Panel A shows a schematic diagram of the experimental workflow for assessing the function of 3A10. Panel B is a line graph showing the dose response. The x-axis represents IL-2 concentration in IU per milliliter, and the y-axis represents pSTAT5 MFI as a percentage of wtIL-2 maximum. Panel C includes scatter plots and a bar graph showing Treg proliferation measured using CTV dilution under different conditions of IL-2 concentration. The scatter plots show Thy1.1 versus CTV, and the bar graph shows the percentage of CTV low cells. Panel D presents histograms showing in vitro suppression assays with different Treg to responder ratios along with a line graph. Panel E is a bar graph showing CD25 expression in NSG mice injected with orthoIL-2R positive Tregs and followed by 7 daily infusions of MSA-fusion proteins or PBS. The x-axis represents different treatment conditions, and the y-axis represents CD25 MFI fold change over PBS. Panel F is a bar graph showing the number of transferred Tregs in the spleens of NSG mice under the same conditions as Panel E. Panel G is a bar graph showing CD25 MFI in NOD.CD28KO mice injected with BDC2.5 orthoIL-2R plus Tregs and followed by 7 daily infusions of MSA-fusion proteins or PBS. Panel H is a bar graph showing the number of transferred Tregs in the spleens of NOD.CD28KO mice under the same conditions as Panel G. Panel I is a Kaplan-Meier survival curve showing the percentage of diabetes-free NOD.CD28KO mice treated with either BDC2.5 Treg plus PBS or BDC2.5 orthoIL-2R plus Tregs plus MSA-3A10 once or twice per week for 15 weeks. The x-axis represents age in weeks, and the y-axis represents the percentage of diabetes-free mice.
OrthoIL-2 3A10 is a selective but weak agonist for orthoIL-2R. (A) Experimental workflow for assessing function of 3A10 in vitro and in vivo. Created in BioRender. Tang (2026) https://BioRender.com/q07c632. (B) Dose response of IL-2–induced pSTAT5 in Tregs transduced with EV or orthoIL-2R with titrated concentrations of wtIL-2 or orthoIL-2 3A10. (C) Treg proliferation was measured using CTV dilution. (D)In vitro suppression. Ordinary two-way ANOVA followed by Tukey’s multiple comparison after test was used to determine the statistical significance of the difference observed. P values on the graph are for comparisons between orthoIL-2R+ Tregs with or without added 100,000 IU/ml 3A10 at each Treg:responder ratio. Results in B–D are a summary of three independent experiments (means ± SD, n = 3). (E and F) NSG mice were injected with orthoIL-2R+ Tregs and followed by seven daily infusions of MSA-fusion proteins (MSA indicated by *) or PBS as shown. CD25 expression (E) and number of transferred Tregs (F) in the spleens normalized to the means of PBS control are summarized (means ± SD, n = 8–10 mice per group from three experiments). Two-way ANOVA followed by Sidak multiple comparison after test was used to assess statistical significance of the difference between orthoIL-2R− and orthoIL-2R+ cells under each treatment condition. (G and H) NOD.CD28KO mice were injected with BDC2.5 orthoIL-2R+ Tregs and followed by seven daily infusions of MSA-fusion proteins or PBS as shown. CD25 MFI and number of transferred Tregs in the spleens are summarized (means ± SD, n = 4–5 mice per group pooled from three experiments). Kruskal–Wallis test was used to determine the statistical significance from the PBS-treated controls. (I) NOD.CD28KO mice were treated with either 2,000 BDC2.5 Treg + PBS or BDC2.5 orthoIL-2R+ Tregs + MSA-3A10 30 μg once or twice per week for 15 wk. Mice with two readings of blood glucose >250 mg/dl were considered diabetic (n = 9–28 mice per group pooled from two experiments). Statistical significance was determined using Kaplan–Meier survival analysis. P values were calculated using Mantel–Cox test comparing each condition with the BDC2.5 Treg-treated group. For D, E, G, and H, only statistically significant P values are listed.