Figure 5. Intracellular expression of IL-17 and IFN-γ and production of IL-17 by ex vivo expanded Tregs.

A. Frequency of IL-17⁺ in CD4⁺CD25⁺FOXP3⁺ Tregs upon isolation and post culture in the presence of rapamycin (day 36) when exposed to two separate mixes of pro-inflammatory cytokines (Mix 1: IL-2, IL1β, IL-6 and TGF-β and Mix 2: IL-2, IL-21, IL-23 and TGF-). B. IL-17 (pg/ml) concentration in a 5-day culture supernatant of the rapamycin expanded Tregs in the presence of Mix1 and Mix2. C. Frequency of IFN-γ⁺ in CD4⁺CD25⁺FOXP3⁺ Tregs upon isolation and post ex vivo expansion in the presence of rapamycin (day 36) when exposed to Mix 1 and Mix 2. D. Gating strategy and Frequency of CD161⁺ Tregs throughout culture. Dot plot depicts expression of CD161 on CD4⁺CD25⁺ Tregs from a representative sample of 9 ARC patients. Graph shows the dynamics of CD161 expressing Tregs throughout culture. E. Gating strategy and Frequency of CCR6⁺CD161⁺ Tregs throughout culture. Dot plot of CD161⁺CCR6⁺ co-expression on CD4⁺CD25⁺ Tregs from a representative sample of 9 ARC patients and graph of percentage CD161⁺CCR6⁺ co-expression throughout culture. n.s.-not significant. *P < 0.05,**P < 0.01. ***P < 0.001. n = 9 ARC patients. Data are represented as mean +/− SEM.