DNA methylation status in the promoter and intronic CpG island region of the <i>Foxp3</i> gene.

<p>(A) The purity of the isolated Treg cells, sorted from the spleens of male mice, was confirmed by staining the intracellular expression of Foxp3 and analysis by flow cytometry. (B) DNA methylation status in the indicated regions was determined by bisulfite sequencing. Each line represents one analyzed clone; open circles, unmethylated CpGs and filled circles, methylated CpGs.</p

Stability of Foxp3 expression during <i>ex vivo</i> culture.

<p>(A) Treg cells, sorted from HBZ-Tg or non-Tg mice, were cultured in the presence of IL-2 for 3 or 7 days. The expression of Foxp3 was analyzed by flow cytometry. (B) Sequential changes of the Foxp3⁻ population are shown. (C) IFN-γ production of <i>ex vivo</i> cultured Foxp3⁺ cells was evaluated by intracellular staining. Sorted Treg cells were cultured for 7 days, and then stimulated for 4 h with PMA/ionomycin and protein transport inhibitor. (D) Foxp3 expression of sorted CD4⁺CD25⁺GITR^highthymocytes from HBZ-Tg mice.</p

Expression of CD11a, CD18 and CD103 in CD4⁺T cells from spleen, lung and LN cells isolated from HBZ-Tg mice.

<p>(A) The expression of CD11a, CD18 and CD103 in CD4⁺ T cells from non-Tg (dashed line) and HBZ-Tg (solid line) mice was analyzed by flow cytometry. Histograms from one representative mouse splenocytes of each group are shown (top panels). The bottom panel shows the results of 4 or 6 mice in each group, each symbol representing an individual mouse. The small horizontal lines indicate the mean. Frozen sections of intestine (B) and lung (C) of non-Tg and HBZ-Tg mice were stained with HE and the indicated antibodies. Original magnification is ×20. Results from one representative mouse of each group are shown. (D) CD11a, CD18 and CD103 expressions are shown on CD4⁺ cells from HDs, CD4⁺Tax⁻ and CD4⁺Tax⁺ cells from HAM/TSP patients.</p

Production of cytokines in HBZ-Tg mice.

<p>(A) Splenocytes of HBZ-Tg mice or non-Tg mice were stimulated with PMA/ionomycin and protein transport inhibitor for 4 h. IFN-γ, IL-17, TNF-α, IL-4 or IL-2 production was analyzed in CD4⁺ T cells by flow cytometry. (B) Cytokine production was analyzed along with Foxp3 expression. (C) Production of cytokines was shown in CD4⁺Foxp3⁺ T cells and CD4⁺Foxp3⁻ T cells. (D) IFN-γ and Foxp3 expression gated on CD4⁺ T cells from PBMC or cells isolated from the lungs were analyzed by flow cytometry. Percentage of IFN-γ⁺ cells in CD4⁺ splenocytes, PBMC and lung cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean.</p