Isotype-matched control antibodies were used for assessment of background fluorescence. Multiple simultaneous cytokine detection for IL-2, IL-4, IL-6, IL-10, IL-17a, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was performed using the human T helper type 1 (Th1)/Th2/Th17 cytokine kit (BD Biosciences) on a MACS QuantTM analyser and MACS Quantify version 2.1 software (Miltenyi Biotec) as well as the FCAP Array software, version 1.0.1

(BD Biosciences). The assays were performed with undiluted supernatants and with supernatants diluted to 1:10 with PBS (Invitrogen). In addition, enzyme-linked immunosorbent assays Etoposide cell line (ELISA, n = 5 per group) were performed with commercial kits for detection of IL-1ra (BioSource Europe

SA, Nivelles, Belgium) and IL-1β and IL-8 (Invitrogen Corporation, Camarillo, CA, USA), according to the manufacturers’ protocols. Statistical analysis was performed using spss software (SPSS Inc., released 2009; PASW Statistics for Windows, version 18.0; SPSS Inc., Chicago, IL, USA). One-way analyses of variance (anova) followed by Bonferroni adjustment were performed to compare the different groups of lymphocyte cultures after creating interindividual differences for each patient. Differences were considered statistically significant for P-values smaller 0·05. Results are shown as Dactolisib order means ± standard deviation (s.d.). An important variation could be observed of Treg percentages after magnetic separation (Fig. 1a). Because of the donor-associated varying baseline

Treg percentages before co-culture, intraindividual differences between the Treg percentages after single- and co-cultures at day 5 and the initial Treg Etomidate percentage (day 0) were calculated in each group. There were no significant differences in CD4 expression (P = 0·522 between the groups) and in the percentages of CD4+CD25+ cells (P = 0·258) between the groups. Tregs were defined as CD4+CD25+CD127– or CD4+CD25+FoxP3 cells, respectively. The gating strategy is demonstrated in Fig. 1b. There was a negative correlation between CD127 and FoxP3 expression, the mean intraindividual difference between CD127– and FoxP3+ cells being 4·62 ± 6·31%. Both B-MSC– and S-MSC–lymphocyte co-cultures showed no significant changes in the Treg proportion, while we observed a significant decrease in the proportion of Treg in T cell monoculture (Fig. 2). This was the case for CD4+CD25+CD127– cells (Fig. 2a, P < 0·001 for both T cell single-culture versus B-MSC/T cell co-culture and S-MSC/T cell co-culture) and CD4+FoxP3+ cells (Fig. 2b, P = 0·006 for T cell single-culture versus B-MSC/T cell and P = 0·005 versus S-MSC/T cell co-cultures). There were no statistical differences between S-MSC/T cell and B-MSC/T cell co-cultures regarding CD127 and FoxP3 expression. The MSC effect on Treg-enriched CD4+ lymphocyte culture was independent of the T cell : MSC ratio (Fig. 2c).