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Clinical management of gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) remains challenging. We recently introduced FMS-like tyrosine kinase 3 ligand (FLT3LG) as a possible biomarker for a proinflammatory tumor microenvironment. Here, we put a spotlight on the quantitative assessment of classical dendritic cells (cDC) and T cells in the context of FLT3LG mRNA levels in a retrospective study on NET G2/ G3 and NEC of pancreatic and gastric origin. Abundance of cDC and T cells and their relevant subpopulations were determined by immunofluorescent staining and correlated with FLT3LG mRNA levels as well as clinical outcome. Immune cell counts attested highly variable infiltration densities. Samples with presence of cDC or high numbers of T cells exhibited increased FLT3LG expression. Abundance of cDC, defined as HLA-DR+CD11c+ cells with CLEC9a (cDC1) or CD1c (cDC2), as well as CD3+ T cell and CTL (CD8+ CD3+) numbers correlated with FLT3LG mRNA levels and predicted disease-specific survival. Combining FLT3LG and T cell counts further improved this prediction. Therefore, tumor-infiltrating cDC and T cells are prognostic markers in NET G2/ G3 or NEC and FLT3LG mRNA may serve as a simple to use biomarker for a quantitative estimate of their abundance, mandating prospective evaluation in the context of immune-targeted therapies.

Transfer does not induce homeostatic inhibition on CD4+ T-cells

T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor–induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT—”immunotransplant”—increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies.Significance:For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant.See related commentary by Ansell, p. 1487.This article is highlighted in the In This Issue feature, p. 1469

Background & Aims: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. Methods: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. Results: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. Conclusion: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. Lay summary: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

BackgroundGastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) are a rare and heterogeneous family of tumors arising from the disseminated neuroendocrine system of the gastrointestinal tract and pancreas. Clinical management of high-grade GEP-NEN is challenging due to disease heterogeneity, illustrating the need for reliable biomarkers facilitating patient stratification and guiding treatment decisions. FMS-like tyrosine kinase 3 ligand (Flt3L) is emerging as a prognostic or predictive surrogate marker of host tumoral immune response and might enable stratification of patients with otherwise comparable tumor features.MethodsWe used RNAseq data from human foregut-derived pancreatic and gastric GEP-NEN to evaluate Flt3L gene expression in tumor tissue. The data set (n=54) represented the full range of NEN grades and differentiation, and expression levels were compared to healthy control tissue as well. We also analyzed circulating Flt3L levels in serum samples of a separate cohort of G2/G3 GEP-NEN (n=59) an healthy controls (n=4). The study was approved by the local ethics committee at Charité Universitätsmedizin Berlin, Germany (ethical approval number EA1/229/17) and patient informed consent was obtained.ResultsWe detected a prominent induction of Flt3L gene expression in individual G2 and G3 NEN, but not in G1 neuroendocrine tumors (NET). Flt3L mRNA expression levels in tumor tissue predicted disease related survival of patients with highly proliferative G2 and G3 NEN more accurately than the conventional criteria of grading or NEC/NET differentiation. High level Flt3L mRNA expression was associated with increased expression of genes related to immunogenic cell death, lymphocyte effector function and dendritic cell maturation, suggesting a less tolerogenic (more proinflammatory) phenotype of tumors with Flt3L induction. Importantly, circulating levels of Flt3L were also elevated in high grade NEN and correlated with patients´ progression-free and disease-related survival, thereby reflecting the results observed in tumor tissue.ConclusionsOur results suggest Flt3L as a surrogate marker of an inflammatory tumor microenvironment. Therefore, we propose Flt3L as a prognostic biomarker for high grade GEP-NEN. Flt3L measurements in serum, which can be easily be incorporated into clinical routine, may hold the promise to guide patient stratification and tailor treatment decisions and should be further evaluated, especially in the context of immunotherapies.Ethics ApprovalThe study was approved by the local ethics committee at Charité Universitätsmedizin Berlin, Germany (ethical approval number EA1/229/17) and patient informed consent was obtained.

Background and aims: Inflammatory cytokines represent diagnostic and prognostic biomarkers in manifold cancers. Recent data suggest a pivotal role of these cytokines in different biological processes involved in the development of neuroendocrine tumors (NETs). However, their role as biomarkers in NETs is only poorly understood. Methods: We analyzed serum concentrations of 13 inflammation-related cytokines at different time points in 43 patients with well-differentiated gastroenteropancreatic NETs (G1/G2) treated at Charité Berlin and compared them to 40 healthy controls. The results were correlated with clinical records. Results: Serum concentrations (Median (Interquartile Range (IQR)) in pg/mL) of IL-1β (124 (82) vs. 68 (61) pg/mL; p = 0.0003), IL-6 (111(122) vs. 88 (32) pg/mL; p = 0.0086), IL-8 (1058 (768) vs. 210 (90) pg/mL; p < 0.0001), IL-18 (2936 (1723) vs. 1590 (704) pg/mL; p < 0.0001), and TNF (271 (260) vs. 42 (25) pg/mL; p < 0.0001) were significantly elevated in NET patients, whereas IL-10 (43 (44) vs. 105 (48) pg/mL; p < 0.0001) showed lower concentrations in NETs when compared to controls. Cytokine levels significantly correlated with tumor grade (IL-6; p = 0.0070), prevalence of distant metastasis (IL-18; p = 0.0313), and disease progression over time (IL-10; p = 0.0033) but not tumor location. Chromogranin A (CgA) and the NETest are currently used to monitor treatment response. A more accurate prediction could possibly be achieved by employing a subset of cytokines. Our data clearly warrants further functional investigation into the role of the immune response and cytokine release in NETs. Conclusion: A biologically plausible panel of cytokines might be added to the diagnostic and prognostic tools currently employed in patients with NETs. Combining different markers into a score would elevate diagnostic accuracy compared to single markers.

Indolent non-Hodgkin’s lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8+ T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial ( NCT01976585 ). Non-responding patients developed a population of PD1+CD8+ T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy. In situ vaccine recruits and activates cross-presenting dendritic cells and augments PD1 blockade efficacy in patients with indolent non-Hodgkin’s lymphoma.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a rare, heterogeneous group of tumors that originate from the endocrine system of the gastrointestinal tract and pancreas. GEP-NENs are subdivided according to their differentiation into well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Since GEP-NENs represent rare diseases, only limited data from large prospective, randomized clinical trials are available, and recommendations for treatment of GEP-NEN are in part based on data from retrospective analyses or case series. In this context, tractable disease models that reflect the situation in humans and that allow to recapitulate the different clinical aspects and disease stages of GEP-NET or GEP-NEC are urgently needed. In this review, we highlight available data on mouse models for GEP-NEN. We discuss how these models reflect tumor biology of human disease and whether these models could serve as a tool for understanding the pathogenesis of GEP-NEN and for disease modeling and pharmacosensitivity assays, facilitating prediction of treatment response in patients. In addition, open issues applicable for future developments will be discussed.

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4 -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Conclusion Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).

T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor–induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT—”immunotransplant”—increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies. Significance: For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant. See related commentary by Ansell, p. 1487. This article is highlighted in the In This Issue feature, p. 1469

In almost all cases, hepatocellular carcinoma (HCC) develops as the endpoint of a sequence that starts with chronic liver injury, progresses to liver cirrhosis, and finally, over years and decades, results in liver cancer. Recently, the role of non-coding RNA such as microRNA (miRNA) has been demonstrated in the context of chronic liver diseases and HCC. Moreover, data from a phase II trial suggested a potential role of microRNAs as therapeutics in hepatitis-C-virus infection, representing a significant risk factor for development of liver cirrhosis and HCC. Despite progress in the clinical management of chronic liver diseases, pharmacological treatment options for patients with liver cirrhosis and/or advanced HCC are still limited. With their potential to regulate whole networks of genes, miRNA might be used as novel therapeutics in these patients but could also serve as biomarkers for improved patient stratification. In this review, we discuss available data on the role of miRNA in the transition from liver cirrhosis to HCC. We highlight opportunities for clinical translation and discuss open issues applicable to future developments.

Vaccination has had a tremendous impact on human health by harnessing the immune system to prevent and eradicate infectious diseases and this same approach might be used in cancer therapy. Cancer vaccine development has been slowed hindered by the paucity of universal tumor-associated antigens and the difficulty in isolating and preparing individualized vaccines ex vivo. Another approach has been to initiate or stimulate an immune response in situ (at the tumor site) and thus exploit the potentially numerous tumor-associated antigens there. Here, we review the many approaches that have attempted to accomplish effective in situ vaccination, using intratumoral administration of immunomodulators to increase the numbers or activation state of either antigen present cells or T cells within the tumor.

The past decade has seen clinical cancer research turn away from refining cytotoxic therapies to identifying immunomodulatory therapies that can harness the immune system to do the work of fighting cancer for us. The first revolution in cancer therapy brought us potent but toxic chemotherapies that were often initially good at controlling tumors; however, in the metastatic setting, recurrence was typically inevitable and more difficult to control. Immunotherapy has now emerged as among the most promising class of therapeutics for the treatment of cancer. These therapies harness the potent tumoricidal potential of cytotoxic effector cells of the patient’s own immune system. Although checkpoint-blocking antibodies approved in the past decade offer great promise, they non-specifically release the brakes on immune cells, and the majority of cancer patients are, so far, non-responders, while some patients experience toxic autoimmune adverse effects. One approach to improve on these limitations is with localized, in situ therapies, inducing a vaccinal response within the tumor microenvironment. These approaches alter the tumor microenvironment by inducing local immunogenic cell death to release tumor antigen, recruiting inflammatory leukocytes, priming of the adaptive immune response, and inhibiting tolerogenic mechanisms through which tumors dampen the immune response. Through a combined approach using some or all of these features, we are working to develop an in situ vaccine strategy that will result in systemic elimination of tumor cells as well as long-term remissions.

As cancer immunotherapy continues to benefit from novel approaches which cut immune 'brake pedals' (e.g. anti-PD1 and anti-CTLA4 antibodies) and push immune cell gas pedals (e.g. IL2, and IFNα) there will be increasing need to develop immune 'steering wheels' such as vaccines to guide the immune system specifically toward tumor associated antigens. Two primary hurdles in cancer vaccines have been: identification of universal antigens to be used in 'off-the-shelf' vaccines for common cancers, and 2) logistical hurdles of ex vivo production of individualized whole tumor cell vaccines. Here we summarize approaches using 'in situ vaccination' in which intratumoral administration of off-the-shelf immunomodulators have been developed to specifically induce (or amplify) T cell responses to each patient's individual tumor. Clinical studies have confirmed the induction of systemic immune and clinical responses to such approaches and preclinical models have suggested ways to further potentiate the translation of in situ vaccine trials for our patients.

While the cellular origin of lymphoma is often characterized by chromosomal translocations and other genetic aberrations, its growth and development into a malignant neoplasm is highly dependent upon its ability to escape natural host defenses. Neoplastic cells interact with a variety of non-malignant cells in the tumor milieu to create an immunosuppressive microenvironment. The resulting functional impairment and dysregulation of tumor-associated immune cells not only allows for passive growth of the malignancy but may even provide active growth signals upon which the tumor subsequently becomes dependent. In the past decade, the success of immune checkpoint blockade and adoptive cell transfer for relapsed or refractory lymphomas has validated immunotherapy as a possible treatment cornerstone. Here, we review the mechanisms by which lymphomas have been found to evade and even reprogram the immune system, including alterations in surface molecules, recruitment of immunosuppressive subpopulations, and secretion of anti-inflammatory factors. A fundamental understanding of the immune evasion strategies utilized by lymphomas may lead to better prognostic markers and guide the development of targeted interventions that are both safer and more effective than current standards of care.

Molecular factors driving immune-mediated inflammation in the liver are incompletely understood. The transcription factor, cyclic adenosine monophosphate-responsive element modulator alpha (CREMα) can endorse differentiation of T lymphocytes toward T-helper (Th)17 cells, thereby promoting autoimmunity in systemic lupus erythematosus or lung inflammation. To investigate the role of CREMα in liver disease, we subjected transgenic (Tg) mice overexpressing CREMα under control of the CD2 promoter (cremtg mice), which restrains expression mainly to lymphocytes (T, natural killer [NK], and NKT cells), to acute and chronic liver injury models. Already in steady state, Tg CREMα overexpression broadly reduced hepatic immune cell numbers by decreasing their viability, but did not affect immune cell migration or the fibrogenic response to chronic liver injury. Strikingly, cremtg mice developed more severe immune-mediated hepatitis with a higher mortality rate, compared to wild-type (wt) mice, upon concanavalin A (ConA) administration. Unlike in T cells from spleen, CREMα overexpression did not induce a predominant Th17 response in intrahepatic T cells, given that hepatic cremtg CD4+ T cells expressed less interleukin (IL)-17 than wt T cells. Reconstitution of Rag1−/− mice with Crem−/− T cells did not ameliorate ConA hepatitis. Overexpression of CREMα did not influence NK and NKT-cell effector functions either. Interestingly, a subset of monocytic myeloid-derived suppressor cells (MDSCs) also expressed CD2 and CREMα. Cremtg MDSCs isolated from liver expressed reduced inducible nitric oxide synthase and arginase 1 and displayed a reduced T-cell suppressive activity. The adoptive transfer of wt MDSCs was capable of reducing the fulminant immune-mediated liver damage in cremtg mice to wt level. Conclusion: These results suggest compartmental differences of T cell activation pathways between liver and other organs in autoimmunity and define a functional role of CREMα in hepatic monocytic MDSCs for the pathogenesis of immune-mediated liver disease. (Hepatology 2015;61:990–1002)

Acute kidney injury (AKI) is associated with high morbidity and mortality. Recent genetic fate mapping studies demonstrated that recovery from AKI occurs from intrinsic tubular cells. It is unresolved whether these intrinsic cells (so-called "scattered tubular cells") represent fixed progenitor cells or whether recovery involves any surviving tubular cell. Here, we show that the doxycycline-inducible parietal epithelial cell (PEC)-specific PEC-reverse-tetracycline transactivator (rtTA) transgenic mouse also efficiently labels the scattered tubular cell population. Proximal tubular cells labeled by the PEC-rtTA mouse coexpressed markers for scattered tubular cells (kidney injury molecule 1, annexin A3, src-suppressed C-kinase substrate, and CD44) and showed a higher proliferative index. The PEC-rtTA mouse labeled more tubular cells upon different tubular injuries but was independent of cellular proliferation as determined in physiological growth of the kidney. To resolve whether scattered tubular cells are fixed progenitors, cells were irreversibly labeled before ischemia reperfusion injury (genetic cell fate mapping). During recovery, the frequency of labeled tubular cells remained constant, arguing against a fixed progenitor population. In contrast, when genetic labeling was induced during ischemic injury and subsequent recovery, the number of labeled cells increased significantly, indicating that scattered tubular cells arise from any surviving tubular cell. In summary, scattered tubular cells do not represent a fixed progenitor population but rather a phenotype that can be adopted by almost any proximal tubular cell upon injury. Understanding and modulating these phenotypic changes using the PEC-rtTA mouse may lead to more specific therapies in AKI.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Chronic Lymphocytic Leukemia (CLL) is the predominant hematologic malignancy in the U.S. BTK inhibitors (BTKi) demonstrate prolonged survival compared to standard chemotherapy, but they also show notable toxicity, including increased risk for infections. While BTKi have shown both immunosuppressive and -stimulatory effects, the overall effect on T cells (TC), including tumor-reactive TC, is not well understood. Because non-BTK TEC-family and homologous kinases have critical roles in immune cells, we tested whether differentially specific BTKi have differing immunomodulatory effects. We treated mice with ibrutinib or acalabrutinib in vivo and analyzed their immune cell repertoire. Mass cytometry revealed that acalabrutinib, but not ibrutinib, increased the number of NK cells and myeloid cells in spleens and tumors of A20-lymphoma bearing mice, while both BTKi increased TC numbers in A20 tumors. Ibrutinib inhibited the activation of CD8 TC and NK cells isolated from BTKi-treated mice and their ability to lyse tumor cells ex vivo, while acalabrutinib improved CD8 TC-mediated cytolysis. A20 tumor growth was not influenced by BTKi in vivo, but ibrutinib inhibited the ability of transferred, antigen-specific TC to induce tumor regression. To translate our findings, we tested the effect of BTKi on immunogenicity of primary human CLL/MCL cells and their ability to stimulate TC. Ibrutinib inhibited upregulation of activation markers on tumor cells to a higher extent than acalabrutinib. Similarly, only ibrutinib inhibited autologous TC activation after co-culture with tumor cells and superantigen. In conclusion, ibrutinib, but not acalabrutinib inhibits the activation and function of anti-tumor T and NK cells in vivo and in vitro.

Oncolytic viruses (OV) represent a novel, mechanistically distinct therapy with significant anti-tumor potential and minimal toxicity. Crucial to OV efficacy is their induction of immunogenic cell death resulting in tumor associated antigen (TAA) uptake by dendritic cells (DCs) and cross-presentation to TAA-reactive T cells. Therefore, we hypothesized that OV efficacy would be increased by increasing intratumoral DCs. Because DCs are potently activated by type I IFNs and Newcastle disease virus (NDV) is a potent type I IFN inducer, we tested our hypothesis by treating A20 lymphoma-bearing mice with intratumoral Flt3L and NDV. In vitro, NDV significantly enhanced the susceptibility of A20 cells to CD8 T cell killing, in a TAA-specific manner. In vivo, the growth of A20 tumors was significantly inhibited by NDV; however NDV alone failed to eradicate some tumors. The addition of Flt3L potentiated the effect of NDV and led to sustained complete remissions in the majority of mice. Ex vivo characterization showed that Flt3L induced a potent accumulation of immature DCs in the tumor, spleen and lymph nodes, and NDV induced the activation of these DCs, as well as activation of NK and T cells. Further, Flt3L enhanced the ability of APCs to cross-prime TAA-specific T cells upon NDV-induced tumor cell death, as shown by increased proliferation and production of IFN-γ and TNF. The therapeutic effect of the Flt3L and NDV combination was completely lost in Batf3−/− mice (which lack cross-presenting DCs), demonstrating the importance of cross-priming in vivo. These findings support the use of NDV and Flt3L as a combined OV-immunotherapy with the potential to activate a powerful anti-tumor immune response and promote long-term immunological memory.

BACKGROUND In situ vaccination (ISV) against lymphoma can be achieved with Flt3-ligand treatment to recruit dendritic cells (DC), radiotherapy to load DC with tumor antigens, and intratumoral injection of a pattern-recognition- receptor agonist (PRRa) to activate antigen-loaded DC. While clinical trials of ISV using synthetic PRRa have yielded remissions, the optimal approach to activate DC is unknown. We hypothesize that ‘natural’ PRRa, such as the attenuated pathogens in common prophylactic vaccines, could target multiple PRR, leading to more robust activation of DC as compared to synthetic PRRa. METHODS 20 FDA-approved vaccines (BCG, MMR, etc.) were screened in vitro, for their effect on DC phenotype and function. DC tumor antigen cross-presentation was assessed using CRISPR gene-edited β2m−/−GFP-lymphoma cells and a GFP-specific CD8 T cell system. Mechanisms of immune activation were interrogated using MyD88, TRIF, MAVS, and IFNAR knockout mice, as well as a library of CRISPR-edited PRR-null Raw264 macrophages. Potent vaccines functioning through distinct mechanisms were evaluated in vivo in an ISV using the A20 murine lymphoma model. RESULTS Several vaccines induced robust DC activation, cross-presentation and increases in T cell activation, proliferation, and tumor killing. Some vaccines were more effective than synthetic PRRa in activating DCs to induce a T cell response. Distinct PRR and signaling pathways were engaged by different vaccines. In vivo, vaccine combination therapy induced tumor regression in a majority of animals. CONCLUSIONS Prophylactic vaccines, used alone or in combination, are effective DC activators and can be repurposed for use in ISV, with immediate translation into the clinic.

Chronic liver injury promotes hepatic inflammation, representing a prerequisite for organ fibrosis. We hypothesized a contribution of chemokine receptor CCR6 and its ligand, CCL20, which may regulate migration of T-helper (Th)17, regulatory, and gamma-delta (γδ) T cells. CCR6 and CCL20 expression was intrahepatically up-regulated in patients with chronic liver diseases (n = 50), compared to control liver (n = 5). Immunohistochemistry revealed the periportal accumulation of CCR6+ mononuclear cells and CCL20 induction by hepatic parenchymal cells in liver disease patients. Similarly, in murine livers, CCR6 was expressed by macrophages, CD4 and γδ T-cells, and up-regulated in fibrosis, whereas primary hepatocytes induced CCL20 upon experimental injury. In two murine models of chronic liver injury (CCl4 and methionine-choline-deficient diet), Ccr6−/− mice developed more severe fibrosis with strongly enhanced hepatic immune cell infiltration, compared to wild-type (WT) mice. Although CCR6 did not affect hepatic Th-cell subtype composition, CCR6 was explicitly required by the subset of interleukin (IL)-17- and IL-22-expressing γδ T cells for accumulation in injured liver. The adoptive transfer of WT γδ, but not CD4 T cells, into Ccr6−/− mice reduced hepatic inflammation and fibrosis in chronic injury to WT level. The anti-inflammatory function of hepatic γδ T cells was independent of IL-17, as evidenced by transfer of Il-17−/− cells. Instead, hepatic γδ T cells colocalized with hepatic stellate cells (HSCs) in vivo and promoted apoptosis of primary murine HSCs in a cell-cell contact-dependent manner, involving Fas-ligand (CD95L). Consistent with γδ T-cell-induced HSC apoptosis, activated myofibroblasts were more frequent in fibrotic livers of Ccr6−/− than in WT mice. Conclusion: γδ T cells are recruited to the liver by CCR6 upon chronic injury and protect the liver from excessive inflammation and fibrosis by inhibiting HSCs. (Hepatology 2014;59:630–642)

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).

Prophylactic vaccinations have been one of the greatest advances in modern medicine, both eradicating disease and reducing mortality. The translation of this advance into cancer therapy has been challenging and dates back to the turn of the twentieth century (Currie, Br J Cancer 26: 141–153, 1972). Cancer cells, derived from an aberrant clone, bear predominantly self-antigens and thus avoid alerting the immune system. In addition, the tumor microenvironment can be severely immunosuppressive; adding an extra layer of protection against the host immune response. Tumor cells can actively suppress immune responses through the downregulation of antigen presentation and the production of membrane-bound and secreted immuneregulatory molecules (Upadhyay et al, Cancers (Basel), 7: 736-62, 2015). To overcome such obstacles, a successful cancer vaccine must be able to induce a powerful immune response against tumor-associated antigens (TAAs) while avoiding normal host cells. This strategy has proven difficult because TAAs are highly variable in their immunogenicity and undergo immune editing to escape recognition. In addition, they can differ between tumor types and more importantly between individuals (Escors, New J Sci, 2014: 25, 2014). The presence of antigen-presenting cells (APCs) is generally low in the tumor microenvironment. Some efficacy in the treatment of cancer has been demonstrated by the use of autologous dendritic cells (DCs) pulsed with tumor cell lysates containing a whole array of antigens as well as single TAAs (Reichardt et al, Blood Rev: 18, 235-43, 2004). DC can be differentiated and expanded from peripheral blood ex vivo, and a resected tumor mass can be used to subsequently load the DC with TAAs. These strategies, while successful in developing a patient-specific vaccine, are labor and time intensive limiting the ability to experiment with numerous iterations to optimize the approach.

It is unclear why kidney dendritic cells attenuate some models of kidney disease but aggravate others. Kidney dendritic cells ameliorate the early phase of nonaccelerated nephrotoxic nephritis, a murine model of crescentic glomerulonephritis, but their effect on the later phase is unknown. Here, we report that kidney dendritic cells at later stages of nephrotoxic nephritis expressed higher levels of costimulatory molecules but lower levels of the cosuppressor molecule ICOS-L and started production of IL-12/23p40 and TNF-α. Furthermore, we noted that kidney dendritic cells captured more filterable antigen in proteinuric mice at late time points of nephrotoxic nephritis and started to capture molecules that were too large for filtration by a healthy kidney. They presented filtered antigen to Th cells, which responded by producing the proinflammatory cytokines IL-2, IFN-γ, TNF-α, IL-6, and IL-17. Notably, production of the suppressive cytokine IL-10 further increased in late nephrotoxic nephritis. Depletion of kidney dendritic cells at a late stage attenuated nephrotoxic nephritis, in contrast to the exacerbation observed with depletion at an early stage, indicating that their acquired proinflammatory phenotype adversely affected disease. These findings indicate that the intrarenal inflammatory microenvironment determines how kidney dendritic cells affect nephritis. In addition, proteinuria may harm the kidney by providing dendritic cells with more antigens to stimulate potentially pathogenic Th cells.

B cell lymphomas are generally incurable, with low response rates to anti-PD1 therapy. Here, we tested the combination of in situ vaccination with PD-1 blockade in a pre-clinical mouse model. A20 lymphoma-bearing mice were treated with a PD1 blocking antibody with or without an in situ vaccine combining intratumoral (it) injections of FMS-like tyrosine kinase-3 ligand (Flt3L), poly-ICLC (pIC) and local irradiation (XRT) of the tumor. Untreated lymphoma tumors contained few dendritic cells (DC) and anti-PD1 treatment did not induce tumor regression. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC in the tumor. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by it DC. Importantly, tumor antigens were taken up mainly by CD103+ DC and not CD103-DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103-subsets. Combination of Flt3L with XRT and pIC induced tumor-reactive T cells, and delayed tumor growth and improved survival in 40% of mice. In situ vaccination also increased expression of PD-L1 on tumor cells and it DC. Consistently, combination of in situ vaccination with anti-PD1 led to complete tumor regression and increased long-term survival in the majority of mice. PD1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. In conclusion, in situ vaccination combining Flt3L, local XRT and pIC improves efficacy of anti-PD1 in checkpoint-unresponsive lymphoma tumors through induction of a highly efficient cross-presenting DC subset.

76 Background: Low-grade non-Hodgkin’s B-cell lymphomas are generally incurable; preliminary results with anti-PD-1 therapy have yielded low response rates. Tumoral DC infiltration correlates with efficacy of checkpoint blockade and tumor-targeted vaccines represent promising, novel treatment strategies to induce anti-tumor T cells. Methods: A20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with an in situ vaccine (ISV) consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC). Results: Untreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC at the tumor site, the draining lymph node and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by DC. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory cross-presenting DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, IFNg-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. ISV also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistently, combination of ISV with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. Conclusions: In situ vaccination can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a cross-presenting DC subset leading to long-term regression of established lymphoma tumors.

123 Background: In patients with low-grade lymphoma, in situ vaccination has yielded both partial and complete remissions in clinical trials. Though clinical responses have been observed with multiple pattern recognition receptor agonists (PRRa), the optimal immune stimulant is unknown. We hypothesize that natural PRRa, such as the attenuated pathogens or subunits found in common prophylactic vaccines, could target multiple PRR in a physiologically relevant context and lead to a more robust activation of dendritic cells (DCs) versus synthetic PRRa. Methods: 20 vaccines, including BCG, Typhim Vi, MMR-II, etc. were screened in vitro, where DC phenotype and function were evaluated by flow cytometry. Flt3L-mobilized DC ability to phagocytose, process, present, and cross-present soluble protein or tumor derived antigen, were assessed using CRISPR gene-edited, β2m(-/-) GFP-lymphoma cells and a novel GFP-specific (‘JEDI’) CD8 T cell system. Vaccine mechanism of immune activation was elucidated using a library of PRR-null macrophage cell lines. Potent vaccines were also evaluated in vivo in a Flt3L-primed in situ vaccination using the A20 murine lymphoma model. Results: Several vaccines induced robust DC activation and several showed significant increases in subsequent T cell activation, proliferation, and tumor killing, suggesting increased antigen processing and cross-presentation by DCs. Some vaccines, either as single agents or in combination, were significantly more effective than synthetic PRRa in activating DCs and inducing a T cell response. In vivo, vaccine combination therapies induced tumor regression in a majority of animals, suggesting synergistic immune activation. Conclusions: This data suggests prophylactic vaccines are effective clinical-grade DC activators and can be repurposed for use in the in situ vaccination maneuver, with immediate translation into the clinic. Additionally, by extensive in vitro evaluation in parallel with in vivo studies, this work aims to identify a predictive in vitro molecular immune signature that correlates closely with adjuvant efficacy in vivo.

World journal of gastrointestinal pathophysiology 6(3), 43-50 (2015). doi:10.4291/wjgp.v6.i3.43, Published by Beijing Baishideng BioMed Scientific Co., Beijing

Background Low-grade non-Hodgkin's B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions. Preliminary results with anti-PD-1 therapy have yielded low response rates, though tumor-targeted vaccines represent promising, novel treatment strategies. In a pre-clinical mouse model, we attempt to develop and optimize an in situ vaccine combining recruitment of dendritic cells (DC) and low-dose local radiotherapy (XRT) with intratumoral (i.t) administration of a toll-like receptor (TLR) agonist Methods A20 lymphoma-bearing mice were injected i.t. with FMS-like tyrosine kinase-3 ligand (Flt3L) daily for 9 days (30ug/mouse), followed by local XRT (9Gy) and i.t. injections of poly-ICLC (50ug/mouse) for 5 days. Leukocyte accumulation in tumors, lymph nodes, and spleens was analyzed by flow cytometry and animals were monitored for tumor growth and survival. To assess uptake of tumor antigens by DC, mCherry-expressing A20 cells were used. For assessment of systemic anti-tumor response tumors were inoculated on both flanks, but only one site was treated as described before. In some groups, anti-PD-1 blocking antibody was injected systemically during vaccination. Results Injection of Flt3L induced potent accumulation of DC at the tumor site, tumor-draining lymph node (TDLN) and the spleen, with intratumoral injection being superior to systemic injection in increasing intratumoral and TDLN DCs. Interestingly, Flt3L-treatment led to an 8-fold increase in TLR3+ DC in the tumor. Local XRT increased the amount of mCherry+ DC in the tumor, indicating enhanced uptake of dying tumor cells. XRT of A20 cells also induced activation of Flt3L-treated splenic DC in vitro. While combination of FLt3L and local XRT was not able to cure established tumors, the combination of Flt3L and XRT with poly-ICLC induced long-lasting tumor regression in 40% of mice as well as regression of untreated tumors. This was accompanied by induction of tumor-reactive, Interferon γ (IFN γ)-producing T cells. Of note, the combination of Flt3L and XRT increased expression of PD-1 and PD-L1 on tumor infiltrating T cells and tumor cells, respectively. Consistently, systemic treatment with a PD-1 blocking antibody significantly enhanced the efficacy of the Flt3L-primed in situ vaccine leading to complete tumor regression at the treated site and a significant survival benefit compared to the in situ vaccine without PD-1 blockade. PD-1 blockade also increased the number of tumor-reactive T cells. Conclusions In situ vaccination combining intratumoral Flt3L injection with local XRT, poly-ICLC and anti-PD-1 induces a potent anti-tumor immune response able to induce long-term regression of established lymphoma tumors. Disclosures Davis: Celldex Therapeutics: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership. Salazar:Oncovir Inc: Employment. Brody:Gilead: Honoraria, Other: Travel expenses, Speakers Bureau; Acerta Pharma: Research Funding; Immunogen: Equity Ownership; Pharmacyclics: Honoraria, Other: Travel expenses, Speakers Bureau; Novavax: Equity Ownership; Merck: Consultancy, Research Funding; Seattle Genetics: Consultancy; Synergy Pharmaceuticals: Equity Ownership.

Chronic liver injury promotes hepatic inflammation, representing a prerequisite for organ fibrosis. We hypothesized a contribution of chemokine receptor CCR6 and its ligand, CCL20, which may regulate migration of T-helper (Th)17, regulatory, and gamma-delta (γδ) T cells. CCR6 and CCL20 expression was intrahepatically up-regulated in patients with chronic liver diseases (n = 50), compared to control liver (n = 5). Immunohistochemistry revealed the periportal accumulation of CCR6+ mononuclear cells and CCL20 induction by hepatic parenchymal cells in liver disease patients. Similarly, in murine livers, CCR6 was expressed by macrophages, CD4 and γδ T-cells, and up-regulated in fibrosis, whereas primary hepatocytes induced CCL20 upon experimental injury. In two murine models of chronic liver injury (CCl4 and methionine-choline-deficient diet), Ccr6−/− mice developed more severe fibrosis with strongly enhanced hepatic immune cell infiltration, compared to wild-type (WT) mice. Although CCR6 did not affect hepatic Th-cell subtype composition, CCR6 was explicitly required by the subset of interleukin (IL)-17- and IL-22-expressing γδ T cells for accumulation in injured liver. The adoptive transfer of WT γδ, but not CD4 T cells, into Ccr6−/− mice reduced hepatic inflammation and fibrosis in chronic injury to WT level. The anti-inflammatory function of hepatic γδ T cells was independent of IL-17, as evidenced by transfer of Il-17−/− cells. Instead, hepatic γδ T cells colocalized with hepatic stellate cells (HSCs) in vivo and promoted apoptosis of primary murine HSCs in a cell-cell contact-dependent manner, involving Fas-ligand (CD95L). Consistent with γδ T-cell-induced HSC apoptosis, activated myofibroblasts were more frequent in fibrotic livers of Ccr6−/− than in WT mice. Conclusion: γδ T cells are recruited to the liver by CCR6 upon chronic injury and protect the liver from excessive inflammation and fibrosis by inhibiting HSCs.

Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6+/gfp and Cxcr6gfp/gfp mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6−/− mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6−/− mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.