Fig. 5

Effects of LV-mediated expression of STAT5B and BACH2 in in vitro-induced Treg cells. a–c Schematic structure of the LVs used to coordinately express the orange fluorescence protein (as marker transgene) and STAT5B (a) or BACH2 (b) or the control vector-expressing GFP and dNGFR c. Transgene transcript is indicated by arrows. d Scheme of the experimental strategy. CD4⁺ naïve T cells from PBMC or cord blood mononuclear cells of HDs were activated by anti-CD3/CD28 beads for 24 h, transduced with the indicated LVs, and cultured in presence of IL-2 and TGF-β for 2 weeks; e Relative percentage of GFP⁺ or Orange⁺ cells (for bidirectional SIN LVs encoding for BACH2 or STAT5B) of naïve CD4⁺ T cells transduced with the indicated vector and analyzed 5 days after transduction, N = 5; f Percentage of CD25⁺FOXP3⁺ cells in CD4⁺ T cells transduced with the indicated vector and untransduced cells after 2 weeks of culture, N = 5; g Inhibition of proliferation (analyzed by flow cytometry for e-Fluor dilution and indicated as % suppression vs. responder alone) of anti-CD3/CD28 activated allogeneic Responder cells (R) cultured in presence of different doses (Ratio R:S were 1:1 and 1:0.25) of CD4⁺CD25⁺ suppressive cells (S) purified from cells transduced or not with the indicated LVs, N = 5; h Proliferation rate (measured by [3 H]-thymidine incorporation) of purified CD25⁺ cells from T cells transduced or not with the indicated vector and activated with anti-CD3 CD28 in the absence (black bar) or presence (orange bar) of IL-2. Fold increase vs. unstimulated control cells (N = 3) is shown, N = 5. Data are represented as Mean ± SEM. Significance was determined by one-way ANOVA with Bonferroni correction (*p < 0.05; **p < 0.01).