Panel A shows a line graph of 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 max. Panel B shows scatter plots of Treg proliferation measured using CTV dilution, with different concentrations of wtIL-2 and 1G12 along with bar graphs. Panel C shows a line graph of an in vitro suppression assay, with the x-axis representing the Treg to responder ratio and the y-axis representing the percentage of suppression. Panel D shows a bar graph of CD25 expression in NSG mice treated with PBS or MSA-fusion proteins, with the x-axis representing different treatment conditions and the y-axis representing CD25 MFI as a fold change over PBS. Panel E shows a bar graph of the total cell number in the same experiment, with the x-axis representing different treatment conditions and the y-axis representing the total cell number as a fold change over PBS. Panel F shows a bar graph of CD25 expression on transferred Tregs in NOD.CD28KO mice, with the x-axis representing different treatment conditions and the y-axis representing CD25 MFI as a fold change over PBS. Panel G shows a bar graph of the number of transferred Tregs in the same experiment, with the x-axis representing different treatment conditions and the y-axis representing the number of Tregs as a fold change over PBS. Panel H shows a bar graph of CD25 MFI on host T cells in the same experiment, with the x-axis representing different treatment conditions and the y-axis representing CD25 MFI as a fold change over PBS. Panel I shows a line graph of diabetes-free survival in NOD.CD28KO mice treated with PBS or MSA-1G12 with Tregs, with the x-axis representing age in weeks and the y-axis representing the percentage of diabetes-free survival. Panel J shows a line graph of diabetes-free survival in NOD.CD28KO mice treated with PBS or MSA-1G12 without Tregs, with the x-axis representing age in weeks and the y-axis representing the percentage of diabetes-free survival.
OrthoIL-2 1G12 has higher potency but cross-reacts on wtIL-2R. (A) Dose response of IL-2–induced pSTAT5 in Tregs transduced with EV or orthoIL-2R. (B) Treg proliferation measured using CTV dilution. The data were collected in the same experiment as shown in Fig. 2 C, thus the wtIL-2 control data are the same. (C)In vitro suppression assay. Results shown in A–C are summaries of three independent experiments (means ± SD, n = 3). 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 with orthoIL-2R+ Tregs without addition of 1G12 at each Treg:responder ratio. (D and E) NSG mice were injected with a mixture of orthoIL-2R–transduced Thy1.1+ and non-transduced Thy1.1− NOD Tregs followed by treatment with PBS or MSA-fusion proteins (MSA indicated by *) at the doses shown on the graph. CD25 expression (D) and number of transferred Tregs (E) in the spleens are summarized. Numbers shown are 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. (F–H) NOD.CD28KO mice were injected with orthoIL-2R+ BDC2.5 Tregs and followed by seven daily injection of PBS or MSA-fusion proteins at the doses indicated. CD25 expression on transferred Tregs (F), number of transferred Tregs (G), and CD25 MFI on host T cells (H) in the spleens are summarized (means ± SD, n = 4–5 mice per group from three experiments). Kruskal–Wallis test was used to determine the statistical significance from the PBS-treated controls. (I and J) NOD.CD28KO mice were treated with PBS or 7.6 μg MSA-1G12 from 5 to 20 wk of age with (I) or without (J) 2,000 orthoIL-2R+ BDC2.5 Tregs. 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 to BDC2.5 Treg-treated group and comparing the same dose of MSA-1G12 with or without BDC2.5 Tregs. For C–H, only statistically significant P values are listed.