Panel A shows a schematic diagram of the experimental workflow. Panel B features a line graph depicting pSTAT5 levels in response to varying concentrations of exogenous IL-2 in Tregs transduced with empty vector (EV), orthoIL-2R, or 3A10t. The x-axis represents rhIL-2 concentration in IU/ml, and the y-axis represents pSTAT5 MFI. Panel C presents a line graph showing in vitro suppression data, with the x-axis indicating the Treg:Responder ratio and the y-axis showing the percentage of suppression. Panel D includes a box plot illustrating the number of transferred Tregs in the spleens of recipient mice, normalized to EV control. The x-axis lists the different groups (EV, OrthoIL-2R, 3A10t), and the y-axis shows the fold change over EV. Panel E contains multiple box plots showing CD25 and Foxp3 expression and the number of transferred Tregs in the spleen, pancreatic lymph nodes (pancLN), and islets. The x-axis lists the different groups (OrthoIL-2R, 3A10t), and the y-axis shows the fold change over OrthoIL-2R. Panel F features a Kaplan-Meier survival curve depicting diabetes-free survival in NOD.CD28KO mice treated with EV- or 3A10t-transduced BDC2.5 Tregs. The x-axis represents age in weeks, and the y-axis shows the percentage of diabetes-free survival. Panel G includes bar graphs showing the number of transferred Thy1.1 plus Tregs recovered in the spleen, pancLN, and islets. The x-axis lists the different groups (EV, 3A10t), and the y-axis shows the number of Tregs. Panel H presents box plots showing the percentages of Thy1.1 plus Tregs among CD4 plus T cells in the spleen, pancLN, and islets. The x-axis lists the different groups (EV, 3A10t), and the y-axis shows the percentage of Thy1.1 plus Tregs. Panel I features a scatter plot and line graphs showing the expression of Foxp3 and CD25 in endogenous Tregs and transferred Thy1.1 plus Tregs in the islets. The x-axis lists the different groups (Host, 3A10t), and the y-axis shows the MFI of Foxp3 and CD25.
Function of Tregs expressing tethered 3A10-orthoIL-2R. (A) Experimental workflow for assessing function of 3A10 expressing Treg in vitro and in vivo. Created in BioRender. Tang (2026) https://BioRender.com/hloc6vm. (B) pSTAT5 in response to titrated concentrations of exogenous IL-2 in Tregs transduced with EV, orthoIL-2R, or 3A10t. Results shown are representative of two independent experiments. (C)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 3A10t+ and orthoIL-2R+ Tregs at each Treg:responder ratio. (D) NSG mice were injected with NOD Tregs transduced with EV, orthoIL-2R, or 3A10t. The numbers of transferred Tregs in the spleens of the recipient mice were analyzed 1 wk after cell injection. Numbers shown were normalized to EV control. Data shown were a summary of three independent experiments (means ± SD, n = 8–10 mice per group). Kruskal–Wallis test was used to determine the statistical significance among the groups. (E) NOD.CD28KO mice were injected with 20,000 FACS-purified Thy1.1+ orthoIL-2R or 3A10t-transduced BDC2.5 Tregs. CD25 and Foxp3 expression (normalized to orthoIL-2R control) in the transferred Tregs and the numbers of transferred Thy1.1+ Tregs in the spleens, pancLN, and islets were analyzed 1 wk later. Data shown are a summary of three independent experiments (n = 7–13 mice per group). Mann–Whitney test was used to determine the statistical significance. (F) NOD.CD28KO mice were treated with either 2,000 EV- or 3A10t-transduced BDC2.5 Tregs. Mice with two readings of blood glucose >250 mg/dl were considered diabetic (n = 14–28 mice per group in two experimental replicates). Statistical significance was determined using Kaplan–Meier survival analysis. P values were calculated using Mantel–Cox test comparing all experimental groups. (G–I) NOD.CD28KO mice were injected with 2,000 FACS-purified Thy1.1+ EV- or 3A10t-transduced BDC2.5 Tregs and analyzed 10 wk later. (G) Numbers of transferred Thy1.1+ Tregs recovered in the spleens, pancLN, and islets. Mann–Whitney test was used to determine the statistical significance. (H) Percentages of Thy1.1+ Tregs among CD4+ T cells. Friedman test with Dunn’s multiple comparison test were used to determine statistical significance. (I) Expression of Foxp3 and CD25 in endogenous Tregs and transferred Thy1.1+ Tregs in the islets. Paired t test was used to determine the significance of the differences. For all panels, only statistically significant P values are listed.