Panel A shows close-up structural views of 3A10 binding to CD25r, CD25f, GS3, and GS4 variants. Panel B shows front, top, and back structural views of the complete 3A10–OrthoIL-2 receptor complex. Panel C is a bar graph showing the expression of Bcl-2 in Tregs 5 days after withdrawal of exogenous IL-2. The y-axis represents Bcl-2 MFI, and the x-axis lists different constructs, including EV, OrthoIL-2R, 3A10-CD25r, 3A10-CD25f, 3A10-GS3, 3A10-GS4, 1G12-CD25r, 1G12-CD25f, 1G12-GS3, and 1G12-GS4. Notable trends include higher Bcl-2 expression in 3A10-CD25r and 3A10-CD25f constructs. Panel D consists of histograms showing the CTV dilution profile of Tregs, comparing untransduced cells, cells with empty vector, and various tethered constructs. The x-axis represents CTV, and the y-axis represents cell count. The histograms indicate differences in cell proliferation among the constructs. Panel E displays flow cytometry profiles of pSTAT5 in Tregs cultured without exogenous IL-2 for 15 to 18 hours. The left side shows representative flow cytometry profiles for EV, 1G12t, and 3A10t constructs, while the right side summarizes pSTAT5 MFI in a bar graph. The y-axis represents pSTAT5 MFI, and the x-axis lists time points.
Structure prediction and function assessment of orthoIL-2–IL-2R constructs (related to Figs. 4 and 5). (A and B) Alpha-fold models showing the distance of 3A10’s side chains from the orthoIL-2Rβ–binding pocket when tethered to all four linkers (A) and 3A10-CD25r and the trimeric IL-2R complex (B). (C and D) Functional assessment of various tethered constructs. (C) Expression of Bcl-2 in Tregs 5 days after withdrawal of exogenous IL-2 (related to Fig 4 G). (D) CTV dilution profile of Fig 4 H. (E) Tregs were removed from exogenous IL-2 immediately after spinfection on day 2 after activation. The cells were cultured in the absence of exogenous IL-2 for 15–18 h before flow cytometric analysis of pSTAT5. Representative flow cytometry profiles of pSTAT5 are shown on the left and pSTAT5 MFI are summarized in the graph on the right. Statistical significance of the difference was determined using two-way ANOVA followed by Tukey’s multiple comparison test.