A novel pro-inflammatory FOXP3+ T cell subpopulation has been identified in patients with Crohn's disease (CD), which exhibits a unique transcriptional signature associated with strong TNFα signaling. Using single-cell RNA sequencing, researchers analyzed CD4+ T cells from ileal biopsies of CD patients and healthy controls, revealing five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy individuals represent the origin of pseudotemporal development into inflammation-associated subtypes. These pro-inflammatory Tregs displayed impaired suppressive activity in vitro and elevated cytokine responses in organoid co-culture systems. The histone deacetylase inhibitor vorinostat normalized gene expression patterns and rescued the suppressive function of FOXP3+ cells in vitro. The study highlights the therapeutic potential of targeting these pro-inflammatory Tregs using vorinostat, an FDA-approved drug. The findings suggest that these Tregs are highly HDAC-dependent and that vorinostat treatment can restore their suppressive function. The results have implications for patient selection for anti-TNF therapy and may inform Treg adoptive cell transfer programs. The study provides a pipeline to specifically target these cells, offering new insights into the pathogenesis of CD and potential therapeutic strategies.A novel pro-inflammatory FOXP3+ T cell subpopulation has been identified in patients with Crohn's disease (CD), which exhibits a unique transcriptional signature associated with strong TNFα signaling. Using single-cell RNA sequencing, researchers analyzed CD4+ T cells from ileal biopsies of CD patients and healthy controls, revealing five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy individuals represent the origin of pseudotemporal development into inflammation-associated subtypes. These pro-inflammatory Tregs displayed impaired suppressive activity in vitro and elevated cytokine responses in organoid co-culture systems. The histone deacetylase inhibitor vorinostat normalized gene expression patterns and rescued the suppressive function of FOXP3+ cells in vitro. The study highlights the therapeutic potential of targeting these pro-inflammatory Tregs using vorinostat, an FDA-approved drug. The findings suggest that these Tregs are highly HDAC-dependent and that vorinostat treatment can restore their suppressive function. The results have implications for patient selection for anti-TNF therapy and may inform Treg adoptive cell transfer programs. The study provides a pipeline to specifically target these cells, offering new insights into the pathogenesis of CD and potential therapeutic strategies.