Abstract Background Isolated lymphoid follicles (ILFs) are organized immune aggregates normally present in the gut. ILFs expand into the submucosa and muscularis propria in inflammatory bowel disease (IBD), including both ulcerative colitis (UC) and Crohn’s disease (CD), potentially sustaining B- and T-cell responses against intestinal antigens. In contrast, granulomas (GAs) are compact macrophage-rich lesions found in CD, but not UC, and whose pathogenesis and role in disease remain unclear. 1,2,3 To understand these complex structures, we used single-cell RNA-seq (scRNA-seq) and Spatial Transcriptomics (ST) and compared healthy and IBD ILFs with CD-associated GAs, revealing their common features, as well as their divergent cellular programs that may drive the formation and maintenance of GA. Methods We analyzed 9 FFPE colonic samples (healthy, UC, CD) using spatial transcriptomics (CosMx-SMI 1k gene panel), involving over 530,000 cells. Cell identities were annotated using an in-house reference scRNA-seq dataset of 92 intestinal biopsies (6 Healthy Controls (HC), 36 UC and 50 CD). To characterize tissue organization, we applied neighborhood analysis, enabling identification of statistically recurrent cellular neighborhoods.4 (Fig. 1A) We also performed SCOTIA to map cell-to-cell interactions in the tissue.5 Results We identified 21 conserved cellular neighborhoods encompassing 94% of colonic cells. From these neighborhoods we were able to detect ILFs, present in both healthy colon and IBD. We also found a CD-specific cluster that coincided with GAs, which we then confirmed by histology. In IBD, but not healthy control samples, ILFs contained inflammation-associated fibroblasts (IAFs) and showed reduced CCL21 expression, suggesting impaired naïve T-cell recruitment, and a significant increase in IGHG1 and IGHG2. On the other hand, GAs formed macrophage-rich cores surrounded by T cells and IAFs, with strong matrix-remodeling signals (MMP1, SPP1) (Fig. 1B). Finally, IBD ILFs displayed fewer cell–cell interactions than healthy ILFs, with increased IAF signaling and loss of FRC interactions. GAs showed an overall interaction decrease, dominated by IAF and SPP1+ macrophage-enhanced inflammation. (Fig. 1C) Conclusion Together, our spatial analysis of ILF- and GA-like regions in IBD reveals distinct cellular ecosystems defined by macrophage subsets and fibroblast populations that likely orchestrate immune activation, tissue remodeling, and granuloma formation. Building on these shifts, we hypothesize that GAs may emerge from dysregulated ILFs, as suggested by the progressive reorganization of interactions and cell populations from healthy ILFs to IBD-associated ILFs and ultimately to GA. References: 1.McNamee, E. N. & Rivera-Nieves, J. Ectopic tertiary lymphoid tissue in inflammatory bowel disease: protective or provocateur? Front. Immunol. 7, 308 (2016). 2.Zhao, L. et al. Tertiary lymphoid structures in diseases: immune mechanisms and therapeutic advances. Signal Transduct. Target. Ther. 9, 225 (2024). 3.Krausgruber, T. et al. Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation. Immunity 56, 289–306.e7 (2023). 4.Cadinu, P. et al. Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling. Cell 187, 2010–2028.e30 (2024). 5.Shiau, C. et al. Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis. bioRxiv (2023). https://doi.org/10.1101/2023.06.28.546848 Conflict of interest: Moro Buendia, Marc: No conflict of interest Sanzo Machuca, Angela: No conflicts. Veny, Marisol: Nothing to declare Sabate, Sofia: No conflict of interest Dotti, Isabella: I declare no conflicts of interest Gudiño, Victoria: No conflicts. Carrasco, Antonio: No conflict of interest Lopez-Prades, Sandra: No conflict of interest Acera, Mario: No conflict of interest Mereu, Elisabetta: No conflict of interest Cuatrecasas, Miriam: No conflict of interest Ricart Gomez, Elena: E. Ricart has received support for congress and conference attendance, speaker fees, research support or consulting fees from MSD, Abbvie, Ferring, Janssen, Otsuka, Pfizer, Takeda, Faes Farma, Galapagos/Alphasigma, Kern Pharma, Lilly, J & J, Dr Falk Pharma, and Fresenius-Kabi. Salas, Azucena: Grant: Takeda, Roche, Nestle, Pfizer, Genentech, AbbVie, GSK, Scipher Medicine, Alimentiv, Inc, Boehringer Ingelheim and Agomab. Personal Fees: Lilly, Johnson & Johnson, Genentech, GSK, Pfizer, Galapagos, AdBio Partners, HotSpot Therapeutics, Alimentiv, Nestle, GoodGut and Agomab.
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