Figure 1.
1,25(OH)2D3 and TX527 reduce T helper cytokines in human T cell cultures.
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. * P<0.05; ** P<0.01; *** P<0.001. All other comparisons were not statistically significant.
Figure 2.
Induction of CCR10 and CXCR6 and reduction of CLA on activated T cells.
Expression of CCR10, CLA or CXCR6 by CD4+ (A) and CD8+ T cells (B) after activation of T cells from control subjects (Control, n = 33) or type 1 diabetes patients (T1D, n = 45) in the presence of vehicle (CTR), 10−8 M 1,25(OH)2D3 (1,25D3) or 10−8 M TX527. On day 8, cells were harvested and stained for flow cytometry. Shown are box and Tukey whisker plot summarizing the frequencies of positive cells in the CD4 or CD8 gate respectively, data are grouped per donor type and treatment, cross-bar indicates median. Significance was calculated using a two-tailed Mann-Whitney test. * P<0.05; ** P<0.01; *** P<0.001. All other comparisons were not statistically significant.
Figure 3.
1,25(OH)2D3 and TX527 trigger a stable Treg phenotype in T cells from human type 1 diabetic patients.
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: A-D) or a cytokine cocktail (right panel: E-H) as described in the methods 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. A: 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: Frequency of CD25highCD127low cells in the CD4+ T cell gate from control subjects (Control, n = 4) and type 1 diabetes patients (T1D, n = 7). CTLA-4 (C) and FOXP3 (D) expression in CD4+CD25highCD127low T cells of control donors (Control, n = 28) and type 1 diabetes patients (T1D, n = 45). E: 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. F: Frequency of CD25highCD127low cells in CD4+ T cells. CTLA-4 (G) and FOXP3 (H) expression in the CD4+ CD25highCD127low 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. * P<0.05; ** P<0.01; *** P<0.001. All other comparisons were not significantly different.
Figure 4.
1,25(OH)2D3 and TX527 reduce IFN-γ, IL-4, and IL-17 but increase IL-10 in expanded human CD4+CD25highCD127low T cells.
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+CD25highCD127low 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. *P<0.05; **P<0.01. All other comparisons were not statistically significant.
Figure 5.
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.
A, 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+ (A) or sorted CD4+CD25highCD127low (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 Methods section) of CD4+ (top) and CD8+ (bottom) T cells without or with Tregs at a 2∶1 (in case of unsorted CD4+ T cells, A) or 1∶1 (in case of sorted CD4+CD25highCD127low T cells, B) Treg:Tresponder ratio. Significance was tested using a two-tailed Mann-Whitney test, all not significantly different. * P<0.05. All other comparisons were not statistically significant.