TCR-triggered IL-15-expanded CD4⁺ T cells remain dependent on IL-15 for expansion and survival.

<p>CD4⁺ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdACs without or with 3 ng/ml IL-15. After 3 days, 50 ng/ml soluble IL-15Rα (sIL-15Rα, T1, 50 ng/ml) or control mutated sIL-15Rα (M4), lacking IL-15 binding capacity, were added. (<b>A</b>) Proliferation of CD4⁺ T cells, as determined by ³H-thymidine incorporation of triplicate cultures on day 5. (<b>B</b>) Viable CD4⁺ cell recovery on day 5, determined by flow cytometry. Bar graphs represent the mean±SEM. Statistical significance was calculated using student t-test: ns = not significant, ** <i>P</i><0.01, *** <i>P</i><0.001. Data are representative of three independent experiments.</p

CD25^High CD4⁺ T cells originate from natural CD25⁺ CD4⁺ T cells.

<p>(<b>A</b>) Histogram showing CD25 expression pattern of unstimulated CD4⁺ T cells (day 0; top). Isolated CD25⁻ and CD25⁺ CD4⁺ T cells were co-cultured in a cross-over setup with only one of the subsets carrying CFSE label. Three days after stimulation with 0.1 µg/ml anti-CD3 mAb and irradiated TdAC, CD25 expression and CFSE dye dilution were measured using flow cytometry and displayed as density plots (bottom). (<b>B</b>) CFSE-labeled total CD4⁺ T cells (top) or CD25⁺-depleted CD4⁺ T cells (bottom) were stimulated as in (<b>A</b>). Shown are flow cytometry density plots of CFSE dye dilution versus CD25 expression gated on viable CD4⁺ T cells. Percentages indicate the percentage of CD25^High or CD25^Int cells within all CD25⁺ positive CD4⁺ T cells. (<b>C</b>) Contour plots showing CD25 versus Foxp3 expression of unstimulated and day 4-stimulated CD4⁺ T cells. Numbers on the right reflect the percentages within the gates 1–5, gated on viable CD4⁺ T cells. (<b>D</b>) CFSE-labeled total CD4⁺ T cells (shaded) or CD25⁺-depleted CD4⁺ T cells (solid line) were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for 3 days. Shown are histogram overlays gated on viable CD4⁺ T cells and bar graph of proliferation index mean±SEM. (<b>E</b>) Total CD4⁺ T cells or CD25-depleted CD4⁺ T cells were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for indicated time and proliferation was measured via ³H-thymidine incorporation. (<b>B, D, E</b>) Bar graphs represent the mean±SEM. Statistical significance was calculated by student t-test, ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. Experiments performed at least 3 times with similar results.</p

IL-15 blocks suppression by CD25^High CD4⁺ T cells.

<p>(A) CD4⁺ T cell numbers and (B) proliferation of total CD4⁺ T cells (white bars) or CD25-depleted CD4⁺ T cells (grey bars) stimulated with anti-CD3 mAb, irradiated TdACs and supplemented or not with IL-15. Data are expressed as absolute values (left panels) or fold increase of IL-15-supplemented versus non-supplemented cultures (right panels; 1 reflects no effect of IL-15 addition). Bar graphs represent the mean ± SEM. Statistical significance was calculated by student t-test, ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. (C) CD25⁻ CD4⁺ responder T cells (Tresp) were co-cultured with CD25^High (left) or CD25^Int (right) CD4⁺ T cells, FACS-purified from activated CD4⁺ T cell cultures as described in Materials and Methods, as putative suppressors at indicated ratios, prior to stimulation with 0.1 µg/ml anti-CD3, irradiated TdACs, and indicated cytokines for 3 days. Proliferation was measured via ³H-thymidine incorporation and the percent suppression was calculated as follows: % suppression = 100×(cpm_Responder – cpm_Coculture)/cpm_Responder. Statistical significance between IL-15-supplemented and non-supplemented cultures was calculated using two-way ANOVA with Bonferroni posttest: ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 (statistical analysis versus IL-2-supplemented cultures not shown). All experiments were performed at least 3 times with similar results.</p

Exogenous IL-15 decreases the fraction but not the absolute number of CD25^High CD4⁺ T cells.

<p>(<b>A</b>) IL-15 alters TCR-induced gene expression of IL-2 and IL-15 receptor subunits. Purified CD4+ T cells were stimulated with 0.1 µg/ml anti-CD3 mAb and irradiated TdACs in the absence (white circles) or presence (black circles) of 3 ng/ml IL-15. At the indicated time points, CD4⁺ T cells were purified for PCR analysis of indicated gene transcripts and expressed as 2^−ΔCt where ΔCt = Ct_{target gene} - mean Ct_{normalization genes}. Statistical significance was calculated using student t-test: ns = not significant, ** <i>P</i><0.01, *** <i>P</i><0.001. (<b>B</b>) Spleen CD4⁺ T cells were left unstimulated or stimulated with anti-CD3 in the presence or absence of IL-15 for two days, as indicated. Shown are histogram overlays of CD25 expression, gated on viable CD4⁺ T cells (d: day). (<b>C</b>) CFSE-labeled CD4⁺ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdAC, without or with 3 ng/ml IL-15 for four or five days. Shown are flow cytometry dot plots of CFSE dye dilution versus CD25 expression in the viable CD4⁺ gate (left, shown percentages were calculated on viable CD4⁺ T cells) and summarizing bar graph of the CD25 high fraction (left). Statistical significance was calculated using student t-test: * <i>P</i><0.05, ** <i>P</i><0.01. (<b>D</b>) Absolute numbers of viable CD25^High, CD25^Int, CD25^Negative and total CD4⁺ T cells, as determined by flow cytometry using micro-beads as reference. White and black bars represent stimulation with anti-CD3 in the absence or presence of 3 ng/ml rhIL-15, respectively. Statistical analysis was calculated using 2-way ANOVA and Bonferroni posttest: ns = not significant, *** <i>P</i><0.001. Data shown are representative of three independent experiments. (<b>E</b>) CFSE-labeled CD4⁺ T cells were stimulated as in <i>B</i>. Shown are histogram overlays (top) of CD25 expression of the last generation of viable CD4⁺ T cells (by CFSE dilution) on day 5 after stimulation in the absence (dashed line) or presence (shaded) of 3 ng/ml rmIL-15. Bar graph (bottom) represents the mean fluorescenceIntensity of CD25 expression in the last generation, as shown in the overlays. Statistical significance was calculated using student t-test: * <i>P</i><0.05, ** <i>P</i><0.01.</p

IL-15 growth factor activity depends on TCR-signals, but is IL-2-dependent only in CD4⁺ T cells.

<p>(<b>A</b>) CD8⁺ or (<b>B</b>) CD4⁺ T cells were stimulated with anti-CD3 mAb and irradiated TdACs in the absence or presence of 3 ng/ml rhIL-15, or 10 µg/ml anti-IL-2 mAb (JES6-5H4) or isotype control, as indicated. On day 4, ³H-thymidine incorporation was measured. Results are from one of five experiments. (<b>C</b>) IL-2^+/+ or IL-2^−/− CD4⁺ cells were stimulated with anti-CD3 mAb and irradiated IL-2^+/+ and IL-2^−/− splenocytes, respectively, in the absence (white circles) or presence of 1 ng/ml IL-2 (crosses) or 3 ng/ml IL-15 (black circles). (<b>D</b>) CD4⁺ cells were stimulated for 5 days with 0.1 µg/ml anti-CD3 and irradiated TdACs. On day 0, 1, 2, or 3, IL-15 alone or IL-15 plus anti-IL-2 mAb was added. On day 4, proliferation was measured by ³H-thymidine incorporation and represented as bar graphs (with left Y-axis) as ratio of cpm_IL-15/cpm_none (black bars) or cpm_{IL-15 plus anti-IL-2}/cpm_none (white bars). The line represents the percentage of reduction of the IL-15 effect by IL-2 depletion as calculated using the following formula: [(cpm_IL-15– cpm_{IL-15 plus anti-IL-2})/cpm_IL-15]×100. Results are from one representative out of three experiments.</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- 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 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