Fig. 1: Functional screening identified Timosaponin AIII impairs Treg differentiation and function.

a Schematic illustrating the generation of the high-throughput screening platform for Foxp3-mediated Il-2 inhibition and the inhibitor screening workflow. b Treg functional inhibitor screening identified Timosaponin AIII (TAIII) as a “hit”. c Human PBMCs were activated through TCR stimulation and treated with 1 μM of the indicated compounds for 48 h. IL-2 secretion levels were measured by ELISA (upper panel). Cell viability was measured using the CellTiter-Glo assay (lower panel). d Chemical structure of TAIII. e FoxP3a-IL-2-Fluc-reporter were generated by introducing IL-2-promoter-Fluc-reporter that containing a FoxP3 binding site, into Jurkat T cells, the monoclonal cells were picked out post-puromycin selection, followed delivery of a FoxP3a over-expression lentiviral construct containing a BSD resistance cassette. Cells were treated with or without (w/o) TAIII, the target effect towards Foxp3a-mediated IL-2 suppression were determined by luciferase assay. Data are presented as mean ± SD (n = 3–4 biological replicates); ordinary one-way ANOVA with Dunnett’s test. f TAIII impairs Treg differentiation and functional marker expression in a dose-dependent manner. Human CD4⁺ Naïve T cells were isolated from umbilical cord blood of healthy donors, and cultured under iTreg polarization conditions for 5 days. iTreg cells were then subjected to TAIII treatment, mRNA levels of indicated genes were assessed post-16 h by qRT-PCR. Data are presented as mean ± SD (n = 3 biological replicates); two-way ANOVA with Dunnett’s test. g–h Naïve human CD4⁺ T cells were obtained and differentiated into the iTreg lineage as in e. Expression levels of Foxp3 were assessed post-72 h treatment w/o TAIII by flow cytometry. Data are presented as mean ± SD Data are presented as mean ± SD (n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. Source data are provided as a Source Data file.