P0106 Profiling inflammation-associated T helper subsets in Inflammatory Bowel Disease
Authors: Jiang Q , Lindelauf C , Ouboter L F , Koning F , van der Meulen-de Jong A E , van Unen V , Pascutti M F 
Published: January 2025

Abstract

Background
Microbiota-specific CD4+ T cells drive chronic inflammation in Inflammatory Bowel Disease (IBD), comprising Crohn’s Disease (CD) and Ulcerative Colitis (UC). In previous projects we have identified several CD4+ T cell subsets enriched in the inflamed mucosa from naïve IBD patients, especially a colon-specific increase of HLA-DR+CD38+CD4+ memory T cells1. However, the functional properties remained to be elucidated. We therefore aimed to characterize CD4+ helper T cell lineage-specifying transcription factor (TF) expression and cytokine profiles in mucosa and circulation from IBD patients.

Methods
We collected intestinal samples and peripheral blood from patients and healthy controls undergoing diagnostic ileocolonoscopy, and subsequently employed 5-laser spectral flow cytometry to analyse single-cell data.

Results
For phenotypic characterization, a 37-antibody panel including TF and chemokine receptors was applied to 70 intestinal samples from 16 CD and 12 UC patients (including 22 non-inflamed, 22 inflamed samples and 16 samples in remission), as well as 5 healthy controls. Unbiased tSNE-based analysis revealed an increase of Foxp3+ Treg cells in inflamed biopsies. Particularly, in Ki-67+RORγt+ cells, we identified a cluster co-expressing Foxp3 and Helios, and a cluster expressed T-bet were enriched in inflamed colonic biopsies. As described before, two clusters of activated RORγt+HLA-DR+CD38+ T cell subset, one CD103+ and another CD103-, were increased in frequency in inflamed biopsies. Both clusters expressed CTLA-4, CD39 and Ki-67. In contrast, the frequencies of RORγt+ NKT-like cells (CD161+CD69+CD103-) were decreased in inflamed tissues.
Next, we measured cytokine production in CD4+ T intestinal cells upon anti-CD3/CD28 stimulation of non-inflamed (healthy controls=8, IBD in active stage=8, IBD in remission=4) and inflamed samples (IBD in active stage=7). Frequencies of multifunctional CD103+ and CD103- CD154+ cells simultaneously producing IFNγ, TNFα and IL-2 were decreased in inflamed biopsies. On the contrary, CXCL-13 expression was elevated in the inflamed group.

Conclusion
In conclusion, we observed that a Th17/Treg and a Th17/Th1 subset were increased in inflamed biopsies, which we hypothesize to represent a transformation of Th17 to Treg or Th1. In addition, the HLA-DR+CD38+ subset displaying a Th17 profile was proliferating in inflamed mucosa, indicative of an active role in the disease pathogenesis. Simultaneously, Tregs were upregulated while CD4+ T cells produced less cytokines in inflamed mucosa, potentially indicating that Treg cells suppress the pro-inflammatory CD4+ T cell response in IBD patients. Thus, our results provide new clues about the functional contribution of CD4+ T cells to inflammation in IBD.

References
1.van Unen V, Ouboter LF, Li N, et al. Identification of a Disease-Associated Network of Intestinal Immune Cells in Treatment-Naive Inflammatory Bowel Disease. Front Immunol. 2022;13:893803. doi:10.3389/fimmu.2022.893803