eight patients with median PFS 1.6 months. In addition, pretreatment blood from the four patients with median PFS 2.3 years had more potent antiviral responses to in vitro lerapolturev challenge compared with eight patients with median PFS 1.6 months.ConclusionAn inflamed pretreatment tumor microenvironment, possibly induced by prior anti-PD-1 therapy and a proficient peripheral blood pretreatment innate immune response (antiviral/interferon signaling) to lerapolturev was associated with long term PFS after intratumoral lerapolturev in a small cohort of patients. These findings imply a link between intratumoral T cell inflammation and peripheral immune function.Trial registration numberNCT03712358.

Patients with a medical history of Inflammatory Bowel Disease are predisposed to developing colitis-associated colorectal cancer (CAC), with greater than 20% developing CAC within 30 years of disease onset. CAC is a form of inflammation-induced tumorigenesis, where unregulated inflammation causes cells in the colon to accumulate mutations poising them for neoplasia. We demonstrate that NF-κB inducing kinase (NIK) is a critical regulator of intestinal epithelial cell (IEC) regeneration. We hypothesize that IECs exposed to chronic inflammatory conditions mitigated by dysregulated noncanonical NF-κB signaling are most prone to mutagenesis and cancer-initiating events termed Top-down tumorigenesis. In CAC, NIK depletion results in the accumulation of mature IECs that are increasingly susceptible to mutation and neoplastic transformation. This suggests that terminally differentiated IECs dedifferentiate into a stem-like phenotype. Our work has clinical relevancy, where human CAC biopsy samples have diminished noncanonical NF-κB signaling and NIK (MAP3K14) gene expression. Murine models with NIK deletion in IECs have increased susceptibility to inflammation-induced tumorigenesis following the chemical induction of AOM/DSS to induce colitis. Our mechanistic studies evaluating crypts demonstrate accumulation of mature, non-dividing IECs, which are more susceptible to mutations and malignant transformation. Here, we present a novel role for noncanonical NF-κB signaling in regulating intestinal epithelial cell turnover in protecting against CAC development. Focusing on the key niche signaling interplay following noncanonical NF-κB dysregulation gives greater insight into the CAC tumor microenvironment. Supported by grants from the iTHRIVE Pilot Translational and Clinical Studies Program, VCOM Center for One Health Research, Virginia Tech Carilion Research Acceleration Program, and the Virginia-Maryland College of Veterinary Medicine.

Immunotherapies are rapidly being integrated into standard of care (SOC) therapy in conjunction with surgery, chemotherapy, and radiotherapy for many cancers and a large number of clinical studies continue to explore immunotherapy alone and as part of combination therapies in patients with cancer. It is evident that clinical effectiveness of immunotherapy is limited to a subset of patients and improving immunotherapy related outcomes remains a major scientific and clinical effort. Understanding the immune cell subset phenotype and activation/functional status (cellular immunome) prior to and post therapy is therefore critical to develop biomarkers that (1) will predict if a patient will respond to immunotherapy and (2) are a result of immunotherapy. In this study, we investigated local (tumor) and peripheral (blood) cellular immunome of patients with melanoma, breast cancer, and brain cancer using a rapid and reliable standardized, multiparameter flow cytometry assay. We used this approach to monitor changes in the peripheral cellular immunome in women with breast cancer undergoing SOC therapy. Our analysis is unique because it is conducted using matched fresh tumor tissue and blood from patients in real-time, within 2-3 h of sample acquisition, and provides insight into the innate and adaptive immune cell profile in blood and tumor. Specific to blood, this approach involves no manipulation and evaluates all immune subsets such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils using 0.5 ml of blood. Analysis of the corresponding tumor provides much needed insight into the phenotype and activation status of immune cells, especially T and B cells, in the tumor microenvironment vs. the periphery. This analysis will be used to assess baseline and therapy-mediated changes in local and peripheral cellular immunome in patients with glioblastoma, breast cancer, and melanoma in planned immunotherapy clinical studies.

Background Sipuleucel-T is an autologous cellular immunotherapy that is FDA approved for the treatment of asymptomatic or minimally symptomatic metastatic castrate-resistant prostate cancer (mCRPC). The IMPACT registry trial demonstrated a 4.1 month survival benefit, but not a consistent PSA response or improvement in progression-free survival. Based upon several factors, including this lack of objective treatment response, sipuleucel-T has been under-utilized in this patient population, despite current NCCN recommendations. Methods In order to explore if delayed treatment response occurs in a subset of patients, we performed a single institutional retrospective analysis of mCRPC patients treated with sipuleucel-T and ongoing ADT alone. Within that group, we then identified a subset of sipuleucel-T-treated men with long-term disease control and no additional interventions. To independently confirm this finding, we evaluated a total of 336 patients from 4 large urology group practices treated with sipuleucel-T between 2010 and 2014 and identified 44 patients who met the same criteria and demonstrated evidence of PSA stabilization post sipuleucel-T treatment. Results For this subgroup of patients, 79% (95% CI: 64.5%, 88.1%) survived 36 months with a median time to subsequent therapy of 17.8 months (95% CI 10.3, 25.3). Conclusions Although patient selection could account for some or all of these results, these data support the utilization of sipuleucel-T alone in select mCRPC patients that is associated with a delay in disease progression and a good overall prognosis.

Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal's ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

Background: Immune cell composition and immune-mediated processes occurring in the sentinel lymph node (SLN) of melanoma patients regulate generation of tumor-specific effector T-cell responses and ultimately tumor control. We hypothesized that comprehensive analysis of these immune-associated processes in the SLN biopsy may identify patients at high risk of melanoma recurrence. Methods: We analyzed 38 formalin-fixed, paraffin-embedded sentinel lymph nodes from melanoma patients undergoing SLN biopsy from 2009-2012 using the NanoString nCounter® PanCancer Immune Profiling panel. The panel included 770 immune related genes. Results: Of 38 patients, 19 were SLN negative and 19 were SLN positive. Among SLN-negative patients (n=19), the median Breslow depth was 1.25 mm, and 21% (n=4) had developed recurrence at median follow-up of 6.2 years. Among SLN-positive patients, the median Breslow depth was 3.35 mm and 37% (n=7) had developed recurrence at median follow-up of 6.5 years. There were 31 genes significantly differentially expressed (P Conclusion: In this retrospective series, we found the immunologic gene expression profile of melanoma SLNs was significantly different in SLN negative versus SLN positive. There were also significant differences in the profile between patients who developed melanoma recurrence versus no recurrence regardless of SLN status. We aim to build on these pilot data. The goal is to use this profile to predict patients at high risk of recurrence after SLNB who may benefit from adjuvant therapy. Citation Format: Georgia M. Beasley, Eda K. Holl, Norma E. Farrow, Margaret G. Leddy, April K. Salama, Brent A. Hanks, Smita K. Nair. The immune profile of sentinel lymph nodes in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B16.

Rationale: Chronic inflammation is a hallmark of cancer. Inflammation, whether it is an underlying baseline condition or induced/exacerbated due to treatment, is a major hurdle that ultimately impacts the efficacy of standard of care therapy. However, no strategy exists to effectively control endogenous or therapy-induced inflammation in women with breast cancer. Moreover, there is limited information on how endogenous or therapy-induced inflammation modulates immune cells in women with breast cancer. Nucleic acid-containing pro-inflammatory complexes, termed Nucleic Acid-containing Damage Associated Molecular Patterns (NA-DAMPs), are released from dead or dying cancer cells or activated immune cells. NA-DAMPs activate nucleic acid sensing toll-like receptors (TLR) and other pattern recognition receptors (PRR). NA-DAMPs promote tumor progression and metastasis by inducing pro-tumorigenic signaling in cancer cells. Excessive and persistent NA-DAMP-mediated PRR signaling in immune cells is associated with immune dysfunction. Nucleic acid binding polymers have been used as vehicles for DNA/RNA delivery. We have discovered that a subset of nucleic acid binding polymers, termed nucleic acid scavengers (NAS), act as anti-inflammatory agents by capturing cancer-promoting pro-inflammatory NA-DAMPs. Therefore, we investigated if a NAS could limit cancer progression and metastasis by capturing and mitigating the effects of NA-DAMPs in immunocompetent murine models of breast cancer. Methods: We tested the use of the NAS PAMAM-G3 in the 1] orthotopic surgical resection 4T1 breast cancer model; 2] intravenous experimental metastasis 4T1 breast cancer model; 3] MMTV-PyMT spontaneous breast cancer model. We also measured changes in inflammation/inflammatory cell profile in tumor and in blood. Next, we investigated the relationship between therapy, NA-DAMPs and immune cell profile in blood from women with breast cancer. We also examined if NA-DAMPs in patient serum induces tumor invasion and if the NAS PAMAM-G3 prevents tumor invasion using an in vitro assay. Results: We have determined that the NAS PAMAM-G3 controls lung metastasis in immunocompetent murine models of breast cancer and our data indicate that: 1] chronic inflammation and inflammatory cells in the tumor and periphery promote cancer invasion and metastasis; 2] modulating inflammation by removing excessive NA-DAMPs using the NAS PAMAM-G3 limits cancer metastasis; 3] scavenging inflammation-associated NA-DAMPs significantly alters immune cell and cytokine/chemokine profile in the tumor and periphery, promotes T cell infiltration in the tumor and induces antitumor CD8+ T cell responses. In experiments conducted using blood collected longitudinally from women undergoing standard of care therapy, we observed that: 1] NA-DAMPs and inflammatory monocytes are elevated in women undergoing standard of care therapy; 2] Post-chemotherapy breast cancer patient serum increases invasion/migration of human breast cancer cells in vitro; 3] PAMAM-G3 abrogates increase in invasion/migration of human breast cancer cells in vitro in response to post-chemotherapy breast cancer patient serum. Conclusions: These results highlight the potential utility of NAS to treat cancer associated inflammation and thereby inhibit cancer metastasis by 1) limiting extracellular NA-DAMP-induced persistent activation of pro-inflammatory signaling, and 2) promoting full engagement of both the innate and adaptive immune systems. These principles will aid in the development of a novel anti-metastatic therapy for women with breast cancer. This work is supported by Department of Defense Breast Cancer Research Program Awards W81XWH-16-1-0512 (Nair) and W81XWH-16-1-0513 (Sullenger). Citation Format: Smita Nair, Eda Holl, Karenia Landa, Victoria Frazier, Elias Eteshola, David Boczkowski, Rachel Rempel, Shelley Hwang, Bruce Sullenger. Modulating inflammation with nucleic acid scavengers to prevent breast cancer metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-03.

RNA vaccines traditionally consist of messenger RNA synthesized byin vitrotranscription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

Tumors thrive in an immunosuppressive microenvironment that impedes antitumor innate and adaptive immune responses. Thus, approaches that can overcome immunosuppression and engage antitumor immunity are needed. This study defines the adjuvant and cancer immunotherapy potential of the recombinant poliovirus/rhinovirus chimera PVSRIPO. PVSRIPO is currently in clinical trials against recurrent World Health Organization grade IV malignant glioma, a notoriously treatment-refractory cancer. Cytopathogenic infection of neoplastic cells releases the proteome and exposes pathogen- and damage-associated molecular patterns. At the same time, sublethal infection of antigen-presenting cells, such as dendritic cells and macrophages, yields potent, sustained type I interferon-dominant activation in an immunosuppressed microenvironment and promotes the development of tumor antigen-specific T cell responses in vitro and antitumor immunity in vivo. PVSRIPO's immune adjuvancy stimulates canonical innate anti-pathogen inflammatory responses within the tumor microenvironment that culminate in dendritic cell and T cell infiltration. Our findings provide mechanistic evidence that PVSRIPO functions as a potent intratumor immune adjuvant that generates tumor antigen-specific cytotoxic T lymphocyte responses.

KAGRA is a 3-km interferometric gravitational wave telescope located in the Kamioka mine in Japan. It is the first km-class gravitational wave telescope constructed underground to reduce seismic noise, and the first km-class telescope to use cryogenic cooling of test masses to reduce thermal noise. The construction of the infrastructure to house the interferometer in the tunnel, and the initial phase operation of the interferometer with a simple 3-km Michelson configuration have been completed. The first cryogenic operation is expected in 2018, and the observing runs with a full interferometer are expected in 2020s. The basic interferometer configuration and the current status of KAGRA are described., Comment: 7 pages, 2 figures. Proceedings for XV International Conference on Topics in Astroparticle and Underground Physics (TAUP2017), Sudbury, July 24-28, 2017

Summary Background Given improvements in multimodality therapy, survival among children with Wilms tumor (WT) exceeds 90%. However, 15% of children with favorable histology and 50% of children with anaplastic WT experience recurrence or progression. Of patients with advanced disease, only 50% survive to adulthood. In adult malignancies (including renal tumors), patient survival has improved with the advent of immunotherapy. However, little is known about the immune microenvironment of WT, making the potential role of immunotherapy unclear. Objective The objective of the study is to perform an exploratory, descriptive analysis of the immune milieu in WT. Study design Between 2016 and 2017, all pediatric patients with WT, some of whom received neoadjuvant chemotherapy, underwent ex vivo wedge biopsy at the time of nephrectomy. The fresh tumor tissue and peripheral blood samples were analyzed for infiltrating immune infiltrate and effector cells using flow cytometry. Immunohistochemistry was performed for CD4, CD8, and PD-L1 expression. Matched blood samples were obtained for each patient, and circulating immune cells were analyzed by flow cytometry. Results A total of six patients were enrolled. One patient with neuroblastoma was excluded. The remaining five patients included the following: two with unilateral WT (resected before chemotherapy), two with bilateral WT (resected after neoadjuvant chemotherapy), and one with Denys–Drash syndrome, end-stage renal disease, and history of WT in the contralateral kidney. Immune analysis showed that WT were infiltrated by immune cells regardless of chemotherapy status. CD8 and CD4 T cells were present in the tumor tissue and exhibited an activated phenotype. Elevated levels of natural killer (NK) cells were observed in the tumors (Figure). Immune checkpoint PD-L1 was also found expressed in one of the tumors stained. Discussion In this pilot study, it was found that WTs were infiltrated by immune cells (CD45+) both before and after chemotherapy. Elevated levels of NK cells infiltrating the tumor specimens, which were quantitatively increased compared with levels of NK cells circulating in the blood, were noted. T cells, particularly CD4+ and CD8+ T cells, were present in tumor specimens. Tumor-infiltrating CD4 and CD8 T cells displayed an activated phenotype as defined by increased expression of human leukocyte antigen-DR isotype (HLA-DR), programmed cell death protein 1 (PD1), and CD57. Together, these findings suggest that WT microenvironment is immune engaged and may be susceptible to immunotherapy similar to other malignancies. Conclusions These pilot data suggest an immune-engaged tumor microenvironment is present within WT. This implies that WT may be susceptible to immunotherapy similar to adult renal tumors and other adult malignancies. Follow-up studies are currently underway. Download : Download high-res image (256KB) Download : Download full-size image Summary Figure

7 Background: Neoadjuvant chemotherapy (NAC) for breast cancer (BC) is being increasingly used in patients with stage I/II disease. Although previously reserved for patients with locally advanced disease, its use in patients with localized disease allows for higher rates of breast-conserving procedures and provides insight into tumor biology. A complete pathologic response (pCR) to NAC correlates with better clinical outcomes; sadly, many patients do not achieve pCR. The advent of immunotherapy has provided cancer patients with additional treatment options. To optimize immunotherapy in BC we must understand the peripheral cellular immunome (immune subsets and activation status) of patients as they undergo standard of care therapy (SOC). Methods: Our population includes patients with stages I-III BC undergoing SOC. Samples were collected pre- and post-NAC, post-surgery and at 2-month follow-up (FU). Results: Flow cytometry analysis for 11 patients was performed at each time-point to examine percentages of circulating immune cells (see Table). We grouped samples in 3 categories: human epidermal growth factor receptor 2 (HER2+) positive, HER2 negative (HER2-) and triple-negative (TN) tumors. 3 out of 11 patients had pCR (1 HER2+, 1 HER2-, 1 TN); they had highest percentage of circulating CD56+ NK cells during treatment course. Conclusions: We observed striking changes in the immunome of women with stage I-III BC undergoing NAC. Although our findings are preliminary, given our sample size, we observed distinct trends within each immune cell population in specific tumor receptor subtypes. These trends could serve to guide our therapies, allow for better patient selection and predict treatment response in patients. [Table: see text]

Plexins and semaphorins are a large family of proteins that are involved in cell movement and response. The importance of plexins and semaphorins has been emphasized by their discovery in many organ systems including the nervous (Nkyimbeng-Takwi and Chapoval, 2011; McCormick and Leipzig, 2012; Yaron and Sprinzak, 2012), epithelial (Miao et al., 1999; Fujii et al., 2002), and immune systems (Takamatsu and Kumanogoh, 2012) as well as diverse cell processes including angiogenesis (Serini et al., 2009; Sakurai et al., 2012), embryogenesis (Perala et al., 2012), and cancer (Potiron et al., 2009; Micucci et al., 2010). Plexins and semaphorins are transmembrane proteins that share a conserved extracellular semaphorin domain (Hota and Buck, 2012). The plexins and semaphorins are divided into four and eight subfamilies respectively based on their structural homology. Semaphorins are relatively small proteins containing the extracellular semaphorin domain and short intra-cellular tails. Plexins contain the semaphorin domain and long intracellular tails (Hota and Buck, 2012). The majority of plexin and semaphorin research has focused on the nervous system, particularly the developing nervous system, where these proteins are found to mediate many common neuronal cell processes including cell movement, cytoskeletal rearrangement, and signal transduction (Choi et al., 2008; Takamatsu et al., 2010). Their roles in the immune system are the focus of this review.

// Eda K. Holl 1 , Michael C. Brown 2 , David Boczkowski 1 , Megan A. McNamara 3 , Daniel J. George 3 , Darell D. Bigner 4 , Matthias Gromeier 2 , Smita K. Nair 1, 4 1 Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA 2 Department of Neurosurgery, Duke University School of Medicine, Durham, NC 27710, USA 3 Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA 4 Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA Correspondence to: Smita K. Nair, email: smita.nair@duke.edu Keywords: oncolytic poliovirus, PVSRIPO, inflammation, innate immunity Received: August 29, 2016 Accepted: October 13, 2016 Published: October 28, 2016 ABSTRACT Intratumoral inoculation of viruses with tumor-selective cytotoxicity may induce cancer cell death and, thereby, shrink neoplastic lesions. It is unlikely, however, that viral tumor cell killing alone could produce meaningful, durable clinical responses, as clinically suitable ‘oncolytic’ viruses are severely attenuated and their spread and propagation are opposed by host immunity. Thus, a more propitious event in this context is the innate antiviral response to intratumoral virus administration, in particular for recruiting durable adaptive immune effector responses. It may represent a double-edged sword, as innate immune activation may eliminate infected tumor cells early, intercept viral spread and block any meaningful therapeutic response. The innate response to viral infection of tumors may be very different from that in non-malignant target tissues, owing to the unusual composition/tissue properties of tumor stroma. In this work, we report investigations of the innate immune response to the oncolytic poliovirus recombinant, PVSRIPO, in two mouse xenotransplantation models for breast and prostate cancer. Our observations indicate short-term virus persistence in infected tumors and virus recovery indicative of modest intratumoral propagation and persistence. Yet, a powerful innate inflammatory response coincided with chemokine induction and myeloid cell infiltration into tumors that was, interestingly, dominated by neutrophils. The combined effect of PVSRIPO tumor infection and the innate response it elicits was significant tumor regression in both models.

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ+ and Igκ+ B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1−/− mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1−/− B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1−/− mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.

The tumor microenvironment favors tumor immune escape by suppressing production, activation and/or function of antitumor T cells. Our group has developed a recombinant Rhino-Poliovirus chimera, PVSRIPO, currently being evaluated in Phase-II clinical trial against recurrent glioma. PVSRIPO therapy has afforded durable clinical responses in recurrent glioma patients. Clinical and pre-clinical findings suggest that PVSRIPO primarily acts through an immunological mechanism, rather than via cancer selective cytotoxicity. Here we define the immune adjuvant potential of intratumoral-delivered PVSRIPO that ultimately leads to the engagement/production of antitumor T cells. Sub-lethal infection of human dendritic cells (DCs) with PVSRIPO yields potent, sustained type I interferon dominant activation. PVSRIPO infection of macrophages induces similar activation; however, virus translation and the resulting type I Interferon/TNF-α production by macrophages is strongly enhanced in the presence of the Th2 cytokine IL-4. Compared to conventional adjuvants, e.g. poly(I:C) and LPS, PVSRIPO stimulation of APCs withstands tumor-associated immunosuppression, particularly in macrophages, and achieves more durable activation of DCs. Therapy of B16 melanoma tumors with PVSRIPO led to early production of type I interferon, IL-12, and IFN-γ; which was associated with the recruitment of neutrophils to the tumor site. Neutrophil influx was followed by DC and T cell infiltration. Mice treated with PVSRIPO developed tumor-antigen specific, cytotoxic T cells that were present in both tumor draining lymph nodes and spleens. These events correlated with delay in tumor growth and increase in survival following PVSRIPO therapy. Thus, PVSRIPO’s immune adjuvancy stimulates canonical innate inflammatory responses within the tumor microenvironment that culminates in tumor antigen-specific T cell responses. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Elena Dobrikova, Mubeen Mosaheb, Vidya Chandramohan, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of antigen-presenting cells and tumor antigen-specific CTLs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A79.

Breast cancer is the most common malignancy among women in the United States and the second leading cause of cancer deaths in women. Disease prognosis, local recurrence rates and response to therapy are all related to receptor status of the individual cancer. Triple negative breast cancer (TNBC; ER-, PR-, and HER2-negative) is a subset of breast cancer with poor prognosis and high rate of local recurrence and metastasis with limited therapeutic options. The oncolytic poliovirus, PVSRIPO, is a recombinant polio:rhinovirus chimera that has been engineered to eliminate neuropathogenicity and has demonstrated promising responses in patients with recurrent glioblastoma. PVSRIPO is oncolytic in tumor cells and enters cells via its receptor CD155, which is overexpressed in solid malignancies, including TNBC. Treatment of primary TNBC tissue with PVSRIPO induces IFN-I and pro-inflammatory cytokines. In a murine immunotherapy model, intratumoral injection of PVSRIPO induces IFN-I, IL12 and IFN-γ and rapid recruitment of neutrophils into the tumor followed by infiltration of DCs, T cells and B cells. Treatment of orthotopic breast tumors with PVSRIPO results in delayed tumor growth and increased survival in an immunocompetent murine immunotherapy model. We have designed a pilot study to examine the effects of intratumoral injection of PVSRIPO into six women with TNBC with at least 1 cm of residual tumor after chemotherapy. The primary exploratory objective is to describe the changes in the amount of tumor infiltrating immune cells in tumor tissue pre- and post-injection of PVSRIPO. We will also collect blood at different time points during PVSRIPO treatment to evaluate inflammatory and immune signatures. We hypothesize that intratumoral administration of PVSRIPO will cause inflammation thus stimulating innate and adaptive immune activation. This study is funded by the Department of Defense Breast Cancer Research Program award W81XWH-16-1-0354 (PI, Smita Nair). Citation Format: Karenia Landa, Eda Holl, Michael Brown, James Herndon, Lars Grimm, Jeremy Force, Darell Bigner, Shelley Hwang, Matthias Gromeier, Smita Nair. Oncolytic poliovirus-mediated inflammation and immunity in breast cancer [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 LB-302.

e12641Background: Breast cancers thrive in an immunosuppressive microenvironment. Checkpoint blockade has had poor results in metastatic breast cancers. Recombinant oncolytic polio:rhinovirus (PVSR...

Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro. We have previously demonstrated the ability of nucleic acid binding polymers (NABPs) to bind and inhibit the pro-inflammatory effects of cfDNA and RNA in models of systemic lupus erythematosus and influenza. Herein, we demonstrate that in tumor bearing mice, levels of cfDNA increase with tumor burden and decline post NABP in vivo administration. Using an orthotopic breast cancer murine model we demonstrate that NABP treatment, following surgical resection, results in reduced or completely absent lung metastasis. In addition, intravenous delivery of breast cancer cells in the presence of NABPs also results in decreased tumor cell lung seeding and lung metastasis. Taken together, our data suggest that NABPs can be used to target cfDNA and inhibit such DAMPs from activating pathological inflammation, an innovative approach to treat cancer progression and metastasis. In particular, NABPs may be useful in the clinical setting following standard of care surgical tumor resection.

Aberrant inflammation has been linked to the development of a diverse spectrum of human diseases. We now appreciate that Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling pathways play an important role in the onset and/or severity of these pathologies, and targeting of these receptors has shown promise in preclinical studies of infectious diseases, autoimmune disorders, tissue injury, and cancer. Recently, clinical trials have been initiated to establish the efficacy of compounds targeting TLR and NLR pathways to improve disease outcomes. Despite advancements in the field, major challenges remain in potential ways to target these receptors at various stages of disease pathogenesis and develop effective therapeutics.

Dysregulation of NF-κB signaling is a common feature in Colorectal Cancer (CRC). While the canonical signaling pathway is well characterized, the non-canonical NF-κB signaling cascade is understudied. Thus, we evaluated the hypothesis that non-canonical NF-κB signaling significantly modulates CRC pathogenesis. To evaluate this hypothesis, we initially conducted a meta-analysis of human gene expression data. Using this approach, we found that non-canonical NF-κB signaling is significantly down-regulated in CRC patients and the extent of pathway attenuation is correlated with disease severity. These findings were confirmed in patient biopsy specimens, where we found significantly reduced expression of ligands, receptors, regulators, and chemokines associated with non-canonical NF-κB signaling. Notably, we observed significantly reduced CCL19, CCL21, CXCL12, and CXCL13 expression in CRC patients compared to healthy controls. To gain mechanistic insight, we utilized genetically modified mice lacking the kinase Map3K14 (NIK), which is essential for non-canonical NF-κB signaling. Nik−/− mice exhibited ablation of non-canonical NF-κB signaling and significantly increased epithelial cell proliferation in the GI tract compared to wild type mice in both in vivo and in vitro studies. Together, our data defines a protective role for non-canonical NF-κB signaling in the gut during CRC and identifies a highly protective role for Map3K14 in attenuating epithelial cell proliferation.

Pathologic cutaneous scarring affects over 40 million people worldwide and costs billions of dollars annually. Understanding mechanisms of fibroblast activation and granulation tissue contraction is the first step toward preventing pathologic scarring. The authors hypothesize that nucleic acids increase fibroblast activation and cause granulation tissue contraction and that sequestration of nucleic acids by application of a nucleic acid scavenger dendrimer, polyamidoamine third-generation dendrimer, will decrease pathologic scarring.In vitro experiments were performed to assess the effect of nucleic acids on pathologic scar-associated fibroblast activity. The effect of nucleic acids on cytokine production and migration on mouse fibroblasts was evaluated. Immunofluorescence microscopy was used to determine the effect of nucleic acids on the differentiation of human primary fibroblasts into myofibroblasts. Using a murine model, the effect of polyamidoamine third-generation dendrimer on granulation tissue contraction was evaluated by gross and histologic parameters.Mouse fibroblasts stimulated with nucleic acids had increased cytokine production (i.e., transforming growth factor-β, monocyte chemotactic protein 1, interleukin-10, tumor necrosis factor-α, and interferon-γ), migration, and differentiation into myofibroblasts. Polyamidoamine third-generation dendrimer blocked cytokine production, migration, and differentiation into myofibroblasts. Using a murine model of granulation tissue contraction, polyamidoamine third-generation dendrimer decreased wound contraction and angiogenesis. Collagen deposition in polyamidoamine third-generation dendrimer-treated tissues was aligned more randomly and whorl-like compared with control tissue.The data demonstrate that nucleic acid-stimulated fibroblast activation and granulation tissue contraction are blocked by polyamidoamine third-generation dendrimer. Sequestration of pathogen-associated molecular patterns may be an approach for preventing pathologic scarring.

The creation of bone marrow and fetal liver chimeric mice has proven to be a valuable tool in the field of immunology. Chimeric mice are used to study the contribution of various cell types of hematopoietic versus non-hematopoietic origin in the course of an immune response. In this chapter, we describe a detailed method to obtain bone marrow or fetal liver chimeric mice and assess the efficiency of donor cells to repopulate the hematopoietic compartment of recipient mice.

Background: The recent clinical success of immune checkpoint inhibitors in melanoma revealed the therapeutic potential of an anti-tumor T cell response. Previous studies using peptide vaccines to elicit anti tumor T cell response have been limited in efficacy. We have discovered that an oncolytic poliovirus leads to persistent IFN dominant activation of antigen presenting cells. Based upon these findings, we propose to use a recombinant poliovirus (PVS-RIPO) expressing tumor antigens to activate antigen presenting cells and elicit an anti tumor T cell response. PVS-RIPO is the live attenuated vaccine (SABIN) type 1 containing a foreign human rhinovirus IRES. Preliminary studies in our lab showed that PVS-RIPO is a potent human dendritic cell and CD8 T cell activator. In addition, PVS-RIPO has recently achieved breakthrough status following a very promising phase I clinical trial targeting recurrent glioblastoma (GBM). The efficacy of PVS-RIPO against GBM is thought to be mediated mainly by an anti-tumor CD8 T cells response. The potent activation of dendritic cells as well as the promising results of the phase I clinical trial makes PVS-RIPO a prime candidate for use as a therapeutic tumor vaccine. The PVS-RIPO vector technology can be adapted to multiple tumor mutations, we are planning to target 2 glioma mutations at this stage: IDH1R132H and EGFRviii mutations. IDH1R132H is a point mutation in the enzyme isocitrate dehydrogenase 1, the mutation is present at high frequency (>70%) in low grade glioma. EGFRviii is a deletion in exon 2-7 of the EGFR gene, it is present in 67% of patients with GBM. Results: Adjuvancy of PVS-RIPO We first tested the immune adjuvancy of PVS-RIPO in an in vitro human system. PVS-RIPO was found to sub lethally infect and activate dendritic cells leading to pro inflammatory cytokine production. In addition, dendritic cell incubated with tumor cell lysate, PVS-RIPO and autologous T cells produced an anti tumor CD8 T cell response. Designing stable PVS-RIPO Vectors Previous attempts to use poliovirus as a vaccine vector were unsuccessful because of its notorious genetic instability. Our lab has developed a novel strategy to generate stable poliovirus vectors: we use the relative genetic plasticity of the IRES (the dispensable stem-loop Domain VI) to encode tumor antigens. We have successfully made stable PVS-RIPO vectors expressing EGFRviii and IDH1R132H antigens. Mouse models to study efficiency of vaccine in vivo Because PVS-RIPO has a rhinovirus IRES, it is incompetent in mouse cells and first had to be adapted for mouse competency. Through murine cell passaging, we have successfully generated a mouse adapted PVS-RIPO virus (mPVS-RIPO) with 5 point mutations in the IRES. We also generated 3 different transgenic (expressing the human poliovirus receptor, CD155) mouse tumor models: 1. Chemically induced model: CT2A-CD155 (C57BL/6-CD155), 2. Genetic model: Neuro Stem Cell-P53fl/fl-mPDGFB-CD155 (C57BL/6-CD155) and 3. Spontaneous tumor model: SMA560-CD155 (VM/DK-CD155). Conclusion: Here we have put together all the required tools to test the antitumor efficacy of a PVS-RIPO vector vaccine in vivo. Given that PVS-RIPO has some replication competence in muscle cells and also in tumor cells, we are planning to administer the mPVS-RIPO vector intramuscularly as well as intratumorally in mice and monitor its anti tumor efficacy. Citation Format: M Mubeen Mosaheb, Elena Dobrikova, Michael C. Brown, Christopher Pirozzi, Vidyalakshmi Chandramohan, Eda Holl, Smita Nair, Matthias Gromeier. Using oncolytic polio expressing tumor as a cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A032.

Introduction: Tumor-targeted therapies that efficiently eliminate malignant cells and in the process engage the innate and adaptive immune system are desirable for preventing cancer recurrence. We have pioneered an oncolytic poliovirus therapy, PVSRIPO, which selectively targets and eliminates malignant cells. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal competence and is devoid of neuropathogenicity after intracerebral inoculation in non-human primates and in humans. PVSRIPO has cancer tropism due to widespread ectopic expression of the poliovirus receptor, CD155, in solid cancers. PVSRIPO kills tumor cells with no effect on normal cells. Unlike other oncolytic viruses, PVSRIPO is effective in the presence of neutralizing antibodies and an innate antiviral response. A first-in-human Phase-1 study with PVSRIPO at Duke University has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) via CD155. Type I IFNs are known to inhibit tumor proliferation, activate innate immune cells and bridge innate and adaptive immunity among other functions. Methods: To understand immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. To investigate if DCs exposed to PVSRIPO-induced dying tumor cells are capable of presenting tumor antigens to T cells, we performed an in vitro human immunotherapy assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. All tumor cell lines used for the human immunotherapy experiments were positive for HLA-A2 expression. The following tumor cell lines were used for this analysis: DM6 (MART+) melanoma cell line, MDA-MB231 (CEA+) triple-negative breast cancer cell line, SUM149 (EGFR+) inflammatory breast cancer cell line and LNCaP (PSA+) prostate cancer cell line. Results: PVSRIPO infection of THP1 macrophages induces Mda5/Stat1 and Stat1 phosphorylation as well as TNFα and IFNβ release. Coculturing of DCs with PVSRIPO-induced tumor cell lysate stimulates DC activation and IL12 production. Human DCs loaded with PVSRIPO-induced tumor cell lysate are capable of stimulating tumor antigen-specific cytotoxic T cells in vitro. Autologous DCs transfected with RNA that encodes for CEA, EGFR, MART or PSA (tumor antigen-expressing cells) and human tumor cell lines were used as targets to assess tumor antigen-specificity of T cells. Conclusion: Our data suggests that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. Importantly, we demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific cytotoxic T cells in an in vitro human immunotherapy assay. In ongoing studies we are analyzing oncolytic poliovirus mediated immune events in syngeneic, immunocompetent murine models of breast cancer and prostate cancer. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Oncolytic poliovirus mediated immune events. [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 A157.

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.

The effect of a non-specific thiol-alkylating agent N-ethylmaleimide (NEM) was studied on neurogenic contractile mechanisms in rat ventral prostate gland.Male Wistar albino rats were used. The rats were killed by cervical dislocation under sevoflurane anesthesia and ventral prostate gland was removed. Two preparations were obtained from each lobe. Neurally evoked isometric contractions were induced using trains of electrical field stimulation (EFS; 0.5, 1, 4, or 8 Hz). The effect of NEM on the contractions to EFS was examined in the absence or presence of adrenergic and/or purinergic antagonists.NEM enhanced the EFS-evoked contractions without altering the basal tone. These effects were significantly suppressed by an α(1) -adrenergic receptor antagonist (prazosin), a P2-purinergic antagonist (suramin), a specific P2X-receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), an ATP analog (α,β-methylene ATP), or a calcium channel blocker (verapamil). This facilitating effect of NEM did not occur following the administration of L-cysteine or glutathione which saturated NEM with excess thiols. However, a thiol-oxidant diamide failed to affect the contractions to EFS. An adrenergic neuron blocker (guanethidine) completely suppressed the responses to NEM. On the other hand, an α(2) -adrenergic receptor blocker (yohimbine), a nitric oxide synthase inhibitor (N(ω) -nitro-L-arginine) or a cholinergic muscarinic receptor antagonist (atropine) did not significantly affect the facilitatory response of NEM.These findings suggest that NEM has a prejunctional facilitatory action on the adrenergic nerves in rat prostate tissue to enhance release of transmitters, noradrenaline, and ATP. NEM sensitive proteins involved in transmitter release mechanisms can play a role in this effect.

Colitis-associated cancer (CAC) is a major complication of inflammatory bowel diseases. We show that components of the inflammasome are protective during acute and recurring colitis and CAC in the dextran sulfate sodium (DSS) and azoxymethane + DSS models. Mice lacking the inflammasome adaptor protein PYCARD (ASC) and caspase-1 demonstrate increased disease outcome, morbidity, histopathology, and polyp formation. The increased tumor burden is correlated with attenuated levels of IL-1β and IL-18 at the tumor site. To decipher the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) component that is involved in colitis and CAC, we assessed Nlrp3 and Nlrc4 deficient mice. Nlrp3−/− mice showed an increase in acute and recurring colitis and CAC, although the disease outcome was less severe in Nlrp3−/− mice than in Pycard−/− or Casp1−/− animals. No significant differences were observed in disease progression or outcome in Nlrc4−/− mice compared with similarly treated wild-type animals. Bone marrow reconstitution experiments show that Nlrp3 gene expression and function in hematopoietic cells, rather than intestinal epithelial cells or stromal cells, is responsible for protection against increased tumorigenesis. These data suggest that the inflammasome functions as an attenuator of colitis and CAC.

The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell–cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor–ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.

SummaryThe nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules mediate host immunity to various pathogenic stimuli. However, in vivo evidence for the involvement of NLR proteins in viral sensing has not been widely investigated and remains controversial. As a test of the physiologic role of the NLR molecule NLRP3 during RNA viral infection, we explored the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, Pycard, or caspase-1, but not Nlrc4, exhibited dramatically increased mortality and a reduced immune response after exposure to the influenza virus. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrated that an NLRP3-mediated response could be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by the influenza virus was dependent on lysosomal maturation and reactive oxygen species (ROS). Inhibition of ROS induction eliminated IL-1β production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.

It has been known for a long time that the fundamental group of the quotient of $\RR ^3$ by the Case-Chamberlin continuum is nontrivial. In the present paper we prove that this group is in fact, uncountable.