What question did this study set out to answer?

The research aims to understand the role of cytotoxic CD4+ T cells in ulcerative colitis and their association with treatment outcomes.

January 23, 2026

OP09Polyfunctional cytotoxic CD4+ T cells associate with mucosal inflammation and predict treatment resistance in Ulcerative Colitis

Key Points

The research aims to understand the role of cytotoxic CD4+ T cells in ulcerative colitis and their association with treatment outcomes.
In vitro functional assays to assess T-cell responses
Multiparameter flow cytometry of colonic samples from patients
Bulk transcriptomics and single-cell RNA sequencing analyses
Single-cell spatial transcriptomics to evaluate tissue context
GZMB+ CD4+ T cells are significantly more abundant in ulcerative colitis compared to controls.
Increased levels of GZMB+ CD4+ T cells correlate with higher endoscopic disease severity.
A cytotoxic CD4+ T-cell gene signature predicts poor response to ustekinumab after 8 weeks.
Higher expression of cytotoxic genes in baseline biopsies is associated with failure to achieve mucosal healing.

Abstract

Abstract Background The contribution of cytotoxic T cells to the pathogenesis and treatment outcomes of ulcerative colitis (UC) remains poorly understood. Methods We integrated in vitro functional assays, multiparameter flow cytometry, bulk transcriptomics, single-cell RNA sequencing (scRNAseq), and single-cell spatial transcriptomics analyses to characterise cytotoxic T-cell responses in UC and evaluate their association with clinical and endoscopic outcomes. Results In vitro stimulation of lamina propria mononuclear cells (LPMCs) from UC patients with agonistic anti-CD3 antibodies upregulated cytotoxic gene programs, including GZMB, indicating enhanced T-cell cytotoxicity. Bulk transcriptomics analysis using the TaMMA resource1 confirmed upregulation of these cytotoxic genes in the UC colonic mucosa compared to non-IBD controls and Crohn’s disease. Multiparameter flow cytometry of 42 UC patients and 22 controls showed increased GZMB+ LPMCs in UC. Surprisingly, CD4+ T cells were a major source of GZMB in UC patients and were significantly more abundant than in controls. These GZMB+ CD4+ T cells exhibited a “hyper-inflammatory” polyfunctional phenotype, co-expressing IFN-γ, IL-13, IL-17, IL-22, and TNFα, with cells producing ≥3 pro-inflammatory cytokines significantly increased in UC compared to controls (p = 0.004). Analysis of UC scRNAseq data2 confirmed that GZMB+ CD4+ T cells co-expressed cytotoxic genes (GZMA, PRF1) and pro-inflammatory mediators (IFNG, IL26, OSM) compared to GZMB- CD4+ T cells. A cytotoxic CD4+ T-cell gene signature derived from these data was strongly enriched in UC compared to controls in a large independent cohort (n = 525)3, increasing with endoscopic disease severity and stratifying UC patients by degree of cytotoxic enrichment (Figure 1). Independent single-cell spatial transcriptomics analysis further validated enrichment of this cytotoxic CD4+ T-cell signature in UC versus non-IBD colonic tissues. This signature was also detected in multiple murine models of colitis, with strongest upregulation observed in T-cell transfer colitis. Notably, higher cytotoxic CD4+ T-cell signature enrichment predicted poor response to ustekinumab at week 8 in the UNIFI trial4 (n = 384) for mucosal healing (OR 0.11, p-value = 0.042), even after adjusting for CRP, faecal calprotectin, Total Mayo Score, disease duration, and prior anti-TNF failure. Similarly, the signature was upregulated (p-value = 0.013) in baseline biopsies from UC patients who failed to achieve mucosal healing to infliximab compared with responders in an independent cohort. Conclusion Pro-inflammatory cytotoxic GZMB+ CD4+ T cells are increased in the colonic mucosa of UC patients, correlate with endoscopic disease severity, and predict resistance to biologic therapies. References: 1.Massimino L, Lamparelli LA, Houshyar Y, et al. The Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) framework. Nature Computational Science 2021 1:8. 2021;1(8):511-515. doi:10.1038/s43588-021-00114-y. 2.Smillie CS, Biton M, Ordovas-Montanes J, et al. Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis. Cell. 2019;178(3):714-730.e22. doi:10.1016/J.CELL.2019.06.029. 3.Argmann C, Hou R, Ungaro RC, et al. Biopsy and blood-based molecular biomarker of inflammation in IBD. Gut. 2023;72(7):1271-1287. doi:10.1136/GUTJNL-2021-326451. 4.Sands BE, Sandborn WJ, Panaccione R, et al. Ustekinumab as Induction and Maintenance Therapy for Ulcerative Colitis. New England Journal of Medicine. 2019;381(13):1201-1214. doi:10.1056/NEJMOA1900750. Conflict of interest: Thomas, John P: Research support from AstraZeneca and Roche Hermangild Kottoor, Sherine: No conflict of interest Ibraheim, Hajir: No conflict of interest Cozzetto, Domenico: No conflict of interest Wooldridge, Tyler: No conflict of interest Madgwick, Matthew: No conflict of interest Olbei, Marton: No conflict of interest Digby-Bell, Jonathan Leith: Speaker fees: AbbVie, Lilly, J&J Conference and Travel Fees: AbbVie, Lilly Advisory board: AbbVie Lo, Jonathan: No conflict of interest Korcsmaros, Tamas: Grant: Unilever, Roche Powell, Nick: Grant: Takeda, BMS, Pfizer, Astra-Zeneca Personal Fees: Abbvie, Abivax, Allergan, Astra-Zeneca, Bristol-Myers Squibb, Celgene, Celltrion, Dr Falk Pharma UK Ltd, Ferring, Galapagos, GSK, Janssen, MSD, Roche, Pfizer, Sobi, Takeda, Tillotts

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