IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Age-dependent inflammatory response is altered in an ex vivo model of bacterial pneumonia.

BACKGROUND: Aging is associated with an increased incidence and mortality of Pseudomonas aeruginosa-induced pneumonias. This might be partly due to age-dependent increases in inflammatory mediators, referred to as inflamm-aging and a decline in immune functions, known as immunosenescence. Still, the impact of dysregulated immune responses on lung infection during aging is poorly understood. Here, we aimed to mimic inflamm-aging using ex vivo precision-cut lung slices (PCLS) and neutrophils - as important effector cells of innate immunity - from young and old mice and investigated the influence of aging on inflammation upon infection with P. aeruginosa bacteria. METHODS: Murine PCLS were infected with the P. aeruginosa standard lab strain PAO1 and a clinical P. aeruginosa isolate D61. After infection, whole-transcriptome analysis of the tissue as well as cytokine expression in supernatants and tissue lysates were performed. Responses of isolated neutrophils towards the bacteria were investigated by quantifying neutrophil extracellular trap (NET) formation, cytokine secretion, and analyzing expression of surface activation markers using flow cytometry. RESULTS: Inflamm-aging was observed by transcriptome analysis, showing an enrichment of biological processes related to inflammation, innate immune response, and chemotaxis in uninfected PCLS of old compared with young mice. Upon P. aeruginosa infection, the age-dependent pro-inflammatory response was even further promoted as shown by increased production of cytokines and chemokines such as IL-1ß, IL-6, CXCL1, TNF-α, and IL-17A. In neutrophil cultures, aging did not influence NET formation or cytokine secretion during P. aeruginosa infection. However, expression of receptors associated with inflammatory responses such as complement, adhesion, phagocytosis, and degranulation was lower in neutrophils stimulated with bacteria from old mice as compared to young animals. CONCLUSIONS: By using PCLS and neutrophils from young and old mice as immunocompetent ex vivo test systems, we could mimic dysregulated immune responses upon aging on levels of gene expression, cytokine production, and receptor expression. The results furthermore reflect the exacerbation of inflammation upon P. aeruginosa lung infection as a result of inflamm-aging in old age.

A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation.

BACKGROUND: The coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyze the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation. RESULTS: Here, we identify and verify general biomarker signatures robustly evaluating T-cell activation in a time-resolved manner. We identify time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. In addition, intermediate and late response activation signatures in CAR T-cell infusion products are correlated to immune effector cell-associated neurotoxicity syndrome. CONCLUSION: This study is the first to describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source, e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies, or assessing dysregulated functions of human T-cell immunity.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

Sensory modulation of airways immunity.

The airways are constantly exposed to a multitude of inhaled particles and, as such, require a finely tuned discrimination between harmful or potentially threatening stimuli, and discrete responses to maintain homeostasis. Both the immune and nervous systems have the ability to sense environmental (and internal) signals, to integrate the obtained information and to initiate a protective reaction. Lung immunity and innervation are known to be individually involved in these processes, but it is becoming clear that they can also influence one another via a multitude of complex mechanisms. Here, we specifically describe how sensory innervation affects airways immunity with a focus on pathological conditions such as asthma or infections, describing cellular and molecular mechanisms, and highlighting potentially novel therapeutic targets.

From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment.

Success in cancer treatment over the last four decades has ranged from improvements in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven beneficial for the treatment of inflammatory and autoimmune diseases. In this review, we report on challenging examples that bridge between treatment of cancer and immune-mediated diseases, addressing mechanisms and experimental models as well as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. However, new developments using human ex vivo approaches modeling cancer, for example in microfluidic human organs-on-chips, promise to identify key molecular, cellular and immunological features of human cancer progression in a fully human setting. Classical drugs which bridge the gap, for instance, include cytotoxic drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals developed for cancer therapy have also shown efficacy in the treatment of autoimmune diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have achieved spectacular remissions in refractory B cell leukemia and lymphoma and are currently under development for tolerance induction using cell-based therapies such as CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from bridging cancer and autoimmune diseases as well as tolerance induction.

Long Noncoding RNA-Enriched Vesicles Secreted by Hypoxic Cardiomyocytes Drive Cardiac Fibrosis.

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance.

Mast cells within cellular networks.

Mast cells are highly versatile in terms of their mode of activation by a host of stimuli and their ability to flexibly release a plethora of biologically highly active mediators. Within the immune system, mast cells can best be designated as an active nexus interlinking innate and adaptive immunity. Here we try to draw an arc from initiation of acute inflammatory reactions to microbial pathogens to development of adaptive immunity and allergies. This multifaceted nature of mast cells is made possible by interaction with multiple cell types of immunologic and nonimmunologic origin. Examples for the former include neutrophils, eosinophils, T cells, and professional antigen-presenting cells. These interactions allow mast cells to orchestrate inflammatory innate reactions and complex adaptive immunity, including the pathogenesis of allergies. Important partners of nonimmunologic origin include cells of the sensory neuronal system. The intimate association between mast cells and sensory nerve fibers allows bidirectional communication, leading to neurogenic inflammation. Evidence is accumulating that this mast cell/nerve crosstalk is of pathophysiologic relevance in patients with allergic diseases, such as asthma.

Human lung tissue provides highly relevant data about efficacy of new anti-asthmatic drugs.

Subgroups of patients with severe asthma are insensitive to inhaled corticosteroids and require novel therapies on top of standard medical care. IL-13 is considered one of the key cytokines in the asthma pathogenesis, however, the effect of IL-13 was mostly studied in rodents. This study aimed to assess IL-13 effect in human lung tissue for the development of targeted therapy approaches such as inhibition of soluble IL-13 or its receptor IL-4Rα subunit. Precision-cut lung slices (PCLS) were prepared from lungs of rodents, non-human primates (NHP) and humans. Direct effect of IL-13 on human lung tissue was observed on inflammation, induction of mucin5AC, and airway constriction induced by methacholine and visualized by videomicroscopy. Anti-inflammatory treatment was evaluated by co-incubation of IL-13 with increasing concentrations of IL-13/IL-13 receptor inhibitors. IL-13 induced a two-fold increase in mucin5AC secretion in human bronchial tissue. Additionally, IL-13 induced release of proinflammatory cytokines eotaxin-3 and TARC in human PCLS. Anti-inflammatory treatment with four different inhibitors acting either on the IL-13 ligand itself (anti-IL-13 antibody, similar to Lebrikizumab) or the IL-4Rα chain of the IL-13/IL-4 receptor complex (anti-IL-4Rα #1, similar to AMG 317, and #2, similar to REGN668) and #3 PRS-060 (a novel anticalin directed against this receptor) could significantly attenuate IL-13 induced inflammation. Contrary to this, IL-13 did not induce airway hyperresponsiveness (AHR) in human and NHP PCLS, although it was effective in rodent PCLS. Overall, this study demonstrates that IL-13 stimulation induces production of mucus and biomarkers of allergic inflammation in human lung tissue ex-vivo but no airway hyperresponsiveness. The results of this study show a more distinct efficacy than known from animals models and a clear discrepancy in AHR induction. Moreover, it allows a translational approach in inhibitor profiling in human lung tissue.

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), and interleukin 1 alpha (IL-1α) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin.

The cellular serine protease TMPRSS2, a member of the type II transmembrane serine protease (TTSP) family, cleaves and activates the hemagglutinin of influenza A viruses (FLUAV) in cell culture and is essential for spread of diverse FLUAV in mice. Non-human primates (NHP), in particular rhesus and cynomolgus macaques, serve as animal models for influenza and experimental FLUAV infection of common marmosets has recently also been reported. However, it is currently unknown whether the NHP orthologues of human TMPRSS2 cleave and activate FLUAV hemagglutinin and contribute to viral spread in respiratory tissue. Here, we cloned and functionally analyzed the macaque and marmoset orthologues of human TMPRSS2. In addition, we analyzed the macaque orthologues of human TMPRSS4 and HAT, which also belong to the TTSP family. We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT cleave and activate HA upon directed expression and provide evidence that endogenous TMPRSS2 is expressed in the respiratory epithelium of rhesus macaques. Finally, we demonstrate that a serine protease inhibitor active against TMPRSS2 suppresses FLUAV spread in precision-cut lung slices of human, macaque and marmoset origin. These results indicate that FLUAV depends on serine protease activity for spread in diverse NHP and in humans. Moreover, our findings suggest that macaques and marmosets may serve as models to study FLUAV activation by TMPRSS2 in human patients.