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Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α+ DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8+ T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity. [ABSTRACT FROM AUTHOR]

Peptides have been established as modular catalysts for various transformations. Still, the vast number of potential amino acid building blocks renders the identification of peptides with desired catalytic activity challenging. Here, we develop a machine-learning workflow for the optimization of peptide catalysts. First-in a hypothetical competition-we challenged our workflow to identify peptide catalysts for the conjugate addition reaction of aldehydes to nitroolefins and compared the performance of the predicted structures with those optimized in our laboratory. On the basis of the positive results, we established a universal training set (UTS) containing 161 catalysts to sample an in silico library of ∼30,000 tripeptide members. Finally, we challenged our machine learning strategy to identify a member of the library as a stereoselective catalyst for an annulation reaction that has not been catalyzed by a peptide thus far. We conclude with a comparison of data-driven versus expert-knowledge-guided peptide catalyst optimization., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Due to the chemical and biological relevance of amino acids, efficient methods for the recognition and separation of their enantiomers are highly sought after. Chiral receptors based on extended molecular scaffolds are typically employed for this purpose. These receptors are often effective only in specific environments and towards a narrow scope of amino acid guests. Recently we reported a simple, glucose-based macrocycle capable of enantioselective binding of a broad range of amino acid methyl esters in water. Herein we demonstrate that the same receptor can be used for chiral recognition of amino acid esters in organic solvents. We show that the binding affinity and selectivity of the receptor are highly dependent on the coordinating strength of the solvent. An in-depth analysis of the receptor's conformation and its interactions with amino acid methyl esters allowed us to propose a binding mode of amino acids to the receptor in CDCl 3 . The binding modes in CDCl 3 and D 2 O were then compared, highlighting the main interactions responsible for binding affinity and selectivity in each solvent. We envision that the insight provided by this study will facilitate the development of further amino acid receptors based on monosaccharides with improved binding affinities and both enantio- as well as chemoselectivities.

Purpose: Physiotherapists work with people with chronic conditions and can act as catalysts for behavioural change. Physiotherapy has also seen a shift to a bio-psychosocial model of health management and interdisciplinary care, which is important in the context of chronic conditions. This scoping review addressed the research question "How do physiotherapists use mental health-based interventions in their treatment of individuals with chronic conditions?" Method: The Embase, MEDLINE, PsycINFO, and CINAHL databases were searched, and a variety of study designs were included. Data were categorized and descriptively analyzed. Results: Data were extracted from 103 articles. Low back pain (43; 41.7%) and non-specified pain (16; 15.5%) were the most commonly researched chronic conditions, but other chronic conditions were also represented. Outpatient facilities were the most common setting for intervention (68; 73.1%). A total of 73 (70.9%) of the articles involved cognitive-behavioural therapy, and 41 (40.0%) included graded exercise or graded activity as a mental health intervention. Conclusions: Physiotherapists can use a variety of mental health interventions in the treatment of chronic conditions. More detailed descriptions of treatment and training protocols would be helpful for incorporating these techniques into clinical practice., (© Canadian Physiotherapy Association.)

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Linder, Bothe, Linder, Schwarzlmueller, Dahlström, Bartenhagen, Dugas, Pandey, Thorn-Seshold, Boehmer, Koenig, Kobold, Schnurr, Raedler, Spielmann, Karimzadeh, Schmidt, Endres and Rothenfusser.)

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r -/- - but not Il-1r -/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8 + T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4 flox . In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.

An exacerbated and unbalanced immune response may account for the severity of COVID-19, the disease caused by the novel severe acute respiratory syndrome (SARS) coronavirus 2, SARS-CoV-2. In this Viewpoint, we summarize recent evidence for the role of neutrophils in the pathogenesis of COVID-19 and propose CXCR2 inhibition as a promising treatment option to block neutrophil recruitment and activation., (© 2020 Koenig et al.)

Background: The tumor microenvironment (TME) combines features of regulatory cytokines and immune cell populations to evade the recognition by the immune system. Myeloid-derived suppressor cells (MDSC) comprise populations of immature myeloid cells in tumor-bearing hosts with a highly immunosuppressive capacity. We could previously identify RIG-I-like helicases (RLH) as targets for the immunotherapy of pancreatic cancer inducing immunogenic tumor cell death and type I interferons (IFN) as key mediators linking innate with adaptive immunity., Methods: Mice with orthotopically implanted Kras G12D p53 fl/R172H Ptf1a-Cre (KPC) pancreatic tumors were treated intravenously with the RLH ligand polyinosinic-polycytidylic acid (poly(I:C)), and the immune cell environment in tumor and spleen was characterized. A comprehensive analysis of the suppressive capacity as well as the whole transcriptomic profile of isolated MDSC subsets was performed. Antigen presentation capability of MDSC from mice with ovalbumin (OVA)-expressing tumors was investigated in T cell proliferation assays. The role of IFN in MDSC function was investigated in Ifnar1 -/- mice., Results: MDSC were strongly induced in orthotopic KPC-derived pancreatic cancer, and frequencies of MDSC subsets correlated with tumor weight and G-CSF serum levels, whereas other immune cell populations decreased. Administration of the RLH-ligand induced a IFN-driven immune response, with increased activation of T cells and dendritic cells (DC), and a reduced suppressive capacity of both polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC fractions. Whole transcriptomic analysis confirmed an IFN-driven gene signature of MDSC, a switch from a M2/G2- towards a M1/G1-polarized phenotype, and the induction of genes involved in the antigen presentation machinery. Nevertheless, MDSC failed to present tumor antigen to T cells. Interestingly, we found MDSC with reduced suppressive function in Ifnar1-deficient hosts; however, there was a common flaw in immune cell activation, which was reflected by defective immune cell activation and tumor control., Conclusions: We provide evidence that the treatment with immunostimulatory RNA reprograms the TME of pancreatic cancer by reducing the suppressive activity of MDSC, polarizing myeloid cells into a M1-like state and recruiting DC. We postulate that tumor cell-targeting combination strategies may benefit from RLH-based TME remodeling. In addition, we provide novel insights into the dual role of IFN signaling in MDSC's suppressive function and provide evidence that host-intrinsic IFN signaling may be critical for MDSC to gain suppressive function during tumor development.

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)-mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4 -/- mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo , a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4 -/- mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies., (Copyright © 2019 Lorenz, Moschovaki-Filippidou, Würf, Metzger, Steiger, Batz, Carbajo-Lozoya, Koziel, Schnurr, Cohen, Schmaderer, Anders, Lindenmeyer and Lech.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with poor prognosis and rising incidence. Late detection and a particularly aggressive biology are the major challenges which determine therapeutic failure. In this review, we present the current status and the recent advances in PDAC treatment together with the biological and immunological hallmarks of this cancer entity. On this basis, we discuss new concepts combining distinct treatment modalities in order to improve therapeutic efficacy and clinical outcome - with a specific focus on protocols involving radio(chemo)therapeutic approaches.

Background: Decreased organ function following liver resection is a major clinical issue. The practical method of ischemic postconditioning (IPostC) has been studied in heart diseases, but no data exist regarding fibrotic livers., Aims: We aimed to determine whether IPostC could protect healthy, fibrotic, and cirrhotic livers from ischemia reperfusion injury (IRI)., Methods: Fibrosis was induced in male SD rats using bile duct ligation (BDL, 4 weeks), and cirrhosis was induced using thioacetamide (TAA, 18 weeks). Fibrosis and cirrhosis were histologically confirmed using HE and EvG staining. For healthy, fibrotic, and cirrhotic livers, isolated liver perfusion with 90 min of warm ischemia was performed in three groups (each with n=8): control, IPostC 8x20 sec, and IPostC 4x60 sec. additionally, healthy livers were investigated during a follow-up study. Lactate dehydrogenase (LDH) and thromboxane B 2 (TXB 2 ) in the perfusate, as well as bile flow (healthy/TAA) and portal perfusion pressure, were measured., Results: LDH and TXB 2 were reduced, and bile flow was increased by IPostC, mainly in total and in the late phase of reperfusion. The follow-up study showed that the perfusate derived from a postconditioned group had much less damaging potential than perfusate derived from the nonpostconditioned group., Conclusion: IPostC following warm ischemia protects healthy, fibrotic, and cirrhotic livers against IRI. Reduced efflux of TXB 2 is one possible mechanism for this effect of IPostC and increases sinusoidal microcirculation. These findings may help to improve organ function and recovery of patients after liver resection.

Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.

Inflammatory bowel disease (IBD) is associated with enhanced levels of the IL-1 family cytokines IL-1β and IL-18, which are activated by the Nlrp3 inflammasome. Here, we investigated the role of inflammasome-driven cytokine release on T cell polarization and DC differentiation in steady state and T cell transfer colitis. In vitro and in vivo data showed that IL-1β induces Th17 polarization and increases GM‑CSF production by T cells. Reduced IL-1β levels in Nlrp3-/- mice correlated with enhanced FLT3L levels and increased frequency of tolerogenic CD103+ DC. In the T cell transfer colitis model, Nlrp3 deficiency resulted in lower IL‑1β levels, reduced Th17 immunity, and less severe colitis. Unaltered IL-18 levels in both mouse strains pointed toward Nlrp3-independent processing. Importantly, cohousing revealed that the gut microbiome had no impact on the observed Nlrp3-/- phenotype. This study demonstrates that NLRP3 acts as a molecular switch of intestinal homeostasis by shifting local immune cells toward an inflammatory phenotype via IL-1β.

Volcanic ash is a heterogeneous mineral dust that is typically composed of a mixture of amorphous (glass) and crystalline (mineral) fragments. It commonly contains an abundance of the crystalline silica (SiO 2 ) polymorph cristobalite. Inhalation of crystalline silica can induce inflammation by stimulating the NLRP3 inflammasome, a cytosolic receptor complex that plays a critical role in driving inflammatory immune responses. Ingested material results in the assembly of NLRP3, ASC, and caspase-1 with subsequent secretion of the interleukin-1 family cytokine IL-1β. Previous toxicology work suggests that cristobalite-bearing volcanic ash is minimally reactive, calling into question the reactivity of volcanically derived crystalline silica, in general. In this study, we target the NLRP3 inflammasome as a crystalline silica responsive element to clarify volcanic cristobalite reactivity. We expose immortalized bone marrow-derived macrophages of genetically engineered mice and primary human peripheral blood mononuclear cells (PBMCs) to ash from the Soufrière Hills volcano as well as representative, pure-phase samples of its primary componentry (volcanic glass, feldspar, cristobalite) and measure NLRP3 inflammasome activation. We demonstrate that respirable Soufrière Hills volcanic ash induces the activation of caspase-1 with subsequent release of mature IL-1β in a NLRP3 inflammasome-dependent manner. Macrophages deficient in NLRP3 inflammasome components are incapable of secreting IL-1β in response to volcanic ash ingestion. Cellular uptake induces lysosomal destabilization involving cysteine proteases. Furthermore, the response involves activation of mitochondrial stress pathways leading to the generation of reactive oxygen species. Considering ash componentry, cristobalite is the most reactive pure-phase with other components inducing only low-level IL-1β secretion. Inflammasome activation mediated by inhaled ash and its potential relevance in chronic pulmonary disease was further evidenced in PBMC using the NLRP3 small-molecule inhibitor CP-456,773 (CRID3, MCC950). Our data indicate the functional activation of the NLRP3 inflammasome by volcanic ash in murine and human macrophages in vitro . Cristobalite is identified as the apparent driver, thereby contesting previous assertions that chemical and structural imperfections may be sufficient to abrogate the reactivity of volcanically derived cristobalite. This is a novel mechanism for the stimulation of a pro-inflammatory response by volcanic particulate and provides new insight regarding chronic exposure to environmentally occurring particles.

IL-22 has been identified as a cancer-promoting cytokine that is secreted by infiltrating immune cells in several cancer models. We hypothesized that IL-22 regulation would occur at the interface between cancer cells and immune cells. Breast and lung cancer cells of murine and human origin induced IL-22 production from memory CD4 + T cells. In the present study, we found that IL-22 production in humans is dependent on activation of the NLRP3 inflammasome with the subsequent release of IL-1β from both myeloid and T cells. IL-1 receptor signaling via the transcription factors AhR and RORγt in T cells was necessary and sufficient for IL-22 production. In these settings, IL-1 induced IL-22 production from a mixed T helper cell population comprised of Th1, Th17, and Th22 cells, which was abrogated by the addition of anakinra. We confirmed these findings in vitro and in vivo in two murine tumor models, in primary human breast and lung cancer cells, and in deposited expression data. Relevant to ongoing clinical trials in breast cancer, we demonstrate here that the IL-1 receptor antagonist anakinra abrogates IL-22 production and reduces tumor growth in a murine breast cancer model. Thus, we describe here a previously unrecognized mechanism by which cancer cells induce IL-22 production from memory CD4 + T cells via activation of the NLRP3 inflammasome and the release of IL-1β to promote tumor growth. These findings may provide the basis for therapeutic interventions that affect IL-22 production by targeting IL-1 activity., Competing Interests: The authors declare no conflict of interest.

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6-24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC-MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled "Mitochondrial adaptation in steatotic mice" (Einer et al., 2017) [1].

Background: A number of new drugs for tumor immunotherapy have been approved in the past few years. They work by activating T cells to combat tumors., Methods: This review is based on publications on recently approved T-cell-activating drugs that were retrieved by a selective search in PubMed., Results: Randomized, controlled trials of "checkpoint" inhibitors, i.e., inhibitory antibodies for use against tumors, have shown that the cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor ipilimumab can prolong the survival of patients with advanced melanoma by 2 to 4 months. No data on median overall survival are yet available for the two programmed-death-1 (PD-1) inhibitors pembrolizumab und nivolumab; the endpoint "tumor response" was achieved in 24% and 32% of patients receiving these drugs, respectively. Grade 3 or 4 adverse effects occurred in 50% of patients receiving ipilimumab and in 12 to 13% of those taking either of the two PD-1-inhibitors. Nivolumab prolonged the median survival of patients with metastatic non-small-cell lung cancer from 6 to 9 months. In refractory or recurrent Philadelphia-chromosome-negative pre-B acute lymphoblastic leukemia (pre-B-ALL), treatment with the bispecific antibody construct blinatumomab led to complete remission in 43% of the patients, while grade 3, 4 or 5 toxicities occurred in 83%., Conclusion: T-cell-directed strategies have been established as a new pillar of treatment in medical oncology. As these drugs have frequent and severe adverse effects, therapeutic decision-making will have to take account not only of the predicted prolongation of survival, but also of the potential for an impaired quality of life while the patient is under treatment.

Background: Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance., Methods: Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided., Results: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors., Conclusions: Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The RIG-I-like helicase melanoma differentiation-associated protein 5 (MDA5) is an innate immune receptor for double-stranded viral RNA (dsRNA) that, upon activation, induces a Type I interferon (IFN)-driven immune response. In the present study, we demonstrate that human und murine pancreatic cancer cells express functional MDA5 and are highly sensitive to MDA5-induced cell death. Activation of MDA5 by cytosolic delivery of the synthetic dsRNA analog poly(I:C) led to phosphorylation of the transcription factor IRF3, IFNβ production and upregulation of MHC-I expression. MDA5 signaling also induced tumor cell apoptosis via the intrinsic pathway and sensitized tumor cells toward extrinsic, Fas-mediated apoptosis. Systemic treatment of orthotopic pancreatic cancer-bearing mice with the MDA5 ligand resulted in activated CD8 + T cell tumor infiltration, an increased frequency of tumor antigen-specific CD8 + T cells and an immunogenic cytokine milieu in the tumor microenvironment. These effects were paralleled by MDA5-induced pronounced tumor cell death in situ and significantly prolonged survival in two different mouse models for pancreatic cancer, an immunotherapeutic response dependent on CD8 + T cells. Treated mice were further protected from subsequent tumor challenge. In summary, we identified MDA5 as a novel therapeutic target for overcoming apoptosis resistance and tumor-mediated immunosuppression in pancreatic cancer. MDA5 ligands link innate with adaptive immune mechanisms for effective tumor control.

Despite continuous progress in the understanding of deregulated pathways in pancreatic cancer cells and development of targeted therapies, therapeutic advances with clinical benefit have been scarce over the last decades. The recent success of immunotherapy for some solid cancers has fueled optimism that this approach might also work for pancreatic cancer. However, a highly immunosuppressive microenvironment mediated by tumor, stromal and immune cells creates a major hurdle for immunotherapy. Mouse models have helped to unravel critical immunosuppressive mechanisms that could serve as novel therapeutic targets. Here we review new promising strategies that alone or in combination with other modalities, such as chemotherapy or irradiation, have the potential to lead to tumor immune control and finally better clinical outcome.

RIG-I-like helicases (RLH) are cytosolic sensors for viral RNA inducing type I interferon production. We found that pancreatic cancer cells express functional RLH and are susceptible to RLH-induced apoptosis via intrinsic and extrinsic pathways. Tumor cells displayed features of immunogenic cell death resulting in dendritic cell activation, enhanced antigen cross-presentation and efficient tumor control in vivo .

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy., Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided., Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells., Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model., Materials and Methods: Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8(+) T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively., Results: Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8(+) T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8(+) T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination., Conclusions: Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon ((129)Xe) atoms in cryptophane-A-monoacid (CrAma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrAma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75-98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Introduction: In lung cancer, interleukin-22 (IL-22) expression within primary tissue has been demonstrated, but the frequency and the functional consequence of IL-22 signaling have not been addressed. This study aims at analyzing the cellular effects of IL-22 on lung carcinoma cell lines and the prognostic impact of IL-22 tissue expression in lung cancer patients., Methods: Biological effects of IL-22 signaling were investigated in seven lung cancer cell lines by Western blot, flow cytometry, real-time polymerase chain reaction, and proliferation assays. Tumor tissue specimens of two cohorts with a total of 2300 lung cancer patients were tested for IL-22 expression by immunohistochemistry. IL-22 serum concentrations were analyzed in 103 additional patients by enzyme-linked immunosorbent assay., Results: We found the IL-22 receptor 1 (IL-22-R1) to be expressed in six of seven lung cancer cell lines. However IL-22 signaling was functional in only four cell lines, where IL-22 induced signal transducer activator of transcription 3 phosphorylation and increased cell proliferation. Furthermore, IL-22 induced the expression of antiapoptotic B-cell lymphoma 2, but did not rescue tumor cells from carboplatin-induced apoptosis. Cisplatin-resistant cell lines showed a significant up-regulation of IL-22-R1 along with a stronger proliferative response to IL-22 stimulation. IL-22 was preferentially expressed in small- and large-cell lung carcinoma (58% and 46% of cases, respectively). However, no correlation between IL-22 expression by immunohistochemistry and prognosis was observed., Conclusion: IL-22 is frequently expressed in lung cancer tissue. Enhanced IL-22-R1 expression and signaling in chemotherapy-refractory cell lines are indicative of a protumorigenic function of IL-22 and may contribute to a more aggressive phenotype.

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that is activated by 5'-triphosphate RNA molecules to induce type I interferon secretion and apoptosis in response to viral infection. We have designed a bifunctional small-interfering RNA that combines transforming growth factor β silencing with RIG-I activation to break tumor-induced immunosuppression. This strategy showed therapeutic efficacy in a murine model of pancreatic cancer.

Deregulated TGF-β signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-β by siRNA. By introducing a triphosphate group at the 5' end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9-mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-β1 (ppp-TGF-β) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-β reduced systemic and tumor-associated TGF-β levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-β significantly prolonged survival as compared with ppp-RNA or TGF-β siRNA alone. Furthermore, we observed the recruitment of activated CD8(+) T cells to the tumor and a reduced frequency of CD11b(+) Gr-1(+) myeloid cells. Therapeutic efficacy was dependent on CD8(+) T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-β gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8(+) T cell suppression.

Densely arranged pericytes engird the endothelial tube of all coronary microvessels. Since the experimental access to these abundant cells in situ is difficult, a prerequisite for broader investigation is the availability of sufficient numbers of fully differentiated pericytes in homogenous culture. To reach this goal, we applied strictly standardized cell isolation techniques, optimized culture methods and specific histological staining. Approximately 1,000-fold enriched pericytes were proteolytically detached from highly purified coronary microvascular networks (density gradient centrifugation) of eight mammalian species including human. Addition of species-autologous fetal or neonatal serum (10-20% vol/vol) was a precondition for longer term survival of homogenous pericyte cultures. This ensured optimal growth (doubling time <14 h) and full expression of pericyte-specific markers. In 3-mo, 10(10) pericytes (15 g) could be cultivated from 1 bovine heart. Pericytes could be stored in liquid N(2), recultured, and passaged repeatedly without loss of typical features. In cocultures with EC or vascular smooth muscle cells, pericytes transferred fluorescent calcein to each other and to EC via their antler-like extensions, organized angiogenetic sprouting of vessels, and rapidly activated coagulation factors X and II via tissue factor and prothrombinase. The interconnected pericytes of the coronary system are functionally closely correlated with the vascular endothelium and may play key roles in the adjustment of local blood flow, the regulation of angiogenic processes, and the induction of procoagulatory processes. Their successful bulk cultivation enables direct experimental access under defined in vitro conditions and the isolation of pericyte specific antigens for the production of specific antibodies.

Background and Aims: Dendritic cell (DC)-based vaccination can induce antitumor T cell responses in vivo. This clinical pilot study examined feasibility and outcome of DC-based tumor vaccination for patients with advanced pancreatic adenocarcinoma., Methods: Tumor lysate of patients with pancreatic carcinoma was generated by repeated freeze-thaw cycles of surgically obtained tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were generated from peripheral blood mononuclear cells (PBMC), loaded with autologous tumor lysate, stimulated with TNF-α and PGE(2) and injected intradermally. All patients received concomitant chemotherapy with gemcitabine. Disease response was the primary endpoint. Individual immunological responses to DC vaccination were analyzed by T cell-based immunoassays using pre- and post-vaccination samples of non-adherent PBMC., Results: Twelve patients received DC vaccination and concomitant chemotherapy. One patient developed a partial remission, and two patients remained in stable disease. Median survival was 10.5 months. No severe side effects were observed. Tumor-reactive T cells could be detected prior to vaccination. DC vaccination increased the frequency of tumor-reactive cells in all patients tested; however, the degree of this increase varied. To quantify the presence of tumor-reactive T cells, stimulatory indices (SI) were calculated as the ratio of proliferation-inducing capacity of lysate-loaded versus -unloaded DC. The patient with longest overall survival of 56 months had a high SI of 6.49, indicating that the presence of a pre-vaccination antitumor T cell response might be associated with prolonged survival. Five patients survived 1 year or more., Conclusion: DC-based vaccination can stimulate an antitumoral T cell response in patients with advanced or recurrent pancreatic carcinoma receiving concomitant gemcitabine treatment.

The ability of dendritic cells (DCs) to cross-present protein tumor antigens to cytotoxic T lymphocytes (CTLs) underpins the success of therapeutic cancer vaccines. We studied cross-presentation of the cancer/testis antigen, NY-ESO-1, and the melanoma differentiation antigen, Melan-A by human DC subsets. Monocyte-derived DCs (MoDCs) efficiently cross-presented human leukocyte associated (HLA)-A2-restricted epitopes from either a formulated NY-ESO-1/ISCOMATRIX vaccine or when either antigen was mixed with ISCOMATRIX adjuvant. HLA-A2 epitope generation required endosomal acidification and was proteasome-independent for NY-ESO-1 and proteasome-dependent for Melan-A. Both MoDCs and CD1c(+) blood DCs cross-presented NY-ESO-1-specific HLA-A2(157-165)-, HLA-B7(60-72)-, and HLA-Cw3(92-100)-restricted epitopes when formulated as an NY-ESO-1/ISCOMATRIX vaccine, but this was limited when NY-ESO-1 and ISCOMATRIX adjuvant were added separately to the DC cultures. Finally, cross-presentation of NY-ESO-1(157-165)/HLA-A2, NY-ESO-1(60-72)/HLA-B7, and NY-ESO-1(92-100)/HLA-Cw3 epitopes was proteasome-dependent when formulated as immune complexes (ICs) but only proteasome-dependent for NY-ESO-1(60-72)/HLA-B7-restricted cross-presentation facilitated by ISCOMATRIX adjuvant. We demonstrate, for the first time, proteasome-dependent and independent cross-presentation of HLA-A-, B-, and C-restricted epitopes within the same full-length tumor antigen by human DCs. Our findings identify important differences in the capacities of human DC subsets to cross-present clinically relevant, full-length tumor antigens and how vaccine formulation impacts CTL responses in vivo.

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal, dermal, and pulmonary SLE manifestations of MRL(Fas)lpr mice. However, even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts, which were significantly reduced in CYC-treated mice. Together, the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence, DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC.

The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.

Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-alpha, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.

Plasmacytoid dendritic cells (PDCs) play powerful regulatory roles in innate and adaptive immune responses and are a major source of type I interferon (IFN) following viral infection. During inflammation and mechanical stress, cells release nucleotides into the extracellular space where they act as signaling molecules via G protein-coupled P2Y receptors. We have previously reported on the regulation of myeloid dendritic cell (DC) function by nucleotides. Here, we report that human PDCs express several subtypes of P2Y receptors and mobilize intracellular calcium in response to nucleotide exposure. As a functional consequence, PDCs acquire a mature phenotype that is further enhanced in the context of CD40 ligation. Strikingly, nucleotides strongly inhibit IFN-alpha secretion induced by influenza virus or CpG-A. This effect is most pronounced for the uridine nucleotides UDP and UTP and the sugar nucleotide UDP-glucose, ligands of P2Y(6), P2Y(4), and P2Y(14), respectively. Nucleotide-induced inhibition of IFN-alpha production is blocked by suramin, a P2Y receptor antagonist. Pharmacological data point toward a role of protein kinase C in the negative regulation of type I IFN. Manipulating PDC function with P2Y receptor agonists may offer novel therapeutic strategies for autoimmune diseases or cancer.

Dendritic cells (DC) generated in vitro have to be viable and phenotypically mature to be capable of inducing T cell-mediated immunity after in vivo administration. To facilitate optimization of DC-based vaccination protocols, we investigated whether the cytokine environment and the mode of activation affect maturation and survival of DC derived from monocytes by a short-term protocol. Monocytes cultured for 24 h with granulocyte macrophage-colony stimulating factor and interleukin-4 were stimulated with proinflammatory mediators for another 36 h to generate mature DC. Additional activation with CD40 ligand and interferon (IFN)-gamma increased viability of DC and promoted definitive maturation as defined by maintenance of a mature phenotype after withdrawal of cytokines. Addition of IFN-alpha to DC cultures prior to stimulation further enhanced definitive maturation: IFN-alpha-primed DC expressed high levels of costimulatory molecules and CC chemokine receptor 7 (CCR7) up to 5 days after cytokine withdrawal. Compared with unprimed DC, IFN-alpha-primed DC displayed equal capacity to migrate upon CCR7 ligation and to prime antigen-specific T helper cell as well as cytolytic T cell responses. In conclusion, we show that optimal maturation and survival of monocyte-derived DC require multiple activation signals. Furthermore, we identified a novel role for IFN-alpha in DC development: IFN-alpha priming of monocytes promotes definitive maturation of DC upon activation.

Phenotypic maturation, cytokine secretion, and migration are distinct functional characteristics of dendritic cells (DCs). These functions are independently regulated by a number of extracellular variables, such as type, strength, and persistence of an array of soluble and membrane-bound mediators. Since the exact composition of these variables in response to infection may differ between individuals, the intracellular signaling pathways activated by these extracellular networks may more closely correlate with DC function and predict the course of adaptive immunity. We found that activation of p38 kinase (p38K), extracellular signal-related kinase 1/2 (ERK1/2), and phosphatidylcholine-specific phospholipase C (PC-PLC) enhanced cytokine secretion, whereas p38K, cyclic adenosine monophosphate (cAMP), and PC-PLC enhanced migration. In contrast, phosphatidylinositol 3-kinase (PI3K)/Akt-1 and cAMP inhibited cytokine secretion while ERK1/2 inhibited migration. Migration and cytokine secretion further differed in their sensitivity to inhibition over time. However, although DCs could be manipulated to express migration, cytokine secretion, or both, the level of activation or persistence of intracellular pathway signaling was not predictive. Our results suggest a modular organization of function. We hypothesize that the expression of specific DC functions integrates a large variety of activating and inhibitory variables, and is represented by the formation of a functional unit of molecular networks-the signal response module (SRM). The combined activities of these modules define the functional outcome of DC activation.