Fig. 3. FOXP3 edited T_regs express FOXP3 protein and display characteristic in vitro phenotype and function.

(A) Quantification of FOXP3 protein expression in the MT-2 T_reg cell line by flow cytometry, showing median fluorescence intensity (MFI) values for one representative experiment out of three performed. (B) FOXP3 homology donor constructs designed to improve FOXP3 protein expression, including further codon optimization (FOXP3^FLco) and addition of a WPRE element (FOXP3^FLcoW). (C) Flow cytometry for FOXP3 in MT-2 cells comparing the different donor constructs. (D) FOXP3 expression in primary T_regs by flow cytometry and quantification represented as ratio of MFI in treated cells versus WT unmodified T_regs [mean ± SD, one-way analysis of variance (ANOVA), Tukey’s multiple comparisons]. Significance comparing FOXP3^FL (n = 2, **P < 0.01) or FOXP3^FLcoW (n = 6, **P < 0.01) to WT mock treated (n = 2). (E) Expression of signature T_reg marker proteins on primary T_regs by flow cytometry and corresponding quantification (mean ± SD, n = 2 to 4). (F) Suppression assay showing unstimulated responders (R), stimulated responders (R*) alone or in coculture with T_regs, and calculated percent suppression. Responders stained with proliferation dye, carboxyfluorescein diacetate succinimidyl ester (CFSE). Average suppressive potential over several independent experiments is quantified (right). Significance comparing the WT T_regs with following T_regs: IPEX, FOXP3 KO, FOXP3^FL edited, FOXP3^FLco edited, and FOXP3^FLcoW edited (mean ± SD, one-way ANOVA, Tukey’s multiple comparisons test, *P < 0.05; ****P < 0.0001; ns, not significant).