Crohn's disease (CD) remains a significant therapeutic challenge, with limited treatment options and heterogeneous clinical responses. The selective JAK1 inhibitor filgotinib has shown great potential in treating ulcerative colitis but failed to demonstrate consistent efficacy in CD, for reasons that remain unclear. Here, we address this gap using an immunocompetent ex vivo model of precision-cut intestinal slices (PCIS) from CD patients, allowing region-specific analysis of ileal and colonic responses. We reveal that colonic PCIS display strong T cell activation and are highly sensitive to filgotinib, with suppression of Th1/Th17 cytokines, oxidative stress, and STAT signaling ex vivo. In contrast, ileal PCIS exhibited predominantly innate and Th17-driven inflammation, and were less sensitive to JAK1 inhibition. These data provide a potential mechanistic explanation for the limited efficacy of filgotinib in CD clinical trials and established PCIS as a human-relevant ex vivo model for region-specific drug responses, underscoring the importance of developing therapies tailored to different segments of the gut in inflammatory bowel disease.

Inflammatory bowel disease (IBD) affects approximately 5 million people worldwide, causing chronic inflammation and increased mortality. Despite advances in therapy, the underlying immune mechanisms remain poorly understood, highlighting the need for human-immunocompetent models to enhance translational research. This study aimed to investigate local immune responses using precision-cut intestinal slices (PCIS) from IBD patients and evaluate immunomodulatory treatment directly in patient tissue ex vivo. PCIS from ileal resections of IBD and non-IBD patients were stimulated with Concanavalin A (ConA) or lipopolysaccharide (LPS). Histological analysis of IBD-derived PCIS showed villus atrophy, infiltration of lymphocytes and macrophages, and RNA analysis revealed upregulation of IL-17 and interferon signaling pathways. LPS- and ConA-induced functional immune responses in the tissue, with IBD tissue exhibiting increased levels of specific cytokines compared with non-IBD tissue, including IL-17F and IL-21 after ConA-stimulation, and IL-22 as well as ENA-78 following LPS-stimulation. Pimecrolimus treatment led to a marked reduction in the release of IL-2, IL-17A, and IFN-γ, and inhibited the IBD supernatant-induced reduction in transepithelial electrical resistance. Our data provide the first in-depth characterization of local tissue immune responses in human PCIS, highlighting the potential of this model to study disease-specific immune activity and evaluate pharmacological interventions ex vivo.

IntroductionCytokine release syndrome (CRS) is a potentially severe systemic inflammatory condition triggered by various immunomodulatory therapies, making understanding its pathogenesis critical for improving patient outcomes.Results/MethodsBy combining immunotoxicology and systems biology approaches, we offer a novel and integrative conceptual model of CRS as an adverse outcome (AO), induced by five different immunomodulatory biotherapies: 1) chimeric antigen receptor (CAR) T cells, 2) checkpoint inhibitors, 3) T cell engaging bispecific modalities, 4) monoclonal antibodies targeting and activating T cell receptors, and 5) FcγR activating monoclonal antibodies. This model uniquely integrates multiple CRS-inducing therapies into a unified framework, offering a comprehensive mechanistic representation of CRS pathophysiology. For that, we built an adverse outcome pathway (AOP) CRS network for these therapies and then developed a systems biology map of molecular mechanisms relevant to the AOP network. The map of mechanisms is made available via a dedicated online platform for exploration and data visualisation. It includes 24 cell types, 425 entities and 430 interactions. DiscussionBeyond a static representation, the CRS Map serves as a dynamic tool for clinical and research applications, allowing researchers and clinicians to explore CRS progression in detail, identify biomarkers, and discover potential therapeutic targets. The map demonstrates stages of CRS progression and shows molecules that can be measured in relevant immunotoxicological assays, as well as potential drug targets for therapeutic intervention of CRS.

This work focuses on the need for modeling and predicting adverse outcomes in immunotoxicology to improve nonclinical assessments of the safety of immunomodulatory therapies. The integrated approach includes, first, the adverse outcome pathway concept established in the toxicology field, and, second, the systems medicine disease map approach for describing molecular mechanisms involved in a particular pathology. The proposed systems immunotoxicology workflow is illustrated with chimeric antigen receptor (CAR) T cell treatment as a use case. To this end, the linear adverse outcome pathway (AOP) is expanded into a molecular interaction model in standard systems biology formats. Then it is shown how knowledge related to immunotoxic events can be integrated, encoded, managed, and explored to benefit the research community. The map is accessible online at https://imsavar.elixir-luxembourg.org via the MINERVA Platform for browsing, commenting, and data visualization. Our work transforms a graphical illustration of an AOP into a digitally structured and standardized form, featuring precise and controlled vocabulary and supporting reproducible computational analyses. Because of annotations to source literature and databases, the map can be further expanded to match the evolving knowledge and research questions.

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.

By combining immunotoxicology and systems biology approaches, we offer a novel conceptual model of cytokine release syndrome (CRS) as an adverse outcome (AO), induced by five different immunomodulatory biotherapies: 1) chimeric antigen receptor (CAR) T cells, 2) checkpoint inhibitors, 3) T cell engaging bispecific modalities, 4) monoclonal antibodies targeting and activating T cell receptors, and 5) FcγR activating monoclonal antibodies. For that, we built an adverse outcome pathway (AOP) CRS network for these therapies and then developed a systems biology map of molecular mechanisms relevant to the AOP network. The map of mechanisms is made available via a dedicated online platform for exploration and data visualisation. It includes 24 cell types, 425 entities and 430 interactions. The map demonstrates stages of the CRS progression and shows molecules that can be measured in relevant immunotoxicological assays, as well as potential drug targets for therapeutic intervention of CRS.

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.

Innovative therapeutics like biologicals that modulate the immune system are on the rise. However, their immune-modulating characteristics can also lead sometimes to the induction of adverse effects, by triggering unintended immune reactions. Due to the complexity and target-specificity of such therapeutics, these drug-induced adverse events could remain undetected during non-clinical development, if the test systems are, for example, animal-based, and only emerge in clinical development when tested in humans and subsequently lead to discontinuance of otherwise promising drug candidates. To identify adverse effects on the human immune system at an early stage, new approaches, assays, and technologies are needed. The Innovative Medicine Initiative (IMI) cooperation Immune Safety Avatar (imSAVAR) project aims to develop a tool for integrated non-clinical safety assessment for immune-modulatory new therapeutic drugs and clinical trial applications. To achieve this goal, imSAVAR has relied on the Adverse Outcome Pathway (AOP) framework to gather knowledge in a structured approach and to design, select or develop, when needed, appropriate test systems for prediction of the immune-related adverse outcomes. So far, the imSAVAR consortium has identified the “mode of action” for certain classes of drugs that needed improved risk assessment, including chimeric antigen receptor T cells (CAR T cells), immune checkpoint inhibitors (ICIs), and recombinant proteins (e.g. interleukin [IL]-2), has linked those to their immune-related adverse outcomes and has formulated literature-based immune-related AOPs (irAOPs). Models to measure those immune-specific perturbations were selected, adjusted, or newly developed. The imSAVAR work described in this special issue of The Journal of Immunotoxicology supports our understanding of immune-mediated adverse effects and their early discovery during development to improve the safety of innovative biomedicals.

Interleukin-2 (IL-2) holds promise for the treatment of cancer and autoimmune diseases, but its high-dose usage is associated with systemic immunotoxicity. Differential IL-2 receptor (IL-2R) regulation might impact function of cells upon IL-2 stimulation, possibly inducing cellular changes similar to patients with hypomorphic IL2RB mutations, presenting with multiorgan autoimmunity. Here, we show that sustained high-dose IL-2 stimulation of human lymphocytes drastically reduces IL-2Rβ surface expression especially on T cells, resulting in impaired IL-2R signaling which correlates with high IL-2Rα baseline expression. IL-2R signaling in NK cells is maintained. CD4+ T cells, especially regulatory T cells are more broadly affected than CD8+ T cells, consistent with lineage-specific differences in IL-2 responsiveness. Given the resemblance of cellular characteristics of high-dose IL-2-stimulated cells and cells from patients with IL-2Rβ defects, impact of continuous IL-2 stimulation on IL-2R signaling should be considered in the onset of clinical adverse events during IL-2 therapy.

The skin is the organ most often affected by adverse drug reactions. Although these cutaneous adverse drug reactions (CADRs) often are mild, they represent a major burden for patients. One of the drugs inducing CADRs is aldesleukin, a recombinant interleukin-2 (recIL-2) originally approved to treat malignant melanoma and metastatic renal cell carcinoma which frequently led to skin rashes when applied in high doses for anti-cancer therapy. Skin rashes and other side effects, together with poor efficacy led to a drawback of the therapeutic, but modified recIL-2 molecules are on the rise to treat both cancer and inflammatory diseases such as autoimmunity. Still, pathophysiological mechanisms of recIL-2-induced skin rashes are not understood. In the study reported here, a hypothetical literature-based immune-related adverse outcome pathway (irAOP) was developed to identify possible key cells and molecules in recIL-2-induced skin rash. Using this approach, a hypothesis was formed that the induced immune response predominantly is Type 2-driven by T-helper and innate lymphoid cells, leading to the occurrence of cutaneous side effects during recIL-2 therapy. This paper further discusses mechanisms beyond the proposed irAOP which might add to the pathology but currently are less-studied. Together, this hypothetic irAOP forms a basis to clarify possible cellular and molecular interactions leading to recIL-2-induced skin rash. This might be used to adapt existing or develop new test systems to help predict and prevent cutaneous side effects in future IL-2-based or similar therapies.

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.

ATMP („advanced therapy medicinal products“) sind eine neue Klasse von Arzneimitteln, die somatische Zelltherapeutika, Gentherapeutika und biotechnologisch bearbeitete Gewebeprodukte umfassen. Bisher sind nur wenige Produkte europaweit zugelassen. Ein Grund dafür ist der langwierige und schwierige Weg ihrer Entwicklung. Vor der Anwendung beim Menschen muss in einer nichtklinischen Phase die Sicherheit des jeweiligen ATMP gezeigt werden, um potenzielle Nebenwirkungen auszuschließen. Da ATMP biologischen Ursprungs sind und somit eine hohe Komplexität und Heterogenität aufweisen, bedarf es individueller Teststrategien. Die Entwicklung von ATMP vom Labormaßstab zur Herstellung unter GMP(Good Manufacturing Practice)-Bedingungen folgt behördlich vorgegebenen Regularien. Um die Behandlung mit ATMP größeren Patientengruppen zu ermöglichen, müssen GMP-Prozesse hochskaliert werden, was eine große logistische und technologische Herausforderung darstellt. Klinische Prüfungen für ATMP sind kostenintensiv und zeitaufwändig, bieten aber auch einzigartige neue Möglichkeiten zur Behandlung schwerer Erkrankungen, wie z. B. Krebs.