For sotrastaurin-treated patients the absolute number of FoxP3+CD127low Tregs remained stable: median numbers were 23, 16 and 28 cells/μl pre-, 3 and 6 months after transplantation (Fig. 4b). In neoral-treated patients, the number of FoxP3+CD127low Tregs decreased significantly at month 3 but returned to levels pretransplantation at month 6 (median check details 12 and 18 cells/μl 3 and 6 months after transplantation, P = 0·008 for neoral 3 months versus pretransplantation (Fig. 4b). Trough levels of sotrastaurin correlated with Treg numbers: the AUC of trough levels at 0–3 months

correlated with the absolute number of FoxP3+CD127lowCD4+CD25high T regulatory cells at 3 months (n = 10, Pearson’s r = 0·65, P = 0·04). The AUC of trough levels at 0–6 months also AZD1208 supplier correlated with Treg numbers at 6 months (n = 10, Pearson’s r = 0·68, P = 0·03) (Fig. 5). The functional capacity of patients’ effector and regulator T cells was tested in MLR. Of two sotrastaurin- and two neoral-treated patients, the number of isolated CD4+CD25high T cells was insufficient to determine their inhibitory capacity at each timepoint. The proliferative response of effector CD25low cells in samples of three patients was <1000 cpm. Co-culture experiments with isolated CD4+CD25high Tregs in a 1 : 10 ratio were performed (n = 4 sotrastaurin and n = 6 neoral). We analysed whether

co-culture with Tregs and time after transplantation influenced the proliferation in sotrastaurin- versus neoral-treated patients. The inhibitory capacity of Tregs in sotrastaurin-treated patients

remained intact: the median percentages of inhibition by Tregs were 82% pretransplantation, 71% at 3 months and 67% at 6 months against donor cells (months 3 and 6, P > 0·05 due to small sample size) (Fig. 6). The protein kinase C inhibitor sotrastaurin is a novel, calcineurin-independent drug in autoimmune disease, oncology and clinical organ transplantation. Currently, the effect of sotrastaurin Chlormezanone on cell populations that control immune responses is unknown. We therefore investigated the number and function of regulatory T cells in samples of healthy volunteers and in renal allograft recipients during sotrastaurin treatment. First, we determined the IC50 of sotrastaurin in MLR to confirm previous reported findings: Evenou et al. have shown that sotrastaurin potently inhibited alloreactivity of mouse and human T cells [6]. In a two-way MLR performed with human T cells, the IC50 of sotrastaurin to inhibit [3H]-thymidine incorporation after 6 days was 37 nM. The studies by Matz et al. also revealed dose-dependent inhibition by sotrastaurin of carboxyfluorescein succinimidyl ester (CFSE)-labelled CD4+ T cells, after allogeneic stimulation [17]. In one-way MLR with irradiated stimulator cells, we demonstrated that sotrastaurin blocked alloreactivity dose-dependently (Fig. 1). In the high concentration of 250 ng/ml, the mean percentage of inhibition was 92%. The mean IC50 of sotrastaurin in our experiments was 89 nM.