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The nutritional requirements of neonates with congenital abdominal wall defects (AWD) remain poorly described. In particular, there is a lack of literature on the calorie, protein and micronutrient needs of those with AWD. Nutritional therapy is a cornerstone of care in patients with burns due to the metabolic consequences of injury to the epithelial layer. Similarly, children with AWD may require specialized nutritional plans to support their growth and wound healing. This case series supports the theory that patients with ruptured omphaloceles may require higher calorie, protein, and micronutrient provisions in comparison to patients with intact omphaloceles, due to increased metabolic demand to support wound healing and skin epithelization.

Supplementary Figures S1-S11

Purpose:While immune checkpoint inhibitors such as anti–PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials.Experimental Design:We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects.Results:A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell–dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti–PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti–PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression.Conclusions:These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080

Supplementary Methods

Parent-education programs in youth sport appear to provide an appropriate avenue to facilitate healthy parental involvement, enhance positive parental support, and help to relieve stressors placed ...

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti–PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo. Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

OBJECTIVES A third of infants with congenital diaphragmatic hernia (CDH) require a gastrostomy tube (GT) for nutritional support. We compared CDH infants who are GT-dependent to those able to meet their nutritional needs orally, to identify factors associated with requiring a GT and evaluate their long-term growth. METHODS Patients with CDH repaired at a single institution between 2012 and 2020 were included. Charts were retrospectively reviewed for demographic, surgical, and post-operative details. Mann-Whitney test and Fischer exact test were performed to compare GT-dependent neonates (n = 38, experimental) with orally fed neonates (n = 63, control). Significance was set at

Purpose: While immune checkpoint inhibitors such as anti–PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. Experimental Design: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. Results: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell–dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti–PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti–PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. Conclusions: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity. See related commentary by Cirella et al., p. 6080

Oncolytic virus (OV) therapy is an emerging approach with the potential to redefine treatment options across a range of cancer indications and in patients who remain resistant to existing standards of care, including immuno-oncology (IO) drugs. MEDI5395, a recombinant Newcastle disease virus (NDV), engineered to express granulocyte–macrophage colony-stimulating factor (GM-CSF), exhibits potent oncolytic activity. It was hypothesized that activation of immune cells by MEDI5395, coupled with its oncolytic activity, would enhance the priming of antitumor immunity. Using MEDI5395 and recombinant NDVs encoding fluorescent reporter genes, we demonstrated preferential virus uptake and non-productive infection in myeloid cells, including monocytes, macrophages, and dendritic cells (DCs). Infection resulted in immune-cell activation, with upregulation of cell surface activation markers (e.g., CD80, PD-L1, HLA-DR) and secretion of proinflammatory cytokines (IFN-α2a, IL-6, IL-8, TNF-α). Interestingly, in vitro M2-polarized macrophages were more permissive to virus infection than were M1-polarized macrophages. In a co-culture system, infected myeloid cells were effective virus vectors and mediated the transfer of infectious NDV particles to tumor cells, resulting in cell death. Furthermore, NDV-infected DCs stimulated greater proliferation of allogeneic T cells than uninfected DCs. Antigens released after NDV-induced tumor cell lysis were cross-presented by DCs and drove activation of tumor antigen-specific autologous T cells. MEDI5395 therefore exhibited potent immunostimulatory activity and an ability to enhance antigen-specific T-cell priming. This, coupled with its tumor-selective oncolytic capacity, underscores the promise of MEDI5395 as a multimodal therapeutic, with potential to both enhance current responding patient populations and elicit de novo responses in resistant patients. Electronic supplementary material The online version of this article (10.1007/s00262-020-02495-x) contains supplementary material, which is available to authorized users.

Audience: The Residents-as-Teachers (RAT) curriculum is designed for emergency medicine (EM) residents of all years (PGY1-4).Length of Curriculum: The curriculum is divided into three hour-long sessions. The entire curriculum can be run as a single block or can be spread out over multiple days. Introduction: The Accreditation Council of Graduate Medical Education (ACGME) and the Liaison Committee on Medical Education (LCME) both require residents to receive training in teaching medical students and junior residents. They also require opportunities for residents to participate in teaching and be assessed on their effectiveness in this role.1,2 However, the ACGME does not provide guidance or require formal curricula on molding residents into effective teachers. Many programs and institutions have incorporated RAT curricula as a solution to provide residents with the skills necessary to create an excellent educational environment for junior learners. These curricula have been embraced by many specialties, including Emergency Medicine (EM).3-6 The effectiveness of the teaching received during the clinical rotations has important long-term effects on medical students, and may impact their future career choices in medicine.6The COVID-19 pandemic has also required education institutions to vastly alter the delivery of their didactics, including moving to a virtual platform. A completely online format has many benefits that extend beyond the pandemic, such as easier access to participants (including those off-service or at remote sites), no requirement for a physical space, and easier recording of sessions.Educational Goals: To provide residents with an introduction to teaching techniques that can be utilized on shift to facilitate an excellent educational experience for junior learners while balancing the resident’s patient care responsibilities.Educational Methods: The educational strategies used in this curriculum include PowerPoint (Redmond, WA) slideshows given by a live presenter via the telecommunications platform Zoom (San Jose, CA), viewing of videos demonstrating curriculum topics, simulation-based learning through role-play, and small-group discussions including simulation debriefing.Research Methods: A survey was distributed to residents before and following the completion of the three training sessions to assess resident satisfaction with the delivery of the content and comfort with the teaching tools discussed. Suggestions on potential improvements were also assessed to inform changes to future iterations of the curriculum. Comfort regarding the included teaching tools was assessed using a five-point Likert scale. After completion of the curriculum, rotating medical students were provided with an evaluation form to assess if residents were teaching using the techniques from the course.Results: Both the pre-curriculum and post-curriculum surveys had a response rate of 61.1%. Student’s t-test showed a statistically significant increase in mean resident comfort level with the teaching strategies postcurriculum (3.05 to 3.83, p < 0.01). Medical student evaluations have shown, overall, that the majority of residents are utilizing the education techniques on-shift. There were no significant differences found in medical student perception of resident use of taught skills between those who had and had not attended the sessions. However, all but one assessed skill showed higher utilization in those who had attended the correlating session.Discussion: The educational content was effective in improving the residents’ comfort with the teaching strategies presented, and residents are utilizing these techniques on-shift. Through implementation, we discovered that presenting a curriculum over video conferencing required additional administrative support to help ensure efficacy of break-out groups. Based on resident feedback after the first session, multiple changes were made, including providing residents with hand-out references for use during the role-playing sessions. The success of this curriculum demonstrated th feasibility and utility of running a RAT curriculum entirely in a virtual format.

A third of infants with congenital diaphragmatic hernia (CDH) require a gastrostomy tube (GT) for nutritional support. We compared CDH infants who are GT-dependent to those able to meet their nutritional needs orally, to identify factors associated with requiring a GT and evaluate their long-term growth.Patients with CDH repaired at a single institution between 2012 and 2020 were included. Charts were retrospectively reviewed for demographic, surgical, and post-operative details. Mann-Whitney test and Fischer exact test were performed to compare GT-dependent neonates (n = 38, experimental) with orally fed neonates (n = 63, control). Significance was set at0.05.Thirty-eight percent received a GT (median 67 days, interquartile range [IQR] 50-88). GT-dependent neonates were significantly more likely to have a lower lung-to-head ratio (median 1.2, IQR 0.9-1.4, vs 1.6, IQR 1.3-2.0, IQR P 0.0001), undergone patch or flap repair (79% vs 33%, P 0.0001), and been hospitalized longer (median 47, IQR 24-75 vs 28 days, P 0.0001). Fourteen of 38 had their GT removed (median 26 months, IQR 14-36). GT-dependent neonates initiated oral feeds (calculated as time since extubation) later (median 21, IQR 8-26, vs 8 days, IQR 4-13, P = 0.006). Height-for-age z scores remained stable after GT removal, while weight-for-age z scores dropped initially and began improving a year later.The need for a gastrostomy for nutritional support is associated with more severe CDH. Over a third of patients no longer needed a GT at a median of 26 months. Linear growth generally remains stable after removal. These results may help counsel parents regarding nutritional expectations.

Infants with congenital diaphragmatic hernia (CDH) who require non-invasive positive pressure ventilation or high flow nasal cannula are at risk for aspiration and delayed initiation of oral feeding. We developed a dysphagia provider-led protocol that involved early consultation with an occupational therapist or speech/language pathologist and modified barium swallow study (MBSS) to assess for readiness for oral feeding initiation/advancement on non-invasive positive pressure ventilation. The objective of this study was to retrospectively compare this intervention cohort to a historical control cohort to evaluate the protocol's impact on the time to initiate oral feeding. We describe the development and implementation of the protocol, the MBSS findings of the intervention cohort, and compared the control (n = 64) and intervention (n = 37) cohorts using Fischer's exact test and Mann-Whitney test. We found that both cohorts had similar prenatal and neonatal characteristics including age at extubation. Significantly more infants in the intervention cohort were on non-invasive positive pressure ventilation or high flow nasal cannula at the time of oral feeding initiation (84% vs. 28%, p 0.0001). None of the control cohort infants underwent MBSS while on respiratory support. Of the intervention cohort, 15 infants underwent a MBSS while on non-invasive positive pressure ventilation; 6 had no evidence of laryngeal penetration and/or aspiration during swallowing. Infants in the control cohort initiated oral feeds significantly sooner after extubation (6 versus 11 days, p = 0.001) and attained full oral feeds earlier (20 days versus 28 days, p = 0.02) than the intervention group. There was no difference in the rate of gastrostomy tube placement (38%). Appropriate monitoring by a dysphagia provider and evaluation with clinical and radiological means are crucial to determine the safety of initiating oral feeding in term infants with CDH. Continued surveillance is needed to determine the long-term impact on oral feeding progression in this population.

The root hair-less brb of Hordeum vulgare L. (bald root barley) mutant was used to assess the significance that root hairs have for the hydraulic properties of roots and response to a limited supply of mineral nutrients in plants grown on hydroponics. The barley brb mutant and its parent wild-type (H. vulgare cv. Pallas) were grown under nutrient sufficient control conditions, and under conditions of low supply of P and N. Plants were analysed when they were 14–18 days old. Root hydraulic conductivity (Lp) was determined for excised root systems and intact transpiring plants, and cell Lp was determined through cell pressure probe measurements. The formation of Casparian bands and suberin lamellae was followed through staining of cross-sections. The presence or absence of root hairs had no effect on the overall hydraulic response of plants to nutritional treatments. Root and cell Lp did not differ between the two genotypes. The most apparent difference between brb and wild-type plants was the consistently reduced formation of apoplastic barriers in brb plants. Any hydraulic function of root hairs can be redundant in barley, at least under the hydroponic conditions tested.

Oncolytic viruses (OV) are defined by their intrinsic or engineered tropism for tumor cells. Understanding the drivers of susceptibility to infection as well as the downstream intracellular signaling and immune activation profile is critical in identifying the patients that will respond to OV therapy. Newcastle Disease Virus (NDV) is a negative stranded RNA avian paramyxovirus, here modified to encode human GM-CSF or IL-12. Patient derived xenografts (PDX) can more accurately reflect the genetic, epigenetic, and architectural heterogeneity of human tumors and were therefore selected to model the in vivo activity of NDV:GM-CSF. Eighty-nine independent models derived from patients with Bladder, Colorectal, Head and Neck, Kidney, Liver, Lung, Ovarian, Pancreatic, and Breast tumors were studied. Mice were dosed intravenously with NDV:GM-CSF, monitored for pharmacodynamic and pharmacokinetic changes 48 hours after the first dose, and followed for tumor growth inhibition. Objective response was observed in 68% of treated mice, with responses observed in all indications. Using total stranded RNAseq, we were able to assess changes in human tumor, murine immune infiltrate, and viral RNA. Results showed that NDV:GM-CSF treatment leads to increases in transcripts of inflammatory chemokines and cytokines such as RANTES and CXCL9, with induction directly correlated to level of viral replication. Additionally, we saw increases in NKp46 and IL-12R, markers of NK cells. To assess if IL-12 would augment immune activation, we treated surgically resected human tumors with NDV:IL-12. NDV:IL-12 was capable of infecting tumor cells, generating IL-12 protein, and driving a strong IFNγ response. Here we show that NDV can infect a wide variety of human tumors across multiple indications and drive a potent immune activation which can be modulated by transgenes. This data provides insight into what types of tumors could be treated with NDV:GM-CSF or NDV:IL-12. Citation Format: Nicholas M. Durham, Kelly McGlinchey, Shannon Burke, Todd Creasy, Nicola Rath, Noel Monks, Ravinder Tammali, Kevin Schifferli, Nick Holoweckyj, Susie Hayes, Emma Jones, Elizabeth J. Kelly, Danielle Carroll, James Harper, Katie Streicher. Newcastle Disease Virus in human derived tumors: Understanding immune recruitment and activation via virally delivered IL-12 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5544.

Yu et al. demonstrate that the transcription factor Bcl11b is specifically expressed in mouse innate lymphoid progenitors committed to the ILC2 lineage and is required for their development. Bcl11b-deficient mice exhibit a complete lack of ILC2 development, which is confirmed by immune challenges with either papain treatment or influenza virus infection., Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.

In this article, we examine responses to the March on Washington from the vantage point of Durham University, Durham, UK. As members of a course titled ‘Feminist Geographies of Intimacy’, we viewed the March on Washington as a prudent and timely event with which to think ‘on’ and ‘with’ intimacy and also to consider on a more personal level the significance of the March to our own feminism/s, feminist geographies and everyday lives. This piece is a collaborative effort written together by two instructors and eight students. We reflected on how we encountered ‘the March’ in the classroom in order to consider the varied locations and forms of protest; the range of issues that were fought for or fought against; the role of humour and other emotions when expressing dissent and/or solidarity; and the exclusion and inclusion of bodies which did or did not protest. The March for us, therefore, became a ‘generative space’ in that it provided a pedagogic tool with which to explore geographies of intimacy.

Patients who respond to PD-L1 / PD-1 immune checkpoint blockade tend to have an inflamed, TH1 polarised tumor microenvironment (TME), characterised by expression of interferon-γ (IFNγ) and PD-L1. Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor immunity. As a central mediator of TH1 immune responses, interleukin 12 (IL-12) directly induces IFNγ release from activated NK, NKT and T cells, and is known to play a key role in driving anti-tumor responses. However systemic recombinant IL-12 was poorly tolerated in early clinical trials. We therefore designed MEDI1191 as a novel IL-12-based therapy designed for injection directly into tumors, composed of a lipid nanoparticle (LNP)-formulated mRNA encoding human IL-12. We previously reported that intratumoral (IT) mouse (m) IL-12 mRNA, the surrogate for MEDI1191, promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models. Here, we demonstrate that IFNγ is also required for the anti-tumor activity of mIL-12 mRNA. A single dose of mIL-12 mRNA significantly increased expression of IFNγ and TH1 genes in MC38 tumor-bearing mice. Treatment with an IFNγ neutralising antibody blocked mIL-12 mRNA anti-tumor activity in this model. In addition, we report here that MC38 tumor rejection in response mIL-12 mRNA / anti-PD-L1 combination therapy correlates with increased cytotoxic T cell infiltration into tumors, and expansion of tumor-reactive T cells in the periphery. We next investigated the pharmocodynamic activity of MEDI1191 in patient tumor-derived models. A single IT dose of MEDI1191 induced human IL-12p70 expression in mice bearing four different patient-derived xenograft tumors. Furthermore, in an ex vivo patient tumor slice culture assay, MEDI1191 induced dose-dependent IL-12 release, IFNγ expression and upregulation of TH1-signature gene expression. IL-12 protein secretion was induced in slices of all patient tumors tested. However, the magnitude of the IFNγ response to MEDI1191 varied between patient tumors. Quantification of the tumoral T cell and NK cell numbers within the patient tumor samples revealed a positive correlation between MEDI1191-induced IFNγ release and baseline tumor NK infiltrate. These preclinical data demonstrate the potential for MEDI1191 to induce IFNγ-dependent TH1 transformation of the TME, and support the development of MEDI1191 as a potential treatment for patients with solid tumors, alone and in combination with inhibitors of the PD-1/PD-L1 T cell checkpoint. Citation Format: Nadia Luheshi, Susannah Hewitt, Fabien Garcon, Shannon Burke, Amanda Watkins, Kristen Arnold, John Zielinski, Philip Martin, Michael Sulikowski, Christopher Bagnall, Jean-Martin Lapointe, Gordon Moody, Han Si, Christopher Morehouse, Robert W. Wilkinson, Ronald Herbst, Joshua Frederick. MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5017.

Immuno-oncology (IO) therapies for cancer have recently delivered step-change improvements in patient outcomes, but the major proportion of patients remain unresponsive or become refractory to IO treatment. Because IO-based approaches necessarily rely on the interplay of multiple cell types, including immune cells, and other elements that constitute the tumor microenvironment (TME), innovative assays are critical to generate further translational insight. Indeed, results from in vivo mouse models and in vitro assays with human cell lines often do not correlate with clinical efficacy and as such, do not always represent relevant avatars. We have leveraged recent advances in tumor slice culture (TSC) techniques to enable in vitro mechanistic and novel biomarker testing/identification studies. TSC involves the in vitro culture of thick slices (200-300 μm) of fresh tissue prepared using a vibratome and is currently the only in vitro assay system which retains the 3-dimensional tissue architecture, complete with cell-cell and cell-matrix interactions. We are now regularly using the TSC together with tumor samples derived from patients and human cancer cell line derived xenograft or syngeneic mouse cancer models, to generate exciting pre-clinical data for several Oncology programs, including oncolytic viruses, modified RNA, bispecific antibodies and antibody-drug conjugates. For instance, the TSC platform has allowed us to analyse the strong type I IFN-driven inflammatory response initiated by infection of tumor nests within patient samples and demonstrate the consequent infiltration of immune cells from the surrounding stroma. Thus, using a tissue phenomics approach incorporating a range of endpoint assays, including multiplex whole-mount imaging, transcriptomics and cytokine expression, the data generated using the TSC platform will inform the underlying biology and translational strategies of multiple therapeutic candidates. Citation Format: Shannon Burke, Fabien Garcon, Kelli Ryan, James Harper, Robert W. Wilkinson. Tissue slice culture platform: A slice of reality in drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1190.

MEDI5395 is a genetically modified attenuated Newcastle disease virus (NDV). A reverse genetic system has overcome environmental and regulatory concerns by uncoupling oncolytic potency from avian pathogenicity. MEDI5395 has the intrinsic ability to infect and kill tumor cells and has been inserted with a GM-CSF transgene to potentiate a stronger adaptive immune response. Described here is an extensive in vitro and in vivo pharmacology package that reveals the broad oncolytic activity and immune-modulatory properties of MEDI5395 in a variety of pre-clinical models. In vitro, MEDI5395 lytic activity in human tumor cells is associated with elevated levels of tumor selective viral replication and expression of the GM-CSF transgene. MEDI5395 infection of immune cells indicated preferential uptake of virus and subsequent self-limiting replication in myeloid cells. Infected myeloid cells expressed cell surface activation markers (e.g. PD-L1) after 24 hours and were effective carriers of virus and mediated the transfer of infectious NDV particles to tumor cells resulting in death through oncolysis. Further mechanistic studies, indicated NDV-killed tumor cells released antigens that were capable of cross-presentation by dendritic cells driving activation of tumor antigen-specific autologous T cells. In murine models, IV delivery of NDV leads to long lasting tumor selective replication and transgene expression. MEDI5395 administration results in significant anti-tumor activity, observed in patient-derived xenograft models, and in murine syngeneic cancer models. Together, the results suggest that MEDI5395 may act to positively transform the tumor microenvironment. This, coupled to its tumor-selective oncolytic capacity, further underscore NDV as a promising multimodal cancer therapeutic platform and why FTIH studies are planned to start in 2019. Citation Format: James A. Harper, Shannon Burke, Andrew Leinster, Nicola Rath, Xing Cheng, Hong Jin, Robert W. Wilkinson, Danielle Carroll. MEDI5395: A recombinant oncolytic virus with oncolytic and immune modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1456.

T-cell development primarily occurs in the thymus and involves in the interactions of many important transcription factors. Until recently, no single transcription factor has been identified to be essential for T-cell lineage commitment or maintenance of T-cell identity. Recent studies have now identified the zinc finger transcription factor Bcl11b to be essential for T-cell development and for maintenance of T-cell identity. Remarkably, T cells acquire NK cell properties upon Bcl11b deletion. These reprogrammed cells have unique properties in proliferation, cytokine dependency and killing target cells, and may therefore provide a new cell source for some cell-based therapies.

Background Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. Results In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a+/+ and Bcl11a−/−). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a−/− HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a−/− HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a−/− HSCs are myeloerythroid-restricted and defective in self-renewal. Conclusions Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0739-5) contains supplementary material, which is available to authorized users.

Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has significant oncolytic activity against mammalian cancers. It is multi-modal in its anti-tumour activity and offers a tumour selective, self-propagating therapeutic with oncolytic activity and immunostimulatory properties. Within the tumour itself, NDV infection is able to modulate the immune suppressive microenvironment and induce positive anti-tumour inflammatory responses. Additionally, we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). To better understand the immune modulatory mechanisms by which NDV infection is able to alter the tumour microenvironment we investigated the responses of normal human PBMCs and isolated immune cell populations following NDV exposure. Using flow cytometry, cytokine and gene expression analysis we demonstrated activation of the innate immune cells and a robust type I IFN and pro-inflammatory response. 24 hours post infection, innate immune cells (macrophages, natural killer cells and dendritic cells) had upregulated cell surface activation markers and secreted high levels of cytokines in a dose-dependent fashion. Furthermore, by using NDV constructs encoding fluorescent proteins we showed, in a specific subset of myeloid cells, the preferential uptake of virus and subsequent self-limiting viral replication. In a co-culture system, these infected myeloid cells were able to function as ‘virus cellular carriers’ and were able to mediate the efficient transfer of infectious NDV to tumour cells resulting in their oncolytic death. To further investigate the ability of NDV to infect, spread and kill tumour cells in vivo, we utilised tissue slice cultures of fresh patient samples. These studies demonstrated virus replication and transgene expression in tumour slices, as well as the uptake of virus in a small fraction of specific cells in slices of normal liver. Moreover, evidence that NDV was able to alter the tumour microenvironment could be demonstrated in such cultures by sampling the culture supernatants over time. By investigating the immune modulatory properties of NDV in vitro and in vivo we will gain greater insight and understanding of the anti-cancer properties of NDV. This work will also help guide the selection of transgenes for next generation approaches to augment the inherent immunostimulatory properties of NDV, and help inform clinical dosing options such as intravenous infusion or cell-based delivery. Citation Format: Shannon Burke, Ruth Franks, James Harper, Jon Travers, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin, Danielle Carroll. Exploring the immune stimulatory properties of oncolytic Newcastle Disease Virus [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 4562. doi:10.1158/1538-7445.AM2017-4562

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell death occurs as a natural consequence of the viral life cycle and newly produced virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of potentially producing novel tumour antigens that can induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of mammalian cancers in PhI clinical studies. Using reverse genetics, we generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. We have additionally inserted the GM-CSF gene in order to maximize anti-tumour immune response and provide an in situ, patient/tumour- specific vaccine, combined with potent oncolysis. We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. In vitro recNDVGM-CSF selectively replicates in and kills a wide variety of tumour cell types. In vivo, using a fibrosarcoma model that supports viral replication, a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. Additionally, following IV administration recNDVGM-CSF localises to and replicates within the tumour leading to a localised GM-CSF production within the tumour. The inherent properties of NDV (self-propagation, tumour-selective replication, immune activation) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: James A. Harper, Jon Travers, Ruth Franks, Shannon Burke, Christel Navarro, Cheng Xing, Weijia Wang, Qi Xu, Robert W. Wilkinson, Hong Jin, Danielle Carroll. Exploring the oncolytic potential of Newcastle Disease Virus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2234.

Bcl11a regulates development of lymphoid cells in adult mice in part by inhibiting expression of p53., Transcription factors play important roles in lymphopoiesis. We have previously demonstrated that Bcl11a is essential for normal lymphocyte development in the mouse embryo. We report here that, in the adult mouse, Bcl11a is expressed in most hematopoietic cells and is highly enriched in B cells, early T cell progenitors, common lymphoid progenitors (CLPs), and hematopoietic stem cells (HSCs). In the adult mouse, Bcl11a deletion causes apoptosis in early B cells and CLPs and completely abolishes the lymphoid development potential of HSCs to B, T, and NK cells. Myeloid development, in contrast, is not obviously affected by the loss of Bcl11a. Bcl11a regulates expression of Bcl2, Bcl2-xL, and Mdm2, which inhibits p53 activities. Overexpression of Bcl2 and Mdm2, or p53 deficiency, rescues both lethality and proliferative defects in Bcl11a-deficient early B cells and enables the mutant CLPs to differentiate to lymphocytes. Bcl11a is therefore essential for lymphopoiesis and negatively regulates p53 activities. Deletion of Bcl11a may represent a new approach for generating a mouse model that completely lacks an adaptive immune system.

One Two TT cells develop in the thymus, where they proceed through several developmental stages, losing alternative lineage potential as they progress. The molecular regulation of this developmental process, however, is not fully understood (see the Perspective byDi Santo).P. Liet al.(p.85, published online 10 June),L. Liet al.(p.89), andIkawaet al.(p.93) now identify expression of the zinc finger transcription factorBcl11bas the earliest checkpoint in T cell development in mice. Genetic deletion ofBcl11bin developing T cells inhibited commitment to the T cell lineage. Under conditions that should have stimulated T lineage differentiation,Bcl11b-deficient T cell progenitors failed to up-regulate genes associated with lineage-committed T cells and maintained stem cell– and progenitor cell–associated gene expression. In both developing and committed T cells, loss ofBcl11bresulted in the generation of cells that resembled natural killer (NK) cells in both phenotype and function. These NK-like cells could be expanded easily in vitro and possessed antitumor cytotoxicity, but they did not exhibit cytotoxicity against normal cells and were not tumorigenic. Because T cells are much easier to obtain from human patients than NK cells, deletion ofBcl11bin T cells may thus provide a source of easy-to-grow NK cells for cell-based antitumor therapies.

Natural killer (NK) cell-based immunotherapies treat hematopoietic malignancies, but are less effective against solid tumors. Here, we review recent data on NK cell recognition of melanoma at various stages of the disease and propose a combinatorial strategy to exploit fully the potential of NK cells. Depending on the stage of melanoma progression, NK cell-based therapies could be combined with pharmacological and T cell-based immunotherapies, to: (i) prevent lymph node metastases by redistributing cytotoxic NK cells; (ii) boost NK cell activity using chemotherapy to upregulate activating ligands on tumor cells; and (iii) target visceral metastases by transfer of autologous or allogeneic NK cells.

Natural killer (NK) cells were discovered in the 1970 s and named after their naturally occurring cytotoxic activity against tumor cells. It has recently become clear that NK cells are not just killers and that malignancy is unlikely to be the selective pressure driving the evolution of NK cells. Indeed, NK cells secrete a host of cytokines and chemokines that contribute to tissue remodeling at the feto-maternal interface and to both innate and adaptive immunity during infection. Moreover, in certain conditions, they cannot deliver functions cell autonomously, as they require priming from other cells, namely dendritic cells. Nevertheless, natural cytotoxicity is still considered an important parameter used to evaluate NK cell biology, both in the clinic and in the research lab. In this chapter we describe a simple method to quantify spontaneous NK cell cytotoxicity in vivo.

Natural killer (NK) cells were discovered in the 1970 s and named after their naturally occurring cytotoxic activity against tumor cells. It has recently become clear that NK cells are not just killers and that malignancy is unlikely to be the selective pressure driving the evolution of NK cells. Indeed, NK cells secrete a host of cytokines and chemokines that contribute to tissue remodeling at the feto-maternal interface and to both innate and adaptive immunity during infection. Moreover, in certain conditions, they cannot deliver functions cell autonomously, as they require priming from other cells, namely dendritic cells. Nevertheless, natural cytotoxicity is still considered an important parameter used to evaluate NK cell biology, both in the clinic and in the research lab. In this chapter we describe a simple method to quantify spontaneous NK cell cytotoxicity in vivo.

Type 1 IFNs, innate cytokines with important effector and immunomodulatory properties, are rapidly induced in the acute phase of many virus infections; however, this is generally a transient response that is not sustained during virus persistence. To gain insight into mechanisms that can contribute to down-regulation of type 1 IFN production during virus persistence, we analyzed type 1 IFN production during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. High-level type 1 IFN production was transiently up-regulated in cells including plasmacytoid and conventional dendritic cells (DCs) following LCMV infection of mice, but LCMV persistence was associated with only low-level type 1 IFN production. Nonetheless, chronically infected mice were able to up-regulate type 1 IFN production in response to TLR3, 7, and 9 ligands, albeit less efficiently than uninfected mice. Splenic DC numbers in mice chronically infected with LCMV were decreased, and the remaining cells exhibited a reduced response to TLR stimulation. LCMV-infected cell lines efficiently up-regulated type 1 IFN production following TLR ligation and infection with a DNA virus, but exhibited a defect in type 1 IFN induction following infection with Sendai, an RNA virus. This block in type 1 IFN production by infected cells, together with abnormalities in DC numbers and functions, likely contribute to the low-level type 1 IFN production in mice chronically infected with LCMV. Impairment of type 1 IFN production may both promote virus persistence and impact on host immunocompetence. Understanding the mechanisms involved may assist in development of strategies for control of virus persistence and superinfection.

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell lysis occurs as a natural consequence of the viral life cycle and released virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of releasing multiple tumour antigens that may further induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of preclinical cancer models and in PhI clinical studies as an oncolytic agent, oncolysate or whole cell vaccine. Using reverse genetics, we have generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. Furthermore we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. RecNDVGM-CSF selectively replicates in and kills a wide variety of human and mouse tumour cell lines. Additionally NDV infection of cancer cells results in the increased production and secretion of pro-inflammatory cytokines and chemokines which are able to recruit mediators of both the innate and adaptive immune responses. NDV is a potent activator of the type I interferon response and is also able to directly activate many immune cell types. In vivo, using a range of syngeneic and xenograft models we have demonstrated that NDV treatment has profound anti-tumour activity. In a HT1080 fibrosarcoma xenograft model a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. In syngeneic mouse tumour models, NDV treatment causes profound changes in the immune suppressive microenvironment and can result in long-lasting anti-tumour immune responses. The inherent properties of NDV (self-propagation, tumour-selective replication, and immunostimulatory properties) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: Danielle K. Carroll, James Harper, Shannon Burke, Jon Travers, Ruth Franks, Christel Navarro, Xing Cheng, Robert W. Wilkinson, Hong Jin. Oncolytic Newcastle disease virus: armed but not dangerous. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5334. doi:10.1158/1538-7445.AM2015-5334