PMC

RLM-11–1 cells were transfected with control vector (pGL3 promoter) or Foxp3-CE vector containing the conserved element (CE). Cells were stimulated with PMA for 24 h. Control cells were left unstimulated. Results given are relative luciferase light units (RLUs) normalized for Renilla luciferase activity (mean ± standard deviation; n = 3). Results are representative for two independent experiments.

(A) CD25⁻CD4⁺ T cells were cultured for 6 d in the presence of TGF-β as described in . On day 6, cultured cells were sorted by FACS for CD25⁺ cells and analyzed for intracellular Foxp3 expression by FACS (upper panel). Sorted CD25⁺ cells from the same TGF-β-cultures were restimulated for another 6 d in the absence of TGF-β. On day 6, cultured cells were analyzed for intracellular Foxp3 expression by FACS (lower panel). Numbers display frequency of cells within indicated populations.
(B) Ex vivo isolated CD25⁺CD4⁺ Tregs were analyzed for intracellular Foxp3 expression before (upper panel) and after 6 d in vitro culture (lower panel) by FACS. Numbers display frequency of cells within indicated populations. One representative experiment out of two individual experiments is shown.

(A) CD25⁺CD4⁺CD8⁻ and CD25⁻CD4⁺CD8⁻ subsets were sorted from thymocytes pooled from 30 male BALB/c mice. FACS analysis shows purity of sorted subsets (upper panel) and Foxp3 expression in gated CD25⁻ and CD25⁺ subsets of CD4⁺ SP thymocytes (lower panel). Numbers display frequency of cells within indicated populations. The bars in the lower graphs indicate the marker gate for Foxp3⁺ cells. The vertical and horizontal lines in the upper graphs indicate the quadrant used to identify the CD4⁺CD25^+/− subsets.
(B) Methylation pattern of selected amplicons of the foxp3 locus in CD25⁺ and CD25⁻ CD4 SP thymocytes. The methylation status of individual CpG motifs within the four amplicons is color coded as described in . One representative experiment out of two individual experiments is shown.

ChIP assays were performed with CD25⁺CD4⁺ Tregs (filled bars) and conventional CD25⁻CD4⁺ T cells (open bars) sorted from spleens and LNs pooled from 20 male BALB/c mice. DNA fragments binding to acetylated or trimethylated histones were immunoprecipitated using antibodies directed against acetylated histone H3, acetylated histone H4, or trimethylated histone H3 at position K4. A rabbit isotype immunoglobulin G (IgG) served as control. Precipitated DNA was quantified by real-time PCR with primers specific for the differentially methylated region of the foxp3 locus. Sample PCR products were set in relation to input DNA. One representative experiment out of two individual experiments is shown.

CD25⁺CD4⁺ Tregs were isolated ex vivo from pooled spleen and LN single-cell suspensions, labeled with CFSE, and then transferred into syngenic 8-wk-old recipients (2 × 10⁶/mouse). Before transfer, sorted cells were analyzed for intracellular Foxp3 expression by FACS. Fourteen days after transfer, splenocytes of recipient mice were stained for CD4, CD25, and Foxp3, and analyzed by FACS. About 0.1% of total splenocytes and, accordingly, 3% of total Foxp3⁺ splenocytes were donor derived (CFSE⁺). Representative dot and histogram plots from four independently analyzed mice were selected. Numbers display frequency of cells within indicated populations. Comparable results were obtained for cells isolated from peripheral or mesenteric LNs (unpublished data). The bars in the left and right graphs indicate the marker gate for Foxp3⁺ cells. The box in the middle graph indicates the region that was used to gate for CD4⁺CFSE⁺ cells. These gated cells (CFSE⁺) are depicted in the right graph.

(A) CD25⁻CD4⁺ T cells sorted from spleens and LNs pooled from 20 male BALB/c mice were cultured for 6 d in the presence of TGF-β. Cells cultured under Th1 conditions were used as control. Within the starting population, less than 1% of the cells were Foxp3⁺ (unpublished data). On day 6, cultured cells were sorted by FACS for CD25⁺ cells. FACS analysis shows the purity of sorted subsets (upper panel) and Foxp3 expression in gated CD25⁺ cells derived from indicated cultures (lower panel). Numbers display frequency of cells within indicated populations. The bars in the lower graphs indicate the marker gate for Foxp3⁺ cells. The vertical and horizontal lines in the upper graphs indicate the quadrant used to identify the CD4⁺CD25^+/− subsets.
(B) Methylation pattern of selected amplicons of the foxp3 locus in sorted CD25⁺ cells derived from indicated cultures. The methylation status of individual CpG motifs within the four amplicons is color coded as described in . One representative experiment out of two individual experiments is shown. n.a., not analyzed.

(A) Schematic view on the foxp3 locus depicts exon-intron structure and position of selected amplicons (Amp 1–4). Shown is the distribution and position of individual CpG motifs within the amplicons.
(B) CD25⁺CD4⁺ and CD25⁻CD4⁺ T cells were sorted from spleens and LNs pooled from 20 male BALB/c mice. FACS analysis shows the sort purity (upper panel) and Foxp3 expression in sorted subsets (lower panel). Numbers display frequency of cells within indicated populations. The bars in the lower graphs indicate the marker gate for Foxp3⁺ cells. The vertical and horizontal lines in the upper graphs indicate the quadrant used to identify the CD4⁺CD25^+/− subsets.
(C) Methylation pattern of selected amplicons of the foxp3 locus in CD25⁺CD4⁺ Tregs and conventional CD25⁻CD4⁺ T cells. The amplicons are subdivided by horizontal lines each representing an individual CpG motif. ESME software [] is used for normalization and quantification of methylation signals from sequencing data by calculating ratios of T and C signals at CpG sites. Data are condensed to methylation information at CpG positions forming matrices of consecutive CpGs. The methylation status of individual CpG motifs within the four amplicons is color coded according to the degree of methylation at that site. The color code ranges from yellow (0% methylation) to blue (100% methylation) according to the color scale on the right. CpG motifs from amplicon 2 overlapping with motifs in amplicon 1 were excluded. Due to sequencing problems, the CpG motif 37 from amplicon 4 is not listed. One representative experiment out of two individual experiments is shown.