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Figure S1. Methylation status of the IFNG gene locus in ex vivo isolated (A) CD8+ T-cell subsets and (B) NK-cells. CD8+ T cells were purified by MACS positive selection using CD8 microbeads, followed by FACS sorting of CD3+ (PE; house conjugate), CD4 (PerCP; BD Biosciences) and DAPI cells. Naïve (CD45RA+CD27+), central memory (CD27+CD45RA), and effector memory (CD45RA+CD27) CD8+ T-cell subsets were further sorted using the following Abs: anti-CD27 FITC (BD Biosciences), -CD45RA allophycocyanin (Caltag). NK-cells were sorted by enrichment of CD56+ cells using a NK cell isolation kit (Milenyi Biotec) and in conjunction with FACS sorting on CD3 (FITC; house conjugate) and CD56+ (PE; Miltenyi) cells. The purity of sorted populations was 95–99%, as assessed by FACSCalibur or LSRII using CellQuest (BD Biosciences) or Flowjo (Tree Star) software. DNA methylation levels were determined as described previously1. Each rectangle represents one individual CpG and each row represents the methylation status of one donor.

Figure S2. Methylation status of a CNS region (2.4 kb upstream from the TSS, CNS-2.4) of IFNG. (A) No differential methylation of CNS-2.4 in ex vivo CD4 and CD8 T cells and NK cells. Cells were purified and methylation status analyzed as described in supplementary figure 1. (B) No dynamic changes in methylation of a CNS-2.4 during early Th1 differentiation. Fraction of cells from the time course analysis (Fig. 3) was subjected to bisulfate clone sequencing. For each time point, 30 to 35 clones were analyzed and data dipicted by using BISMA software (http://biochem.jacobs-university.de/BDPC/BISMA/).

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