Development of a unique epigenetic signature during in vivo Th17 differentiation

abstract: ...Activated naive CD4 + T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN- (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4 + T cells, Th1 and Th17 cells. We could demonstrate that naive CD4 + T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.

..., Th1 and Th17 cells. We could demonstrate that naive CD4 + T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex

...-seq data sets. Next, we screened the MBD-seq data for regions showing valid signals in all samples except Th17 cells to identify Th17-specific demethylated regions. Note that 195 candidate regions were tested by MS-HRM analyses (data not shown). Highest melting-curve differences between Th17 cells and all other T cell subsets were observed for regions within Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 (Figure 5 ). Most of these regions are located in introns, except for the region from Acsbg1 , which covers transcribed exons, and from Ccr6 that spans promoter, start site and the first non-translated exon. To get more detailed information on the methylation status of individual CpG motifs, we performed pyrosequencing for the regions within Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 and could observe a more pronounced demethylation in Th17 cells when compared to naive T cells, Th1 cells and Tregs (Figure 6A , Supplementary Figures S5 and Supplementary Table S3). Interestingly, the regions within Il17a, Zfp362, Ccr6, Dpp4 and Rora showed a more or less homogeneous demethylation of the majority of CpG motifs analyzed, whereas the demethylation within Acsbg1 and Dclk1 is restricted to a subpart of the region or even to a single CpG motif, respectively. It is worth mentioning that also ex vivo isolated Th1 cells and Tregs showed signs of demethylation in some of the regions analyzed, but less pronounced when compared to Th17 cells and restricted to individual CpG motifs only (Figure 6A , Supplementary Figure S5 and Supplementary Table S3). Interestingly, in vitro generated Th17 cells did not show any demethylation within Ccr6, Acsbg1, Dpp4, Rora and Dclk1 when compared to naive T cells (Figure 6A and B and Supplementary Table S3). Only Il17a and Zfp362 showed some signs of demethylation albeit less pronounced when compared to ex vivo isolated Th17 cells ( Il17a ) or restricted to only subparts of the region ( Zfp362 ). In vitro generated Th1 cells and Tregs, which were analyzed as controls, showed a methylation pattern comparable to naive T cells in any of the regions being analyzed (Figure 6A and B and Supplementary Table S3). Together, these data suggest that ex vivo isolated and in vitro generated Th17 cells represent different developmental states. Figure 5. Candidate regions Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 are differentially methylated. Locus organization and CpG motif distribution are shown for six candidate regions. The methylation status of marked

... performed by pyrosequencing using genomic DNA from the indicated (A) ex vivo isolated and (B) in vitro generated T cell subsets. The methylation rates were translated into a color code from yellow (0%) via green (50%) up to blue (100%). Each rectangle represents the methylation of one CpG motif. Data from three independently performed cell sorts and cultures are depicted. To unravel whether the demethylation of regions within Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 in ex vivo isolated Th17

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