1). The concurrent expressions of CD25 and FOXP3, following expansion of CD4+CD25+CD127lo/− and CD4+CD25− cultures, were analysed and compared to each other, as seen in Fig. 5a–c. The cut-offs for the gates were set after the fluorescence of a biologically FOXP3 negative and CD25 negative population. Data was analysed using the FlowJo software (Tree Star) and expressed as mean fluorescence intensity (MFI; geometrical and standard mean) and percentages of cells expressing each marker. Tregs were PR-171 in vivo expanded according to a protocol adapted from Putnam et al.

[24]. Briefly, on day 0 sorted cells were resuspended in AIM-V medium (Gibco/Invitrogen) containing 10% HS and amphotericin B and plated according to Table 2. Dynabeads® Human Treg Expander anti-CD3/anti-CD28 coated microbeads (Invitrogen; catalogue number 111.61D) were added at a 1:1 bead to cell ratio. When Treg numbers were lower than 40.000, Anti-infection Compound Library 96-well flat-bottomed plates were used. The cell culture volume was doubled at day 2 and IL-2 (Proleukin, Chiron Therapeutics) added at a final concentration of 300 U/ml.

On days 5 and 7, cells were counted, washed in AIM-V and resuspended as above, adding fresh IL-2. Restimulation with anti-CD3/anti-CD28 coated microbeads, was performed on day 9, as described for day 0. Cells were counted again on day 11 and 13, washed, resuspended according to Table 2, and supplemented with fresh IL-2. Cultures were terminated on day 15 and cells stained for FOXP3 analysis. CD4+CD25− cells were expanded according to a scheme similar to Tregs, with the following alterations. As anti-CD3/anti-CD28 coated microbeads caused overstimulation and activation-induced apoptosis, CD4+CD25− cells were expanded using anti-CD3 (OKT3, 10 μg/ml) coated culturing vessels (Table 3) and soluble anti-CD28 (1 μg/ml). IL-2 addition was added at a concentration of 30 U/ml. As expression of Treg-markers was not normally distributed, two-group comparisons were performed with the Mann–Whitney U-test, while three or more groups were compared using the Kruskal–Wallis

test for unpaired observations. For pair-wise comparisons, Wilcoxon signed rank test was almost used. A probability level <0.05 was considered statistically significant. Calculations were performed using the statistical package GraphPad Prism version 5.01 for Windows (GraphPad Software, Inc.). The study was approved by the Research Ethics Committee of the Faculty of Health Sciences, Linköping University. Informed consent was obtained from all volunteers and/or their parents. Before sorting of Tregs, a small pre-study of healthy volunteering adults was performed assessing the stability of Treg-markers through cryopreservation and thawing. We did not find cryopreservation and thawing of PBMC to yield any differences in the percentage of FOXP3 expressing cells or the FOXP3 MFI, in the CD4+CD25hi cell population (Fig. 2a,b).