Figure 1.

Generation of Tregs stably expressing NIS-GFP

(A) Sketch of the lentiviral construct. SFFV, spleen focus-forming virus promoter. (B) Representative example of Treg transduction with the NIS-GFP reporter. Post-transduction FACS yielded more than 99% pure NIS-GFP⁺ Tregs. (C) Fluorescence microscopy revealed NIS-GFP expression in Tregs to be predominantly membranous. Scale bars, 10 μm and 5 μm (inset). (D) Flow cytometry analysis of NIS-GFP⁺ Tregs in the weeks after transduction alongside corresponding untransduced Tregs (numbers indicate mean fluorescence intensity [MFI]). One representative of three experiments is shown. (E) Transduced NIS-FP⁺ Tregs were sorted by FACS (cf. B) and expanded further for subsequent experiments over a period of ten days. Fold expansion of these cells during this period was quantified and compared with corresponding untransduced parental Tregs. Shown is a box-and-whisker plot with median and 25^th–75^th percentiles indicated as well as min and max values drawn as whiskers; n = 9. There was no significant difference between the cell types. (F) NIS-GFP function as quantified per [^99mTc]TcO₄⁻ uptake in untransduced and NIS-GFP⁺ Tregs (gray). NaClO₄ block reports NIS specificity (black). n = 3, error bars indicate SD; ANOVA with Tukey multiple comparisons correction.