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Blood, Vol. 112, Issue 6, 2340-2352, September 15, 2008
Human CD25highFoxp3pos regulatory T cells differentiate into IL-17–producing cells
Blood Koenen et al.
112: 2340
Supplemental materials for: Koenen et al
Files in this Data Supplement:
- Figure S1. Dotplots show forward and side scatter plots of cultured CD25highCD27posTreg for the experiments displayed in Fig. 4A (A) and Fig. 6A (B) (JPG, 112 KB)
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The live cell percentages based on cell-size are given.
- Figure S2. TGFβ does not enhance differentiation of CD25highCD27posTreg into IL-17 producing cells (JPG, 128 KB)
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Flow cytometry of sorted CD25highCD27posCD45RAneg CD4pos (CD25highCD27pos) Treg and CD25negCD27posCD45RAneg CD4pos (CD25negCD27pos) Teff that were stimulated with allogeneic PBMC in the absence or presence of rIL-2, rTGFβ, rIL-6, or combinations thereof. Density plots show intracellular IL-17 and Foxp3 expression at day 8 of the cultures after re-stimulation of the cells with PMA plus ionomycin in the presence of Brefeldin A. Numbers at the top of the plots indicate the percentages of IL-17 producing cells. Data are shown from 2 different donors.
- Figure S3. Explanatory schematic overview for Fig. 7D and 7F (JPG, 80.9 KB)
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- Figure S4. Schematic representation and mechanistic explanation of our findings (JPG, 42.8 KB)
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Illustration summarizes the essence of our findings. In short, the differentiation of Treg into CCR6 positive IL-17 producing cells in our hands requires T-cell receptor mediated activation by antigen presenting cells, especially monocytes, in the presence of rIL-2 and/or rIL-15. This process is enhanced by the addition of exogenous IL-1β, IL-23 and IL-21 (IL-21 was effective in 50% of our experiments). In contrast, the addition of IL-1 receptor antagonist (IL-1RA) led to a decrease in the number of IL-17 producing cells. Addition of rIL-6 or rTGFβ to these cultures did not affect the emergence of IL-17 producing cells. Interestingly, in our standard culture system using stimulation with allogeneic PBMC and rIL-2+rIL-15, neutralization of IL-23 by an anti-IL23 antibody (mAb) did not have an effect on the number of IL-17 producing cells generated. When an HDAC inhibitor (TSA) was evaluated, we found a profound negative effect on the emergence of IL-17 producing cells from Treg, implying that Treg differentiation into IL-17 producing cells depends on histone/protein deacetylase (HDAC) activity. Indeed, the data suggest that epigenetic modification underlies the phenomenon of Treg plasticity here described.
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