Abstract Background Advanced Combination Treatment (ACT) aims to overcome the limited efficacy of biologic monotherapy in inflammatory bowel disease (IBD) by simultaneously targeting complementary inflammatory pathways. We investigated, at single-cell resolution, the transcriptional landscape and T-cell receptor (TCR) repertoire remodelling induced by ACT with distinct mechanisms of action. Methods Peripheral CD3⁺ T cells were isolated from patients with IBD receiving anti-TNF plus anti–IL-23 or anti-TNF plus anti-α4β7, and from healthy controls (n = 6 per group). Single-cell RNA sequencing with paired TCR sequencing was integrated using Harmony, achieving excellent donor mixing without residual batch effects. Cell states were assigned using canonical markers and validated by deconvolution with the scIBD ileal atlas, confirming robust annotation of CD4⁺ and CD8⁺ subsets. TCR diversity indices, V-gene usage, clonotype composition and public antigen mapping were analysed using Immunarch and McPAS-TCR. Results Integration revealed stable clustering of major CD3⁺ populations, including naïve, central memory, effector memory, regulatory and cytotoxic subsets, with comparable CD4⁺ and CD8⁺ distributions across groups. Anti-TNF plus anti–IL-23 was associated with reduced richness, contraction of rare clonotypes, dominance of hyperexpanded effector clones and skewed TRBV usage, particularly TRBV20-1, TRBV7-2 and TRBV6-5, resulting in lower true diversity and D50 indices. In contrast, anti-TNF plus anti-α4β7 preserved low-frequency clonotypes, maintained balanced V-gene distribution and exhibited markedly higher evenness, true diversity and rarefaction-based richness. CDR3-length profiles remained physiological in all groups, and public antigen mapping showed no enrichment of known viral or autoimmune specificities, indicating that clonal dynamics reflected treatment-driven rather than pathogen-driven effects. Conclusion Single-cell profiling demonstrates that ACT induces mechanism-specific remodelling of the peripheral T-cell repertoire. Anti-TNF plus anti–IL-23 promotes oligoclonal focusing and effector dominance, whereas anti-TNF plus anti-α4β7 maintains a diverse, homeostatic-like repertoire enriched in rare clonotypes. These findings support the concept that dual-pathway blockade exerts synergistic, non-additive effects on clonal architecture and identify TCR diversity as a potential biomarker of immune recalibration in IBD. References: Danese S, Solitano V, Jairath V, et al. The future of drug development for inflammatory bowel disease: the need to ACT (advanced combination treatment). Gut. 2022 Dec;71(12):2380-2387. Solitano V, Hanžel J, Ma C, et al. An international consensus on the design of clinical trials for advanced combination treatment (ACT) in inflammatory bowel disease. EClinicalMedicine. 2025 Oct 23;89:103582. Conflict of interest: Dr. Solitano, Virginia: Speaker’s fees from Pfizer, Takeda, Giuliani, Tillotts Pharma consulting fees from J&J travel grant from Abbvie Massimino, Luca: No conflict of interest Parigi, Tommaso Lorenzo: Tommaso L Parigi declares no relevant conflicts of interest Spano’, Salvatore: No conflict of interest Cagliani, Stefania: No conflict of interest Errico, Carmela: No conflict of interest Nicolo’, Sabrina: No conflict of interest D’Amico, Ferdinando: Grant:ECCO fellowship grant 2020 ECCO grant 2021 Personal Fees: F D’Amico has served as a speaker for Abbvie, Alfasigma, Ferring, Lilly, Sandoz, Janssen, Fresenius Kabi, Galapagos, Giuliani, MSD, Pfizer, Takeda, Tillotts, and Omega Pharma he also served as an advisory board member for Abbvie, AnaptysBio, Ferring, Fresenius Kabi, Galapagos, Janssen, Lilly, MSD, Takeda, and Nestlè. Allocca, Mariangela: Personal Fees: consulting fees from Nikkiso Europe, Mundipharma, Janssen, Abbvie, Pfizer, Ferring, Galapagos, Sandoz, Lilly and Alfasigma Jairath, Vipul: Consulting Fees: Abbvie, Alimentiv, Amgen, Anaptys Bio, Asahi Kasei, Asieris, Astra Zeneca, Attovia, Blackbird Labs, BMS, Boehringer Ingleheim, Biomebank, Caldera, Calluna, Catalytic Health, Celltrion, Ensho, Enthera, Exeliome Biosciences, Ferring, Fresenius Kabi, Gilead, Granite Bio, GSK, Janssen, Lilly, Merck, Mountainfield, MRM Health, Nxera, Organon, OSE Immunotherapeutics, Pendopharm, Pioneering Medicine, Pfizer, Prometheus, Roche/Genentech, Sanofi, SCOPE, Shattuck Labs, Sorriso, Spyre, Synedgen, Takeda, Teva, Tillotts, Union Therapeutics, Ventus, Ventyx, Vividion, Xencor, Zealand Pharma. Ungaro, Federica: No conflict of interest Danese, Silvio: Personal Fees: AbbVie, Alimentiv, Allergan, Amgen, Applied Molecular Transport, AstraZeneca, Athos Therapeutics, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Dr Falk Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals Inc., Gilead, Hospira, Inotrem, Janssen, Johnson & Johnson, Morphic, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, Teladoc Health, TiGenix, UCB Inc., Vial, Vifor Lecture fees from Abbvie, Amgen, Ferring Pharmaceuticals Inc., Gilead, Janssen, Mylan, Pfizer, Takeda
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