Figure 5.

The impact of rapamycin concentrations and α‐CD3/C28 bead ratios on iTreg functionality. Following a 7‐day stimulation and a 3‐day rest, cells were stimulated using a 1:10 α‐CD3/CD28 beads and low‐dose IL‐2. After 3 days of stimulation, (a) CLTA4 gene expression, (b) IL2 gene expression, production of (c) active TGF‐β, (d) IL‐4, IL‐6, IL‐10, TNF‐α, IFN‐γ and IL‐17A were measured. For (c and d), the cells were stimulated with PMA and ionomycin for 6 hours prior to collection of cell supernatants. Cell‐free medium was used as a control to measure background cytokine levels in the medium which was supplemented with heat‐inactivated human serum. Background cytokine levels in the medium were negligible (below the lowest standards). Data are represented as mean ± sem, N = 3 in three independent experiments (a and b) or in one independent experiment (c and d). For each donor (N), technical triplicates (a and b) or technical duplicates (c and d) were utilised, and the average of technical replicates was used for each datapoint. Statistical significance identified by RM one‐way Dunnett’s multiple comparisons test: *P < 0.05, **0.01, ***0.001, ****0.0001.