Fig 1.
Foxp3 expression upon use of different protocols for human iTreg differentiation.
(A) Experimental setup: Human naïve CD4 T cells were cultured for 6 days in serum-free medium under the indicated conditions. T cells were stimulated with anti-CD3 and anti-CD28 antibodies plus 100 U/ml IL-2 (“Stim.”). Where indicated, TGF-β1, rapamycin (Rapa), all-trans retinoic acid (ATRA) or butyrate were added. nTreg (ex vivo isolated peripheral blood CD25 high cells) were left unstimulated and used as positive control. (B) The histogram shows representative Foxp3 stainings for one donor, gated on CD4+ cells in the upper panel or on CD25++CD4+ cells in the lower panel. The striped line represents an isotype control staining (exemplarily shown for the condition “stim. + IL-2 + TGF- β + ATRA + Rapa“). Unstim. = unstimulated naïve T cells. (C) The boxplot shows percentages of Foxp3 positive cells measured by FACS as in (B) for n = 13 to 23 different donors in 8 to 13 independent experiments (n numbers are indicated below plot). The upper panel shows percentage of Foxp3+ cells from CD4+ cells, the lower panel of Foxp3+ cells from CD4+CD25++ cells. NA = not applicable. Significance was calculated by paired t test. n.s.: not significant, *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
Fig 2.
Expression of Treg signature molecules in human iTregs.
(A) CD25 surface expression was measured by surface staining and flow cytometry on day 6 of culture under the indicated conditions, gated on live CD4+ cells. Shown are mean +/- SEM values for n = 9 to 15 donors (n number indicated below plot). Significance was calculated with paired t test. The right panel shows representative CD25 histogram overlays; color code as in the left panel. (B) CTLA-4 expression (surface and intracellular) was quantified by staining of permeabilized cells and flow cytometry on day 6 of culture under the indicated conditions, gated on live CD4+ cells. Shown are mean +/- SEM values for n = 7 to 10 donors. Significance was calculated with paired t test. The right panel shows representative CTLA-4 histogram overlays; color code as in the left panel. Dashed line represents CTLA-4 isotype control (isotype example shown for stim. + IL-2 + TGF-β + ATRA sample). (C) IKZF4 (EOS) mRNA expression in naive T cells cultured 6 days under the indicated iTreg or control conditions. nTregs and unstimulated naive T cells were sampled on day 0. mRNA was quantified by Taqman assay, normalized to RPL13A expression. IKZF4 mRNA expression in unstimulated naive T cells from the corresponding donor was set to 1, and fold change of IKZF4 mRNA was calculated. Shown are mean +/- SEM values for n = 9to 10 donors. Significance was calculated with paired t test. (D) SATB1 mRNA expression in naive T cells cultured 6 days under the indicated iTreg or control conditions. nTregs and unstimulated naive T cells were sampled on day 0. mRNA was quantified by Taqman assay as described in (C). Shown are mean +/- SEM values for n = 4 to 6 donors. Significance was calculated with paired t test. n.s.: not significant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
Fig 3.
Cytokine production by human iTregs.
(A) IFN-γ expression was measured by intracellular staining after 4 hours of PMA/ionomycin pulse on day 6 of culture under the indicated conditions, gated on live CD4+ cells. Shown are mean +/- SEM values for n = 7 to 15 donors (n numbers indicated below the bar chart). Significance was calculated with paired t test. (B) IFNG mRNA expression in naive T cells cultured 6 days under the indicated iTreg or control conditions. nTregs and unstimulated naive T cells were sampled on day 0. mRNA was quantified by Taqman assay, normalized to RPL13A expression. IFNG mRNA expression in unstimulated naive T cells from the corresponding donor was set to 1, and fold change of IFNG mRNA was calculated. Shown are mean +/- SEM values for n = 7 to 11 donors (n numbers indicated below the bar chart). Significance was calculated with paired t test. (C) IL10 mRNA expression in naive T cells cultured 6 days under the indicated iTreg or control conditions. nTregs and unstimulated naive T cells were sampled on day 0. mRNA was quantified by Taqman assay as described in (B). Shown are mean +/- SEM values for n = 4 to 7 donors (n numbers indicated below the bar chart). Significance was calculated with paired t test. n.s.: not significant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.
Fig 4.
Analysis of Foxp3 stability and TSDR demethylation in human iTregs.
(A) iTregs were induced for 6 days under the indicated conditions, with 100 U/ml IL-2 if not otherwise stated. Foxp3 protein expression on day 6 is shown as solid, thin line. After 6 days, iTregs were either further cultured 2 days (thick line) or washed and rested in medium containing only IL-2, either 25 U/ml (dotted lines) or 50 U/ml (dashed lines). Left panels show the percentage of Foxp3+ cells, right panels show FACS histogram overlays for corresponding Foxp3 stainings. The “standard”conditions (100 U/ml IL-2 for induction, 50 U/ml IL-2 for resting) are marked by grey arrows. Gate: Live CD4+ cells. Shown is a representative donor (out of 4 to 6 for standard conditions; out of 2 for IL-2 titrations during induction). (B) TSDR DNA demethylation was analyzed at day 6 of Treg induction under the indicated conditions (left panel), or on day 8 after resting (two lanes on the right). Tnaive and nTreg DNA was isolated on day 0 as control. T cells were isolated from a male donor, and a representative donor out of 2 to 4 is shown. The color scale indicates 0 to 100% methylation at the given CpG nucleotides in the Foxp3 locus. (C) iTregs or control cells were cultured for 6 days under the indicated conditions, then rested 2 days, and further cultured for 5 days with restimulation, or without restimulation and without or with IL-2 (upper, middle and lower panel, respectively). nTregs were cryopreserved after isolation, thawed on day 6 and rested 2 days, and then treated in parallel with iTregs as a control. Intracellular Foxp3 expression was measured by flow cytometry, gated on live CD4+ cells. Shown is a representative donor out of 4.
Fig 5.
Comparative analysis of in vitro suppressive function of human iTregs generated with different iTreg-inducing protocols.
iTregs were induced 6 days under the indicated conditions, washed, rested 2 days, washed again, and then used to analyze their suppressive capacity towards Tresp. Suppression assays with iTregs or control cells were performed with CFSE-labeled CD3+CD25- Tresp. Tresp and iTregs (or control stimulated mock suppressor cells, grey line) were cocultured at the indicated ratios, or Tresp cultured alone (black) in different densities. nTregs (yellow) were used as control. Cultures were stimulated for 4 to 5 days and then analyzed by FACS. (A) shows a representative donor. It was gated on CFSE+ CD4+ Tresp (left panel) or CFSE+CD8+ Tresp (right panel). The upper plots show mean +/- SD of wells plated in replicate. The lower panel shows overlays of CFSE histograms for the 1:0.5 ratio. Unstim. = unstimulated; others are stimulated for 5 days with pb anti-CD3 and soluble anti-CD28 antibodies. Dotted black lines = 2:0 Tresp, solid black lines = 1:0 Tresp, filled histogram = unstimulated Tresp. Lines in same color represent cells plated in replicate wells. The gate for determining the percentage of proliferated cells is indicated by the horizontal line. (B) Shows percent suppression calculated from CFSE proliferation as in (A), with 2:0 Tresp set to 100% proliferation (0% suppression). Shown is the compiled data (mean +/- SEM) for n = 2 to 6 donors (n = 2 for 1:2 ratio; n = 4 for “butyrate”iTreg, all other iTregs: n = 6 donors). Significance was calculated by paired t test, comparing suppression by iTreg populations to mock suppressor cells (stimulated with anti-CD3/-CD28 and IL-2 only; grey line) within each donor at 1:1; 1:0.5 or 1:0.25 cell ratios. Asterisks indicate significant differences and are depicted in the color of the respective iTreg condition. *: p<0.05; **: p<0.01.
Fig 6.
Analysis of suppressive function of human iTregs in vivo in xenogeneic GvHD.
iTregs were induced 6 days under the indicated conditions, then washed and rested for 2 days. Allogeneic PBMCs were isolated and CD25-depleted the day before injection. NOG mice were irradiated and injected intravenously on the same day with 20x106 PBMCs alone or together with 5x106 iTregs or mock control T cells or expanded nTregs in PBS. As control, irradiated mice received PBS only. Mice were weighed every 1 to 3 days. (A) Shows percentage of mouse weight as mean +/- SEM for n = 4 to 8 mice per iTreg group combined from 2 independent experiments; mouse numbers (n) are indicated in the figure (B). (B) Shows the corresponding survival curves for the data from (A). There were no significant differences when comparing each iTreg group to either PBMC only or mock T cell groups.