1,25(OH)₂D₃- or TX527-exposed human T cells from control donors and type 1 diabetes patients can suppress autologous CD4 and CD8 T cell responses.

<p><b>A, B</b>: CFSE-labeled responder cells from control (Control, n = 5–7) and type 1 diabetes (T1D, n = 7–10) donors were stimulated for 4 days with anti-CD3/CD28 mAbs and co-cultured with autologous unsorted CD4⁺ (<b>A</b>) or sorted CD4⁺CD25^highCD127^low (<b>B</b>) T cell populations (day 8) from control-, 1,25(OH)₂D₃- or TX527-treated cultures, as indicated. Shown are bar graphs summarizing the percentage suppression of proliferation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109194#s2" target="_blank">Methods</a> section) of CD4⁺ (top) and CD8⁺ (bottom) T cells without or with Tregs at a 2∶1 (in case of unsorted CD4⁺ T cells, <b>A</b>) or 1∶1 (in case of sorted CD4⁺CD25^highCD127^low T cells, <b>B</b>) Treg:Tresponder ratio. Significance was tested using a two-tailed Mann-Whitney test, all not significantly different. * <i>P</i><0.05. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 reduce T helper cytokines in human T cell cultures.

<p>Human peripheral blood CD3⁺ T cells, isolated from control subjects (n = 19) and type 1 diabetes patients (n = 20), were activated using anti-CD3/CD28 and treated with vehicle (CTR), 10⁻⁸ M 1,25(OH)₂D₃ (1,25D₃) or 10⁻⁸ M TX527. Concentrations of indicated cytokines were determined in the supernatant of day 8 cultures. Results are shown as bar graphs of mean ± SEM, data are grouped per treatment and donor type. Significance was calculated using a two-tailed Mann-Whitney test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 reduce IFN-γ, IL-4, and IL-17 but increase IL-10 in expanded human CD4⁺CD25^highCD127^low T cells.

<p>Peripheral blood CD3⁺ T cells from control donors (n = 5) or type 1 diabetes patients (n = ) were cultured for 8 days in the presence of 10^-8 M 1,25(OH)₂D₃ (1,25D₃) or TX527 or corresponding concentration of vehicle (CTR). CD4⁺CD25^highCD127^low T cells were sort-purified and mRNA expression of IFN-γ, IL-4, IL-17, and IL-10 was quantified by real-time RT-PCR using B2M and RPL27 as normalization genes. Bar graphs represent the mean ± SEM. Significance was tested using a two-tailed Mann-Whitney test. *<i>P</i><0.05; **<i>P</i><0.01. All other comparisons were not statistically significant.</p

1,25(OH)₂D₃ and TX527 trigger a stable Treg phenotype in T cells from human type 1 diabetic patients.

<p>T cells from control subjects (Control) or type 1 diabetes patients (T1D) were cultured in the presence of vehicle (CTR; white boxes), 10⁻⁸ M 1,25(OH)₂D₃ (1,25D₃, grey boxes) or 10⁻⁸ M TX527 (black boxes). On day 6, the T cell cultures were exposed to normal T cell medium (left panel: <b>A-D</b>) or a cytokine cocktail (right panel: <b>E-H</b>) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109194#s2" target="_blank">methods</a> section. T cells were harvested 48 h later and stained for flow cytometry. Box and Tukey wisker plot summarizes the frequencies of positive cells in the CD4+ T cell gate. <b>A</b>: Surface expression of OX-40 (CD134) by activated CD4⁺ T cells of control donors (Control, n = 43) or type 1 diabetes patients (T1D, n = 58). <b>B</b>: Frequency of CD25^highCD127^low cells in the CD4⁺ T cell gate from control subjects (Control, n = 4) and type 1 diabetes patients (T1D, n = 7). CTLA-4 (<b>C</b>) and FOXP3 (<b>D</b>) expression in CD4⁺CD25^highCD127^low T cells of control donors (Control, n = 28) and type 1 diabetes patients (T1D, n = 45). <b>E</b>: Frequency of OX-40 expression on CD4⁺ T cells from control subjects (Control, n = ) or type 1 diabetes patients (T1D, n = 7) after additional stimulation with a cytokine cocktail. <b>F</b>: Frequency of CD25^highCD127^low cells in CD4⁺ T cells. CTLA-4 (<b>G</b>) and FOXP3 (<b>H</b>) expression in the CD4⁺ CD25^highCD127^low T cell gate. Data are grouped per donor type and treatment, cross-bars indicate median ± SEM. Significance was calculated using a two-tailed Mann-Whitney test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. All other comparisons were not significantly different.</p