Fig. 4: TAIII activates T cell immuno-response through impairing Tregs in A2AR-dependent manner.

a Human PBMCs (1 × 10⁶) were activated with anti-CD3/CD28 beads for 4 days, then treated with NECA ± TAIII for 24 h. Bulk RNA sequencing was performed, and a heatmap displays selected mRNAs and surface markers of Treg and Teff subsets. b PBMCs were stimulated with CD3/CD28 beads and treated with indicated compounds for 48 h. IFN-γ and IL-2 in supernatants were measured by ELISA. Data represent mean ± SEM of three independent experiments, each in triplicate; analyzed by ordinary one-way ANOVA with Dunnett’s test. c T cells were generated as in (a). IFN-γ, IL-2, and FoxP3 transcription (c) and FoxP3 expression during 10-day induced Treg (iTreg) differentiation (d) were determined by qRT-PCR. Data are presented as mean ± SD (n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. e–f Human CD4⁺ naive T cells were skewed to Tregs for 7 days. Suppressive function was assessed by co-culture with CFSE-labeled responder Teff cells; CFSE dilution in CD4⁺CFSE⁺ Teff cells was analyzed by flow cytometry to determine the percentage of undivided Teff cells. Data are presented as mean ± SD (n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g–h iTreg cells (4-day differentiation) were transduced with shCK or shA2AR constructs (shA2AR-2, -3), and mRNA levels of indicated genes, including FoxP3, were measured by qRT-PCR. Data are presented as mean ± SD (n = 3 biological replicates); One-way ANOVA with Dunnett’s test. i iTreg cells were generated and the recruitment change of CREB on FoxP3a (FoxP3) promoter w/o TAIII treatment was confirmed by CUT & RUN assay. Data are presented as mean ± SD (n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test.