Figure 7. Conditional Treg secrete very low levels of proinflammatory cytokines.

CD4⁺CD25⁻ cells purified from B6 spleens were transduced with cFoxp3 or empty vector ‘empty’. For comparison, Foxp3⁺ nTreg were isolated from B6.Foxp3^hCD2 mice by MoFlo sorting based on hCD2 expression. Negative control cells ‘Ctr’ were untransfected CD4⁺CD25⁻ cells. Transduced CD4⁺ cells were MoFlo sorted based on GFP expression 40 h after the onset of transduction and plated at 2×10⁵ cells/well in fresh medium on anti-CD3 coated 96-well plates with (black bars) or without (white bars) 4HT for a further 48 h. Supernatant was collected and analysed using a custom Bio-Plex Multiplex Cytokine Assay. For statistical analysis, one-way ANOVA with Dunnett’s comparison test was used (all groups compared to untransduced cells). *≤0.05, ***≤0.001. “>” equals more than 10⁵ pg/ml. Data represent technical triplicates plus and minus standard error of the mean. Data are representative of two separate experiments.