Ustekinumab Resistance in Individuals with Ulcerative Colitis is Associated with an Alteration of a Subset of Pro-inflammatory Mucosal Regulatory T cells

Background & Aims: Ustekinumab is a monoclonal antibody therapy targeting interleukin 12 and 23 for the treatment of inflammatory bowel diseases, including ulcerative colitis (UC). While these pathways remain quite attractive for UC therapy, response to ustekinumab can be variable. There is an urgent need to better understand the underlying mucosal immune alterations associated with treatment to guide therapy decisions. This study aims to examine the mucosal immune signatures in individuals with UC with variable treatment response to ustekinumab. Methods: Sigmoid colon tissue from individuals treated with ustekinumab were analyzed using a multi-modal approach. Single-cell RNA and T cell receptor sequencing was performed on mucosal biopsies. In a subset of these patents, multiparameter flow cytometry and spatial transcriptomics was also completed on matched pre- and post-treatment tissue samples. Key findings were also validated on a larger cohort using immunohistochemistry. Results: Ustekinumab significantly altered the frequency and phenotype of mucosal regulatory T cells (Tregs). Non-responders to ustekinumab had a higher frequency of Tregs that expressed OX40 and GITR, which is associated with decreased suppressive abilities. In contrast, responders had Tregs with elevated GPR15 and reduced expression of the kinase PIM2, which can alter Treg stability and function. Additionally, Th17 cells in non-responders demonstrated an enhanced pro-inflammatory gene expression profile. Conclusion: Non-response to ustekinumab in UC is linked to a mucosal immune environment enriched with pro-inflammatory T cell phenotypes and impaired regulatory T cell function. These findings suggest that Tregs are both targets and potential biomarkers of ustekinumab response, with their phenotypic and transcriptional features providing insight into mechanisms of therapeutic resistance.