Panel A shows a line graph depicting diabetes-free survival percentages over days post cell transfer. The x-axis represents days post cell transfer, and the y-axis represents diabetes-free survival percentage. Two lines are shown: one for empty vector (EV) and one for 3A10t, with a p-value of 0.0026 indicating statistical significance. Panel B presents a schematic diagram of the 3A10t and vector knock-in strategy, detailing the Foxp3 gene locus and the insertion points. Panel C displays two PCR fragment gels. The left gel shows fragments from exon 13 to Thy1.1, and the right gel shows fragments from exon 13 to 3A10t. Four lanes are present in each gel, representing different conditions: no RNP or AAV5, RNP only, RNP AAV5-Vector, and RNP AAV5-3A10t. Panel D contains four flow cytometry scatter plots showing Thy1.1 expression in Tregs and Tconvs 4 days after editing. The x-axis represents Thy1.1, and the y-axis represents forward scatter area (FSC-A). Panel E outlines an experimental workflow diagram for evaluating the function of 3A10t Foxp3 knock-in in NOD Tregs, detailing steps from Treg activation to in vitro cell enrichment and phenotyping. Panel F includes a histogram and a bar graph. The histogram shows pSTAT5 levels in 3A10t and vector conditions, while the bar graph quantifies pSTAT5 mean fluorescence intensity (MFI) with statistical significance indicated. Panel G presents four flow cytometry scatter plots showing fold change in Thy1.1 plus Treg percentages cultured without exogenous IL-2 from day 6 to day 10 after activation. The x-axis represents Thy1.1, and the y-axis represents FSC-A. A bar graph quantifies the percentage of Thy1.1 plus enrichment with statistical significance. Panel H displays two flow cytometry scatter plots and four bar graphs. The scatter plots show expression of Bcl-2, CD25, Foxp3, and Thy1.1 by Thy1.1 plus cells in vector knock-in and 3A10t knock-in Tregs cultured without IL-2. The bar graphs quantify the mean fluorescence intensity (MFI) of Bcl2, CD25, Foxp3, and Thy1.1, with statistical significance indicated.
Safe engineering of the tethered orthoIL-2–IL-2R system. (A) Diabetes development in NOD.CD28KO mice that received 20,000 CD4+ BDC2.5Tg TCR transgenic T cells transduced with EV or 3A10t. Results shown are a summary of two independent experiments. Statistical significance was determined using Kaplan–Meier survival analysis. P values were calculated using Mantel–Cox test. (B) Schematic of 3A10t and vector knock-in strategy. Created in BioRender. Tang (2026) https://BioRender.com/nq4hkzh. (C) PCR of genomic DNA showing vector and 3A10t insertion into the Foxp3 locus. Uncropped gel images are available online. (D) Flow cytometric analysis of Thy1.1 expression in Tregs and Tconvs 4 days after editing, day 6 after activation. (E) Experimental workflow for evaluating the function of 3A10t Foxp3 knock-in in NOD Tregs. (F) pSTAT5 1 day after IL-2 withdrawal. (G) Fold change Thy1.1+ Treg percentages cultured 4 days without exogenous IL-2 from day 6 to day 10 after activation. The 3A10t Tregs in this panel are the same as the 3A10t Tregs shown in D. (H) Expression of Bcl2, CD25, Foxp3, and Thy1.1 by Thy1.1+ cells in vector knock-in and 3A10t knock-in Tregs cultured without IL-2. Statistical significance was determined using Mann–Whitney test. Only statistically significant P values are shown. Source data are available for this figure: SourceData F7.