Generation of human eTregs by transduction with FOXP3 and/or Helios. (A) Illustration of SFG retroviral vector containing genes of interest and transduction surface markers. (B-E) Transduction marker (ΔCD34 and ΔCD19), Helios, and FOXP3 protein expression in eTregs was assessed via surface and intracellular transcription factor staining and flow cytometry. Cells were assessed after the second transduction and magnetic bead purification for CD19. Graphs represent a summary of the percentage of eTregs positive for ΔCD34, ΔCD19, FOXP3, and Helios of total live cells (B) and geometric mean fluorescent intensity (GMFI) of FOXP3 and Helios of transduced cells gated on ΔCD19 expression (C); n = 3 to 9, and 6 different donors. Histograms and dot plots are representative figures of ΔCD34 and ΔCD19 expression following the second transduction and CD19 bead purification (D) and FOXP3 and Helios expression in ΔCD19⁺ transduced cells (E). (F) Representative figure of Helios messenger RNA expression assessed via real-time polymerase chain reaction and visualized via gel electrophoresis.*P ≤ .05 compared with no eTregs based on a 1-tailed Mann-Whitney U test.