Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a CD19 CAR-T cells were generated from human T cells as described in the Methods. After expansion, CAR-T cells were treated with DMSO or TAIII for 72 h. The proportion of FoxP3 + Treg in CD4 + T was then analyzed by flow cytometry. Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. b – c CAR-T and Untransduced T cells (UNT) cells were treated with TAIII (0, 0.5, 1, 2 µM) for 24 and 72 h under non-stimulatory conditions (without tumor cells). T cell proliferation and cell viability/apoptosis were assessed using CellTiter-Glo assays and flow cytometry (PI/Annexin V/7-AAD staining). Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t-test or ordinary one-way ANOVA with Dunnett’s test. CAR-T cells were co-cultured with Raji-Luc ( d ) or Nalm6-Luc ( e ) cells at effector-to-target (E:T) ratios of 1:2 or 1:4 under indicated treatments. Cytotoxicity was measured by residual luciferase activity, and IFN-γ, IL-2, and TNF-α levels were determined by ELISA. UNT cells were included as negative controls; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. f Real-time cytotoxicity of CAR-T cells co-cultured with Raji cells was monitored over 36 h using Incucyte® cytolight red and annexin V staining; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. g IFN-γ and TNF-α protein levels in CAR-T–Raji co-cultures after 16 h were measured by ELISA; Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. h FoxP3a (FoxP3) mRNA levels in CAR-T–Raji co-cultures were determined by qRT-PCR. Data are presented as mean ± SD from independent experiments ( n = 3–4 biological replicates); two-sided unpaired t -test.

Article Snippet: For this assay, Raji cells were stained with Incucyte® cytolight rapid red dye (Sartorius, #4706) prior to seeding at a density of 10,000 cells per well in a 96-well flat-bottom plate.

Techniques: Generated, Flow Cytometry, Staining, Cell Culture, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR