Periodic Reporting for period 1 - EDIOM (Elucidating drivers of human Inflammatory Bowel Disease by next-generation organoid modeling)
Periodo di rendicontazione: 2023-11-01 al 2025-10-31
Inflammatory Bowel Disease (IBD), including Crohn’s disease and ulcerative colitis, is a chronic condition in which the immune system mistakenly attacks the intestine. The resulting inflammation causes recurring symptoms and long-term tissue damage, and many patients require lifelong treatment. Although several anti-inflammatory and immunosuppressive medicines are available, a substantial proportion of people do not respond well or lose response over time. This reflects a core challenge: IBD is biologically diverse, and multiple inflammatory pathways can be active in different patients and at different stages of disease.
The project was motivated by the need for a human-tissue–based understanding of these pathways, because animal models and simplified cell cultures cannot fully capture the complexity of the human gut immune system. In particular, CD4⁺ “helper” T cells are central coordinators of immunity and are strongly implicated in IBD, including responses to harmless gut microbes (the commensal microbiota). Yet it has remained unclear how inflammatory helper T-cell programs, tissue-resident states, and regulatory programs coexist, change during disease activity, and interact with other immune and non-immune cells within the intestinal environment.
The overall objective was therefore to identify, and then experimentally test, the key cellular networks and cell-to-cell communication pathways that drive human intestinal inflammation. To achieve this, the project combined three elements into a single workflow: immune-competent “mini-gut” organoids grown from patient biopsies (air–liquid interface cultures) that preserve multiple tissue compartments, high-dimensional immune profiling to capture many features of T-cell state in one experiment (including a 37-marker spectral flow cytometry assay), and spatial plus computational approaches to place immune states in their tissue context and integrate cellular, protein and spatial measurements into disease-associated interaction networks.
Overall objectives:
1) Build and validate immune-competent patient-derived intestinal organoids as an ex vivo model for IBD.
2) Profile CD4⁺ T helper cell programs in gut tissue at high resolution, including functional outputs and the capacity to shift between inflammatory and regulatory states (“plasticity”).
3) Add spatial context to determine where disease-linked immune states arise and which cell types interact most frequently within inflamed tissue.
4) Integrate cellular, protein and spatial measurements with computational modelling to identify disease-enriched cellular networks and actionable cell-to-cell signalling pathways.
5) Functionally test top candidate drivers in organoids (by targeted perturbation) to distinguish association from mechanism, prioritise therapeutic targets, and define measurable biomarkers of disease-relevant immune states.
Pathway to impact: by connecting realistic human tissue models with multi-modal readouts and data integration, the project aims to deliver a practical pipeline for identifying causal drivers of inflammation, selecting druggable targets, and developing biomarkers that can help stratify patients and support more targeted, durable treatment strategies; ultimately reducing the burden of IBD on patients and healthcare systems.
The project was motivated by the need for a human-tissue–based understanding of these pathways, because animal models and simplified cell cultures cannot fully capture the complexity of the human gut immune system. In particular, CD4⁺ “helper” T cells are central coordinators of immunity and are strongly implicated in IBD, including responses to harmless gut microbes (the commensal microbiota). Yet it has remained unclear how inflammatory helper T-cell programs, tissue-resident states, and regulatory programs coexist, change during disease activity, and interact with other immune and non-immune cells within the intestinal environment.
The overall objective was therefore to identify, and then experimentally test, the key cellular networks and cell-to-cell communication pathways that drive human intestinal inflammation. To achieve this, the project combined three elements into a single workflow: immune-competent “mini-gut” organoids grown from patient biopsies (air–liquid interface cultures) that preserve multiple tissue compartments, high-dimensional immune profiling to capture many features of T-cell state in one experiment (including a 37-marker spectral flow cytometry assay), and spatial plus computational approaches to place immune states in their tissue context and integrate cellular, protein and spatial measurements into disease-associated interaction networks.
Overall objectives:
1) Build and validate immune-competent patient-derived intestinal organoids as an ex vivo model for IBD.
2) Profile CD4⁺ T helper cell programs in gut tissue at high resolution, including functional outputs and the capacity to shift between inflammatory and regulatory states (“plasticity”).
3) Add spatial context to determine where disease-linked immune states arise and which cell types interact most frequently within inflamed tissue.
4) Integrate cellular, protein and spatial measurements with computational modelling to identify disease-enriched cellular networks and actionable cell-to-cell signalling pathways.
5) Functionally test top candidate drivers in organoids (by targeted perturbation) to distinguish association from mechanism, prioritise therapeutic targets, and define measurable biomarkers of disease-relevant immune states.
Pathway to impact: by connecting realistic human tissue models with multi-modal readouts and data integration, the project aims to deliver a practical pipeline for identifying causal drivers of inflammation, selecting druggable targets, and developing biomarkers that can help stratify patients and support more targeted, durable treatment strategies; ultimately reducing the burden of IBD on patients and healthcare systems.
The project established a human intestinal workflow to resolve CD4⁺ T-cell states and tissue interaction networks linked to IBD, and to enable downstream causal testing in advanced ex vivo models. A core output was a 37-color spectral flow cytometry panel measuring CD4⁺ T helper lineage identity, memory/tissue residency, activation, and a broad set of cytokines in a single assay. This tool was applied to intestinal biopsies and matched blood from IBD patients to generate high-dimensional functional immune profiles in relevant human tissues.
Computational analyses defined inflammation-associated CD4⁺ programs, including expansion of RORγt⁺ cells with increased T-bet and emergence of Foxp3⁺RORγt⁺ cells, consistent with regulatory–Th17 plasticity in the inflamed niche. Trajectory visualization supported a branched differentiation landscape toward regulatory versus tissue-resident Th17-like endpoints enriched for activation/proliferation features. Network analyses connected pro-inflammatory CD4⁺ states with T-bet⁺Granzyme-B⁺ CD8⁺ subsets, supported by histological evidence of CD4–CD8 proximity. Functional stimulation assays showed that active disease can feature broad suppression of CD4⁺ inflammatory cytokines, while an HLA-DR⁺CD38⁺ memory subset retains strong multifunctional cytokine production.
To add spatial context (ongoing work toward a planned 2026 manuscript), we successfully implemented Xenium high-plex spatial transcriptomics in IBD tissue and, in parallel, established imaging mass cytometry (IMC) for multiplex protein mapping across an IBD cohort, enabling integrated spatial RNA–protein network discovery. Simultaneously, we advanced air–liquid interface (ALI) organoid cultures from patient biopsies, improving preservation and functionality of tissue-resident immune cells. Ongoing work focuses on perturbing prioritised inflammatory drivers in the enhanced organoid model to test causality and prioritise therapeutic targets.
Main achievements
1) 37-color spectral flow panel built and deployed in IBD biopsies/blood.
2) Disease-linked CD4⁺ states, plasticity, and differentiation structure resolved.
3) Coordinated CD4–CD8 inflammatory networks identified and supported by histology.
4) Xenium spatial transcriptomics and IMC implemented on IBD cohorts (manuscript in preparation).
5) Immune-competent ALI organoids improved; causal perturbation studies initiated.
Computational analyses defined inflammation-associated CD4⁺ programs, including expansion of RORγt⁺ cells with increased T-bet and emergence of Foxp3⁺RORγt⁺ cells, consistent with regulatory–Th17 plasticity in the inflamed niche. Trajectory visualization supported a branched differentiation landscape toward regulatory versus tissue-resident Th17-like endpoints enriched for activation/proliferation features. Network analyses connected pro-inflammatory CD4⁺ states with T-bet⁺Granzyme-B⁺ CD8⁺ subsets, supported by histological evidence of CD4–CD8 proximity. Functional stimulation assays showed that active disease can feature broad suppression of CD4⁺ inflammatory cytokines, while an HLA-DR⁺CD38⁺ memory subset retains strong multifunctional cytokine production.
To add spatial context (ongoing work toward a planned 2026 manuscript), we successfully implemented Xenium high-plex spatial transcriptomics in IBD tissue and, in parallel, established imaging mass cytometry (IMC) for multiplex protein mapping across an IBD cohort, enabling integrated spatial RNA–protein network discovery. Simultaneously, we advanced air–liquid interface (ALI) organoid cultures from patient biopsies, improving preservation and functionality of tissue-resident immune cells. Ongoing work focuses on perturbing prioritised inflammatory drivers in the enhanced organoid model to test causality and prioritise therapeutic targets.
Main achievements
1) 37-color spectral flow panel built and deployed in IBD biopsies/blood.
2) Disease-linked CD4⁺ states, plasticity, and differentiation structure resolved.
3) Coordinated CD4–CD8 inflammatory networks identified and supported by histology.
4) Xenium spatial transcriptomics and IMC implemented on IBD cohorts (manuscript in preparation).
5) Immune-competent ALI organoids improved; causal perturbation studies initiated.
This project goes beyond current IBD immune profiling by combining (i) high-dimensional functional phenotyping, (ii) spatially resolved RNA/protein measurements in intact tissue, and (iii) an immune-preserving patient-derived organoid system that enables mechanistic follow-up.
Overview of results:
1) High-dimensional CD4⁺ T-cell profiling tool: A validated 37-color spectral flow cytometry panel measuring CD4⁺ T helper lineage identity, activation, memory/tissue residency, and multi-cytokine function in one assay, applicable to intestinal biopsies and blood.
2) More precise model of pathogenic T-cell activity: Identification of co-existing CD4⁺ programs in inflamed gut, including helper states associated with inflammation, evidence for regulatory–Th17 plasticity, and an activation-defined subset retaining strong multifunctionality even when broader cytokine responses are suppressed.
3) Spatial multi-modal mapping in IBD tissue: Successful implementation of Xenium high-plex spatial transcriptomics and imaging mass cytometry (IMC) across IBD cohorts, enabling integrated spatial RNA–protein network discovery and localisation of inflammatory niches.
4) Immune-competent ex vivo testing platform: Improved air–liquid interface (ALI) organoid cultures with enhanced preservation and functionality of tissue-resident immune cells, creating a bridge from tissue associations to causal testing.
Indicative potential impacts:
1) Faster target prioritisation: A workflow that connects tissue-embedded immune states and spatial networks to functional perturbation capacity reduces reliance on purely associative findings.
2) Improved stratification/monitoring: Phenotype-defined immune states provide candidates for translational biomarkers and therapy-relevant patient stratification.
3) Broad applicability: The combined platform is transferable to other immune-mediated inflammatory diseases beyond IBD.
Key needs for further uptake and success:
1) Causality demonstration: Complete ongoing perturbation studies in immune-competent organoids to validate candidate drivers and actionability.
2) Scale and robustness: Extend to larger and longitudinal cohorts, including multi-centre validation.
3) Standardisation: Harmonise protocols and QC for spectral flow, Xenium and IMC to support reproducibility and future clinical-grade translation.
4) Accessible analytics: Package and document integration pipelines for spatial and network analyses to enable adoption by other research and clinical teams.
Overview of results:
1) High-dimensional CD4⁺ T-cell profiling tool: A validated 37-color spectral flow cytometry panel measuring CD4⁺ T helper lineage identity, activation, memory/tissue residency, and multi-cytokine function in one assay, applicable to intestinal biopsies and blood.
2) More precise model of pathogenic T-cell activity: Identification of co-existing CD4⁺ programs in inflamed gut, including helper states associated with inflammation, evidence for regulatory–Th17 plasticity, and an activation-defined subset retaining strong multifunctionality even when broader cytokine responses are suppressed.
3) Spatial multi-modal mapping in IBD tissue: Successful implementation of Xenium high-plex spatial transcriptomics and imaging mass cytometry (IMC) across IBD cohorts, enabling integrated spatial RNA–protein network discovery and localisation of inflammatory niches.
4) Immune-competent ex vivo testing platform: Improved air–liquid interface (ALI) organoid cultures with enhanced preservation and functionality of tissue-resident immune cells, creating a bridge from tissue associations to causal testing.
Indicative potential impacts:
1) Faster target prioritisation: A workflow that connects tissue-embedded immune states and spatial networks to functional perturbation capacity reduces reliance on purely associative findings.
2) Improved stratification/monitoring: Phenotype-defined immune states provide candidates for translational biomarkers and therapy-relevant patient stratification.
3) Broad applicability: The combined platform is transferable to other immune-mediated inflammatory diseases beyond IBD.
Key needs for further uptake and success:
1) Causality demonstration: Complete ongoing perturbation studies in immune-competent organoids to validate candidate drivers and actionability.
2) Scale and robustness: Extend to larger and longitudinal cohorts, including multi-centre validation.
3) Standardisation: Harmonise protocols and QC for spectral flow, Xenium and IMC to support reproducibility and future clinical-grade translation.
4) Accessible analytics: Package and document integration pipelines for spatial and network analyses to enable adoption by other research and clinical teams.