Our center participated in a randomized, multi-center trial comparing sotrastaurin and the calcineurin inhibitor neoral in de novo renal transplant recipients [15]. We conducted an ex vivo study on patient samples (stage 1 phase) to investigate the frequency and function of FoxP3+CD4+CD25high T cells. We also performed in vitro functional studies on samples of blood bank volunteers to study the different effects of sotrastaurin on T effector and regulatory cells. Twenty-one patients were randomized to receive either sotrastaurin 300 mg twice daily (n = 14) or neoral [starting dose 4 mg/kg/day, aimed trough levels 100–200 ng/ml (month 1), 75–150 ng/ml (months 2–3), 50–100 ng/ml (months 4–5) and 25–50 ng/ml

(months 6–12), n = 7] 1 day after living (un)related de novo kidney transplantation. This cohort involved PCI-32765 molecular weight all (adult) patients in our center participating in an open-label, multi-centre, randomized Phase II trial [15] (trial number CAEB071A2206, stage 1) (Table 1). Both regimens included steroids, basiliximab [anti-CD25 monoclonal antibody (mAb)] and the mTOR-inhibitor everolimus [starting dose 1·5 mg twice daily, aimed trough levels 4–8 ng/ml)]. Patient blood CHIR-99021 clinical trial samples were collected pre-, 2, 3 and 6 months after transplantation. Blood sampling was approved by the local ethical committee on human research. All patients

gave written informed consent (Medical Ethic Committee number MEC-2007-219). Donor age in years median (range) Type of transplantation LR : LUR HLA mismatch mean ± s.e.m. A: 0·79 (0·15) B: 1·0 (0·21) DR: 1·07 (0·22) A: 0·71 (0·36) B: 0·57 (0·20) DR: 1·0 (0·22) Peripheral blood mononuclear cells (PBMC) from patient heparinized blood samples were isolated by density gradient using Ficoll-Paque (density gradient 1077 g/ml).

After isolation the PBMC samples were frozen in 10% dimethylsulphoxide (DMSO) (Merck, Schuchardt, IMP dehydrogenase Germany) and stored at −140°C until analysis. PBMC from healthy blood bank donors were also isolated and served as control. Neoral infusion (SandImmune®; Novartis Pharma, Switzerland) and sotrastaurin (Novartis Pharma) powder were dissolved in RPMI-1640 (Gibco BRL, Paisley, UK) and DMSO, respectively, and stored at −80°C until use. On the day of the experiment, stock solutions were dissolved in RPMI-1640. Defrosted PBMC were resuspended in cold magnetic-activated cell sorting (MACS) buffer according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) and supplemented with 7 μl CD25-microbeads (directed against epitope A of the CD25 molecule; Miltenyi Biotec)/107 PBMCs to isolate the CD25high T cells. After 15 min at 4°C, the cells were washed with MACS buffer and resuspended in 1 ml MACS buffer. Subsequently, the POSSEL-D protocol was performed on the autoMACS (Miltenyi Biotec). The CD4+CD25high population was defined as cells with high CD25 expression with a slightly lower CD4 expression.