Panel A shows a schematic of engineered IL-2 ligand variants fused to the OrthoIL-2 receptor beta chain. Panel B shows structural models comparing 3A10 antibody binding to CD25r, CD25f, GS3, and GS4 variants. Panel C shows histograms of phosphorylated STAT5 signalling in Thy1.1-positive and Thy1.1-negative cells. Panel D shows bar graphs quantifying phosphorylated STAT5 mean fluorescence intensity across engineered ligand and receptor constructs. Panel E shows a bar graph comparing Thy1.1-positive to Thy5.1 cell ratios following construct expression. Panel F shows phosphorylated AKT histograms and quantification following recombinant human IL-2 stimulation over multiple time points. Panel G shows flow cytometry contour plots of Bcl-2 expression versus Thy1.1 in engineered cell populations. Panel H shows a bar graph of CellTrace Violet dilution, indicating proliferation across different engineered constructs.
Engineering a tethered orthoIL-2–IL-2Rβ system. (A) Schematic of the tethered IL-2–IL-2R components tested. Created in BioRender. Tang (2026) https://BioRender.com/dayo897. (B) Visualization of the four linkers used to tether orthoIL-2 3A10 to orthoIL-2R using PyMOL. (C and D) Flow cytometric plots of pSTAT5 in transduced Tregs (Thy1.1+, left) or not (Thy1.1−, right) with various constructs indicated. Representative flow plots are shown. (C) Results were repeated in three independents experiments. (D) Data were expressed as percentages of pSTAT5 MFI relative to that induced in orthoIL-2R–transduced Tregs stimulated with 100 IU/ml mouse IL-2. (E) Enrichment of Thy1.1+ Tregs on day 5 versus day 1 after exogenous IL-2 withdrawal. Results shown are a summary of three independent experiments (mean ± SD, n = 3–6). (F) pAKT in 3A10-CD25r (3A10t), 1G12-CD25r (1G12t), and orthoIL-2R engineered Tregs. Aliquots of orthoIL-2R Tregs were stimulated with 2,000 IU/ml rhIL-2 for the indicated duration as a reference. (G) Representative flow cytometric plots of Bcl-2 expression in Tregs transduced with 3A10t and 1G12t constructs. The results shown are representative of three independent experiments. (H) On day 5 after activation, Thy1.1+ Tregs were purified, labelled with CTV, and cultured without TCR activation or exogenous IL-2. CTV dilution was measured on day 14. For D–F and H, Kruskal–Wallis test was used to determine the statistical significance when compared with the orthoIL-2R controls. For all panels, only statistically significant P values are shown.